Electrophysiology of the GABAergic synapses

GABA is the most widely distributed inhibitory transmitter in the brain, and it acts commonly by augmenting chloride permeability. When resting membrane potential (Vm) is equal to chloride equilibrium potential (EC1) GABA tends to keep Vm to its resting value. In some synapses, a pump carries chloride actively out of the cell and EC1 is different from Vm. An increased permeability leads to a hyperpolarization. In presynaptic inhibition a chloride pump brings chloride ions actively inside the cell and an increased permeability leads to a depolarization. GABAA receptors are associated with chloride channels, whereas GABAB receptors cause a selective decrease of a voltage sensitive calcium channels which operates at synaptic terminals.     

Localization of the clustering protein gephyrin at GABAergic synapses in the main olfactory bulb of the rat

The tubulin-binding protein gephyrin is essential for the formation of postsynaptic glycine-receptor clusters in cultured spinal neurons. In addition, there is increasing evidence that gephyrin can also be present at nonglycinergic synapses. Here we analyzed immunocytochemically the subcellular localization of gephyrin in the main olfactory bulb of the rat and compared its distribution with that of γ-aminobutyric acid (GABA) and of two major GABA_A-receptor subunits. Gephyrin was selectively localized to the postsynaptic side of symmetric synaptic junctions, where the presynaptic terminals contained GABA. Moreover, gephyrin colocalized extensively with the α1 and γ2 subunits of the GABA_A receptor. In contrast, gephyrin was not detected at presumed glutamatergic synapses. These results indicate that gephyrin is not uniquely associated with glycine receptors, but can also be found at distinct GABAergic synapses. Thus, they raise the possibility that gephyrin is involved in anchoring certain GABA_A-receptor subtypes in the postsynaptic membrane. J. Comp. Neurol. 395:231–244, 1998. © 1998 Wiley-Liss, Inc.     

Formation of GABAergic synapses in the cerebellum

In the adult central nervous system (CNS), gamma-amino butyric acid (GABA) is a predominant inhibitory neurotransmitter, and is involved in the expression of various higher brain functions. In the cerebellum, formation of GABAergic synapses is crucial for cerebellar functions. However, it is not fully understood how GABAergic synapses and networks are formed. We are morphologically investigating the developmental changes in GABAergic signaling and the mechanisms underlying the assembly of GABAergic synapses using the cerebellum, which provides an ideal system for the investigation of brain development. The anatomy and development of GABAergic synapses and networks in the cerebellar cortex are reviewed, the key factors for the formation of GABAergic synapses are addressed, and the mechanisms underlying the formation of cerebellar GABAergic networks are discussed.     

Stathmin is required for stability of the Drosophila neuromuscular junction

Synaptic connections can be stably maintained for prolonged periods, yet can be rapidly disassembled during the developmental refinement of neural circuitry and following cytological insults that lead to neurodegeneration. To date, the molecular mechanisms that determine whether a synapse will persist versus being remodeled or eliminated remain poorly understood. Mutations in Drosophila stathmin were isolated in two independent genetic screens that sought mutations leading to impaired synapse stability at the Drosophila neuromuscular junction (NMJ). Here we demonstrate that Stathmin, a protein that associates with microtubules and can function as a point of signaling integration, is necessary to maintain the stability of the Drosophila NMJ. We show that Stathmin protein is widely distributed within motoneurons and that loss of Stathmin causes impaired NMJ growth and stability. In addition, we show that stathmin mutants display evidence of defective axonal transport, a common feature associated with neuronal degeneration and altered synapse stability. The disassembly of the NMJ in stathmin includes a predictable sequence of cytological events, suggesting that a common program of synapse disassembly is induced following the loss of Stathmin protein. These data define a required function for Stathmin during synapse maintenance in a model system in which there is only a single stathmin gene, enabling future genetic investigation of Stathmin function with potential relevance to the cause and progression of neuromuscular degenerative disease.     

