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γ-Aminobutyric acid (GABA) is thought to play a paracrine role in adrenal medullary chromaffin (AMC) cells. Comparative physiological and immunocytochemical approaches were used to address the issue of how the paracrine function of GABA in AMC cells is established. GABA A receptor Cl - channel activities in AMC cells of rats and mice, where corticosterone is the major glucocorticoid, were much smaller than those in AMC cells of guinea-pigs and cattle, where cortisol is the major. The extent of enhancement of GABA A receptor α3 subunit expression in rat pheochromocytoma (PC12) cells by cortisol was larger than that by corticosterone in parallel with their glucocorticoid activities. Thus, the species difference in GABA A receptor expression may be ascribed to a difference in glucocorticoid activity between corticosterone and cortisol. GABA A receptor Cl - channel activity in mouse AMC cells was enhanced by allopregnanolone, as noted with that in guinea-pig AMC cells, and the enzymes involved in allopregnanolone production were immunohistochemically detected in the zona fasciculata in both mice and guinea pigs. The expression of glutamic acid decarboxylase 67 (GAD67), one of the GABA synthesizing enzymes, increased after birth, whereas GABA A receptors already developed at birth. Stimulation of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, but not nicotinic or muscarinic receptors, in PC12 cells, resulted in an increase in GAD67 expression in a protein-kinase A-dependent manner. The results indicate that glucocorticoid and PACAP are mainly responsible for the expressions of GABA A receptors and GAD67 involved in GABA signaling in AMC cells, respectively., (© 2021 International Society for Neurochemistry.)

Endocannabinoids (eCBs) act as ubiquitous modulators of synaptic transmission via the activation of cannabinoid receptors (CBRs). Cerebellar Purkinje cells (PCs) make strong inhibitory synaptic contacts not only with neurons in the deep cerebellar nuclei (DCN) but also with Lugaro cells and globular cells, whose cell bodies are located underneath the PC layer. However, little is known about the modulatory actions of eCBs on GABA release from PC axon terminals. Here, we examined the effects of eCBs on the GABAergic transmission at PC-globular cell synapses and PC-large DCN neuron synapses electrophysiologically using mouse cerebellar slices. We showed that the types 1 and 2 CBR agonist WIN55212 did not affect either spontaneous or miniature inhibitory postsynaptic currents (IPSCs) in globular cells under control conditions and in a state of enhanced synaptic activity. By contrast, another G i/o protein-coupled receptor agonist, baclofen, significantly reduced the miniature IPSC frequency in globular cells. WIN55212 had no effects on IPSCs in large DCN neurons. A type 2 CBR agonist, HU308, also had no effects on IPSCs in either globular cells or large DCN neurons. Moreover, the PCs' target neurons did not elicit depolarization-induced suppression of inhibition. These results suggest the lack of a functional role of CBRs at PCs' axon terminals. This is in sharp contrast to the fact that PCs receive abundant excitatory and inhibitory inputs that are under eCB-mediated presynaptic inhibitory modulation. The actions of eCBs are selective to distinct synapses and possibly contribute to information processes and rigorous signal transmission in the cerebellum., (© 2020 Wiley Periodicals LLC.)

The GABAergic system is thought to play an important role in the control of cognition and emotion, such as fear, and is related to the pathophysiology of psychiatric disorders. For example, the expression of the 67-kDa isoform of glutamate decarboxylase (GAD67), a GABA-producing enzyme, is downregulated in the postmortem brains of patients with major depressive disorder and schizophrenia. However, knocking out the Gad1 gene, which encodes GAD67, is lethal in mice, and thus, the association between Gad1 and cognitive/emotional functions is unclear. We recently developed Gad1 knockout rats and found that some of them can grow into adulthood. Here, we performed fear-conditioning tests in adult Gad1 knockout rats to assess the impact of the loss of Gad1 on fear-related behaviors and the formation of fear memory. In a protocol assessing both cued and contextual memory, Gad1 knockout rats showed a partial antiphase pattern of freezing during training and significantly excessive freezing during the contextual test compared with wild-type rats. However, Gad1 knockout rats did not show any synchronous increase in freezing with auditory tones in the cued test. On the other hand, in a contextual memory specialized protocol, Gad1 knockout rats exhibited comparable freezing behavior to wild-type rats, while their fear extinction was markedly impaired. These results suggest that GABA synthesis by GAD67 has differential roles in cued and contextual fear memory., (© 2020 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

GABA and glycine are inhibitory neurotransmitters. However, the mechanisms underlying the formation of GABAergic and glycinergic synapses remain unclear. The influence of GABAergic input deprivation on inhibitory terminal formation was investigated using Purkinje cell (PC)-specific vesicular GABA transporter (VGAT) knockout (L7-VGAT) mice, in which GABA release from PCs diminishes in an age-dependent manner. We compared the late development of GABAergic and glycinergic terminals in the cerebellar nucleus (CN) between control and L7-VGAT mice. In the control CN, the density of glutamate decarboxylase (GAD)-positive dots remained unchanged between postnatal 2 months (P2M) and 13 months (P13M), whereas glycine transporter 2 (GlyT2)-positive dots increased in density during this time frame. No difference in the density of GlyT2-positive dots was observed between control and L7-VGAT mice at P2M, but the density was significantly higher in the L7-VGAT fastigial nuclei (FN) than the control FN at P13M. When VGAT was absent from PC terminals, GlyT2-positive dots included GAD and VGAT and formed synapses. These results indicated that GABAergic terminals were formed by P2M, glycinergic terminals were actively formed after P2M, and more glycinergic terminals were formed in the L7-VGAT FN than in the control FN, suggesting that the increased glycinergic terminals may derive from interneurons within the FN and may also release GABA. These results suggest that the deprivation of GABAergic inputs from PCs may accelerate the formation of co-releasing terminals derived from interneurons and that the inhibitory terminal numbers and types may be regulated by the quantity of functional GABAergic inputs., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

In mammals, the circadian rhythms are regulated by the central clock located in the hypothalamic suprachiasmatic nucleus (SCN), which is composed of heterogeneous neurons with various neurotransmitters. Among them an inhibitory neurotransmitter, γ-Amino-Butyric-Acid (GABA), is expressed in almost all SCN neurons, however, its role in the circadian physiology is still unclear. Here, we show that the SCN of fetal mice lacking vesicular GABA transporter (VGAT -/- ) or GABA synthesizing enzyme, glutamate decarboxylase (GAD65 -/- /67 -/- ), shows burst firings associated with large Ca 2+ spikes throughout 24 hours, which spread over the entire SCN slice in synchrony. By contrast, circadian PER2 rhythms in VGAT -/- and GAD65 -/- /67 -/- SCN remain intact. SCN-specific VGAT deletion in adult mice dampens circadian behavior rhythm. These findings indicate that GABA in the fetal SCN is necessary for refinement of the circadian firing rhythm and, possibly, for stabilizing the output signals, but not for circadian integration of multiple cellular oscillations., Competing Interests: Competing interestsThe authors declare no competing interests.

The supramammillary nucleus (SuM) of the hypothalamus projects to the dentate gyrus (DG) and the CA2 region of the hippocampus. Although the SuM-to-hippocampus circuits have been implicated in spatial and emotional memory formation, little is known about precise neural connections between the SuM and hippocampus. Here, we report that axons of SuM neurons make monosynaptic connections to granule cells (GCs) and GABAergic interneurons, but not to hilar mossy cells, in the DG and co-release glutamate and γ-aminobutyric acid (GABA) at these synapses. Although inputs from the SuM can excite some interneurons, the inputs alone fail to generate spikes in GCs. However, despite the insufficient excitatory drive and GABAergic co-transmission, SuM inputs have net excitatory effects on GCs and can potentiate GC firing when temporally associated with perforant path inputs. Our results indicate that the SuM influences DG information processing by modulating GC outputs., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

In mammals, circadian rhythms, such as sleep/wake cycles, are regulated by the central circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN consists of thousands of individual neurons, which exhibit circadian rhythms. They synchronize with each other and produce robust and stable oscillations. Although several neurotransmitters are expressed in the SCN, almost all SCN neurons are γ-amino butyric acid (GABA)-ergic. Several studies have attempted to understand the roles of GABA in the SCN; however, precise mechanisms of the action of GABA in the SCN are still unclear. GABA exhibits excitatory and/or inhibitory characteristics depending on the circadian phase or region in the SCN. It can both synchronize and destabilize cellular circadian rhythms in individual SCN cells. Differing environmental light conditions, such as a long photoperiod, result in the decoupling of circadian oscillators of the dorsal and ventral SCN. This is due to high intracellular chloride concentrations in the dorsal SCN. Because mice with functional GABA deficiency, such as vesicular GABA transporter- and glutamate decarboxylase-deficient mice, are neonatal lethal, research has been limited to pharmacological approaches. Furthermore, different recording methods have been used to understand the roles of GABA in the SCN. The excitability of GABAergic neurons also changes during the postnatal period. Although there are technical difficulties in understanding the functions of GABA in the SCN, technical developments may help uncover new roles of GABA in circadian physiology and behavior.

