Activity-Dependent Bidirectional Regulation of GAD Expression in a Homeostatic Fashion Is Mediated by BDNF-Dependent and Independent Pathways

Homeostatic synaptic plasticity, or synaptic scaling, is a mechanism that tunes neuronal transmission to compensate for prolonged, excessive changes in neuronal activity. Both excitatory and inhibitory neurons undergo homeostatic changes based on synaptic transmission strength, which could effectively contribute to a fine-tuning of circuit activity. However, gene regulation that underlies homeostatic synaptic plasticity in GABAergic (GABA, gamma aminobutyric) neurons is still poorly understood. The present study demonstrated activity-dependent dynamic scaling in which NMDA-R (N-methyl-D-aspartic acid receptor) activity regulated the expression of GABA synthetic enzymes: glutamic acid decarboxylase 65 and 67 (GAD65 and GAD67). Results revealed that activity-regulated BDNF (brain-derived neurotrophic factor) release is necessary, but not sufficient, for activity-dependent up-scaling of these GAD isoforms. Bidirectional forms of activity-dependent GAD expression require both BDNF-dependent and BDNF-independent pathways, both triggered by NMDA-R activity. Additional results indicated that these two GAD genes differ in their responsiveness to chronic changes in neuronal activity, which could be partially caused by differential dependence on BDNF. In parallel to activity-dependent bidirectional scaling in GAD expression, the present study further observed that a chronic change in neuronal activity leads to an alteration in neurotransmitter release from GABAergic neurons in a homeostatic, bidirectional fashion. Therefore, the differential expression of GAD65 and 67 during prolonged changes in neuronal activity may be implicated in some aspects of bidirectional homeostatic plasticity within mature GABAergic presynapses.     

GABAergic inhibition regulates developmental synapse elimination in the cerebellum

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.     

Proteolytic degradation of glutamate decarboxylase mediates disinhibition of hippocampal CA3 pyramidal cells in cathepsin D-deficient mice

Although of clinical importance, little is known about the mechanism of seizure in neuronal ceroid lipofuscinosis (NCL). In the present study, we have attempted to elucidate the mechanism underlying the seizure of cathepsin D-deficient (CD-/-) mice that show a novel type of lysosomal storage disease with a phenotype resembling late infantile NCL. In hippocampal slices prepared from CD-/- mice at post-natal day (P)24, spontaneous burst discharges were recorded from CA3 pyramidal cells. At P24, the mean amplitude of IPSPs after stimulation of the mossy fibres was significantly smaller than that of wild-type mice, which was substantiated by the decreased level of gamma-aminobutyric acid (GABA) contents in the hippocampus measured by high-performance liquid chromatography (HPLC). At this stage, activated microglia were found to accumulate in the pyramidal cell layer of the hippocampal CA3 subfield of CD-/- mice. However, there was no significant change in the numerical density of GABAergic interneurons in the CA3 subfield of CD-/- mice at P24, estimated by counting the number of glutamate decarboxylase (GAD) 67-immunoreactive somata. In the hippocampus and the cortex of CD-/- mice at P24, some GABAergic interneurons displayed extremely high somatic granular immunoreactivites for GAD67, suggesting the lysosomal accumulation of GAD67. GAD67 levels in axon terminals abutting on to perisomatic regions of hippocampal CA3 pyramidal cells was not significantly changed in CD-/- mice even at P24, whereas the total protein levels of GAD67 in both the hippocampus and the cortex of CD-/- mice after P24 were significantly decreased as a result of degradation. Furthermore, the recombinant human GAD65/67 was rapidly digested by the lysosomal fraction prepared from the whole brain of wild-type and CD-/- mice. These observations strongly suggest that the reduction of GABA contents, presumably because of lysosomal degradation of GAD67 and lysosomal accumulation of its degraded forms, are responsible for the dysfunction of GABAergic interneurons in the hippocampal CA3 subfield of CD-/- mice.     

