Jean-Marc Fritschy

Benzodiazepine actions mediated by specific gamma-aminobutyric acidA receptor subtypes

GABAA (γ-aminobutyric acidA) receptors are molecular substrates for the regulation of vigilance, anxiety, muscle tension, epileptogenic activity and memory functions, which is evident from the spectrum of actions elicited by clinically effective drugs acting at their modulatory benzodiazepine-binding site. Here we show, by introducing a histidine-to-arginine point mutation at position 101 of the murine α1-subunit gene, that α1-type GABAA receptors, which are mainly expressed in cortical areas and thalamus, are rendered insensitive to allosteric modulation by benzodiazepine-site ligands, whilst regulation by the physiological neurotransmitter γ-aminobutyric acid is preserved. α1(H101R) mice failed to show the sedative, amnesic and partly the anticonvulsant action of diazepam. In contrast, the anxiolytic-like, myorelaxant, motor-impairing and ethanol-potentiating effects were fully retained, and are attributed to the nonmutated GABAA receptors found in the limbic system (α2, α5), in monoaminergic neurons (α3) and in motoneurons (α2, α5). Thus, benzodiazepine-induced behavioural responses are mediated by specific GABAA receptor subtypes in distinct neuronal circuits, which is of interest for drug design.

Postsynaptic clustering of major GABA A receptor subtypes requires the γ2 subunit and gephyrin

Most fast inhibitory neurotransmission in the brain is mediated by GABA A receptors, which are mainly postsynaptic and consist of diverse α and ß subunits together with the γ2 subunit. Although the γ2 subunit is not necessary for receptor assembly and translocation to the cell surface, we show here that it is required for clustering of major postsynaptic GABAA receptor subtypes. Loss of GABAA receptor clusters in mice deficient in the γ2 subunit, and in cultured cortical neurons from these mice, is paralleled by loss of the synaptic clustering molecule gephyrin and synaptic GABAergic function. Conversely, inhibiting gephyrin expression causes loss of GABAA receptor clusters. The γ2 subunit and gephyrin are thus interdependent components of the same synaptic complex that is critical for postsynaptic clustering of abundant subtypes of GABAA receptors in vivo.

Trace fear conditioning involves hippocampal α5 GABAA receptors

The heterogeneity of γ-aminobutyric acid type A (GABAA) receptors contributes to the diversity of neuronal inhibition in the regulation of information processing. Although most GABAA receptors are located synaptically, the small population of α5GABAA receptors is largely expressed extrasynaptically. To clarify the role of the α5GABAA receptors in the control of behavior, a histidine-to-arginine point mutation was introduced in position 105 of the murine α5 subunit gene, which rendered the α5GABAA receptors diazepam-insensitive. Apart from an incomplete muscle relaxing effect, neither the sedative, anticonvulsant, nor anxiolytic-like activity of diazepam was impaired in α5(H105R) mice. However, in hippocampal pyramidal cells, the point mutation resulted in a selective reduction of α5GABAA receptors, which altered the drug-independent behavior. In line with the role of the hippocampus in certain forms of associative learning, trace fear conditioning, but not delay conditioning or contextual conditioning, was facilitated in the mutant mice. Trace fear conditioning differs from delay conditioning in that the conditioned and unconditioned stimulus are separated by a time interval. Thus, the largely extrasynaptic α5GABAA receptors in hippocampal pyramidal cells are implicated as control elements of the temporal association of threat cues in trace fear conditioning.

NMDA receptor heterogeneity during postnatal development of the rat brain : Differential expression of the NR2A, NR2B, and NR2C subunit proteins

Abstract: Changes in the expression of the NMDA receptor subunits (NRs) NR2A, 2B, and 2C were investigated in histo blots of the developing rat brain with subunit‐specific antisera. At birth, the NR2B subunit was detected almost ubiquitously, the NR2A subunit staining was faint and restricted to the hippocampus, cerebral cortex, and striatum, and no NR2C subunit immunoreactivity was detected. During the first 3 postnatal weeks, the NR2B subunit became confined to forebrain structures, whereas the NR2A immunoreactivity became abundantly expressed throughout the brain. The NR2C immunoreactivity emerged 5 days after birth in the olfactory bulb, thalamus, and vestibular nuclei and became very intense after 10 days in cerebellar granule cells, its primary site of expression in adulthood. After 3 weeks, NR2A and NR2B immunoreactivity decreased to adult levels, whereas NR2C immunoreactivity remained unchanged. The patterns of distribution of the subunit proteins were in agreement with those of their corresponding mRNAs, as monitored by in situ hybridization histochemistry, although the mRNA translation appeared to be delayed by several days in certain areas. Our results reveal a progressive increase in the heterogeneity of NMDA receptors due to the comparably late onset of NR2A and NR2C subunit expression and by the area‐specific rearrangement of NR2B subunit expression following birth.

