metabotropic Glutamate receptor: Biological Overview | References
Gene name - metabotropic Glutamate receptor
Cytological map position - 102F1-102F3
Function - G-protein coupled receptor
Symbol - mGluR
FlyBase ID: FBgn0019985
Genetic map position - chr4:957468-967451
Classification - GPCR
Cellular location - surface transmembrane
In vertebrates, several groups of metabotropic glutamate receptors (mGluRs) are known to modulate synaptic properties. In contrast, the Drosophila genome encodes a single functional mGluR (DmGluRA), an ortholog of vertebrate group II mGluRs, greatly expediting the functional characterization of mGluR-mediated signaling in the nervous system. This study shows that DmGluRA is expressed at the glutamatergic neuromuscular junction (NMJ), localized in periactive zones of presynaptic boutons but excluded from active sites. Null DmGluRA mutants are completely viable, and all of the basal NMJ synaptic transmission properties are normal. In contrast, DmGluRA mutants display approximately a threefold increase in synaptic facilitation during short stimulus trains. Prolonged stimulus trains result in very strongly increased (approximately 10-fold) augmentation, including the appearance of asynchronous, bursting excitatory currents never observed in wild type. Both defects are rescued by expression of DmGluRA only in the neurons, indicating a specific presynaptic requirement. These phenotypes are reminiscent of hyperexcitable mutants, suggesting a role of DmGluRA signaling in the regulation of presynaptic excitability properties. The mutant phenotypes could not be replicated by acute application of mGluR antagonists, suggesting that DmGluRA regulates the development of presynaptic properties rather than directly controlling short-term modulation. DmGluRA mutants also display mild defects in NMJ architecture: a decreased number of synaptic boutons accompanied by an increase in mean bouton size. These morphological changes bidirectionally correlate with DmGluRA levels in the presynaptic terminal. These data reveal the following two roles for DmGluRA in presynaptic mechanisms: (1) modulation of presynaptic excitability properties important for the control of activity-dependent neurotransmitter release and (2) modulation of synaptic architecture (Bogdanik, 2004).
DmGluRA is expressed at the NMJ, although at relatively low abundance. Transgenic RNAi constructs expressed in either the presynaptic or postsynaptic cell revealed that DmGluRA is primarily presynaptic, consistent with localization of its vertebrate orthologs, group II mGluRs (mGlu2/3). A presynaptic localization is also consistent with Zhang (1999), who reported a group II agonist-induced presynaptic enhancement of synaptic transmission at the Drosophila NMJ of first instar larvae (Bogdanik, 2004).
DmGluRA receptors were not distributed homogeneously in synaptic boutons but rather appear in discrete patches that do not overlap with ionotropic glutamate receptors inhabiting the postsynaptic specialization apposing presynaptic glutamate release sites. Thus, DmGluRA receptors appear to be mostly excluded from release sites and rather inhabit the periactive zone. These results fit well with the reported ultrastructural localization of vertebrate group II mGluRs, which are known to be excluded from glutamate release zones. This distribution suggests that DmGluRA receptors are positioned to function as presynaptic autoreceptors, to sense glutamate spilling out of the active signaling zone (Bogdanik, 2004).
DmGluRA receptors are well positioned to modulate presynaptic release properties. Numerous reports have shown that the application of group II mGluR agonists inhibits synaptic transmission at various glutamatergic synapses, suggesting that mGluR mutants display enhanced synaptic transmission. However, mGluR2 knock-out mice show no modifications of basal synaptic transmission or paired-pulse facilitation at mossy fiber-CA3 synapses. Similarly, electrophysiological characterization of DmGluRA mutants revealed no changes in basal synaptic transmission or paired-pulse facilitation at the Drosophila NMJ. Thus, mGluR signaling is not involved in maintenance-regulation of basal neurotransmission. Nevertheless, mGluRs may mediate modulation processes at elevated activity levels. Indeed, activation of postsynaptic group I mGluRs critically depends on duration and frequency of synaptic activity. Likewise, activity-dependent activation of presynaptic mGluR2 on mossy fibers caused by glutamate spillover from adjacent synapses has been proposed, suggesting that presynaptic mGluRs are activated only during elevated activity. Nevertheless, synaptic transmission in DmGluRA mutants was unchanged at higher stimulation frequencies (1-20 Hz) in physiological conditions. The possibility cannot be excluded that differences in the performance of mutant synapses may arise under specific stimulation conditions. For example, in the lateral perforant path of the dentate gyrus, application of a group III agonist influences synaptic depression only during prolonged stimulation at frequencies higher than 5 Hz. Thus, presynaptic mGluRs may regulate neurotransmission only during certain, tightly defined conditions (Bogdanik, 2004).
A general feature of many synapses, including the Drosophila NMJ, is that low extracellular Ca2+ concentration (e.g., 0.15 mm) alleviates synaptic depression and permits the manifestation of synaptic facilitation. Under this condition, we observed a strong requirement for mGluR signaling: in DmGluRA mutants, a striking, approximately threefold increase in facilitation during short trains of 10 Hz stimulation was observed. In general, facilitation is thought to be caused by presynaptic accumulation of Ca2+, which strengthens synaptic transmission by promoting Ca2+-sensitive steps of exocytosis. However, altering Ca2+ entry or Ca2+ sensitivity should affect basal transmission and paired-pulse facilitation, but both parameters were unchanged in DmGluRA mutants. Thus, other defective mechanisms must account for the facilitation defect. The most probable explanation is altered presynaptic excitability properties. Hyperexcitable Drosophila mutants, including Hyperkinetic, bemused, frequenin, and para-overexpressing flies have all been demonstrated to exhibit increased onset rates during facilitation, indicating that facilitation is significantly altered by excitability changes. The hypothesis that DmGluRA signaling regulates presynaptic excitability seems particularly intriguing when considering the described changes in response patterns observed in mutants during more prolonged (1 min, 5 Hz) stimulation: DmGluRA mutants show a striking threshold-like change in response pattern, resulting in extraordinarily increased EJC amplitudes that have an asynchronous event probability. The sharp transition between the initially relatively mild enhancement of facilitation and the abruptly 10-fold increased responses is most readily explained by a change in excitability properties, allowing the generation of multiple spikes after a single stimuli. Most interestingly, identically changed response patterns have been reported in frequenin-overexpressing larvae, which have been termed 'large facilitated responses.' Frequenin overexpression modulates the properties of presynaptic potassium channels, making these channels a likely target for DmGluRA regulation. Similarly, in vertebrates, activation of mGluRs can induce the modulation of presynaptic voltage-gated ion channels (Bogdanik, 2004).
Putative excitability changes in DmGluRA mutants was studied by extracellularly recording endogenous action potential trains in the motor nerve using physiological conditions (1.8 mm Ca2+). Spike trains in mutants were similar to controls and did not contain multiple, closely successive spike events that would be indicative of hyperexcitability. Therefore, generally elevated excitability is unlikely. DmGluRA-dependent changes in excitability might be restricted to synaptic boutons in which DmGluRA is localized. In addition, elevated nerve excitability may occur in DmGluRA mutants only with reduced Ca2+, when the excitability of motor nerves is already increased by changing the surface charge as a result of the removal of divalent cations. Unfortunately, technical limitations have so far prevented assaying of action potentials under the same conditions found to promote the synaptic phenotype. Interestingly, it was found that DmGluRA mutants fired action potentials within trains with a significantly reduced frequency. This change may reflect altered excitability within the motor neuron or changed activity patterns within the CNS. DmGluRA is expressed in the ventral nerve cord neuropil, in which it could potentially modulate neuronal activity (Bogdanik, 2004).
The dramatic changes in neurotransmitter release during high-frequency stimulation are most readily explained by abolishment of a critical presynaptic negative feedback mechanism in mutant animals. However, phenotypes could not be mimiced by acute application of mGluR antagonists (MPPG or LY341495). A particular important caveat is that pharmacological inhibition may simply not be effective enough to fully recapitulate phenotypes observed in null mutants; therefore, the possibility of an acute feedback mechanism cannot be excluded. From this perspective, it is noted that results presented in this study from third instar contradict a study by Zhang (1999) in the early first instar describing acute increase in miniature frequency induced by group II agonists and partial blockade of evoked excitatory junction currents (EJCs) by antagonists. This difference may be attributable to the absence of the convoluted subsynaptic reticulum in the first instar, which may be causing a significant experimental limitation to drug application in the third instar. Indeed, it wasobserved that the iGluR blocker argiotoxin-636 results in a 70% block in receptor function in the third instar, whereas the same toxin eliminates iGluR function in the more accessible first instar. These caveats aside, a developmental role for mGluR signaling is certainly possible. For example, thalamic mGluR1 shows a developmental reorganization, in which its subcellular distribution changes. Intriguingly, this reorganization is correlated to a change in the properties of glutamatergic synapses at maturing thalamic neurons (Bogdanik, 2004).
Drosophila NMJ structure is sculpted by developmental cues and neuronal activity. Increased neuronal activity positively correlates with an increased number of synaptic boutons, as illustrated by eag Sh or Hk eag hyperexcitable mutants. However, other hyperexcitable mutants, such as frequenin-overexpressing larvae, display an opposite phenotype, i.e., a reduction in bouton number and an increase in bouton size. Thus, different mechanisms must be involved in linking activity to synaptic morphology (Bogdanik, 2004).
This study shows that DmGluRA null mutants display altered presynaptic excitability and an ~10%-20% decreased number of synaptic boutons. It is debatable whether and how structural and physiological phenotypes in DmGluRA mutants are mechanistically linked. It is uncertain whether the morphological changes significantly influence the functional properties or whether the suspected excitability change is responsible for the subtle structural changes. Experiments using DmGluRA overexpression and DmGluRA-RNAi indicate a significant correlation between presynaptic DmGluRA level and bouton differentiation. Moreover, DmGluRA mutants exhibit an increased bouton size, compensating for the decreased bouton number. Thus, DmGluRA modulates bouton number but has no detectable impact on the overall bouton area of the NMJ. Consistently, the frequency of spontaneous mEJCs was unchanged in DmGluRA mutants, suggesting that functional release site density is normal. Such a role of a presynaptic group II mGluR in the fine control of synaptic architecture was still unknown. It could be of growing interest, because human group II/III mGluRs have been shown to be drug targets in numerous psychiatric and neurological disorders, such as schizophrenia and seizure disorders. Interestingly, such disorders are characterized by the presence of subtle synaptic abnormalities (Bogdanik, 2004).