GABAergic signaling in the pulmonary neuroepithelial body microenvironment: functional imaging in GAD67-GFP mice

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system (CNS) of vertebrates, but has also been reported in multiple cell types outside the CNS. A GABAergic system has been proposed in neuroepithelial bodies (NEBs) in monkey lungs. Pulmonary NEBs are known as complex intraepithelial sensory airway receptors and are part of the NEB microenvironment. Aim of the present study was to unravel a GABAergic signaling system in the NEB microenvironment in mouse lungs, enabling the use of genetically modified animals for future functional studies. Immunostaining of mouse lungs revealed that glutamic acid decarboxylase 65/67 (GAD65/67), a rate-limiting enzyme in the biosynthesis of GABA, and the vesicular GABA transporter (VGAT) were exclusively expressed in NEB cells. In GAD67-green fluorescent protein (GFP) knock-in mice, all pulmonary NEBs appeared to express GFP. For confocal live cell imaging, ex vivo vibratome lung slices of GAD67-GFP mice can be directly loaded with fluorescent functional probes, e.g. a red-fluorescent calcium dye, without the necessity of time-consuming prior live visualization of NEBs. RT-PCR of the NEB microenvironment obtained by laser microdissection revealed the presence of both GABA_A and GABA_B (R1 and R2) receptors, which was confirmed by immunostaining. In conclusion, the present study not only revealed the presence of a GABAergic signaling pathway, but also the very selective expression of GFP in pulmonary NEBs in a GAD67-GFP mouse model. Different proof of concept experiments have clearly shown that adoption of the GAD67-GFP mouse model will certainly boost future functional imaging and gene expression analysis of the mouse NEB microenvironment.     

The spatiotemporal segregation of GAD forms defines distinct GABA signaling functions in the developing mouse olfactory system and provides novel insights into the origin and migration of GnRH neurons

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. Develop Neurobiol 75: 249–270, 2015     

Characterization of GABAergic Neurons in the Mouse Lateral Septum: A Double Fluorescence In Situ Hybridization and Immunohistochemical Study Using Tyramide Signal Amplification

Gamma-aminobutyric acid (GABA) neurotransmission in the lateral septum (LS) is implicated in modulating various behavioral processes, including emotional reactivity and maternal behavior. However, identifying the phenotype of GABAergic neurons in the CNS has been hampered by the longstanding inability to reliably detect somal immunoreactivity for GABA or glutamic acid decarboxylase (GAD), the enzyme that produces GABA. In this study, we designed unique probes for both GAD65 (GAD2) and GAD67 (GAD1), and used fluorescence in Situ hybridization (FISH) with tyramide signal amplification (TSA) to achieve unequivocal detection of cell bodies of GABAergic neurons by GAD mRNAs. We quantitatively characterized the expression and chemical phenotype of GABAergic neurons across each subdivision of LS and in cingulate cortex (Cg) and medial preoptic area (MPOA) in female mice. Across LS, almost all GAD65 mRNA-expressing neurons were found to contain GAD67 mRNA (approximately 95-98%), while a small proportion of GAD67 mRNA-containing neurons did not express GAD65 mRNA (5-14%). Using the neuronal marker NeuN, almost every neuron in LS (> 90%) was also found to be GABA-positive. Interneuron markers using calcium-binding proteins showed that LS GABAergic neurons displayed immunoreactivity for calbindin (CB) or calretinin (CR), but not parvalbumin (PV); almost all CB- or CR-immunoreactive neurons (98-100%) were GABAergic. The proportion of GABAergic neurons immunoreactive for CB or CR varied depending on the subdivisions examined, with the highest percentage of colocalization in the caudal intermediate LS (LSI) (approximately 58% for CB and 35% for CR). These findings suggest that the vast majority of GABAergic neurons within the LS have the potential for synthesizing GABA via the dual enzyme systems GAD65 and GAD67, and each subtype of GABAergic neurons identified by distinct calcium-binding proteins may exert unique roles in the physiological function and neuronal circuitry of the LS.     

