Region-specific expression of messenger RNAs encoding GABAA receptor subunits in the developing rat brain.

The distribution and levels of messenger RNAs encoding the alpha 1, beta 1, beta 2, beta 3, and gamma 2 subunits of the GABAA receptor in the developing and adult rat brain were investigated using quantitative in situ hybridization histochemistry and subunit-specific probes. Regional localization of the subunit messenger RNAs was determined with film autoradiography and expression in identified neuronal cell populations was examined using higher resolution techniques. Each of the GABAA receptor subunit messenger RNAs exhibits a distinct pattern of localization in the developing and adult brain. Of the subunits examined, the alpha 1, beta 2, and gamma 2 are the most abundant and are found in many brain regions, including the olfactory bulb, cortex, hippocampus, thalamic nuclei, and inferior colliculus. In addition, these subunit messenger RNAs are prominent in the cerebellum where virtually all cells of the deep cerebellar nuclei and Purkinje cell layer are labeled. The levels of most of the subunit messenger RNAs, with the exception of that encoding the beta 1 subunit, increase during postnatal development. While the alpha 1, beta 2, and gamma 2 subunit messenger RNAs rise in parallel in many regions and identified cell populations, different subsets of receptor subunit messenger RNAs are co-ordinately expressed at other sites. The greatest increases in subunit messenger RNA levels occur in the cerebellar cortex during the second postnatal week, a period coincident with cerebellar maturation. The co-distribution of different GABAA receptor subunit messenger RNAs in various regions of the developing and adult nervous systems supports the hypothesis that multiple receptor compositions exist. Moreover, that different subunit messenger RNAs exhibit coordinate changes in expression in different regions and cell populations suggests that receptor gene expression is modulated by cell type-specific signals. The temporal changes in subunit messenger RNA levels in the cerebellum raise the possibility that synaptogenesis may play a role in receptor gene regulation in this brain region.     

大鼠神经系统内5-羟色胺2A受体亚型的免疫组织化学定位

目的：观察大鼠神经系统内5－羟色胺受体2A亚型（5－HT2AR）免疫组织化学染色阳性结构的分布。方法：5－HT2AR特异性抗体的免疫组织化学染色。结果：5－HT2AR阳性细胞体主要分布于嗅球的小球层和僧帽细胞层、海马始层、外侧缰核、丘脑背外侧核、下丘脑室旁核、中脑中央灰质、小脑浦肯野细胞、脑干运动核和感觉神经节等；5－HT2AR阳性纤维和终末主要分面于嗅球的小球层和内丛层、大脑皮质、外侧隔核、杏仁外侧核、丘脑网状核、腹侧顶盖区、桥核、下橄榄及脑干运动核等。结论：5－HT2AR亚型阳性结构广泛分布于大鼠神经系统，它们可能介导5－HT在神经系统中的多种生理功能。     

Mapping of benzodiazepine-like immunoreactivity in the rat brain as revealed by a monoclonal antibody to benzodiazepines.

A monoclonal antibody against benzodiazepines (21-7F9) was used to study the distribution of benzodiazepine-like immunoreactivity in the rat brain. Immunodensitometry in combination with image analysis were used for quantification. The results showed a ubiquitous distribution of benzodiazepine-like immunoreactivity throughout the brain. Very high levels of benzodiazepine-like immunoreactivity were found in the Purkinje cell layer of the cerebellum, in the primary olfactory cortex, in the stratum pyramidale of the hippocampus and in the mitral cell layer of the olfactory bulb. High densities of benzodiazepine-like immunoreactivity were found in the granule cell layer of the cerebellum, the pyramidal cell layer of the olfactory tubercle, the granule layer of the dentate gyrus, the arcuate nucleus of the hypothalamus, the mammillary bodies, the interstitial nucleus of Cajal and superficial grey layer of superior colliculus. The substantia nigra pars compacta, the islands of Calleja and layers II, III, V and VI of the cerebral cortex had moderate levels of benzodiazepine-like immunoreactivity. Lower densities were found in the internal granular layer and the external plexiform layer of the olfactory bulb, in the molecular layer of the dentate gyrus, in layers I and IV of the cerebral cortex, in the nucleus caudate-putamen and most of the thalamic nuclei. The lowest density of immunoreactivity was found in the globus pallidus, and the strata radiatum, oriens and lacunosum-moleculare of the hippocampus. The distribution of endogenous benzodiazepine-like immunoreactivity was compared with the distribution of the GABA/benzodiazepine receptor by using both immunocytochemistry and receptor autoradiography. Our studies have shown a clear mismatch between the localization of the benzodiazepine-like immunoreactivity and the GABA/benzodiazepine receptors.     