DmGluRA mutants share both electrophysiological and morphological phenotypes with frequenin-overexpressing larvae. This study has shown that the eag Sh structural phenotype does not depend on the presence of DmGluRA. This suggests that similar signaling pathways are affected in DmGluRA and frequenin mutants and that these pathways differ from those affected in eag Sh mutants. We started to dissect the transduction pathway activated by DmGluRA: here, we show that the Gβ13F subunit is required to mediate the morphological changes (Bogdanik, 2004).
Ligand activation of the metabotropic glutamate receptor (mGluR) activates the lipid kinase PI3K in both the mammalian central nervous system and Drosophila motor nerve terminal. In several subregions of the mammalian brain, mGluR-mediated PI3K activation is essential for a form of synaptic plasticity termed long-term depression (LTD), which is implicated in neurological diseases such as fragile X and autism. In Drosophila larval motor neurons, ligand activation of DmGluRA, the sole Drosophila mGluR, similarly mediates a PI3K-dependent downregulation of neuronal activity. The mechanism by which mGluR activates PI3K remains incompletely understood in either mammals or Drosophila. This study identified CaMKII and the nonreceptor tyrosine kinase DFak as critical intermediates in the DmGluRA-dependent activation of PI3K at Drosophila motor nerve terminals. Transgene-induced CaMKII inhibition or the DFakCG1 null mutation each block the ability of glutamate application to activate PI3K in larval motor nerve terminals, whereas transgene-induced CaMKII activation increases PI3K activity in motor nerve terminals in a DFak-dependent manner, even in the absence of glutamate application. It was also found that CaMKII activation induces other PI3K-dependent effects, such as increased motor axon diameter and increased synapse number at the larval neuromuscular junction. CaMKII, but not PI3K, requires DFak activity for these increases. It is concluded that the activation of PI3K by DmGluRA is mediated by CaMKII and DFak (Lin, 2011).
Metabotropic glutamate receptors (mGluRs), G protein-coupled receptors for which glutamate is ligand, mediate aspects of synaptic plasticity in several systems. In several regions of the mammalian brain, including the hippocampus, the cerebellum, the prefrontal cortex, and others, ligand activation of group I mGluRs induces a long-term depression of synaptic activity, termed mGluR-mediated long-term depression (LTD). Induction of mGluR-mediated LTD both activates and requires the activation of the lipid kinase PI3 kinase (PI3K) and the downstream kinase Tor (Hou, 2004). Several genetic diseases of the nervous system are predicted to increase sensitivity to activation of mGluR-mediated LTD. For example, increased sensitivity to induction of mGluR-mediated LTD has been observed in the mouse model for fragile X (Bear, 2004). Furthermore, the genes affected in tuberous sclerosis (Tsc1 and Tsc2) and neurofibromatosis (Nf1) encode proteins that downregulate Tor activity. These observations raise the possibility that hyperactivation of mGluR-mediated LTD plays a causal role in the neurological phenotypes of fragile X, neurofibromatosis and tuberous sclerosis. Because these diseases are each associated with an extremely high incidence of autism spectrum disorders (ASDs), and because several lines of evidence suggest that elevated PI3K activity is associated with ASDs, it has been hypothesized that hyperactivation of this pathway might be responsible for ASDs as well. Thus it would be of interest to identify additional molecular components by which mGluR activation activates PI3K, and yet despite recent advances, this mechanism remains incompletely understood (Lin, 2011).
In Drosophila larval motor neurons, glutamate activation of the single mGluR, called DmGluRA, downregulates neuronal excitability (Bogdanik, 2004); glutamate both activates PI3K and requires PI3K activity for this downregulation (Howlett, 2008). Because glutamate is the excitatory neurotransmitter at the Drosophila neuromuscular junction (NMJ), it was hypothesized that this DmGluRA-mediated downregulation of neuronal excitability carried out a negative feedback on activity: glutamate released from motor nerve terminals would activate DmGluRA autoreceptors, which would then depress excitability (Lin, 2011).
This study identified additional molecular components that mediate the activation of PI3K by DmGluRA in Drosophila larval motor nerve terminals. It was found that activity of the calcium/calmodulin-dependent kinase II (CaMKII) is necessary for glutamate application to activate PI3K, and expression of the constitutively active CaMKIIT287D is sufficient both to activate PI3K even in the absence of glutamate and to confer several other neuronal phenotypes consistent with PI3K hyperactivation. It was also found that CaMKIIT287D requires the nonreceptor tyrosine kinase DFak for this PI3K activation: the DFakCG1 null mutation blocks the ability of glutamate application to activate PI3K and prevents CaMKIIT287D from hyperactivating PI3K. Finally, CaMKIIT287D expression completely suppresses the hyperexcitability conferred by the DmGluRA null mutation DmGluRA112b. It is concluded that ligand activation of DmGluRA activates PI3K via CaMKII and DFak (Lin, 2011).
In both mammalian central synapses and Drosophila larval motor neurons, activation by glutamate of the metabotropic glutamate receptor (mGluR) activates the lipid kinase PI3K, but the mechanism by which this activation occurs has not been elucidated. This study identified CaMKII as a critical intermediate in the ability of the single Drosophila mGluR (DmGluRA) to activate PI3K and shows that the ability of both activated DmGluRA and CaMKII to activate PI3K requires the nonreceptor tyrosine kinase, DFak (see A proposed mechanism for the DmGluRA-dependent activation of PI3K via CaMKII and DFak). These results provide novel insights into the mechanism by which DmGluRA activation triggers the observed downregulation of subsequent neuronal activity in Drosophila motor neurons. These results might also be relevant to the mechanism by which mGluR activates PI3K in mammalian central synapses, a process implicated in fragile X, ASDs, neurofibromatosis, and tuberous sclerosis (Lin, 2011).
How might CaMKII lead to the DFak-dependent activation of PI3K? Although the ability of CaMKII to activate PI3K has only recently been reported, it has been well established in mammals that CaMKII phosphorylates both Fak and Pyk2 on multiple serines on the C terminus. These phosphorylation events can activate Pyk2 by enabling subsequent tyrosine phosphorylations (particularly at Tyr402) via mechanisms that are incompletely understood. It has also been well established that Fak and Pyk2, when activated by tyrosine phosphorylation, are each able to activate PI3K: tyrosine-phosphorylated Fak binds p85, the PI3K regulatory subunit, via both the SH3 and SH2 domains. In addition, both tyrosine-phosphorylated Fak and Pyk2 are capable of activating Ras via the conserved Grb2-SoS pathway, which could in principle lead to the Ras-dependent, p85-independent activation of PI3K. These observations raise the possibility that Drosophila CaMKII might similarly activate PI3K by directly phosphorylating and activating DFak. Alternatively, DFak might function in a more indirect fashion, perhaps as a scaffold linking CaMKII and PI3K in a signaling complex. This alternative possibility would suggest that additional intermediates linking CaMKII and PI3K activation exist but are currently unidentified (Lin, 2011).
The observation that DmGluRA-mediated activation of PI3K requires CaMKII implies that DmGluRA activation increases intracellular Ca2+ levels in Drosophila motor nerve terminals as a necessary step in PI3K activation. The source of Ca2+ for this activation is not known. However in mammals, activation of group I mGluRs, which are responsible for mGluR-mediated LTD in the hippocampus and cerebellum, induce phospholipase C and IP3-mediated Ca2+ transients, which are essential intermediates in cerebellar mGluR-mediated LTD. Although the Drosophila DmGluRA is most similar to mammalian group II mGluRs, which are not known to activate Ca2+ transients, given that DmGluRA is the sole mGluR in Drosophila, it seems possible that DmGluRA might carry out many of the functions carried out by each of the three groups of mGluRs in mammals, as suggested previously (Pan, 2008). Alternatively, it is possible that DmGluRA activation might increase intracellular Ca2+ via the ryanodine receptor, which was previously shown to be an essential activator of CaMKII in Drosophila larval motor nerve terminals (Lin, 2011).
The ability of CaMKII to activate PI3K requires the nonreceptor tyrosine kinase DFak; the DFakCG1 null mutation completely blocks the ability of glutamate applied to motor nerve terminals to activate PI3K, completely suppresses the increase in basal p-Akt levels conferred by CaMKIIT287D, and blocks the ability of CaMKIIT287D to confer two additional PI3K-dependent phenotypes: increased synapse number at the NMJ and increased motor axon diameter. These results identify DFak as an essential intermediate in PI3K activation by DmGluRA and CaMKII. However, DFakCG1 mutants fail to exhibit other phenotypes conferred by decreased PI3K activity: in an otherwise wild-type background, DFakCG1 larvae exhibit only minor effects on NMJ synapse number or motor axon diameters, which are each significantly decreased by decreased PI3K. These results raise the possibility that, whereas PI3K activation by DmGluRA and CaMKII is blocked in DFakCG1, total PI3K activity is not strongly decreased because other significant routes to PI3K activation are DFak independent. Alternatively, DFak might participate in signaling pathways distinct from the CaMKII-DFak-PI3K pathway identified in this study that would oppose the effects of PI3K on synapse number and axon diameter. In this view, CaMKII would preferentially promote the ability of DFak to activate PI3K, rather than other DFak-dependent pathways (Lin, 2011).
In several subregions of the mammalian brain, ligand activation of group I mGluRs induces LTD, a type of synaptic plasticity. This induction both activates and requires the activity of PI3K as well as the PI3K-activated kinase Tor (Hou, 2004; Ronesi, 2008). Several lines of evidence have led to the proposal that increased sensitivity to mGluR-mediated LTD induction might underlie specific neurogenetic disorders. In particular, mice null for the gene affected in fragile X, which is associated with an extremely high incidence of autism as well as other cognitive deficits, exhibit increased sensitivity to mGluR-mediated LTD induction in both the hippocampus. Furthermore, the genes identified in two additional diseases associated with a high incidence of autism, neurofibromatosis (Nf1) and tuberous sclerosis (Tsc1 and Tsc2), each encode negative regulators of the PI3K pathway: Nf1 encodes a Ras GTPase activator, which inhibits the PI3K activator Ras, whereas the Tsc proteins are Tor inhibitors that are in turn inhibited by PI3K activity. Thus loss of Nf1 or Tsc might also increase sensitivity to mGluR-mediated LTD. Finally, several lines of direct evidence suggest that PI3K hyperactivation plays a causal role in autism. For example, DNA copy number variants observed in individuals with autism but not unaffected individuals identify at high frequency PI3K subunits or regulators, and each genetic change is predicted to elevate PI3K activity. In addition, a translocation that increases expression of the translation factor eIF-4E, which is known to be activated by the PI3K pathway, plays a direct, causal role in autism. The potential involvement of mGluR-mediated LTD in these neurogenetic disorders increases interest in identifying the molecular intermediates that participate in this pathway, but these intermediates are for the most part unidentified. Thus, the possibility that CaMKII and Fak might participate in mGluR-mediated PI3K activation in mammals as well as Drosophila might have significant medical interest (Lin, 2011).
