A reduction in hippocampal GABAA receptor alpha5 subunits disrupts the memory for location of objects in mice.

The memory for location of objects, which binds information about objects to discrete positions or spatial contexts of occurrence, is a form of episodic memory particularly sensitive to hippocampal damage. Its early decline is symptomatic for elderly dementia. Substances that selectively reduce alpha5-GABA(A) receptor function are currently developed as potential cognition enhancers for Alzheimer's syndrome and other dementia, consistent with genetic studies implicating these receptors that are highly expressed in hippocampus in learning performance. Here we explored the consequences of reduced GABA(A)alpha5-subunit contents, as occurring in alpha5(H105R) knock-in mice, on the memory for location of objects. This required the behavioral characterization of alpha5(H105R) and wild-type animals in various tasks examining learning and memory retrieval strategies for objects, locations, contexts and their combinations. In mutants, decreased amounts of alpha5-subunits and retained long-term potentiation in hippocampus were confirmed. They exhibited hyperactivity with conserved circadian rhythm in familiar actimeters, and normal exploration and emotional reactivity in novel places, allocentric spatial guidance, and motor pattern learning acquisition, inhibition and flexibility in T- and eight-arm mazes. Processing of object, position and context memories and object-guided response learning were spared. Genotype difference in object-in-place memory retrieval and in encoding and response learning strategies for object-location combinations manifested as a bias favoring object-based recognition and guidance strategies over spatial processing of objects in the mutants. These findings identify in alpha5(H105R) mice a behavioral-cognitive phenotype affecting basal locomotion and the memory for location of objects indicative of hippocampal dysfunction resulting from moderately decreased alpha5-subunit contents.

Distribution of soluble and microsomal epoxide hydrolase in the mouse brain and its contribution to cerebral epoxyeicosatrienoic acid metabolism.

Epoxide hydrolases comprise a family of enzymes important in detoxification and conversion of lipid signaling molecules, namely epoxyeicosatrienoic acids (EETs), to their supposedly less active form, dihydroxyeicosatrienoic acids (DHETs). EETs control cerebral blood flow, exert analgesic, anti-inflammatory and angiogenic effects and protect against ischemia. Although the role of soluble epoxide hydrolase (sEH) in EET metabolism is well established, knowledge on its detailed distribution in rodent brain is rather limited. Here, we analyzed the expression pattern of sEH and of another important member of the EH family, microsomal epoxide hydrolase (mEH), in mouse brain by immunohistochemistry. To investigate the functional relevance of these enzymes in brain, we explored their individual contribution to EET metabolism in acutely isolated brain cells from respective EH -/- mice and wild type littermates by mass spectrometry. We find sEH immunoreactivity almost exclusively in astrocytes throughout the brain, except in the central amygdala, where neurons are also positive for sEH. mEH immunoreactivity is abundant in brain vascular cells (endothelial and smooth muscle cells) and in choroid plexus epithelial cells. In addition, mEH immunoreactivity is present in specific neuronal populations of the hippocampus, striatum, amygdala, and cerebellum, as well as in a fraction of astrocytes. In freshly isolated cells from hippocampus, where both enzymes are expressed, sEH mediates the bulk of EET metabolism. Yet we observe a significant contribution of mEH, pointing to a novel role of this enzyme in the regulation of physiological processes. Furthermore, our findings indicate the presence of additional, hitherto unknown cerebral epoxide hydrolases. Taken together, cerebral EET metabolism is driven by several epoxide hydrolases, a fact important in view of the present targeting of sEH as a potential therapeutic target. Our findings suggest that these different enzymes have individual, possibly quite distinct roles in brain function and cerebral EET metabolism.

Activin tunes GABAergic neurotransmission and modulates anxiety-like behavior.