GABAA and GABAB receptor site distribution in the rat central nervous system

An autoradiographic procedure has been used to determine the quantitative distributions of gamma-aminobutyric acid (GABAA and GABAB) receptor subtypes in rat brain. Although the concentrations of both receptor binding sites were similar in some brain regions GABAA sites generally outnumbered GABAB sites. The highest concentration of GABAA sites were detected in the frontal cortex, the granule cell layer of the cerebellum, the olfactory bulb and the thalamic medial geniculate. The highest concentration of GABAB sites occurred in the molecular layer of the cerebellum, the interpeduncular nucleus, frontal cortex, anterior olfactory nucleus and thalamic nuclei. In addition the globus pallidus, temporal cortex, lateral posterior thalamus, superior colliculus, pontine nucleus, raphe magnus, spinal trigeminal tract and substantia gelatinosa contained significantly more GABAB sites than GABAA sites. The physiological and pharmacological significance of this heterogeneity has yet to be determined.     

The differential expression patterns of messenger RNAs encoding K-Cl cotransporters (KCC1,2) and Na-K-2Cl cotransporter (NKCC1) in the rat nervous system.

Cation-chloride cotransporters have been considered to play pivotal roles in controlling intracellular and extracellular ionic environments of neurons and hence controlling neuronal function. We investigated the total distributions of K-Cl cotransporter 1 (KCC1), KCC2 (KCC2), and Na-K-2Cl cotransporter 1 (NKCC1) messenger RNAs in the adult rat nervous system using in situ hybridization histochemistry. KCC2 messenger RNA was abundantly expressed in most neurons throughout the nervous system. However, we could not detect KCC2 messenger RNA expression in the dorsal root ganglion and mesencephalic trigeminal nucleus, where primary sensory neurons show depolarizing responses to GABA, suggesting that the absence of KCC2 is necessary for this phenomenon. Furthermore, KCC2 messenger RNA was also not detected in the dorsolateral part of the paraventricular nucleus, dorsomedial part of the suprachiasmatic nucleus, and ventromedial part of the supraoptic nucleus where vasopressin neurons exist, and in the reticular thalamic nucleus. As vasopressin neurons in the suprachiasmatic nucleus and neurons in the reticular thalamic nucleus produce their intrinsic rhythmicity, the lack of KCC2 messenger RNA expression in these regions might be involved in the genesis of rhythmicity through the control of intracellular chloride concentration. The expression levels of KCC1 and NKCC1 messenger RNAs were relatively low, however, positive neurons were observed in several regions, including the olfactory bulb, hippocampus, and in the granular layer of the cerebellum. In addition, positive signals were seen in the non-neuronal cells, such as choroid plexus epithelial cells, glial cells, and ependymal cells, suggesting that KCC1 and NKCC1 messenger RNAs were widely expressed in both neuronal and non-neuronal cells in the nervous system.These results clearly indicate a wide area- and cell-specific variation of cation chloride cotransporters, emphasizing the central role of anionic homeostasis in neuronal function and communication.     

Localization of cytosolic phospholipase A2 messenger RNA mainly in neurons in the rat brain.