Neuroligin-3 is a neuronal adhesion protein at GABAergic and glutamatergic synapses

Synaptic adhesion molecules are thought to play a critical role in the formation, function and plasticity of neuronal networks. Neuroligins (NL1-4) are a family of presumptive postsynaptic cell adhesion molecules. NL1 and NL2 isoforms are concentrated at glutamatergic and GABAergic synapses, respectively, but the cellular expression and synaptic localization of the endogenous NL3 and NL4 isoforms are unknown. We generated a panel of NL isoform-specific antibodies and examined the expression, developmental regulation and synaptic specificity of NL3. We found that NL3 was enriched in brain, where NL3 protein levels increased during postnatal development, coinciding with the peak of synaptogenesis. Subcellular fractionation revealed a concentration of NL3 in synaptic plasma membranes and postsynaptic densities. In cultured hippocampal neurons, endogenous NL3 was highly expressed and was localized at both glutamatergic and GABAergic synapses. Clustering of NL3 in hippocampal neurons by neurexin-expressing cells resulted in coaggregation of NL3 with glutamatergic and GABAergic scaffolding proteins. Finally, individual synapses contained colocalized NL2 and NL3 proteins, and coimmunoprecipitation studies revealed the presence of NL1-NL3 and NL2-NL3 complexes in brain extracts. These findings suggest that rodent NL3 is a synaptic adhesion molecule that is a shared component of glutamatergic and GABAergic synapses.     

Collybistin is required for both the formation and maintenance of GABAergic postsynapses in the hippocampus

Collybistin (Cb) is a brain-specific guanine nucleotide exchange factor, which interacts with the inhibitory receptor anchoring protein gephyrin. In the hippocampus of constitutively Cb-deficient adult mice, gephyrin and gephyrin-dependent GABA(A) receptors (GABA(A)Rs) are lost from postsynaptic sites. Here, we used a Cre-loxP system to inactivate the Cb gene in the forebrain at different developmental stages. Deletion of Cb during embryonic development prevented gephyrin clustering during synaptogenesis and caused an accumulation of gephyrin aggregates in the cell body of CA1 pyramidal neurons. Inactivation of the Cb gene during the third postnatal week resulted in a protracted loss of postsynaptic gephyrin clusters and the appearance of cytoplasmic gephyrin aggregates. These changes in gephyrin distribution were accompanied by a similar reduction in synaptically localized GABA(A)R gamma2-subunit immunoreactivity. Our data show that Cb is required for both the initial localization and maintenance of gephyrin and gephyrin-dependent GABA(A)Rs at inhibitory postsynaptic membrane specializations in the hippocampus.     

Compartmentalization of the GABAB receptor signaling complex is required for presynaptic inhibition at hippocampal synapses

Presynaptic inhibition via G-protein-coupled receptors (GPCRs) and voltage-gated Ca(2+) channels constitutes a widespread regulatory mechanism of synaptic strength. Yet, the mechanism of intermolecular coupling underlying GPCR-mediated signaling at central synapses remains unresolved. Using FRET spectroscopy, we provide evidence for formation of spatially restricted (<100 ?) complexes between GABA(B) receptors composed of GB(1a)/GB(2) subunits, Gα(o)β(1)γ(2) G-protein heterotrimer, and Ca(V)2.2 channels in hippocampal boutons. GABA release was not required for the assembly but for structural reorganization of the precoupled complex. Unexpectedly, GB(1a) deletion disrupted intermolecular associations within the complex. The GB(1a) proximal C-terminal domain was essential for association of the receptor, Ca(V)2.2 and Gβγ, but was dispensable for agonist-induced receptor activation and cAMP inhibition. Functionally, boutons lacking this complex-formation domain displayed impaired presynaptic inhibition of Ca(2+) transients and synaptic vesicle release. Thus, compartmentalization of the GABA(B1a) receptor, Gβγ, and Ca(V)2.2 channel in a signaling complex is required for presynaptic inhibition at hippocampal synapses.     

Endocannabinoid-dependent plasticity at GABAergic and glutamatergic synapses in the striatum is regulated by synaptic activity