Activin, a member of the transforming growth factor-beta superfamily, affords neuroprotection in acute brain injury, but its physiological functions in normal adult brain are largely unknown. Using transgenic (tg) mice expressing a dominant-negative activin receptor mutant under the control of the CaMKIIalpha promoter in forebrain neurons, we identified activin as a key regulator of gamma-aminobutyric acid (GABA)ergic synapses and anxiety-like behavior. In the open field, wild-type (wt) and tg mice did not differ in spontaneous locomotion and exploration behavior. However, tg mice visited inner fields significantly more often than wt mice. In the light-dark exploration test, tg mice made more exits, spent significantly more time on a well-lit elevated bar and went farther away from the dark box as compared to wt mice. In addition, the anxiolytic effect of diazepam was abrogated in tg mice. Thus the disruption of activin receptor signaling produced a low-anxiety phenotype that failed to respond to benzodiazepines. In whole-cell recordings from hippocampal pyramidal cells, enhanced spontaneous GABA release, increased GABA tonus, reduced benzodiazepine sensitivity and augmented GABA(B) receptor function emerged as likely substrates of the low-anxiety phenotype. These data provide strong evidence that activin influences pre- and postsynaptic components of GABAergic synapses in a highly synergistic fashion. Given the crucial role of GABAergic neurotransmission in emotional states, anxiety and depression, dysfunctions of activin receptor signaling could be involved in affective disorders: and drugs affecting this pathway might show promise for psychopharmacological treatment.

Normal sleep homeostasis and lack of epilepsy phenotype in GABA A receptor alpha3 subunit-knockout mice.

Thalamo-cortical networks generate specific patterns of oscillations during distinct vigilance states and epilepsy, well characterized by electroencephalography (EEG). Oscillations depend on recurrent synaptic loops, which are controlled by GABAergic transmission. In particular, GABA A receptors containing the alpha3 subunit are expressed predominantly in cortical layer VI and thalamic reticular nucleus (nRT) and regulate the activity and firing pattern of neurons in relay nuclei. Therefore, ablation of these receptors by gene targeting might profoundly affect thalamo-cortical oscillations. Here, we investigated the role of alpha3-GABA A receptors in regulating vigilance states and seizure activity by analyzing chronic EEG recordings in alpha3 subunit-knockout (alpha3-KO) mice. The presence of postsynaptic alpha3-GABA A receptors/gephyrin clusters in the nRT and GABA A-mediated synaptic currents in acute thalamic slices was also examined. EEG spectral analysis showed no difference between genotypes during non rapid-eye movement (NREM) sleep or at waking-NREM sleep transitions. EEG power in the spindle frequency range (10-15 Hz) was significantly lower at NREM-REM sleep transitions in mutant compared with wild-type mice. Enhancement of sleep pressure by 6 h sleep deprivation did not reveal any differences in the regulation of EEG activities between genotypes. Finally, the waking EEG showed a slightly larger power in the 11-13-Hz band in alpha3-KO mice. However, neither behavior nor the waking EEG showed alterations suggestive of absence seizures. Furthermore, alpha3-KO mice did not differ in seizure susceptibility in a model of temporal lobe epilepsy. Strikingly, despite the disruption of postsynaptic gephyrin clusters, whole-cell patch clamp recordings revealed intact inhibitory synaptic transmission in the nRT of alpha3-KO mice. These findings show that the lack of alpha3-GABA(A) receptors is extensively compensated for to preserve the integrity of thalamo-cortical function in physiological and pathophysiological situations.

Cortical glutamatergic neurons mediate the motor sedative action of diazepam.