Ca2(+)-sensitive 85,000 mol. wt cytosolic phospholipase A2 plays an essential role in the selective and stimulus-dependent release of arachidonic acid from membrane phospholipids. Cytosolic phospholipase A2-catalysed lipid mediators including arachidonic acid and its metabolites have been suggested to be involved in a variety of neuronal functions in the CNS. Since the cellular localization of cytosolic phospholipase A2 is still controversial and obscure, we tried an improved method of rapid processing of each specimens and succeeded in obtaining intense signals of cytosolic phospholipase A2 messenger RNA in the normal rat brain by northern blot analysis and in situ hybridization. Northern blot analysis showed the abundant distribution of cytosolic phospholipase A2 messenger RNA in most regions of the brain, with intense signals observed in the pineal gland and pons. Macroautoradiographs prepared after in situ hybridization with three different antisense riboprobes gave essentially similar patterns of localization; significant signals were widely detected in the gray matter of various regions, i.e. the olfactory bulb, cerebral cortex, hippocampus, amygdala, several thalamic and hypothalamic nuclei and cerebellum. Microautoradiographs showed that most of the intense signals were predominant in neurons, and that faint signals were from glial cells and other non-neuronal cells in the choroid plexus, inner surface cells of veins and the leptomeninges. In addition, the cycloheximide treatment increased the cytosolic phospholipase A2 messenger RNA level in the same cell populations originally possessing messenger RNA signals. Predominant expression of cytosolic phospholipase A2 messenger RNA in neurons may provide the basis for the contribution of cytosolic phospholipase A2-catalysed lipid mediators to a variety of neurotransmission and synaptic functions in the CNS.     

Expression and localization of the LGN protein in the mouse brain with reference to its relationship with Gai2

It has been suggested that the LGN protein is associated with G!_i2 by the yeast two-hybrid system and in vitro pull-down assay. To determine the functions of LGN in the central nervous system, we examined the expression and localization of LGN in mouse brain by immunoblotting and immunofluorescence microscopy. By immunoblotting, almost similar amounts of LGN were detected in the olfactory bulb, cerebral cortex, hippocampus, and cerebellum of the adult mouse brain, and the levels of the postnatal LGN expression in the whole brain were fairly constant. Immunofluorescence microscopy showed that LGN is localized in nuclei of the neurons in the olfactory bulb, cerebral cortex, and hippocampus, but in both nuclei and cytoplasm of Purkinje cells in the cerebellum. On the other hand, G!_i2 was distributed throughout the neuronal elements except for the nuclei. Thus, LGN and G!_i2 were colocalized in the cytoplasm of Purkinje cells, but not in other neurons examined. These results suggest that LGN may be involved not only in the G!_i2-mediated signaling but also in other signaling pathways.     

Hippocampus-specific deficiency in RNA editing of GluA2 in Alzheimer's disease

Adenosine to inosine (A-to-I) RNA editing is a base recoding process within precursor messenger RNA, catalyzed by members of the adenosine deaminase acting on RNA (ADAR) family. A notable example occurs at the Q/R site of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptor subunit GluA2. Abnormally, low editing at this site leads to excessive calcium influx and cell death. We studied hippocampus and caudate samples from Alzheimer's disease (AD) patients and age-matched healthy controls, using direct sequencing and a high accuracy primer-extension technique to assess RNA editing at the Q/R GluA2 site. Both techniques revealed lower, more variable RNA editing in AD, specific to the hippocampus and the GluA2 site. Deficient editing also characterized the hippocampus of apolipoprotein ε4 allele carriers, regardless of clinical diagnosis. In AD, messenger RNA expression of neuronal markers was decreased in the hippocampus, and expression of the Q/R-site editing enzyme ADAR2 was decreased in caudate. These findings provide a link between neurodegenerative processes and deficient RNA editing of the GluA2 Q/R site, and may contribute to both diagnosis and treatment of AD.     