Glutamate Decarboxylase Distribution in Discrete Motor Nuclei in the Cat Brain

Abstract: The distribution of activity of glutamate decarboxylase (GAD), the enzyme synthesising γ-aminobutyric acid (GABA), was measured in the cat brain by means of microdissection of the structures from frozen slices and a radioisotopic assay for the enzyme. About 20 cerebral regions were chosen for study because of their role in sensorimotor integration. GAD presented an uneven distribution among these areas. Highest activities were found in the basal ganglia, particularly in the substantia nigra and in the globus pallidus, and to a lesser extent in the cerebellum. Relatively low levels of the enzyme were found in the thalamus and in the cerebral motor cortex. Special detailed studies were made in the caudate nucleus, the substantia nigra, and in the red nucleus for the purpose of defining the intranuclear distribution of their GABAergic innervation. There were only small differences in the rostro-caudal distribution of the enzyme in the head of the caudate nucleus but GAD activity was higher in the ventral than in the dorsal part of the structure. In the substantia nigra, GAD activity was high in both the medial and intermediate thirds of the structure. The GAD activity decreased from the caudal to the rostral part of the nucleus. GAD levels were lower in the caudal part of the red nucleus than in the rostral part. These results indicate that GABA would be present as a putative neurotransmitter in many motor nuclei of the cat brain. In view of the general inhibitory action of this amino acid, this could be related to the presence of inhibitory responses widely distributed in these nuclei as identified by mean of electrophysiological studies. The origin of these GABAergic innervations in many cases remains to be determined.     

Post-translational modification of glutamic acid decarboxylase 67 by intermittent hypoxia: evidence for the involvement of dopamine D1 receptor signaling

Intermittent hypoxia (IH) associated with sleep apnea leads to cardio-respiratory morbidities. Previous studies have shown that IH alters the synthesis of neurotransmitters including catecholamines and neuropeptides in brainstem regions associated with regulation of cardio-respiratory functions. GABA, a major inhibitory neurotransmitter in the CNS, has been implicated in cardio-respiratory control. GABA synthesis is primarily catalyzed by glutamic acid decarboxylase (GAD). In this study, we tested the hypothesis that IH like its effect on other transmitters also alters GABA synthesis. The impact of IH on GABA synthesis was investigated in pheochromocytoma 12 cells, a neuronal cell line which is known to express active form of GAD67 in the cytosolic fraction and also assessed the underlying mechanisms contributing to IH-evoked response. Exposure of cell cultures to IH decreased GAD67 activity and GABA level. IH-evoked decrease in GAD67 activity was caused by increased cAMP - protein kinase A (PKA) - dependent phosphorylation of GAD67, but not as a result of changes in either GAD67 mRNA or protein expression. PKA inhibitor restored GAD67 activity and GABA levels in IH treated cells. Pheochromocytoma 12 cells express dopamine 1 receptor (D1R), a G-protein coupled receptor whose activation increased adenylyl cyclase activity. Treatment with either D1R antagonist or adenylyl cyclase inhibitor reversed IH-evoked GAD67 inhibition. Silencing D1R expression with siRNA reversed cAMP elevation and GAD67 inhibition by IH. These results provide evidence for the role of D1R-cAMP-PKA signaling in IH-mediated inhibition of GAD67 via protein phosphorylation resulting in down-regulation of GABA synthesis.     

Two distinct mechanisms target GAD67 to vesicular pathways and presynaptic clusters

The inhibitory neurotransmitter γ-amino butyric acid (GABA) is synthesized by two isoforms of the enzyme glutamic acid decarboxylase (GAD): GAD65 and GAD67. Whereas GAD67 is constitutively active and produces >90% of GABA in the central nervous system, GAD65 is transiently activated and augments GABA levels for rapid modulation of inhibitory neurotransmission. Hydrophobic lipid modifications of the GAD65 protein target it to Golgi membranes and synaptic vesicles in neuroendocrine cells. In contrast, the GAD67 protein remains hydrophilic but has been shown to acquire membrane association by heterodimerization with GAD65. Here, we identify a second mechanism that mediates robust membrane anchoring, axonal targeting, and presynaptic clustering of GAD67 but that is independent of GAD65. This mechanism is abolished by a leucine-103 to proline mutation that changes the conformation of the N-terminal domain but does not affect the GAD65-dependent membrane anchoring of GAD67. Thus two distinct mechanisms target the constitutively active GAD67 to presynaptic clusters to facilitate accumulation of GABA for rapid delivery into synapses.     

