InteractiveFly: GeneBrief

Resistant to dieldrin: Biological Overview | References


Gene name - Resistant to dieldrin

Synonyms -

Cytological map position - 67A1-67A1

Function - channel

Keywords - GABA-A receptor, memory acquisition, sleep

Symbol - Rdl

FlyBase ID: FBgn0004244

Genetic map position - 3L:9,143,695..9,170,704 [-]

Classification - Neurotransmitter-gated ion-channel ligand binding domain

Cellular location - transmembrane



NCBI link: EntrezGene
Rdl orthologs: Biolitmine

Recent literature
Tachibana, S., Touhara, K. and Ejima, A. (2015). Modification of male courtship motivation by olfactory habituation via the GABAA receptor in Drosophila melanogaster. PLoS One 10: e0135186. PubMed ID: 26252206
Summary:
A male-specific component, 11-cis-vaccenyl acetate (cVA) works as an anti-aphrodisiac pheromone in Drosophila melanogaster. The presence of cVA on a male suppresses the courtship motivation of other males and contributes to suppression of male-male homosexual courtship, while the absence of cVA on a female stimulates the sexual motivation of nearby males and enhances the male-female interaction. However, little is known how a male distinguishes the presence or absence of cVA on a target fly from either self-produced cVA or secondhand cVA from other males in the vicinity. This study demonstrates that male flies have keen sensitivity to cVA; therefore, the presence of another male in the area reduces courtship toward a female. This reduced level of sexual motivation, however, could be overcome by pretest odor exposure via olfactory habituation to cVA. Real-time imaging of cVA-responsive sensory neurons using the neural activity sensor revealed that prolonged exposure to cVA decreased the levels of cVA responses in the primary olfactory center. Pharmacological and genetic screening revealed that signal transduction via GABAA receptors contributed to this olfactory habituation. It was also found that the habituation experience increased the copulation success of wild-type males in a group. In contrast, transgenic males, in which GABA input in a small subset of local neurons was blocked by RNAi, failed to acquire the sexual advantage conferred by habituation. Thus, this study illustrates a novel phenomenon in which olfactory habituation positively affects sexual capability in a competitive environment.

Mohammad, F., Aryal, S., Ho, J., Stewart, J. C., Norman, N. A., Tan, T. L., Eisaka, A. and Claridge-Chang, A. (2016). Ancient anxiety pathways influence Drosophila defense behaviors. Curr Biol 26: 981-986. PubMed ID: 27020741
Summary:
Anxiety helps us anticipate and assess potential danger in ambiguous situations; however, the anxiety disorders are the most prevalent class of psychiatric illness. Emotional states are shared between humans and other animals, as observed by behavioral manifestations, physiological responses, and gene conservation. Anxiety research makes wide use of three rodent behavioral assays-elevated plus maze, open field, and light/dark box-that present a choice between sheltered and exposed regions. Exposure avoidance in anxiety-related defense behaviors was confirmed to be a correlate of rodent anxiety by treatment with known anxiety-altering agents and is now used to characterize anxiety systems. Modeling anxiety with a small neurogenetic animal would further aid the elucidation of its neuronal and molecular bases. Drosophila neurogenetics research has elucidated the mechanisms of fundamental behaviors and implicated genes that are often orthologous across species. In an enclosed arena, flies stay close to the walls during spontaneous locomotion, a behavior proposed to be related to anxiety. This study tested this hypothesis with manipulations of the GABA receptor, serotonin signaling, and stress. The effects of these interventions were strikingly concordant with rodent anxiety, verifying that these behaviors report on an anxiety-like state. Application of this method was able to identify several new fly anxiety genes. The presence of conserved neurogenetic pathways in the insect brain identifies Drosophila as an attractive genetic model for the study of anxiety and anxiety-related disorders, complementing existing rodent systems.
Raccuglia, D., Yan McCurdy, L., Demir, M., Gorur-Shandilya, S., Kunst, M., Emonet, T. and Nitabach, M. N. (2016). Presynaptic GABA receptors mediate temporal contrast enhancement in Drosophila olfactory sensory neurons and modulate odor-driven behavioral kinetics. eNeuro 3 [Epub ahead of print]. PubMed ID: 27588305
Summary:
Contrast enhancement mediated by lateral inhibition within the nervous system enhances the detection of salient features of visual and auditory stimuli, such as spatial and temporal edges. However, it remains unclear how mechanisms for temporal contrast enhancement in the olfactory system can enhance the detection of odor plume edges during navigation. To address this question, pulses of high odor intensity that induce sustained peripheral responses in olfactory sensory neurons (OSNs) were delivered to Drosophila melanogaster flies. Optical electrophysiology was used to directly measure electrical responses in presynaptic terminals, and it was demonstrated that sustained peripheral responses are temporally sharpened by the combined activity of two types of inhibitory GABA receptors, ionotropic GABAA and metabotropic GABAB inhibitory receptors to generate contrast-enhanced voltage responses in central OSN axon terminals. Furthermore, it was shown how these GABA receptors modulate the time course of innate behavioral responses after odor pulse termination, demonstrating an important role for temporal contrast enhancement in odor-guided navigation.
Seugnet, L., Dissel, S., Thimgan, M., Cao, L. and Shaw, P. J. (2017). Identification of genes that maintain behavioral and structural plasticity during sleep loss. Front Neural Circuits 11: 79. PubMed ID: 29109678
Summary:
Although patients with primary insomnia experience sleep disruption, they are able to maintain normal performance on a variety of cognitive tasks. This observation suggests that insomnia may be a condition where predisposing factors simultaneously increase the risk for insomnia and also mitigate against the deleterious consequences of waking. To gain insight into processes that might regulate sleep and buffer neuronal circuits during sleep loss, three genes, fat facet (faf), highwire (hiw) and the GABA receptor Resistance to dieldrin (Rdl), were manipulated that were differentially modulated in a Drosophila model of insomnia. The results indicate that increasing faf and decreasing hiw or Rdl within wake-promoting large ventral lateral clock neurons (lLNvs) induces sleep loss. As expected, sleep loss induced by decreasing hiw in the lLNvs results in deficits in short-term memory and increases of synaptic growth. However, sleep loss induced by knocking down Rdl in the lLNvs protects flies from sleep-loss induced deficits in short-term memory and increases in synaptic markers. Surprisingly, decreasing hiw and Rdl within the Mushroom Bodies (MBs) protects against the negative effects of sleep deprivation (SD) as indicated by the absence of a subsequent homeostatic response, or deficits in short-term memory. Together these results indicate that specific genes are able to disrupt sleep and protect against the negative consequences of waking in a circuit dependent manner.
Li, Q., Li, Y., Wang, X., Qi, J., Jin, X., Tong, H., Zhou, Z., Zhang, Z. C. and Han, J. (2017). Fbxl4 serves as a clock output molecule that regulates sleep through promotion of rhythmic degradation of the GABAA receptor. Curr Biol [Epub ahead of print]. PubMed ID: 29174887
Summary:
The timing of sleep is tightly governed by the circadian clock, which contains a negative transcriptional feedback loop and synchronizes the physiology and behavior of most animals to daily environmental oscillations. However, how the circadian clock determines the timing of sleep is largely unclear. In vertebrates and invertebrates, the status of sleep and wakefulness is modulated by the electrical activity of pacemaker neurons that are circadian regulated and suppressed by inhibitory GABAergic inputs. This study showed that Drosophila GABAA receptors undergo rhythmic degradation in arousal-promoting large ventral lateral neurons (lLNvs) and their expression level in lLNvs displays a daily oscillation. This study also demonstrated that the E3 ligase Fbxl4 promotes GABAA receptor ubiquitination and degradation and revealed that the transcription of fbxl4 in lLNvs is CLOCK dependent. Finally, it was demonstrated that Fbxl4 regulates the timing of sleep through rhythmically reducing GABA sensitivity to modulate the excitability of lLNvs. This study uncovered a critical molecular linkage between the circadian clock and the electrical activity of pacemaker neurons and demonstrated that CLOCK-dependent Fbxl4 expression rhythmically downregulates GABAA receptor level to increase the activity of pacemaker neurons and promote wakefulness.
Gowda, S. B. M., Paranjpe, P. D., Reddy, O. V., Thiagarajan, D., Palliyil, S., Reichert, H. and VijayRaghavan, K. (2018). GABAergic inhibition of leg motoneurons is required for normal walking behavior in freely moving Drosophila. Proc Natl Acad Sci U S A 115(9): E2115-e2124. PubMed ID: 29440493
Summary:
Walking is a complex rhythmic locomotor behavior generated by sequential and periodical contraction of muscles essential for coordinated control of movements of legs and leg joints. Studies of walking in vertebrates and invertebrates have revealed that premotor neural circuitry generates a basic rhythmic pattern that is sculpted by sensory feedback and ultimately controls the amplitude and phase of the motor output to leg muscles. However, the identity and functional roles of the premotor interneurons that directly control leg motoneuron activity are poorly understood. This study took advantage of the powerful genetic methodology available in Drosophila to investigate the role of premotor inhibition in walking by genetically suppressing inhibitory input to leg motoneurons. For this, an algorithm was developed for automated analysis of leg motion to characterize the walking parameters of wild-type flies from high-speed video recordings. Further, genetic reagents were used for targeted RNAi knockdown of inhibitory neurotransmitter receptors in leg motoneurons together with quantitative analysis of resulting changes in leg movement parameters in freely walking Drosophila. The findings indicate that targeted down-regulation of the GABAA receptor Rdl (Resistance to Dieldrin) in leg motoneurons results in a dramatic reduction of walking speed and step length without the loss of general leg coordination during locomotion. Genetically restricting the knockdown to the adult stage and subsets of motoneurons yields qualitatively identical results. Taken together, these findings identify GABAergic premotor inhibition of motoneurons as an important determinant of correctly coordinated leg movements and speed of walking in freely behaving Drosophila.
Li, X., Ishimoto, H. and Kamikouchi, A. (2018). Auditory experience controls the maturation of song discrimination and sexual response in Drosophila. Elife 7. PubMed ID: 29555017
Summary:
In birds and higher mammals, auditory experience during development is critical to discriminate sound patterns in adulthood. However, the neural and molecular nature of this acquired ability remains elusive. In fruit flies, acoustic perception has been thought to be innate. This study reports, surprisingly, that auditory experience of a species-specific courtship song in developing Drosophila shapes adult song perception and resultant sexual behavior. Preferences in the song-response behaviors of both males and females were tuned by social acoustic exposure during development. This study examined the molecular and cellular determinants of this social acoustic learning and found that GABA signaling acting on the GABAA receptor Rdl in the pC1 neurons, the integration node for courtship stimuli, regulated auditory tuning and sexual behavior. These findings demonstrate that maturation of auditory perception in flies is unexpectedly plastic and is acquired socially, providing a model to investigate how song learning regulates mating preference in insects.
Yamada, D., Ishimoto, H., Li, X., Kohashi, T., Ishikawa, Y. and Kamikouchi, A. (2018). GABAergic local interneurons shape female fruit fly response to mating songs. J Neurosci 38(18): 4329-4347. PubMed ID: 29691331
Summary:
Many animals use acoustic signals to attract a potential mating partner. In fruit flies (Drosophila melanogaster), the courtship pulse song has a species-specific interpulse interval (IPI) that activates mating. Although a series of auditory neurons in the fly brain exhibit different tuning patterns to IPIs, it is unclear how the response of each neuron is tuned. This study examined the neural circuitry regulating the activity of antennal mechanosensory and motor center (AMMC)-B1 neurons, key secondary auditory neurons in the excitatory neural pathway that relay song information. By performing Ca(2+) imaging in female flies, it as found that the IPI selectivity observed in AMMC-B1 neurons differs from that of upstream auditory sensory neurons [Johnston's organ (JO)-B]. Selective knock-down of a GABAA receptor subunit in AMMC-B1 neurons increased their response to short IPIs, suggesting that GABA suppresses AMMC-B1 activity at these IPIs. Connection mapping identified two GABAergic local interneurons that synapse with AMMC-B1 and JO-B. Ca(2+) imaging combined with neuronal silencing revealed that these local interneurons, AMMC-LN and AMMC-B2, shape the response pattern of AMMC-B1 neurons at a 15 ms IPI. Neuronal silencing studies further suggested that both GABAergic local interneurons suppress the behavioral response to artificial pulse songs in flies, particularly those with a 15 ms IPI. Altogether, this study identified a circuit containing two GABAergic local interneurons that affects the temporal tuning of AMMC-B1 neurons in the song relay pathway and the behavioral response to the courtship song. These findings suggest that feedforward inhibitory pathways adjust the behavioral response to courtship pulse songs in female flies.
Lee, J., Iyengar, A. and Wu, C. F. (2019). Distinctions among electroconvulsion- and proconvulsant-induced seizure discharges and native motor patterns during flight and grooming: quantitative spike pattern analysis in Drosophila flight muscles. J Neurogenet: 1-18. PubMed ID: 30982417
Summary:
In Drosophila, high-frequency electrical stimulation across the brain triggers a highly stereotypic repertoire of spasms. These electroconvulsive seizures (ECS) manifest as distinctive spiking discharges across the nervous system and can be stably assessed throughout the seizure repertoire in the large indirect flight muscles dorsal longitudinal muscles (DLMs) to characterize modifications in seizure-prone mutants. However, the relationships between ECS-spike patterns and native motor programs, including flight and grooming, are not known and their similarities and distinctions remain to be characterized. This study employed quantitative spike pattern analyses for the three motor patterns including: (1) overall firing frequency, (2) spike timing between contralateral fibers, and (3) short-term variability in spike interval regularity (CV2) and instantaneous firing frequency (ISI(-1)). This base-line information from wild-type (WT) flies facilitated quantitative characterization of mutational effects of major neurotransmitter systems: excitatory cholinergic (ChAT), inhibitory GABAergic (Rdl) and electrical (ShakB) synaptic transmission. The results provide an initial glimpse on the vulnerability of individual motor patterns to different perturbations. Marked alterationsof ECS discharge spike patterns were found in terms of either seizure threshold, spike frequency or spiking regularity. In contrast, no gross alterations during grooming and a small but noticeable reduction of firing frequency during Rdl mutant flight were found, suggesting a role for GABAergic modulation of flight motor programs. Picrotoxin (PTX), a known pro-convulsant that inhibits GABAA receptors, induced DLM spike patterns that displayed some features, e.g. left-right coordination and ISI(-1) range, that could be found in flight or grooming, but distinct from ECS discharges. These quantitative techniques may be employed to reveal overlooked relationships among aberrant motor patterns as well as their links to native motor programs.
Kim, J. H., Ki, Y., Lee, H., Hur, M. S., Baik, B., Hur, J. H., Nam, D. and Lim, C. (2020). The voltage-gated potassium channel Shaker promotes sleep via thermosensitive GABA transmission. Commun Biol 3(1): 174. PubMed ID: 32296133
Summary:
Genes and neural circuits coordinately regulate animal sleep. However, it remains elusive how these endogenous factors shape sleep upon environmental changes. This study demonstrates that Shaker (Sh)-expressing GABAergic neurons projecting onto dorsal fan-shaped body (dFSB) regulate temperature-adaptive sleep behaviors in Drosophila. Loss of Sh function suppressed sleep at low temperature whereas light and high temperature cooperatively gated Sh effects on sleep. Sh depletion in GABAergic neurons partially phenocopied Sh mutants. Furthermore, the ionotropic GABA receptor, Resistant to dieldrin (Rdl), in dFSB neurons acted downstream of Sh and antagonized its sleep-promoting effects. In fact, Rdl inhibited the intracellular cAMP signaling of constitutively active dopaminergic synapses onto dFSB at low temperature. High temperature silenced GABAergic synapses onto dFSB, thereby potentiating the wake-promoting dopamine transmission. We propose that temperature-dependent switching between these two synaptic transmission modalities may adaptively tune the neural property of dFSB neurons to temperature shifts and reorganize sleep architecture for animal fitness.
Driscoll, M., Buchert, S. N., Coleman, V., McLaughlin, M., Nguyen, A. and Sitaraman, D. (2021). Compartment specific regulation of sleep by mushroom body requires GABA and dopaminergic signaling. Sci Rep 11(1): 20067. PubMed ID: 34625611
Summary:
Sleep is a fundamental behavioral state important for survival and is universal in animals with sufficiently complex nervous systems. Biogenic amines like dopamine, serotonin and norepinephrine have been shown to be critical for sleep regulation across species but the precise circuit mechanisms underlying how amines control persistence of sleep, arousal and wakefulness remain unclear. The fruit fly, Drosophila melanogaster, provides a powerful model system for the study of sleep and circuit mechanisms underlying state transitions and persistence of states to meet the organisms motivational and cognitive needs. In Drosophila, two neuropils in the central brain, the mushroom body (MB) and the central complex (CX) have been shown to influence sleep homeostasis and receive aminergic neuromodulator input critical to sleep-wake switch. Dopamine neurons (DANs) are prevalent neuromodulator inputs to the MB but the mechanisms by which they interact with and regulate sleep- and wake-promoting neurons within MB are unknown. This study investigated the role of subsets of PAM-DANs that signal wakefulness and project to wake-promoting compartments of the MB. This study found that PAM-DANs are GABA responsive and require GABA(A)-Rdl receptor in regulating sleep. In mapping the pathways downstream of PAM neurons innervating γ5 and β'2 MB compartments it was found that wakefulness is regulated by both DopR1 and DopR2 receptors in downstream Kenyon cells (KCs) and mushroom body output neurons (MBONs). Taken together, a dopamine modulated sleep microcircuit has been identified within the mushroom body that has previously been shown to convey information about positive and negative valence critical for memory formation. These studies will pave the way for understanding how flies balance sleep, wakefulness and arousal (Buchert, 2021).
Coquerel, Q. R. R., Ddmares, F., Geldenhuys, W. J., Le Ray, A. M., Brdard, D., Richomme, P., Legros, C., Norris, E. and Bloomquist, J. R. (2021).. Toxicity and mode of action of the aporphine plant alkaloid liriodenine on the insect GABA receptor. Toxicon 201: 141-147. PubMed ID: 34474068
Summary:
Liriodenine is a biologically active plant alkaloid with multiple effects on mammals, fungi, and bacteria, but has never been evaluated for insecticidal activity. Accordingly, liriodenine was applied topically in ethanolic solutions to adult female Anopheles gambiae, and found to be mildly toxic. Its lethality was synergized in mixtures with dimethyl sulfoxide and piperonyl butoxide. Recordings from the ventral nerve cord of larval Drosophila melanogaster showed that liriodenine was neuroexcitatory and reversed the inhibitory effect of 1 mM GABA at effective concentrations of 20-30 μM. GABA antagonism on the larval nervous system was equally expressed on both susceptible and cyclodiene-resistant rdl preparations. Acutely isolated neurons from Periplaneta americana were studied under patch clamp and inhibition of GABA-induced currents with an IC(50) value of about 1 μM were observed. In contrast, bicuculline did not reverse the effects of GABA on cockroach neurons, as expected. In silico molecular models suggested reasonable structural concordance of liriodenine and bicuculline and isosteric hydrogen bond acceptor sites. This study is the first assessing of the toxicology of liriodenine on insects and implicates the GABA receptor as one likely neuronal target, where liriodenine might be considered an active chemical analog of bicuculline.
Eick, A. K., Ogueta, M., Buhl, E., Hodge, J. J. L. and Stanewsky, R. (2022). The opposing chloride cotransporters KCC and NKCC control locomotor activity in constant light and during long days. Curr Biol 32(6): 1420-1428.e1424. PubMed ID: 35303416
Summary:
Cation chloride cotransporters (CCCs) regulate intracellular chloride ion concentration ([Cl(-)](i)) within neurons, which can reverse the direction of the neuronal response to the neurotransmitter GABA. Na(+) K(+) Cl(-) (NKCC) and K(+) Cl(-) (KCC) cotransporters transport Cl(-) into or out of the cell, respectively. When NKCC activity dominates, the resulting high [Cl(-)](i) can lead to an excitatory and depolarizing response of the neuron upon GABA(A) receptor opening, while KCC dominance has the opposite effect. This inhibitory-to-excitatory GABA switch has been linked to seasonal adaption of circadian clock function to changing day length, and its dysregulation is associated with neurodevelopmental disorders such as epilepsy. In Drosophila melanogaster, constant light normally disrupts circadian clock function and leads to arrhythmic behavior. This study demonstrates a function for CCCs in regulating Drosophila locomotor activity and GABA responses in circadian clock neurons because alteration of CCC expression in circadian clock neurons elicits rhythmic behavior in constant light. The same effects were observed after downregulation of the Wnk and Fray kinases, which modulate CCC activity in a [Cl(-)](i)-dependent manner. Patch-clamp recordings from the large LNv clock neurons show that downregulation of KCC results in a more positive GABA reversal potential, while KCC overexpression has the opposite effect. Finally, KCC and NKCC downregulation reduces or increases morning behavioral activity during long photoperiods, respectively. In summary, these results support a model in which the regulation of [Cl(-)](i) by a KCC/NKCC/Wnk/Fray feedback loop determines the response of clock neurons to GABA, which is important for adjusting behavioral activity to constant light and long-day conditions.
Chaturvedi, R., Stork, T., Yuan, C., Freeman, M. R. and Emery, P. (2022). Astrocytic GABA transporter controls sleep by modulating GABAergic signaling in Drosophila circadian neurons. Curr Biol. PubMed ID: 35303417
Summary:
A precise balance between sleep and wakefulness is essential to sustain a good quality of life and optimal brain function. GABA is known to play a key and conserved role in sleep control, and GABAergic tone should, therefore, be tightly controlled in sleep circuits. This study examined the role of the astrocytic GABA transporter (Gat) in sleep regulation using Drosophila melanogaster. A hypomorphic Gat mutation (Gat33-1) increased sleep amount, decreased sleep latency, and increased sleep consolidation at night. Interestingly, sleep defects were suppressed when Gat33-1 was combined with a mutation disrupting wide-awake (wake), a gene that regulates the cell-surface levels of the GABA(A) receptor Resistance to dieldrin (Rdl) in the wake-promoting large ventral lateral neurons (l-LNvs). Moreover, RNAi knockdown of Rdl and its modulators dnlg4 and wake in these circadian neurons also suppressed Gat33-1 sleep phenotypes. Brain immunohistochemistry showed that GAT-expressing astrocytes were located near RDL-positive l-LNv cell bodies and dendritic processes. It is concluded that astrocytic GAT decreases GABAergic tone and RDL activation in arousal-promoting LNvs, thus determining proper sleep amount and quality in Drosophila.
Chen, S. L., Liu, B. T., Lee, W. P., Liao, S. B., Deng, Y. B., Wu, C. L., Ho, S. M., Shen, B. X., Khoo, G. H., Shiu, W. C., Chang, C. H., Shih, H. W., Wen, J. K., Lan, T. H., Lin, C. C., Tsai, Y. C., Tzeng, H. F. and Fu, T. F. (2022). WAKE-mediated modulation of cVA perception via a hierarchical neuro-endocrine axis in Drosophila male-male courtship behaviour. Nat Commun 13(1): 2518. PubMed ID: 35523813
Summary:
The nervous and endocrine systems coordinate with each other to closely influence physiological and behavioural responses in animals. This study shows that Wake (encoded by wide awake) modulates membrane levels of GABA(A) receptor Resistance to Dieldrin (Rdl), in insulin-producing cells of adult male Drosophila melanogaster. This results in changes to secretion of insulin-like peptides which is associated with changes in juvenile hormone biosynthesis in the corpus allatum, which in turn leads to a decrease in 20-hydroxyecdysone levels. A reduction in ecdysone signalling changes neural architecture and lowers the perception of the male-specific sex pheromone 11-cis-vaccenyl acetate by odorant receptor 67d olfactory neurons. These finding explain why WAKE-deficient in Drosophila elicits significant male-male courtship behaviour.
Xie, N. and Gross, A. D. (2022). Muscarinic acetylcholine receptor activation synergizes the knockdown and toxicity of GABA-gated chloride channel insecticides. Pest Manag Sci 78(11): 4599-4607. PubMed ID: 35841135
Summary:
Pest management requires continual identification of new physiological targets and strategies to control pests affecting agriculture and public/animal health. It is proposed that the muscarinic system is a target for agrochemicals because of its physiological importance. Unlike the muscarinic system, gamma-amino butyric acid (GABA) receptors are an established insecticide target. This study investigated target-site synergism using small molecule probes (agonist and antagonist) against the muscarinic system and their ability to enhance the toxicity of GABAergic insecticides in Drosophila melanogaster. Oral delivery of pilocarpine (muscarinic agonist) enhanced the toxicity of dieldrin, fipronil, and lindane, resulting in synergist ratios (SRs) between 4-32-fold (orally delivered) or between 2-67-fold when insecticides were topically applied. The synergism between pilocarpine and the GABA-insecticides was greater than the synergism observed with atropine (muscarinic antagonist), and was greater, or comparable, to the synergism observed with the metabolic inhibitor piperonyl butoxide. In addition to lethality, pilocarpine increased the knockdown of lindane. The mechanism of synergism was also investigated in the central nervous system using extracellular electrophysiology, where pilocarpine (3 μmo/L) lowered the half-maximal inhibitory concentration (IC(50)) of lindane and fipronil's IC(50). It is concluded that convergence of the cellular function between the muscarinic and GABAergic systems enhanced the insecticidal activity of GABA receptor blocking insecticides through the modulation of the central nervous system (CNS).
Braun, A., Borst, A. and Meier, M. (2023). Disynaptic inhibition shapes tuning of OFF-motion detectors in Drosophila. Curr Biol 33(11): 2260-2269. PubMed ID: 37236181
Summary:
The circuitry underlying the detection of visual motion in Drosophila melanogaster is one of the best studied networks in neuroscience. Lately, electron microscopy reconstructions, algorithmic models, and functional studies have proposed a common motif for the cellular circuitry of an elementary motion detector based on both supralinear enhancement for preferred direction and sublinear suppression for null-direction motion. In T5 cells, however, all columnar input neurons (Tm1, Tm2, Tm4, and Tm9) are excitatory. So, how is null-direction suppression realized there? Using two-photon calcium imaging in combination with thermogenetics, optogenetics, apoptotics, and pharmacology, this study discovered that it is via CT1, the GABAergic large-field amacrine cell, where the different processes have previously been shown to act in an electrically isolated way. Within each column, CT1 receives excitatory input from Tm9 and Tm1 and provides the sign-inverted, now inhibitory input signal onto T5. Ablating CT1 or knocking down GABA-receptor subunit Rdl significantly broadened the directional tuning of T5 cells. It thus appears that the signal of Tm1 and Tm9 is used both as an excitatory input for preferred direction enhancement and, through a sign inversion within the Tm1/Tm9-CT1 microcircuit, as an inhibitory input for null-direction suppression.
Guo, L., Qiao, X., Haji, D., Zhou, T., Liu, Z., Whiteman, N. K. and Huang, J. (2023). Convergent resistance to GABA receptor neurotoxins through plant-insect coevolution. Nat Ecol Evol. PubMed ID: 37460839
Summary:
The molecular mechanisms of coevolution between plants and insects remain elusive. GABA receptors are targets of many neurotoxic terpenoids, which represent the most diverse array of natural products known. Over deep evolutionary time, as plant terpene synthases diversified in plants, so did plant terpenoid defence repertoires. This study shows that herbivorous insects and their predators evolved convergent amino acid changing substitutions in duplicated copies of the Resistance to dieldrin (Rdl) gene that encodes the GABA receptor, and that the evolution of duplicated Rdl and terpenoid-resistant GABA receptors is associated with the diversification of moths and butterflies. These same substitutions also evolved in pests exposed to synthetic insecticides that target the GABA receptor. In vivo genome editing in Drosophila melanogaster was used to evaluate the fitness effects of each putative resistance mutation, and pleiotropy was found to both facilitate and constrain the evolution of GABA receptor resistance. The same genetic changes that confer resistance to terpenoids across 300 Myr of insect evolution have re-evolved in response to synthetic analogues over one human lifespan.
Zhang, Y., Huang, Q., Sheng, C., Liu, G., Zhang, K., Jia, Z., Tang, T., Mao, X., Jones, A. K., Han, Z. and Zhao, C. (2023). G3'MTMD3 in the insect GABA receptor subunit, RDL, confers resistance to broflanilide and fluralaner. PLoS Genet 19(6): e1010814. PubMed ID: 37384781
Summary:
Meta-diamides (e.g. broflanilide) and isoxazolines (e.g. fluralaner) are novel insecticides that target the resistant to dieldrin (RDL) subunit of insect γ-aminobutyric acid receptors (GABARs). This study used in silico analysis to identify residues that are critical for the interaction between RDL and these insecticides. Substitution of glycine at the third position (G3') in the third transmembrane domain (TMD3) with methionine (G3'M TMD3), which is present in vertebrate GABARs, had the strongest effect on fluralaner binding. This was confirmed by expression of RDL from the rice stem borer, Chilo suppressalis (CsRDL) in oocytes of the African clawed frog, Xenopus laevis, where the G3'MTMD3 mutation almost abolished the antagonistic action of fluralaner. Subsequently, G3'MTMD3 was introduced into the Rdl gene of the fruit fly, Drosophila melanogaster, using the CRISPR/Cas9 system. Larvae of heterozygous lines bearing G3'MTMD3 did not show significant resistance to avermectin, fipronil, broflanilide, and fluralaner. However, larvae homozygous for G3'MTMD3 were highly resistant to broflanilide and fluralaner whilst still being sensitive to fipronil and avermectin. Also, homozygous lines showed severely impaired locomotivity and did not survive to the pupal stage, indicating a significant fitness cost associated with G3'MTMD3. Moreover, the M3'GTMD3 mutation in the mouse Mus musculus α1β2 GABAR increased sensitivity to fluralaner. Taken together, these results provide convincing in vitro and in vivo evidence for both broflanilide and fluralaner acting on the same amino acid site, as well as insights into potential mechanisms leading to target-site resistance to these insecticides. In addition, these findings could guide further modification of isoxazolines to achieve higher selectivity for the control of insect pests with minimal effects on mammals.