Long-term depression (LTD) at striatal synapses is mediated by postsynaptic endocannabinoid (eCB) release and presynaptic cannabinoid 1 receptor (CB₁R) activation. Previous studies have indicated that eCB mobilization at excitatory synapses might be regulated by afferent activation. To further address the role of neuronal activity in synaptic plasticity we examined changes in synaptic strength induced by the L-type calcium channel activator 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester (FPL 64176, FPL) at glutamatergic and γ-aminobutyric acid (GABA)ergic synapses in the striatum. We found that the basic mechanisms for FPL-mediated eCB signaling are the same at glutamatergic and GABAergic synapses. FPL-induced LTD (FPL-LTD) was blocked in slices treated with the CB₁R antagonist AM251 (2 μ m ), but established depression was not reversed by AM251. FPL-LTD was temperature dependent, blocked by protein translation inhibitors and prevented by intracellular loading of the anandamide transporter inhibitor VDM11 (10 μ m ) at both glutamatergic and GABAergic synapses. FPL-LTD at glutamatergic synapses required paired-pulse afferent stimulation, while FPL-LTD at GABAergic synapses could be induced even in the absence of explicit afferent activation. By evaluating tetrodotoxin-insensitive spontaneous inhibitory postsynaptic currents we found that neuronal firing is vital for eCB release and LTD induction at GABAergic synapses, but not for short-term depression induced by CB₁R agonist. The data presented here suggest that the level of neuronal firing regulates eCB signaling by modulating release from the postsynaptic cell, as well as interacting with presynaptic mechanisms to induce LTD at both glutamatergic and GABAergic synapses in the striatum.     

DN-cadherin is required for spatial arrangement of nerve terminals and ultrastructural organization of synapses

We studied roles of DN-cadherin, the Drosophila major neuronal cadherin, in neuronal connections in the visual system. In DN-cadherin mutants, axon terminals of a large subset of photoreceptor cells reached and associated with their target interneurons, but their characteristic spatial arrangement was disrupted as synaptogenesis proceeded. Although synapses were formed at contact sites between the axon terminals and target neurons, underlying cytoplasmic structures were not fully specialized at both pre- and postsynaptic terminals and synaptic vesicles appeared to accumulate at the presynapses. These results suggest that the cadherin adhesion system is required for interaction between pre- and postsynaptic terminals and for generation of the mature synaptic structures.     

BDNF is required for the induction of a presynaptic component of the functional conversion of silent synapses

Long‐term potentiation (LTP) has received attention because of its proposed role in learning and memory. Despite substantial effort the pre‐ or postsynaptic expression site of LTP remains unsettled. It has been proposed that LTP is expressed postsynaptically through the functional conversion of “silent synapses.” We had shown that Schaffer collateral (SC) silent and “functional synapses,” which lack and express AMPA receptors, respectively exhibit distinct transmitter release properties. Therefore the functional conversion of silent synapses with LTP should be associated with presynaptic modifications. We now show that the pairing‐induced LTP at SC synapses is mediated by combined pre‐ and postsynaptic modifications involving the postsynaptic emergence of an AMPA response coupled with an enhanced glutamate release. BDNF replicates the changes associated with this LTP by activating TrkBRs, suggesting that the neurotrophin is required for the coordinated changes on both sides of the synaptic cleft. © 2010 Wiley‐Liss, Inc.     

Electrical synapses define networks of neocortical GABAergic neurons

Recent work using paired recording has provided a direct demonstration of functional electrical synapses between neocortical neurons of both juvenile and adult animals. Electrical synapses have been found among GABAergic interneurons but not pyramidal cells. Interestingly, necortical electrical synapses almost exclusively connect GABAergic neurons belonging to the same class. So far, at least five different neocortical networks defined by extensive and selective electrical coupling have been studied in the neocortex. These results could provide important clues to the understanding of functional cortical circuitry.     

Calcium-binding proteins in GABAergic calyciform synapses of the reticular nucleus

Large calyciform synapses in the rat reticular thalamic nucleus are characterized by the presence of gamma-aminobutyric acid. Presynaptic terminals are also loaded with calcium-binding proteins such as parvalbumin, calbindin, calretinin and calcineurin. The number of calyciform terminals containing gamma-aminobutyric acid and parvalbumin is 2005 in young adult rats; calbindin is present in 1,500, calretinin in 850 and calcineurin in 560 calyciform terminals. Developmental studies revealed that gamma-aminobutyric acid and calcium-binding proteins are virtually absent from calyciform terminals at birth but their occurrence increased considerably during postnatal life, suggesting increasing regulation of presynaptic calcium signaling during postnatal life. It is concluded that synaptic activity of large calyciform gamma-aminobutyric acid-containing synapses of the reticular thalamic nucleus is mediated, regulated or accompanied by calcium ions.     

Immunocytochemical demonstration of GABAergic synapses on identified rubrospinal neurons

GABAergic synapses on rubrospinal neurons were demonstrated with immunocytochemistry combined with intracellular injection of horseradish peroxidase. Sections containing red nucleus neurons were processed for glutamic acid decarboxylase (GAD) immunohistochemistry. GAD-immunoreactive synaptic endings formed synaptic contacts with somata and dendrites of red nucleus neurons and identified rubrospinal neurons. Our observation provides further evidence that GABA acts as an inhibitory transmitter mediating cortically evoked inhibitory postsynaptic potentials in red nucleus neurons.     