Differential messenger RNA distribution of lactate dehydrogenase LDH-1 and LDH-5 isoforms in the rat brain

The role of lactate in brain energy metabolism has recently received renewed attention. Although blood-borne monocarboxylates such as lactate poorly cross the blood-brain barrier in the adult brain, lactate produced within the brain parenchyma may be a suitable substrate for brain cells. Lactate dehydrogenase is crucial for both the production and utilization of lactate. In this article, we report the regional distribution of the messenger RNAs for lactate dehydrogenase isoforms 1 and 5 in the adult rat brain using in situ hybridization histochemistry with specific [alpha-(35)S]dATP 3' end-labeled oligoprobes. The autoradiographs revealed that the lactate dehydrogenase-1 messenger RNA is highly expressed in a variety of brain structures, including the main olfactory bulb, the piriform cortex, several thalamic and hypothalamic nuclei, the pontine nuclei, the ventral cochlear nucleus, the trigeminal nerve and the solitary tractus nucleus. In addition, the granular and Purkinje cell layers of the cerebellum showed a strong labeling. The neocortex (e.g., cingular, retrosplenial and frontoparietal cortices) often exhibits a marked laminar pattern of distribution of lactate dehydrogenase-1 messenger RNA (layers II/III, IV and VI being most strongly labeled). In contrast, expression of the lactate dehydrogenase-5 messenger RNA generally seemed more diffusely distributed across the different brain regions. Expression was particularly strong in the hippocampal formation (especially in Ammon's horn and dentate gyrus) and in the cerebral cortex, where no laminar pattern of distribution was observed. Overall, these data are consistent with the emerging idea that lactate is an important energy substrate produced and consumed by brain cells.     

Cellular and subcellular localization of an octadecaneuropeptide derived from diazepam binding inhibitor: immunohistochemical studies in the rat brain

Immunocytochemical methods, both light and electron microscopic, were used to identify the cellular and subcellular locations of octadecaneuropeptide-like immunoreactivity (ODN-LI) in rat brains serially sectioned in total. ODN-LI includes a newly discovered family of rat brain neuropeptides that are processing products of a common endogenous neuropeptide precursor, diazepam binding inhibitor (DBI). The members of this neuropeptide family have been shown to displace benzodiazepines and beta carbolines from their specific recognition sites located on the allosteric modulatory centers of GABAA receptors. We have previously examined the distribution of DBI-LI in rat brain. The anti-ODN antiserum used in this study does not cross-react with rat DBI, and thus allows a distinct analysis of ODN-LI as opposed to DBI-LI, in rat brain. Neuronal perikarya with ODN-LI were located in many brain nuclei, such as the pontine n., reticular thalamic n., subgeniculate n., supraoptic n. and suprachiasmatic n., and also in brain areas such as cerebral and cerebellar cortex, hippocampus, inferior colliculus, olfactory bulb and subiculum. In addition to perikaryal labelling, a punctate or diffuse immunostaining with ODN antibodies was detected in many brain regions such as cerebellum, hippocampus, amygdaloid area, olfactory tubercle, some of the deep cerebellar nuclei and some circumventricular organs. At the electron microscopic level ODN-LI was identified in neuronal perikarya, processes and terminals. In the axon terminals, ODN-LI appears to be associated with synaptic vesicles. Whenever ODN-LI was detected within neurons, DBI-LI was also found in identical cells. In addition to neurons, DBI-LI was found in glia or glial-like cells, while ODN-LI was not found in these cells. Our findings are consistent with the hypothesis that ODN may be a neuron-specific processing product of DBI and that ODN-like peptides may act as putative endogenous allosteric modulators of various GABAA receptor subtypes.     

Regulation of brain-derived neurotrophic factor messenger RNA levels in avian hypothalamic slice cultures