Objective: Mutations of the KCNQ2 gene, which encodes the K v 7.2 subunit of voltage-gated M-type potassium channels, have been associated with epilepsy in the neonatal period. This developmental stage is unique in that the neurotransmitter gamma aminobutyric acid (GABA), which is inhibitory in adults, triggers excitatory action due to a reversed chloride gradient., Methods: To examine whether KCNQ2-related neuronal hyperexcitability involves neonatally excitatory GABA, we examined 1-week-old knockin mice expressing the K v 7.2 variant p.Tyr284Cys (Y284C)., Results: Brain slice electrophysiology revealed elevated CA1 hippocampal GABAergic interneuron activity with respect to presynaptic firing and postsynaptic current frequency. Blockade with the GABA A receptor antagonist bicuculline decreased ictal-like bursting in brain slices with lowered divalent ion concentration, which is consistent with GABA mediating an excitatory function that contributes to the hyperexcitability observed in mutant animals., Significance: We conclude that excitatory GABA contributes to the phenotype in these animals, which raises the question of whether this special type of neurotransmission has broader importance in neonatal epilepsy than is currently recognized., (Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.)

GABA acts as the major inhibitory neurotransmitter in the mammalian brain, shaping neuronal and circuit activity. For sustained synaptic transmission, synaptic vesicles (SVs) are required to be recycled and refilled with neurotransmitters using an H(+) electrochemical gradient. However, neither the mechanism underlying vesicular GABA uptake nor the kinetics of GABA loading in living neurons have been fully elucidated. To characterize the process of GABA uptake into SVs in functional synapses, we monitored luminal pH of GABAergic SVs separately from that of excitatory glutamatergic SVs in cultured hippocampal neurons. By using a pH sensor optimal for the SV lumen, we found that GABAergic SVs exhibited an unexpectedly higher resting pH (∼6.4) than glutamatergic SVs (pH ∼5.8). Moreover, unlike glutamatergic SVs, GABAergic SVs displayed unique pH dynamics after endocytosis that involved initial overacidification and subsequent alkalization that restored their resting pH. GABAergic SVs that lacked the vesicular GABA transporter (VGAT) did not show the pH overshoot and acidified further to ∼6.0. Comparison of luminal pH dynamics in the presence or absence of VGAT showed that VGAT operates as a GABA/H(+) exchanger, which is continuously required to offset GABA leakage. Furthermore, the kinetics of GABA transport was slower (τ > 20 s at physiological temperature) than that of glutamate uptake and may exceed the time required for reuse of exocytosed SVs, allowing reuse of incompletely filled vesicles in the presence of high demand for inhibitory transmission., Competing Interests: The authors declare no conflict of interest.

Corticotropin-releasing hormone (CRH), which is synthesized in the paraventricular nucleus (PVN) of the hypothalamus, plays an important role in the endocrine stress response. The excitability of CRH neurons is regulated by γ-aminobutyric acid (GABA)-containing neurons projecting to the PVN. We investigated the role of GABA in the regulation of CRH release. The release of CRH was impaired, accumulating in the cell bodies of CRH neurons in heterozygous GAD67-GFP (green fluorescent protein) knock-in mice (GAD67(+/GFP)), which exhibited decreased GABA content. The GABAA receptor (GABAAR) and the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1), but not the K(+)-Cl(-) cotransporter (KCC2), were expressed in the terminals of the CRH neurons at the median eminence (ME). In contrast, CRH neuronal somata were enriched with KCC2 but not with NKCC1. Thus, intracellular Cl(-) concentrations ([Cl(-)]i) may be increased at the terminals of CRH neurons compared with concentrations in the cell body. Moreover, GABAergic terminals projecting from the arcuate nucleus were present in close proximity to CRH-positive nerve terminals. Furthermore, a GABAAR agonist increased the intracellular calcium (Ca(2+)) levels in the CRH neuron terminals but decreased the Ca(2+) levels in their somata. In addition, the increases in Ca(2+) concentrations were prevented by an NKCC1 inhibitor. We propose a novel mechanism by which the excitatory action of GABA maintains a steady-state CRH release from axon terminals in the ME.

Gamma-aminobutyric acid (GABA) has a dual role as an inhibitory neurotransmitter in the adult central nervous system (CNS) and as a signaling molecule exerting largely excitatory actions during development. The rate-limiting step of GABA synthesis is catalyzed by two glutamic acid decarboxylase isoforms GAD65 and GAD67 coexpressed in the GABAergic neurons of the CNS. Here we report that the two GADs show virtually nonoverlapping expression patterns consistent with distinct roles in the developing peripheral olfactory system. GAD65 is expressed exclusively in undifferentiated neuronal progenitors confined to the proliferative zones of the sensory vomeronasal and olfactory epithelia In contrast GAD67 is expressed in a subregion of the nonsensory epithelium/vomeronasal organ epithelium containing the putative Gonadotropin-releasing hormone (GnRH) progenitors and GnRH neurons migrating from this region through the frontonasal mesenchyme into the basal forebrain. Only GAD67+, but not GAD65+ cells accumulate detectable GABA. We further demonstrate that GAD67 and its embryonic splice variant embryonic GAD (EGAD) concomitant with GnRH are dynamically regulated during GnRH neuronal migration in vivo and in two immortalized cell lines representing migratory (GN11) and postmigratory (GT1-7) stage GnRH neurons, respectively. Analysis of GAD65/67 single and double knock-out embryos revealed that the two GADs play complementary (inhibitory) roles in GnRH migration ultimately modulating the speed and/or direction of GnRH migration. Our results also suggest that GAD65 and GAD67/EGAD characterized by distinct subcellular localization and kinetics have disparate functions during olfactory system development mediating proliferative and migratory responses putatively through specific subcellular GABA pools., (© 2014 Wiley Periodicals, Inc.)

Activation of mu-opioid receptor (MOR) disinhibits dopaminergic neurons in the ventral tegmental area (VTA) through inhibition of γ-aminobutyric acid (GABA)ergic neurons. This mechanism is thought to play a pivotal role in mediating reward behaviors. Here, we characterised VTA-projecting enkephalinergic neurons in the anterior division of the bed nucleus of the stria terminalis (BST) and investigated their targets by examining MOR expression in the VTA. In the BST, neurons expressing preproenkephalin mRNA were exclusively GABAergic, and constituted 37.2% of the total GABAergic neurons. Using retrograde tracer injected into the VTA, 21.6% of VTA-projecting BST neurons were shown to express preproenkephalin mRNA. Enkephalinergic projections from the BST exclusively formed symmetrical synapses onto the dendrites of VTA neurons. In the VTA, 74.1% of MOR mRNA-expressing neurons were GABAergic, with the rest being glutamatergic neurons expressing type-2 vesicular glutamate transporter mRNA. However, MOR mRNA was below the detection threshold in dopaminergic neurons. By immunohistochemistry, MOR was highly expressed on the extrasynaptic membranes of dendrites in GABAergic VTA neurons, including dendrites innervated by BST-VTA projection terminals. MOR was also expressed weakly on GABAergic and glutamatergic terminals in the VTA. Given that GABAA α1 is expressed at GABAergic BST-VTA synapses on dendrites of GABAergic neurons [T. Kudo et al. (2012) J. Neurosci., 32, 18035-18046], our results collectively indicate that the BST sends dual inhibitory outputs targeting GABAergic VTA neurons; GABAergic inhibition via 'wired' transmission, and enkephalinergic inhibition via 'volume' transmission. This dual inhibitory system provides the neural substrate underlying the potent disinhibitory control over dopaminergic VTA neurons exerted by the BST., (© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Although focal cortical malformations are considered neuronal migration disorders, their formation mechanisms remain unknown. We addressed how the γ-aminobutyric acid (GABA)ergic system affects the GABAergic and glutamatergic neuronal migration underlying such malformations. A focal freeze-lesion (FFL) of the postnatal day zero (P0) glutamic acid decarboxylase-green fluorescent protein knock-in mouse neocortex produced a 3- or 4-layered microgyrus at P7. GABAergic interneurons accumulated around the necrosis including the superficial region during microgyrus formation at P4, whereas E17.5-born, Cux1-positive pyramidal neurons outlined the GABAergic neurons and were absent from the superficial layer, forming cell-dense areas in layer 2 of the P7 microgyrus. GABA imaging showed that an extracellular GABA level temporally increased in the GABAergic neuron-positive area, including the necrotic center, at P4. The expression of the Cl(-) transporter KCC2 was downregulated in the microgyrus-forming GABAergic and E17.5-born glutamatergic neurons at P4; these cells may need a high intracellular Cl(-) concentration to induce depolarizing GABA effects. Bicuculline decreased the frequency of spontaneous Ca(2+) oscillations in these microgyrus-forming cells. Thus, neonatal FFL causes specific neuronal accumulation, preceded by an increase in ambient GABA during microgyrus formation. This GABA increase induces GABAA receptor-mediated Ca(2+) oscillation in KCC2-downregulated microgyrus-forming cells, as seen in migrating cells during early neocortical development.