The neuronal circuits mediating the sedative action of diazepam are unknown. Although the motor-depressant action of diazepam is suppressed in alpha1(H101R) homozygous knockin mice expressing diazepam-insensitive alpha1-GABA(A) receptors, global alpha1-knockout mice show greater motor sedation with diazepam. To clarify this paradox, attributed to compensatory up-regulation of the alpha2 and alpha3 subunits, and to further identify the neuronal circuits supporting diazepam-induced sedation, we generated Emx1-cre-recombinase-mediated conditional mutant mice, selectively lacking the alpha1 subunit (forebrain-specific alpha1(-/-)) or expressing either a single wild-type (H) or a single point-mutated (R) alpha1 allele (forebrain-specific alpha1(-/H) and alpha1(-/R) mice, respectively) in forebrain glutamatergic neurons. In the rest of the brain, alpha1(-/R) mutants are heterozygous alpha1(H101R) mice. Forebrain-specific alpha1(-/-) mice showed enhanced diazepam-induced motor depression and increased expression of the alpha2 and alpha3 subunits in the neocortex and hippocampus, in comparison with their pseudo-wild-type littermates. Forebrain-specific alpha1(-/R) mice were less sensitive than alpha1(-/H) mice to the motor-depressing action of diazepam, but each of these conditional mutants had a similar behavioral response as their corresponding control littermates. Unexpectedly, expression of the alpha1 subunit was reduced in forebrain, notably in alpha1(-/R) mice, and the alpha3 subunit was up-regulated in neocortex, indicating that proper alpha1 subunit expression requires both alleles. In conclusion, conditional manipulation of GABA(A) receptor alpha1 subunit expression can induce compensatory changes in the affected areas. Specifically, alterations in GABA(A) receptor expression restricted to forebrain glutamatergic neurons reproduce the behavioral effects seen after a global alteration, thereby implicating these neurons in the motor-sedative effect of diazepam.

GABAergic phenotype of periglomerular cells in the rodent olfactory bulb.

Periglomerular (PG) cells in the rodent olfactory bulb are heterogeneous anatomically and neurochemically. Here we investigated whether major classes of PG cells use gamma-aminobutyric acid (GABA) as a neurotransmitter. In addition to three known subtypes of PG cells expressing tyrosine hydroxylase (TH), calbindin D-28k (CB), and calretinin (CR), we identified a novel PG cell population containing the GABAA receptor alpha5 subunit. Consistent with previous studies in the rat, we found that TH-positive cells were also labeled with antibodies against GABA, whereas PG cells expressing CB or the alpha5 subunit were GABA-negative. Using GAD67-GFP knockin mice, we found that all PG cell subtypes expressed GAD67-GFP. Calretinin labeled the major fraction (44%) of green fluorescent protein (GFP)-positive cells, followed by TH (16%), CB (14%), and the alpha5 subunit (13%). There was no overlap between these neuronal populations, which accounted for approximately 85% of GAD67-GFP-positive cells. We then demonstrated that PG cells labeled for TH, CB, or CR established dendrodendritic synapses expressing glutamic acid decarboxylase (GAD) or the vesicular inhibitory amino acid transporter, VGAT, irrespective of their immunoreactivity for GABA. In addition, CB-, CR-, and TH-positive dendrites were apposed to GABAA receptor clusters containing the alpha1 or alpha3 subunits, which are found in mitral and tufted cells, and the alpha2 subunit, which is expressed by PG cells. Together, these findings indicate that all major subtypes of PG cells are GABAergic. In addition, they show that PG cells provide GABAergic input to the dendrites of principal neurons and are interconnected with other GABAergic interneurons, which most likely are other PG cells.

Intestinal expression of cytochrome P450 enzymes and ABC transporters and carbamazepine and phenytoin disposition.