The GAD65 knock out mouse – a model for GABAergic processes in fear‐ and stress‐induced psychopathology

The γ‐amino butyric acid (GABA) synthetic enzyme glutamic acid decarboxylase (GAD)65 is critically involved in the activity‐dependent regulation of GABAergic inhibition in the central nervous system. It is also required for the maturation of the GABAergic system during adolescence, a phase that is critical for the development of several neuropsychiatric diseases. Mice bearing a null mutation of the GAD65 gene develop hyperexcitability of the amygdala and hippocampus, and a phenotype of increased anxiety and pathological fear memory reminiscent of posttraumatic stress disorder. Although genetic association of GAD65 in human has not yet been reported, these findings are in line with observations of reduced GABAergic function in these brain regions of anxiety disorder patients. The particular value of GAD65(?/?) mice thus lies in modeling the effects of reduced GABAergic function in the mature nervous system. The expression of GAD65 and a second GAD isozyme, GAD67, are differentially regulated in response to stress in limbic brain areas suggesting that by controlling GABAergic inhibition these enzymes determine the vulnerability for the development of pathological anxiety and other stress‐induced phenotypes. In fact, we could recently show that GAD65 haplodeficiency, which results in delayed postnatal increase of GABA levels, provides resilience to juvenile‐stress‐induced anxiety to GAD65(+/?) mice thus foiling the increased fear and anxiety in homozygous GAD65(?/?) mice.     

Identification of rat ventral tegmental area GABAergic neurons

The canonical two neuron model of opioid reward posits that mu opioid receptor (MOR) activation produces reward by disinhibiting midbrain ventral tegmental area (VTA) dopamine neurons through inhibition of local GABAergic interneurons. Although indirect evidence supports the neural circuit postulated by this model, its validity has been called into question by growing evidence for VTA neuronal heterogeneity and the recent demonstration that MOR agonists inhibit GABAergic terminals in the VTA arising from extrinsic neurons. In addition, VTA MOR reward can be dopamine-independent. To directly test the assumption that MOR activation directly inhibits local GABAergic neurons, we investigated the properties of rat VTA GABA neurons directly identified with either immunocytochemistry for GABA or GAD65/67, or in situ hybridization for GAD65/67 mRNA. Utilizing co-labeling with an antibody for the neural marker NeuN and in situ hybridization against GAD65/67, we found that 23±3% of VTA neurons are GAD65/67(+). In contrast to the assumptions of the two neuron model, VTA GABAergic neurons are heterogeneous, both physiologically and pharmacologically. Importantly, only 7/13 confirmed VTA GABA neurons were inhibited by the MOR selective agonist DAMGO. Interestingly, all confirmed VTA GABA neurons were insensitive to the GABA(B) receptor agonist baclofen (0/6 inhibited), while all confirmed dopamine neurons were inhibited (19/19). The heterogeneity of opioid responses we found in VTA GABAergic neurons, and the fact that GABA terminals arising from neurons outside the VTA are inhibited by MOR agonists, make further studies essential to determine the local circuit mechanisms underlying VTA MOR reward.     