BIOLOGICAL OVERVIEW

In both mammals and insects, neurons involved in learning are strongly modulated by the inhibitory neurotransmitter GABA. The GABAA receptor, Resistance to dieldrin (Rdl), is highly expressed in the Drosophila mushroom bodies (MBs), a group of neurons playing essential roles in insect olfactory learning. Flies with increased or decreased expression of Rdl in the MBs were generated. Olfactory associative learning tests showed that Rdl overexpression impaired memory acquisition but not memory stability. This learning defect is due to disrupting the physiological state of the adult MB neurons rather than causing developmental abnormalities. Remarkably, Rdl knockdown enhanced memory acquisition but not memory stability. Functional cellular imaging experiments showed that Rdl overexpression abolished the normal calcium responses of the MBs to odors while Rdl knockdown increased these responses. Together, these data suggest that RDL negatively modulates olfactory associative learning, possibly by gating the input of olfactory information into the MBs (Liu, 2007).

Neurons comprising the neural circuits that mediate learning are modulated by the inhibitory neurotransmitter γ-amino butyric acid (GABA). For instance, the hippocampus, which is involved in the formation of multiple types of memories in mammalian organisms, is densely innervated by GABAergic interneurons. The insect mushroom bodies (MBs), which similarly are involved in the formation of multiple types of memories, are also subject to GABAergic modulation (Perez-Orive, 2002; Yasuyama, 2002). These and other similar observations make it clear that a deep understanding of the molecular and systems neuroscience properties that underlie memory formation will not emerge until a detailed knowledge of when and where GABAergic modulation occurs and how this modulation alters the function of the cells and networks that mediate memory formation.

GABAA receptors are GABA-gated chloride channels. Accumulating pharmacological and genetic evidence suggests that GABAA receptors participate in the cellular and circuit mechanisms underlying learning and memory, but the current information is inconsistent and lacks depth. Several prior studies have used intraperitoneal or intracerebroventricular injection of GABAA receptor agonists or antagonists and monitored effects on behavior. However, the widespread effects caused by this approach make it impossible to assign behavioral changes to any specific population of neurons. Better spatial resolution for the pharmacological effects has been achieved by injecting drugs into specific brain regions either before or after training or just prior to testing, and in several cases, receptor agonists have inhibited behavioral performance, and antagonists have facilitated it. However, these studies fail to provide information regarding the specific cell type affected within the targeted region and the identity of the targeted GABA receptor. Furthermore, they provide no information about how the pharmacological agents affect the information processing relevant to learning mechanisms by the neurons. Moreover, the simplistic idea that GABAA receptor agonists and antagonists/inverse agonists may decrease and increase behavioral performance, respectively, remains controversial because of reports to the contrary (Liu, 2007 and references therein).

Genetic dissections of GABAA receptor function using viable knockouts have provided more specific information regarding the receptor type involved, but they lack information about how information processing is altered, the neurons involved in the behavior being tested, and whether the behavioral results are due to a physiological disruption of GABAA function or a developmental insult secondary to the developmental loss of the receptor. Moreover, the controversies regarding the direction of behavioral change (improve versus impair) with decreased receptor function remain. For instance, DeLorey (1998) reported that GABAAβ3 knockout mice showed impaired performance several days after training in a step-through passive avoidance task and contextual fear conditioning, but Collinson (2002) and Crestani (2002) reported GABAAα5 mutant mice to have enhanced performance in a match-to-place version of the water maze test and in trace fear conditioning, respectively. Although genetic dissections point to the inadequacy of pharmacological manipulations by emphasizing receptor-specific functions, the use of whole-animal knockouts fails to offer reliable conclusions about where and how GABAA receptors influence the complex neural circuitry underlying learning and memory (Liu, 2007).

This study probed the role of GABAergic modulation using Drosophila olfactory learning as a model because of the ability to bidirectionally alter the expression of specific GABAA receptors in identified populations of neurons of the adult and to probe how these modulations alter the information processing capabilities of the neurons. In Drosophila, at least three genes are thought to encode GABAA receptors: resistance to dieldrin (Rdl), GABA and glycine-like receptor of Drosophila (Grd), and ligand-gated chloride channel homologue 3 (Lcch3). Rdl is by far the best characterized of the three molecularly and through functional expression experiments (Hosie, 1997; Buckingham, 2005). RDL also has an important role in insecticide resistance (ffrench-Constant, 2004). This receptor is highly expressed in the Drosophila antennal lobes (ALs) and the MBs (Harrison, 1996), both of which are essential structures required for the acquisition, storage, and retrieval of olfactory memory (Liu, 2007).

One attractive idea for the role of GABAergic inhibition of neurons involved in learning is that the inhibition serves to sparsen sensory representations to make learning easier and recall faster (Perez-Orive, 2002; Olshausen, 2004). The projection neurons in the insect AL receive information about odors from olfactory receptor neurons in the antennae and transmit this information to higher-order structures including the MBs and the lateral horn (LH). The AL projection neurons exhibit robust firing when the animal senses an odor, but the robustness of the response in the postsynaptic MB neurons is sparsened because of postulated feedforward GABAergic inhibition received from the LH (Perez-Orive, 2002). Unfortunately, there remains no direct experimental evidence in favor of or against this hypothesis (Liu, 2007).

In this study, flies were generated with elevated or decreased expression of Rdl in the MBs and the learning performance of these flies was assayed along with the calcium responses in the MBs produced by odor and electric shock stimulation. The results indicate that the level of memory acquisition is inversely related to the level of RDL expression in the MB neurons, indicating that RDL in the MBs inhibits olfactory learning. This inhibition of learning is due to the expression level of Rdl at the time of learning, rather than to developmental alterations that may occur in the neural circuit from perturbing Rdl expression during development. Furthermore, the calcium response of MB neurons to odor stimulation is also inversely related to the level of Rdl expression in these neurons, indicating that the expression level of Rdl gates the receipt of information about the conditioned stimulus during olfactory learning (Liu, 2007).

The reported expression pattern (Harrison, 1996) of Rdl in the adult Drosophila brain was verified. A polyclonal antibody was developed that recognizes RDL protein by immunoblotting. The abundance of this protein was reduced in flies heterozygous for two different null and homozygous lethal alleles of Rdl, Rdl1 and Rdlf02994, confirming the ability of this antibody to detect the RDL protein. The expression pattern of Rdl in the central brain by was characterized by immunohistochemistry. The RDL protein was detected throughout the ALs, the MBs, and the central complex. In the MBs, RDL was detected both in the dendrites (calyces) and the axons (α, α′, β, β′, γ lobes and peduncles), but no RDL signal was observed in the cell bodies of MB neurons (Liu, 2007).

Both overexpression and knockdown strategies with tissue and time-specific control were used to probe the role of the GABAA receptor RDL in olfactory learning. The abundant endogenous expression of Rdl in the olfactory nervous system strongly suggests a critical role in odor perception, discrimination, and learning. The results conclusively show a physiological role for Rdl in olfactory learning. Overexpression of Rdl in the MBs impaired learning, while knockdown of Rdl in the same neurons enhanced learning. The data also show that RDL is involved in memory acquisition but not memory stability. These behavioral data along with functional imaging results indicate that the GABAergic system inhibits olfactory learning, probably by gating the level of olfactory information into the MBs (Liu, 2007).

The Rdl gene exhibits extensive alternative splicing. Exons 3 and 6 of the Rdl gene have two alternative splice forms each, so that the Rdl gene encodes four different isoforms, all of which are found in RNA isolated from early embryos (ffrench-Constant, 1993a). When expressed in Xenopus oocytes, the proteins produced from alternative splicing show differential responses to agonists (Hosie, 2001), suggesting different physiological properties for the isoforms in vivo. Since the detailed temporal and spatial expression pattern of each isoform in the adult fly brain has not been reported, the antigen and all RNAi constructs against sequences common to all known isoforms were designated. Therefore those isoform(s) that are expressed in the adult MBs and those that are responsible for inhibiting olfactory learning cannot be identify (Liu, 2007).

Prior studies have shown that GABAergic inhibition shapes odor-evoked spatiotemporal activity patterns in the Drosophila ALs (Wilson, 2005). GABA receptor function in the honeybee AL has also been shown to be required for fine, but not coarse, odor discrimination, by using picrotoxin to inhibit AL GABA receptors (Stopfer, 1997). These observations raise the issue of why the c772-Gal4, MB {Gal80}; UAS-Rdli flies exhibit normal olfactory learning. Although it is possible that c772-Gal4 drives expression in AL interneurons other than those involved in olfactory discrimination, which would explain the observation, the more likely explanation is that the odors used in this study are quite disparate, allowing for the normal learning of these odors. This predicts that a phenotype may emerge in tests of these flies for fine odor discrimination (Liu, 2007).

One possible role for the GABAergic inhibition of the MB neurons is to sparsen the odor representations (Perez-Orive, 2002). Sparsening of sensory representations has been proposed as a simplification that the nervous system makes to allow easier and faster encoding and retrieval of memories (Olshausen and Field, 2004). In its simplest form, the sparsening hypothesis for GABAergic inhibition of the MBs predicts that lessening the inhibition by reducing Rdl expression should make the representations more complex and more difficult to learn, whereas enhanced acquisition with reduced Rdl expression was observed. Rather than facilitating and enhancing memory formation by the sparsening of representations, these results are more consistent with the alternative idea that the GABAergic system inhibits learning (Liu, 2007).

What is the purpose of a neural system that inhibits learning? One possibility is that this inhibitory system may provide a necessary balance for the acquisition of different forms of memory. Extinction is an active form of learning occurring when the repeated presentation of a CS alone causes a gradual decrease in the conditioned response in a previously conditioned animal. The surface expression of the GABAA receptor and the expression level of gephyrin, a protein involved in GABAA receptor clustering, have been reported to decrease in the basolateral amygdala of the rat after fear conditioning, yet these GABAergic markers significantly increase after extinction training (Chhatwal, 2005), suggesting that the GABAergic system has opposing roles for conditioning and extinction. Preliminary data also show that Rdl knockdown reduced extinction, supporting the hypothesis that the GABAergic system inhibits conditioning while enhancing extinction (Liu, 2007).

Second, this inhibitory system could serve as a noise filter for information transmission from the ALs to the MBs. The projection neurons of the ALs convey olfactory information to at least two third-order olfactory areas: the MBs and the lateral horns. The MBs are required for olfactory learning, and the lateral horns are thought to be involved in establishing odor identity. Excitatory local neurons were discovered in the ALs. These neurons may be involved in signal amplification by providing cross excitation to projection neurons that are innervated by olfactory receptor neurons that are not responsive to the test odor. The net result is enhanced and more generalized output from the ALs. While this signal amplification could potentially be beneficial for odor detection and discrimination in the lateral horns, it could also introduce extra noise and be detrimental to the MBs for learning about odors in a specific way relative to their importance. By reducing the activity of the MB neurons, the GABAergic system could potentially reduce the time window for coincidence detection in the MBs, thus inhibiting generalized learning and facilitating selective learning. Thus, the GABAergic system may be a noise filter needed by the MBs for optimal learning (Liu, 2007).

Finally, the inhibitory system on the MBs may allow learning to occur through the mechanism of inhibiting the inhibition. There are two major questions of focus for future investigations relative to this idea. One key question is whether learning alters the abundance or function of RDL receptors in the MB neurons. This change could serve to lessen the inhibitory constraints on MB neurons postconditioning, thus potentiating the effect of the trained odor. The surface expression of GABAA receptors has been reported to decrease in the basolateral amygdala after fear conditioning in rodents (Chhatwal, 2005). A related question is whether there are learning-induced changes that occur in the presynaptic GABAergic extrinsic neurons that innervate the MBs. Although these neurons and potential learning-induced changes are yet to be identified in Drosophila, it has been reported that classical olfactory discrimination conditioning of the mouse alters the release of neurotransmitters in the olfactory bulb, including the release of GABA (Brennan, 1998). Presynaptic changes in the release of GABA due to conditioning might also have similar effects by potentiating CS responses after learning. Glutamate released by repetitive activation of the Schaffer collateral triggers a heterosynaptic and persistent depression of GABA release onto CA1 pyramidal neurons (Chevaleyre, 2003). The appropriate paired stimulation of MB neurons by CS and US could in a related way produce a retrograde signal to depress GABA release and thus potentiate learning mediated by the MB neurons (Liu, 2007).

The GABAA receptor RDL suppresses the conditioned stimulus pathway for olfactory learning

Assigning a gene's function to specific pathways used for classical conditioning, such as conditioned stimulus (CS) and unconditioned stimulus (US) pathway, is important for understanding the fundamental molecular and cellular mechanisms underlying memory formation. Prior studies have shown that the GABA receptor RDL inhibits aversive olfactory learning via its role in the Drosophila mushroom bodies (MBs). This study describes the results of further behavioral tests to further define the pathway involvement of RDL. The expression level of Rdl in the MBs influenced both appetitive and aversive olfactory learning, suggesting that it functions by suppressing a common pathway used for both forms of olfactory learning. Rdl knock down failed to enhance learning in animals carrying mutations in genes of the cAMP signaling pathway, such as rutabaga and NF1, suggesting that RDL works up stream of these functions in CS/US integration. Finally, knocking down Rdl or over expressing the dopamine receptor dDA1 in the MBs enhanced olfactory learning, but no significant additional enhancement was detected with both manipulations. The combined data suggest that RDL suppresses olfactory learning via CS pathway involvement (Liu, 2009b).

The level of Rdl expression in the MBs affects the calcium response observed in these neurons when animals are presented with odor but not shock stimulus. This provided the basis for hypothesizing that RDL might specifically regulate the CS pathway for olfactory learning. Data presented in this study shows that the level of Rdl expression the MBs influences both aversive and appetitive olfactory learning, which share a common CS pathway. Thus, these observations are consistent with the CS pathway-specific hypothesis. Rdl knock down failed to produce enhanced learning when combined with mutations of either the rut or NF1 gene, both of which may be involved in the process of integration of CS and US information. This observation argues against the possibility that RDL acts downstream of CS/US integration, providing further support for RDL's role in the CS pathway (Liu, 2009b).

Prior experiments have shown that blocking neurotransmitter release from dopaminergic neurons impairs aversive olfactory learning but not appetitive olfactory learning, while blocking the synthesis of octopamine impairs appetitive olfactory learning but not aversive olfactory learning. This is consistent with the simple model that the neuromodulators are involved in US pathways for learning, with octopamine delivering only appetitive US (sugar) and dopamine delivering only aversive US (electric shock). This model also suggests that increasing the expression level of dDA1 will increase aversive US input, and thereby enhance aversive learning, as long as other factors such as dopamine release are not limiting. This possibility was tested, and evidence is provided for increased performance with increased expression of dDA1 in the MBs. Since knocking down Rdl increases the CS signal, it follows that combining over-expression of dDA1 with knock down of Rdl might enhance learning synergistically, and produce an even greater enhancement of learning. However, no synergism between these two was detected: although dDA1 over-expression alone and Rdl knock down alone both enhance olfactory learning, the combined treatments failed to produce a significantly higher performance score than either treatment alone. Two possible hypotheses can account for these results. The learning enhancement of either treatment produces performance close to ceiling levels, where no further enhancement can be detected. Alternatively, the dDA1 receptor, and thus the dopamine system, plays some role in the CS pathway that overlaps with RDL, such that the two learning enhancing effects do not sum. The authors prefer the later possibility for two reasons. (1) Functional imaging of the dopaminergic neurons projecting to the MBs using calcium reporters has revealed that these neurons respond not only to shock stimuli presented to the fly, but also to odor stimuli (Riemensperger, 2005). This indicates that the response properties of these neurons are not specific to the US pathway, which is predicted by the 'US pathway only' hypothesis. Rather, dopaminergic neurons respond to the CS and are therefore intertwined in some way with the CS pathway. (2) Flies mutant for the dDA1 gene exhibit impairment in both aversive and appetitive olfactory learning, both of which can be rescued by expressing dDA1 in the MBs (Kim, 2007). This observation suggests that dDA1 may play a role in the CS pathway like RDL. An overriding conclusion is that the model envisioning aversive and appetitive specific US pathway roles for dopamine and octopamine, respectively, is overly simplistic (Liu, 2009b).

The results suggest that the GABAA receptor RDL regulates the CS pathway in Drosophila olfactory learning. The conclusion that the GABAA receptor modulates the CS pathway for learning is not limited to either insects or learning supported by olfactory cues. During taste aversion learning in mice, pre-exposure to the CS of the tastant alone causes latent inhibition where the mice show reduced learning to the CS after pairing the CS with the US. This phenomenon is distinctly absent in male mice carrying a point mutation in the α5 subunit of the GABAA receptor, which is highly expressed in the hippocampus (Gerdjikov, 2008). Since CS information is the only stimulus presented during the pre-exposure period, these results support the role of GABAA receptors in regulating the CS pathway. Extinction is another type of learning where repeated exposure to the CS alone after CS/US conditioning reduces the CR. Systemic administration of a GABAA receptor antagonist blocks the development and expression of extinction in rats during contextual fear learning (Harris, 1998). Since extinction trials are composed of the CS exposure by itself, these results also indicate that GABAA receptors modulate the CS pathway. Moreover, other studies have shown that the surface expression of GABAA receptors increases in the basolateral amygdala after extinction trials following fear conditioning (Chhatwal, 2005). These results indicate that CS exposure alone during extinction is sufficient to modulate the cellular trafficking of GABAA receptors, again indicating a role for GABAA receptors in the CS pathway. The current results, together with these previous studies, strongly indicate that GABAA receptors regulate the CS pathway for associative learning (Liu, 2009b).

A role for GABAA receptors in suppressing learning by regulating the CS pathway has at least two broad implications. (1) It suggests that the receptors provide a gate to the association center (MBs). Other molecules may also provide similar gates, but learning must overcome this negative influence for memory formation to occur. This gate is probably nonspecific relative to odor type, that is, the GABAA receptor gate suppresses learning to most or all odors. It follows that learning must mobilize cellular mechanisms for overriding the gate. These could be at the level of the presynaptic GABAergic neurons, such that the presynaptic neurons release less neurotransmitter after learning, or they could be at the level of the postsynaptic receptor, with receptor expression, sensitivity, or conductance altered by learning. Evidence has been provided for a reduced presynaptic release following learning (Liu, 2009b), but postsynaptic mechanisms may occur as well (Chhatwal, 2005). (2) Events or processes that alter the salience of the CS and its ability to enter into associations might function via altering the presynaptic GABAergic release or the postsynaptic GABAA receptors. For instance, spaced conditioning is generally more effective in producing long-lasting memories compared with massed conditioning. It is possible that the rest period between spaced conditioning trials allows for receptor desensitization, producing a more effective subsequent training trial. Memory acquisition becomes more difficult with age. It could be that aging alters the fluidity of the GABAA receptor gate, making acquisition more difficult (Liu, 2009b).

Central synaptic mechanisms underlie short-term olfactory habituation in Drosophila larvae

Naive Drosophila larvae show vigorous chemotaxis toward many odorants including ethyl acetate (EA). Chemotaxis toward EA is substantially reduced after a 5-min pre-exposure to the odorant and recovers with a half-time of ~20 min. An analogous behavioral decrement can be induced without odorant-receptor activation through channelrhodopsin-based, direct photoexcitation of odorant sensory neurons (OSNs). The neural mechanism of short-term habituation (STH) requires the (1) Rutabaga adenylate cyclase; (2) transmitter release from predominantly GABAergic local interneurons (LNs); (3) GABA-A receptor function in projection neurons (PNs) that receive excitatory inputs from OSNs; and (4) NMDA-receptor function in PNs. These features of STH cannot be explained by simple sensory adaptation and, instead, point to plasticity of olfactory synapses in the antennal lobe as the underlying mechanism. These observations suggest a model in which NMDAR-dependent depression of the OSN-PN synapse and/or NMDAR-dependent facilitation of inhibitory transmission from LNs to PNs contributes substantially to short-term habituation (Larkin, 2010).