Loss of the RNA-binding fragile X protein [fragile X mental retardation protein (FMRP)] results in a spectrum of cognitive deficits, the fragile X syndrome (FXS), while aging individuals with decreased protein levels present with a subset of these symptoms and tremor. The broad range of behavioral deficits likely reflects the ubiquitous distribution and multiple functions of the protein. FMRP loss is expected to affect multiple neuronal proteins and intracellular signaling pathways, whose identity and interactions are essential in understanding and ameliorating FXS symptoms. Heterozygous mutants and targeted RNA interference-mediated abrogation were used in Drosophila to uncover molecular pathways affected by FMRP reduction. Evidence that FMRP loss results in excess metabotropic glutamate receptor (mGluR) activity, attributable at least in part to elevation of the protein in affected neurons. Using high-resolution behavioral, genetic, and biochemical analyses, evidence is presented that excess mGluR upon FMRP attenuation is linked to the cAMP decrement reported in patients and models, and underlies olfactory associative learning and memory deficits. Furthermore, the data indicate positive transcriptional regulation of the fly fmr1 gene by cAMP, via protein kinase A, likely through the transcription factor CREB. Because the human Fmr1 gene also contains CREB binding sites, the interaction of mGluR excess and cAMP signaling defects presented in this study suggests novel combinatorial pharmaceutical approaches to symptom amelioration upon FMRP attenuation (Kanellopoulos, 2012).
This study has demonstrated robust learning and LTM deficits associated with 50% reduction in dFMRP and mapped the phenotype to adult MB α/β lobes with spatiotemporally controlled RNAi-mediated abrogation. The learning deficit of dfmr13-null allele heterozygotes was quantitatively similar to that of animals lacking significantly more of the protein due to RNAi-mediated dFMRP abrogation either pan-neuronally or specifically in the MBs. This is surprising given the efficient attenuation of dFMRP with UAS-dfmr-R. Interestingly, a similar learning deficit was independently reported for null homozygotes as well (Kanellopoulos, 2012).
The lack of enhanced learning and memory deficits upon further reduction than 50% in the heterozygotes indicates that the activity of the remaining dFMRP in these animals may be reduced to levels functionally approaching those in null homozygotes. This suggests that an essential posttranslational modification may be suppressed in the heterozygotes, effectively further reducing functional dFMRP. Ser499 phosphorylation by S6K1 is essential for the translational repressor function of vertebrate FMRP and phosphate removal or suppression inactivates it. Moreover, phosphorylation by casein kinase II at dFMRP Ser406 has been reported functionally important. Hence, altered dFMRP phosphorylation in dfmr13/+ is a plausible explanation for the phenotypes in the heterozygotes, and this hypothesis is currently under investigation. Because near-complete abrogation of the protein does not completely eliminate olfactory learning, dFMRP does not appear to play an essential role in all molecular processes engaged within the MBs for associative olfactory learning (Kanellopoulos, 2012).
Processes requiring normal dFMRP function appear to be involved in learning rate, because dfmr13 heterozygotes and animals with attenuated protein in the MBs reach asymptotic performance levels like controls, but require additional US/CS pairings. The aberrantly elevated responses after training with two and four pairings may be akin to the heightened arousal reported to result in increased activity and exaggerated responses during the initial sessions of a learning task in mice lacking the Fmr1 gene. In addition, synaptic hyperexcitability, especially upon high-frequency stimulation, at the neuromuscular junction was reported for dfmr1-null larvae. These phenomena may be related to the elevated emotional reactivity and anxiety often associated with FXS patients. Excess DmGluRA activity appears to be involved in this exaggerated response, because it was eliminated after feeding mutant flies with MPEP. The known anxiolytic and antidepressant properties of Rolipram and other PDE inhibitors support the interpretation that the aberrantly elevated responses after few US/CS pairings may reflect anxiety in flies as in mice. The mechanism of this performance enhancement is unclear at the moment but will be investigated in detail in the future (Kanellopoulos, 2012).
Initially, tests were performed to see whether the mGluR elevation proposed to underlie many FXS behavioral deficits and validated in Drosophila with mutant homozygotes was also applicable when dFMRP was reduced, but not eliminated. The results indicate that, in mutant heterozygotes, it is not solely the activity of the mGluRA receptor that is increased but also the levels of the protein itself. Because no evidence was uncover suggesting increased DmGluRA transcripts, this evidence suggests that it is translation of the receptor likely regulated by dFMRP. In the Drosophila larval NMJ, dFMRP has been reported to regulate the abundance of ionotropic glutamate receptor subclasses, and similarly it may regulate the levels of DmGluRA in the adult CNS. Moreover, a recent report indicated that several mGluRs are targets of FMRP-dependent translational regulation (Darnell, 2011). These results are consistent with the RNAi-mediated attenuation of the receptor, which reversed both learning and memory deficits in animals with abrogated dFMRP. Therefore, dFMRP function appears dosage sensitive since 50% reduction suffices to elevate DmGluRA (Kanellopoulos, 2012).
DmGluRA is present in the MB dendrites. Therefore, dFMRP abrogation in these neurons is expected to result in elevation of the receptor within them. The fact that feeding MPEP reverses the learning deficits of animals with abrogated dFMRP specifically in the MBs strongly suggests that the pharmaceutical reaches these neurons and acts on the locally elevated DmGluRA (Kanellopoulos, 2012).
Interestingly, inhibiting DmGluRA with MPEP or abrogating the receptor in the adult fly CNS rescued the low cAMP levels in dfmr13/+. This indicates that cAMP levels are directly influenced by the level of DmGluRA. Furthermore, cAMP levels appear to lie downstream of the receptor, because elevation of the nucleotide by reducing the dosage of the PDE Dnc, or administration of Rolipram resulted in complete reversal of the learning and LTM phenotypes of dfmr13/+. Consistent with this observation, the mGluR antagonists LY341495, MPPG, and MTPG, previously used to rescue the courtship learning defect of dfmr13 homozygotes, are known to also increase cAMP signaling in Drosophila. Hence, the low cAMP levels reported for fly, mouse, and humans with compromised FMRP function are likely a consequence of enhanced levels of a Gi/o-linked glutamate receptor, which is thought to be the DmGluRA in Drosophila. These data strongly suggest this as the mechanism linking mGluR overactivity and/or levels and the proposed FMRP regulation of cAMP levels. Consistently, use of group II mGluR antagonists rescued the LTD phenotype in a mouse FXS model. Therefore, at least in the Drosophila model and with respect to associative learning and memory, the mGluR theories of accounting for the behavioral deficits of FXS seem to converge and describe different points of the same molecular interaction network (Kanellopoulos, 2012).
The behavioral effects of reducing the dosage of Dnc by 50% are also noteworthy. First, dnc1 heterozygosity precipitates small but significant effects on learning, and much larger effects on LTM, suggesting exaggerated effects of elevated cAMP on consolidated memory. The significance of reestablishing cAMP balance within the MBs is likely reflected in the surprising complete rescue of the dfmr13/+ LTM defect by reducing the dosage of the PDE, thus elevating cAMP and dFMRP levels (Kanellopoulos, 2012).
Interestingly, pharmacological or genetic manipulations that raised cAMP levels in dfmr13/+ animals also resulted in increased levels of dFMRP. This response appears to be mediated entirely by a cAMP-dependent increase in dfmr1 transcription, is apparently transduced via PKA signalin, and likely engages the transcription factor CREB. A correlation between cAMP and FMRP levels had been noted previously with respect to regional variation in brain areas suggestive of interactions in a developmental context. In contrast, the current results demonstrate an acute transcriptional response, as elevated cAMP and dfmr1 transcripts are apparent within a few hours of pharmaceutical administration. Therefore, dFMRP levels respond acutely to cAMP in the fly CNS and appear to reflect the abundance or activity of DmGluRA as a negative-feedback loop. In support of this notion, dFMRP levels were reduced in homozygous mutants for the Rut adenylyl cyclase, a situation where DmGluRA levels are presumed normal. Furthermore, given the function of dFMRP as a translational repressor, it is of interest to consider its elevation in dnc heterozygous and further increase in homozygous mutants. Is then the etiology of the learning and memory deficits in dnc mutants cAMP elevation, or exaggerated translational repression within their MBs because of enhanced dFMRP levels therein (Kanellopoulos, 2012)?
Collectively, these results suggest that pharmaceuticals that modulate cAMP signaling are promising routes to effectively ameliorate behavioral symptoms of 'premutation' carriers. Amelioration due to transcriptional upregulation of FMRP in response to cAMP elevation is not possible in patients and models harboring deletions or functional silencing of the gene. However, point mutations that do not affect transcription of the gene, but rather particular functional domains were identified recently. Because these appear associated with some but not all behavioral deficits, they likely do not represent null but rather hypomorphic alleles. Perhaps Rolipram and MPEP-mediated increases in cAMP levels and FMRP transcription may be beneficial to such patients as well as those presenting FXTAS symptoms. Like in Drosophila, the human gene also appears to contain CREB sites in its putative promoter area, indicating that this approach is at least feasible (Kanellopoulos, 2012).
