GABAergic Innervation Research Articles

Neuronal Dystroglycan Is Necessary for Formation and Maintenance of Functional CCK-Positive Basket Cell Terminals on Pyramidal Cells

Dystroglycan, an extracellular and transmembrane protein of the dystrophin-glycoprotein complex, is at the center of molecular studies of muscular dystrophies. Although its synaptic distribution in cortical brain regions is long established, function of dystroglycan in the synapse remained obscure. Using mice that selectively lack neuronal dystroglycan, we provide evidence that a subset of GABAergic interneurons requires dystroglycan for formation and maintenance of axonal terminals on pyramidal cells. As such, dystroglycan is the first postsynaptic GABAergic protein for which an interneuron terminal-specific function could be shown. Our findings also offer a new perspective on the mechanisms that lead to intellectual disability in muscular dystrophies without associated brain malformations.

Development of an excitatory kisspeptin projection to the oxytocin system in late pregnancy.

Oxytocin release from the posterior pituitary gland stimulates uterine contraction during birth but the central mechanisms that activate oxytocin neurones for birth are not well characterized. We found that that kisspeptin fibre density around oxytocin neurones increases in late-pregnant rats. These kisspeptin fibres originated from hypothalamic periventricular nucleus neurones that upregulated kisspeptin expression in late pregnancy. Oxytocin neurones were excited by central kisspeptin administration in late-pregnant rats but not in non-pregnant rats or early- to mid-pregnant rats. Our results reveal the emergence of a new excitatory kisspeptin projection to the oxytocin system in late pregnancy that might contribute to oxytocin neurone activation for birth. The hormone oxytocin promotes uterine contraction during parturition. Oxytocin is synthesized by magnocellular neurones in the hypothalamic supraoptic and paraventricular nuclei and is released into the circulation from the posterior pituitary gland in response to action potential firing. Systemic kisspeptin administration increases oxytocin neurone activity to elevate plasma oxytocin levels. Here, immunohistochemistry revealed that rats on the expected day of parturition (day21 of gestation) had a higher density of kisspeptin-positive fibres in the perinuclear zone surrounding the supraoptic nucleus (which provides dense glutamatergic and GABAergic innervation to the supraoptic nucleus) than was evident in non-pregnant rats. Retrograde tracing showed the kisspeptin projections to the perinuclear zone originated from the hypothalamic periventricular nucleus. Quantitative RT-PCR showed that kisspeptin receptor mRNA, Kiss1R mRNA, was expressed in the perinuclear zone-supraoptic nucleus and that the relative Kiss1R mRNA expression does not change over the course of pregnancy. Finally, intracerebroventricular administration of kisspeptin increased the firing rate of oxytocin neurones in anaesthetized late-pregnant rats (days 18-21 of gestation) but not in non-pregnant rats, or in early- or mid-pregnant rats. Taken together, these results suggest that kisspeptin expression is upregulated in the periventricular nucleus projection to the perinuclear zone of the supraoptic nucleus towards the end of pregnancy. Hence, this input might activate oxytocin neurones during parturition.

Variant BDNF-Val66Met Polymorphism is Associated with Layer-Specific Alterations in GABAergic Innervation of Pyramidal Neurons, Elevated Anxiety and Reduced Vulnerability of Adolescent Male Mice to Activity-Based Anorexia.

Previously, we determined that rodents' vulnerability to food restriction (FR)-evoked wheel running during adolescence (activity-based anorexia, ABA) is associated with failures to increase GABAergic innervation of hippocampal and medial prefrontal pyramidal neurons. Since brain-derived neurotrophic factor (BDNF) promotes GABAergic synaptogenesis, we hypothesized that individual differences in this vulnerability may arise from differences in the link between BDNF bioavailability and FR-evoked wheel running. We tested this hypothesis in male BDNF-Val66Met knock-in mice (BDNFMet/Met), known for reduction in the activity-dependent BDNF secretion and elevated anxiety-like behaviors. We found that 1) in the absence of FR or a wheel (i.e., control), BDNFMet/Met mice are more anxious than wild-type (WT) littermates, 2) electron microscopically verified GABAergic innervations of pyramidal neurons of BDNFMet/Met mice are reduced at distal dendrites in hippocampal CA1 and medial prefrontal cortex, 3) following ABA, WT mice exhibit anxiety equal to those of the BDNFMet/Met mice and have lost GABAergic innervation along distal dendrites, 4) BDNFMet/Met mice show blunted ABA vulnerability, and 5) unexpectedly, GABAergic innervation is higher at somata of BDNFMet/Met mice than of WT. We conclude that lamina-specific GABAergic inhibition is important for regulating anxiety, whether arising from environmental stress, such as food deprivation, or genetically, such as BDNFMet/Met single nucleotide polymorphism.