Experience-induced plasticity of synapses is believed to be a fundamental mechanism of learning and memory. However, central synaptic changes that underlie memory have not been clearly defined, even for relatively simple nonassociative learning processes such as habituation (Larkin, 2010).

During habituation, unreinforced exposure to a repeated or prolonged stimulus results in a reversible decrease in response to that stimulus. Habituation probably serves as an important building block for more complex cognitive function. By allowing unchanging or irrelevant stimuli to be ignored, it allows cognitive resources to be focused on more salient stimuli (Larkin, 2010 and references therein).

The neural basis of short-term habituation (STH) is best studied in the marine snail, Aplysia californica. Here STH (lasting ~30 min) of the defensive gill-withdrawal reflex in response to tactile stimulation of the siphon is thought to arise from presynaptic depression of transmitter release at sensorimotor synapses. However, even here, presynaptic plasticity may not be cell-autonomous, potentially requiring, for instance, activity of yet-to-be-identified interneurons (Larkin, 2010).

Several forems of habituation have been described in Drosophila and are often shown to require the function of genes that regulate cAMP-dependent forms of associative memory. For instance, habituation of proboscis extension reflex as well as odor-evoked startle reflex in adult Drosophila requires rutabaga (rut)-encoded Ca2+/calmodulin-sensitive adenylyl cyclase. In addition, habituation of the ethanol-induced startle response requires the shaggy/GSK-3 signaling pathway. Despite such pioneering observations, the mechanisms of these various forms of habituation, even whether the primary neuronal changes are purely sensory or involve plasticity of central synapses (involving centrally located interneurons that may integrate various different kinds of modulatory, inhibitory, and excitatory inputs), remain poorly understood (Larkin, 2010).

Recent advances in understanding the circuitry that underlies Drosophila olfactory behavior, as well as the development of new tools to perturb identified neurons in vivo, has opened the opportunity for understanding mechanisms of olfactory habituation at the level of the underlying neural circuitry (Larkin, 2010).

In the larval olfactory system, 21 olfactory sensory neurons (OSNs), each expressing a single odorant receptor (together with the broadly expressed Or83b co-receptor), synapse, respectively, onto 21 cognate projection neurons (PNs) within 21 glomeruli in the larval antennal lobe (AL). Local, predominantly GABAergic interneurons (LNs) synapse widely within the antennal lobe, interlinking different glomeruli. Various neuromodulatory synapses also form on the larval antennal lobe and mushroom body. Thus, odorant-stimulated signals in sensory neurons are processed in the antennal lobe, modulated by motivational or emotional states, and relayed through projection neurons to higher brain centers (Larkin, 2010).

Previous work has shown that in Drosophila larvae, olfactory chemotaxis decreases after odorant pre-exposure. This study shows that this behavioral habituation, alternatively referred to as 'adaptation' by some previous investigators, arises from mechanisms of synaptic plasticity. This study demonstrates that odorant receptor activation is not necessary for olfactory habituation; however, local interneuron activity and projection neuron signaling is necessary. These observations suggest a model in which habituation occurs by a pathway in which NMDA receptors in projection neurons signal depression of OSN-PN synapses and/or facilitation of LN-PN synapses (Larkin, 2010).

Previous studies have not clearly discriminated between peripheral and central mechanisms. Indeed, the term 'adaptation,' better applied to sensory neuron changes such as receptor desensitization, has often been used interchangeably with the term 'habituation', which is usually restricted to behavioral changes arising from central synaptic mechanisms (Larkin, 2010).

The form of larval olfactory STH characterized in this study displays at least some of the defining behavioral characteristics of habituation. First, there is a behavioral decrement in response to repeated or sustained application of a particular stimulus. Second, STH shows spontaneous recovery with time in the absence of the habituating stimulus. And third, STH is susceptible to dishabituation when habituated larvae are presented with of a strong or noxious stimulus. The property of dishabituation is particularly significant, as an important way of distinguishing between habituation and either fatigue or sensory adaptation. Dishabituation shows that the habituated animal retains the capability to respond and suggests that the attenuated behavioral response arises from some form of active suppression. Thus, the behavioral data suggest (1) that the term 'habituation' may be better used in place of 'adaptation,' while referring to the behavioral phenomenon that was studied; and (2) that STH probably arises from central synaptic mechanisms, rather than sensory neuron adaptation (Larkin, 2010).

Three main lines of data support the conclusion that STH arises from a central synaptic mechanism that resides in the antennal lobe, rather than from adaptation of olfactory receptor signaling in the OSN. First, behavioral decrements similar to STH can be induced by direct depolarization of OSNs, indicating that STH may potentially be induced by processes stimulated by activation action-potential firing in OSNs, independently of olfactory receptor activation. Second, and more striking, STH requires synaptic-vesicle exocytosis from local interneurons during the process of odorant exposure, when STH is being established. This requirement is incompatible with an exclusively sensory mechanism. Third, STH requires the function of NMDA receptors on postsynaptic projection neurons. This last observation also provides a particularly strong argument for a synaptic mechanism, indicating a need for plasticity of OSN and/or LN synapses made onto dendrites of projection neurons in the antennal lobe. Given that OSNs are excitatory and LNs are primarily inhibitory, it appears most likely that NMDAR functions in PNs to depress excitatory OSN-PN synapses and/or to potentiate inhibition by strengthening the LN-PN synapse. It is suggestd that the LN-PN mechanism may be involved because (1) LN transmission seems necessary for both induction and expression of habituation; and (2) the process of dishabituation could be attractively explained as arising from the inhibition of local inhibitory synapses through descending neuromodulation. A requirement for facilitation of the LN-PN synapse would be consistent with previous studies (Sachse, 2007) showing that adult-long-term olfactory habituation is associated with an increase in odor-evoked calcium fluxes in GABAergic processes within the Drosophila antennal lobe (Larkin, 2010).

Based both on experimental and theoretical arguments, a simple model is suggested for short-term olfactory habituation. Since this is a model, no claim is being made to to having ruled out additional major contributing mechanisms, It is suggested that during initial odorant pre-exposure, dendritic NMDA receptors on projection neurons detect and respond to membrane depolarization occurs coincident with transmitter release from LNs. Calcium entry through dendritic NMDA receptors may trigger a local retrograde signal required for facilitation of transmitter release from the LNs. Although existing data do not rule out functions for rutabaga in higher larval brain centers, it is suggested that either the generation of a retrograde signal in PN dendrites or the presynaptic response of LNs to this signal could be dependent on the rut adenylate cyclase. In habituated animals, facilitation of GABA release would reduce odor-evoked projection neuron outputs to higher brain centers, thereby reducing olfactory behavior. As NMDAR signaling would only occur at active glomeruli, this mechanism can account not only for the observed odor selectivity of habituation, but also the instances of cross-habituation (Larkin, 2010).

Such a model also naturally suggests a hypothesis for the mechanism of dishabituation: namely, that dishabituating stimuli cause release of neuromodulators that act to reduce GABA release from local inhibitory synapses (Larkin, 2010).

Given the remarkable similarities in the anatomical organization of insect and mammalian olfactory systems, a significant conservation of olfactory mechanisms would be expected. In rodents, at least two forms of habituation have been described, lasting 2-3 and 30-60 min, respectively: the latter equivalent in timescale to larval STH described in this study. Consistent with a similar underlying mechanism, the more persistent form of olfactory habituation can be blocked by an N-methyl-D-aspartate (NMDA) receptor antagonist in the olfactory bulb, a structure homologous to the insect antennal lobe. Thus, larval STH described in this study has some similarities to a previously characterized form of mammalian olfactory habituation. Analysis of the underlying mechanisms is therefore likely to provide directly transferable insights in mammalian olfaction. The data make the prediction that the activity of mammalian olfactory interneurons, either periglomerular or granule cells, is critical for the establishment and display of at least one timescale of olfactory habituation (Larkin, 2010).

In addition to providing some insight into mechanisms of olfactory habituation in mammals, it possible that circuit mechanisms of larval olfactory habituation are relevant to other forms of behavioral habituation. In at least three previous instances, increased inhibition has been associated with attenuated behavior. For example, habituation of an escape reflex mediated by the lateral giant fibers in the crayfish has been associated with enhanced GABAergic transmission onto giant fibers. Similarly, LTP of inhibitory synapses controlling excitability of the Mauthner cell has been associated with reduced escape behavior in goldfish. Furthermore, ethanol, a potentiator of GABA synapses, has been shown to enhance habituation of a motor pathway in the frog spinal cord. Could these different instances of habituation all involve circuit mechanisms similar to those used in Drosophila larval olfactory behavior (Larkin, 2010)?

In all brain regions, principal/projection neurons are subject to inhibitory feedback modulation and a pathway that has been appreciated as potentially essential for neuronal homeostasis. Potentiation of inhibitory feedback triggered by the pattern of principle cell activation would be predicted to preferentially dampen this particular output pattern. Thus, the circuit mechanism suggest in this study is theoretically generalizable to other and more complex forms of habituation. Further experiments will be required to determine the validity of this very testable hypothesis (Larkin, 2010).

The importance of habituation has been underlined by the fact that deficits in sensory gating and pre-pulse inhibition (PPI), processes with similarities to habituation, have been linked with various neurological problems, including autism and schizophrenia. Indeed, a circuit model for understanding schizophrenia has specifically proposed that altered negative feedback in the hippocampus may underlie both positive and negative symptoms of schizophrenia (Larkin, 2010).

In addition, defects in habituation or habituation-like processes have been described in Fragile X syndrome and migraines. It has also been shown to have important effects relating to learning disabilities, age-related changes in learning, and substance abuse. If mechanisms of olfactory habituation prove to be general, then studies of olfactory plasticity may prove relevant for other forms of cognition as well as for human neurological disease (Larkin, 2010).

Plasticity of local GABAergic interneurons drives olfactory habituation

Despite its ubiquity and significance, behavioral habituation is poorly understood in terms of the underlying neural circuit mechanisms. This study presents evidence that habituation arises from potentiation of inhibitory transmission within a circuit motif commonly repeated in the nervous system. In Drosophila, prior odorant exposure results in a selective reduction of response to this odorant. Both short-term (STH) and long-term (LTH) forms of olfactory habituation require function of the rutabaga-encoded adenylate cyclase in multiglomerular local interneurons (LNs) that mediate GABAergic inhibition in the antennal lobe; LTH additionally requires function of the cAMP response element-binding protein (CREB2) transcription factor in LNs. The odorant selectivity of STH and LTH is mirrored by requirement for NMDA receptors and GABAA receptors in odorant-selective, glomerulus-specific projection neurons (PNs). The need for the vesicular glutamate transporter in LNs indicates that a subset of these GABAergic neurons also releases glutamate. LTH is associated with a reduction of odorant-evoked calcium fluxes in PNs as well as growth of the respective odorant-responsive glomeruli. These cellular changes use similar mechanisms to those required for behavioral habituation. Taken together with the observation that enhancement of GABAergic transmission is sufficient to attenuate olfactory behavior, these data indicate that habituation arises from glomerulus-selective potentiation of inhibitory synapses in the antennal lobe. It is suggested that similar circuit mechanisms may operate in other species and sensory systems (Das, 2011)

A key observation is that rut function is uniquely required in adult-stage GABAergic local interneurons for STH and LTH. This observation contrasts with the rut requirement in mushroom-body neurons for olfactory aversive memory. The demonstration of fundamentally different neural mechanisms used in olfactory habituation and olfactory-associative memory elegantly refutes a proposal of the Rescorla-Wagner model that habituation (and extinction) may be no more than associations made with an unconditioned stimulus of zero intensity (Das, 2011)

The requirement for rut in inhibitory LNs also indicates that intrinsic properties of multiglomerular LNs change during habituation. However, logic, as well as anatomical and functional imaging data, indicate that glomerulus-selective plasticity must be necessary if LN changes produce odorant-selective habituation. A potentially simple mechanism for glomerulus-specific potentiation of LN terminals is suggested by the specific requirement for postsynaptic NMDAR in odorant-responsive glomeruli (Das, 2011)

The observation that LTH and STH show similar dependence on rut, NMDAR, VGLUT, GABAA receptors, and transmitter release from LN1 cells indicates a substantially shared circuit mechanism for the two timescales of habituation. The data point to a model in which transient facilitation of GABAergic synapses underlies STH; long-lasting potentiation of these synapses through CREB and synaptic growth-dependent processes underlies LTH. This finding differs in three ways from synaptic facilitation that underlies Aplysia sensitization. First, it refers to inhibitory synapses, with potentiation that may involve a specific heterosynaptic mechanism similar to that used for inhibitory Long Term Potentiation (iLTP) in the rodent ventral tegmentum. Second, by presenting evidence for necessary glutamate corelease from GABAergic neurons, it proposes the involvement of a relatively recently discovered synaptic mechanism for plasticity. Third, it posits an in vivo mechanism to enable glomerulus- specific plasticity of LN terminals (Das, 2011)

It is pleasing that, in all instances tested, physiological and structural plasticity induced by 4-d odorant exposure requires the same mechanisms required for behavioral LTH. When taken together, these different lines of experimental evidence come close to establishing a causal connection between behavioral habituation and accompanying synaptic plasticity in the antennal lobe (Das, 2011)

It is important to acknowledge that, although the current experiments show that plasticity of LN-PN synapses contributes substantially to the process of behavioral habituation, it remains possible that plasticity of other synapses, such as of recently identified excitatory inputs made onto inhibitory LNs, also accompany and contribute to olfactory habituation (Das, 2011)

The conserved organization of olfactory systems suggests that mechanisms of olfactory STH and LTH could be conserved across species. Although this prediction remains poorly tested, early observations indicate that a form of pheromonal habituation in rodents, termed the Bruce effect, may arise from enhanced inhibitory feedback onto mitral cells in the vomeronasal organ (Das, 2011)

Less obviously, two features of the circuit mechanism that we describe suggest that it is scalable and generalizable. First, selective strengthening of inhibitory transmission onto active glomeruli can be used to selectively dampen either uniglomerular (CO2) or multiglomerular (EB) responses; thus, the mechanism is scalable. Second, the antennal lobe/olfactory bulb uses a circuit motif commonly repeated throughout the brain, in which an excitatory principal cell activates not only a downstream neuron but also local inhibitory interneurons, which among other things, limit principal cell excitation (Das, 2011)

It is possible that, in nonolfactory regions of the brains, a sustained pattern of principal neuron activity induced by a prolonged, unreinforced stimulus could similarly result in the specific potentiation of local inhibition onto these principal neurons. Subsequently, the pattern of principal cell activity induced by a second exposure to a now familiar stimulus would be selectively gated such that it would create only weak activation of downstream neurons. In this manner, the circuit model that is proposed for olfactory habituation could be theoretically generalized. More studies are expected to test the biological validity of this observation (Das, 2011)

Calcium-stores mediate adaptation in axon terminals of olfactory receptor neurons in Drosophila

In vertebrates and invertebrates, sensory neurons adapt to variable ambient conditions, such as the duration or repetition of a stimulus, a physiological mechanism considered as a simple form of non-associative learning and neuronal plasticity. Although various signaling pathways, as cAMP, cGMP, and the inositol 1,4,5-triphosphate receptor (InsP3R) play a role in adaptation, their precise mechanisms of action at the cellular level remain incompletely understood. In Drosophila odor-induced Ca2+-response in axon terminals of olfactory receptor neurons (ORNs) has been shown to be related to odor duration. In particular, a relatively long odor stimulus (such as 5 s) triggers the induction of a second component involving intracellular Ca2+-stores. A recently developed in-vivo bioluminescence imaging approach was used to quantify the odor-induced Ca2+-activity in the axon terminals of ORNs. Using either a genetic approach to target specific RNAs, or a pharmacological approach, this study showed that the second component, relying on the intracellular Ca2+-stores, is responsible for the adaptation to repetitive stimuli. In the antennal lobes (a region analogous to the vertebrate olfactory bulb) ORNs make synaptic contacts with second-order neurons, the projection neurons (PNs). These synapses are modulated by GABA, through either GABAergic local interneurons (LNs) and/or some GABAergic PNs. Application of GABAergic receptor antagonists, both GABAA or GABAB, abolishes the adaptation, while RNAi targeting the GABABR (a metabotropic receptor) within the ORNs, blocks the Ca2+-store dependent component, and consequently disrupts the adaptation. These results indicate that GABA exerts a feedback control. Finally, at the behavioral level, using an olfactory test, genetically impairing the GABABR or its signaling pathway specifically in the ORNs disrupts olfactory adapted behavior. Taken together, these results indicate that a relatively long lasting form of adaptation occurs within the axon terminals of the ORNs in the antennal lobes, which depends on intracellular Ca2+-stores, attributable to a positive feedback through the GABAergic synapses (Murmu, 2011).

This study provides evidence that the bioluminescent (GFP-aequorin) Ca2+-sensor is sensitive enough to monitor the Ca2+-response following various protocols (duration and repetition-frequency) of odor application. 1 s of odor induces a response which does not significantly decrease if repeated every 5 min, whereas a longer stimulus, such as 5 s, is sufficient to induce a decrease in response following repeated odor stimulations (adaptation). Similarly, using a 5 s odor stimulation and increasing the frequency of repetition to 1-min intervals also induces, in an odor specific manner, a faster adaptation. It was also demonstrated that prolonged odor application (up to 2 min) generates a sustained Ca2+-response within the ORN axon terminals, indicating that the ORNs are capable of responding as long as the odor is presented, of even longer. This work also indicates that the GFP-aequorin probe is not a limiting factor for the detection of the Ca2+-activity. These physiological results (reduction of the Ca2+-activity according to prolonged/sustained odor duration and/or odor repetition) are consistent with previous studies which report that adaptation depends both on the duration of a stimulus and on the frequency of its repetition (Murmu, 2011).

Different physiological approaches, based either on fluorescence brain imaging or electrophysiological techniques have previously reported odor-induced activity in different interconnected neurons in the antennal lobes of different invertebrate model organisms, including honeybees with the goal of deciphering the neural odor code. However, except for one study performed in locusts, which indirectly described a form of adaptation, long-lasting forms of adaptation within ORNs such as that described in this study have not been reported. This is likely due to the experimental design of these previous studies, which either generally took into account the odor-induced signal solely after the response was stabilized (generally after about 5 successive odor applications), or used a shorter odor stimulation duration (< = 1 s), which as demonstrated in this study, is not sufficient to induce detectable and reliable adaptation. Additionally, others have relied on extracellular recordings of the sensillae of the antennae which reflects the activity occurring in the cell-bodies of the ORNs. In this study, monitoring the axon terminals of the ORNs, 5 s odor stimulations, repeated at 5-min intervals, induced a relatively long-lasting adaptation that resembles in term of kinetics, the long-lasting adaptation (LLA) reported in ORNs in salamanders. Indeed and interestingly, the recovery time (15 min for spearmint and octanol and 30 min for citronella) occurs over a similar time scale in salamander ORNs (which are different from the long-lasting olfactory adaptation described in C. elegans. However, in contrast to long-lasting adaptation, which was reported in isolated ORNs, the adaptation described in this study seems to rely on different mechanisms, since it is sensitive to a 'feedback control' provided by GABAergic synaptic transmission within the antennal lobes (Murmu, 2011).

In Drosophila, mutants lacking InsP3R are defective in olfactory adaptive behavior. In vertebrates, different forms of olfactory adaptation have also been reported in the ORNs. First, this study shows in Drosophila that an adaptation mechanism occurs in axon terminal of the ORNs in the antennal lobes. Second, using two independent approaches, pharmacological and genetic, it was shown that odor-induced specific adaptation relies principally on InsP3R and RyR. When these two different receptors are blocked or knocked-down, although some difference (variability) can be observed between different conditions, overall the odor-induced Ca2+-response no longer adapts or is severely affected. More specifically, it seems that the lack of adaptation is due to the non-induction of the 'second delayed and slow rising component' of the Ca2+-response, which is triggered in particular and specific conditions: when the duration of an odor stimulation is relatively long (1 s does not induce it, while 5 s induces an important second component. Alternatively, the second component of the response is also induced and visible particularly on the first and to a lesser extent, on the second odor applications, especially when the odor is successively repeated. This second component gradually vanishes with sequential repetition. That is, adaptation is not directly due to a decrease in the response, but rather indirectly to a defect in presynaptic Ca2+-increase, due to a lack of triggering release of intracellular Ca2+-stores, normally occurring in the first and successive responses following either a sufficiently strong (long stimulus) or repeated stimuli. These results suggest that one of the major intracellular mechanisms of adaptation depends on internal Ca2+-stores. In brief, the intracellular mechanism was blocked that allows the cell to adapt to long lasting or repetitive stimuli. Interestingly, in mammals, in hippocampal CA3 pyramidal neurons, intracellular Ca2+-stores, which are controlled by InsP3R and/or RyR at the presynaptic terminal, have been previously implicated in neurotransmitter release as well as in synaptic plasticity (Murmu, 2011).

In vertebrates, neuronal plasticity related to odor representation occurs at the synapse between the ORNs and the second-order neurons in the olfactory bulb glomeruli, a region analogous to the invertebrate antennal lobes. At this synapse, signal transmission is modulated presynaptically by several mechanisms, a major one being via the metabotropic GABAB receptors. This suppresses presynaptic Ca2+-influx and subsequently transmitter release from the receptor neurons terminal. At least two kinds of presynaptic inhibition (intra- and interglomerular) are mediated by GABAB receptors. Intraglomerular presynaptic inhibition seems to control input sensitivity, while interglomerular presynaptic inhibition seems to increase the contrast of sensory input (although the two studies addressing this question in-vivo show contradictory results). In Drosophila, a similar mechanism seems to occur, as interglomerular presynaptic inhibition, mediated by both ionotropic and metabotropic receptors on the same axon terminal of the ORNs, mediate gain control mechanism, serving to adjust the gain of PN in response to ORN stimulation (Olsen, 2008). Yet another study has suggested that GABAB but not GABAA receptors are involved in presynaptic inhibition (Root, 2008) yielding a contradiction. In this study, by monitoring the Ca2+-release from the axon terminals of ORNs, in experimental conditions that generate a long-lasting form of adaptation, it was shown that GABAergic synaptic transmission plays a role in adaptation. Both ionotropic GABAAR antagonists, bicuculline and picrotoxin, block partially or completely the Ca2+-response, while, CGP54626, a metabotropic GABABR antagonist, also blocks the adaptation, albeit not completely. It should be mentioned that application of picrotoxin per se induces a strong transient Ca2+-release within the axon terminals of the ORNs, even without odor application. This 'transient release effect' likely disturbs the resting state of the neurons, which probably accounts for the important reduction observed in the amplitude of the odor-induced response. Nevertheless, these results suggest that both types of GABA receptors (A and B) are involved in adaptation. Moreover, as proposed by the study of Olsen and Wilson (2008), it cannot be ruled-out that ORNs also express different subtypes of GABAAR (homo- and/or heteromultimers), since the results showed that picrotoxin and particularly bicuculline, two distinct inhibitors of GABAAR, block adaptation. Another possibility is that the effect of the two GABAAR antagonists results from the blockage of GABAAR on other neurons in the antennal lobes, as the LNs or certain PNs (which have not yet been demonstrated). Lastly and unfortunately, this pharmacological approach does not allow distinguishing by which precise neurons this GABAergic-dependent adaptation is mediated. With the goal of clarifying precisely in which neurons GABAergic transmission acts, the metabotropic GABABR (GABABR2-RNAi) or its signaling pathway (UAS-PTX) were blocked directly within ORNs. This yields defects in long-lasting adaptation for several conditions, seemingly in an odor specific manner. Therefore, although GABAergic effects have been described in ORNs of both Drosophila and mammals, to support 'feedback inhibition', this study reports that in different experimental conditions such as a long odor duration (5 s) and/or repetition of the stimulus, it also participates in the adaptation process. Indeed, the results suggest that GABA signaling support a positive (excitatory) feedback control instead of an inhibitory feedback, as formerly reported by other studies. Though these results seem to be contradictory, some explanations can be provided. First, as aforementioned, the experimental conditions are different: this study used a relatively high odor concentration with relatively long odor duration (5 s). In addition, recordings were taken immediately from the first odor application and the successive one, while in the experimental protocol of certain studies, the odor is generally presented several times (priming) before the beginning of recording. Consequently, it seems that these previous studies were performed on already adapted ORNs. This implies that the neuronal network in the antennal lobes was already stimulated, and therefore its dynamics was probably already modified, since as described in this study, an important effect occurs immediately after the first odor application. Moreover, GFP-aequorin allows monitoring, in continuity over a long time period, the intracellular level of calcium with high sensitivity to [Ca2+] (from ~ 10-7 to 10-3). In addition, although it is not possible to precisely assign which glomeruli are activated, this approach allows visualizing simultaneously the odor-induced Ca2+-activity from the entire antennal lobes (the overall depth). Therefore, the outcome of the overall response of the antennal lobes is being monitored, instead of the response from single or a few glomeruli. Finally, in vertebrates it has been reported that in certain experimental conditions, GABA could be excitatory, although this contradiction cannot yet be precisely explained. Furthermore, it seems that a given synapse can display inhibitory effects under one protocol and an excitatory effect with another. Notably, it has been reported that a short stimulation of GABA is inhibitory, while during a long stimulation, the GABA effect can switch from inhibitory to excitatory. Interestingly, this particular 'switching effect' could potentially explain the current 'contradictory' situation reported in this study: in the current experimental conditions, in which a relatively long odor stimulus (5 s) was used, GABA generates an excitatory effect, whereas in previous studies based on short (<1 s) odor stimuli, GABA seems to be inhibitor. This difference in the duration of stimuli could perhaps account for such inverted or 'switching effects' (Murmu, 2011).