GABAA receptors can initiate the formation of functional inhibitory GABAergic synapses

The mechanisms that underlie the selection of an inhibitory GABAergic axon's postsynaptic targets and the formation of the first contacts are currently unknown. To determine whether expression of GABA_A receptors (GABA_ARs) themselves – the essential functional postsynaptic components of GABAergic synapses – can be sufficient to initiate formation of synaptic contacts, a novel co‐culture system was devised. In this system, the presynaptic GABAergic axons originated from embryonic rat basal ganglia medium spiny neurones, whereas their most prevalent postsynaptic targets, i.e. α1/β2/γ2‐GABA_ARs, were expressed constitutively in a stably transfected human embryonic kidney 293 (HEK293) cell line. The first synapse‐like contacts in these co‐cultures were detected by colocalization of presynaptic and postsynaptic markers within 2 h. The number of contacts reached a plateau at 24 h. These contacts were stable, as assessed by live cell imaging; they were active, as determined by uptake of a fluorescently labelled synaptotagmin vesicle‐luminal domain‐specific antibody; and they supported spontaneous and action potential‐driven postsynaptic GABAergic currents. Ultrastructural analysis confirmed the presence of characteristics typical of active synapses. Synapse formation was not observed with control or N‐methyl‐d ‐aspartate receptor‐expressing HEK293 cells. A prominent increase in synapse formation and strength was observed when neuroligin‐2 was co‐expressed with GABA_ARs, suggesting a cooperative relationship between these proteins. Thus, in addition to fulfilling an essential functional role, postsynaptic GABA_ARs can promote the adhesion of inhibitory axons and the development of functional synapses.     

Muscarinic signaling is required for spike-pairing induction of long-term potentiation at rat Schaffer collateral-CA1 synapses

Cholinergic input from the basal forebrain and septum to the hippocampus is well known to be critical in learning and memory. Muscarinic induction of theta-frequency oscillations may synchronize pre- and postsynaptic firing and thereby enhance plasticity in the hippocampus. Previous studies have demonstrated that muscarinic activation facilitates long-term potentiation (LTP) induced with tetanus in vitro. In the present study, we tested the role of muscarinic receptor activity in the induction of LTP beyond effects on spike timing by using a spike-pairing (SP) method at Schaffer collateral-CA1 synapses in rat hippocampal slices. Pairings of pre- and postsynaptic action potentials (APs) have been shown to induce LTP when the presynaptic AP precedes the postsynaptic AP by 5-15 ms, but contribution of muscarinic co-activation has not been ruled out. We demonstrate that the mAChR antagonist atropine abolishes LTP induction by SP. Surprisingly, prolonged exposure to the mAChR agonist carbachol inhibits LTP induction by SP, perhaps because of receptor desensitization. These results demonstrate an essential role of cholinergic signaling in this form of hippocampal plasticity.     

Semaphorin 3C is not required for the establishment and target specificity of the GABAergic septohippocampal pathway in vitro

The septohippocampal (SH) pathway comprises cholinergic and GABAergic fibers. Whereas the former establish synaptic contacts with all types of hippocampal neurons, the latter form complex baskets specifically on interneurons. The GABAergic SH function is associated with the control of hippocampal synchronous networks. Little is known about the mechanisms involved in the formation of the GABAergic SH pathway. Semaphorin (Sema) 3C is expressed in most hippocampal interneurons targeted by these axons. To ascertain whether Sema 3C influences the formation of the SH pathway, we analyzed the development of this connection in Sema 3C-deficient mice. As these animals die at birth, we developed an in vitro organotypic co-culture model reproducing the postnatal development of the SH pathway. In these SH co-cultures, the GABAergic SH pathway developed with target specificity similar to that present in vivo. SH axons formed incipient baskets on several types of hippocampal interneurons at 7 days in vitro, which increased their complexity by 18-25 days in vitro. These SH fibers formed symmetric synaptic contacts on GABAergic interneurons. This synaptic specificity was not influenced by the absence of entorhinal afferents. Finally, the absence of Sema 3C in target neurons or its blockage by neuropilin-1 and -2 ectodomains in slice co-cultures did not lead to major changes in either the target specificity of the GABAergic SH pathway or its density of innervation. We conclude that the formation and synaptic specificity of the GABAergic SH pathway relies on robust molecular mechanisms, independent of Sema 3C, that are retained in our in vitro co-culture model.     