The lipid kinase PI3K plays key roles in cellular responses to activation of receptor tyrosine kinases or G protein coupled receptors such as the metabotropic glutamate receptor (mGluR). Activation of the PI3K catalytic subunit p110 occurs when the PI3K regulatory subunit p85 binds to phosphotyrosine residues present in upstream activating proteins. In addition, Ras is uniquely capable of activating PI3K in a p85-independent manner by binding to p110 at amino acids distinct from those recognized by p85. Because Ras, like p85, is activated by phosphotyrosines in upstream activators, it can be difficult to determine if particular PI3K-dependent processes require p85 or Ras. This study asked if PI3K requires Ras activity for either of two different PI3K-regulated processes within Drosophila larval motor neurons. To address this question, the effects on each process were determined of transgenes and chromosomal mutations that decrease Ras activity, or mutations that eliminate the ability of PI3K to respond to activated Ras. It was found that PI3K requires Ras activity to decrease motor neuron excitability, an effect mediated by ligand activation of the single Drosophila mGluR DmGluRA. In contrast, the ability of PI3K to increase nerve terminal growth is Ras-independent. These results suggest that distinct regulatory mechanisms underlie the effects of PI3K on distinct phenotypic outputs (Johnson, 2012).
Synchronized neuronal activity is vital for complex processes like behavior. Circadian pacemaker neurons offer an unusual opportunity to study synchrony as their molecular clocks oscillate in phase over an extended timeframe (24 h). To identify where, when, and how synchronizing signals are perceived, the minimal clock neural circuit in Drosophila larvae were studied, manipulating either the four master pacemaker neurons (LNvs) or two dorsal clock neurons (DN1s). Unexpectedly, it was found that the PDF Receptor (PdfR) is required in both LNvs and DN1s to maintain synchronized LNv clocks. It was also found that glutamate is a second synchronizing signal that is released from DN1s and perceived in LNvs via the metabotropic glutamate receptor (mGluRA). Because simultaneously reducing Pdfr and mGluRA expression in LNvs severely dampened Timeless clock protein oscillations, it is concluded that the master pacemaker LNvs require extracellular signals to function normally. These two synchronizing signals are released at opposite times of day and drive cAMP oscillations in LNvs. Finally it was found that PdfR and mGluRA also help synchronize Timeless oscillations in adult s-LNvs. It is proposed that differentially timed signals that drive cAMP oscillations and synchronize pacemaker neurons in circadian neural circuits will be conserved across species (Collins, 2014: PubMed).
Loss of the mRNA-binding protein FMRP results in the most common inherited form of both mental retardation and autism spectrum disorders: fragile X syndrome (FXS). The leading FXS hypothesis proposes that metabotropic glutamate receptor (mGluR) signaling at the synapse controls FMRP function in the regulation of local protein translation to modulate synaptic transmission strength. In this study, the Drosophila FXS disease model was used to test the relationship between Drosophila FMRP (dFMRP) and the sole Drosophila mGluR (dmGluRA) in regulation of synaptic function, using two-electrode voltage-clamp recording at the glutamatergic neuromuscular junction (NMJ). Null dmGluRA mutants show minimal changes in basal synapse properties but pronounced defects during sustained high-frequency stimulation(HFS). The double null dfmr1;dmGluRA mutant shows repression of enhanced augmentation and delayed onset of premature long-term facilitation (LTF) and strongly reduces grossly elevated post-tetanic potentiation (PTP) phenotypes present in dmGluRA-null animals. Null dfmr1 mutants show features of synaptic hyperexcitability, including multiple transmission events in response to a single stimulus and cyclic modulation of transmission amplitude during prolonged HFS. The double null dfmr1;dmGluRA mutant shows amelioration of these defects but does not fully restore wildtype properties in dfmr1-null animals. These data suggest that dmGluRA functions in a negative feedback loop in which excess glutamate released during high-frequency transmission binds the glutamate receptor to dampen synaptic excitability, and dFMRP functions to suppress the translation of proteins regulating this synaptic excitability. Removal of the translational regulator partially compensates for loss of the receptor and, similarly, loss of the receptor weakly compensates for loss of the translational regulator (Repicky, 2008).
Drosophila is an excellent, simplified genetic system for comprehensively testing interactions between mGluR signaling and FMRP function in the nervous system. There is a single Drosophila homolog of the three member mammalian FMR gene family (dFMRP) and a single Drosophila homolog of the eight member mammalian mGluR family (dmGluRA). Antagonists of different mammalian mGluR classes have been shown to rescue several dfmr1 null phenotypes (McBride, 2005; Pan, 2008), suggesting that dmGluRA signaling does indeed have a mechanistic connection with dFMRP function. More importantly, the double mutant combination of the two Drosophila null alleles provides an excellent opportunity to test genetic relationships between all FMR family function and all mGluR signaling, particularly in the regulation of synapse development, function, and plasticity. Indeed, it has been shown, using double mutants, that dFMRP and dmGluRA interact in the regulation of ionotropic glutamate receptor trafficking at the NMJ synapse (Pan, 2007), as well as in the modulation of movement behavior and the control of NMJ gross architecture and synaptic ultrastructure (Pan, 2008) (Repicky, 2008).
The goal of this study was to examine the key question of the role of dFMRP in synaptic transmission properties and to determine whether a genetic block in dmGluRA signaling would modulate functional defects caused by loss of dFMRP. All work was done in low external Ca2+ concentrations, as required to permit amplitude facilitation driven by high-frequency stimuli. Previous research has shown that dmGluRA is not required to maintain basal neurotransmission, but is critical for the regulation of activity-dependent synaptic plasticity processes, particularly in establishing the threshold for LTF and limiting the expression of PTP (Bogdanik, 2004). Null dmGluRA phenotypes resemble the consequences of applying K+ channel blockers to wildtype synapses, as well as mutants that either increase Na+ currents (pumilio) or decrease K+ currents (hyperkinetic, frequenin). Interestingly, the pumilio gene encodes an RNA-binding translational suppressor, like dFMRP, whose activity down-regulates the paralytic RNA encoding voltage-gated Na+ channels. The hyperkinetic gene encodes a K+ channel β subunit, and frequenin regulates the function of K+ channels. Thus loss of dmGluRA generates synaptic phenotypes caused by increased neuronal membrane excitability. These comparisons suggest that the dmGluRA receptor monitors glutamate release, particularly during periods of high activity, to feedback and down-regulate the membrane excitability controlling Ca2+ influx and glutamate release, thus dictating neurotransmission strength. There are clear indications that dmGluRA interacts with dFMRP in synapse regulation (Pan, 2007; Pan, 2008), but dFMRP has not previously been shown to control synaptic excitability. This study aimed at discovering whether or not dFMRP and dmGluRA interact in the regulation of activity-dependent synaptic modulation, particularly through mechanisms of altered synaptic excitability (Repicky, 2008).
No striking difference was found in basal transmission properties at low external Ca2+ concentrations in either dfmr1 or dmGluRA single mutants or the dfmr1;dmGluRA double mutant. There is a clear tendency of increased basal transmission strength, particularly in dfmr1, as well as shifts in the power relationship of Ca2+-dependent synaptic vesicle release. Together these changes explain the elevated dfmr1 synaptic strength and more rapid presynaptic vesicle cycle at higher Ca2+ concentrations. Nevertheless, under the low [Ca2+] conditions used in this study, it can be infered that altered neurotransmission in response to HFS must be caused by activity-dependent changes in transmission probability in these genotypes. During short HFS trains, the single null mutants behave similar to control, but there is strongly elevated STF in the dfmr1;dmGluRA double null mutant. Similarly, previous work on synaptic structure also has shown that some phenotypes interact in a synergistic fashion (Pan, 2008). The STF defect shows a clear interaction between dmGluRA-dependent glutamatergic signaling and the requirement for dFMRP function at the synapse, although it does not necessarily demonstrate that the two proteins work in the same pathway(s) in the manifestation of short-term changes in synapse function. It is possible that mutation of the two genes concurrently shows an overlapping function that is not evident otherwise (Repicky, 2008).
The interaction between dmGluRA and dFMRP in long-term, activity-dependent changes in neurotransmission strength is more extensive and informative. During prolonged HFS, dmGluRA null mutants display a profound increase in augmentation, suggesting loss of a glutamate negative feedback loop to reign in glutamate release. Interestingly, removal of dFMRP in the dfmr1;dmGluRA double mutant modifies this defect during the early stages of HFS, extending the time it takes to elevate response amplitudes to the fully augmented level. However, as HFS continues, the full augmentation defect is expressed in the dfmr1;dmGluRA double mutant, indicating that other, dFMRP-independent pathways play a large role in the dmGluRA augmentation phenotype. Following prolonged HFS, wild-type synapses exhibit low-level, persistent PTP, but dmGluRA mutants show grossly elevated PTP (>5-fold normalized amplitude). Importantly, removal of dFMRP in dfmr1;dmGluRA double mutants produces nearly complete loss of this potentiation defect. This restoration toward wild-type seems to indicate converging pathways for dFMRP and dmGluRA in the regulation of activity-dependent synaptic potentiation. The third plasticity defect apparent in dmGluRA nulls is the premature, sudden step-wise appearance of long-term facilitation (LTF). The dfmr1;dmGluRA animals retain this lowered LTF threshold phenotype, but aspects of the defect are reduced by co-removal of dFMRP. First, the time of LTF onset is longer in double mutants compared with dmGluRA (11.8 vs. 9.7 s). Second, the dmGluRA null always shows an abrupt, single step increase in response amplitude between two consecutive stimulations in the HFS train, whereas dfmr1;dmGluRA characteristically oscillates between augmented and LTF amplitudes multiple times before finally succumbing to LTF. These data suggest that removal of dFMRP alleviates the consequences of lost mGluR signaling, albeit insufficiently to block presentation of enhanced transmission phenotypes (Repicky, 2008).
Taken together, the above results show clearly that removal of dFMRP can modulate defects caused by loss of dmGluRA. Removal of dFMRP by itself fails to present any defects in assayed forms of activity-dependent plasticity. However, two new phenotypes were identified in the dfmr1 null synapse. First, during prolonged HFS, dfmr1 mutants fail to maintain consistent transmission amplitudes, but rather manifest striking and characteristic cycling of amplitudes between a low and high transmission state. This cycling presents with sudden, drastic changes in amplitude size in bursts of quite regular periodicity. This is a novel phenotype without clear comparisons in the literature. Second, dfmr1 mutants display multiple excitatory junction current (EJC) events in response to single nerve stimuli during and after HFS. Similar hyperactivity is characteristic of Shaker K+ channel mutants, and double mutation combinations with ether-a-go-go, acting synergistically to increase membrane excitability. Interestingly, synaptic hyperexcitability in Shaker, and also bang-senseless mutants, is rescued with loss of no action potential (nap), to reduce Na+ channels. The similar phenotypes of these mutants compared with dfmr1 suggests dFMRP regulates synaptic excitability, perhaps via regulating membrane excitability, providing a clear mechanistic relationship with mGluR-mediated negative feedback control (Repicky, 2008).