To explore the behavioral and functional consequences of disturbing the GABAergic signaling pathway, flies were studied with a GABABR2 (RNAi) ORN-specific genetic knockdown, as well those with a component of its signaling pathway, the G-protein, blocked by the pertussis toxin. Both groups of flies present strong behavioral deficits, as adaptation-disrupted flies are not able to discern between odors and air after 5-min of exposure to odor. Interestingly, control flies reverse their choice preferring odor after a 5-min pre-exposure (adaptation) suggesting that in these experimental conditions, the meaning of the odor changes in the fly's adapted state. These results are consistent with previous studies suggesting that adaptation could serve to extend the operating range of sensory systems over different stimulus intensities. In other terms, adaptation modifies the sensitivity (threshold) to the odor, as previously reported in different organisms, such as C. elegans and vertebrates including humans. This phenomenon is similar to that in other sensory modalities, as in visual system, where light adaptation in photoreceptors sets the gain, allowing vision at both high and low light levels. As previously reported, odors could be repulsive (at high concentrations) or attractive (at low concentrations). In the current experimental conditions in control flies the odors are repulsive. However, after 5-min of preexposure, the flies adapt to this odor concentration, and when tested at the same concentration odors are then likely only weakly perceived and therefore might correspond to an attractive 'weak-odor concentration'. In a former study in similar experimental conditions, it was reported that the flies are attracted by each of these three odors for a weak odor concentration. Interestingly, reverse odor preference has already been reported in C. elegans, resulting from presynaptic changes involving a receptor-like guanylate cyclase (GCY-28) via the diacylglycerol/protein kinase C pathway. Finally, the fact that without pre-exposure all groups of flies preferred the control arm and were repelled by the odorants indicates that the odor acuity of these flies is intact. In other words, odor-adaptation and not odor-acuity is affected in each of these groups of flies. These results strengthen the idea that odor perception and adaptation are indeed two distinct and separable processes (Murmu, 2011).

This study has demonstrated that the adaptation process occurring specifically in the axon terminals of the ORNs depends on intracellular Ca2+-stores, through InsP3 and ryanodine receptors. Moreover, evidence is provided that this Ca2+-release requires synaptic transmission, since it does not occur when the cholinergic receptors are blocked (α-bungarotoxin experiment). It also requires a feedback control through GAB A, since blocking GABAB signaling within ORNs prevents or strongly affects adaptation, suggesting that a local neuronal network mediated by GABAergic neurons is involved (for a more complete overview, see the schematic model of synaptic interactions within the antennal lobes. In complement to the brain imaging data, knocking-down the metabotropic GABABR2, or its signaling pathway specifically in the ORNs, yields olfactory functional behavioral deficits. These results, combined with the results of blocking the InsP3R or RyR suggest that a crucial olfactory integration process that can be ascribed to a form of neuronal plasticity and/or short-term memory occurs directly in the ORNs immediately after the first odor application or during a prolonged odor application. Thus, this effect could resemble the long-lasting form of odor adaptation described previously at the cellular and systems levels in vertebrates, including humans. By extension, it is hypothesized that in humans, the well-known 'odor-specific transient functional anosmia' following a prolonged odor exposure, which results from an adaptation, may also rely on intracellular Ca2+-stores (Murmu, 2011).

Suppression of inhibitory GABAergic transmission by cAMP signaling pathway: alterations in learning and memory mutants

The cAMP signaling pathway mediates synaptic plasticity and is essential for memory formation in both vertebrates and invertebrates. In the fruit fly Drosophila melanogaster, mutations in the cAMP pathway lead to impaired olfactory learning. These mutant genes are preferentially expressed in the mushroom body (MB), an anatomical structure essential for learning. While cAMP-mediated synaptic plasticity is known to be involved in facilitation at the excitatory synapses, little is known about its function in GABAergic synaptic plasticity and learning. Using whole-cell patch-clamp techniques on Drosophila primary neuronal cultures, this study demonstrates that focal application of an adenylate cyclase activator forskolin (FSK) suppressed inhibitory GABAergic postsynaptic currents (IPSCs). A dual regulatory role of FSK on GABAergic transmission was observed, where it increases overall excitability at GABAergic synapses, while simultaneously acting on postsynaptic GABA receptors to suppress GABAergic IPSCs. Further, it was shown that cAMP decreased GABAergic IPSCs in a PKA-dependent manner through a postsynaptic mechanism. PKA acts through the modulation of ionotropic GABA receptor sensitivity to the neurotransmitter GABA. This regulation of GABAergic IPSCs is altered in the cAMP pathway and short-term memory mutants dunce and rutabaga, with both showing altered GABA receptor sensitivity. Interestingly, this effect is also conserved in the MB neurons of both these mutants. Thus, this study suggests that alterations in cAMP-mediated GABAergic plasticity, particularly in the MB neurons of cAMP mutants, account for their defects in olfactory learning (Ganguly, 2013).

Ca2+/CaM dependent adenylate cyclase (AC) produces cAMP and is also known to function as a co-incidence detector during learning in both Drosophila and Aplysia. In addition, AC-dependent cAMP activation changes the strength of Drosophila excitatory synapses which may be the cellular mechanism underlying learning and memory. Although inhibitory synaptic transmission is equally important for proper neuronal communication, the effects of cAMP at the inhibitory GABAergic synapses have remained unexplored. This study shows that forskolin (FSK), an activator of cAMP, suppresses the frequency of inhibitory GABAergic IPSCs in Drosophila primary neuronal cultures. A concentration dependent effect of FSK on GABAergic IPSCs was observed in the same physiological range as described in recent imaging studies in intact fly brains. Further cAMP was shown to decrease GABAergic IPSCs in a PKA-dependent manner through a postsynaptic mechanism (Ganguly, 2013).

Sparsening of odor representation through GABAergic inhibition in the mushroom body (MB) neurons is thought to be a possible mechanism for information storage in locusts (Perez-Orive, 2002). GABAergic local neurons are known to be involved in olfactory information processing in Drosophila (Wilson, 2005; Olsen, 2008) indicating that GABAergic transmission plays a crucial role in shaping odor response. The MB shows extensive GABAergic innervation in both locusts (Perez-Orive., 2002) and Drosophila (Yasuyama, 2002). This, along with the observation that cAMP pathway genes like dunce and rutabaga are preferentially expressed in the MB (Davis, 2011), indicates that cAMP mediated GABAergic plasticity may be important for learning in Drosophila. Consistent with this hypothesis, this study observed altered cAMP mediated GABAergic IPSCs in the cAMP mutants dnc1 and rut1. The effect of cAMP on suppression of GABAergic currents was less pronounced in the mutants. This suggests that the altered inhibition contributes to their observed learning defects. In fact, recent studies have shown that GABAA RDL receptors expressed in the MB and GABAergic neurons projecting to the MB are essential for olfactory learning (Liu, 2007; Liu, 2009). It is thus possible that altered cAMP mediated GABAergic plasticity at the MB neurons may account for some forms of the learning defects in Drosophila (Ganguly, 2013).

GABAergic IPSCs are known to act through picrotoxin-sensitive postsynaptic GABA receptors in both Drosophila embryonic and pupal neuronal cultures. This study observed that the suppression of GABAergic IPSCs by FSK is completely abolished in the presence of a membrane impermeable PKA inhibitor restricted to the postsynaptic neuron. This indicates that PKA may modulate GABAergic IPSCs by regulating GABA receptor sensitivity by phosphorylation, similar to what has been suggested in the mammalian hippocampus (Ganguly, 2013).

There are three known ionotrophic GABA receptor gene homologs in Drosophila - RDL, LCCH3 and GRD. Amongst them, the GABA RDL subunit is widely expressed in several regions of the Drosophila brain and its expression in the MB is inversely correlated to olfactory learning. Therefore, RDL-containing GABA receptors may play an important role in cAMP-dependent synaptic plasticity and thus be involved in learning and memory. The data suggests that the majority of synaptic GABA receptors contain the RDL subunit while a small fraction of synaptic GABA receptors lack RDL, providing evidence of heterogeneous synaptic GABA receptors in Drosophila for the first time. However, it is still not known what particular subunit of GABA receptors is involved in regulation of cAMP-dependent GABAergic plasticity. Based on the observation that RDL containing GABA receptors mediate the majority of GABAergic IPSCs in Drosophila primary neuronal cultures, the action of FSK on GABAergic IPSCs is probably through the GABA RDL subunit. While the detailed molecular mechanism remains to be explored, it is proposed that PKA-mediated phosphorylation of RDL subunits and subsequent GABA receptor internalization may occur in the postsynaptic region. In this scenario, the only functional synaptic GABA receptors will be those lacking the RDL subunit at the postsynaptic regions. This will account for a decrease in mIPSC frequency with response to FSK, while leaving mIPSC amplitude almost unchanged (Ganguly, 2013).

Although GABA receptor subunits would be the target of cAMP-PKA signaling the possibility that other molecules can be phosphorylated and then indirectly regulate GABA receptor subunits can still not be rule out. Future work using heterologous expression systems for GABA subunits will help to determine whether GABA receptors are directly phosphorylated (Ganguly, 2013).

Recordings from embryonic and pupal MB neurons of both dunce and rutabaga mutants show a defect in ionotropic GABA receptor response in the presence of FSK. Interestingly, this response to FSK is similar in both the mutants despite their contrasting levels of cellular cAMP. Recent imaging studies in the rutabaga mutant have shown that AC is required for co-incidence detection in the MB neurons. FSK application also fails to increase PKA to wild-type levels in the MB neurons of rutabaga. Thus in the current experiments, the changes in receptor response in rutabaga can be explained by a lack of increase in cAMP/PKA levels due to defects in FSK-mediated AC activation (Ganguly, 2013).

The dunce mutants with high levels of cAMP also show defects in short-term memory due to alterations in the spatiotemporal restriction of dunce phosphodiesterase to the Drosophila MB. Further, the dunce MB neurons show an increase in PKA levels on FSK application similar to the wild-type strains. These findings suggest that FSK-mediated inhibition of GABA receptor should be greater in dunce neurons. However, in the current results the dunce and rutabaga mutants, despite having opposing effects on cellular cAMP levels, showed very similar FSK mediated effects on GABAergic IPSCs. Several other studies have also shown that dunce and rutabaga have similar defects in growth cone motility, excitatory synaptic plasticity and more importantly, short-term memory. Even though the effect of FSK on GABAergic IPSCs in dunce and rutabaga mutants is similar, it is very likely that the molecular mechanisms underlying these responses differ in the two mutants. It has been shown that elevated cAMP signaling reduces phosphorylation in rat kidney cells through activation of protein phosphatase 2A. In addition, increased PKA activity in mouse hippocampus hyper-phosphorylates several downstream molecular targets including a tyrosine phosphatase (STEP), correlates with decreased phosphodiesterase protein (PDE4) levels and results in memory defects. Therefore, it is tempting to speculate that high levels of cAMP due to the dunce mutation leads to the activation of phosphatase(s) and thus reduces the effects of FSK as seen in the current study. Taken together, all these findings strongly suggest that the disruption of cellular cAMP homeostasis can alter inhibitory GABAergic synaptic plasticity and hence cause defects in olfactory learning, although the underlying mechanisms leading to this effect can be different (e.g. reduced PKA activity in rut1 versus increased phosphatase activity in dnc1) (Ganguly, 2013).

Strengthening in the efficacy of excitatory transmission underlies enhanced synaptic plasticity such as hippocampal long-term potentiation (LTP) and facilitation (LTF) in Aplysia. It is thus possible that the suppression of inhibitory transmission by a common second messenger like cAMP, which can enhance excitatory synaptic transmission, may lead to synaptic strengthening. Previous work has shown that the cAMP activator FSK increases excitability at the cholinergic synapses in Drosophila primary neuronal cultures. However the effect of FSK on other synapses like the GABAergic synapses has not been explored. This study shows that FSK elevates overall cellular excitability at GABAergic synapses as demonstrated by the increase in spontaneous AP frequency. Moreover, when PKA in the postsynaptic neuron is completely blocked by an inhibitor, an increase is seen in the frequency of GABAergic IPSCs. Together with previous studies on cholinergic synapses (Yuan, 2007), the current results indicate that FSK/cAMP act as common molecules regulating globally presynaptic excitability at both the cholinergic as well as GABAergic synapses. It is also noted that FSK inhibits the response of postsynaptic GABA receptors in a specific manner leading to a decrease in GABAergic synaptic strength. These studies demonstrate a novel dual regulatory role of cAMP by showing that it increases overall presynaptic function on one hand; and, acts specifically on postsynaptic GABA receptors to decrease GABAergic plasticity on the other. This action of cAMP could result in global increases in excitability and learning (Ganguly, 2013).

The role of Rdl in resistance to phenylpyrazoles in Drosophila melanogaster

Extensive use of older generation insecticides may result in pre-existing cross-resistance to new chemical classes acting at the same target site. Phenylpyrazole insecticides block inhibitory neurotransmission in insects via their action on ligand-gated chloride channels (LGCCs). Phenylpyrazoles are broad-spectrum insecticides widely used in agriculture and domestic pest control. So far, all identified cases of target site resistance to phenylpyrazoles are based on mutations in the Rdl (Resistance to dieldrin) LGCC subunit, the major target site for cyclodiene insecticides. This study examined the role that mutations in Rdl have on phenylpyrazole resistance in Drosophila melanogaster, exploring naturally occurring variation, and generating predicted resistance mutations by mutagenesis. Natural variation at the Rdl locus in inbred strains of D. melanogaster included gene duplication, and a line containing two Rdl mutations found in a highly resistant line of Drosophila simulans. These mutations had a moderate impact on survival following exposure to two phenylpyrazoles, fipronil and pyriprole. Homology modelling suggested that the Rdl chloride channel pore contains key residues for binding fipronil and pyriprole. Mutagenesis of these sites and assessment of resistance in vivo in transgenic lines showed that amino acid identity at the Ala301 site influenced resistance levels, with glycine showing greater survival than serine replacement. It was confirmed that point mutations at the Rdl 301 site provide moderate resistance to phenylpyrazoles in D. melanogaster. The beneficial aspects of testing predicted mutations in a whole organism is emphasized to validate a candidate gene approach (Remnant, 2014).

GABAA receptor-expressing neurons promote consumption in Drosophila melanogaster

Feeding decisions are highly plastic and bidirectionally regulated by neurons that either promote or inhibit feeding. In Drosophila melanogaster, recent studies have identified GABAergic interneurons that act as critical brakes to prevent incessant feeding. These GABAergic neurons may inhibit target neurons that drive consumption. This study tested this hypothesis by examining GABA receptors and neurons that promote consumption. Resistance to dieldrin (RDL), a GABAA type receptor, is required for proper control of ingestion. Knockdown of Rdl in a subset of neurons causes overconsumption of tastants. Acute activation of these neurons is sufficient to drive consumption of appetitive substances and non-appetitive substances and acute silencing of these neurons decreases consumption. Taken together, these studies identify GABAA receptor-expressing neurons that promote Drosophila ingestive behavior and provide insight into feeding regulation (Cheung, 2017).

The dissection of neural circuits that underlie consumption remains an important challenge toward understanding the regulation of feeding behavior. This study identifies neurons that regulate the consumption of non-appetitive and appetitive substances, and depend on the expression of RDL receptor for proper regulation of consumption. These RDL receptor-expressing neurons are able to orchestrate consumption regardless of taste quality, as knockdown of Rdl expression within these neurons not only causes overconsumption of sugar, bitter, and water substances, but tasteless substances as well. Acute activation of these neurons also caused overconsumption of sweet, bitter and water substances, whereas blocking neurotransmission of these neurons results in decreased sucrose consumption in starved flies. These studies reveal a subset of neurons that play a critical role in promoting consumption (Cheung, 2017).

Previous studies have identified two different classes of interneurons that trigger sucrose consumption. FDG neurons are located in the SEZ and respond to sugar stimulation on the proboscis and the cholinergic IN1 neurons respond to sugar stimulation of the internal mouthparts. These two classes of neurons respond selectively to sucrose, suggesting that there is a pathway selective for regulating sucrose consumption. Similarly, ectopic activation of these neurons increased consumption of sucrose but not water or bitter. These studies indicate that consumption of sucrose is regulated independently of consumption of water or bitter and argue for distinct circuits mediating consumption for each class of tastant. The RDL-expressing neurons differ from previously identified consumption neurons because either knockdown of Rdl or optogenetic activation of these neurons elicited consumption not only of appetitive substances, but also of non-appetitive substances. One model suggested by these studies that bears testing is that there may be distinct circuits for sweet, water, and bitter food sources that all converge on the RDL-expressing neurons (Cheung, 2017).

Knockdown of Rdl results in increased consumption of water, sucrose and bitter substances. These RDL neurons may be inhibited by GABAergic neurons such as DSOG1. Previous studies indicate that DSOG1 neurons act as a tonic inhibitor of consumption. Flies with silenced DSOG1 neurons overconsume all taste substances independent of taste quality and nutritional state, very similar to the phenotype observed when activating the RDL neurons in this study. An attractive model is that GABA release from DSOG1 inhibits the RDL neurons, restricting consumption. Indeed, 'studies show that RDL neuronal silencing is able to suppress the DSOG1-silencing phenotype. Although the data are consistent with the model that DSOG1 acts on the RDL neurons, it remains possible that the RDL neurons and DSOG1 influence parallel pathways. Further characterization of the RDL neurons that promote consumption and the DSOG1 neurons that inhibit consumption will enable distinguishing of these models (Cheung, 2017).

This study demonstrates that RDL function in a subset of neurons is critical for the regulation of consumption of all substances, regardless of taste modality. Further studies characterizing these neurons and their interactions with the different neurons that regulate feeding will provide insight into the temporal dynamics and plasticity in feeding decisions (Cheung, 2017).

Intra-neuronal competition for synaptic partners conserves the amount of dendritic building material

Brain development requires correct targeting of multiple thousand synaptic terminals onto staggeringly complex dendritic arbors. The mechanisms by which input synapse numbers are matched to dendrite size, and by which synaptic inputs from different transmitter systems are correctly partitioned onto a postsynaptic arbor, are incompletely understood. By combining quantitative neuroanatomy with targeted genetic manipulation of synaptic input to an identified Drosophila neuron, this study shows that synaptic inputs of two different transmitter classes locally direct dendrite growth in a competitive manner. During development, the relative amounts of GABAergic and cholinergic synaptic drive shift dendrites between different input domains of one postsynaptic neuron without affecting total arbor size. Therefore, synaptic input locally directs dendrite growth, but intra-neuronal dendrite redistributions limit morphological variability, a phenomenon also described for cortical neurons. Mechanistically, this requires local dendritic Ca2+ influx through Dα7 nAChRs or through low-voltage-activated channels following GABAA receptor-mediated depolarizations (Ryglewski, 2017).

These results are consistent with competition of GABAergic and cholinergic inputs for a given amount of dendrites. It is proposed that total dendrite size is determined by global cues, such as the transcriptional identity of the neuron, hormone signals, and overall firing activity, all of which regulate dendritic length and branch numbers in many types of neurons. Manipulations that increase MN5 firing activity cause dendrite overgrowth via activation of CaMKII- and AP1-dependent transcription. MN5 firing causes global Ca2+ influx into the soma and the dendrites. Conversely, knockdown of the Drosophila Cav2 homolog cacophony reduces somatic and dendritic Ca2+ influx upon firing and decreases dendrite arbor size. Therefore, overall MN5 firing activity and global Ca2+ influx regulate total dendrite length, likely together with steroid hormones. This study showa that the total amount of available dendrite becomes locally redistributed within the arbor by competitive synaptic mechanisms. A mechanistic separation between global overall growth and local fine branching control might enable the formation and maintenance of dendritic branches at sites of newly formed input synapses while preventing dendrite overgrowth, thus enabling opportunistic functional connections with minimal dendrites (Ryglewski, 2017).

Competitive interactions between GABAergic and cholinergic inputs may optimize dendritic gestalt to partition synaptic inputs from different transmitter systems to segregated postsynaptic input domains. Whenever synaptic input to the GABAergic or to the cholinergic domain is increased, dendrites shift toward this domain and away from the other one. Vice versa decreased input to one domain causes dendrite shift to the other domain. Increasing input to both domains has no effect, thus indicating synaptic activity-dependent competition. Although regulation of dendrite growth by synaptic activity has previously been reported for excitatory cholinergic and glutamatergic, as well as excitatory and inhibitory GABAergic synapses, intra-neuronal dendrite shifts that reshape the entire tree have not been demonstrated (Ryglewski, 2017).

However, it must be noted that not all neurons contain distinctly different input domains, but some receive intermingled inputs of different transmitter classes. In addition, this analysis focused on the most abundant excitatory and inhibitory inputs to MN5, which segregate to different input domains (Kuehn, 2013). MN5 is a flight motorneuron that is monopolar with more than 4000 dendritic branches. MN5 likely receives also glutamatergic inputs and potentially also aminergic modulatory inputs. It remains unclear whether such input classes are also targeted to specific input domains, or whether these also compete for dendrites. Nonetheless, the current findings are likely of general interest, because segregated input domains for different transmitter classes also exist in mammalian pyramidal neurons. Although the underlying mechanisms have not been addressed in the mammalian brain, size reductions in pyramidal neuron apical dendrites are accompanied by size increases of the basal dendrites and vice versa. Moreover, the relative sizes of pyramidal neuron dendritic sub-trees are counterbalanced and stabilize total dendritic length. Similarly, for the Drosophila MN5, previously studies have reported high size variability of individual dendritic sub-trees while counterbalance between sub-tree sizes keeps total dendrite size variability low. This study provides evidence that competitive interactions between different synaptic inputs locally direct arbor growth in different input domains. This may be a general means to fine tune the gestalt of neurons with segregated input domains. The data on an identified Drosophila motoneuron may provide an entry point into understanding the mechanism by which presynaptic partners may instruct the re-distribution of postsynaptic dendrites between different input domains in a 'synaptotropic' manner (Ryglewski, 2017).

Although the mechanisms by which competition between GABAergic and cholinergic inputs locally directs dendrite growth require further studies, the current data indicate that both competitors employ the same intracellular signaling mechanism. In retinal ganglion cells, newly formed dendritic branches become selectively stabilized at sites of cholinergic input synapses from amacrine cells, and strictly local dendritic Ca2+ signals are required for this process. This study also found local dendritic Ca2+ signals at sites of cholinergic and at sites of GABAergic input, although there is no direct live imaging evidence of local branch stabilization by these signals. However, any manipulation, be it presynaptic or postsynaptic, that affects synaptic input to either the cholinergic or the GABAergic domain changes arbor size in that domain. Local dendritic Ca2+ signals at cholinergic synapses are expected, because the Dα7nAChR conducts Ca2+. By contrast, the Rdl GABAAR is a chloride channel. During vertebrate embryonic development, GABAAR activation is initially excitatory because of elevated intracellular chloride concentrations. This study found that such a developmental shift of the chloride reversal potential to more negative values at mature stages likely also exists in insect neurons and thus, may be more conserved than previously known (Ryglewski, 2017).

As a result, GABAAR activation during Drosophila pupal stages causes depolarization, which in turn, induces local dendritic Ca2+ influx through DmαG LVA Ca2+ channels. LVA channels are required for GABA-mediated dendrite redistribution, because GABAergic input fails to direct dendrites to the GABAergic domain in DmαG null mutants. This interpretation is consistent with a recent study demonstrating that depolarizing GABAergic input selectively promotes apical dendrite growth via Ca2+ influx during neonatal cortical neuron development. Therefore, directing dendrite arbor growth via GABA-mediated Ca2+ signals takes place in flies and mammals (Ryglewski, 2017).