Synaptic Adhesion Molecules Regulate the Integration of New Granule Neurons in the Postnatal Mouse Hippocampus and their Impact on Spatial Memory.

Postnatal hippocampal neurogenesis induces network remodeling and may participate to mechanisms of learning. In turn, the maturation and survival of newborn neurons is regulated by their activity. Here, we tested the effect of a cell-autonomous overexpression of synaptic adhesion molecules on the maturation and survival of neurons born postnatally and on hippocampal-dependent memory performances. Families of adhesion molecules are known to induce pre- and post-synaptic assembly. Using viral targeting, we overexpressed three different synaptic adhesion molecules, SynCAM1, Neuroligin-1B and Neuroligin-2A in newborn neurons in the dentate gyrus of 7- to 9-week-old mice. We found that SynCAM1 increased the morphological maturation of dendritic spines and mossy fiber terminals while Neuroligin-1B increased spine density. In contrast, Neuroligin-2A increased both spine density and size as well as GABAergic innervation and resulted in a drastic increase of neuronal survival. Surprisingly, despite increased neurogenesis, mice overexpressing Neuroligin-2A in new neurons showed decreased memory performances in a Morris water maze task. These results indicate that the cell-autonomous overexpression of synaptic adhesion molecules can enhance different aspects of synapse formation on new neurons and increase their survival. Furthermore, they suggest that the mechanisms by which new neurons integrate in the postnatal hippocampus conditions their functional implication in learning and memory.

Receptor tyrosine kinase EphA7 is required for interneuron connectivity at specific subcellular compartments of granule cells

Neuronal transmission is regulated by the local circuitry which is composed of principal neurons targeted at different subcellular compartments by a variety of interneurons. However, mechanisms that contribute to the subcellular localisation and maintenance of GABAergic interneuron terminals are poorly understood. Stabilization of GABAergic synapses depends on clustering of the postsynaptic scaffolding protein gephyrin and its interaction with the guanine nucleotide exchange factor collybistin. Lentiviral knockdown experiments in adult rats indicated that the receptor tyrosine kinase EphA7 is required for the stabilisation of basket cell terminals on proximal dendritic and somatic compartments of granular cells of the dentate gyrus. EphA7 deficiency and concomitant destabilisation of GABAergic synapses correlated with impaired long-term potentiation and reduced hippocampal learning. Reduced GABAergic innervation may be explained by an impact of EphA7 on gephyrin clustering. Overexpression or ephrin stimulation of EphA7 induced gephyrin clustering dependent on the mechanistic target of rapamycin (mTOR) which is an interaction partner of gephyrin. Gephyrin interactions with mTOR become released after mTOR activation while enhanced interaction with the guanine nucleotide exchange factor collybistin was observed in parallel. In conclusion, EphA7 regulates gephyrin clustering and the maintenance of inhibitory synaptic connectivity via mTOR signalling.

Enrichment of GABAA Receptor α-Subunits on the Axonal Initial Segment Shows Regional Differences.

Although it is generally recognized that certain α-subunits of γ-aminobutyric acid type A receptors (GABAARs) form enriched clusters on the axonal initial segment (AIS), the degree to which these clusters vary in different brain areas is not well known. In the current study, we quantified the density, size, and enrichment ratio of fluorescently labeled α1-, α2-, or α3-subunits aggregates co-localized with the AIS-marker ankyrin G and compared them to aggregates in non-AIS locations among different brain areas including hippocampal subfields, basal lateral amygdala (BLA), prefrontal cortex (PFC), and sensory cortex (CTX). We found regional differences in the enrichment of GABAAR α-subunits on the AIS. Significant enrichment was identified in the CA3 of hippocampus for α1-subunits, in the CA1, CA3, and BLA for α2-subunits, and in the BLA for α3-subunits. Using α-subunit knock-out (KO) mice, we found that BLA enrichment of α2- and α3-subunits were physiologically independent of each other, as the enrichment of one subunit was unaffected by the genomic deletion of the other. To further investigate the unique pattern of α-subunit enrichment in the BLA, we examined the association of α2- and α3-subunits with the presynaptic vesicular GABA transporter (vGAT) and the anchoring protein gephyrin (Geph). As expected, both α2- and α3-subunits on the AIS within the BLA received prominent GABAergic innervation from vGAT-positive terminals. Further, we found that the association of α2- and α3-subunits with Geph was weaker in AIS versus non-AIS locations, suggesting that Geph might be playing a lesser role in the enrichment of α2- and α3-subunits on the AIS. Overall, these observations suggest that GABAARs on the AIS differ in subunit composition across brain regions. As with somatodendritic GABAARs, the distinctive expression pattern of AIS-located GABAAR α-subunits in the BLA, and other brain areas, likely contribute to unique forms of GABAergic inhibitory transmission and pharmacological profiles seen in different brain areas.