The electrophysiological properties and functional role of GABAergic signal transmission from neurons to the gap junction-coupled astrocytic network are still unclear. GABA-induced astrocytic Cl⁻ flux has been hypothesized to affect the driving force for GABAergic transmission by modulating [Cl⁻]o. Thus, revealing the properties of GABA-mediated astrocytic responses will deepen our understanding of GABAergic signal transmission. Here, we analysed the Cl⁻ dynamics of neurons and astrocytes in CA1 hippocampal GABAergic tripartite synapses, using Cl⁻ imaging during GABA application, and whole cell recordings from interneuron-astrocyte pairs in the stratum lacunosum-moleculare. Astrocytic [Cl⁻]i was adjusted to physiological conditions (40 mm). Although GABA application evoked bidirectional Cl⁻ flux via GABAA receptors and mouse GABA transporter 4 (mGAT4) in CA1 astrocytes, a train of interneuron firing induced only GABAA receptor-mediated inward currents in an adjacent astrocyte. A GAT1 inhibitor increased the interneuron firing-induced currents and induced bicuculline-insensitive, mGAT4 inhibitor-sensitive currents, suggesting that synaptic spillover of GABA predominantly induced the astrocytic Cl⁻ efflux because GABAA receptors are localized near the synaptic clefts. This GABA-induced Cl⁻ efflux was accompanied by Cl⁻ siphoning via the gap junctions of the astrocytic network because gap junction inhibitors significantly reduced the interneuron firing-induced currents. Thus, Cl⁻ efflux from astrocytes is homeostatically maintained within astrocytic networks. A gap junction inhibitor enhanced the activity-dependent depolarizing shifts of reversal potential of neuronal IPSCs evoked by repetitive stimulation to GABAergic synapses. These results suggest that Cl⁻ conductance within the astrocytic network may contribute to maintaining GABAergic synaptic transmission by regulating [Cl⁻]o.

Unlabelled: The rostroventromedial medulla (RVM) is an important area of the endogenous pain-regulating system, in which 5-hydroxytryptamine (5-HT) and gamma-aminobutyric acid (GABA) are 2 main transmitters involved in pain modulation. However, whether 5-HT and GABA are colocalized is poorly understood. By using glutamate decarboxylase 67-green fluorescence protein (GAD67-GFP) knock-in mouse, we confirmed the colocalization of 5-HT and GABA in the RVM, with a main distribution in the raphe magnus nucleus and paragigantocellular reticular nucleus. Interestingly, more than half (51.6%) of the 5-HT/GABA-immunoreactive (ir) neurons expressed neurokinin-1 receptors (NK-1R) and one-third (30.1%) of the 5-HT/GABA/NK-1R-ir neurons projected to the spinal cord, suggesting that substance P (SP) should regulate the activity of 5-HT/GABA-ir spinal cord-projecting neurons. By combining retrograde and anterograde tracing methods, we observed that the cuneiform nucleus, dorsal raphe nucleus, and lateral periaqueductal gray are the main origin nuclei for the SP-ir fibers and terminals in the RVM. Finally, after formalin injection into the mice hind paw, 29.2% SP-ir RVM-projecting neurons from supra-RVM nuclei and 33.1% NK-1R-ir spinal cord-projecting neurons in the RVM were activated. The present study provides potent morphological evidence that 5-HT and GABA are coexistent in RVM-spinal cord-projecting neurons that are also regulated by SPergic projections., Perspective: The results will greatly enhance our understanding for the modulation of nociceptive information in the descending pain-regulating system., (Copyright © 2013 American Pain Society. Published by Elsevier Inc. All rights reserved.)

Central nervous system GABAergic and glycinergic synaptic activity switches from postsynaptic excitation to inhibition during the stage when motor neuron numbers are being reduced, and when synaptic connections are being established onto and by motor neurons. In mice this occurs between embryonic (E) day 13 and birth (postnatal day 0). Our previous work on mice lacking glycinergic transmission suggested that altered motor neuron activity levels correspondingly regulated motor neuron survival and muscle innervation for all respiratory and non respiratory motor neuron pools, during this period of development [1]. To determine if GABAergic transmission plays a similar role, we quantified motor neuron number and the extent of muscle innervation in four distinct regions of the brain stem and spinal cord; hypoglossal, phrenic, brachial and lumbar motor pools, in mice lacking the enzyme GAD67. These mice display a 90% drop in CNS GABA levels ( [2]; this study). For respiratory-based motor neurons (hypoglossal and phrenic motor pools), we have observed significant drops in motor neuron number (17% decline for hypoglossal and 23% decline for phrenic) and muscle innervations (55% decrease). By contrast for non-respiratory motor neurons of the brachial lateral motor column, we have observed an increase in motor neuron number (43% increase) and muscle innervations (99% increase); however for more caudally located motor neurons within the lumbar lateral motor column, we observed no change in either neuron number or muscle innervation. These results show in mice lacking physiological levels of GABA, there are distinct regional changes in motor neuron number and muscle innervation, which appear to be linked to their physiological function and to their rostral-caudal position within the developing spinal cord. Our results also suggest that for more caudal (lumbar) regions of the spinal cord, the effect of GABA is less influential on motor neuron development compared to that of glycine.

Functional neural circuit formation during development involves massive elimination of redundant synapses. In the cerebellum, one-to-one connection from excitatory climbing fiber (CF) to Purkinje cell (PC) is established by elimination of early-formed surplus CFs. This process depends on glutamatergic excitatory inputs, but contribution of GABAergic transmission remains unclear. Here, we demonstrate impaired CF synapse elimination in mouse models with diminished GABAergic transmission by mutation of a single allele for the GABA synthesizing enzyme GAD67, by conditional deletion of GAD67 from PCs and GABAergic interneurons or by pharmacological inhibition of cerebellar GAD activity. The impaired CF synapse elimination was rescued by enhancing GABA(A) receptor sensitivity in the cerebellum by locally applied diazepam. Our electrophysiological and Ca2+ imaging data suggest that GABA(A) receptor-mediated inhibition onto the PC soma from molecular layer interneurons influences CF-induced Ca2+ transients in the soma and regulates CF synapse elimination from postnatal day 10 (P10) to around P16., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Inhibitory neurons are an essential element of the locomotor network in the mammalian spinal cord. However, little is known about the firing pattern and synaptic modulation during locomotion in the majority of them. In this study, we performed whole cell recording in visually identified ventrolaterally located GABAergic neurons (VL-GNs) in the rostral (L2 segment) and caudal (L5 segment) lumbar cord using isolated spinal cord preparations taken from glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mouse neonates. These neurons did not respond to electrical stimulation of the ventral root, indicating that they were not Renshaw cells. Ninety-five percent of VL-GNs in the L2 segment and fifty percent of those in the L5 segment showed significant rhythmic firing during locomotor-like rhythmic activity induced by bath application of 5-HT and NMDA. Seventy percent of these neurons fired mainly during the extensor phase, and twenty-five percent fired mainly during the flexor phase. Voltage-clamp recordings revealed that most of these neurons received rhythmic inhibition during the nonfiring phase and excitatory synaptic inputs during the firing phase. Morphological examination of recorded neurons filled with neurobiotin showed that their soma was located lateral to the motoneuron pool and that they extended their processes into the local ipsilateral ventromedial region and dorsal regions. The present study indicates that these GABAergic interneurons located in the ventrolateral region adjacent to the motoneuron pool are rhythmically active during locomotion and involved in the inhibitory modulation of local locomotor network in the lumbar spinal cord.