Consistent with this feedback loop, co-removal of mGluR signaling appreciably diminishes these dfmr1 defects. The dfmr1;dmGluRA double null still displays the EJC response amplitude cycling, and requires a similar duration of HFS prior to the onset of cycling. However, the cycling defect is present in far fewer dfmr1;dmGluRA animals compared with the dfmr1 single mutant, and the limited cycling manifest in the double mutants has a slower cycling period, showing partial alleviation of the phenotype in the double mutant. The threshold for manifestation of this intriguing synaptic modulation is clearly lowered by removal of dFMRP but raised again by co-removal of mGluR signaling, albeit not to wild-type levels. In dfmr1 mutants, multiple separate and distinct EJCs occur in response to a single stimulus during HFS. This hyperexcitability defect is effectively lowered in dfmr1;dmGluRA double mutants. Indeed, the hyperactive response was recorded only in two isolated incidents in two separate dfmr1;dmGluRA animals. In dfmr1 mutants, the hyperexcitable responsiveness persists following the HFS train during basal stimulation, but post-HFS hyperexcitability was never observed in dfmr1;dmGluRA animals. Thus co-removal of dmGluRA does indeed diminish the consequences of loss of dFMRP, only partially in the case of the cyclic transmission defect, but quite strongly to block dfmr1 hyperexcitability. Together, these data support the conclusion of a partial co-dependency of dmGluRA receptor signaling on dFMRP regulative function, and vice versa in a feedback loop, to modulate synapse properties critical for the maintenance of transmission fidelity and activity-dependent plasticity (Repicky, 2008).
Both rescue and synergistic interactions of dfmr1 and dmGluRA null mutations in a range of synaptic mechanisms has been shown (Pan, 2007; Pan, 2008). However, a major, persistent limitation has been the lack of any functional data on synaptic transmission, a primary focus of FXS dysfunction. This crucial question has similarly not as yet been addressed in the mouse fmr1 KO model, despite evidence of rescue in other fmr1 defects. This study has shown that activity-dependent synaptic plasticity defects in dmGluRA nulls, including elevated augmentation, potentiation, and premature LTF, are each reduced by the co-removal of dFMRP. Similarly, the synaptic defects in dfmr1 nulls, including transmission amplitude cycling during HFS and multiple EJCs in response to a single stimulus, are decreased by the co-removal of dmGluRA, and hence loss of all mGluR signaling at the synapse. The striking exception to this trend is STF, which is somehow enhanced in the dfmr1;dmGluRA double null compared with both single mutants. These interactions clearly support the conclusion of a relationship between dFMRP function and dmGluRA signaling, but argue against a simple direct signaling cascade. Rather, dFMRP function is likely controlled by several converging signaling pathways, of which dmGluRA-mediated glutamatergic synaptic signaling is only one (Repicky, 2008).
Fragile X syndrome is caused by loss of the FMRP translational regulator. A current hypothesis proposes that FMRP functions downstream of mGluR signaling to regulate synaptic connections. Using the Drosophila disease model, relationships between dFMRP and the sole Drosophila mGluR (DmGluRA) were tested by assaying protein expression, behavior and neuron structure in brain and NMJ; in single mutants, double mutants and with an mGluR antagonist. At the protein level, dFMRP is upregulated in dmGluRA mutants, and DmGluRA is upregulated in dfmr1 mutants, demonstrating mutual negative feedback. Null dmGluRA mutants display defects in coordinated movement behavior, which are rescued by removing dFMRP expression. Null dfmr1 mutants display increased NMJ presynaptic structural complexity and elevated presynaptic vesicle pools, which are rescued by blocking mGluR signaling. Null dfmr1 brain neurons similarly display increased presynaptic architectural complexity, which is rescued by blocking mGluR signaling. These data show that DmGluRA and dFMRP convergently regulate presynaptic properties (Pan, 2008).
A current hypothesis proposes that fragile X mental retardation protein (FMRP), an RNA-binding translational regulator, acts downstream of glutamatergic transmission, via metabotropic glutamate receptor (mGluR) Gq-dependent signaling, to modulate protein synthesis critical for trafficking ionotropic glutamate receptors (iGluRs) at synapses. However, direct evidence linking FMRP and mGluR function with iGluR synaptic expression is limited. This study used the Drosophila fragile X model to test this hypothesis at the well characterized glutamatergic neuromuscular junction (NMJ). Two iGluR classes reside at this synapse, each containing common GluRIIC (III), IID and IIE subunits, and variable GluRIIA (A-class) or GluRIIB (B-class) subunits. In Drosophila fragile X mental retardation 1 (dfmr1) null mutants, A-class GluRs accumulate and B-class GluRs are lost, whereas total GluR levels do not change, resulting in a striking change in GluR subclass ratio at individual synapses. The sole Drosophila mGluR, DmGluRA, is also expressed at the NMJ. In dmGluRA null mutants, both iGluR classes increase, resulting in an increase in total synaptic GluR content at individual synapses. Targeted postsynaptic dmGluRA overexpression causes the exact opposite GluR phenotype to the dfmr1 null, confirming postsynaptic GluR subtype-specific regulation. In dfmr1; dmGluRA double null mutants, there is an additive increase in A-class GluRs, and a similar additive impact on B-class GluRs, toward normal levels in the double mutants. These results show that both dFMRP and DmGluRA differentially regulate the abundance of different GluR subclasses in a convergent mechanism within individual postsynaptic domains (Pan, 2007).
The finding of elevated group I mGluR5-dependent hippocampal LTD in the fmr1 knock-out mouse has elicited a great deal of attention and excitement. This type of LTD is caused by loss of surface expression of AMPA GluRs, in a mechanism requiring protein synthesis. Because synaptic protein translation is regulated by FMR, these observations suggest a mechanistic connection between mGluR signaling and FMRP translation regulation in the control of GluR expression at the synapse and indicate that this pathway may be a critical regulator of functional synaptic plasticity. This idea has been formally expressed as the mGluR theory of FXS (Bear, 2004). This study directly investigates this hypothesized connection between mGluR signaling, FMRP regulatory function, and the synaptic expression of GluRs using the Drosophila FXS model. In Drosophila, there is a single FMR1 protein (dFMRP) and a single mGluR (DmGluRA). dFMRP structure, expression, and regulative functions closely resemble mammalian FMRP. In contrast, DmGluRA is more homologous to mammalian group II/III mGluRs, not the group I mGluRs implicated in the FMRP mechanism. However, these mGluR class distinctions may mean little in Drosophila, with its single mGluR. The mammalian group I mGluR antagonist MPEP rescues morphological and behavioral phenotypes in dfmr1 null mutants (McBride, 2005
Both dfmr1 and dmGluRA mutants have been shown to have strong defects in glutamatergic synaptic function at the Drosophila NMJ. Neurotransmission at this synapse is mediated by A- and B-class AMPA-type GluRs, which have distinctive functional properties and subsynaptic distributions and are regulated by distinct mechanisms. This study shows in dfmr1 null mutants that A-class GluRs accumulate and B-class GluRs are lost. The total GluR content does not change, but rather there is a striking shift in the GluR class ratio within single postsynaptic domains. This subclass-specific regulation of GluRs is a novel finding. In dmGluRA null mutants, it was shown that both GluR classes, and therefore the total GluR population, are significantly increased. This is a novel finding for DmGluRA but consistent with findings in mammals showing that GluR1 AMPA receptors are decreased in synaptic terminals when mGluR activity is induced. Moreover, this study showed that postsynaptic overexpression of DmGluRA induces exactly opposite changes of A- and B-class GluRs compared with dfmr1 null mutants. By testing active zone density and targeted presynaptic rescue of dFMRP in the dfmr1 null, we show that the regulatory function of dFMRP on the GluR classes is a postsynaptic mechanism. Finally, it was show in dfmr1; dmGluRA double null mutants that both GluR class phenotypes are additive; A-class GluRs increase further with the additive increases of dfmr1 and dmGluRA single mutants, and B-class GluRs tend toward normal levels, with the additive downregulation in the dfmr1 single mutant and upregulation in the dmGluRA single mutant. These results suggest that DmGluRA signaling and dFMRP function converge to regulate the synaptic expression of these two GluR classes but that independent pathways of DmGluRA signaling and dFMRP function also exist (Pan, 2007).
This study suggests that dFMRP and DmGluRA perform in both overlapping and independent pathways in the regulation of postsynaptic GluR classes. Targeted presynaptic expression of dFMRP in the dfmr1 null fails to provide any rescue of class-specific GluR misregulation, showing that the dFMRP requirement is in the postsynaptic compartment. Consistently, targeted postsynaptic overexpression of DmGluRA causes the opposite class-specific GluR misregulation of the dfmr1 null, suggesting an intersection of DmGluRA signaling and dFMRP function in the postsynaptic compartment. In the dfmr1 null, quantal size is increased, a hallmark postsynaptic defect. A mechanistic cause suggested by this study is the elevated A-class GluR level, consistent with former reports that GluRIIA overexpression increases quantal size. Moreover, GluRIIA overexpression increases active zone number per bouton, based on the NC82/bruchpilot probe, but does not alter active zone density, which is identical to the phenotype reported in this study for dfmr1 mutants. These results support the conclusion that both dFMRP and DmGluRA function in the postsynaptic domain in class-specific GluR regulation and that this mechanism may feedback to alter presynaptic properties (Pan, 2007).
In addition to the postsynaptic mechanism, there appears to also be presynaptic roles of both dFMRP and DmGluRA that can impact the postsynaptic GluR domains. It was shown previously that both proteins are expressed in the presynaptic neuron of the Drosophila NMJ. Single null mutants show differential misregulation, with the dfmr1 null displaying the class-specific change reflecting its postsynaptic function but the dmGluRA null increasing both GluR classes in common. This must reflect a presynaptic function for DmGluRA. Consistently, presynaptic overexpression of DmGluRA depresses the level of both A- and B-class GluRs, the opposite phenotype as the dmGluRA null. Likewise, presynaptic overexpression of dFMRP also reduces B-class GluR expression, although it does not change the abundance of A-class receptors. Presumably, these presynaptic roles reflect the know functions of dFMRP and DmGluRA in regulating presynaptic glutamate release properties, and therefore the GluR changes reflect transynaptic signaling in a homeostatic mechanism (Pan, 2007).