Synaptic Targets of Medial Septal Projections in the Hippocampus and Extrahippocampal Cortices of the Mouse.

Temporal coordination of neuronal assemblies among cortical areas is essential for behavioral performance. GABAergic projections from the medial septum and diagonal band complex exclusively innervate GABAergic interneurons in the rat hippocampus, contributing to the coordination of neuronal activity, including the generation of theta oscillations. Much less is known about the synaptic target neurons outside the hippocampus. To reveal the contribution of synaptic circuits involving the medial septum of mice, we have identified postsynaptic cortical neurons in wild-type and parvalbumin-Cre knock-in mice. Anterograde axonal tracing from the septum revealed extensive innervation of the hippocampus as well as the subiculum, presubiculum, parasubiculum, the medial and lateral entorhinal cortices, and the retrosplenial cortex. In all examined cortical regions, many septal GABAergic boutons were in close apposition to somata or dendrites immunopositive for interneuron cell-type molecular markers, such as parvalbumin, calbindin, calretinin, N-terminal EF-hand calcium-binding protein 1, cholecystokinin, reelin, or a combination of these molecules. Electron microscopic observations revealed septal boutons forming axosomatic or axodendritic type II synapses. In the CA1 region of hippocampus, septal GABAergic projections exclusively targeted interneurons. In the retrosplenial cortex, 93% of identified postsynaptic targets belonged to interneurons and the rest to pyramidal cells. These results suggest that the GABAergic innervation from the medial septum and diagonal band complex contributes to temporal coordination of neuronal activity via several types of cortical GABAergic interneurons in both hippocampal and extrahippocampal cortices. Oscillatory septal neuronal firing at delta, theta, and gamma frequencies may phase interneuron activity.

The different effects of high-frequency stimulation of the nucleus accumbens shell and core on food consumption are possibly associated with different neural responses in the lateral hypothalamic area

Obesity may result from dysfunction of the reward system, especially in the nucleus accumbens (Acb). Based on this hypothesis, many researchers have tested the effect of high-frequency stimulation (HFS) of the Acb shell (Acb-Sh) and/or core (Acb-Co) on ingestive behaviors, but few studies have explored the possible mechanisms involved in the differences between the Acb-Sh and Acb-Co. The present study tested effects of HFS of the Acb-Sh and Acb-Co on high-fat food (HFF) consumption in rats after 24h of food deprivation. Microdialysis and electrophysiological experiments were carried out in awake rats to explore potential mechanisms. The results showed that the Acb-Sh decreased HFF consumption after food deprivation both during and post-HFS. However, HFS of the Acb-Co did not induce similar changes in food consumption. HFS of the Acb-Sh (Sh-HFS) induced an increase in GABA level in the lateral hypothalamic area (LHA) during both phases, whereas HFS of the Acb-Co (Co-HFS) did not exhibit similar effects. The electrophysiological experiment showed that nearly all the LHA neurons were inhibited by Sh-HFS, and the mean firing rate decreased significantly both during and post-HFS. In contrast, the mean firing rate of the LHA neurons did not exhibit clear changes during Co-HFS, although some individual neurons appeared to exhibit responses to Co-HFS. Considering all the data, we postulated that Sh-HFS, rather than Co-HFS, might inhibit palatable food consumption after food deprivation by decreasing the reward value of that food, which suggested that it might also disturb the process of developing obesity. The mechanisms involved in the different effects of Sh-HFS and Co-HFS on food consumption may be associated with different neural responses in the LHA. The Acb-Sh has abundant GABAergic projections to the LHA, whereas the Acb-Co has few or no GABAergic innervations to the LHA. Thus, neural activity in the LHA exhibits different responses to Sh-HFS and Co-HFS.