OBJECTIVES: Interindividual variability in intestinal absorption and bioavailability might contribute to inadequate control of seizures under treatment with carbamazepine and phenytoin. We therefore correlated intestinal expression levels and genetics of CYP3A4, CYP2C9/19, MDR1 and MRP2 with dose requirement and plasma levels of carbamazepine and phenytoin. MATERIALS AND METHODS: Epileptic patients on carbamazepine (n = 29) or phenytoin (n = 15) were stratified into a 'high'-dose (carbamazepine > or =800 mg/day, phenytoin > or =300 mg/day) and a 'low'-dose group (carbamazepine < or =600 mg/day, phenytoin < or =200 mg/day). Duodenal biopsies and DNA were obtained for Western blotting and genotyping studies. RESULTS: Low carbamazepine plasma levels showed a trend towards higher intestinal MDR1 expression (P = 0.06). Furthermore, carbamazepine dose was positively correlated with MRP2 expression (P = 0.1). Moreover, MDR1 expression and carbamazepine and phenytoin dose requirement was influenced by the genotype in position 2677 and 3435 of the MDR1 gene. CONCLUSION: Differences in intestinal MDR1 and MRP2 expression may influence carbamazepine and phenytoin disposition and may account for interindividual pharmacokinetic variability.

Shift of adenosine kinase expression from neurons to astrocytes during postnatal development suggests dual functionality of the enzyme.

Adenosine is a potent modulator of excitatory neurotransmission, especially in seizure-prone regions such as the hippocampal formation. In adult brain ambient levels of adenosine are controlled by adenosine kinase (ADK), the major adenosine-metabolizing enzyme, expressed most strongly in astrocytes. Since ontogeny of the adenosine system is largely unknown, we investigated ADK expression and cellular localization during postnatal development of the mouse brain, using immunofluorescence staining with cell-type specific markers. At early postnatal stages ADK immunoreactivity was prominent in neurons, notably in cerebral cortex and hippocampus. Thereafter, as seen best in hippocampus, ADK gradually disappeared from neurons and appeared in newly developed nestin- and glial fibrillary acidic protein (GFAP)-positive astrocytes. Furthermore, the region-specific downregulation of neuronal ADK coincided with the onset of myelination, as visualized by myelin basic protein staining. After postnatal day 14 (P14), the transition from neuronal to astrocytic ADK expression was complete, except in a subset of neurons that retained ADK until adulthood in specific regions, such as striatum. Moreover, neuronal progenitors in the adult dentate gyrus lacked ADK. Finally, recordings of excitatory field potentials in acute slice preparations revealed a reduced adenosinergic inhibition in P14 hippocampus compared with adult. These findings suggest distinct roles for adenosine in the developing and adult brain. First, ADK expression in young neurons may provide a salvage pathway to utilize adenosine in nucleic acid synthesis, thus supporting differentiation and plasticity and influencing myelination; and second, adult ADK expression in astrocytes may offer a mechanism to regulate adenosine levels as a function of metabolic needs and synaptic activity, thus contributing to the differential resistance of young and adult animals to seizures.

Role of dystrophin and utrophin for assembly and function of the dystrophin glycoprotein complex in non-muscle tissue.

The dystrophin glycoprotein complex (DGC) is a multimeric protein assembly associated with either the X-linked cytoskeletal protein dystrophin or its autosomal homologue utrophin. In striated muscle cells, the DGC links the extracellular matrix to the actin cytoskeleton and mediates three major functions: structural stability of the plasma membrane, ion homeostasis, and transmembrane signaling. Mutations affecting the DGC underlie major forms of congenital muscle dystrophies. The DGC is prominent also in the central and peripheral nervous system and in tissues with a secretory function or which form barriers between functional compartments, such as the blood-brain barrier, choroid plexus, or kidney. A considerable molecular heterogeneity arises from cell-specific expression of its constituent proteins, notably short C-terminal isoforms of dystrophin. Experimentally, the generation of mice carrying targeted gene deletions affecting the DGC has clarified the interdependence of DGC proteins for assembly of the complex and revealed its importance for brain development and regulation of the 'milieu intérieur. Here, we focus on recent studies of the DGC in brain, blood-brain barrier and choroid plexus, retina, and kidney and discuss the role of dystrophin isoforms and utrophin for assembly of the complex in these tissues.

Differential, strain-specific cellular and subcellular distribution of multidrug transporters in murine choroid plexus and blood-brain barrier.