Glycine-receptor immunoreactivity in retinal bipolar cells is postsynaptic to glycinergic and GABAergic amacrine cell synapses.

Glycinergic innervation of the synaptic terminals of mixed rod-cone bipolar cells in the goldfish retina was investigated by electron microscopical immunocytochemistry with presynaptic and postsynaptic markers for glycinergic neurons: a monoclonal antibody (mAb 7A) against the 93 kDa subunit of the strychnine-sensitive glycine receptor and polyclonal antisera against a glycine/BSA conjugate. Conventional "glycinergic" synaptic contacts, made by amacrine cell processes, accounted for 7-10% of the input to the bipolar cell terminals, whether determined by glycine receptor immunoreactivity (GlyR-IR) or glycine-IR. In addition to the conventional synapses, the large bipolar cell terminals in the proximal inner plexiform layer (type Mb) gave rise to spinules (spine-like protrusions) that invaginated into presynaptic amacrine cell processes. Although 85% of the spinules were GlyR-IR, no spinules were postsynaptic to glycine-IR processes; yet 86% of the spinules were postsynaptic to GAD-IR processes, suggesting that the GlyR-IR spinules were postsynaptic to GABAergic terminals. Furthermore, a single amacrine cell process could make two synapses with an Mb terminal: a GlyR-IR contact onto a spinule and a conventional synapse that was not GlyR-IR. We suggest that glycinergic innervation of bipolar cell terminals involves conventional glycinergic synapses as well as an unconventional situation in which GABA and glycine may interact in as yet undetermined manner, perhaps by potentiation.     

Rafts are required for acetylcholine receptor clustering

Cholesterol-sphingolipid microdomains, or lipid rafts, are major regulators of molecular interactions in membrane organization. Because lipid rafts can move laterally and cluster into larger patches, they have been proposed to play a role in the redistribution of specific molecules to specialized cellular structures. Rafts have been shown to favor formation and maintenance of synaptic receptor clusters in neurons of the central nervous system. However, little is known about their role in formation of the neuromuscular junction (NMJ). To determine whether lipid rafts are involved in acetylcholine receptor (AChR) cluster formation and stabilization in myogenic cells, two standard tools were employed: (1) Perturbation of lipid rafts by drugs that deplete membrane cholesterol was carried out to verify that cholesterol is required for AChR clustering in agrin-treated C2C12 myotubes; and (2) detergent resistance of lipid-ordered domains was also used to demonstrate that AChRs, as well as key components of the postsynaptic membrane of the NMJ, are associated with rafts.     

Neural cell adhesion molecule is required for stability of reinnervated neuromuscular junctions

Studies examining the etiology of motoneuron diseases usually focus on motoneuron death as the defining pathophysiology of the disease. However, impaired neuromuscular transmission and synapse withdrawal often precede cell death, raising the possibility that abnormalities in synaptic function contribute to disease onset. Although little is known about the mechanisms maintaining the synaptic integrity of neuromuscular junctions (NMJs), Drosophila studies suggest that Fasciclin II plays an important role. Inspired by these studies we used a reinnervation model of synaptogenesis to analyze neuromuscular function in mice lacking neural cell adhesion molecule (NCAM), the Fasciclin II vertebrate homolog. Our results showed that the recovery of contractile force was the same in wild‐type and NCAM?/? mice at 1 month after nerve injury, indicating that endplates were appropriately reformed. This normality was only transient because the contractile force and myofiber number decreased at 3 months after injury in NCAM?/? mice. Both declined further 3 months later. Myofibers degenerated, not because motoneurons died but because synapses were withdrawn. Although neurotransmission was initially normal at reinnervated NCAM?/? NMJs, it was significantly compromised 3 months later. Interestingly, the selective ablation of NCAM from motoneurons, or muscle fibers, did not mimic the deficits observed in reinnervated NCAM?/? mice. Taken together, these results indicate that NCAM is required to maintain normal synaptic function at reinnervated NMJs, although its loss pre‐synaptically or post‐synaptically is not sufficient to induce synaptic destabilization. Consideration is given to the role of NCAM in terminal Schwann cells for maintaining synaptic integrity and how NCAM dysfunction may contribute to motoneuron disorders.