Interneurons and oligodendrocyte progenitors form a structured synaptic network in the developing neocortex.

NG2 cells, oligodendrocyte progenitors, receive a major synaptic input from interneurons in the developing neocortex. It is presumed that these precursors integrate cortical networks where they act as sensors of neuronal activity. We show that NG2 cells of the developing somatosensory cortex form a transient and structured synaptic network with interneurons that follows its own rules of connectivity. Fast-spiking interneurons, highly connected to NG2 cells, target proximal subcellular domains containing GABAA receptors with γ2 subunits. Conversely, non-fast-spiking interneurons, poorly connected with these progenitors, target distal sites lacking this subunit. In the network, interneuron-NG2 cell connectivity maps exhibit a local spatial arrangement reflecting innervation only by the nearest interneurons. This microcircuit architecture shows a connectivity peak at PN10, coinciding with a switch to massive oligodendrocyte differentiation. Hence, GABAergic innervation of NG2 cells is temporally and spatially regulated from the subcellular to the network level in coordination with the onset of oligodendrogenesis.

In vivo clonal overexpression of neuroligin 3 and neuroligin 2 in neurons of the rat cerebral cortex: Differential effects on GABAergic synapses and neuronal migration.

We studied the effect of clonal overexpression of neuroligin 3 (NL3) or neuroligin 2 (NL2) in the adult rat cerebral cortex following in utero electroporation (IUEP) at embryonic stage E14. Overexpression of NL3 leads to a large increase in vesicular gamma-aminobutyric acid (GABA) transporter (vGAT) and glutamic acid decarboxylase (GAD)65 in the GABAergic contacts that the overexpressing neurons receive. Overexpression of NL2 produced a similar effect but to a lesser extent. In contrast, overexpression of NL3 or NL2 after IUEP does not affect vesicular glutamate transporter 1 (vGlut1) in the glutamatergic contacts that the NL3 or NL2-overexpressing neurons receive. The NL3 or NL2-overexpressing neurons do not show increased innervation by parvalbumin-containing GABAergic terminals or increased parvalbumin in the same terminals that show increased vGAT. These results indicate that the observed increase in vGAT and GAD65 is not due to increased GABAergic innervation but to increased expression of vGAT and GAD65 in the GABAergic contacts that NL3 or NL2-overexpressing neurons receive. The majority of bright vGAT puncta contacting the NL3-overexpressing neurons have no gephyrin juxtaposed to them, indicating that many of these contacts are nonsynaptic. This contrasts with the majority of the NL2-overexpressing neurons, which show plenty of synaptic gephyrin clusters juxtaposed to vGAT. Besides having an effect on GABAergic contacts, overexpression of NL3 interferes with the neuronal radial migration, in the cerebral cortex, of the neurons overexpressing NL3.

If I had a million neurons: Potential tests of cortico-hippocampal theories.

Considerable excitement surrounds new initiatives to develop techniques for simultaneous recording of large populations of neurons in cortical structures. This chapter focuses on the potential value of large-scale simultaneous recording for advancing research on current issues in the function of cortical circuits, including the interaction of the hippocampus with cortical and subcortical structures. The review describes specific research questions that could be answered using large-scale population recording, including questions about the circuit dynamics underlying coding of dimensions of space and time for episodic memory, the role of GABAergic and cholinergic innervation from the medial septum, the functional role of spatial representations coded by grid cells, boundary cells, head direction cells, and place cells, and the fact that many models require cells coding movement direction.

Ring Finger Protein 34 (RNF34) Interacts with and Promotes γ-Aminobutyric Acid Type-A Receptor Degradation via Ubiquitination of the γ2 Subunit