Multidrug transporters of the ATP-binding cassette family play an important role in regulating drug distribution and efflux in the brain, owing to their selective distribution in microvessels and choroid plexus. Their expression pattern and cellular distribution remain controversial, in part due to technical difficulties in localizing these membrane proteins in closely associated cells, such as endothelial cells and astrocytic end-feet at the blood-brain barrier. Here, we used high-resolution immunofluorescence staining with cell-type specific markers to investigate the distribution of major ATP-binding cassette transporters in mouse brain. We report that four ATP-binding cassette transporters, Mdr1, Mrp1, Mrp2 and Mrp5 can be detected in brain endothelial cells, forming three distinct layers, with Mdr1 and Mrp5 being located on the luminal side, Mrp1 on the abluminal (basal) side, and Mrp2 in between. Mrp3 and Mdr3 were undetectable. In choroid plexus, only Mrp1, Mrp2 and Mrp3 were detected, again with a differential distribution. Mrp1 was targeted basolaterally in epithelial cells, Mrp2 was restricted to endothelial cells, and Mrp3 was co-localized with zonula occludens-1 at tight junctions. Analysis of Mdr1a(0/0) and Mrp1(0/0) mice, generated in the FVB strain, revealed no major alteration in expression of the remaining transporters. An unexpected strain difference was unraveled, with wildtype FVB mice selectively lacking Mrp2 protein in brain, but not liver. In conclusion, these results indicate that ATP-binding cassette transporters provide multiple penetration barriers in the blood-brain barrier and choroid plexus, with a selective cellular and subcellular distribution, emphasizing their potential role for drug resistance in neurological disorders, such as epilepsy.

A schizophrenia-related sensorimotor deficit links alpha 3-containing GABAA receptors to a dopamine hyperfunction.

Overactivity of the dopaminergic system in the brain is considered to be a contributing factor to the development and symptomatology of schizophrenia. Therefore, the GABAergic control of dopamine functions was assessed by disrupting the gene encoding the alpha3 subunit of the GABA(A) receptor. alpha3 knockout (alpha3KO) mice exhibited neither an obvious developmental defect nor apparent morphological brain abnormalities, and there was no evidence for compensatory up-regulation of other major GABA(A)-receptor subunits. Anxiety-related behavior in the elevated-plus-maze test was undisturbed, and the anxiolytic-like effect of diazepam, which is mediated by alpha2-containing GABA(A) receptors, was preserved. As a result of the loss of alpha3 GABA(A) receptors, the GABA-induced whole-cell current recorded from midbrain dopamine neurons was significantly reduced. Spontaneous locomotor activity was slightly elevated in alpha3KO mice. Most notably, prepulse inhibition of the acoustic startle reflex was markedly attenuated in the alpha3KO mice, pointing to a deficit in sensorimotor information processing. This deficit was completely normalized by treatment with the antipsychotic D2-receptor antagonist haloperidol. The amphetamine-induced hyperlocomotion was not altered in alpha3KO mice compared with WT mice. These results suggest that the absence of alpha3-subunit-containing GABA(A) receptors induces a hyperdopaminergic phenotype, including a severe deficit in sensorimotor gating, a common feature among psychiatric conditions, including schizophrenia. Hence, agonists acting at alpha3-containing GABA(A) receptors may constitute an avenue for an effective treatment of sensorimotor-gating deficits in various psychiatric conditions.

Pathophysiology and pharmacology of GABA(A) receptors.

By controlling spike timing and sculpting neuronal rhythms, inhibitory interneurons play a key role in brain function. GABAergic interneurons are highly diverse. The respective GABA(A) receptor subtypes, therefore, provide new opportunities not only for understanding GABA-dependent pathophysiologies but also for targeting of selective neuronal circuits by drugs. The pharmacological relevance of GABA(A) receptor subtypes is increasingly being recognized. A new central nervous system pharmacology is on the horizon. The development of anxiolytic drugs devoid of sedation and of agents that enhance hippocampus-dependent learning and memory has become a novel and highly selective therapeutic opportunity.