Biochemical and Immunochemical Studies on the GABAergic System in the Rat Fallopian Tube and Ovary

gamma-Aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD) activities were measured in the ovary and the Fallopian tube of rats and compared with brain values. GABA levels in the Fallopian tube were about twice as high as in the brain, while in the ovary they represented only about 5% of the amino acid content of the CNS. In vitro decarboxylation of glutamate, measured via CO2 formation, occurred both in the Fallopian tube and in the ovary. These two organs contained, respectively, 10% and 1% of brain GAD activity. However, the actual formation of GABA from glutamate in a high-speed supernatant was detectable only in the Fallopian tube, where it represented about 5% of brain GAD activity. In contrast with the enzyme present in ovary, liver, anterior pituitary, and kidney, that in the Fallopian tube was quantitatively precipitated by a specific antiserum directed against rat neuronal GAD. Moreover, subcutaneous transplantation resulted in a quantitative decrease of both GABA levels and GAD activity in the Fallopian tube while no change occurred in the ovary, and vagus nerve section induced a 50% decrease of GAD activity in the Fallopian tube, although GABA levels were not significantly altered. The findings suggest an extrinsic GABAergic innervation in the rat Fallopian tube but not in the ovary.     

GABA Synthesis in Astrocytes After Infection with Defective Herpes Simplex Virus Vectors Expressing Glutamic Acid Decarboxylase 65 or 67

Abstract: Defective herpes simplex virus (HSV) vectors containing glutamic acid decarboxylase (GAD) cDNAs, either GAD65 or GAD67, were used to examine GAD function and GABA synthesis in rat cortical astrocytes, CNS cells that do not endogenously synthesize GABA. GAD vector infection resulted in isoform-specific expression of GAD as determined by western blotting and immunohistochemistry. Astrocytes infected with a β-galactosidase vector or uninfected expressed no GAD and contained no detectable GABA. GABA was detected in glial fibrillary acid protein-expressing cells after GAD65 vector infection. Significant amounts of GABA, as determined by HPLC, were synthesized in cultures infected with either GAD vector. The levels of GABA in GAD67 vector-infected cells were almost twofold higher than in GAD65 vector-infected cells. Vector infection did not alter levels of other intracellular amino acids. GABA was tonically released from astrocytes infected with the GAD67 vector, but no increase in release could be detected after treatment of the cells with K⁺, veratridine, glutamate, or bradykinin. The ability to transduce astrocytes so that they express GAD and thereby increase GABA levels provides a potential strategy for the treatment of neurologic disorders associated with hyperexcitable or diminished inhibitory activity.     

Mammalian retinal horizontal cells are unconventional GABAergic neurons

Lateral interactions at the first retinal synapse have been initially proposed to involve GABA by transporter-mediated release from horizontal cells, onto GABA(A) receptors expressed on cone photoreceptor terminals and/or bipolar cell dendrites. However, in the mammalian retina, horizontal cells do not seem to contain GABA systematically or to express membrane GABA transporters. We here report that mouse retinal horizontal cells express GAD65 and/or GAD67 mRNA, and were weakly but consistently immunostained for GAD65/67. While GABA was readily detected after intracardiac perfusion, it was lost during classical preparation for histology or electrophysiology. It could not be restored by incubation in a GABA-containing medium, confirming the absence of membrane GABA transporters in these cells. However, GABA was synthesized de novo from glutamate or glutamine, upon addition of pyridoxal 5'-phosphate, a cofactor of GAD65/67. Mouse horizontal cells are thus atypical GABAergic neurons, with no functional GABA uptake, but a glutamate and/or glutamine transport system allowing GABA synthesis, probably depending physiologically from glutamate released by photoreceptors. Our results suggest that the role of GABA in lateral inhibition may have been underestimated, at least in mammals, and that tissue pre-incubation with glutamine and pyridoxal 5'-phosphate should yield a more precise estimate of outer retinal processing.     

Transient increase in expression of a glutamate decarboxylase (GAD) mRNA during the postnatal development of the rat striatum.