We have found that the large intracellular loop of the γ2 GABAA receptor (R) subunit (γ2IL) interacts with RNF34 (an E3 ubiquitin ligase), as shown by yeast two-hybrid and in vitro pulldown assays. In brain extracts, RNF34 co-immunoprecipitates with assembled GABAARs. In co-transfected HEK293 cells, RNF34 reduces the expression of the γ2 GABAAR subunit by increasing the ratio of ubiquitinated/nonubiquitinated γ2. Mutating several lysines of the γ2IL into arginines makes the γ2 subunit resistant to RNF34-induced degradation. RNF34 also reduces the expression of the γ2 subunit when α1 and β3 subunits are co-assembled with γ2. This effect is partially reversed by leupeptin or MG132, indicating that both the lysosomal and proteasomal degradation pathways are involved. Immunofluorescence of cultured hippocampal neurons shows that RNF34 forms clusters and that a subset of these clusters is associated with GABAergic synapses. This association is also observed in the intact rat brain by electron microscopy immunocytochemistry. RNF34 is not expressed until the 2nd postnatal week of rat brain development, being highly expressed in some interneurons. Overexpression of RNF34 in hippocampal neurons decreases the density of γ2 GABAAR clusters and the number of GABAergic contacts that these neurons receive. Knocking down endogenous RNF34 with shRNA leads to increased γ2 GABAAR cluster density and GABAergic innervation. The results indicate that RNF34 regulates postsynaptic γ2-GABAAR clustering and GABAergic synaptic innervation by interacting with and ubiquitinating the γ2-GABAAR subunit promoting GABAAR degradation.

Thorough GABAergic innervation of the entire axon initial segment revealed by an optogenetic 'laserspritzer'.

GABAergic terminals of chandelier cells exclusively innervate the axon initial segment (AIS) of excitatory neurons. Although the anatomy of these synapses has been well-studied in several brain areas, relatively little is known about their physiological properties. Using vesicular γ-aminobutyric acid transporter-channelrhodopsin 2-enhanced yellow fluorescence protein (VGAT-ChR2-YFP)-expressing mice and a novel fibreoptic 'laserspritzer' approach that we developed, we investigated the physiological properties of axo-axonic synapses (AASs) in brain slices from the piriform cortex (PC) of mice. AASs were in close proximity to voltage-gated Na(+) (NaV) channels located at the AIS. AASs were selectively activated by a 5 μm laserspritzer placed in close proximity to the AIS. Under a minimal laser stimulation condition and using whole-cell somatic voltage-clamp recordings, the amplitudes and kinetics of IPSCs mediated by AASs were similar to those mediated by perisomatic inhibitions. Results were further validated with channelrhodopsin 2-assisted circuit mapping (CRACM) of the entire inhibitory inputs map. For the first time, we revealed that the laserspritzer-induced AAS-IPSCs persisted in the presence of TTX and TEA but not 4-AP. Next, using gramicidin-based perforated patch recordings, we found that the GABA reversal potential (EGABA) was -73.6 ± 1.2 mV when induced at the AIS and -72.8 ± 1.1 mV when induced at the perisomatic site. Our anatomical and physiological results lead to the novel conclusions that: (1) AASs innervate the entire length of the AIS, as opposed to forming a highly concentrated cartridge, (2) AAS inhibition suppresses action potentials and epileptiform activity more robustly than perisomatic inhibitions, and (3) AAS activation alone can be sufficient to inhibit action potential generation and epileptiform activities in vitro.

Defects in dendrite and spine maturation and synaptogenesis associated with an anxious-depressive-like phenotype of GABAA receptor-deficient mice

Mice that were rendered heterozygous for the γ2 subunit of GABAA receptors (γ2+/− mice) have been characterized extensively as a model for major depressive disorder. The phenotype of these mice includes behavior indicative of heightened anxiety, despair, and anhedonia, as well as defects in hippocampus-dependent pattern separation, HPA axis hyperactivity and increased responsiveness to antidepressant drugs. The γ2+/− model thereby provides strong support for the GABAergic deficit hypothesis of major depressive disorder. Here we show that γ2+/− mice additionally exhibit specific defects in late stage survival of adult-born hippocampal granule cells, including reduced complexity of dendritic arbors and impaired maturation of synaptic spines. Moreover, cortical γ2+/− neurons cultured in vitro show marked deficits in GABAergic innervation selectively when grown under competitive conditions that may mimic the environment of adult-born hippocampal granule cells. Finally, brain extracts of γ2+/− mice show a numerical but insignificant trend (p = 0.06) for transiently reduced expression of brain derived neurotrophic factor (BDNF) at three weeks of age, which might contribute to the previously reported developmental origin of the behavioral phenotype of γ2+/− mice. The data indicate increasing congruence of the GABAergic, glutamatergic, stress-based and neurotrophic deficit hypotheses of major depressive disorder.This article is part of the Special Issue entitled ‘GABAergic Signaling in Health and Disease’.