Mechanisms regulating the expression of brain-derived neurotrophic factor, a member of the neurotrophin family, have been extensively studied in the rat cerebral cortex, hippocampus and cerebellum. In contrast, little is known regarding the regulation of this growth factor in the hypothalamus. Here we present an analysis of the regulation of brain-derived neurotrophic factor messenger RNA levels in chick embryo hypothalamic slice cultures following exposure to potassium chloride, glutamate agonists and sex steroids. Following a week in chemically-defined media the tissue was depolarized by exposure to 50 mM potassium chloride for 6h, resulting in a significant 4.2-fold increase in the level of brain-derived neurotrophic factor messenger RNA. This result is consistent with studies of other brain regions. Similar 6-h acute exposures of the hypothalamic cultures to 25 microM N-methyl-D-aspartic acid, 25 microM kainic acid and 25 microM alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid also significantly increased messenger RNA levels 2.5-, 2.1- and 1.4-fold, respectively. It was previously reported that brain-derived neurotrophic factor levels within the rat cerebral cortex, olfactory bulb and hippocampus are altered by exposure to 17beta-estradiol. Here we show that in hypothalamic slice cultures neither acute nor chronic treatments with 10 and 100 nM 17beta-estradiol and 10nM testosterone significantly altered the steady-state level of this growth factor.These findings show that neuronal activity, induced by glutamate agonists and potassium chloride, can regulate brain-derived neurotrophic factor messenger RNA levels within embryonic hypothalamic slice cultures. This regulation could play a critical role in the modulation of programmed cell death and synaptic maturation during development of the hypothalamus.     

Immunocytochemical evidence for the involvement of glycine in sensory centers of the rat brain.

Glycine-like immunoreactivity was localized to a number of sites in the rat brain which are involved in processing sensory information. In the auditory and vestibular systems, glycine immunoreactivity was seen in dorsal and ventral cochlear nuclei, superior olive, trapezoid body, medial and lateral vestibular nuclei, and inferior colliculus. Staining in the visual system was seen in retina, dorsal lateral geniculate nucleus, and superior colliculus. The olfactory system exhibited staining in the olfactory bulb and accessory olfactory formation. Somatosensory centers with glycine immunoreactivity included the dorsal column nuclei, spinal trigeminal nucleus, principal sensory nucleus of V, reticular formation, and periaqueductal gray. Glycine-immunoreactive neurons were also seen in cerebellar cortex, deep cerebellar nuclei, hippocampus, cerebral cortex, and striatum. The distribution of staining indicates that glycine plays a major role in sensory centers with actions at both strychnine-sensitive and strychnine-insensitive receptors.     

Nitric oxide synthase protein levels, not the mRNA, are downregulated in olfactory bulb interneurons of reeler mice

Homozygous mutations in the Reelin gene result in severe disruption of brain development. The histogenesis of layered regions, like the neocortex, hippocampus and the cerebellum, is most notably affected in mouse reeler mutants and similar traits are also present in mice lacking molecular components of the Reelin signalling pathway. Moreover, there is evidence for an additional role of Reelin in sustaining synaptic plasticity in adult networks. Nitric oxide is an important gaseous messenger that can modulate neuronal plasticity both in developing and mature synaptic networks and has been shown to facilitate synaptic changes in the hippocampus, cerebellum and olfactory bulb. We studied the distribution and content of neuronal nitric oxide synthase in the olfactory bulbs of reeler and wildtype mice. Immunocytochemistry reveals that Reelin and neuronal nitric oxide synthase containing interneurons are two distinct, non overlapping cell populations of the olfactory bulb. We show by in situ hybridization that both nitrergic and Reelin expressing cells represent only a subset of olfactory bulb GABAergic neurons. Immunoblots show that neuronal nitric oxide synthase protein content is decreased by two thirds in reeler mice causing a detectable loss of immunolabelled cells throughout the olfactory bulb of this strain. However, neuronal nitric oxide synthase mRNA levels, essayed by quantitative real-time RT-PCR, are unaffected in the reeler olfactory bulb. Thus, disruption of the Reelin signalling pathway may modify the turnover of neuronal nitric oxide synthase in the olfactory bulb and possibly affects nitric oxide functions in reeler mice.     