The role of utrophin and Dp71 for assembly of different dystrophin-associated protein complexes (DPCs) in the choroid plexus and microvasculature of the brain.

In the brain, utrophin is present in the choroid plexus epithelium and vascular endothelial cells, whereas the short C-terminal isoform of dystrophin (Dp71) is localized in the glial end-feet surrounding blood vessels. Both proteins serve as anchors for the so-called dystrophin-associated protein complex (DPC), composed of isoforms of syntrophin, dystroglycan and dystrobrevin. Numerous transporter proteins and channels have a polarized distribution in vascular endothelial cells and in glial end-feet, suggesting an association with the DPC. We investigated the composition and localization of the DPC in dependence on the anchoring proteins in mice lacking either utrophin (utrophin0/0) or dystrophin isoforms (mdx3Cv). Three distinct complexes were identified: (i) associated with utrophin in the basolateral membrane of the choroid plexus epithelium, (ii) associated with utrophin in vascular endothelial cells, and (iii) associated with Dp71 in the glial end-feet. Upon ablation of utrophin or Dp71, the corresponding DPCs were disrupted and no compensation of the missing protein by its homologue was observed. Association of the water channel aquaporin 4 with the glial DPC likewise was disrupted in mdx3Cv mice. These results demonstrate the essential role of utrophin and Dp71 for assembly of the DPC and suggest that these proteins contribute to the proper functioning of the cerebrospinal fluid and blood-brain barriers.

Specific GABA(A) circuits in brain development and therapy.

GABAergic interneurons are highly diverse and operate with a corresponding diversity of GABA(A) receptor subtypes in controlling behaviour. In this article, we review the significance of GABA(A) receptor heterogeneity for neural circuit development and central nervous system pharmacology. GABA(A) receptor subtypes were identified as selective targets for behavioural actions of benzodiazepines and of selected intravenous anesthetic agents using point mutations which render a specific receptor subtype insensitive to the action of the respective drugs and also by novel subtype-selective ligands. The pharmacological separation of anxiolysis and sedation guides the development of novel anxiolytics, while inverse agonism at extrasynaptic GABA(A) receptors involved in learning and memory is currently being evaluated as a novel therapeutic principle for symptomatic memory enhancement.

Cellular localization of GABA receptor alpha subunit immunoreactivity in the rat hypothalamus: relationship with neurones containing orexigenic or anorexigenic peptides.

gamma-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the brain, acts via two different type of GABA receptors. GABA(A) receptors are composed of five subunits that belong to eight different classes. Depending on their subunit composition, distinct pharmacological and electrophysiological properties are obtained. GABA is produced in certain hypothalamic neurones known to be involved in control of feeding behaviour. We report the detailed immunohistochemical localization of four GABA(A)R alpha subunits in hypothalamic regions associated with the regulation of feeding behaviour. Immunoreactive structures for all studied GABA(A)R alpha subunits were observed in the hypothalamus, but with subunit-specific staining patterns. GABA(A)R alpha(1) immunoreactivity was most prominent in the dorsomedial hypothalamic nucleus and in the lateral hypothalamic area (LHA), whereas GABA(A)R alpha(2), alpha(3) and alpha(5) subunits exhibited particularly strong immunoreactivity in the ventromedial hypothalamic nucleus. In comparison, GABA(A)R alpha subunit immunoreactivities were generally weak in the arcuate nucleus. In the ventromedial part of the arcuate nucleus, neuropeptide Y- and agouti-related peptide-containing cell bodies, which also are known to be GABAergic, were immunoreactive for only the GABA(A)R alpha(3) subunit, whereas pro-opiomelanocortin- and cocaine- and amphetamine-regulated transcript- containing cell bodies located in the ventrolateral subdivision of the arcuate nucleus, showed GABA(A)R alpha(1), alpha(2) and alpha(3) subunit immunoreactivity. In the LHA, GABA(A)R alpha(3) subunit immunoreactivity was demonstrated in both melanin-concentrating hormone (MCH) and orexin-containing neurones. In addition, MCH neurones contained GABA(A)R alpha(2) immunoreactivity. In neurones of the tuberomammillary nucleus, GABA(A)R alpha(2) and alpha(5) subunits were colocalized with histidine decarboxylase, a marker for histamine-containing neurones.