By strict genetic criteria, the prediction for the interaction of two proteins within a common regulatory pathway is that the mutant phenotype for the gene product downstream in the pathway should be epistatic to that of the gene product upstream in the pathway. Clearly, the FMRP translation regulatory activity should be downstream of mGluR surface glutamate reception. Such a strict epistatic relationship is not observed for DmGluRA and dFMRP in the control of GluR expression. Rather, the null mutant phenotypes are obviously additive in double mutants. The A-class GluR goes up in both single mutants and goes up further in the double mutant. The B-class GluR goes down in dfmr1 and up in dmGluRA and shows an intermediate, additive level in the double mutant. Such additive phenotypes show that dFMRP and DmGluRA have overlapping functions but can be operating in the independent pathways. Together, these results suggest that dFMRP and DmGluRA pathways converge on the regulation of GluR synaptic expression and that this involves both presynaptic and postsynaptic interactions (Pan, 2007).
Regulating GluR class composition in the postsynaptic domain is an important mechanism controlling neurotransmission strength and synaptic plasticity properties. The subunit composition of mammalian NMDA and AMPA receptors are both known to be regulated in this manner. Similarly at the Drosophila NMJ, the independent regulation of GluR classes is critical, because each receptor class has distinct functional properties, e.g., the A-class specifically is negatively regulated by protein kinase A phosphorylation, is modulated by atypical protein kinase C, is important in retrograde signaling, and mediates larger, slower-decaying transmission events with a smaller single channel conductance. The molecular mechanisms for controlling each GluR therefore must be distinct and, indeed, distinct mechanisms have been identified. For example, the PDZ [postsynaptic density 95 (PSD-95)/Discs Large (DLG)/zona occludens-1]-domain scaffold DLG, a PSD-95 homolog, is involved in the localization of many synaptic proteins but plays a specific role in B-class GluR regulation: GluRIIB abundance correlates with DLG level, but GluRIIA localization is unaffected in dlg mutants. Similarly, the Rho-type guanine nucleotide exchange factor dPix (the Drosophila homolog of the Pak interacting exchange factor), its interacting Drosophila p-21 activated kinase (dPak), a serine threonine kinase activated by GTPases Rac and cell division cycle 42, and a dPak binding partner, the adaptor Dreadlocks (Dock; Nck homolog), are all required to facilitate synaptic expression of A-class GluRs, but GluRIIB is reportedly not affected in mutants of this pathway. Trafficking mechanisms likely involve GluR tethering to the cytoskeleton. The actin-interacting Coracle (mammalian brain 4.1 protein) binds only GluRIIA to specifically regulate its abundance, with no role in B-class GluR tethering. Thus, separable mechanisms for A- and B-class GluR regulation clearly exist (Pan, 2007).
FMRP/dFMRP is an RNA-binding protein and a regulator of protein translation at the synapse. Although FMRP/dFMRP is best defined as a negative regulator of translation, it may also positively regulate the translation of a distinct set of synaptic mRNAs. Presumably, these translation regulation mechanisms underlie the differential, and opposing, regulation of A- and B-class GluRs by dFMRP. Some newly synthesized proteins may promote synaptic expression of A-class GluRs, whereas others promote the diminution of B-classes GluRs. One protein whose synaptic translation is regulated by both mGluR signaling and FMRP function is PSD-95, implicated in both NMDA and AMPA GluR synaptic expression. Thus, altered regulation of the Drosophila PSD-95 homolog DLG is an attractive candidate mechanism for GluR phenotypes discovered in this study. In addition, the synaptic cytoskeleton affects synaptic expression of both NMDA and AMPA GluRs. Notably, a key function of FMRP/dFMRP is regulating microtubule and actin filament dynamics via regulating expression of key cytoskeleton-binding proteins, such as Futsch/microtubule-associated protein 1b. The future goal of these studies will be to test these candidate downstream factors as regulators of GluR synaptic expression in Drosophila, downstream of both DmGluRA signaling and dFMRP translational control (Pan, 2007).
Fragile X syndrome (FXS), caused by loss of FMR1 gene function, is the most common heritable cause of intellectual disability and autism spectrum disorders. The FMR1 protein (FMRP) translational regulator mediates activity-dependent control of synapses. In addition to the metabotropic glutamate receptor (mGluR) hyperexcitation FXS theory, the GABA theory postulates that hypoinhibition is causative for disease state symptoms. This study uses the Drosophila FXS model to assay central brain GABAergic circuitry, especially within the Mushroom Body (MB) learning center. All 3 GABAA receptor (GABAAR) subunits are reportedly downregulated in dfmr1 null brains. Parallel downregulation of glutamic acid decarboxylase (GAD), the rate-limiting GABA synthesis enzyme, is demonstrated although GABAergic cell numbers appear unaffected. Mosaic analysis with a repressible cell marker (MARCM) single-cell clonal studies show that dfmr1 null GABAergic neurons innervating the MB calyx display altered architectural development, with early underdevelopment followed by later overelaboration. In addition, a new class of extra-calyx terminating GABAergic neurons is shown to include MB intrinsic α/β Kenyon Cells (KCs), revealing a novel level of MB inhibitory regulation. Functionally, dfmr1 null GABAergic neurons exhibit elevated calcium signaling and altered kinetics in response to acute depolarization. To test the role of these GABAergic changes, pharmacological restoration of GABAergic signaling is attempted and effects on the compromised MB-dependent olfactory learning in dfmr1 mutants are assayed, but no improvement is found. These results show that GABAergic circuit structure and function are impaired in the FXS disease state, but that correction of hypoinhibition alone is not sufficient to rescue a behavioral learning impairment (Gatto, 2014).
The lion's share of FXS neuronal studies have focused on glutamatergic hyperexcitation, but there is clear evidence that GABAergic hypoinhibition may also be important in both Drosophila and mouse disease models. This study probed GABAergic circuitry in the Drosophila brain, especially in relation to the MB olfactory learning and memory center, and assessed GABAergic modulation as an FXS intervention strategy. In dfmr1 null mutants, depressed GAD expression, altered GABAergic neuron architecture and developmental refinement, and altered GABAergic neuron calcium signaling function within the MB circuit were found, consistent with GABAergic dysfunction contributing to compromised MB-dependent learning in the Drosophila FXS model. However, pharmacological attempts to alleviate GABAergic signaling defects, modeled on previously reported successful intervention strategies feeding GABA and a GABA reuptake inhibitor, NipA, did not improve the behavioral output in dfmr1 null mutants. Taken together, this study reveals GABAergic impairments in the FXS disease state, but does not show that GABAergic hypoinhibition is a strong determinant of the learning component of FXS cognitive compromise (Gatto, 2014).
Results from this study support the reported loss of gad mRNA in the Drosophila FXS model and reduced GAD protein in the amygdala basolateral nucleus in the mouse FXS model. Importantly, FMRP has been suggested to bind gad mRNA directly based on cross-linking immunoprecipitation (HITS-CLIP). However, given FMRP is best defined as a translational repressor (e.g. MAP1B/futsch, profilin/chickadee etc.), this direct interaction would predict GAD elevation, not the reported loss in the FXS disease state. Alternatively, FMRP may confer transcript stability, such that in its absence gad mRNA would be subject to degradation, causing subsequent loss of the GAD protein. During development, loss of GABA production may have consequences unrelated to its mature inhibitory function, as GABA can serve as an excitatory neurotransmitter in immature stages owing to the high intracellular chloride concentration maintained by Na+/K+/Cl− co-transporter NKCC1. In Drosophila, however, early GABA roles in synaptogenesis, circuit establishment, and transmission are unclear, as detectable levels of GABA are reported only late in development, since regulatory mechanisms reportedly suppress GAD activity during earlier maturation phases. Developmental roles for GABA will be an area of future investigation in the Drosophila FXS model (Gatto, 2014).
GAD-positive neurons have been reported to innervate the Drosophila MB calyx, with GAD-Gal4-driven synaptobrevin-GFP punctae adjacent to the inner rim of the actin-rich microglomerular rings, establishing GABAergic synaptic input on KCs and/or PNs. This GABAergic innervation appears grossly normal in dfmr1 mutants, with a normal array of cell bodies and projection pathways. dBrainbow subdivision of the complex GABAergic circuitry suggests that GABAergic lineages are also normal in dfmr1 nulls. Therefore, there is no evidence that FMRP is required for the generation, placement, or maintenance of GABAergic neurons in the Drosophila brain. However, MARCM clonal analyses employed for single-cell resolution to test cell-autonomous FMRP requirements show that dfmr1 null MB-innervating GABAergic neurons display early undergrowth in complex cells followed by later overgrowth in simple cells. Thus, the developmental trajectory of GABAergic innervation is dependent upon FMRP. The GABAergic neuron undergrowth opposes the overgrowth seen in excitatory KCs at the same period of MB development, consistent with theories of GABAergic circuit hypoinhibition in the presence of hyperexcitation in the FXS disease state. In MB-extrinsic GABAergic neurons, FMRP may enhance the expression of targets involved in synapse assembly/maintenance early in development and repress them later, while the converse occurs in MB-intrinsic excitatory KCs. The cell-type-specific translation roles for FMRP in these inhibitory vs. excitatory neurons will be an area of future investigation in the Drosophila FXS model (Gatto, 2014).
In addition to MB-extrinsic GABAergic neurons, it was discovered that a subset of MB-intrinsic KCs is also GABAergic, as confirmed by co-labeling for both GAD and GABA. These newly-defined GABAergic neurons provide a potentially self-regulating inhibitory component within the MB circuit proper, which has not previously been recognized. This late-born class of MB-intrinsic GABAergic neurons can be MARCM marked with HS induction applied as late as pupal day 4 (9d AEL), giving them a birthdate easily distinguished from the early-born MB-extrinsic GABAergic neurons. Importantly, late-born MB core neurons play a permissive role in long-term memory formation, able to both facilitate and limit memory consolidation. These core KCs transiently express glutamate during early MB development, but then transition to a different neurotransmitter output (Gatto, 2014).
