Dietmar Benke

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.

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.

Adjacent phosphorylation sites on GABA A receptor β subunitsdetermine regulation by cAMP-dependent protein kinase

Activation of cAMP-dependent protein kinase (PKA) can enhance or reduce the function of neuronal GABAA receptors, the major sites of fast synaptic inhibition in the brain. This differential regulation depends on PKA-induced phosphorylation of adjacent conserved sites in the receptor β subunits. Phosphorylation of β3 subunit-containing receptors at S408 and S409 enhanced the GABA-activated response, whereas selectively mutating S408 to alanine converted the potentiation into an inhibition, comparable to that of β1 subunits, which are phosphorylated solely on S409. These distinct modes of regulation were interconvertible between β1 and β3 subunits and depended upon the presence of S408 in either subunit. In contrast, β2 subunit-containing receptors were not phosphorylated or affected by PKA. Differential regulation by PKA of postsynaptic GABAA receptors containing different β subunits may have profound effects on neuronal excitability.

Neuron-specific expression of GABAA-receptor subtypes: Differential association of theα1- andα3-subunits with serotonergic and gabaergic neurons

Abstract GABAA-receptors in the brain display a striking structural heterogeneity, which is based on a multiplicity of diverse subunits. The allocation of GABAA-receptor subtypes to identified neurons is essential for an analysis of the functional significance of receptor heterogeneity. Among GABA-receptive neurons, well-characterized examples include the serotonergic and GABAergic neurons in the raphe nuclei. The GABAA-receptor subtypes expressed in these two types of neurons were analysed using antisera which recognize selectively theα1- andα3-subunits, and their co-localization with serotonin and glutamate decarboxylase was assessed by confocal laser microscopy in double and triple immunofluorescence staining in the rat. The vast majority of serotonergic neurons express strongα3-subunit-immunoreactivity, but are devoid ofα1-subunit staining. In contrast, both theα1- andα3-subunit-immunoreactivities are present in glutamate decarboxylase-positive neurons. Thus, serotonergic and GABAergic neurons selectively express distinct patterns of α subunits, suggesting that they possess distinct subtypes of GABAA-receptors. The occurrence of neuron-specific GABAA-receptor subtypes may open new possibilities for the targeting of drugs with selective therapeutic actions.

The γ2 Subunit of the GABAA Receptor is Concentrated in Synaptic Junctions Containing the α1 and β23 Subunits in Hippocampus, Cerebellum and Globus Pallidus

Abstract The γ2 subunit is necessary for the expression of the full benzodiazepine pharmacology of GABAA receptors and is one of the major subunits in the brain. In order to determine the location of channels containing the γ2 subunit in relation to GABA-releasing terminals on the surface of neurons, a new polyclonal antipeptide antiserum was developed to the γ2 subunit and used in high resolution, postembedding, immunoelectron-microscopic procedures. Dual immunogold labelling of the same section for two subunits, and up to three sections of the same synapse reacted for different subunits, were used to characterize the subunit composition of synaptic receptors. The γ2 subunit was present in type 2, “symmetrical” synapses in each of the brain areas studied, with the exception of the granule cell layer of the cerebellum. The γ2 subunit was frequently co-localized in the same synaptic junction with the α1 and β 2 3 subunits. The immunolabelling of synapses was coincident with the junctional membrane specialization of the active zone. Immunolabelling for the receptor often occurred in multiple clusters in the synapses. In the hippocampus, the γ2 subunit was present in basket cell synapses on the somata and proximal dendrites and in axo-axonic cell synapses on the axon initial segment of pyramidal and granule cells. Some synapses on the dendrites of GABAergic interneurones were densely labelled for the γ2, α1 and β 2 3 subunits. In the cerebellum, the γ2 subunit was present in both distal and proximal Purkinje cell dendritic synapses established by stellate and basket cells, respectively. On the soma of Purkinje cells, basket cell synapses were only weakly labelled. Synapses on interneuron dendrites were more densely labelled for the γ2, α1 and β 2 3 subunits than synapses on Purkinje or granule cells. Although immunoperoxidase and immunofluorescence methods show an abundance of the γ2 subunit in granule cells, the labelling of Golgi synapses was much weaker with the immunogold method than that of the other cell types. In the globus pallidus, many type 2 synapses were labelled for the γ2 subunit together with α1 and β 2 3 subunits. The results show that γ2 subunit-containing receptor channels are highly concentrated in GABAergic synapses that also contain the α1 and β 2 3 subunits. Channels containing the γ2 subunit are expressed in synapses on functionally distinct domains of the same neuron receiving GABA from different presynaptic sources. There are quantitative differences in the density of GABAA receptors at synapses on different cell types in the same brain area. Copyright

Distribution of NMDA receptor subunit proteins NR2A, 2B, 2C and 2D in rat brain.