Recent studies have suggested that extrasynaptic GABA(A) receptors, which contribute tonic conductance, are important targets for general anesthetics. We tested the hypothesis that manipulations designed to alter ambient GABA concentrations (tonic conductance) would affect hypnotic (as indicated by loss of righting reflex, LORR) and immobilizing (as indicated by loss of tail-pinch withdrawal reflex, LTWR) actions of sevoflurane, propofol, and midazolam. Two manipulations studied were 1) the genetic absence of glutamate decarboxylase (GAD) 65 gene (GAD65-/-), which purportedly reduced ambient GABA concentrations, and 2) the pharmacological manipulation of GABA uptake using GABA transporter inhibitor (NO-711). The influence of these manipulations on cellular and behavioral responses to the anesthetics was studied using behavioral and electrophysiological assays. HPLC revealed that GABA levels in GAD65-/- mice were reduced in the brain (76.7% of WT) and spinal cord (68.5% of WT). GAD65-/- mice showed a significant reduction in the duration of LORR and LTWR produced by propofol and midazolam, but not sevoflurane. NO-711 (3 mg/kg, ip) enhanced the duration of LORR and LTWR by propofol and midazolam, but not sevoflurane. Patch-clamp recordings revealed that sevoflurane (0.23 mM) slightly enhanced the amplitude of tonic GABA current in the frontal cortical neurons; however, these effects were not strong enough to alter discharge properties of cortical neurons. These results demonstrate that ambient GABA concentration is an important determinant of the hypnotic and immobilizing actions of propofol and midazolam in mice, whereas manipulations of ambient GABA concentrations minimally alter cellular and behavioral responses to sevoflurane., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

N-methyl-D-aspartate (NMDA) receptors (NMDARs) are implicated in synaptic plasticity and modulation of glutamatergic excitatory transmission. Effect of NMDAR activation on inhibitory GABAergic transmission remains largely unknown. Here, we report that a brief application of NMDA could induce two distinct actions in CA1 pyramidal neurons in mouse hippocampal slices: 1) an inward current attributed to activation of postsynaptic NMDARs; and 2) fast phasic synaptic currents, namely spontaneous inhibitory postsynaptic currents (sIPSCs), mediated by GABA(A) receptors in pyramidal neurons. The mean amplitude of sIPSCs was also increased by NMDA. This profound increase in the sIPSC frequency and amplitude was markedly suppressed by the sodium channel blocker TTX, whereas the frequency and mean amplitude of miniature IPSCs were not significantly affected by NMDA, suggesting that NMDA elicits repetitive firing in GABAergic interneurons, thereby leading to GABA release from multiple synaptic sites of single GABAergic axons. We found that the NMDAR open-channel blocker MK-801 injected into recorded pyramidal neurons suppressed the NMDA-induced increase of sIPSCs, which raises the possibility that the firing of interneurons may not be the sole factor and certain retrograde messengers may also be involved in the NMDA-mediated enhancement of GABAergic transmission. Our results from pharmacological tests suggest that the nitric oxide signaling pathway is mobilized by NMDAR activation in CA1 pyramidal neurons, which in turn retrogradely facilitates GABA release from the presynaptic terminals. Thus NMDARs at glutamatergic synapses on both CA1 pyramidal neurons and interneurons appear to exert feedback and feedforward inhibition for determining the spike timing of the hippocampal microcircuit.

In the embryonic neocortex, neuronal precursors are generated in the ventricular zone (VZ) and accumulate in the cortical plate. Recently, the subventricular zone (SVZ) of the embryonic neocortex was recognized as an additional neurogenic site for both principal excitatory neurons and GABAergic inhibitory neurons. To gain insight into the neurogenesis of GABAergic neurons in the SVZ, we investigated the characteristics of intermediate progenitors of GABAergic neurons (IPGNs) in mouse neocortex by immunohistochemistry, immunocytochemistry, single-cell RT-PCR and single-cell array analysis. IPGNs were identified by their expression of some neuronal and cell cycle markers. Moreover, we investigated the origins of the neocortical IPGNs by Cre-loxP fate mapping in transgenic mice and the transduction of part of the telencephalic VZ by Cre-reporter plasmids, and found them in the medial and lateral ganglionic eminence. Therefore, they must migrate tangentially within the telencephalon to reach the neocortex. Cell-lineage analysis by simple-retrovirus transduction revealed that the neocortical IPGNs self-renew and give rise to a small number of neocortical GABAergic neurons and to a large number of granule and periglomerular cells in the olfactory bulb. IPGNs are maintained in the neocortex and may act as progenitors for adult neurogenesis.

Sensory experience influences brain organization and function. A particularly striking example is in the olfactory bulb where reduction of odorant sensory signals profoundly down-regulates dopamine in glomerular neurons. There are two large populations of glomerular inhibitory interneurons: (1) GABAergic periglomerular (PG) cells, whose processes are limited to a single glomerulus, regulate intraglomerular processing and (2) DAergic-GABAergic short axon (SA) cells, whose processes contact multiple glomeruli, regulate interglomerular processing. The inhibitory neurotransmitter GABA is synthesized from L-glutamic acid by the enzyme glutamic acid decarboxylase (GAD) of which there are two major isoforms: GAD65 and GAD67. GAD65 is expressed in uniglomerular PG cells. GAD67 is expressed by SA cells, which also co-express the rate-limiting enzyme for dopamine synthesis, tyrosine hydroxylase (TH). Deafferentation or sensory deprivation decreases TH expression but it is not known if sensory input alters GAD isoforms. Here we report that either deafferentation or reduction of sensory input by nares occlusion significantly reduced GAD67 protein and the number of SA cells expressing GAD67. However, neither manipulation altered GAD65 protein or the number of GAD65 PG cells. These findings show that sensory experience strongly impacts transmitter regulation in the circuit that controls neural processing across glomeruli but not in the circuit that regulates intraglomerular processing., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The direction and amplitude of saccadic eye movements are determined by the location of the center of gravity of burst activity over a neuronal population on the spatial map of the intermediate gray layer (SGI) of the superior colliculus (SC). GABAergic interneurons might play critical roles in shaping the activation field on the topographical map but, to understand the mechanism, basic information on the organization of inhibitory circuits is essential. In the present study, we investigated the electrophysiological and morphological properties of GABAergic neurons in SGI by whole-cell patch-clamp recordings and intracellular staining using biocytin in GAD67-GFP knock-in mice (PND17-22), in which GABAergic neurons specifically express GFP fluorescence. The most common firing properties among these GABAergic neurons (n=231) were fast spiking (58%), followed by burst spiking (29%), late spiking (8%) and, the least common, regular spiking (2%) and rapid spike inactivation (3%). Morphological analysis of axonal trajectories of intracellularly-labeled GABAergic neurons revealed three major subclasses: (i) intralaminar interneurons, which were further divided into two subclasses, local and horizontal interneurons; (ii) interlaminar interneurons; and (iii) commissural and tectofugal neurons. These results reveal distinct subsets of GABAergic neurons including neurons that mediate local and long-range inhibition in the SC, neurons that potentially modulate visual and other sensory inputs to the SC, and neurons that project to nuclei outside the SC., (© 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Cortical GABAergic interneurons originate from ganglionic eminences and tangentially migrate into the cortical plate at early developmental stages. To elucidate the characteristics of this migration of GABAergic interneurons in living animals, we established an experimental design specialized for in vivo time-lapse imaging of the neocortex of neonate mice with two-photon laser-scanning microscopy. In vesicular GABA/glycine transporter (VGAT)-Venus transgenic mice from birth (P0) through P3, we observed multidirectional tangential migration of genetically-defined GABAergic interneurons in the neocortical marginal zone. The properties of this migration, such as the motility rate (distance/hr), the direction moved, and the proportion of migrating neurons to stationary neurons, did not change through P0 to P3, although the density of GABAergic neurons at the marginal zone decreased with age. Thus, the characteristics of the tangential motility of individual GABAergic neurons remained constant in development. Pharmacological block of GABA(A) receptors and of the Na⁺-K⁺-Cl⁻ cotransporters, and chelating intracellular Ca²⁺, all significantly reduced the motility rate in vivo. The motility rate and GABA content within the cortex of neonatal VGAT-Venus transgenic mice were significantly greater than those of GAD67-GFP knock-in mice, suggesting that extracellular GABA concentration could facilitate the multidirectional tangential migration. Indeed, diazepam applied to GAD67-GFP mice increased the motility rate substantially. In an in vitro neocortical slice preparation, we confirmed that GABA induced a NKCC sensitive depolarization of GABAergic interneurons in VGAT-Venus mice at P0-P3. Thus, activation of GABA(A)R by ambient GABA depolarizes GABAergic interneurons, leading to an acceleration of their multidirectional motility in vivo.