Cerebellar Inhibitory Input to the Inferior Olive Decreases Electrical Coupling and Blocks Subthreshold Oscillations

GABAergic projection neurons in the cerebellar nuclei (CN) innervate the inferior olive (IO) that in turn is the source of climbing fibers targeting Purkinje neurons in the cerebellar cortex. Anatomical evidence suggests that CN synapses modulate electrical coupling between IO neurons. Invivo studies indicate that they are also involved in controlling synchrony and rhythmicity of IO neurons. Here, we demonstrate using virally targeted channelrhodopsin in the cerebellar nucleo-olivary neurons that synaptic input can indeed modulate both the strength and symmetry of electrical coupling between IO neurons and alter network activity. Similar synaptic modifications of electrical coupling are likely to occur in other brain regions, where rapid modification of the spatiotemporal features of the coupled networks is needed to adequately respond to behavioral demands.

Innervation by a GABAergic Neuron Depresses Spontaneous Release in Glutamatergic Neurons and Unveils the Clamping Phenotype of Synaptotagmin-1

The role of spontaneously occurring release events in glutamatergic and GABAergic neurons and their regulation is intensely debated. To study the interdependence of glutamatergic and GABAergic spontaneous release, we compared reciprocally connected "mixed" glutamatergic/GABAergic neuronal pairs from mice cultured on astrocyte islands with "homotypic" glutamatergic or GABAergic pairs and autaptic neurons. We measured mEPSC and mIPSC frequencies simultaneously from both neurons. Neuronal pairs formed both interneuronal synaptic and autaptic connections indiscriminately. We find that whereas mEPSC and mIPSC frequencies did not deviate between autaptic and synaptic connections, the frequency of mEPSCs in mixed pairs was strongly depressed compared with either autaptic neurons or glutamatergic pairs. Simultaneous imaging of synapses, or comparison to evoked release amplitudes, showed that this decrease was not caused by fewer active synapses. The mEPSC frequency was negatively correlated with the mIPSC frequency, indicating interdependence. Moreover, the reduction in mEPSC frequency was abolished when established pairs were exposed to bicuculline for 3 d, but not by long-term incubation with tetrodotoxin, indicating that spontaneous GABA release downregulates mEPSC frequency. Further investigations showed that knockout of synaptotagmin-1 did not affect mEPSC frequencies in either glutamatergic autaptic neurons or in glutamatergic pairs. However, in mixed glutamatergic/GABAergic pairs, mEPSC frequencies were increased by a factor of four in the synaptotagmin-1-null neurons, which is in line with data obtained from mixed cultures. The effect persisted after incubation with BAPTA-AM. We conclude that spontaneous GABA release exerts control over mEPSC release, and GABAergic innervation of glutamatergic neurons unveils the unclamping phenotype of the synaptotagmin-1-null neurons.

Temporal redistribution of inhibition over neuronal subcellular domains underlies state-dependent rhythmic change of excitability in the hippocampus

The behaviour-contingent rhythmic synchronization of neuronal activity is reported by local field potential oscillations in the theta, gamma and sharp wave-related ripple (SWR) frequency ranges. In the hippocampus, pyramidal cell assemblies representing temporal sequences are coordinated by GABAergic interneurons selectively innervating specific postsynaptic domains, and discharging phase locked to network oscillations. We compare the cellular network dynamics in the CA1 and CA3 areas recorded with or without anaesthesia. All parts of pyramidal cells, except the axon initial segment, receive GABA from multiple interneuron types, each with distinct firing dynamics. The axon initial segment is exclusively innervated by axo-axonic cells, preferentially firing after the peak of the pyramidal layer theta cycle, when pyramidal cells are least active. Axo-axonic cells are inhibited during SWRs, when many pyramidal cells fire synchronously. This dual inverse correlation demonstrates the key inhibitory role of axo-axonic cells. Parvalbumin-expressing basket cells fire phase locked to field gamma activity in both CA1 and CA3, and also strongly increase firing during SWRs, together with dendrite-innervating bistratified cells, phasing pyramidal cell discharge. Subcellular domain-specific GABAergic innervation probably developed for the coordination of multiple glutamatergic inputs on different parts of pyramidal cells through the temporally distinct activity of GABAergic interneurons, which differentially change their firing during different network states.