Beyond their architecture, dfmr1 GABAergic neurons display altered calcium signaling dynamics in response to depolarization, showing elevated and prolonged responses. This signaling change may serve as a compensatory mechanism, as GAD activity can be upregulated by increasing intracellular calcium. In Drosophila embryos, negative regulation of GAD can be overridden by agents that elevate free calcium (i.e., thapsigargin and monensin). It is unclear whether a similar calcium-dependent modulation of GAD might occur in the brain, but this possibility suggests a prospective means to circumvent GAD depression in the FXS disease state. Elevated calcium signaling could also drive direct enhancement of GABA release. In the mouse FXS model striatal region, spontaneous miniature inhibitory synaptic event frequency is elevated, presumably from increased GABA release, even with a lower density of GABAergic synapses. In contrast, GABAergic tonic inhibition is decreased in pyramidal cells, although phasic currents remain unchanged, implicating GABA receptor not neurotransmitter insufficiencies. However, the mouse amygdala exhibits decreased frequency/amplitude of both tonic and phasic inhibitory currents, linked to decreased GABAergic synapse number and reduced GAD65/67 levels. These findings reveal brain-region-specific changes in the mouse FXS model (Gatto, 2014).
How might GABAergic circuit dysfunction be corrected in the FXS disease state? Earlier studies report that feeding with GABA or the GABA reuptake inhibitor NipA is remarkably effective in correcting a range of dfmr1 null mutant phenotypes, including glutamate toxicity, Futsch/MAP1B over-expression, MB growth, and courtship behavioral memory impairments. Subsequent studies in the mouse FXS model likewise indicate correction of mutant defects using GABAAR-targeted reagents. For example, the GABAAR agonist gaboxadol/THIP restores disinhibition-related principal neuron excitability deficits in the amygdala and significantly attenuates hyperactivity and reduced prepulse inhibition, although it fails to reverse deficits in cued fear or startle response. Moreover, GABAAR modulation via benzodiazepine diazepam or neuroactive steroid alphaxalone rescues audiogenic seizures in the mouse FXS model. Clinically, an open-label FXS trial employing riluzole, hypothesized to have an inhibitory effect on glutamate release, block excitotoxic effects of glutamate, and potentiate postsynaptic GABAAR function, shows behavioral improvement assessed via the ADHD Rating Scale-IV, with significant correction of the ERK activation FXS biomarker in all subjects. Together, these studies suggest that elevating GABAergic function should be an effective strategy for treating the FXS disease state (Gatto, 2014).
Nevertheless, GABAergic augmentation fails to provide any improvement in MB-dependent olfactory learning defects in the Drosophila FXS model, despite partial correction of MB structural defects. MB over-expression of the RDL GABAAR impairs olfactory learning, suggesting that elevated GABAergic signaling could be counterproductive; however, given the RDL reduction in dfmr1 mutants, the intervention was aimed only at restoration. Interestingly, RDL knockdown fails to enhance learning in cAMP signaling pathway mutants, such as rutabaga and NF1, suggesting that RDL works upstream of cAMP signaling driving learning. This connection is particularly relevant to FXS, as cAMP is reduced in patient platelets, human neural progenitor cells, and brains in both mouse and Drosophila disease models. Moreover, FMR1 overexpression in the HN2 mammalian cell line and dfmr1 overexpression in the null mutant both increase cAMP production. Finally, electrophysiological studies in Drosophila primary neuronal cultures demonstrate that application of an adenylate cyclase activator suppresses inhibitory GABAergic postsynaptic currents via tempering of GABAAR receptor sensitivity. Taken together, these findings suggest a cascade interaction between FMRP, GABAARs, and cAMP signaling during learning (Gatto, 2014).
CIDE-N domains mediate interactions between the DNase Dff40/CAD and its inhibitor Dff45/ICAD. This study reports that the CIDE-N protein DNA fragmentation factor-related protein 2 (Drep-2) is a novel synaptic protein important for learning and behavioral adaptation. Drep-2 was found at synapses throughout the Drosophila brain and was strongly enriched at mushroom body input synapses. It was required within Kenyon cells for normal olfactory short- and intermediate-term memory. Drep-2 colocalized with metabotropic glutamate receptors (mGluRs). Chronic pharmacological stimulation of mGluRs compensated for drep-2 learning deficits, and drep-2 and mGluR learning phenotypes behaved non-additively, suggesting that Drep 2 might be involved in effective mGluR signaling. In fact, Drosophila fragile X protein mutants, shown to benefit from attenuation of mGluR signaling, profited from the elimination of drep-2. Thus, Drep-2 is a novel regulatory synaptic factor, probably intersecting with metabotropic signaling and translational regulation (Andlauer, 2014: PubMed).
The mushroom bodies (MBs) are paired brain centers located in the insect protocerebrum involved in olfactory learning and memory and other associative functions. Processes from the Kenyon cells (KCs), their intrinsic neurons, form the bulk of the MB's calyx, pedunculus and lobes. In young adult Drosophila, the last-born KCs extend their processes in the α/β lobes as a thin core (α/β cores) that is embedded in the surrounding matrix of other mature KC processes. A high level of L-glutamate (Glu) immunoreactivity is present in the α/β cores (α/βc) of recently eclosed adult flies. In a Drosophila model of fragile X syndrome, the main cause of inherited mental retardation, treatment with metabotropic Glu receptor (mGluR) antagonists can rescue memory deficits and MB structural defects. To address the role of Glu signaling in the development and maturation of the MB, this study has compared the time course of Glu immunoreactivity with the expression of various glutamatergic markers at various times, that is, 1 hour, 1 day and 10 days after adult eclosion. It was observed that last-born α/βc KCs in young adult as well as developing KCs in late larva and at various pupal stages transiently express high level of Glu immunoreactivity in Drosophila. One day after eclosion, the Glu level was already markedly reduced in the α/βc neurons. Glial cell processes expressing glutamine synthetase and the Glu transporter dEAAT1 were found to surround the Glu-expressing KCs in very young adults, subsequently enwrapping the α/β lobes to become distributed equally over the entire MB neuropil. The vesicular Glu transporter DVGluT was detected by immunostaining in processes that project within the MB lobes and pedunculus, but this transporter is apparently never expressed by the KCs themselves. The NMDA receptor subunit dNR1 is widely expressed in the MB neuropil just after eclosion, but was not detected in the α/βc neurons. In contrast, evidence is provided that DmGluRA, the only Drosophila mGluR, is specifically expressed in Glu-accumulating cells of the MB α/βc immediately and for a short time after eclosion. It is concluded that the distribution and dynamics of glutamatergic markers indicate that newborn KCs transiently accumulate Glu at a high level in late pupal and young eclosed Drosophila, and may locally release this amino acid by a mechanism that would not involve DVGluT. At this stage, Glu can bind to intrinsic mGluRs abundant in the α/βc KCs, and to NMDA receptors in the rest of the MB neuropil, before being captured and metabolized in surrounding glial cells. This suggests that Glu acts as an autocrine or paracrine agent that contributes to the structural and functional maturation of the MB during the first hours of Drosophila adult life (Sinakevitch, 2010).
DmGluRA is the only G-protein-coupled mGluR in Drosophila. This receptor has been shown to localize in the presynaptic site at the neuromuscular junction (Bogdanik, 2002) and it is also expressed in the brain - for example, in clock neurons, where it regulates circadian locomotor behavior (Hamasaka, 2007). The GAL4 enhancer trap technique is a widely used method for analyzing tissue-specific gene expression patterns in Drosophila. To generate the insertion of a GAL4-containing P element in the regulatory region of the DmGluRA gene, localized on the fourth chromosome, a targeted transposition strategy was used. This technique induces the precise replacement of one P element for another. Starting from the line 39C42, in which an outmoded P element lacking any expression reporter is inserted 5.94 kb upstream of the translation initiation codon of DmGluRA, a new strain was generated in which this P element was replaced with a P(Gal4) enhancer trap element, here called DmGluRA-GAL4. This driver line was used to monitor the expression of the DmGluRA receptor in the MBs, with mCD8::GFP as a reporter gene. Strikingly, it was found that the last-born KCs located in the inner part of the α/βc neurons express GFP in their cell bodies, dendrites, and axons in the pedunculus and lobes. According to their position, these cells undoubtedly correspond to Glu-expressing immature α/βc neurons. Although expression of the GAL4 reporter may, in part, differ from the mGluR pattern, such a precise localization suggests that the new born KCs express the DmGluRA receptor as well (Sinakevitch, 2010).
Twenty-four hours after eclosion, GFP immunostaining is still bright in labeled α/βc cell bodies and axons and individual axonal branches are clearly visible at the end of the α and β lobes. All around the labeled axons in the lobes, one can notice GFP-positive dots, which may represent sections of smaller axonal branches derived from the main axons. Ten days later, GFP expression is dramatically reduced in the MB α/βc neurons. Remarkably, only two bundles out of initially four are still GFP-positive. GFP immunoreactivity associated with dendritic fields in the calyx and axons in the lobes is dramatically reduced. In addition, any bright axonal branches and processes have vanished, compared to the staining observed in newly eclosed flies. Such a dramatic change suggests that DmGluRA expression in α/βc neurons is transient and does not persist for long after eclosion. The immunoreactivity still detectable at 10 days might be the remnant of earlier expression and accumulation of the stable GFP protein when the DmGluRA promoter was still active (Sinakevitch, 2010).
Metabotropic glutamate receptors (mGluRs) are responsible for the effects of glutamate in slow synaptic transmission, and are implicated in the regulation of many processes in the CNS. Recently, the expression and purification of a mGluR from Drosophila melanogaster (DmGluRA), a homologue of mammalian group II mGluRs, has been reported. Ligand binding to reconstituted DmGluRA requires the presence of ergosterol in the liposomes [Eroglu, 2002). This study demonstrates that the receptor exists in different affinity states for glutamate, depending on the membrane composition. The receptor is in a high-affinity state when associated with sterol-rich lipid microdomains (rafts), and in a low-affinity state out of rafts. Enrichment of the membranes with cholesterol shifts the receptor into the high-affinity state, and induces its association with rafts. The receptor was crosslinked to photocholesterol. These data suggest that sterol-rich lipid rafts act as positive allosteric regulators of DmGluRA (Eroglu, 2004).