The mechanisms underlying volatile anesthesia agents are not well elucidated. Emerging researches have focused on the participation of γ-aminobutyric acid (GABA) neurons but there still lacks morphological evidence. To elucidate the possible activation of GABAergic neurons by sevoflurane inhalation in morphology, Fos (as neuronal activity marker) and GABA neurons double labeling were observed on the brain of glutamic acid decarboxylase (GAD) 67-GFP knock-in mice after sevoflurane inhalation. Twenty GAD67-GFP knock-in mice were divided into three groups: S1 group: incomplete anesthesia state induced by sevoflurane; S2 group: complete anesthesia state induced by sevoflurane; control(C) group. Sevoflurane induced a significant increase of Fos expression in the dorsomedial hypothalamic nucleus (DM), periaqueductal grey (PAG), hippocampus (CA1, DG), paraventricular thalamic nucleus (PV), lateral septal nucleus (LS), and cingulate cortex (Cg1 and Cg2) in S1 group compared to C group, and increase of Fos expression in S2 group compared to S1 group. In S2 group, Fos was only expressed in the medial amygdaloid nucleus (MeA), Edinger-Westphal (E-W) nucleus, arcuate hypothalamic nucleus (Arc) and the ventral part of paraventricular hypothalamic nucleus (PaV). Double immunofluroscent staining indicated that in LS, almost all Fos were present in GABAergic neurons. In CA1, DG, DM, cg1, cg2, and PAG, Fos was expressed as well, but only few were present in GABAergic neurons. Fos expression was very high in thalamus, but no coexistence were found as no GABAergic neuron was detected in this area. Our results provided morphological evidence that GABAergic transmission in specific brain areas may participate in the sevoflurane-induced anesthesia.

Mature neurons polarize by extending an axon and dendrites. In vitro studies of dissociated neurons have demonstrated that axons are initiated from a nonpolarized stage. Dissociated hippocampal neurons form four to five minor neurites shortly after plating but then one of them starts to elongate rapidly to become the future axon, whereas the rest constitutes the dendrites at later stages. However, neuroepithelial cells as well as migrating neurons in vivo are already polarized, raising the possibility that mature neurons inherit the polarities of immature neurons of neuroepithelial or migrating neurons. Here we show that the axon of interneurons in mouse cortical explant emerges from a morphologically nonpolarized shape. The morphological maturation of cortical interneurons labeled by electroporation at an embryonic stage was analyzed by time-lapse imaging during the perinatal stage. In contrast to earlier stages, most interneurons at this stage show sea urchin-like nonpolarized shapes with alternately extending and retracting short processes. Abruptly, one of these processes extends to give rise to an outstandingly long axon-like process. Given that the interneurons exhibit typical polarized shapes during embryonic development, the present results suggest that axon-dendrite polarity develops from a nonpolarized intermediate stage.

The Kv2 voltage-gated potassium channels, Kv2.1 and Kv2.2, are important regulators of neuronal excitability in mammalian brain. It has been shown that Kv2.1 channels are expressed in virtually all neurons in the brain. However, the cellular localization of Kv2.2 has not been fully elucidated. In this article we report that Kv2.2 is highly expressed in a subset of neurons in the magnocellular preoptic nucleus (MCPO) and the horizontal limb of the diagonal band of Broca (HDB) of the basal forebrain complex, which are areas highly implicated in the regulation of cortical activity and the sleep/wake cycle. It has been shown that MCPO and HDB contain distinct populations of neurons that differ in their neurochemicals, cholinergic, glutamatergic, and gamma-aminobutyric acid (GABA)ergic neurons. Using specific immunolabeling and knockin mice in which green fluorescent protein (GFP) is expressed in GABAergic neurons, we found that Kv2.2 is abundantly expressed in a large subpopulation of the GABAergic neurons in the MCPO and HDB. These data offer Kv2.2 as a molecular target to study the role of the specific subpopulation of basal forebrain GABAergic neurons., (© 2010 Wiley-Liss, Inc.)

The developmental period when neuronal responses are modified by visual experience is reported to start and end earlier in layer 4 than in layer 2/3 of the visual cortex, and the maturation of GABAergic inhibitory circuits is suggested to determine the timing of this period. Here, we examine whether the laminar difference in such timing corresponds to a difference in the time course of the functional maturation of GABAergic synaptic transmission to star pyramidal and pyramidal cells in layers 4 and 2/3, respectively, of the mouse visual cortex and whether the development of the strength of GABAergic transmission is affected by visual deprivation in a laminar-specific manner. Our analysis of developmental changes in inhibitory postsynaptic currents of star pyramidal and pyramidal cells evoked by electrical stimulation of afferents or action potentials of fast-spiking GABAergic neurons revealed that there was a sequential maturation of GABAergic function from layers 4 to 2/3. The maturation of inhibition in layer 4 occurred at postnatal week 3, which preceded by 1 week that of layer 2/3. Visual deprivation by dark rearing arrested the functional development of GABAergic transmission in layer 2/3, whereas dark rearing was not so effective in layer 4. GABAergic synapses in layer 2/3 were sensitive to an agonist for cannabinoid type 1 receptors and not normally matured in receptor knock-out mice, whereas those in layer 4 were not so. These results suggest laminar-specific maturation of inhibition and susceptibility to visual deprivation, which may be related to the laminar difference in sensitivity to endocannabinoids.

Glutamatergic synapses form onto both glutamatergic and GABAergic neurons. These two types of glutamatergic synapses differ in their electrical responses to high-frequency stimulation and postsynaptic density protein composition. However, it is not known whether they differ in the actin cytoskeleton composition. In the present study, we used hippocampal neuronal cultures prepared from glutamate decarboxylase 67 (GAD67)-GFP knock-in mice and analyzed the differences in the actin cytoskeleton at glutamatergic synapses contacting GABAergic and glutamatergic neurons. Drebrin-binding actin filaments enriched in dendritic spines are known to play a pivotal role in spine formation. Immunocytochemical analyses demonstrated that drebrin accumulated at glutamatergic synapses on GABAergic neurons as well as at those on glutamatergic neurons. However, the density of drebrin clusters along dendrites in GABAergic neurons was significantly lower than those of glutamatergic neurons. Furthermore, the level of drebrin accumulating at glutamatergic synapses was lower on GABAergic neurons than on glutamatergic neurons. In neurons overexpressing drebrin, drebrin cluster density and accumulation levels in GABAergic and glutamatergic neurons were similar, suggesting that the low drebrin levels in the glutamatergic postsynaptic sites on GABAergic neurons may be because GABAergic neurons express low levels of drebrin. On the other hand, pharmacological analysis demonstrated that the postsynaptic localization of drebrin depended on actin cytoskeleton organization in both GABAergic and glutamatergic neurons. Together these results indicated that, although GABAergic and glutamatergic neurons share common regulatory systems affecting drebrin localization, the density of drebrin-positive glutamatergic synapses formed on GABAergic neurons is lower than those on glutamatergic neurons. This is probably due to the low expression of drebrin in GABAergic neurons., ((c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

GABA plays an important role in inhibitory neurotransmission. In the developing brain, GABA also acts as a paracrine chemical mediator. To evaluate the ambient GABA gradients in the brain, an enzyme-linked imaging system that consisted of GABase and NADP(+) was developed. In rat cerebellar slices, GABA release was observed in the layers containing GABAergic neurons. In telencephalic slices from embryonic GAD67-GFP knock-in mice, ambient GABA levels were high in the ganglionic eminence, where GABA cells are generated, but missing in homozygotes. This study indicates that this method will be useful to study the topography and dynamics of ambient GABA concentrations., (Copyright 2010 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

Neuronal circuits in the cerebral cortex consist mainly of glutamatergic/excitatory and GABAergic/inhibitory neurons. In the visual cortex, the binocular responsiveness of neurons is modified by monocular visual deprivation during the critical period of postnatal development. Although GABAergic neurons are considered to play a key role in the expression of the critical period, it is not known whether their binocular responsiveness and ocular dominance plasticity are different from those of excitatory neurons. Recently, the end of the critical period was found to be not strict so that cortical neurons in the adult still have some ocular dominance plasticity. It is not known, however, which type of neurons or both maintain such plasticity in adulthood. To address these issues, we applied in vivo two-photon functional Ca(2+) imaging to transgenic mice whose GABAergic neurons express a yellow fluorescent protein called Venus. We found that GABAergic neurons are more binocular than excitatory neurons in the normal visual cortex, and both types of neurons show the same degree of modifiability to monocular visual deprivation during the critical period, but the modifiability of GABAergic neurons is stronger than that of excitatory neurons after the end of the critical period.