Homeostatic Competition between Phasic and Tonic Inhibition

The GABAA receptors are the major inhibitory receptors in the brain and are localized at both synaptic and extrasynaptic membranes. Synaptic GABAA receptors mediate phasic inhibition, whereas extrasynaptic GABAA receptors mediate tonic inhibition. Both phasic and tonic inhibitions regulate neuronal activity, but whether they regulate each other is not very clear. Here, we investigated the functional interaction between synaptic and extrasynaptic GABAA receptors through various molecular manipulations. Overexpression of extrasynaptic α6β3δ-GABAA receptors in mouse hippocampal pyramidal neurons significantly increased tonic currents. Surprisingly, the increase of tonic inhibition was accompanied by a dramatic reduction of the phasic inhibition, suggesting a possible homeostatic regulation of the total inhibition. Overexpressing the α6 subunit alone induced an up-regulation of δ subunit expression and suppressed phasic inhibition similar to overexpressing the α6β3δ subunits. Interestingly, blocking all GABAA receptors after overexpressing α6β3δ receptors could not restore the synaptic GABAergic transmission, suggesting that receptor activation is not required for the homeostatic interplay. Furthermore, insertion of a gephyrin-binding-site (GBS) into the α6 and δ subunits recruited α6(GBS)β3δ(GBS) receptors to postsynaptic sites but failed to rescue synaptic GABAergic transmission. Thus, it is not the positional effect of extrasynaptic α6β3δ receptors that causes the down-regulation of phasic inhibition. Overexpressing α5β3γ2 subunits similarly reduced synaptic GABAergic transmission. We propose a working model that both synaptic and extrasynaptic GABAA receptors may compete for limited receptor slots on the plasma membrane to maintain a homeostatic range of the total inhibition.

GABAergic inputs from direct and indirect striatal projection neurons onto cholinergic interneurons in the primate putamen.

Striatal cholinergic interneurons (ChIs) are involved in reward-dependent learning and the regulation of attention. The activity of these neurons is modulated by intrinsic and extrinsic γ-aminobutyric acid (GABA)ergic and glutamatergic afferents, but the source and relative prevalence of these diverse regulatory inputs remain to be characterized. To address this issue, we performed a quantitative ultrastructural analysis of the GABAergic and glutamatergic innervation of ChIs in the postcommissural putamen of rhesus monkeys. Postembedding immunogold localization of GABA combined with peroxidase immunostaining for choline acetyltransferase showed that 60% of all synaptic inputs to ChIs originate from GABAergic terminals, whereas 21% are from putatively glutamatergic terminals that establish asymmetric synapses, and 19% from other (non-GABAergic) sources of symmetric synapses. Double pre-embedding immunoelectron microscopy using substance P and Met-/Leu-enkephalin antibodies to label GABAergic terminals from collaterals of "direct" and "indirect" striatal projection neurons, respectively, revealed that 47% of the indirect pathway terminals and 36% of the direct pathway terminals target ChIs. Together, substance P- and enkephalin-positive terminals represent 24% of all synapses onto ChIs in the monkey putamen. These findings show that ChIs receive prominent GABAergic inputs from multiple origins, including a significant contingent from axon collaterals of direct and indirect pathway projection neurons.

Developmental Regulation and Activity-Dependent Maintenance of GABAergic Presynaptic Inhibition onto Rod Bipolar Cell Axonal Terminals

Presynaptic inhibition onto axons regulates neuronal output, but how such inhibitory synapses develop and are maintained invivo remains unclear. Axon terminals of glutamatergic retinal rod bipolar cells (RBCs) receive GABAA and GABAC receptor-mediated synaptic inhibition. We found that perturbing GABAergic or glutamatergic neurotransmission does not prevent GABAergic synaptogenesis onto RBC axons. But, GABA release is necessary for maintaining axonal GABA receptors. This activity-dependent process is receptor subtype specific: GABAC receptors are maintained, whereas GABAA receptors containing α1, but not α3, subunits decrease over time in mice with deficient GABA synthesis. GABAA receptor distribution on RBC axons is unaffected in GABAC receptor knockout mice. Thus, GABAA and GABAC receptor maintenance are regulated separately. Although immature RBCs elevate their glutamate release when GABA synthesis is impaired, homeostatic mechanisms ensure that the RBC output operates within its normal range after eye opening, perhaps to regain proper visual processing within the scotopic pathway.