Synapse‐specific localization of NMDA and GABAA receptor subunits revealed by antigen‐retrieval immunohistochemistry

Conventional immunohistochemistry provides little evidence for the synaptic localization of ionotropic neurotransmitter receptors, suggesting that their epitopes are not readily accessible in situ. Here, we have adapted antigen retrieval procedures based on microwave irradiation to enhance the immunohistochemical staining of γ‐aminobutyric acid type A (GABAA) and N‐methyl‐D‐aspartate (NMDA) receptor subunits in rat brain tissue. Microwave irradiation of fixed tissue produced a marked reduction of nonspecific staining, allowing an improved detection of GABAA receptor subunits. However, staining of NMDA receptor subunits remained suboptimal. In contrast, microwave irradiation of cryostat sections prepared from fresh tissue resulted in a major enhancement of both NMDA and GABAA receptor subunit staining. The diffuse, partially intracellular signals were largely replaced by numerous, intensely immunoreactive puncta outlining neuronal somata and dendrites, highly suggestive of synaptic receptors. In hippocampus CA1–CA3 fields, the NR2A and NR2B subunit positive puncta exhibited an extensive colocalization in the stratum oriens and radiatum, whereas pyramidal cell bodies, which receive no excitatory synapses, were unstained. In addition, the NR2A subunit, but not the NR2B subunit, was selectively detected on pyramidal cell dendrites in the stratum lucidum of CA3, suggesting a selective targeting to sites of mossy fiber input. For the GABAA receptor subunits, the most striking change induced by this protocol was the selective staining of the axon initial segment of cortical and hippocampal pyramidal cells. The α2 subunit immunoreactivity was particularly prominent in these synapses. In control experiments, the staining of cytoskeletal proteins (neurofilaments, glial fibrillary acid protein) was not influenced by prior microwave irradiation. The enhancement of cell‐surface–associated staining is therefore strongly suggestive of an ‘unmasking’ of subunit epitopes by the microwave treatment. These results reveal a remarkable specificity in the synaptic targeting of NMDA and GABAA receptor subunits in hippocampal and neocortical neurons, suggesting that individual neurons can express multiple receptor subtypes in functionally distinct synapses. J. Comp. Neurol. 390:194–210, 1998.

Reversal of pathological pain through specific spinal GABAA receptor subtypes

Inflammatory diseases and neuropathic insults are frequently accompanied by severe and debilitating pain, which can become chronic and often unresponsive to conventional analgesic treatment. A loss of synaptic inhibition in the spinal dorsal horn is considered to contribute significantly to this pain pathology. Facilitation of spinal γ-aminobutyric acid (GABA)ergic neurotransmission through modulation of GABAA receptors should be able to compensate for this loss. With the use of GABAA-receptor point-mutated knock-in mice in which specific GABAA receptor subtypes have been selectively rendered insensitive to benzodiazepine-site ligands, we show here that pronounced analgesia can be achieved by specifically targeting spinal GABAA receptors containing the α2 and/or α3 subunits. We show that their selective activation by the non-sedative (‘α1-sparing’) benzodiazepine-site ligand L-838,417 (ref. 13) is highly effective against inflammatory and neuropathic pain yet devoid of unwanted sedation, motor impairment and tolerance development. L-838,417 not only diminished the nociceptive input to the brain but also reduced the activity of brain areas related to the associative-emotional components of pain, as shown by functional magnetic resonance imaging in rats. These results provide a rational basis for the development of subtype-selective GABAergic drugs for the treatment of chronic pain, which is often refractory to classical analgesics.

Formation and plasticity of GABAergic synapses: physiological mechanisms and pathophysiological implications

gamma-Aminobutyric acid(A) (GABA(A)) receptors mediate most of the fast inhibitory neurotransmission in the CNS. They represent a major site of action for clinically relevant drugs, such as benzodiazepines and ethanol, and endogenous modulators, including neuroactive steroids. Alterations in GABA(A) receptor expression and function are thought to contribute to prevalent neurological and psychiatric diseases. Molecular cloning and immunochemical characterization of GABA(A) receptor subunits revealed a multiplicity of receptor subtypes with specific functional and pharmacological properties. A major tenet of these studies is that GABA(A) receptor heterogeneity represents a key factor for fine-tuning of inhibitory transmission under physiological and pathophysiological conditions. The aim of this review is to highlight recent findings on the regulation of GABA(A) receptor expression and function, focusing on the mechanisms of sorting, targeting, and synaptic clustering of GABA(A) receptor subtypes and their associated proteins, on trafficking of cell-surface receptors as a means of regulating synaptic (and extrasynaptic) transmission on a short-time basis, on the role of endogenous neurosteroids for GABA(A) receptor plasticity, and on alterations of GABA(A) receptor expression and localization in major neurological disorders. Altogether, the findings presented in this review underscore the necessity of considering GABA(A) receptor-mediated neurotransmission as a dynamic and highly flexible process controlled by multiple mechanisms operating at the molecular, cellular, and systemic level. Furthermore, the selected topics highlight the relevance of concepts derived from experimental studies for understanding GABA(A) receptor alterations in disease states and for designing improved therapeutic strategies based on subtype-selective drugs.