c－fos基因在听源性惊厥易感大鼠脑的表达──原位杂交组织化学法研究

运用原位杂交技术，以遗传性听源性惊厥易感大鼠Ｐ７７ＰＭＣ为对象，发现听源性惊厥可诱导大鼠脑内ｃ－ｆｏｓ基因快速、大量、短暂性表达。ｃ－ｆｏｓｍＲＮＡ分布于大脑皮层、梨状皮层、杏仁复合体、海马齿状回、上丘脑、背侧丘脑、下丘脑部分核团、下丘、蜗神经核、蓝斑及小脑等处。惊厥后皮层下结构中ｃ－ｆｏｓ基因表达变化程度超过皮层的变化，尤其是下丘、蜗神经核与惊厥时程有明显关系。推测皮层下结构对听源性惊厥的发生有重要意义。Ｐ＜０．０１讨论本文结果说明听源性惊厥同其它因素诱导的惊厥一样［３］，可诱导大鼠脑内ｃ－ｆｏｓ基因的表达，表达涉及到大脑皮层、海马齿状回、丘脑、下丘、蜗神经核等结构，其中以皮层下结构如丘脑、下丘、蜗神经核表达变化最显著。原位杂交显示的ｃ－ｆｏｓ基因表达特征类似于Ｎｏｒｔｈｅｒｎ杂交结果即快速、大量和短暂性。由于不同部位在惊厥活动中的作用差别，因此用原位杂交可以显示每一结构内ｃ－ｆｏｓ基因表达特点。如在惊厥后３０ｍｉｎ，海马齿状回中７０％以上的神经元单位胞质面积上银粒计数超过２０个，而梨状皮层及运动皮层仅占５～１３．８％。有报告指出海马齿状回为钙离子通道和ＮＭＤＡ受体高密度区域［４］，推测Ｃａ２＋和ＮＭＤＡ?     

Comparison of neuronal inositol 1,4,5-trisphosphate 3-kinase and receptor mRNA distributions in the adult rat brain using in situ hybridization histochemistry.

As a result of its interaction with a specific receptor, inositol 1,4,5-trisphosphate mobilizes intracellular calcium. The metabolism of inositol 1,4,5-trisphosphate is rather complex: inositol 1,4,5-trisphosphate 3-kinase produces inositol 1,3,4,5-tetrakisphosphate, a putative second messenger. In order to elucidate inositol 1,3,4,5-tetrakisphosphate function, a comparative in situ hybridization study of the distributions of inositol 1,4,5-trisphosphate 3-kinase and receptor mRNAs was performed in the adult rat brain using oligonucleotides derived from their cDNA sequences. The neuronal distributions of the mRNA for the receptor were larger than for the kinase. Highest levels of both mRNAs were found in the cerebellar Purkinje cells, where they were enriched in their neuronal perikarya and to a lesser extent in their dendrites. In addition to the cerebellum, mRNAs were mainly detected in the hippocampal pyramidal cells of the CA1 sector of the Ammon's horn and in the granule cells of the dentate gyrus, and also in a majority of the neurons in the cortical layers II-III and V, especially in the frontal cortex and cingulate cortex; caudate-putamen, accumbens, olfactory tubercle and Calleja islets; claustrum; anterior olfactory nucleus; taenia tecta; piriform cortex; dorsolateral septum; bed nucleus stria terminalis; amygdala; hippocampal CA2-4 sectors and subiculum. The inositol 1,4,5-trisphosphate receptor mRNA but not kinase mRNA was found in a majority of the neurons in the thalamus, especially in the parafascicular nucleus; hypothalamus, especially the medial hypothalamus; substantia nigra pars compacta and ventral tegmental area; superior colliculus; lateral interpeduncular nucleus and central gray. Taking into account the limitation in sensitivity of the technique, both mRNAs were not detected in glial cells and in the olfactory bulb; basal nucleus of Meynert, diagonal band nuclei; medial septal nucleus; substantia innominata; globus pallidus; entopeduncular nucleus; substantia nigra pars reticulata; ventral pallidum; subthalamic nucleus; spinal cord and dorsal root ganglia. In conclusion, cerebellum and hippocampus appear to contain almost similar levels of kinase mRNA. This is in contrast to receptor mRNA levels which were at much higher levels in the cerebellum when compared with the hippocampus. For this reason, we have chosen hippocampal CA1 pyramidal cells and dentate gyrus granule cells for studying inositol 1,4,5-trisphosphate 3-kinase function.     