Organization of GABA receptor alpha-subunit clustering in the developing rat neocortex and hippocampus.

We compared the expression and co-expression of alpha1, alpha2, alpha3, and alpha5-subunit protein clusters of the gamma-aminobutyric acid (GABA)(A) receptor in the neocortex and hippocampus of rat at postnatal days (PND) 5-10 and 30-40 in order to understand how inhibitory receptors reorganize during brain maturation. The size, intensity, density and pattern of co-localization of fluorescently tagged subunit clusters were determined in deconvolved digital images using a novel 2D cross-correlational analysis. The cross-correlation analysis allowed an unbiased identification of GABA(A) receptor subunit clusters based on staining intensity. Cluster size increased through development; only the alpha2 clusters in dentate gyrus (DG) decreased in size. alpha5-subunit cluster density either increased or decreased with maturation depending on the brain region. For the other subunits, the cluster density remained rather constant, with noted exceptions (increase in alpha2 clusters in cortical layer 5 but a decrease of alpha3 clusters in hilus). The co-localization of alpha1-subunit with the others was unique and not correlated to overall changes in subunit abundance between developmental époques. So, although alpha2-subunit expression went up in the DG, the clusters became less co-localized with alpha1. In contrast, alpha5-subunit clusters became more co-localized with alpha1 as the alpha5-subunit expression declined in cortex and CA1. The co-localization of alpha3 with alpha1 also became greater in layer 6. In the adult brain not all clustering was associated with synapses, as many alpha-subunit clusters did not co-localize with synaptophysin. Overall, these data indicate that the regulation of GABA(A) receptor clustering is both synaptic and extrasynaptic, presumably reflecting complex cellular trafficking mechanisms.

Presynaptic membrane of inhibitory crayfish axon terminals is stained by antibodies raised against mammalian GABA(A) receptor subunits alpha3 and beta(2/3).

The opener muscle of the dactyl of the walking leg of crayfish is innervated by one excitatory axon releasing glutamate and one inhibitory axon releasing GABA. Functional GABA(A) receptors are present postsynaptically on the muscle and presynaptically on terminals and release boutons of the excitatory axon, whereas presynaptic GABA(A) autoreceptors have not been reported on terminals or release boutons of the inhibitory axon. Using antibodies raised against mammalian GABA(A) receptor subunits alpha3 and beta(2/3), we obtained highly specific staining of the presynaptic membrane of the inhibitory bouton and of the postsynaptic membrane of the muscle. Using pre- and postembedding techniques, staining was localized to only presynaptic and postsynaptic membranes of synaptic active zones. We also found extrasynaptic receptor subunit immunoreactivity near (up to 100 nm) to the active zones. Staining with antibodies for the alpha3 and beta(2/3) subunits showed colocalization of particles of the two subunits. We suggest that presynaptic inhibitory boutons of the crayfish possess GABA(A)-like autoreceptors composed of at least the alpha3 and beta(2/3) subunits.

Pre- and post-synaptic mechanisms regulating the clustering of type A gamma-aminobutyric acid receptors (GABAA receptors).