We recently reported that the mammalian brain has two forms of the GABA synthetic enzyme glutamate decarboxylase (GAD, E.C. 4.1.1.15), which are the products of two genes. The two forms, which we call GAD65 and GAD67, differ from each other in sequence, molecular size, subcellular distribution, and interactions with the cofactor pyridoxal phosphate (PLP), with GAD65 activity more dependent than that of GAD67 on the continued presence of exogenous PLP. The existence of two GAD genes suggests that individual GABA neurons may be subject to differential regulation of GABA production. We have examined the expression of these two forms of GAD during postnatal development of the rat striatum to determine whether different classes of GABA neurons selectively express different amounts of the two GAD mRNAs. Here we present evidence for a dramatic developmental difference in the expression of the two mRNAs during postnatal development of the rat striatum. Using in situ hybridization to the two GAD mRNAs, we observed a selective increase in GAD65 mRNA during the second postnatal week, at the time when striatal matrix neurons innervate the substantia nigra (SN). PLP-dependent enzyme activity in the midbrain increases in parallel with increased expression of GAD65 mRNA in the striatum. We hypothesize that the innervation of the SN by striatal neurons triggers an increase in GAD65. The changing ratios of GAD65 and GAD67 in the striatum may contribute to the well-documented changes in seizure susceptibility that occur in early life.     

First direct demonstration of extensive GABA synthesis in mouse cerebellar neuronal cultures

Culturing mouse cerebellar neurones (predominantly glutamatergic) in the presence of [1-(13)C]glucose for 7 days resulted in a surprisingly extensive labelling of the inhibitory neurotransmitter GABA, the average content and labelling of which were 20 +/- 4 nmol/mg protein and 20 +/- 4%, respectively. Cultures of neocortical neurones (predominantly GABAergic) had under similar conditions a GABA content and labelling of 32 +/- 2 nmol/mg protein and 21 +/- 2%. The cerebellar cultures contained only 6% glutamate decarboxylase (GAD)-positive neurones when immunolabelled using a GAD67 antibody, while a dense network of neurones in the neocortical cultures stained positively for GAD67. Exposure of the cerebellar cultures to 50 microm kainic acid (KA) which is known to eliminate vesicular release of GABA, only marginally affected GABA labelling and cellular content. Likewise this treatment had no effect on the number of GAD67-positive neurones but a massive punctate immunostaining observed in control cultures was essentially eliminated. Increasing the KA concentration to 0.5 mm in the culture medium for 7 days led to a reduction of GABA labelling and content compared to cerebellar cultures not exposed to KA. Although it is likely that this large capacity for GABA synthesis resides in the relatively few GAD-positive neurones, it seems unlikely that they could account for the large average GABA content in the cultures. Therefore it must be concluded that the newly synthesized GABA is redistributed among the majority of the cells in these cultures, i.e. the glutamatergic neurones.     

GAD65 is essential for synthesis of GABA destined for tonic inhibition regulating epileptiform activity

GABA is synthesized from glutamate by glutamate decarboxylase (GAD), which exists in two isoforms, that is, GAD65 and GAD67. In line with GAD65 being located in the GABAergic synapse, several studies have demonstrated that this isoform is important during sustained synaptic transmission. In contrast, the functional significance of GAD65 in the maintenance of GABA destined for extrasynaptic tonic inhibition is less well studied. Using GAD65-/- and wild type GAD65+/+ mice, this was examined employing the cortical wedge preparation, a model suitable for investigating extrasynaptic GABA(A) receptor activity. An impaired tonic inhibition in GAD65-/- mice was revealed demonstrating a significant role of GAD65 in the synthesis of GABA acting extrasynaptically. The correlation between an altered tonic inhibition and metabolic events as well as the functional and metabolic role of GABA synthesized by GAD65 was further investigated in vivo. Tonic inhibition and the demand for biosynthesis of GABA were augmented by injection of kainate into GAD65-/- and GAD65+/+ mice. Moreover, [1-(13) C]glucose and [1,2-(13) C]acetate were administered to study neuronal and astrocytic metabolism concomitantly. Subsequently, cortical and hippocampal extracts were analyzed by NMR spectroscopy and mass spectrometry, respectively. Although seizure activity was induced by kainate, neuronal hypometabolism was observed in GAD65+/+ mice. In contrast, kainate evoked hypermetabolism in GAD65-/- mice exhibiting deficiencies in tonic inhibition. These findings underline the importance of GAD65 for synthesis of GABA destined for extrasynaptic tonic inhibition, regulating epileptiform activity.