GABA(A) receptors: immunocytochemical distribution of 13 subunits in the adult rat brain

GABA(A) receptors are ligand-operated chloride channels assembled from five subunits in a heteropentameric manner. Using immunocytochemistry, we investigated the distribution of GABA(A) receptor subunits deriving from 13 different genes (alpha1-alpha6, beta1-beta3, gamma1-gamma3 and delta) in the adult rat brain. Subunit alpha1-, beta1-, beta2-, beta3- and gamma2-immunoreactivities were found throughout the brain, although differences in their distribution were observed. Subunit alpha2-, alpha3-, alpha4-, alpha5-, alpha6-, gamma1- and delta-immunoreactivities were more confined to certain brain areas. Thus, alpha2-subunit-immunoreactivity was preferentially located in forebrain areas and the cerebellum. Subunit alpha6-immunoreactivity was only present in granule cells of the cerebellum and the cochlear nucleus, and subunit gamma1-immunoreactivity was preferentially located in the central and medial amygdaloid nuclei, in pallidal areas, the substantia nigra pars reticulata and the inferior olive. The alpha5-subunit-immunoreactivity was strongest in Ammon's horn, the olfactory bulb and hypothalamus. In contrast, alpha4-subunit-immunoreactivity was detected in the thalamus, dentate gyrus, olfactory tubercle and basal ganglia. Subunit alpha3-immunoreactivity was observed in the glomerular and external plexiform layers of the olfactory bulb, in the inner layers of the cerebral cortex, the reticular thalamic nucleus, the zonal and superficial layers of the superior colliculus, the amygdala and cranial nerve nuclei. Only faint subunit gamma3-immunoreactivity was detected in most areas; it was darkest in midbrain and pontine nuclei. Subunit delta-immunoreactivity was frequently co-distributed with alpha4 subunit-immunoreactivity, e.g. in the thalamus, striatum, outer layers of the cortex and dentate molecular layer. Striking examples of complementary distribution of certain subunit-immunoreactivities were observed. Thus, subunit alpha2-, alpha4-, beta1-, beta3- and delta-immunoreactivities were considerably more concentrated in the neostriatum than in the pallidum and entopeduncular nucleus. In contrast, labeling for the alpha1-, beta2-, gamma1- and gamma2-subunits prevailed in the pallidum compared to the striatum. With the exception of the reticular thalamic nucleus, which was prominently stained for subunits alpha3, beta1, beta3 and gamma2, most thalamic nuclei were rich in alpha1-, alpha4-, beta2- and delta-immunoreactivities. Whereas the dorsal lateral geniculate nucleus was strongly immunoreactive for subunits alpha4, beta2 and delta, the ventral lateral geniculate nucleus was predominantly labeled for subunits alpha2, alpha3, beta1, beta3 and gamma2; subunit alpha1- and alpha5-immunoreactivities were about equally distributed in both areas. In most hypothalamic areas, immunoreactivities for subunits alpha1, alpha2, beta1, beta2 and beta3 were observed. In the supraoptic nucleus, staining of conspicuous dendritic networks with subunit alpha1, alpha2, beta2, and gamma2 antibodies was contrasted by perykarya labeled for alpha5-, beta1- and delta-immunoreactivities. Among all brain regions, the median emminence was most heavily labeled for subunit beta2-immunoreactivity. In most pontine and cranial nerve nuclei and in the medulla, only subunit alpha1-, beta2- and gamma2-immunoreactivities were strong, whereas the inferior olive was significantly labeled only for subunits beta1, gamma1 and gamma2. In this study, a highly heterogeneous distribution of 13 different GABA(A) receptor subunit-immunoreactivities was observed. This distribution and the apparently typical patterns of co-distribution of these GABA(A) receptor subunits support the assumption of multiple, differently assembled GABA(A) receptor subtypes and their heterogeneous distribution within the adult rat brain.