InteractiveFly: GeneBrief

Resistant to dieldrin: Biological Overview | References

In both mammals and insects, neurons involved in learning are strongly modulated by the inhibitory neurotransmitter GABA. The GABA_A 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.

GABA_A receptors are GABA-gated chloride channels. Accumulating pharmacological and genetic evidence suggests that GABA_A 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 GABA_A 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 GABA_A 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 GABA_A 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 GABA_A 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 GABA_Aβ3 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 GABA_Aα5 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 GABA_A 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 GABA_A 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 GABA_A 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 Rdl^f02994, 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 GABA_A 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 we observed enhanced acquisition with reduced Rdl expression. Rather than facilitating and enhancing memory formation by the sparsening of representations, our 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 GABA_A receptor and the expression level of gephyrin, a protein involved in GABA_A 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 GABA_A 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).

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 GABA_B 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 GABA_B receptor immunoreactivity is seen on these neurons as well as the Cha-Tan neurons. Based on an rdl-Gal4 line, the ionotropic GABA_A 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 GABA_A receptor desensitization uncouples sleep onset and maintenance in Drosophila

Many lines of evidence indicate that GABA and GABA_A 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 GABA_A receptor, Rdl^A302S, 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 Rdl^A302S mutant flies are resistant to the effects of CBZ on sleep latency and that mutant RDL^A302S 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 GABA_A receptor signaling dictate sleep latency (Agosto, 2008).

gamma-Aminobutyric acid (GABA) signaling components in Drosophila: immunocytochemical localization of GABA_B receptors in relation to the GABA_A 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 (GABA_AR) for fast inhibitory transmission and on G-protein-coupled receptors (GABA_BR) for slow and modulatory action. Immunocytochemistry was used to map GABA_BR sites in the Drosophila CNS, and the distribution was compared with that of the GABA_AR 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. GABA_BR-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 GABA_BR-IR punctates. However, the mushroom body lobes displayed RDL-IR but not GABA_BR-IR material, and there were subtle differences in other areas. The vGAT antiserum labeled punctates in the same areas as the GABA_BR and appeared to display presynaptic sites of GABAergic neurons. Various GAL4 drivers were used to analyze the relation between GABA_BR 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).

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 GABA_A 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, RDL_ac and RDL_bd (DRC17-1-2), differ in their apparent agonist affinity. This paper reports the cloning of a third splice variant of Rdl, RDL_ad. Two-electrode voltage clamp electrophysiology was used to investigate agonist pharmacology of this expressed subunit following cRNA injection into Xenopus laevis oocytes. The EC_so values for GABA and its analogues isoguvacine, muscimol, isonipecotic acid and 3-amino sulphonic acid on the RDL_ad homomeric receptor differed from those previously described for RDL_ac 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 GABA_A 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 GABA_A 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 RDL_ac and RDL_bd 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 RDL_ac from RDL_ad, 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 GABA_A and nicotinic receptors (Hosie, 2001).

The known determinants of agonist potency on GABA_A receptors align closely to domains A-D of nicotinic receptors. In GABA_A 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 GABA_A receptors. With this in mind, it is interesting that the differences in the agonist potency and kinetics of recombinant rat GABA_A 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).

Search PubMed for articles about Drosophila Rdl

Agosto, J., et al. (2008). Modulation of GABAA receptor desensitization uncouples sleep onset and maintenance in Drosophila. Nat. Neurosci. 11(3): 354-9. PubMed citation: 18223647

Aronstein, K., Auld, V., ffrench-Constant, R. (1996). Distribution of two GABA receptor-like subunits in the Drosophila CNS. Invert Neurosci 2: 115-120. PubMed citation: 9372158

Ben-Ari, Y., Khazipov, R., Leinekugel, X., Caillard, O. and Gaiarsa, J.-L. (1997). GABA_A, NMDA and AMPA receptors: a developmentally regulated 'menage a trois.' Trends Neurosci 20: 523-529. PubMed citation: 9364667

Brennan, P. A., et al. (1998). Changes in neurotransmitter release in the main olfactory bulb following an olfactory conditioning procedure in mice. J. Neuroscience 87: 583-590. PubMed citation: 9758225

Buckingham, S. D., et al. (2005). Insect GABA receptors: splicing, editing, and targeting by antiparasitics and insecticides. Mol. Pharm. 68: 942-951. PubMed citation: 16027231

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date revised: 20 July 2008

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