Inhibitory neurons play important roles in a number of brain functions. They are composed of GABAergic neurons and glycinergic neurons, and vesicular GABA transporter (VGAT) is specifically expressed in these neurons. Since the inhibitory neurons are scattered around in the CNS, it is difficult to identify these cells in living brain preparations. The glutamate decarboxylase (GAD) 67-GFP knock-in mouse has been widely used for the identification of GABAergic neurons, but their GAD67 expression was decreased compared to the wild-type mice. To overcome such a problem and to highlight the function and morphology of inhibitory neurons, we generated four lines of VGAT-Venus transgenic mice (lines #04, #29, #39 and #49) expressing Venus fluorescent protein under the control of mouse VGAT promoter. We found higher expression level of Venus transcripts and proteins as well as brighter fluorescent signal in line #39 mouse brains, compared to brains of other lines examined. By Western blots and spectrofluorometric measurements of forebrain, the line #39 mouse showed stronger GFP immunoreactivity and brighter fluorescent intensity than the GAD67-GFP knock-in mouse. In addition, Venus was present not only in somata, but also in neurites in the line #39 mouse by histological studies. In situ hybridization analysis showed that the expression pattern of Venus in the line #39 mouse was similar to that of endogenous VGAT. Double immunostaining analysis in line #39 mouse showed that Venus-expressing cells are primarily immunoreactive for GABA in cerebral cortex, hippocampus and cerebellar cortex and for GABA or glycine in dorsal cochlear nucleus. These results demonstrate that the VGAT-Venus line #39 mouse should be useful for studies on function and morphology of inhibitory neurons in the CNS.

Analgesic effects of serotonin (5-hydroxytryptamine [5-HT]) type 3 (5-HT3) receptors may involve the release of gamma-aminobutyric acid (GABA) in the spinal dorsal horn. However, the precise synaptic mechanisms for 5-HT3 receptor-mediated spinal analgesia are not clear. In this study, we investigated whether GABAergic neurons in the superficial dorsal horn (SDH) express functional 5-HT3 receptors and how these 5-HT3 receptors affect GABAergic inhibitory synaptic transmission in the SDH, by using slice preparations from adult glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mice. Tight-seal whole cell recordings from GFP-positive and -negative neurons showed that 5-HT3 receptor-specific agonist 2-methyl-serotonin (2-Me-5-HT) induced inward currents in a substantial population of both GFP-positive and -negative neurons. Additionally, we confirmed expression of 5-HT3 receptors in both types of neurons by single-cell reverse transcription-polymerase chain reaction (RT-PCR) analysis. Further, GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs)-both those evoked by electrical stimulation and those occurring spontaneously in tetrodotoxin (i.e., miniature IPSCs [mIPSCs])-were recorded from GFP-negative neurons. 2-Me-5-HT increased the amplitude of the evoked IPSCs and the frequency of mIPSCs. The amplitude of mIPSCs was not affected by 2-Me-5-HT, suggesting that 5-HT augments GABAergic synaptic transmission via presynaptic mechanisms. The present observations indicate that 5-HT3 receptors are expressed on both somadendritic regions and presynaptic terminals of GABAergic neurons and regulate GABAA receptor-mediated inhibitory synaptic transmission in the SDH. Taken together, these results provide clues for the underlying mechanisms of the antinociceptive actions of 5-HT3 receptors in the spinal dorsal horn.

In most CNS regions, the variety of inhibitory interneurons originates from separate pools of progenitors residing in discrete germinal domains, where they become committed to specific phenotypes and positions during their last mitosis. We show here that GABAergic interneurons of the rodent cerebellum are generated through a different mechanism. Progenitors for these interneurons delaminate from the ventricular neuroepithelium of the embryonic cerebellar primordium and continue to proliferate in the prospective white matter during late embryonic and postnatal development. Young postmitotic interneurons do not migrate immediately to their final destination, but remain in the prospective white matter for several days. The different interneuron categories are produced according to a continuous inside-out positional sequence, and cell identity and laminar placement in the cerebellar cortex are temporally related to birth date. However, terminal commitment does not occur while precursors are still proliferating, and postmitotic cells heterochronically transplanted to developing cerebella consistently adopt host-specific phenotypes and positions. However, solid grafts of prospective white matter implanted into the adult cerebellum, when interneuron genesis has ceased, produce interneuron types characteristic of the donor age. Therefore, specification of cerebellar GABAergic interneurons occurs through a hitherto unknown process, in which postmitotic neurons maintain broad developmental potentialities and their phenotypic choices are dictated by instructive cues provided by the microenvironment of the prospective white matter. Whereas in most CNS regions the repertoire of inhibitory interneurons is produced by recruiting precursors from different origins, in the cerebellum it is achieved by creating phenotypic diversity from a single source.

GABAergic interneurons play central roles in the regulation of neuronal activity in the basolateral nucleus of the amygdala (BLA). They are also suggested to be the principal targets of the brainstem noradrenergic afferents which are involved in the enhancement of the BLA-related memory. In addition, behavioral stress has been shown to impair noradrenergic facilitation of GABAergic transmission. However, the noradrenaline (NA) effects in the BLA have not been differentiated among medium- to large-sized GABAergic neurons and principal cells, and remain to be elucidated in terms of their underlying mechanisms. Glutamate decarboxylase 67 (GAD67) is a biosynthetic enzyme of GABA and is specifically expressed in GABAergic neurons. To facilitate the study of the NA effects on GABAergic neurons in live preparations, we generated GAD67-green fluorescent protein (GFP) knock-in mice, in which GFP was expressed under the control of an endogenous GAD67 gene promoter. Here, we show that GFP was specifically expressed in GABAergic neurons in the BLA of this GAD67-GFP knock-in mouse. Under whole-cell patch-clamp recordings in vitro, we identified a certain subpopulation of GABAergic neurons in the BLA chiefly on the basis of the electrophysiological properties. When depolarized by a current injection, these neurons, which are referred to as type A, generated action potentials at relatively low frequency. We found that NA directly excited type-A cells via alpha1-adrenoceptors, whereas its effects on the other types of neurons were negligible. Two ionic mechanisms were involved in this excitability: the activation of nonselective cationic conductance and the suppression of the resting K+ conductance. NA also increased the frequency of spontaneous IPSCs in the principal cells of the BLA. It is suggested that the NA-dependent excitation of type-A cells attenuates the BLA output for a certain period.

Gamma-aminobutyric acid (GABA)ergic neurons are a diverse group of inhibitory neurons playing crucial roles in information processing. We analyzed the gene expression of regionally defined GABAergic neurons from the cortex, olfactory bulb, striatum, and cerebellum of glutamate decarboxylase 67-green fluorescence protein (GAD67-GFP) knock-in mice. We introduce a generally applicable method for singularization of brain cells, flow cytometric enrichment, and global mRNA amplification for sensitive gene expression profiling. Systematic quantification elicited a high dynamic range of GABAergic cell numbers in different brain regions. Clustering of our gene expression results revealed major differences between hind and forebrain GABAergic neurons indicating that the development of GABAergic neurons depends on their regional location. While GABAergic neurons of the forebrain are characterized by three main groups of transcription factors of the Distal-less-family, the POU-family, and ETS/FOX family, specific members of the ZIC- and LHX-family of transcription factors appear to define hindbrain inhibitory neurons.