In agreement with findings that basic flight motoneuron function is retained even with a minimal set of dendrites, intra-neuronal dendrite shifts as caused by either Rdl or nAChR overexpression did not alter firing rates or wingbeat frequencies during tethered flight. By contrast, adaptive adjustments of motoneuron firing frequencies in response to visual input during flight behavior were impaired. The defect was more severe with increased excitation and dendrite shifts to the cholinergic domain than with increased inhibition and dendrite shifts to the GABAergic domain. By contrast, motoneuron firing frequency adjustments were not affected following conditional receptor expression in mature neurons that caused no dendrite shifts. This suggested that the impairment was at least in part caused by dendrite redistribution, although this interpretation requires caution. First, possible indirect effects on network properties to result from receptor overexpression in MN5 through development cannot be completely excluded. Second, although the amount of receptor overexpression can be estimated, light microscopy analysis cannot rule out potential differences between conditional and chronic receptor expression. However, the data indicate that the balance between excitatory cholinergic input to the proximal dendritic domain and inhibitory GABAergic input to the distal domain may play a critical role for adaptive adjustments of motoneuron firing rates during behavior (Ryglewski, 2017).

Directing more dendrites to the input domain of increased GABAergic synaptic input and away from the domain of decreased cholinergic input, or vice versa, seems to contradict the concept of homeostasis. Why to shift more dendrites to sites with many synaptic partners, but away from sites with lower synapse densities? First, a mechanism that locally matches the amount of available dendritic surface to the amount of synapses is in accordance to minimizing cost. Maximum synapse numbers on minimal dendritic length has been proposed as a general principle in cortex. Second, redistribution of dendrites to domains with high synapse density may prevent superfluous increases of total arbor length. Naming dendrite redistribution while maintaining total length 'dendritic conservation' is suggested. Can dendritic conservation function in parallel to homeostatic control of excitability? Tight regulation of excitation-inhibition ratios is essential for normal circuit function. Providing more dendrites to inhibitory or to excitatory input domains, depending on GABAergic or cholinergic synaptic activity during arbor growth does not preclude compensatory adjustments of neuronal excitability on the levels of synapse strength or ion channel expression, both of which have been reported conserved mechanisms from invertebrates to mammals. In fact, even with forced receptor overexpression or massive thermogenetic stimulation of synaptic inputs through development, both of which caused dendrite shifts, multiple aspects of basic flight motor performance were normal. Therefore, at least to a certain degree, intra-neuronal dendrite shifts may operate in concert with homeostatic adjustments to keep overall excitability in check (Ryglewski, 2017).

GABAergic inhibition of leg motoneurons is required for normal walking behavior in freely moving Drosophila

Walking is a complex rhythmic locomotor behavior generated by sequential and periodical contraction of muscles essential for coordinated control of movements of legs and leg joints. Studies of walking in vertebrates and invertebrates have revealed that premotor neural circuitry generates a basic rhythmic pattern that is sculpted by sensory feedback and ultimately controls the amplitude and phase of the motor output to leg muscles. However, the identity and functional roles of the premotor interneurons that directly control leg motoneuron activity are poorly understood. This study took advantage of the powerful genetic methodology available in Drosophila to investigate the role of premotor inhibition in walking by genetically suppressing inhibitory input to leg motoneurons. For this, an algorithm was developed for automated analysis of leg motion to characterize the walking parameters of wild-type flies from high-speed video recordings. Further, genetic reagents were used for targeted RNAi knockdown of inhibitory neurotransmitter receptors in leg motoneurons together with quantitative analysis of resulting changes in leg movement parameters in freely walking Drosophila. The findings indicate that targeted down-regulation of the GABAA receptor Rdl (Resistance to Dieldrin) in leg motoneurons results in a dramatic reduction of walking speed and step length without the loss of general leg coordination during locomotion. Genetically restricting the knockdown to the adult stage and subsets of motoneurons yields qualitatively identical results. Taken together, these findings identify GABAergic premotor inhibition of motoneurons as an important determinant of correctly coordinated leg movements and speed of walking in freely behaving Drosophila (Gowda, 2018).

This report investigated the role of inhibitory premotor input to leg motoneurons in the walking behavior of freely moving Drosophila by suppression of GABAergic inhibitory input to leg motoneurons. The findings indicate that the reduction of inhibitory GABAergic input to leg motoneurons caused by targeted Rdl down-regulation has marked effects on walking behavior. Thus, walking speed was markedly slower compared with controls, and correlated with this, the amplitudes of the leg protraction (swing) and retraction (stance) phases were significantly smaller and the durations of protraction (swing) and retraction (stance) phases were significantly higher than in controls. Moreover, the concurrency state, in which three legs swing together in a tripod gait, was proportionally reduced compared with controls. These prominent effects on walking parameters are similar regardless of whether Rdl down-regulation occurs throughout development or whether it is restricted to the adult stage. Taken together, these findings reveal a prominent, albeit highly specific role of GABAergic premotor inhibitory input to leg motoneurons in the control of normal walking behavior (Gowda, 2018).

The insight into the role of premotor inhibition in walking control reported in this stuyd is the result of two key experimental methodologies. The first is the highly specific genetic access to identified neuronal populations that can now be attained in the Drosophila model system. This is made possible through remarkable targeted expression systems, which together with the availability of libraries of genetically encoded drivers and reporters for molecular manipulation, make it possible to selectively up or down-regulate gene expression in highly specific neuronal populations in intact and freely behaving animals. This study used the Gal4/UAS expression system to achieve targeted genetic access to leg motoneurons and down-regulate GABAergic premotor input to these motoneurons by Rdl RNAi expression. In addition different forms of the Gal80 repressor were used to limit Gal4/UAS targeted expression to adult stages or to specific regions of the central nervous system and, hence, refine the spatiotemporal specificity of the resulting genetic access to motoneurons. Given the wealth of Gal4 drivers and UAS-RNAi reporters currently available, it will be possible to use similar transgenic technology to manipulate other inhibitory and excitatory neurotransmitter receptors in future studies of interneuronal components of the walking circuitry (Gowda, 2018).

The second key method is the development of an advanced automated high-speed video recording and analysis technique that makes it possible to record the protraction (swing) and retraction (stance) phases at high spatiotemporal resolution for each leg in freely walking flies. With this technique, a quantitative assessment of leg motion parameters in freely walking animals can be carried out that can reveal subtle differences in amplitude and phase of movements of individual legs. This quantitative assessment has been critical for uncovering the role of premotor inhibition in walking behavior. Indeed, since the overall leg coordination during walking is unaffected by the reduction of GABAergic input to leg motoneurons, a more conventional qualitative behavioral analysis is unlikely to discern differences in walking between experimental and control animals. Recently, comparable high-resolution recording and analysis methods have been used to quantify leg movement parameters in freely walking flies. These studies have provided important information on walking speed, interleg coordination, and other locomotor parameters and have also documented a role of sensory proprioceptive input to step precision during walking. The fact that these methods for high-resolution recording and analysis are currently available for studying leg movement parameters in freely walking flies should accelerate understanding of walking behavior and the neuronal circuitry involved in its control in Drosophila (Gowda, 2018).

Given the prominent role of inhibitory premotor input to leg motoneurons reported in this study, it will now be important to identify and genetically access the premotor interneurons that provide this inhibitory input. While there is currently little information on the identity of the premotor interneurons that control the activity of leg motoneurons in adult flies, insight into inhibitory premotor interneurons has recently been obtained in larval stages. Thus, in Drosophila larva, a set of inhibitory local interneurons, termed PMSI neurons, have been identified that control the speed of axial locomotion by limiting the burst duration of motoneurons involved in peristaltic locomotion (Kohsaka, 2014). Moreover, a second set of inhibitory premotor interneurons called GVLI neurons have been reported that may be part of a feedback inhibition system involved in terminating each of the waves of motor activity that underlie larval peristalsis (Itakura, 2015). Finally, a pair of segmentally repeated GABAergic interneurons termed GDL neurons have been identified that are necessary for the coordinated propagation of peristaltic motor waves during both forward and backward crawling movements of larvae (Gjorgjieva, 2013). Whether or not these inhibitory premotor interneurons persist into the adult stage and act in the control of walking behavior is not known. Glutamate has previously been suggested to act as an inhibitory neurotransmitter in the Drosophila CNS. To investigate the likelihood that centrally labeled OK371 nonmotoneurons might also play any role in the leg motoneuron inhibition a set of preliminary experiments were carried out involving knockdown of GluCl channels in motoneurons. These experiments reveal enhanced defects in walking behavior with loss of coordination, suggesting there could be a possible role of OK371-labeled glutamatergic interneurons in leg motoneuron inhibition (Gowda, 2018).

In general terms, there are fundamental similarities in the principle mechanisms of locomotion in insects and vertebrates. These mechanistic similarities might also reflect similar motor circuit properties. For example, much like the PMSI neurons in Drosophila, which control the speed of locomotion by limiting motoneuron burst duration, the premotor V1 spinal interneurons in mammals are involved in the regulation of leg motoneuron burst and step cycle duration and thus also likely control the speed of walking movements. Hence, a characterization of the behavioral effects of inhibitory input to leg motoneurons in Drosophila, notably in freely walking flies, is likely to provide useful comparative information for understanding the functional role, and possibly the evolutionary origin, of premotor inhibition in vertebrate locomotory circuitry (Gowda, 2018).

ON selectivity in Drosophila vision is a multisynaptic process involving both glutamatergic and GABAergic inhibition

Sensory systems sequentially extract increasingly complex features. ON and OFF pathways, for example, encode increases or decreases of a stimulus from a common input. This ON/OFF pathway split is thought to occur at individual synaptic connections through a sign-inverting synapse in one of the pathways. This study showed that ON selectivity is a multisynaptic process in the Drosophila visual system. A pharmacogenetics approach demonstrates that both glutamatergic inhibition through GluCl&alpha; and GABAergic inhibition through Rdl mediate ON responses. Although neurons postsynaptic to the glutamatergic ON pathway input L1 lose all responses in GluClalpha mutants, they are resistant to a cell-type-specific loss of GluClα. This shows that ON selectivity is distributed across multiple synapses, and raises the possibility that cell-type-specific manipulations might reveal similar strategies in other sensory systems. Thus, sensory coding is more distributed than predicted by simple circuit motifs, allowing for robust neural processing (Molina-Obando, 2019).

Animals rely on their sensory systems to process behaviorally relevant information. One common feature of sensory systems is the sequential processing of information to extract complex features from simple inputs. For example, in the visual system, photoreceptors detect light and then downstream neurons progressively extract distinct features, such as contrast, direction of motion, form, or specific objects. Sensory pathways diverge into pathways that become selective for increasingly specific features (Molina-Obando, 2019).

One prominent example is the split into ON and OFF pathways, where individual neurons become selective to either increases (ON) or decreases (OFF) in a signal. Such an ON/OFF dichotomy enables more efficient coding of stimuli in the visual system and occurs across many different species and sensory modalities, such as vision, olfaction, audition, thermosensation, and electrolocation. Examples of how the split into ON and OFF pathways is implemented in sensory information processing have already been described. In the vertebrate retina, ON and OFF pathways split downstream of glutamatergic photoreceptors where ionotropic glutamate receptors on OFF bipolar cells maintain the sign of the response in the OFF pathway, and the metabotropic glutamate receptor mGluR6, located on ON bipolar cells, inverts the sign in the ON pathway. In the olfactory system of C. elegans, an odor response can be split into parallel pathways in which glutamate-gated chloride channels mediate the ON response. While these transformations are thought to occur at specific synapses, connectomics data reveals that neural circuits are intricate and that many of the possible neuronal connections are realized. This argues that important signal transformations might actually be distributed across wider circuit motifs (Molina-Obando, 2019).

In the Drosophila visual system, ON and OFF pathways functionally split in the first order lamina interneurons, but the physiological specialization occurs one synaptic layer further downstream. In brief, information travels from the retina, which houses the photoreceptors, through three optic ganglia: the lamina, the medulla, and the lobula complex, comprising lobula and lobula plate (see Figure 1 at the following site: ON pathway medulla neurons that receive graded, glutamatergic input). Contrast is encoded by the transient response of photoreceptors, and downstream lamina neurons amplify the contrast-sensitive signal component. Then, distinct ON and OFF pathways are required to detect contrast increments and decrements, respectively. In the lamina, L1 is the major input to the ON pathway, whereas L2 and L3 feed into the OFF pathway. The assignment of L1, L2, and L3 to ON and OFF pathways originates from neuronal silencing studies. However, all lamina neurons receiving direct input from photoreceptors depolarize to the offset of light and hyperpolarize to the onset of light, thus passing on information about both ON and OFF. Voltage or calcium signals in most downstream medulla neurons then selectively report only one type of contrast polarity. The major ON pathway medulla neurons Mi1 and Tm3, for example, selectively respond with depolarization or an increase in calcium signal to ON. In the OFF pathway, most neurons instead selectively respond to OFF stimuli, retaining the response polarity of their lamina inputs. Therefore, ON selectivity requires a sign inversion between the L1 input and its postsynaptic partners Mi1 and Tm3. Previous work suggested that the L1 input to the ON pathway is glutamatergic, whereas L2 and L3, the two major inputs to the OFF pathway, are cholinergic. This suggests that glutamate might also be used as an inhibitory neurotransmitter to implement ON/OFF dichotomy in the fly visual system. However, the molecular and cellular mechanisms implementing this signal transformation are not known in Drosophila visual circuitry (Molina-Obando, 2019).

This study has identified the mechanisms underlying splitting of the ON and OFF pathways in the Drosophila visual system. As expected from the major input to the ON pathway being glutamatergic, broad GluClα function is required for all ON responses in medulla neurons or downstream direction-selective cells. However, individual cell types downstream of the glutamatergic L1 input are resilient to a cell-type-specific loss of GluClα, demonstrating that ON selectivity is computed in a distributed manner. This study further showed that both the glutamate-gated chloride channel GluClα and the GABA-gated chloride channel Rdl are widely expressed in the visual system and together mediate ON responses. Thus, ON selectivity is a multisynaptic computation that is established across distributed circuits (Molina-Obando, 2019).

This work shows that visual responses in the first ON-selective neuron of the Drosophila visual system uses a combination of GluClα and Rdl receptors. This reveals a new biophysical mechanism through which ON and OFF pathway dichotomy can be established. While pharmacology can be used to deduce the function of specific molecular mechanisms, these approaches are often not specific to one protein. GluCls and GABARs belong to the same receptor family of ligand-gated chloride channels and have closely related structure and phylogeny. All known noncompetitive antagonists like Picrotoxin, γ-HCH, dieldrin, EBOB and fibronil target both receptor types although the actions are weaker in GluCls compared to GABARs. Along these lines, PTX was thought to affect GABAA receptor at low concentrations, and additionally affect GluCls at high concentrations in vitro and in vivo. This study use of PTX-insensitive alleles for glutamate and GABA-gated chloride channels making possible the deduction that, in vivo, GluClα is already blocked by PTX at lower concentrations than previously thought, and that both GluClα and Rdl play critical roles for ON responses in the Drosophila visual system. These pharmacogenetic experiments using toxin-insensitive alleles prove to be a powerful tool to unambiguously assign specific effects to individual channels (Molina-Obando, 2019).

One benefit of the use of two inhibitory transmitter systems might be the distribution of sensory coding across parallel synapses. GluClα and Rdl also appear to have very different channel dynamics. Interestingly, PTX-insensitive GluClα and Rdl alleles predominantly rescue different aspects of the visual responses. Whereas GluClαS278T predominantly rescued the peak response in all medulla layers, RdlMDRR mainly rescued the plateau response. This is consistent with the results and with previous oocyte recordings revealing that GluClα is fast desensitizing. It is also consistent with in vivo recordings of inhibitory glutamate currents in the honeybee. In contrast, GABA receptors stay open throughout the period in which the transmitter is present. Thus, the use of different inhibitory receptors might allow different aspects of a temporally structured stimulus to be encoded. This is consistent with the finding that two different types of inhibition are also in place in the vertebrate retina. There, GABAergic and glycinergic inhibition diversify the response properties of bipolar cells through a direct influence on temporal and spatial features (Molina-Obando, 2019).

While both receptors appear to be broadly expressed in many cell types of the visual system, they could be co-expressed with different transporters and channels, and interact with different molecular partners, further diversifying their role. Another common strategy to generate functional diversity is the bringing together of different receptor subunits with certain homology. Both mammalian GlyR and GABAA receptors can function as hetero-oligomers made up of different subunits and thus generating functional diversity. There are at least three different GluCl subtypes in C. elegans that can be combined. In Drosophila, only one gene coding for a glutamate-gated chloride channel has been identified. Although alternative splicing and post-transcriptional modifications could alter channel function, all known isoforms are identical in their functional domains. However, heteropentameric channels composed of mixed Rdl and GluClα subunits have been suggested biochemically. Such a potential presence of hybrid channels might also explain the higher in vivo sensitivity of GluClα to PTX in some cell types. Finally, two distinct inhibitory transmitter systems might be suitable for individual changes during evolution, allowing for adaptation to specific contextual constraints (Molina-Obando, 2019).

The current experiments revealed that GluClα is not exclusively required in a cell-autonomous manner for ON responses, since loss of GluClα function in Mi1 or Tm3 individually does not lead to a loss of ON responses. It is unlikely that this is due to an incomplete loss of function, since independent genetic tools (FlpStop and RNAi) that both disrupted GluClα expression substantially at the mRNA level gave the same result. Furthermore, the same FlpStop allele effectively abolished all ON responses when GluClα function was disrupted within its entire expression pattern. Additionally, a PTX-resistant Rdl channel can mediate ON responses in a PTX background, although L1 is not GABAergic. Together, these results suggest that ON selectivity is not a monosynaptic computation, but that parallel functional pathways can even compensate for the loss of the major synaptic connection that links L1 directly to Mi1 or Tm3. Thus, the emergence of ON selectivity is more distributed than suggested by minimal core circuit motifs. One synaptic layer further downstream, optogenetic activation of Mi1 and Tm3 most strongly contributes to T4/T5 responses. However, the current data further show that T4/T5 neurons still respond to ON stimuli when both Mi1 and Tm3 responses are completely blocked by PTX, arguing that other neurons also significantly contribute to T4/T5 responses under visual stimulation and suggesting that coding is again more distributed at this stage (Molina-Obando, 2019).

Based on connectomics, one can speculate about candidates for the implementation of these parallel circuit motifs between L1 and Mi1 and Tm3. The lamina neuron L5 and the GABAergic feedback neurons C2 and C3 receive L1 inputs and could be part of an interconnected local microcircuit. Intercolumnar neurons, not present in the current connectome datasets, like Pm or Dm neurons, might also be involved and are likely glutamatergic. In fact, there are close to 100 cell types in the visual system and ~60 medulla neurons, but their role is so far unknown. Sensory pathway splits in the periphery are one of the most fundamental steps in sensory processing. Turning this into a process that parallel pathways can achieve might make this important feature extraction step robust to perturbations (Molina-Obando, 2019).

T4 flash responses in a GluClα-deficient background show an increase in calcium signal during the OFF epoch and a decrease during the ON epoch. For a long time, the mechanisms that generate direction-selective responses in T4/T5 neurons were thought to rely on feedforward excitatory mechanisms. Recently, it was suggested that these direction-selective cells in the fly visual system also implement mechanisms that rely on null-direction suppression. Whereas electrophysiological recordings showed inhibition in T4 when the trailing edge of the receptive field was specifically stimulated, whole-cell recording experiments of T4/T5 neurons are daunting and this is the first time that calcium imaging data directly reveals inhibition in response to single ON flashes. Since glutamatergic inhibition via GluClα was disrupted in this experimental context, the data suggests that this is due to GABAergic inhibition. Several neuronal candidates could make inhibitory synapses onto T4 dendrites. Based on connectomics and neurotransmitter identity, neurons like Mi4, C3, CT1 or TmY15 give direct input and are GABAergic. Alternatively, this decrease in calcium signal in T4 might come from a lack of excitatory inputs in a GluClα mutant background. Interestingly, Mi1 and Tm3 themselves show inhibition in response to light when GluClα is blocked. However, this effect is more pronounced at their dendrites than in their output layer and shows different kinetics. The current work might thus help uncover a GABAergic inhibitory input to T4 that is more strongly apparent in the absence of Mi1 and Tm3 excitation, and could ultimately reveal the circuit implementation for the inhibitory component of T4/T5 receptive fields. Furthermore, the data also reveals an increase in calcium during OFF stimulation. The major inputs to T4 are themselves rectified. However, rectification in T4 might not be purely inherited by its inputs but also further strengthened at the T4 dendrites. The current findings thus suggest that glutamatergic inhibition contributes to establishing or maintaining contrast selectivity in T4 (Molina-Obando, 2019).

Both GluClα and Rdl are ionotropic ligand-gated receptors. While ionotropic receptors also implement the ON and OFF pathway split in C. elegans chemosensation, examples in vertebrate vision, olfaction and gustation require metabotropic receptors. Ionotropic receptors appear to be more common in insects than in vertebrates. Furthermore, glutamate-gated chloride channels have independently arisen three times within invertebrate clades and are present in arthropods, molluscs and flatworms, arguing for a strong evolutionary benefit. Ionotropic receptors mediate rapid transduction events at scales smaller than a millisecond, whereas metabotropic ones are in the millisecond to second range and last longer, from seconds to several minutes, due to an enzymatic secondary cascade previous to channel opening (Betz, 1990; Shiells, 1994). The evolutionary choice of the specific glutamatergic inhibitory system needs to match the sensory processing speed required for accurate behavioral responses in these species. For example, at the photoreceptor level, invertebrate phototransduction is faster than vertebrate phototransduction thanks to sophisticated molecular strategies. Also, the latency of olfactory sensory neurons responses in mammals is longer than that observed in insects. One advantage that metabotropic receptors have over ionotropic receptors is further amplification of the signal. The distributed circuit architecture proposed in this study might therefore strengthen signaling in a system that uses ionotropic signaling (Molina-Obando, 2019).

This study has shown that ON selectivity is not a monosynaptic process as described in other systems. Although acute pharmacological block or a systemic loss of function of GluClα abolished all ON responses in different neurons, cell-type-specific mutants retained intact ON responses, revealing that sensory coding is distributed in the fly visual system. This not only highlights the power of fly genetics but sheds new light onto the mechanisms of ON selectivity in other systems, since conclusions about ON and OFF pathway splits being mediated by specific monosynaptic processes in systems such as the vertebrate retina or the C. elegans chemosensory system relied on systemic loss-of-function approaches. Several of these systems allow for cell-type-specific manipulations using genetic approaches. It will be interesting to revisit these systems and ask if coding is similarly distributed across multiple synapses in different sensory systems and organisms (Molina-Obando, 2019).

Inhibitory interactions and columnar inputs to an object motion detector in Drosophila

The direction-selective T4/T5 cells innervate optic-flow processing projection neurons in the lobula plate of the fly that mediate the visual control of locomotion. In the lobula, visual projection neurons coordinate complex behavioral responses to visual features, however, the input circuitry and computations that bestow their feature-detecting properties are less clear. A highly specialized small object motion detector, LC11, was studied, and its responses were shown to be suppressed by local background motion. LC11 expresses GABA-A receptors that serve to sculpt responses to small objects but are not responsible for the rejection of background motion. Instead, LC11 is innervated by columnar T2 and T3 neurons that are themselves highly sensitive to small static or moving objects, insensitive to wide-field motion and, unlike T4/T5, respond to both ON and OFF luminance steps (Keles, 2020).

The cellular mechanisms of motion vision have become rapidly advanced owing to genetic, optogenetic, and in vivo imaging tools developed in Drosophila melanogaster. As in the mammalian retina, the fly optic lobe segregates ON and OFF polarity luminance changes into parallel cellular pathways. The ON- and OFF-selective pathways supply directionally selective columnar T4 and T5 neurons, respectively. The terminals of these small-field retinotopic motion detectors innervate the third optic ganglion, the lobula plate, where their synaptic output is integrated within the large planar dendrites of projection neurons that map specific wide-field patterns of optic flow onto descending pre-motor neurons to coordinate visual behavior (Keles, 2020).

In parallel to the motion vision pathway of the lobula plate, projection neurons identified in the lobula have been shown to encode moving features such as edges or objects to influence complex visual behaviors. Roughly 20 classes of lobula columnar neurons (LCs) project to the protocerebrum where axon terminals of each class form tight glomerular neuropils. Individual LC11 neurons as well as the glomerular ensemble are highly responsive to small contrasting objects moving in any direction across the ipsilateral field of view. Unlike the output cell types of the lobula plate, little is known about how the receptive field properties of LC11 arise. This study investigated the interactions between background motion and object responses in LC11, has identified a role for GABA-mediated inhibition in shaping object detection by LC11, and identifies presynaptic inputs to LC11. Columnar neurons T2 and T3 projecting from the medulla and terminating in the second and third layers of the lobula overlap with LC11 dendrites. T2 and T3 synapse with dendrites of LC11, and T3 supplies excitatory input to LC11. Finally, it was demonstrated that T2 and T3 neurons are highly selective for small objects, are suppressed by wide-field background motion, and unlike T4/T5, show full-wave rectified ON-OFF excitatory responses to rapid transitions in luminance (Keles, 2020).