G-protein-coupled receptors (GPCRs) form one of the largest superfamilies of membrane proteins. Obtaining high yields of GPCRs remains one of the major factors limiting a detailed understanding of their structure and function. Photoreceptor cells (PRCs) contain extensive stacks of specialized membranes where high levels of rhodopsins are naturally present, which makes them ideal for the overexpression of GPCRs. Transgenic flies expressing a number of GPCRs in the PRCs have been generated. Drosophila melanogaster metabotropic glutamate receptor (DmGluRA) expressed by this novel strategy was purified to homogeneity, giving at least 3-fold higher yields than conventional baculovirus expression systems due to the higher membrane content of the PRCs. Pure DmGluRA was then reconstituted into liposomes of varying composition. Interestingly, glutamate binding was strictly dependent on the presence of ergosterol (Eroglu, 2004).
Mammalian metabotropic glutamate receptors (mGluRs) are classified into three groups based on their sequence similarity and ligand recognition selectivity. A Drosophila mGluR (DmGluAR) has been identified that is about equidistant, phylogenetically, from the three mGluR groups. However, both the G-protein coupling selectivity and the pharmacological profile of DmGluAR, as analysed with mutated G-proteins and a few compounds, look similar to those of mammalian group-II mGluRs. The present study carefully examined the pharmacological profile of DmGluAR, and compared it to those of the rat mGlu1a, mGlu2 and mGlu4a receptors, representative of group-I, II and III respectively. The pharmacological profile of DmGluAR was found to be similar to that of mGlu2R, and only very small differences could be identified at the level of their pharmacophore models. These data strongly suggest that the binding sites of these two receptors are similar. To further document this idea, a 3D model of the mGlu2 binding domain was constructed based on the low sequence similarity with periplasmic amino acid binding proteins, and was used to identify the residues that possibly constitute the ligand recognition pocket. Interestingly, this putative binding pocket was found to be very well conserved between DmGluAR and the mammalian group-II receptors. These data indicate that there has been a strong selective pressure during evolution to maintain the ligand recognition selectivity of mGluRs (Parmentier, 2000).
Neural circuits formed during postnatal development have to be maintained stably thereafter, but their mechanisms remain largely unknown. This study reports that the metabotropic glutamate receptor subtype 1 (mGluR1) is essential for the maintenance of mature synaptic connectivity in the dorsal lateral geniculate nucleus (dLGN). In mGluR1 knockout (mGluR1-KO) mice, strengthening and elimination at retinogeniculate synapses occurred normally until around postnatal day 20 (P20). However, during the subsequent visual-experience-dependent maintenance phase, weak retinogeniculate synapses were newly recruited. These changes were similar to those of wild-type (WT) mice that underwent visual deprivation or inactivation of mGluR1 in the dLGN from P21. Importantly, visual deprivation was ineffective in mGluR1-KO mice, and the changes induced by visual deprivation in WT mice were rescued by pharmacological activation of mGluR1 in the dLGN. These results demonstrate that mGluR1 is crucial for the visual-experience-dependent maintenance of mature synaptic connectivity in the dLGN (Narushima, 2016).
The stress associated with starvation is accompanied by compensatory behaviours that enhance foraging efficiency and increase the probability of encountering food. However, the molecular details of how hunger triggers changes in the activity of neural circuits to elicit these adaptive behavioural outcomes remains to be resolved. This study shows that AMP-activated protein kinase (AMPK; see Drosophila AMPKα) regulates neuronal activity to elicit appropriate behavioural outcomes in response to acute starvation, and this effect is mediated by the coordinated modulation of glutamatergic inputs. AMPK targets both the AMPA-type glutamate receptor GLR-1 (see Drosophila Glu-RIIA) and the metabotropic glutamate receptor MGL-1 (see Drosophila mGluR) in one of the primary circuits that governs behavioural response to food availability in C. elegans. Overall, this study suggests that AMPK acts as a molecular trigger in the specific starvation-sensitive neurons to modulate glutamatergic inputs and to elicit adaptive behavioural outputs in response to acute starvation (Ahmadi, 2016).
Search PubMed for articles about Drosophila mGluR
Ahmadi, M. and Roy, R. (2016). AMPK acts as a molecular trigger to coordinate glutamatergic signals and adaptive behaviours during acute starvation. Elife 5. PubMed ID: 27642785
Andlauer, T. F., Scholz-Kornehl, S., Tian, R., Kirchner, M., Babikir, H. A., Depner, H., Loll, B., Quentin, C., Gupta, V. K., Holt, M. G., Dipt, S., Cressy, M., Wahl, M. C., Fiala, A., Selbach, M., Schwarzel, M. and Sigrist, S. J. (2014). Drep-2 is a novel synaptic protein important for learning and memory. Elife 3 [Epub ahead of print]. PubMed ID: 25392983
Bear, M. F., Huber, K. M. and Warren, S. T. (2004). The mGluR theory of fragile X mental retardation. Trends Neurosci. 27: 370-377. PubMed ID: 15219735
Bogdanik, L., et al. (2004). The Drosophila metabotropic glutamate receptor DmGluRA regulates activity-dependent synaptic facilitation and fine synaptic morphology. J. Neurosci. 24(41): 9105-16. PubMed ID: 15483129
Collins, B., Kaplan, H. S., Cavey, M., Lelito, K. R., Bahle, A. H., Zhu, Z., Macara, A. M., Roman, G., Shafer, O. T. and Blau, J. (2014). Differentially timed extracellular signals synchronize pacemaker neuron clocks. PLoS Biol 12: e1001959. PubMed ID: 25268747
Darnell, J. C. (2011). Defects in translational regulation contributing to human cognitive and behavioral disease. Curr. Opin. Genet. Dev. 21(4): 465-73. PubMed ID: 21764293
Eroglu, C., Cronet, P., Panneels, V., Beaufils, P. and Sinning, I. (2002). Functional reconstitution of purified metabotropic glutamate receptor expressed in the fly eye. EMBO Rep. 3(5): 491-6. PubMed ID: 11964379
Eroglu, C., Brugger, B., Wieland, F. and Sinning, I. (2004). Glutamate-binding affinity of Drosophila metabotropic glutamate receptor is modulated by association with lipid rafts. Proc. Natl. Acad. Sci. 100(18): 10219-24. PubMed ID: 12923296
Gatto, C.L., Pereira, D. and Broadie, K. (2014). GABAergic circuit dysfunction in the Drosophila Fragile X syndrome model. Neurobiol Dis 65: 142-159. PubMed ID: 24423648
Hou, L. and Klann, E. (2004). Activation of the phosphoinositide 3-kinase-Akt-mammalian target of rapamycin signaling pathway is required for metabotropic glutamate receptor-dependent long-term depression. J. Neurosci. 24: 6352-6361. PubMed ID: 15254091
Howlett, E., Lin, C. C., Lavery, W. and Stern, M. (2008). A PI3-kinase-mediated negative feedback regulates neuronal excitability. PLoS Genet. 4: e1000277. PubMed ID: 19043547
Johnson, C., Chun-Jen Lin, C. and Stern, M. (2012). Ras-dependent and Ras-independent effects of PI3K in Drosophila motor neurons. Genes Brain Behav. 11(7): 848-58. PubMed ID: 22783951
Kanellopoulos, A. K, et al. (2012). Learning and memory deficits consequent to reduction of the fragile X mental retardation protein result from metabotropic glutamate receptor-mediated inhibition of cAMP signaling in Drosophila. J. Neurosci. 32(38): 13111-24. PubMed ID: 22993428
Lin, C. C.-J., Summerville, J. B., Howlett, E. and Stern, M. (2011). The metabotropic glutamate receptor activates the lipid kinase PI3K in Drosophila motor neurons through the calcium/calmodulin-dependent protein kinase II and the nonreceptor tyrosine protein kinase DFak. Genetics 188(3): 601-13. PubMed ID: 21515581
McBride, S. M., et al. (2005). Pharmacological rescue of synaptic plasticity, courtship behavior, and mushroom body defects in a Drosophila model of fragile X syndrome. Neuron 45(5): 753-64. PubMed ID: 15748850
Narushima, M., Uchigashima, M., Yagasaki, Y., Harada, T., Nagumo, Y., Uesaka, N., Hashimoto, K., Aiba, A., Watanabe, M., Miyata, M. and Kano, M. (2016). The metabotropic glutamate receptor subtype 1 mediates experience-dependent maintenance of mature synaptic connectivity in the visual thalamus. Neuron 91(5):1097-109. PubMed ID: 27545713
Pan, L. and Broadie, K. S. (2007). Drosophila fragile X mental retardation protein and metabotropic glutamate receptor A convergently regulate the synaptic ratio of ionotropic glutamate receptor subclasses. J. Neurosci. 27(45): 12378-89. PubMed ID: 17989302
Pan, L., Woodruff, E., Liang, P. and Broadie, K. (2008). Mechanistic relationships between Drosophila fragile X mental retardation protein and metabotropic glutamate receptor A signaling. Mol. Cell. Neurosci. 37: 747-760. PubMed ID: 18280750
Parmentier, M. L., et al. (2000). Conservation of the ligand recognition site of metabotropic glutamate receptors during evolution. Neuropharmacology 39(7): 1119-31. PubMed ID: 10760355
Repicky, S. and Broadie, K. (2009). Metabotropic glutamate receptor-mediated use-dependent down-regulation of synaptic excitability involves the fragile X mental retardation protein. J. Neurophysiol. 101(2): 672-87. PubMed ID: 19036865
Ronesi J. A. and Huber K. M. (2008). Homer interactions are necessary for metabotropic glutamate receptor-induced long-term depression and translational activation. J. Neurosci. 28: 543-547. PubMed ID: 18184796
Sinakevitch, I., Grau, Y., Strausfeld, N. J. and Birman, S. (2010). Dynamics of glutamatergic signaling in the mushroom body of young adult Drosophila. Neural Dev. 5: 10. PubMed ID: 20370889
Zhang, D., Kuromi, H. and Kidokoro, Y. (1999). Activation of metabotropic glutamate receptors enhances synaptic transmission at the Drosophila neuromuscular junction. Neuropharmacology 38(5): 645-57. PubMed ID: 10340302
date revised: 25 March 2015
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