Background: The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, comprised of SNAP-25, syntaxin 1A, and VAMP-2, has been shown to be responsible for action potential (AP)-dependent, calcium-triggered release of several neurotransmitters. However, this basic fusogenic protein complex may be further specialized to suit the requirements for different neurotransmitter systems, as exemplified by neurons and neuroendocrine cells. In this study, we investigate the effects of SNAP-25 ablation on spontaneous neuronal activity and the expression of functionally distinct isoforms of this t-SNARE in GABAergic and glutamatergic neurons of the adult brain., Results: We found that neurons cultured from Snap25 homozygous null mutant (Snap25-/-) mice failed to develop synchronous network activity seen as spontaneous AP-dependent calcium oscillations and were unable to trigger glial transients following depolarization. Voltage-gated calcium channel (VGCC) mediated calcium transients evoked by depolarization, nevertheless, did not differ between soma of SNAP-25 deficient and control neurons. Furthermore, we observed that although the expression of SNAP-25 RNA transcripts varied among neuronal populations in adult brain, the relative ratio of the transcripts encoding alternatively spliced SNAP-25 variant isoforms was not different in GABAergic and glutamatergic neurons., Conclusion: We propose that the SNAP-25b isoform is predominantly expressed by both mature glutamatergic and GABAergic neurons and serves as a fundamental component of SNARE complex used for fast synaptic communication in excitatory and inhibitory circuits required for brain function. Moreover, SNAP-25 is required for neurons to establish AP-evoked synchronous network activity, as measured by calcium transients, whereas the loss of this t-SNARE does not affect voltage-dependent calcium entry.

The current dominant concept for the control of saccadic eye movements by the basal ganglia is that release from tonic GABAergic inhibition by the substantia nigra pars reticulata (SNr) triggers burst firings of intermediate gray layer (SGI) neurons in the superior colliculus (SC) to allow saccade initiation. This hypothesis is based on the assumption that SNr cells inhibit excitatory projection neurons in the SGI. Here we show that nigrotectal fibers are connected to local GABAergic neurons in the SGI with a similar frequency to non-GABAergic neurons. This was accomplished by applying neuroanatomical tracing and slice electrophysiological experiments in GAD67-green fluorescent protein (GFP) knock-in mice, in which GABAergic neurons specifically express GFP. We also found that GABA(A), but not GABA(B), receptors subserve nigrotectal transmission. The present results revealed a novel aspect on the role of the basal ganglia in the control of saccades, e.g., the SNr not only regulates burst initiation but also modulates the spatiotemporal properties of premotor neurons via connections to local GABAergic neurons in the SC.

To obtain viable GABAergic interneurons in cerebral cortical slices of adult mice, we investigated the effects of slice cutting solutions on the viability of green fluorescent protein (GFP)-expressing cortical neurons in GAD67-GFP knock-in mice. Almost no nuclei of GFP-positive neurons were labeled with propidium iodide in incubated slices, suggesting that GFP fluorescence was a useful indicator for the viability of GABAergic interneurons. When several cutting solutions were compared with saline-based solution, N-methyl-d-glucamine-based sodium-free solution was most effective to keep the number of GFP-positive neurons near the level of perfusion-fixed brain. GFP-positive neurons in slices cut with sodium-free solution were more numerous in cortical layers V-VI, at 30-60 microm depth from the cut surface and 1-6h after cutting than those with saline-based solution. Furthermore, the number of GFP-positive neurons decreased in the cutting condition of high calcium concentration (5mM) or high temperature (37 degrees C), and GFP fluorescence decreased when cut at 0 degrees C. The present results indicate that cutting the brain at 20 degrees C in sodium-free solution is a method for preparing cortical slices with GABAergic interneurons viable. This method would thus be useful for electrophysiological and morphological studies of cortical interneurons in slice preparations of the adult brain.

gamma-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter and also presumed to be a neurotrophic factor. GABA is synthesized by glutamate decarboxylase (GAD). A mouse lacking a 67kDa isoform of GAD (GAD67) has a reduced GABA level in its brain at birth and does not survive postnatally because of cleft palate. In this study, to investigate the functional and developmental roles of GABA in the postnatal cerebellum, selective GAD67 deletion was achieved using a Cre-loxP strategy. In this mouse, GABA level was reduced to 16-44% in the cerebellum but not in the cerebrum. Inhibitory synaptic transmission to Purkinje cells was seriously impaired. However, the morphology of Purkinje cells and the density of synaptic terminals in the cerebellar cortex appeared unaffected, suggesting that GABA does not participate in cerebellar development substantially.

Purpose: Although the presence of gamma-aminobutyrate acid (GABA) in amacrine cells and its co-localization with other neuronal substances is well known, there exists only little information about their quantitative distribution in the mouse eye. The aim of the present study was to characterize GABA-ergic amacrine cells in the retina of the recently introduced glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mouse., Methods: Whole mounts of the retina were prepared and the GFP-positive neurons quantified. Immunofluorescence staining was performed with antibodies against GABA, calbindin (CB), calretinin (CR), parvalbumin (PV), choline acetyl transferase (ChAT), tyrosine hydroxylase (TH), vesicular glutamate transporter (VGluT) 1, VGluT2 and VGluT3., Results: Displaced GABA-ergic amacrine cells in the ganglion cell layer (GCL) showed a density of 1006 +/- 170 cells/mm(2). In the inner nuclear layer (INL), the density of amacrine cells was 8821 +/- 448 cells/mm(2) in the central region and 6825 +/- 408 cells/mm(2) in the peripheral region. GFP-positive amacrine cells co-localized with GABA (99%), CR (INL 18%, GCL 71.3%), CB (INL 6.3%), bNOS (INL 1%, GCL 4%), and ChAT (INL 17%, GCL 92.6%). No co-localization was seen with antibodies against PV, TH, and VGluT 1-3., Conclusions: This study presents the first quantitative data concerning the co-localization of GABA-ergic neurons in the mouse retina with various neuronal markers.

gamma-Aminobutyric acid-ergic (GABAergic) neurons are major inhibitory interneurons that are widely distributed in the central nervous system. The caudal nucleus tractus solitarius (cNTS), which plays a key role in respiratory, cardiovascular, and gastrointestinal function, contains GABAergic neurons for regulation of neuronal firing. In the present study, GABAergic neuronal organization was analyzed in relation to the location of subnuclei in the mouse cNTS. According to the differential expression of glutamate decarboxylase 67 (GAD67), vesicular glutamate transporter 2 (VGLUT2), calbindin, and tyrosine hydroxylase (TH) mRNAs, the cNTS was divided into four subnuclei: the subpostrema, dorsomedial, commissural, and medial subnuclei. The numerical density and size of soma in the four subnuclei were then quantified by an unbiased dissector analysis. Calbindin-positive cells constituted subpopulations of small non-GABAergic neurons preferentially localized in the subpostrema subnucleus. TH-positive cells constituted large neurons preferentially localized in the medial subnucleus. GABAergic neurons constituted a subpopulation of small neurons, preferentially localized in the commissural and medial subnuclei, which represented > or =50% of small cells in these subnuclei. Thus, the GABAergic small neurons were located around TH-positive large cells in the ventrolateral portion of the cNTS. This finding, in combination with results of previous studies in the rat cNTS showing that large cells originate efferents from the cNTS, suggests that GABAergic small neurons in the commissural and medial subnuclei might regulate output from the cNTS.

The mammalian central nervous system is populated with various types of neurons and glia. To investigate the functions and development of individual cells requires gene-expression analysis at the single-cell level. Here, we developed a microarray-based method for the gene-expression profiling of single cells and tested it for GABAergic neuron progenitors. Single GABAergic neuron progenitors were collected from the neocortex of GAD67-GFP knock-in mice by dissociation followed by the aspiration of GFP-positive cells. Complementary DNA from the single cells was amplified by a method in which Super SMART PCR and T7 RNA polymerase amplification were combined at a optimized condition. The cRNA was subjected to microarray hybridization and analysis, which yielded reliable and reproducible results.

Gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter in the brain, is also located in many peripheral nonneuronal tissues. The glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mouse is a useful model for studying the distribution of GABAergic cells in many tissues and organs. The lungs of these mice contain cells with an intense GFP signal exclusively in the airway epithelium. We aimed to characterize the GFP-positive cells and to clarify their relationship with the GABAergic system. We identified the GFP-positive cells as pulmonary neuroendocrine cells (PNECs) by immunohistochemistry for the protein gene product 9.5 and calcitonin gene-related peptide and by ultrastructural analysis. Immunohistochemistry for GADs and GABA revealed GAD65/67 and GABA in GFP-positive PNECs. Reverse transcription-polymerase chain reaction analyses revealed mRNAs encoding the GABA(B) receptor subunits necessary for the assembly of functional receptors, R1 and R2, in the lung. GABA(B) receptor subunit R1 and R2 proteins were expressed in many airway epithelial cells including alveolar epithelial cells other than GFP-positive PNECs. The present findings demonstrated that PNECs in the airway epithelium have a GABA production system and indicated that GABA plays functional roles in airway epithelial cells through GABA(B) receptors.