In vertebrates, neurons in the retina partially encode object information, but fail to discriminate flicker from coherent motion. Yet, higher-order neurons in the mouse superior colliculus respond strongly only to moving stimuli. Similarly, this study found that T2/T3 neurons are selective for small objects, but respond to ON and OFF flicker as well, whereas downstream LC11 is responsive to object motion, not stationary flicker. It is proposed that LC11 computes continuous object motion from local ON-OFF transients conveyed by T2/T3. Future work on examining the spatiotemporal patterning of columnar inputs to LC11, as well as the cognate neurotransmitters and receptors should reveal how these computations are achieved (Keles, 2020).

In prior work (Keles, 2017), it was demonstrated that bath applied PTX, which selectively blocks chloride currents carried by GABA-A channels or glutamate channels, resulted in LC11 displaying uncharacteristic responses to elongated bars and gratings. This result was predicted under the presumption that inhibition actively filtered wide-field input from LC11. Curiously, in the same preparation, the small object responses for LC11 were essentially eradicated. How can global loss of inhibition by bath applied PTX explain both enhanced wide-field responses and diminished small object responses in LC11? Several lines of evidence suggest that postsynaptic inhibitory neuromodulation acts on LC11 in a center-surround fashion. LC11 expresses both acetylcholine receptors and a GABA-gated chloride channel subunit Rdl. Blocking GABA-A mediated synaptic currents by genetic disruption of Rdl specifically in LC11 neurons results in a decrease in response amplitude to the smallest object tested, and yet, importantly, had no effect on the normal attenuated responses to bars or normal absence of wide-field grating responses. The results support a working model in which Rdl knockout unmasks an ON-pathway input while decreasing the normal OFF object response of LC11. These properties could be explained by an ON-pathway GABAergic input to LC11 through Rdl that normally occludes ON excitation and disinhibits OFF responses. The corollary is that suppression of responses to large objects or wide-field motion occurs upstream of these object detectors. Indeed, LC11 appears to inherit its sensitivity to small object motion from excitatory T2/T3 inputs, perhaps themselves having surround inhibition mechanisms similar to T4/T5. There appears to be two mechanisms of action that are disrupted by PTX application on LC11 receptive field properties (Keles, 2017): crossover inhibition in T2/T3, which would explain their size-tuning, and local inhibition on LC11 that normally enhances small object responses. Thus, it is proposed that upon PTX delivery abnormal bar and wide-field motion responses are conveyed from T2/T3 to LC11 and small object responses are no longer boosted (Keles, 2020).

The importance of dynamic, stimulus-specific inhibition for spatial vision has been elucidated by other studies. In mice, cortical V1 center-surround receptive fields reveal stronger inhibitory currents than excitatory currents in both the surround and center, while inhibitory currents are spread more laterally than excitatory currents. In a visual collision detection circuit in the locust, feedforward inhibitory neurons actively encode dynamical variables such as object angular size. The inhibitory GABA-A receptor subunit Rdl is expressed by nearly all neurons of the fly visual system so far tested, highlighting the ubiquity and importance of inhibition for spatial vision (Keles, 2020).

T2 and T3 neurons share several key features with LC11. First, both show significantly larger responses to small solid objects than to single object edges or elongated bars, with virtually no response to moving wide-field gratings. In the large calliphorid fly Phaenicia sericata, T2 neurons have been examined with intracellular sharp electrodes, which showed that these columnar neurons depolarize to the OFF-phase of flicker, and hyperpolarize to the ON-phase. This contrasts to the GCaMP6f recordings in Drosophila, in which T2 is excited by both ON and OFF luminance transitions. Additionally, in Phaenicia T2 responded robustly to 80 x 62° moving gratings, whereas in Drosophila no response was observed in either T2 or T3 to gratings that filled the 108 x 63° display. The T2a cell type, with similar anatomy but different presynaptic inputs to T2, may show responses more closely matching those from larger flies (Keles, 2020).

An important feature of Drosophila T2/T3 neurons is that unlike T4 and T5 columnar motion detectors, which act as half-wave rectifiers that segregate ON and OFF edge stimuli, respectively, both T2 and T3 neurons show full-wave rectification in that they are excited by both ON and OFF phases of flicker. Notably, T5 shows similar amplitude responses to the OFF edges generated by either a solid two-edged dark object or a single moving OFF edge, whereas T3 responses are markedly stronger for the solid object presenting an OFF-ON sequence than to a single progressing OFF edge. T3 appears to receive input from a combination of neurons that reside in the ON and OFF pathways, including Mi1 and Tm3, providing a possible explanation for this result. Full-wave rectification of ON and OFF stimuli is consistent with single point correlation computations proposed to comprise elementary small target motion detectors (ESTMDs), which underlie the high performance object detection seen in lobula wide-field STMD neurons of hoverflies and dragonflies. Future work must explore the mechanisms that shape responses in T2 and T3, and how the spatiotemporal patterns of input from T2 and T3 confer discrimination of object motion from flicker in LC11 (Keles, 2020).

A feedforward circuit regulates action selection of pre-mating courtship behavior in female Drosophila

In the early phase of courtship, female fruit flies exhibit an acute rejection response to avoid unfavorable mating. This pre-mating rejection response is evolutionarily paralleled across species, but the molecular and neuronal basis of that behavior is unclear. This study shows that a putative incoherent feedforward circuit comprising ellipsoid body neurons, cholinergic R4d, and its repressor GABAergic R2/R4m neurons regulates the pre-mating rejection response in the virgin female Drosophila melanogaster. Both R4d and R2/R4m are positively regulated, via specific dopamine receptors, by a subset of neurons in the dopaminergic PPM3 cluster. Genetic deprivation of GABAergic signal via GABAA receptor RNA interference in this circuit induces a massive rejection response, whereas activation of GABAergic R2/R4m or suppression of cholinergic R4d increases receptivity. Moreover, glutamatergic signaling via N-methyl-d-aspartate receptors induces NO-mediated retrograde regulation potentially from R4d to R2/R4m, likely providing flexible control of the behavioral switching from rejection to acceptance. This study elucidates the molecular and neural mechanisms regulating the behavioral selection process of the pre-mating female (Ishimoto, 2020).

Overall, the present findings provide evidence for a neural relation that regulates the action selection of pre-mating behaviors in female Drosophila. The PPM3, a subset of DA neurons, forms a circuit with the R neurons R2/R4m and R4d in the EB. These different types of R neurons require different types of DA receptors, Dop1R1/D2R and Dop1R2. The knockdown of each DA receptor type indicates that all of these receptors are required to activate the expressing neurons, although the D2R conventionally inhibits D2R-expressing neurons. R2/R4m and R4d are GABAergic and cholinergic, respectively. Synaptic GRASP analysis revealed a neuronal connection from R2/R4m to R4d. R4d inhibition via the GABAA receptor is required for the proper reduction of pre-mating rejection. In addition to DA regulation of R2/R4m, the potential retrograde regulation may facilitate the GABA transmission of R2/R4m, depending on the R4d activity, with the production of NO via NMDAR/NOS signaling. This potentiation-like regulation provides the activation order of each R neuron with flexibility for the neural circuit output, and therefore to the rejection response for controlling the pre-mating behavioral kinetics (Ishimoto, 2020).

The pre-mating rejection should continue if the encounter does not match the female's criteria. Pheromones are important sexual cues provided by the male. Fruit flies produce cuticular hydrocarbons as pheromonal substances. cVA is a male-specific pheromonal cue that elicits female sexual arousal via the olfactory sensory system. This cVA signal activates pCd via a third-order olfactory interneuron, aSP-g. The SMP region contains aSP-g, pCd, and PPM3, although the direct connection between them has not been demonstrated. This aggregation of important components for female mating behavior suggests that pCd potentially integrates and verifies male information to execute the final decision for initiating the copulation. The female must resist the encounter until the evaluation process is complete-that is, the pre-mating rejection response controlled by the circuit found in the present study. These considerations lead to an assumption that the pCd and PPM3/R neuron circuits execute pre-mating computation in parallel. Many sexually dimorphic behaviors are reportedly mediated by sex-specific neural circuits (e.g., pCd, pC1). PPM3 and R neurons do not express fruitless or dsx, and no morphological sexual dimorphism has been detected. Thus, a non-sexually dimorphic circuit modulating sex-specific pre-mating behavior is proposed(Ishimoto, 2020).

Genetic manipulations of R neurons altered the pre-mating kinetics in virgin female flies, leading to the question of whether the modulation of R neurons also affects post-mating behavior, which is induced by injection of the seminal fluid sex peptide from the male fly and sustained for several days. One of the remarkable characteristics of post-mating rejection is ovipositor extrusion controlled by a distinct class of neurons in post-mating females, but either suppression of R2/R4m neurons or activation of R4d neurons, both of which reduced receptivity, rarely induced ovipositor extrusion. Mated females with R4d suppression, which makes virgin females highly receptive, exhibited a large rejection response similar to that in mated control females, and no copulation was observed during the 1-h observation time. In addition to these observations, the virgin females with Rdl knockdown in R4d reduced their walking speed with the progression of the male courtship; however, the walking speed of the mated females increases with the progression of the male courtship. Although further studies are required to elucidate the whole picture of rejection control in females, neuronal regulation of the post-mating response is presumably parallel to the pre-mating rejection response modulated by R neurons in the EB (Ishimoto, 2020).

This study found circuit functions that contain PPM3 DA subcluster neurons, and R neurons R2/R4m and R4d, were involved in the regulation of the kinetics of pre-mating behavior in virgin females. Pre-mating rejection is acutely elicited and then gradually decreased. A circuital feature of PPM3/R neurons may provide a theoretical action for controlling these pre-mating kinetics. PPM3 sends DA signals to both R2/R4m and R4d, and this recurrent circuit forms a feedforward motif with a repressor, the so-called incoherent type 1 feedforward loop (I1-FFL). I1-FFL is one of the most common network motifs implicated in gene and protein regulation networks, metabolic pathways, and neural networks from bacteria to humans. In the I1-FFL circuit, an activator X (e.g., PPM3) activates a target Z (e.g., R4d) and simultaneously activates another target Y (e.g., R2/R4m) that inhibits the target Z. Previous studies demonstrated that the I1-FFL accelerates the response of target Z, with a shorter response time. Moreover, upon input from X, Z activity increases and then, depending on the Y activity, it decreases toward basal levels. The I1-FFL circuit comprising PPM3, R2/R4m, and R4d would thus theoretically generate the acute activation of R4d, which promotes the pre-mating rejection behavior of females that would later be gradually attenuated by GABAergic signals from R2/R4m. This schema is consistent with the current findings, in which the circuit of PPM3/R neurons plays an important role in the action selection of pre-mating behavior (Ishimoto, 2020).

Genetic analysis indicates that retrograde signals from R4d to R2/R4m, mediated by NO and NMDAR, would add some flexibility to the Y-to-Z regulation in the I1-FFL circuit, contributing to progressive attenuation of the rejection response in virgin females. Depolarization of R4d activates NMDAR by the coincident input of glutamate from R2/R4m. The retrograde signals induced by NMDAR/NOS/sGC probably facilitate the GABA transmission of R2/R4m, which progressively suppresses R4d activity via the GABAA receptors. Because NMDARs require simultaneous activation by glutamate and depolarization, the NMDAR pore opening may be insufficient until the activity of R4d neurons will reach a sufficient level. This may lead to keeping the GABAergic R2/R4m function at a lower level that is under a certain threshold required for GABA release, and it may induce a continuous pre-mating rejection response. Recently, new subdivision of R neurons (R5 and R6) were classified by clonal morphological analysis and cell lineage analysis. These new subclasses of R neurons may provide a novel function of the putative I1-FFL. To investigate this further, the physiological properties of each R neuron should be analyzed (Ishimoto, 2020).

The distribution pattern of neurotransmitters in EB neurons has been documented. In the R2/R4m axonal ring structure, the glutamatergic population seems to be smaller than the GABAergic population, suggesting that the neuronal population in the R2/R4m is heterologous and contains few, if any, neurons that co-express both glutamate and GABA. In the vertebrate system, heterosynaptic regulation of GABAergic transmission is incorporated into inhibitory long-term potentiation. Inhibitory long-term potentiation of GABAergic neurons is induced at heterosynaptic sites containing glutamatergic and GABAergic neurons as presynaptic cells. In GABAergic inhibitory long-term potentiation, NO induced by glutamatergic activation of the NMDAR/NOS pathway retrogradely activates sGC, which augments cGMP levels to enhance GABA release. These intriguing analogies between vertebrate findings and the current results suggest that a similar molecular machinery potentiates GABAergic subsets of the R2/R4m neurons for attenuating the pre-mating rejection response, and hence for the action selection of pre-mating kinetics. Regarding the action selection for adaptive behaviors, others have proposed a correspondence of functions and neural architectures between vertebrate basal ganglia and the insect central complex, which contains EB neurons. In the basal ganglia, DA activates the neurons of the nucleus accumbens with modulation of glutamate and GABA release. The neurochemicals in the nucleus accumbens released by sexual interaction regulate female action selection for the affiliation of monogamous prairie voles (Microtus ochrogaster). The current genetic manipulations of particular DA, cholinergic, glutamatergic, and GABAergic systems in the central complex significantly affected the action selection for female fruit fly pre-mating behaviors, implying that the mechanisms are similar to those in the vertebrate system, which sheds light on the evolutionary parallels and diversities across the animal kingdom (Ishimoto, 2020).

Parallel Visual Conditional protein tagging methods reveal highly specific subcellular distribution of ion channels in motion-sensing neurons

Neurotransmitter receptors and ion channels shape the biophysical properties of neurons, from the sign of the response mediated by neurotransmitter receptors to the dynamics shaped by voltage-gated ion channels. Therefore, knowing the localizations and types of receptors and channels present in neurons is fundamental to understanding of neural computation. This study developed two approaches to visualize the subcellular localization of specific proteins in Drosophila: the flippase-dependent expression of GFP-tagged receptor subunits in single neurons and 'FlpTag', a versatile new tool for the conditional labelling of endogenous proteins. Using these methods, the subcellular distribution of the receptors GluClα, Rdl, and Dα7 and the ion channels Para and Ih in motion-sensing T4/T5 neurons of the Drosophila visual system was investigated. A strictly segregated subcellular distribution of these proteins a sequential spatial arrangement of glutamate, acetylcholine, and GABA receptors was discovered along the dendrite that matched the previously reported EM-reconstructed synapse distributions (Fendl, 2020).

How neural circuits implement certain computations in order to process sensory information is a central question in systems neuroscience. In the visual system of Drosophila, much progress has been made in this direction: numerous studies examined the response properties of different cell-types in the fly brain and electron microscopy studies revealed the neuronal wiring between them. However, one element crucial to understanding is still missing; these are the neurotransmitter receptors used by cells at the postsynaptic site. This knowledge is essential since neurotransmitters and corresponding receptors define the sign and the time-course of a connection, that is whether a synapse is inhibitory or excitatory and whether the signal transduction is fast or slow. The same neurotransmitter can act on different receptors with widely differing effects for the postsynaptic neuron. Glutamate for instance is mainly excitatory, however, in invertebrates it can also have inhibitory effects when it acts on a glutamate-gated chloride channel, known as GluClα. Recently, it has also been shown that acetylcholine, usually excitatory, might also be inhibitory in Drosophila, if it binds to the muscarinic mAChR-A receptor. Hence, knowledge inferring the type of transmitter receptor at a synapse is essential for understanding of the way neural circuits process information (Fendl, 2020).

Moreover, voltage-gated ion channels shape synaptic transmission and the integration of synaptic inputs by defining the membrane properties of every neural cell type. The voltage-gated calcium channel cacophony, for instance, mediates influx of calcium ions that drives synaptic vesicle fusion at presynaptic sites. Voltage-gated sodium channels like Paralytic (Para) are important for the cell's excitability and the generation of sodium-dependent action potentials. The voltage-gated channel Ih influences the integration and kinetics of excitatory postsynaptic potentials. However, only little is known about how these channels are distributed in neurons and how this shapes the neural response properties (Fendl, 2020).

One of the most extensively studied neural circuits in Drosophila is the motion vision pathway in the optic lobe and the underlying computation for direction-selectivity. The optic lobe comprises four neuropils: lamina, medulla, lobula, and lobula plate. As in the vertebrate retina, the fly optic lobe processes information in parallel ON and OFF pathways. Along the visual processing chain, T4/T5 neurons are the first neurons that respond to visual motion in a direction selective way. T4 dendrites reside in layer 10 of the medulla and compute the direction of moving bright edges (ON-pathway). T5 dendrites arborize in layer 1 of the lobula and compute the direction of moving dark edges (OFF-pathway). The four subtypes of T4/T5 neurons (a, b, c, d), project axon terminals to one of the four layers in the lobula plate, each responding only to movement in one of the four cardinal directions, their preferred direction (Fendl, 2020).

How do T4/T5 neurons become direction-selective? Both T4 and T5 dendrites span around eight columns collecting signals from several presynaptic input neurons, each of which samples information from visual space in a retinotopic manner. The functional response properties of the presynaptic partners of T4/T5 have been described in great detail along with their neurotransmitter phenotypes. T4 dendrites receive glutamatergic, GABAergic and cholinergic input, whereas T5 dendrites receive GABAergic and cholinergic input only. These input synapses are arranged in a specific spatial order along T4/T5 dendrites (Fendl, 2020).

Which receptors receive this repertoire of different neurotransmitters at the level of T4/T5 dendrites? Recently, several RNA-sequencing studies described the gene expression pattern of nearly all cell-types in the optic lobe of the fruit fly including T4/T5 neurons. T4/T5 neurons were found to express numerous receptor subunits of different transmitter classes and voltage-gated ion channels at various expression strengths. However, RNA-sequencing studies do not unambiguously answer the above question for two reasons: mRNA and protein levels are regulated in complex ways via post-transcriptional, translational, and protein degradation mechanisms making it difficult to assign protein levels to RNA levels. Secondly, standard RNA-sequencing techniques cannot provide spatial information about receptor localizations, hence, they are not sufficient to conclude which transmitter receptors receive which input signal. Both shortcomings could in principle be overcome by antibody staining since immunohistochemical techniques detect neurotransmitter receptors at the protein level and preserve spatial information. However, high-quality antibodies are not available for every protein of interest and may have variable affinity due to epitope recognition. Furthermore, labeling ion channels via antibodies and ascribing expression of a given channel to a cell-type in dense neuronal tissue remains challenging. The disadvantages of the above techniques highlight the need for new strategies for labeling neurotransmitter receptors in cell types of interest (Fendl, 2020).

This study employed existing and generated new genetic methods to label and visualize ion channels in Drosophila. For endogenous, cell-type-specific labeling of proteins, a generalizable method called FlpTag was developed that expresses a GFP-tag conditionally. Using these tools, the subcellular distribution was determined of the glutamate receptor subunit GluClα, the acetylcholine receptor subunit Dα7, and the GABA receptor subunit Rdl in motion-sensing T4/T5 neurons. These receptor subunits were differentially localized between dendrites and axon terminals. Along the dendrites of individual T4/T5 cells, the receptor subunits GluClα, Rdl, and Dα7 reveal a distinct distribution profile that can be assigned to specific input neurons forming synapses in this area. Furthermore, it was demonstrated the generalizability of the FlpTag approach by generating lines for the metabotropic GABA receptor subunit Gaba-b-r1 and the voltage-gated ion channels para and Ih. The strategies described in this study can be applied to other cells as well as other proteins to reveal the full inventory and spatial distribution of the various ion channels within individual neurons (Fendl, 2020).

Neurotransmitter receptors are essential neuronal elements that define the sign and temporal dynamics of synaptic connections. For understanding of complex neural circuits, it is indispensable to examine which transmitter receptor types are used by the participating neurons and to which compartment they localize. This study developed FlpTag, a generalizable method for endogenous, cell-type-specific labeling of proteins. Alongside several GFP-tagged UAS-lines, the newly developed FlpTag lines were developed to explore the distribution of receptor subunits GluClα, Rdl, Dα7, Gaba-b-r1 and voltage-gated ion channels Para and Ih in motion-sensing T4/T5 neurons of the visual system of Drosophila. These ion channels were found to be localized to either the dendrite, the axonal fiber or the axon terminal. Even at the level of individual dendrites, GluClα, Rdl and Dα7 were differentially distributed precisely matching the locations where T4 and T5 neurons sample signals from their glutamatergic, cholinergic, or GABAergic input neurons, respectively (Fendl, 2020).

Working with Drosophila as model organism bears some unrivaled advantages when it comes to genetic tools. The MiMIC and FlyFos libraries, for instance, are large-scale approaches of enormous value for the fly community as they provide GFP-tagged protein lines for thousands of Drosophila genes including several neurotransmitter receptors and voltage-gated ion channels. Recently, Kondo expanded these existing libraries with T2A-Gal4 insertions in 75 neurotransmitter receptor genes that can also be exchanged by the fluorescent protein tag Venus (Kondo, 2020). While all these approaches tag genes at their endogenous locus, none of them are conditional, for example they cannot be applied in a cell-type-specific manner. Hence, ascribing the expression of the pan-neuronally tagged proteins to cell-types of interest are challenging in dense neuronal tissue (Fendl, 2020).

To overcome these difficulties, two conditional strategies were used for the investigation of membrane protein localizations in the cell types of interest, T4 and T5 neurons. First, GFP-tagged UAS-lines were developed for GluClα and Rdl, and an existing UAS-Dα7::GFP line was tested. As stated above, aberrant localization of overexpressed proteins can occur, however, this is not always the case. Overexpression of UAS-GluClα::GFP shows a similar receptor localization pattern as both MiMIC and FlpTag endogenous lines, thus, validating the use of UAS-GluClα::GFP for studying receptor distribution. Additionally, previous studies reported that the UAS-Dα7::GFP line showed proper localization of the acetylcholine receptor to endogenous synapses when compared to antibody stainings or endogenous Bruchpilot (Brp) puncta. This study confirmed confirmed this finding and further showed that Dα7::GFP presumably localizes only to cholinergic synapses. Overexpressing Dα7::GFP in a medulla neuron that is devoid of endogenous Dα7 demonstrated that Dα7::GFP localized to apparent cholinergic synapses. Hence, the UAS-Dα7::GFP line can be used to study the distribution of cholinergic synapses, but not the exact composition of cholinergic receptor subunits. A recent study showed that quantitatively the levels of the postsynaptic density protein PSD95 change when overexpressed, but qualitatively the localization is not altered. Altogether, this suggests that tagged overexpression lines can be used for studying protein localizations, but they have to be controlled carefully and drawn conclusions might be different for every line (Fendl, 2020).

Ideally, a tool for protein tagging should be both endogenous and conditional. This can be achieved by introducing an FRT-flanked STOP cassette upstream of the gene of interest which was engineered with an epitope tag or fluorescent protein. Only upon cell-type specific expression of Flp, the tagged protein will be expressed in a cell-type specific manner. This genetic strategy was utilized by two independent studies to label the presynaptic protein Brp, the histamine channel Ort and the vesicular acetylcholine transporter VAChT. Recently, a new approach based on the split-GFP system was utilized for endogenous, conditional labeling of proteins in two independent studies. However, all these aforementioned approaches are not readily generalizable and easily applicable to any gene of interest (Fendl, 2020).

The FlpTag strategy presented in this study overcomes these caveats by allowing for endogenous, conditional tagging of proteins and by offering a generalizable toolbox for targeting many genes of interest. Similar to the conditional knock-out tools FlpStop and FlipFlop, FlpTag utilizes a FLEx switch to conditionally control expression of a reporter gene, in this case GFP. Likewise, FlpTag also easily integrates using the readily available intronic MiMIC insertions. This study attempted to generate FlpTag lines for six genes, GluClα, Rdl, Dα7, Gaba-b-r1, para and Ih. Four out of these six lines yielded conditional GFP-tagged protein lines (GluClα, Gaba-b-r1, para, Ih). The FlpTag cassette was injected in MI02620 for Rdl and MI12545 for Dα7, but no GFP expression was detected across the brain. The MiMiC insertion sites used for Rdl and Dα7 seem to be in a suboptimal location for tagging the protein (Fendl, 2020).

As of now, there are MiMIC insertions in coding introns for more than 2800 genes available, which covers approximately 24% of neuronal genes. Additionally, the attP insertion sites generated in the study by Kondo provide possible landing sites for the FlpTag cassette for 75 neurotransmitter receptor genes (Kondo, 2020). Transmembrane proteins such as neurotransmitter receptors form complex 3D structures making fluorescent tagging especially difficult. Neither the MiMIC insertion sites, nor the target sites of the Kondo study at the C-terminus of several transmitter receptor genes, ensure a working GFP-tagged protein line. For genes of interest lacking a suitable MiMIC insertion site a homology directed repair (HDR) cassette was generated that utilizes CRISPR/Cas9-mediated gene editing to integrate the FlpTag cassette in any desired gene locus. The plasmid consists of the FlpTag cassette flanked by multiple cloning sites for the insertion of homology arms (HA). Through HDR the FlpTag cassette can be knocked-in into any desired locus. Taken together, the FlpTag cassette is a generalizable tool that can be integrated in any available attP-site in genes of interest or inserted by CRISPR-HDR into genes lacking attP landing sites. This allows for the investigation of the endogenous spatial distributions of proteins, as well as the correct temporal dynamics of protein expression (Fendl, 2020).