Postsynaptic clustering of GABAA (type A gamma-aminobutyric acid) receptors is essential to ensure proper function of GABAergic synapses. This process is initiated during synapse formation and is maintained throughout life. The tubulin-associated protein gephyrin is required for clustering of GABAA receptors, but its specific role in this process is not understood. A second protein associated selectively with GABAA receptors at postsynaptic sites is dystrophin. It is present in a subset of GABAergic synapses along with several partners, forming the dystrophin-associated protein complex. In this review, we discuss recent advances in the role of neuronal activity and trans-synaptic signaling for the clustering of gephyrin and dystrophin during synaptogenesis and on the role of these proteins for plasticity and maintenance of mature synapses.

Presynaptic and postsynaptic GABAA receptors in rat suprachiasmatic nucleus.

The mammalian suprachiasmatic nucleus (SCN), the brain's circadian clock, is composed mainly of GABAergic neurons, that are interconnected via synapses with GABA(A) receptors. Here we report on the subcellular localization of these receptors in the SCN, as revealed by an extensively characterized antibody to the alpha 3 subunit of GABA(A) receptors in conjunction with pre- and postembedding electron microscopic immunocytochemistry. GABA(A) receptor immunoreactivity was observed in neuronal perikarya, dendritic processes and axonal terminals. In perikarya and proximal dendrites, GABA(A) receptor immunoreactivity was expressed mainly in endoplasmic reticulum and Golgi complexes, while in the distal part of dendrites, immunoreaction product was associated with postsynaptic plasma membrane. Many GABAergic axonal terminals, as revealed by postembedding immunogold labeling, displayed GABA(A) receptor immunoreactivity, associated mainly with the extrasynaptic portion of their plasma membrane. The function of these receptors was studied in hypothalamic slices using whole-cell patch-clamp recording of the responses to minimal stimulation of an area dorsal to the SCN. Analysis of the evoked inhibitory postsynaptic currents showed that either bath or local application of 100 microM of GABA decreased GABAergic transmission, manifested as a two-fold increase in failure rate. This presynaptic effect, which was detected in the presence of the glutamate receptor blocker 6-cyano-7-nitroquinoxaline-2,3-dione and the selective GABA(B) receptor blocker CGP55845A, appears to be mediated via activation of GABA(A) receptors. Our results thus show that GABA(A) receptors are widely distributed in the SCN and may subserve both pre- and postsynaptic roles in controlling the mammalian circadian clock.

GABAergic and glutamatergic terminals differentially influence the organization of GABAergic synapses in rat cerebellar granule cells in vitro.

Synapse formation in CNS neurons requires appropriate sorting and clustering of neurotransmitter receptors and associated proteins at postsynaptic sites. In GABAergic synapses, clustering of GABA(A) receptors requires gephyrin, but it is not known whether presynaptic signals are also involved in this process. To investigate this issue, we analyzed the subcellular distribution of GABA(A) receptors and gephyrin in primary cultures of cerebellar granule cells, by comparing cells receiving GABAergic input with cells devoid of such afferents. Using immunofluorescence staining, we show that the GABA(A) receptor alpha1 and gamma2 subunit, but not alpha6 or delta subunit, form clusters co-localized with gephyrin in granule cell neurites, irrespective of the presence of GABAergic axons. GABAergic terminals typically were surrounded by groups of gephyrin clusters, pointing to the presence of multiple synaptic sites. In contrast, in neurites devoid of GABAergic input, gephyrin clusters were distributed at random and apposed to glutamatergic terminals, suggesting the formation of mismatched synapses. Both populations of gephyrin clusters were co-localized with GABA(A) receptor subunits, indicating that these proteins are associated also in non-GABAergic synapses. To determine whether signaling mediated by GABA(A) receptors is required for the formation of appropriately matched gephyrin clusters, cultures were treated chronically with bicuculline, or with either muscimol or 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol. All these treatments failed to influence the distribution of gephyrin clusters. We conclude that although GABAergic presynaptic terminals have a preponderant influence on the distribution of gephyrin clusters in dendrites of cerebellar granule cells, GABA transmission is dispensable for postsynaptic clustering of gephyrin and GABA(A) receptors and for the formation of appropriately matched GABAergic synapses.