Consumption of alcohol (ethanol) during pregnancy can lead to developmental defects in the offspring, the most devastating being the constellation of symptoms collectively referred to as fetal alcohol syndrome (FAS). In the brain, a hallmark of FAS is abnormal cerebral cortical morphology consistent with insult during corticogenesis. Here, we report that exposure to a relatively low level of ethanol in utero (average maternal and fetal blood alcohol level of 25 mg/dl) promotes premature tangential migration into the cortical anlage of primordial GABAergic interneurons, including those originating in the medial ganglionic eminence (MGE). This ethanol-induced effect was evident in vivo at embryonic day 14.5 (E14.5) in GAD67 knock-in and BAC-Lhx6 embryos, as well as in vitro in isotypic telencephalic slice cocultures obtained from E14.5 embryos exposed to ethanol in utero. Analysis of heterotypic cocultures indicated that both cell-intrinsic and -extrinsic factors contribute to the aberrant migratory profile of MGE-derived cells. In this light, we provide evidence for an interaction between ethanol exposure in utero and the embryonic GABAergic system. Exposure to ethanol in utero elevated the ambient level of GABA and increased the sensitivity to GABA of MGE-derived cells. Our results uncovered for the first time an effect of ethanol consumption during pregnancy on the embryonic development of GABAergic cortical interneurons. We propose that ethanol exerts its effect on the tangential migration of GABAergic interneurons extrinsically by modulating extracellular levels of GABA and intrinsically by altering GABA(A) receptor function.

Although neocortical GABAergic (gamma-aminobutyric acidergic) interneurons have been the focus of intense study, especially in the rat, a consensus view of the functional diversity and organization of inhibitory cortical neurons has not yet been achieved. To better analyze GABAergic neurons in the rat, we used a bacterial artificial chromosome (BAC) construct and established 2 lines of transgenic rats that coexpress Venus, a yellow fluorescent protein, with the vesicular GABA transporter. The brain GABA content from both transgenic lines was similar to the level found in wild-type rats. In the frontal cortex, Venus was expressed in >95% of GABAergic neurons, most of which also expressed at least one of 6 biochemical markers, including alpha-actitin-2, which preferentially labeled late-spiking neurogliaform cells. Taking advantage of the fact that Venus expression allows for targeted recording from all classes of nonpyramidal cells, irrespective of their somatic morphologies, we demonstrated that fast-spiking neurons, which were heterogeneous in somatic size as well as vertical dendritic projection, had relatively uniform horizontal dimensions, suggesting a cell type-specific columnar input territory. Our data demonstrate the benefits of VGAT-Venus rats for investigating GABAergic circuits, as well as the feasibility of using BAC technology in rats to label subsets of specific, genetically defined neurons.

Long-term potentiation (LTP) of excitatory synapses on GABAergic neurons in layer II/III of visual cortical slices was examined in GAD67-GFP knock-in mice by whole-cell recordings of EPSPs evoked by layer IV stimulation. Theta burst stimulation (TBS) paired with postsynaptic depolarization induced LTP in 14 of 19 fast-spiking GABAergic (FS-GABA) neurons, whereas only in 6 of 17 non-FS GABAergic neurons. The mean magnitude of LTP in the former cell group was larger than that in the latter. The paired-pulse stimulation protocol and coefficient of variation analysis indicated that LTP of excitatory synapses on FS-GABA neurons may be postsynaptic in origin. Filling postsynaptic cells with a Ca2+-chelator blocked the induction of LTP, suggesting an involvement of postsynaptic Ca2+ rise. The developmental analysis of LTP indicated that almost the same magnitude of LTP was induced after postnatal day 17 to the young adulthood, suggesting no age dependence after eye opening. This form of LTP was dependent neither on NMDA receptors nor voltage-gated Ca2+ channels (L and T types). An antagonist for type 5 metabotropic glutamate receptors (mGluR5) blocked this form of LTP, whereas an antagonist for mGluR1 was not effective. An agonist for mGluR1/5 induced potentiation of EPSPs of FS-GABA neurons in concentration- and use-dependent manners. This potentiation and TBS-induced LTP occluded each other. Further pharmacological analyses suggested that this form of LTP at FS-GABA neurons is induced through an activation of mGluR5, which triggers Ca2+ release from internal stores via activations of phospholipase C and inositol triphosphate.

We investigated the CO2 responsiveness of GABAergic neurons in the ventral medullary surface (VMS), a putative chemoreceptive area using a 67-kDa isoform of GABA-synthesizing enzyme (GAD67)-green fluorescence protein (GFP) knock-in neonatal mouse, in which GFP is specifically expressed in GABAergic neurons. The slice was prepared by transversely sectioning at the level of the rostral rootlet of the XII nerve and the rostral end of the inferior olive in mock cerebrospinal fluid (CSF). Each medullary slice was continuously superfused with hypocapnic CSF. GFP-positive neurons in the VMS were selected by using fluorescent optics and their membrane potentials and firing activities were analyzed with a perforated patch recording technique. Thereafter, superfusion was changed from hypocapnic to hypercapnic CSF. In 4 out of 8 GABAergic neurons in the VMS, perfusion with hypercapnic CSF induced more than a 20% decrease in the discharge frequency and hyperpolarized the neurons. The remaining 4 GFP-positive neurons were CO2-insensitive. GABAergic neurons in the VMS have chemosensitivity. Inhibition of chemosensitive GABAergic neural activity in the VMS may induce increases in respiratory output in response to hypercapnia.

Recent experiments suggest that brainstem GABAergic neurons may control rapid-eye-movement (REM) sleep. However, understanding their pharmacology/physiology has been hindered by difficulty in identification. Here we report that mice expressing green fluorescent protein (GFP) under the control of the GAD67 promoter (GAD67-GFP knock-in mice) exhibit numerous GFP-positive neurons in the central gray and reticular formation, allowing on-line identification in vitro. Small (10-15 microm) or medium-sized (15-25 microm) GFP-positive perikarya surrounded larger serotonergic, noradrenergic, cholinergic and reticular neurons, and > 96% of neurons were double-labeled for GFP and GABA, confirming that GFP-positive neurons are GABAergic. Whole-cell recordings in brainstem regions important for promoting REM sleep [subcoeruleus (SubC) or pontine nucleus oralis (PnO) regions] revealed that GFP-positive neurons were spontaneously active at 3-12 Hz, fired tonically, and possessed a medium-sized depolarizing sag during hyperpolarizing steps. Many neurons also exhibited a small, low-threshold calcium spike. GFP-positive neurons were tested with pharmacological agents known to promote (carbachol) or inhibit (orexin A) REM sleep. SubC GFP-positive neurons were excited by the cholinergic agonist carbachol, whereas those in the PnO were either inhibited or excited. GFP-positive neurons in both areas were excited by orexins/hypocretins. These data are congruent with the hypothesis that carbachol-inhibited GABAergic PnO neurons project to, and inhibit, REM-on SubC reticular neurons during waking, whereas carbachol-excited SubC and PnO GABAergic neurons are involved in silencing locus coeruleus and dorsal raphe aminergic neurons during REM sleep. Orexinergic suppression of REM during waking is probably mediated in part via excitation of acetylcholine-inhibited GABAergic neurons.

GABAergic neurons are the major inhibitory interneurons that are widely distributed in the central nervous system. It is well established that they originate from a focal region in the embryonic forebrain during development, and then migrate to other regions such as the neocortex. However, the migration of GABAergic neurons remains obscure in other axial levels of the brain. We examined the early development of myelencephalic GABAergic neurons using glutamate decarboxylase 67 / green fluorescent protein (GAD67-GFP) knocking mice. Observation of fixed tissues in coronal sections and flat whole-mount preparations indicated that, while GFP-positive cells are restricted to the subpial region in the ventral aspect of the myelencephalon at an early stage, they spread dorsally and eventually occupy the entire region of the myelencephalon as development proceeds. We developed a flat-mount in vitro preparation in which these patterns of development could be recapitulated. Transplantation of dorsal myelencephalic tissue of a wildtype embryo to a corresponding region of GAD67-GFP mouse embryos clearly demonstrated invasion of dorsally oriented GABAergic neurons from host to donor tissue. These results indicate that ventral-to-dorsal tangential migration of GABAergic neurons takes place in the myelencephalon. Our results extend the observations in the forebrain that inhibitory and excitatory neurons in a specific brain compartment take distinct migratory paths., ((c) 2007 Wiley-Liss, Inc.)