Further, the FlyFos project demonstrated that most fly lines with an extra copy of GFP-tagged protein-coding genes worked normally and GFP-tagged proteins could be imaged in living fly embryos and pupae. In principle, live-imaging of the GFP-tagged lines that were created could be performed during different developmental stages of the fruit fly. In general, the tools generated in this study can be used as specific postsynaptic markers, visualizing glutamatergic, GABAergic, and cholinergic synapses with standard confocal light microscopy. This extends the existing toolbox of Drosophila postsynaptic markers for studying the localization and development of various types of synapses (Fendl, 2020).

T4/T5 neurons combine spatiotemporal input from their presynaptic partners, leading to selective responses to one of the four cardinal directions. Numerous studies investigated the mechanisms underlying direction-selective responses in T4/T5 neurons, yet the computation is still not fully understood. At an algorithmic level, a three-arm detector model is sufficient to describe how direction-selective responses in T4/T5 neurons arise. This model relies on the comparison of signals originating from three neighboring points in space via a delay-and-compare mechanism. The central arm provides fast excitation to the neuron. While one flanking arm amplifies the central signal for stimuli moving along the preferred direction, the other inhibits the central signal for stimuli moving along the null direction of the neuron. Exploring the neurotransmitter receptors and their distribution on T4/T5 dendrites allows defining the sign as well as the temporal dynamics of some of the input synapses to T4/T5 (Fendl, 2020).

According to the algorithmic model, an excitatory, amplifying input signal on the distal side of T4/T5 dendrites was expected. This study found that T4 cells receive an inhibitory, glutamatergic input from Mi9 via GluClα, which, at first sight, seems to contradict expectation. However, since Mi9 has an OFF-center receptive field, this glutamatergic synapse will invert the polarity from Mi9-OFF to T4-ON. Theoretically, in darkness, Mi9 inhibits T4 via glutamate and GluClα, and this inhibition is released upon an ON-edge moving into its receptive field. The concomitant closure of chloride channels and subsequent increased input resistance in T4 cells results in an amplification of a subsequent excitatory input signal from Mi1 and Tm3. As shown by a recent modeling study, this biophysical mechanism can indeed account for preferred direction enhancement in T4 cells (Borst, 2018). Some studies failed to detect preferred direction enhancement in T4/T5 neurons and they proposed that the enhanced signal in PD seen in GCaMP recordings could be a result from a non-linear calcium-to-voltage transformation. If this was really the case, the role of Mi9 and GluClα must be reconsidered and future functional experiments will shed light onto this topic (Fendl, 2020).

Nevertheless, Strother (2017) showed that the RNAi- knock-down of GluClα in T4/T5 neurons leads to enhanced turning responses on the ball set-up for faster speeds of repeating ON and OFF edges (Strother, 2017). Although this observation cannot answer the question about preferred direction enhancement in T4 cells, it indicates that both T4 and T5 receive inhibitory input and that removal of such create enhanced turning responses at the behavioral level. In line with these observations, the glutamate receptor GluClα was also found in T4/T5 axon terminals. A possible functional role of these inhibitory receptors in the axon terminals could be a cross-inhibition of T4/T5 cells with opposite preferred directions via lobula plate intrinsic neurons (LPis). Glutamatergic LPi neurons are known to receive a cholinergic, excitatory signal from T4/T5 neurons within one layer and to inhibit lobula plate tangential cells, the downstream postsynaptic partners of T4/T5 neurons, via GluClα in the adjacent oppositely tuned layer. This mechanism induces a motion opponent response in lobula plate tangential cells and increases their flow-field selectivity. In addition, LPi neurons could also inhibit T4/T5 neurons presynaptically at their axon terminals via GluClα in order to further sharpen the flow-field selectivity of lobula plate tangential cells. Taken together, exploring the subcellular distribution of GluClα in T4/T5 neurons highlights its differential functional roles in different parts of these cell types (Fendl, 2020).

Secondly, the Dα7 signal in the center of T4/T5 dendrites discovered in this study, corresponds to ionotropic, cholinergic input from Mi1 and Tm3 for T4, and Tm1, Tm2 and Tm4 for T5. These signals correspond to the central, fast, excitatory arm of the motion detector model. As T4 and T5 express a variety of different ACh receptor subunits, the exact subunit composition and underlying biophysics of every cholinergic synapse on T4/T5 dendrites still awaits further investigations (Fendl, 2020).

Third, inhibition via GABA plays an essential role in creating direction-selective responses in both T4 and T5 neurons by providing null direction suppression. Computer simulations showed that direction selectivity decreases in T4/T5 motion detector models without this inhibitory input on the null side of the dendrite. This study shows that T4 and T5 neurons possess the inhibitory GABA receptor subunit Rdl mainly on the proximal base on the null side of their dendrites, providing the synaptic basis for null direction suppression. The metabotropic GABA receptor subunit Gaba-b-r1 was not detected in T4/T5 neurons using the newly generated FlpTag Gaba-b-r1 line. Finally, all of the receptor subunits GluClα, Rdl and Dα7 investigated in this study are ionotropic, fast receptors, which presumably do not add a temporal delay at the synaptic level. In the detector model described above, the two outer arms provide a slow and sustained signal, and such properties are already intrinsic properties of these input neurons. However, it cannot be excluded that slow, metabotropic receptor subunits for acetylcholine or GABA (e.g. Gaba-br2) which are also present in T4/T5 and could induce additional delays at the synaptic level (Fendl, 2020).

Furthermore, the subcellular distribution was investigated of the voltage-gated ion channels Para and Ih in T4/T5 neurons. Para, a voltage-gated sodium channel, was found to be distributed along the axonal fibers of both T4 and T5 neurons. As Para is important for the generation of sodium-dependent action potentials, it will be interesting for future functional studies to investigate, if T4/T5 really fire action potentials and how this shapes their direction-selective response. Further, Ih, a voltage-gated ion channel permeable for several types of ions, was detected in T4/T5 dendrites using the FlpTag strategy. Ih channels are activated at negative potentials below -50 mV and as they are permeable to sodium and potassium ions, they can cause a depolarization of the cell after hyperpolarization. Loss-of-function studies will unravel the functional role of the Ih channel for direction-selective responses in T4/T5 neurons (Fendl, 2020).

Since the ability to combine synaptic inputs from different neurotransmitters at different spatial sites is common to all neurons, the approaches described in this study represent an important future perspective for other circuits. The tools can be used to study the ion channels GluClα, Rdl, Dα7, Gaba-b-r1, para and Ih in any given Drosophila cell-type and circuit. Furthermore, the FlpTag tool box can be used to target many genes of interest and thereby foster molecular questions across fields (Fendl, 2020).

The techniques described in this study can be transferred to other model organisms as well, to study the distribution of different transmitter receptors. For instance, in the mouse retina - similar to motion-sensing T4/T5 neurons in the fruit fly - so-called On-Off direction-selective ganglion cells receive asymmetric inhibitory GABAergic inputs from presynaptic starburst amacrine cells during null-direction motion. A previous study investigated the spatial distribution of GABA receptors of these direction-selective ganglion cells using super-resolution imaging and antibody staining. Additionally, starburst amacrine cells also release ACh onto ganglion cells which contributes to the direction-selective responses of ganglion cells. Thus, mapping the distribution of ACh receptors on direction-selective ganglion cells will be the next important step to further investigate cholinergic transmission in this network (Fendl, 2020).

Overall, this study has demonstrated the importance of exploring the distributions of neurotransmitter receptors and ion channels for systems neuroscience. The distinct distributions in T4/T5 neurons discovered in this study and the resulting functional consequences expand knowledge of the molecular basis of motion vision. Although powerful, recent RNAseq studies lacked information about spatial distributions of transmitter receptors which can change the whole logic of wiring patterns and underlying synaptic signs. Future studies can use this knowledge to target these receptors and directly probe their role in functional experiments or incorporate the gained insights into model simulations. However, this study is only highlighting some examples of important neural circuit components: expanding the approaches described in this study to other transmitter receptors and ion channels, as well as gap junction proteins will reveal the full inventory and the spatial distributions of these decisive determinants of neural function within an individual neuron (Fendl, 2020).

The GABAA receptor RDL acts in peptidergic PDF neurons to promote sleep in Drosophila

Sleep is regulated by a circadian clock that times sleep and wake to specific times of day and a homeostat that drives sleep as a function of prior wakefulness. Flies display the core behavioral features of sleep, including relative immobility, elevated arousal thresholds, and homeostatic regulation. Sleep-wake modulation was assessed by a core set of circadian pacemaker neurons that express the neuropeptide PDF. It was found that disruption of PDF function increases sleep during the late night in light:dark and the first subjective day of constant darkness. Flies deploy genetic and neurotransmitter pathways to regulate sleep that are similar to those of their mammalian counterparts, including GABA. RNA interference-mediated knockdown of the GABAA receptor gene, Resistant to dieldrin (Rdl), in PDF neurons reduces sleep, consistent with a role for GABA in inhibiting PDF neuron function. Patch-clamp electrophysiology reveals GABA-activated picrotoxin-sensitive chloride currents on PDF+ neurons. In addition, RDL is detectable most strongly on the large subset of PDF+ pacemaker neurons. These results suggest that GABAergic inhibition of arousal-promoting PDF neurons is an important mode of sleep-wake regulation in vivo (Chung, 2009).

It is proposed that GABA release inhibits large LNv output and PDF release to reduce wake, suggesting an important role for GABA inhibition. In this model, the circadian clock times PDF neuron activation and PDF release during the late night and following day to promote waking behavior. Of note, a similar arousal-promoting function for circadian pacemaker neurons has been described in mammals. This is also approximately the time when the large LNv have been shown to be more depolarized and have higher levels of spontaneous activity. RDL receptors on LNv soma and on fibers in the accessory medulla suggest that GABA may regulate LNv excitability. It is interesting that GABA is also an important neurotransmitter in mammalian circadian pacemaker neurons, capable of reducing their spontaneous activity. In addition, RDL receptors on PDF varicosities in the optic lobe may function presynaptically to regulate PDF release. GABA may also act through metabotropic GABAB receptors, which have been described in the sLNv, but their function in circadian or sleep behavior is unknown. GABAergic signaling may affect the function of the transcription factor ATF2, which is important for PDF neuron function in sleep. Changes in PDF neuron function may in turn act by antagonizing sleep-promoting circuits that exist within the mushroom bodies as well as the pars intercerebralis (PI). Of note, the PI appears to express the PDF receptor. Identifying the anatomic targets of PDF as well as the neural sources of GABAergic inputs will be important for further defining sleep-wake circuits in Drosophila (Chung, 2009).

Female contact modulates male aggression via a sexually dimorphic GABAergic circuit in Drosophila

Intraspecific male-male aggression, which is important for sexual selection, is regulated by environment, experience and internal states through largely undefined molecular and cellular mechanisms. To understand the basic neural pathway underlying the modulation of this innate behavior, a behavioral assay was established in Drosophila melanogaster, and the relationship between sexual experience and aggression was investigated. In the presence of mating partners, adult male flies exhibited elevated levels of aggression, which was largely suppressed by prior exposure to females via a sexually dimorphic neural mechanism. The suppression involved the ability of male flies to detect females by contact chemosensation through the pheromone-sensing ion channel Ppk29 and was mediated by male-specific GABAergic neurons acting on the GABAA receptor RDL in target cells. Silencing or activating this circuit led to dis-inhibition or elimination of sex-related aggression, respectively. It is proposed that the GABAergic inhibition represents a critical cellular mechanism that enables prior experience to modulate aggression (Yuan, 2013).

Aggression is a complex behavior that is regulated by various internal and external stimuli. To date, however, studies have remained largely focused on the sensory pathways involved in regulating baseline aggression, with less examination of the central components of the underlying neural pathway. Moreover, the close relationship between sex and aggression has been a fascinating topic in both biology and literature, but their intertwined nature and the underlying neurobiological basis have remained elusive. Using a behavioral genetics approach, this study identified a previously unknown neural pathway that underlies the modulation of sex-related male-male aggression in Drosophila by prior contacts with females (Yuan, 2013).

These results suggest that prior female encounter through direct physical contacts activates the pheromone-sensing ppk29 neurons, resulting in inhibition of the central aggression circuit via GABAergic mechanisms involving the RDL GABAA receptor, thereby suppressing the behavioral output for male-male aggression. The three levels of the neural pathway involved in this experience-dependent behavior modification all exhibited sexual dimorphism, consistent with the notion that morphological differences in male and female brains correlate with their distinct behavioral needs. It was possible to modify the aggressive behavior output by manipulating the circuit at each of these three steps, which possibly represented the sequence of the information relay involved in the native behaviors, the sensory input, the information processing and the execution of the behavior. However, it is recognized that the circuit components elucidated by these experiments are clearly only parts of the machinery responsible for aggression modulation. In addition, by identifying RDL as a molecular target for aggression regulation, this study provides an entry point for characterizing the missing link of aggression studies, namely the central neurons that respond to experience-dependent modulation and mediate the execution of aggressive behaviors. Thus, this work provides new insights regarding the intricate interactions between sexual experience and aggression and delineates the underlying mechanisms to inform potential means to suppress excessive aggression (Yuan, 2013).

The female contact-dependent suppression of male aggression may also be viewed as a form of learning-induced plasticity. The learning procedure in this case requires extended physical interactions between the male and the female (over 10 h), which could consist of repeated sessions of male courtship attempts and female rejection. As no obvious defects were observed in aggression suppression in genetic mutants with deficits in courtship conditioning, such as homer, eag, Shaker and orb2 mutants, this experience-induced suppression of aggression is likely different from conventional courtship conditioning. Another interesting feature of this suppression is that it is long term, yet reversible, lasting up to 2 d after the female encounter. However, it also differs from well-studied long-term memory formation, as no defect was observed in amn mutants, which is required for long-term memory formation. The results implicate the fru+ d5-HT1B+ and GABA+ cluster of neurons in the central brain as the regulators of this suppression, but it remains to be determined whether these neurons are involved in the initiation, acquisition, execution or consolidation phase(s) of this behavior, what takes form as the underlying 'memory trace' and whether plasticity is manifested at the level of the number of neurons activated, neurite arborization, neuronal activity or some other aspect of neuronal signaling (Yuan, 2013).

Notwithstanding the emergence of Drosophila as a successful genetic model for aggression studies and the extensive characterization of its stereotypical motor display of aggression, the strong influence of genetic background over baseline aggression and locomotor activity often complicates Drosophila aggression studies. The current assay avoids such difficulties by consistently eliciting aggression in naive male flies in the presence of females and by inducing a strong suppression of aggression in males with prior female encounter. The small variations among different genetic backgrounds in the behavioral assay make it possible to identify critical cellular and molecular components involved in the regulation of aggression by experience (Yuan, 2013).

One purpose of studying aggression regulation in animal models is to eventually understand the basis of human violence and establish venues to reduce or prevent it. Psychophysiological studies suggest that the failure to maintain an appropriate level of aggression in humans is associated with impaired executive cognitive processes or emotion registration. As an innate behavior built largely on predetermined neural pathways, aggression in Drosophila males can be modulated by prior exposure to females through GABAergic inhibition. This study raises the possibility that an ancient and basic machinery of the central neuronal circuitry, GABAergic inhibition, could be part of a conserved mechanism to modulate the level of aggression in males and ensure proper balance between reproductive competition and individual survival (Yuan, 2013).

Wide awake mediates the circadian timing of sleep onset

How the circadian clock regulates the timing of sleep is poorly understood. This study identifies a Drosophila mutant, wide awake (wake), that exhibits a marked delay in sleep onset at dusk. Loss of Wake in a set of arousal-promoting clock neurons, the large ventrolateral neurons (l-LNvs), impairs sleep onset. Wake levels cycle, peaking near dusk, and the expression of Wake in l-LNvs is Clock dependent. Strikingly, Clock and cycle mutants also exhibit a profound delay in sleep onset, which can be rescued by restoring Wake expression in LNvs. Wake interacts with the GABAA receptor Resistant to Dieldrin (Rdl), upregulating its levels and promoting its localization to the plasma membrane. In wake mutant l-LNvs, GABA sensitivity is decreased and excitability is increased at dusk. It is proposed that Wake acts as a clock output molecule specifically for sleep, inhibiting LNvs at dusk to promote the transition from wake to sleep (Liu, 2014).

The molecular pathways by which the circadian clock modulates the timing of sleep are unknown. This study identified a molecule, Wide Awake, that promotes sleep and is required for circadian timing of sleep onset. The data argue for a direct role for the circadian oscillator in regulating sleep and support a model whereby Wake acts as a molecular intermediary between the circadian clock and sleep. In this model, Wake transmits timing information from the circadian clock to inhibit arousal circuits at dusk, thus facilitating the transition from wake to sleep. wake is transcriptionally upregulated by Clk activity, specifically in LNv clock neurons. Wake levels in l-LNvs rise during the day and peak at the early night, near the wake/sleep transition. This increase in Wake levels upregulates Rdl in l-LNvs, enhancing their sensitivity to GABA signaling and serving to inhibit the l-LNv arousal circuit. In this manner, cycling of Wake promotes cycling of the excitability of l-LNv cells. In wake mutants, l-LNvs lose this circadian electrical cycling; the higher firing rate of these cells at dusk leads to increased release of Pdf, which would act on Pdfr on downstream neurons to inhibit sleep onset. The identity of the GABAergic neurons signaling to the l-LNvs is currently unknown, but if they serve to convey information about sleep pressure from homeostatic circuits, the l-LNvs could serve as a site of integration for homeostatic and circadian sleep regulatory signals (Liu, 2014).

Although Wake is expressed in clock neurons and its levels vary throughout the day, Wake itself is not a core clock molecule, since period length and activity rhythm strength are intact in wake mutants in constant darkness. The effects of Wake on sleep latency are not attributable to alterations in core clock function. In addition, because locomotor rhythm strength is intact in wake mutants, Wake is not a clock output molecule for locomotor rhythms. Rather, Wake is the first clock output molecule shown to specifically regulate sleep timing (Liu, 2014).

Previous studies have demonstrated that Rdl in LNvs regulates sleep in Drosophila. This work further implicates Rdl as a key factor in the circadian modulation of sleep. In mammals, the localization and function of GABAA receptors are regulated by a variety of cytosolic accessory proteins, some of which are associated with the plasma membrane and cytoskeletal elements. The data suggest that Wake acts as an accessory protein for Rdl, upregulating its levels and promoting its targeting to the plasma membrane. Rdl is broadly expressed throughout the adult Drosophila brain, whereas Wake appears more spatially restricted. It is likely that Rdl is regulated by Wake in specific cells (e.g., Wake+ cells), while in other cells that express Rdl but not Wake, other factors are involved. Together, these data suggest a model in which increased GABA sensitivity is required in specific arousal circuits to facilitate rapid and complete switching between sleep/wake states at the appropriate circadian time (Liu, 2014).

Intriguingly, the data, as well as data from the Allen Brain Atlas, suggest that the putative mouse homolog of Wake (ANKFN1) is enriched in the mouse SCN, the master circadian pacemaker in mammals. Specifically, ANKFN1 is expressed in the 'core' region of the SCN, which is analogous to the large LNvs in flies, in that it receives light input and its molecular oscillator does not cycle or cycles weakly in DD. These observations support a potential conservation of Wake function in regulating clock-dependent timing of sleep onset, which will be evaluated by ongoing genetic analysis in mice. The pronounced difficulty of wake flies to fall asleep at lights off is reminiscent of sleep-onset insomnia in humans. Moreover, the most widely used medications for the treatment of insomnia are GABA agonists. Thus, the identification of a molecule that mediates circadian timing of sleep onset by promoting GABA signaling may lead to a deeper understanding of mechanisms underlying insomnia and its potential therapies (Liu, 2014).

Glutamate, GABA and Acetylcholine Signaling Components in the Lamina of the Drosophila Visual System

Synaptic connections of neurons in the Drosophila lamina, the most peripheral synaptic region of the visual system, have been comprehensively described. Although the lamina has been used extensively as a model for the development and plasticity of synaptic connections, the neurotransmitters in these circuits are still poorly known. Thus, to unravel possible neurotransmitter circuits in the lamina of Drosophila, Gal4 driven green fluorescent protein in specific lamina neurons was combined with antisera to gamma-aminobutyric acid (GABA), glutamic acid decarboxylase, a GABAB type of receptor, L-glutamate, a vesicular glutamate transporter (vGluT), ionotropic and metabotropic glutamate receptors, choline acetyltransferase and a vesicular acetylcholine transporter. It is suggested that acetylcholine may be used as a neurotransmitter in both L4 monopolar neurons and a previously unreported type of wide-field tangential neuron (Cha-Tan). GABA is the likely transmitter of centrifugal neurons C2 and C3 and GABAB receptor immunoreactivity is seen on these neurons as well as the Cha-Tan neurons. Based on an rdl-Gal4 line, the ionotropic GABAA receptor subunit RDL may be expressed by L4 neurons and a type of tangential neuron (rdl-Tan). Strong vGluT immunoreactivity was detected in a-processes of amacrine neurons and possibly in the large monopolar neurons L1 and L2. These neurons also express glutamate-like immunoreactivity. However, antisera to ionotropic and metabotropic glutamate receptors did not produce distinct immunosignals in the lamina. In summary, this paper describes novel features of two distinct types of tangential neurons in the Drosophila lamina and assigns putative neurotransmitters and some receptors to a few identified neuron types (Kolodziejczyk, 2008; full text of article).

Modulation of GABAA receptor desensitization uncouples sleep onset and maintenance in Drosophila

Many lines of evidence indicate that GABA and GABAA receptors make important contributions to human sleep regulation. Pharmacological manipulation of these receptors has differential effects on sleep onset and sleep maintenance insomnia. Sleep is regulated by GABA in Drosophila; a mutant GABAA receptor, RdlA302S, specifically decreases sleep latency, the length of time that it takes to accomplish the transition from full wakefulness to sleep. The drug carbamazepine (CBZ) has the opposite effect on sleep; it increases sleep latency as well as decreasing sleep. Behavioral and physiological experiments indicated that RdlA302S mutant flies are resistant to the effects of CBZ on sleep latency and that mutant RDLA302S channels are resistant to the effects of CBZ on desensitization, respectively. These results suggest that this biophysical property of the channel, specifically channel desensitization, underlies the regulation of sleep latency in flies. These experiments uncouple the regulation of sleep latency from that of sleep duration and suggest that the kinetics of GABAA receptor signaling dictate sleep latency (Agosto, 2008).

gamma-Aminobutyric acid (GABA) signaling components in Drosophila: immunocytochemical localization of GABAB receptors in relation to the GABAA receptor subunit RDL and a vesicular GABA transporter

γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in insects and is widely distributed in the central nervous system (CNS). GABA acts on ion channel receptors (GABAAR) for fast inhibitory transmission and on G-protein-coupled receptors (GABABR) for slow and modulatory action. Immunocytochemistry was used to map GABABR sites in the Drosophila CNS, and the distribution was compared with that of the GABAAR subunit RDL. To identify GABAergic synapses, an antiserum was raised to the vesicular GABA transporter (vGAT). For general GABA distribution, an antiserum to glutamic acid decarboxylase (GAD1) and a gad1-GAL4 was used to drive green fluorescent protein. GABABR-immunoreactive (IR) punctates were seen in specific patterns in all major neuropils of the brain. Most abundant labeling was seen in the mushroom body calyces, ellipsoid body, optic lobe neuropils, and antennal lobes. The RDL distribution is very similar to that of GABABR-IR punctates. However, the mushroom body lobes displayed RDL-IR but not GABABR-IR material, and there were subtle differences in other areas. The vGAT antiserum labeled punctates in the same areas as the GABABR and appeared to display presynaptic sites of GABAergic neurons. Various GAL4 drivers were used to analyze the relation between GABABR distribution and identified neurons in adults and larvae. These findings suggest that slow GABA transmission is very widespread in the Drosophila CNS and that fast RDL-mediated transmission generally occurs at the same sites (Enell, 2007).

gamma-glutamyl transpeptidase 1 specifically suppresses green-light avoidance via GABA receptors in Drosophila

Drosophila larvae innately show light-avoidance behavior. Compared with robust blue-light avoidance, larvae exhibit relatively weaker green-light responses. In a previous screening for genes involved in larval light-avoidance, compared with control w1118 larvae, larvae with γ-glutamyl transpeptidase 1 (Ggt-1) knockdown or Ggt-1 mutation were found to exhibit higher percentage of green-light avoidance which was mediated by Rh6 photoreceptors. However, their responses to blue light did not change significantly. By adjusting the expression level of Ggt-1 in different tissues, it was found that Ggt-1 in malpighian tubules is both necessary and sufficient for green-light avoidance. These results showed that glutamate levels were lower in Ggt-1 null mutants compared with controls. Feeding Ggt-1 null mutants glutamate can normalize green-light avoidance, indicating that high glutamate concentrations suppressed larval green-light avoidance. However, rather than directly, glutamate affected green-light avoidance indirectly through GABA, the level of which was also lower in Ggt-1 mutants compared with controls. Mutants in glutamate decarboxylase 1, which encodes GABA synthase, and knockdown lines of the GABAA receptor, both exhibit elevated levels of green-light avoidance. Thus, these results elucidate the neurobiological mechanisms mediating green-light avoidance, which was inhibited in wild-type larvae (Liu, 2014).