The regional distribution of the NMDA receptor subunits NR2A, 2B, 2C and 2D was visualized in adult rat brain using the histo-blot technique with newly developed subunit-specific antisera. NR2A immunoreactivity was found in almost all regions of the brain, whereas NR2B staining was restricted to forebrain, and NR2D immunoreactivity to diencephalic, mesencephalic and brain stem structures. NR2C staining was confined to cerebellum, thalamus and olfactory bulb. Thus, NMDA receptors containing the NR2A subunit are likely to represent a receptor subtype predominant throughout the brain, while those containing the NR2B, NR2C or NR2D subunit represent more region-specific receptor subtypes. The regionally overlapping distribution of certain NR2 subunits points to the existence of NMDA receptors containing more than one NR2 subunit variant.

gamma-aminobutyric acid type B receptor splice variant proteins GBR1a and GBR1b are both associated with GBR2 in situ and display differential regional and subcellular distribution.

The subunit architecture of γ-aminobutyric acid, type B (GABAB), receptors in situis largely unknown. The GABAB receptor variants, characterized by the constituents GBR1a and GBR1b, were therefore analyzed with regard to their subunit composition as well as their regional and subcellular distribution in situ. The analysis was based on the use of antisera recognizing selectively GBR1a, GBR1b, and GBR2. Following their solubilization, GBR1a and GBR1b were both found by immunoprecipitation to occur as heterodimers associated with GBR2. Furthermore, monomers of GBR1a, GBR1b, or GBR2 were not detectable, suggesting that practically all GABAB receptors are heterodimers in situ. Finally, there was no evidence for an association of GBR1a with GBR1b indicating that these two constituents represent two different receptor populations. A size determination of solubilized GABAB receptors by sucrose density centrifugation revealed two distinct peaks of which one corresponded to dimeric receptors, and the higher molecular weight peak pointed to the presence of yet unknown receptor-associated proteins. The distribution and relative abundance of GBR2 immunoreactivity corresponded in all brain regions to that of the sum of GBR1a and GBR1b, supporting the view that most if not all GBR1 proteins are associated with GBR2. However, GBR1a was present preferentially at postsynaptic densities, whereas GBR1b may be mainly attributed to presynaptic or extrasynaptic sites. Thus, GBR1a and GBR1b are both associated with GBR2 to form heterodimers at mainly different subcellular locations where they are expected to subserve different functions.

Developmental and Regional Expression of NMDA Receptor Subtypes Containing the NR2D Subunit in Rat Brain

Abstract: The regional and developmental expression of NMDA receptors containing the NR2D subunit was analyzed on the level of the subunit mRNA and protein in rat brain. RNase protection experiments indicated that among two proposed splice variants of the NR2D subunit, only the NR2D‐2 subunit is expressed. The regional distribution of the NR2D subunit protein was visualized with a newly developed NR2D‐2 subunit‐specific antiserum on brain sections using the histoblot technique. In adult brain, NR2D immunoreactivity was mainly restricted to diencephalic, mesencephalic, and brainstem structures. During postnatal development, the NR2D subunit was detected transiently in certain regions, such as the ventro‐basal complex of the thalamus, hippocampus, inferior colliculus, and brainstem reticular formation, suggesting that NR2D subunit‐containing receptors play a role in these brain areas only during development. The level of NR2D subunit mRNA and protein decreased during late postnatal development. However, significant levels of NR2D subunit mRNA and protein were present in adulthood, in particular, in the globus pallidus, thalamus, subthalamic nuclei, and superior colliculus. These results indicate a functional relevance for NMDA receptors containing the NR2D subunit in the developing and adult brain, although its expression in the adult brain is less prominent and restricted to a few brain areas.