Localization of the organic anion transporting polypeptide 2 (Oatp2) in capillary endothelium and choroid plexus epithelium of rat brain.

In this study we investigated the distribution of a recently cloned polyspecific organic anion transporting polypeptide (Oatp2) in rat brain by nonradioactive in situ hybridization histochemistry and immunofluorescence microscopy. The results demonstrate that Oatp 2 is expressed in brain capillary and in plexus epithelial cells. At the blood-brain barrier (BBB), Oatp 2 expression could be co-localized with the endothelial marker vWF (von Willebrand factor) but not with the astrocyte marker GFAP (glial fibrillary acidic protein). In choroid plexus epithelial cells, Oatp 2 could be localized to the basolateral cell pole, whereas the first member of the Oatp gene family of membrane transporters to be cloned (Oatp 1) co-localized with the α1subunit of Na,K-ATPase at the apical plasma membrane domain. Because Oatp 1 and Oatp 2 have been previously shown to mediate transmembrane transport of a wide variety of amphipathic organic compounds, including many drugs and other xenobiotics, the histochemical localization of Oatp 2 at the BBB and of Oatp 1 and Oatp 2 in the choroid plexus imply a role for these transporters in the active exchange of amphipathic solutes between the blood, brain, and cerebrospinal fluid compartments.

Intact sorting, targeting, and clustering of γ-aminobutyric acid a receptor subtypes in hippocampal neurons in vitro

The cellular and subcellular distribution of four GABAA receptor subtypes, identified by the presence of the α1, α2, α3, or α5 subunit, was investigated immunocytochemically in dissociated cultures of hippocampal neurons. We addressed the questions whether (1) cell‐type specific expression, (2) axonal/somatodendritic targeting, and (3) synaptic/extrasynaptic clustering of GABAA receptor subtypes was retained in vitro. For comparison, the in vivo distribution pattern was assessed in sections from adult rat brain. The differential expression of GABAA receptor subunits allowed to identify five morphologically distinct cell types in culture: the α1 subunit was observed in glutamic acid decarboxylase–positive interneurons, the α2 and α5 subunits marked pyramidal‐like cells, and the α3 subunit labeled three additional cell types, including presumptive hilar cells. All subunits were found in the somatodendritic compartment. In addition, appropriate axonal targeting was evidenced by the intense α2, and sometimes α3 subunit labeling of axon‐initial segments (AIS) of pyramidal cells and hilar cells, respectively. Accordingly, both receptor subtypes were targeted to AIS in vivo, as well. Synaptic receptors were identified by colocalization with gephyrin, a postsynaptic clustering protein, and apposition to presynaptic terminals labeled with synapsin I. In vitro and in vivo, α1‐ and α2‐receptor subtypes formed numerous synaptic clusters, α3‐GABAA receptors were located either synaptically or extrasynaptically depending on the cell type, whereas α5‐GABAA receptors were extrasynaptic. We conclude that receptor targeting to broad subcellular locations does not require specific GABAergic innervation patterns, which are disturbed in vitro, but depends on protein‐protein interactions in the postsynaptic cell that are both subunit‐ and neuron‐specific. J. Comp. Neurol. 443:43–55, 2002.

Bidirectional Alterations of GABAA Receptor Subunit Peptide Levels in Rat Cortex During Chronic Ethanol Consumption and Withdrawal

Abstract: The pharmacological properties of γ‐aminobutyric acidA (GABAA) receptors are altered by prolonged exposure to ethanol both in vivo and in vitro. We have shown previously that prolonged ethanol exposure elicits selective alterations in various GABAA receptor subunit mRNA levels in rat cerebral cortex. Some of these effects are rapidly reversed during ethanol withdrawal. The present study was conducted to determine the effects of prolonged ethanol exposure (dependence) and ethanol withdrawal on cerebral cortical peptide expression for several subunits. GABAA receptor α1 subunit peptide levels were decreased by nearly 40%, whereas α4 subunit peptide levels were increased by 27% in both ethanol‐dependent and withdrawn rats. These changes correlate well with observed alterations in mRNA levels following prolonged ethanol exposure in dependent rats, but do not match the effects on mRNA levels during ethanol withdrawal. β2/3 subunit peptide levels increased by ∼32% in both ethanol‐dependent rats and rats undergoing ethanol withdrawal. We observed a 30–60% increase in γ1 subunit peptide levels in both dependent rats and those undergoing withdrawal, also correlating with the previous report on ethanol‐induced alterations in mRNA levels. Peptide levels for γ2 subunits did not differ from control values in either condition. These findings show that specific alterations in GABAA receptor subunit peptide levels are associated with ethanol dependence in rats. GABAA receptor subunit peptide expression is more stable than mRNA expression, and mRNA levels are not representative of peptide expression during ethanol withdrawal. These findings are consistent with the suggestion that alterations in GABAA receptor gene expression underlie the functional properties of GABAA receptors in ethanol‐dependent rats and those undergoing ethanol withdrawal.