The ADAR RNA editing enzyme controls neuronal excitability in Drosophila melanogaster

RNA editing by deamination of specific adenosine bases to inosines during pre-mRNA processing generates edited isoforms of proteins. Recoding RNA editing is more widespread in Drosophila than in vertebrates. Editing levels rise strongly at metamorphosis, and Adar5G1 null mutant flies lack editing events in hundreds of CNS transcripts; mutant flies have reduced viability, severely defective locomotion and age-dependent neurodegeneration. On the other hand, overexpressing an adult dADAR isoform with high enzymatic activity ubiquitously during larval and pupal stages is lethal. Advantage was taken of this to screen for genetic modifiers; Adar overexpression lethality is rescued by reduced dosage of the Rdl (Resistant to dieldrin), gene encoding a subunit of inhibitory GABA receptors. Reduced dosage of the Gad1 gene encoding the GABA synthetase also rescues Adar overexpression lethality. Drosophila Adar5G1 mutant phenotypes are ameliorated by feeding GABA modulators. This study demonstrates that neuronal excitability is linked to dADAR expression levels in individual neurons; Adar-overexpressing larval motor neurons show reduced excitability whereas Adar5G1 null mutant or targeted Adar knockdown motor neurons exhibit increased excitability. GABA inhibitory signalling is impaired in human epileptic and autistic conditions, and vertebrate ADARs may have a relevant evolutionarily conserved control over neuronal excitability (Li, 2013).

Actions of agonists, fipronil and Ivermectin on the predominant in vivo splice and edit variant (RDLbd, I/V) of the Drosophila GABA receptor expressed in Xenopus laevis oocytes

Ionotropic GABA receptors are the targets for several classes of insecticides. One of the most widely-studied insect GABA receptors is RDL (resistance to dieldrin), originally isolated from Drosophila melanogaster. RDL undergoes alternative splicing and RNA editing, which influence the potency of GABA. Most work has focussed on minority isoforms. This study reports the first characterisation of the predominant native splice variant and RNA edit, combining functional characterisation with molecular modelling of the agonist-binding region. The relative order of agonist potency is GABA> muscimol> TAC>> beta-alanine. The I/V edit does not alter the potency of GABA compared to RDLbd. Docking calculations suggest that these agonists bind and activate RDLbdI/V through a similar binding mode. TACA and beta-alanine are predicted to bind with lower affinity than GABA, potentially explaining their lower potency, whereas the lower potency of muscimol and isoguvacine cannot be explained structurally from the docking calculations. The A301S (resistance to dieldrin) mutation reduced the potency of antagonists picrotoxin, fipronil and pyrafluprole but the I/V edit had no measurable effect. Ivermectin suppressed responses to GABA of RDLbdI/V, RDLbd and RDLbdI/VA301S. The dieldrin resistant variant also showed reduced sensitivity to Ivermectin. This study of a highly abundant insect GABA receptor isoform will help the design of new insecticides (Lees, 2014).

Ivermectin and nodulisporic acid receptors in Drosophila melanogaster contain both gamma-aminobutyric acid-gated Rdl and glutamate-gated GluCl alpha chloride channel subunits

35S-labeled derivatives of the insecticides nodulisporic acid and ivermectin were synthesized and demonstrated to bind with high affinity to a population of receptors in Drosophila head membranes that were previously shown to be associated with a glutamate-gated chloride channel. Nodulisporic acid binding was modeled as binding to a single population of receptors. Ivermectin binding was composed of at least two kinetically distinct receptor populations, only one of which was associated with nodulisporic acid binding. The binding of these two ligands was modulated by glutamate, ivermectin, and antagonists of invertebrate gamma-aminobutyric acid (GABA)ergic receptors. Because solubilized nodulisporic acid and ivermectin receptors comigrated as 230-kDa complexes by gel filtration, antisera specific for both the Drosophila glutamate-gated chloride channel subunit GluCl alpha (DmGluCl alpha) and the GABA-gated chloride channel subunit Rdl (DmRdl) proteins were generated and used to examine the possible coassembly of these two subunits within a single receptor complex. DmGluCl alpha antibodies immunoprecipitated all of the ivermectin and nodulisporic acid receptors solubilized by detergent from Drosophila head membranes. DmRdl antibodies also immunoprecipitated all solubilized nodulisporic receptors, but only approximately 70% of the ivermectin receptors. These data suggest that both DmGluCl alpha and DmRdl are components of nodulisporic acid and ivermectin receptors, and that there also exists a distinct class of ivermectin receptors that contains the DmGluCl alpha subunit but not the DmRdl subunit. This co-association of DmGluCl alpha and DmRdl represents the first biochemical and immunological evidence of coassembly of subunits from two different subclasses of ligand-gated ion channel subunits (Ludmerer, 2002).

GABA receptors containing Rdl subunits mediate fast inhibitory synaptic transmission in Drosophila neurons

GABAergic inhibition in Drosophila, as in other insects and mammals, is important for regulation of activity in the CNS. However, the functional properties of synaptic GABA receptors in Drosophila have not been described. This study reports that spontaneous GABAergic postsynaptic currents (sPSCs) in cultured embryonic Drosophila neurons are mediated by picrotoxin-sensitive chloride-conducting receptors. A rapid increase in spontaneous firing in response to bath application of picrotoxin demonstrates that these GABA receptors mediate inhibition in the neuronal networks formed in culture. Many of the spontaneous GABAergic synaptic currents are sodium action potential independent [miniature IPSCs (mIPSCs)] but are regulated by external calcium levels. The large variation in mIPSC frequency, amplitude, and kinetics properties between neurons suggests heterogeneity in GABA receptor number, location, and/or subtype. A decrease in the mean mIPSC decay time constant between 2 and 5 d, in the absence of a correlated change in rise time, demonstrates that the functional properties of the synaptic GABA receptors are regulated during maturation in vitro. Finally, neurons from the GABA receptor subunit mutant Rdl exhibit reduced sensitivity to picrotoxin blockade of the mIPSCs and resistance to picrotoxin-induced increases in spontaneous firing frequency. This demonstrates that Rdl containing GABA receptors play a direct role in mediating synaptic inhibition in Drosophila neural circuits formed in culture (Lee, 2003).

The abundant expression of acetylcholine and GABA, throughout the Drosophila CNS, suggests that these classic neurotransmitters play a major role in mediating fast synaptic transmission. Recent electrophysiological studies have provided insights into the functional aspects of excitatory cholinergic transmission in Drosophila neurons. However, virtually nothing was known about the properties of inhibitory GABAergic synaptic transmission. The current data demonstrate that the receptors mediating GABAergic currents in Drosophila neurons are PTX sensitive, heterogeneous with respect to their biophysical properties, and regulated during maturation in culture. The GABAergic currents are inhibitory, and Rdl encoded subunits contribute to the population of receptors meditating fast inhibitory synaptic transmission in neuronal networks formed in culture (Lee, 2003).

A reversal potential near the chloride equilibrium potential and blockade by low (1 µM) concentrations of PTX, a potent antagonist of insect GABA receptors (Sattelle, 1990), suggested that the spontaneous IPSCs recorded in the embryonic neurons are mediated by GABA-gated chloride channels. This conclusion is also supported by the finding that, although puffing of GABA or glutamate evokes chloride currents in the cultured neurons, as predicted from a study of larval motor neurons (Rohrbough, 2002), only the GABA-evoked currents are effectively blocked by 1 µM PTX. This PTX concentration does not significantly reduce the glutamate-evoked currents. These data strongly support the hypothesis that the receptors mediating spontaneous synaptic currents are GABA-gated, as opposed to glutamate-gated, chloride channels (Lee, 2003).

GABA acts primarily as an inhibitory neurotransmitter in the adult CNS of both vertebrates and invertebrates (Mody, 1994; Hosie, 1997). However, there is abundant evidence that GABA can be excitatory during early development (Ben-Ari, 1997). Recently, it has been reported that a blockade of GABAA receptors in the neonatal rodent brain can induce increases in neuronal excitation. This indicates that GABAergic transmission can also serve an inhibitory role in hippocampal and cortical circuits during early development in mammals (Lamsa, 2000; Palva, 2000; Wells, 2000). Using a similar strategy, blocking GABA receptors by bath application of PTX, an increase was observed in spontaneous neuronal firing in the Drosophila cultures. This demonstrates that embryonic neurons form spontaneously active circuits in culture and GABAergic transmission can mediate inhibition in these networks, even at this early developmental stage in Drosophila. Future studies will be necessary to determine whether GABA can be depolarizing and/or elicit action potentials, as observed in some neurons from the early postnatal rodent brain, in subpopulations of Drosophila neurons (Lee, 2003).

At many chemical synapses, some portion of the vesicular release of neurotransmitter is action potential (AP) independent. In cultured embryonic Drosophila neurons, much of the spontaneous release of GABA at synapses appears to occur in the absence of sodium spikes in presynaptic neurons. A blockade of the mIPSCs by removal of external calcium or the addition of cobalt demonstrates that these events are dependent on flux of calcium through voltage-gated channels in the presynaptic neurons. Spontaneously occurring calcium-dependent APs, could result in transient changes in levels of calcium in the presynaptic terminals that would in turn regulate vesicular release of GABA. However, this seems unlikely in the Drosophila cultures because, although many of the neurons fire spontaneous sodium APs in normal saline, regenerative spikes have not been observed in the presence of TTX. Alternatively, small fluctuations in the resting membrane potential could regulate the opening of voltage-gated calcium channels in the presynaptic terminals. The resulting changes in intracellular calcium levels would in turn influence the frequency of spontaneous fusion events. This seems plausible, given the relatively depolarized resting potential of the embryonic neurons and calcium channels that activate at voltages as low as -60 mV. In a similar manner, the unexpected appearance of mIPSCs in bursts could arise from regular oscillations in the membrane potential of a population of presynaptic GABAergic neurons in which peaks, associated with high intracellular calcium levels, may trigger the release of multiple quanta. Calcium imaging studies, in combination with electrophysiological recording, should be useful in elucidating the mechanisms underlying the AP-independent burst activity observed in the embryonic Drosophila neurons (Lee, 2003).

In mammals, there is a high degree of heterogeneity in GABA receptor properties in neurons from different regions of the CNS. Factors influencing functional heterogeneity include receptor subunit composition and desensitization rates (Vicini, 1999). In addition, the functional properties of receptors mediating synaptic currents in individual neurons can change during development. For example, there is a maturational progression from mIPSCs with slow to more rapid decay kinetics correlated with changes in receptor subunit composition and populations of cerebellar, hippocampal, cortical, and thalamic neurons. The heterogeneity in the decay kinetics of currents recorded from embryonic Drosophila neurons during the first week in culture is consistent with the expression of multiple receptor subtypes. This is not surprising, given that the cultures are prepared from whole embryos and therefore contain neurons from all parts of the nervous system. The shift in mean decay time constant, 1.5- to 2-fold during the first week in culture, suggests that functional properties of receptors mediating the GABAergic mIPSCs in Drosophila neurons are also subject to regulation during maturation. In rodent cortical neurons, a decrease in mPSC decay time constant, both in vivo and in dissociated cell culture, demonstrated that signals necessary for initiating the changes in GABA receptor function can be retained in dissociated cell culture. Although no parallel study has been conducted on GABAergic mIPSCs in Drosophila in vivo, preliminary data suggest that the GABAergic mIPSC decay rate in CNS neurons cultured from late-stage pupae are faster than those seen even in the older embryo cultures. This is consistent with the changes occurring over time in culture representing maturation that normally occurs in the animal. The evolutionary conservation of this change supports the hypothesis that alterations in GABA receptor kinetics play an important role in shaping early neural circuitry (Lee, 2003).

Cloning and expression studies have been important in defining the role of two Drosophila GABA receptor subunit genes, Rdl and LCCH3, in the formation of functional GABA-gated ion channels (Hosie, 1997). Pharmacological analysis of wild-type and Rdl mutant neurons now provide the first insights into the subunit composition of synaptic GABA receptors in Drosophila. The PTX-sensitive mIPSCs in wild-type neurons are not blocked by bicuculline methylchloride (BMC). This pharmacological profile is similar to homomultimeric GABA-gated chloride channels encoded by the Rdl GABA receptor subunit gene when expressed in Xenopus oocytes (ffrench-Constant, 1993b; full text of article ) and Sf2 cells (Zhang, 1995). A significant reduction of the sensitivity of the mIPSCs to blockade by PTX in Rdl mutant versus wild-type neurons confirmed that Rdl-encoded subunits contribute to the population of functionally active synaptic GABA receptors (Lee, 2003).

The Rdl mutant neurons in culture exhibit a 5- to 10-fold reduction in PTX sensitivity based on the comparison with the wild-type dose-response curve. In contrast, the GABA-evoked currents mediated by mutant Rdl channels expressed in oocytes are ~100-fold less sensitive to PTX blockade than the wild-type Rdl channels (ffrench-Constant, 1993b; full text of article). This suggests that synaptic GABA receptors containing Rdl in the neurons are heteromultimers rather than homomultimers. It does not seem likely that the receptors are Rdl- and LCCH3-encoded heteromultimers because expression studies indicate that these form PTX-insensitive, BMC-sensitive receptors (Zhang, 1995). In addition, antibody staining has shown that Rdl protein is localized in the synaptic neuropil in embryos and larval CNS, whereas LCCH3 is found primarily in the cell bodies, making it unlikely that they interact in vivo (Aronstein, 1996). Therefore, the synaptic receptors may include additional subunits, perhaps encoded by GRD (Harvey, 1994) or other as yet uncharacterized GABA receptor genes (Lee, 2003).

The resistance to PTX-induced increases in neuronal firing rates in Rdl mutant cultures demonstrates that Rdl subunit-containing GABA receptors actively mediate synaptic inhibition in Drosophila neural circuits. Therefore, it is possible that synaptically localized Rdl-containing receptors are involved in higher-order functions such as GABA receptor-mediated synchronization of neural activity known to be important in olfactory information-processing locusts (MacLeod, 1996). Manipulation of Rdl expression in Drosophila should make it possible to test this hypothesis (Lee, 2003).

Alternative splicing of a Drosophila GABA receptor subunit gene identifies determinants of agonist potency

Alternative splicing of the Drosophila Rdl gene yields four ionotropic GABA receptor subunits. The two Rdl splice variants cloned to date, RDLac and RDLbd (DRC17-1-2), differ in their apparent agonist affinity. This paper reports the cloning of a third splice variant of Rdl, RDLad. Two-electrode voltage clamp electrophysiology was used to investigate agonist pharmacology of this expressed subunit following cRNA injection into Xenopus laevis oocytes. The ECso values for GABA and its analogues isoguvacine, muscimol, isonipecotic acid and 3-amino sulphonic acid on the RDLad homomeric receptor differed from those previously described for RDLac and DRC17-1-2 receptors. In addition to providing a possible physiological role for the alternative splicing of Rdl, these data delineate a hitherto functionally unassigned region of the N-terminal domain of GABA receptor subunits, which affects agonist potency and aligns closely with known determinants of potency in nicotinic acetylcholine receptors. Thus, using expression in Xenopus oocytes, differences have been demonstrated in agonist potency for the neurotransmitter GABA (and four analogues) between splice variant products of the Drosophila melanogaster Rdl gene encoding homomer-forming GABA receptor subunits (Hosie, 2001).

There is mounting evidence that subunits encoded by insect Rdl genes underly the characteristic pharmacology of the bicuculline-insensitive GABA receptors that predominate in insect nervous systems. cRNAs exhibiting a high identity (>80%) with those encoding Drosophila RDL subunits, have been cloned from a variety of insect species; these subunits are widely distributed in insect CNS. Although the intron-exon structures of Rdl genes from insect species other than Drosophila melanogaster and Aedes aegyptae remain to be determined, there is evidence to suggest that some of these may also be alternatively spliced. To date, multiple RDL isoforms have been isolated in Drosophila and in other insects such as Blatella germanica (the German cockroach, where four homologues have been identified (Kaku, 1994); all differ in the region corresponding to that encoded by exon 6 of the Drosophila Rdl gene. Indeed, in some cases, the putative D. melanogaster and B. germanica subunits differ at homologous residues and the identities of these variant residues are preserved in different species (Hosie, 2001).

It is, therefore, possible that Rdl genes are alternatively spliced in a number of insect species. The present study demonstrates that the 10 amino acid differences, which result from the alternative splicing of exon 6 of D. melanogaster Rdl, confer a three-fold change in the potencies of GABA and its analogues. Thus, by generating small changes in agonist sensitivity, the alternative splicing of Rdl genes may serve to increase functional diversity in insect GABA receptors. Such a mechanism would distinguish insects from vertebrates, where the principal mechanism for increasing GABA receptor diversity appears to be the co-assembly of different subunit classes and isoforms, which are encoded by separate genes. While the difference in agonist potency conferred by splice variants of D. melanogaster Rdl is not great, it is similar to that observed for certain recombinant GABAA receptors containing different α subunit isoforms. Just how significant this is depends very much on the synaptic concentration of GABA; high micromolar concentrations would saturate Rdl variants. The physiological role of Rdl splice variants may, therefore, be to yield GABA receptors with differing single-channel kinetics. In line with this, the kinetics of recombinant GABAA receptor channels depends on their subunit composition, and is affected by the presence of different α isoforms. While the M2 domain of α subunits is highly conserved, the most variant region in the N-terminal domain of α isoforms corresponds to the region of RDL subunits encoded by exon 6. With this in mind, it may be worth performing a detailed study of the kinetics of different RDL receptors. Such a detailed comparison of kinetics has yet to be undertaken; however, no differences in the single-channel conductances of RDLac and RDLbd have been observed (Hosie, 2001).

The alternative splicing of D. melanogaster Rdl provides further evidence for a conserved structure-function relationship for the cys-loop family of neurotransmitter receptors, which includes nAChRs and strychnine-sensitive glycine receptors. In the best studied members of this family, nAChRs of vertebrate muscle, the agonist binding sites are considered to lie at the interfaces of two subunit pairs and are composed of a number of discrete regions of the N-terminal domain of each subunit. Six such regions have been identified, loops A-F although further determinants lying outside these domains have also been identified. Loops A, B and C lie on the nicotinic α subunit, while the remainder lie on the adjacent face of another subunit, normally a non-α subunit. The subunits encoded by the Rdl gene, like the α7-9 nAChR subunits of vertebrates, are capable of forming functional homomers. The residues, which differentiate RDLac from RDLad, lie between loops B and C and thus encompass a region equivalent to loop E of nAChRs. In nAChRs, the residues of loop E appear to interact directly with agonists and their substitution confers marked changes in agonist potency. Although the majority of the substitutions in the RDL subunit variants are not conservative they affect relatively small changes in agonist potency, suggesting that rather than interacting directly with the agonist molecule they may influence the shape of the binding site and/or its coupling to the channel gate. However, the changes in RDL are naturally occurring, with a possible physiological role, and could therefore be expected to produce subtle differences in receptor function rather than the dramatic changes associated with site-directed mutagenesis of vertebrate GABAA and nicotinic receptors (Hosie, 2001).

The known determinants of agonist potency on GABAA receptors align closely to domains A-D of nicotinic receptors. In GABAA receptors, residues homologous to domains B and C have been identified on the β subunit while a residue in the homologue of domain D has been identified in the α subunit. Similarly, determinants of agonist potency are located in bicuculline-insensitive vertebrate GABA receptor ρ subunits at positions homologous to domains A, B and C. However, an equivalent of domain E has yet to be recognised in vertebrate ionotropic GABA receptors. The results of the present study demonstrate that naturally occurring substitutions of amino acids in this region affect the potency of GABA on RDL homomers. Whether this region of the N-terminal domain also affects the potency of agonists on vertebrate GABA receptors remains to be determined. However, with the exception of the signal peptide and the extreme N-terminus of the mature subunit, this is the least conserved region in the N-terminal domain of invertebrate α subunit isoforms, and it is the α subunits which would be expected to contribute domain E to the agonist binding site of GABAA receptors. With this in mind, it is interesting that the differences in the agonist potency and kinetics of recombinant rat GABAA receptors containing α1 or α3 subunits could be accounted for primarily by differences in the transition rates underlying agonist association and dissociation (Hosie, 2001).

The present study demonstrates that a naturally occurring substitution of residues in the extracellular region of Drosophila GABA receptors underlies moderate changes in the potency of the natural agonist, GABA, and may therefore serve a physiological role. The region containing these changes aligns with determinants of agonist potency on nAChRs. It is therefore possible that this region may also affect either the agonist responses of vertebrate GABA receptors, or the kinetics of channel opening (Hosie, 2001).

Functions of Rdl orthologs in other species

The netrin receptor UNC-40/DCC assembles a postsynaptic scaffold and sets the synaptic content of GABAA receptors
Increasing evidence indicates that guidance molecules used during development for cellular and axonal navigation also play roles in synapse maturation and homeostasis. In C. elegans the netrin receptor UNC-40/DCC (see Drosophila Frazzled) controls the growth of dendritic-like muscle cell extensions towards motoneurons and is required to recruit type A GABA receptors (GABAARs; see Drosophila Rdl) at inhibitory neuromuscular junctions. This study show that activation of UNC-40 assembles an intracellular synaptic scaffold by physically interacting with FRM-3, a FERM protein orthologous to FARP1/2. FRM-3 then recruits LIN-2, the ortholog of CASK (see Drosophila Cask), that binds the synaptic adhesion molecule NLG-1/Neuroligin (see Drosophila Neuroligin) and physically connects GABAARs to prepositioned NLG-1 clusters. These processes are orchestrated by the synaptic organizer CePunctin/MADD-4 (a member of the ADAMTS family of proteases), which controls the localization of GABAARs by positioning NLG-1/neuroligin at synapses and regulates the synaptic content of GABAARs through the UNC-40-dependent intracellular scaffold. Since DCC is detected at GABA synapses in mammals, DCC might also tune inhibitory neurotransmission in the mammalian brain (Zhou, 2020).

GABA promotes the competitive selection of dendritic spines by controlling local Ca2+ signaling

Activity-dependent competition of synapses plays a key role in neural organization and is often promoted by GABA; however, its cellular bases are poorly understood. Excitatory synapses of cortical pyramidal neurons are formed on small protrusions known as dendritic spines, which exhibit structural plasticity. This study used two-color uncaging of glutamate and GABA in rat hippocampal CA1 pyramidal neurons and found that spine shrinkage and elimination were markedly promoted by the activation of GABAA receptors shortly before action potentials. GABAergic inhibition suppressed bulk increases in cytosolic Ca2+ concentrations, whereas it preserved the Ca2+ nanodomains generated by NMDA-type receptors, both of which were necessary for spine shrinkage. Unlike spine enlargement, spine shrinkage spread to neighboring spines (<15 μm) and competed with their enlargement, and this process involved the actin-depolymerizing factor ADF/cofilin. Thus, GABAergic inhibition directly suppresses local dendritic Ca2+ transients and strongly promotes the competitive selection of dendritic spines (Hayama, 2013).

Activity-dependent switch of GABAergic inhibition into glutamatergic excitation in astrocyte-neuron networks

Interneurons are critical for proper neural network function and can activate Ca2+ signaling in astrocytes. However, the impact of the interneuron-astrocyte signaling into neuronal network operation remains unknown. Using the simplest hippocampal Astrocyte-Neuron network, i.e., GABAergic interneuron, pyramidal neuron, single CA3-CA1 glutamatergic synapse, and astrocytes, this study found that interneuron-astrocyte signaling dynamically affected excitatory neurotransmission in an activity- and time-dependent manner, and the sign (inhibition vs potentiation) of the GABA-mediated effects were determined. While synaptic inhibition was mediated by GABAA receptors (see Drosophila Rdl), potentiation involved astrocyte GABAB receptors, astrocytic glutamate release, and presynaptic metabotropic glutamate receptors. Using conditional astrocyte-specific GABAB receptor (Gabbr1; see Drosophila metabotropic GABA-B receptor subtype 1) knockout mice, the glial source of the interneuron-induced potentiation was confirmed, and the involvement of astrocytes in hippocampal theta and gamma oscillations in vivo was demonstrated. Therefore, astrocytes decode interneuron activity and transform inhibitory into excitatory signals, contributing to the emergence of novel network properties resulting from the interneuron-astrocyte interplay (Perea, 2016).


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