Distribution of somatostatin in the rat brain: Telencephalon and diencephalon

The distribution of somatostatin (SRIF) was examined using the unlabeled antibody enzyme method of immunocytochemistry on thick 30-50 microns Vibratome sections. The greatest population of SRIF-neurons was observed along the ventricular wall in the preoptic area and anterior hypothalamus. Dense accumulations of fibers were observed in the suprachiasmatic, ventromedial and arcuate nuclei, the internal and external zone of the median eminence, the organum vasculosum of the lamina terminalis and the subfornical organ. Extrahypothalamic sites of SRIF-containing neurons and fibers were also observed throughout the telencephalon. The widespread distribution of SRIF is consistent with radioimmunoassay data and suggests a diverse physiological role for somatostatin.     

GABAA receptor function and regional analysis of subunit mRNAs in long-sleep and short-sleep mouse brain.

The greater sensitivity of long-sleep (LS), as compared with short-sleep (SS), mice to ethanol is due in part to differences in GABAA receptor function in specific brain regions. To determine if differences in subunit composition of GABAA receptors contribute to this differential sensitivity, we measured alpha 1 and gamma 2 subunit mRNAs with Northern analysis and in situ hybridization and gamma 2S, gamma 2L and alpha 6 subunit mRNAs with polymerase chain reaction (PCR) amplification. No differences in mRNAs in whole brain were apparent by Northern analysis. In situ hybridization revealed that alpha 1 and gamma 2 subunit mRNAs were co-localized in many brain regions but that they still had distinct patterns of hybridization. However, the few differences observed between LS and SS mice in the levels of hybridization for these subunits did not show a regional distribution consistent with ethanol sensitivity differences. Similar ratios of gamma 2L, and gamma 2S subunit mRNAs were found in LS and SS mouse cerebral cortex and hippocampus, and both mouse lines expressed essentially only gamma 2L subunit mRNA in cerebellum. mRNA for the alpha 6 subunit was detected only in cerebellum and also was qualitatively similar between LS and SS mice. Studies of muscimol-stimulated 36Cl- uptake by cortical membrane vesicles confirmed earlier findings that ethanol does not enhance function of GABAA receptors in SS mice when assayed at 30 degrees C. However, at 34 degrees C ethanol did increase this function in SS mice although the enhancement remained greater in LS mice. These functional results, together with the results showing similar levels of alpha 1, gamma 2S, gamma 2L and alpha 6 subunits in LS and SS mice, suggest that the ethanol-insensitivity of SS mouse GABAA receptors cannot be due solely to lack of subunits required for ethanol action and further suggest that differences in catalytic mechanisms affecting post-translational processing may account for some genetic differences in ethanol sensitivity of GABAA receptors.     

Persistent elevation in GABAA receptor subunit mRNAs following social stress.

Stress is associated with alterations in GABA/benzodiazepine binding and function. We evaluated effects of social stress on GABAA receptor subunit (alpha 1 and gamma 2) mRNAs by Northern hybridization. In cortex, no change was observed in either subunit mRNA immediately after stress, but a 4 hours mRNAs for both subunits were increased. These changes persisted for 72 hours after stress, and returned to baseline levels at 7 days. No changes in mRNAs were observed in sham-treated mice. No changes in either subunit mRNA were observed in stressed or sham-treated mice in cerebellum or hippocampus. In undefeated resident mice, mRNAs for both subunits in cortex were unaffected at 24 hours after the stress episode. Social stress is associated with increases in GABAA receptor alpha 1 and gamma 2 subunit mRNAs in cortex.     

Developmental profile of polyadenylated and non-polyadenylated GABAA receptor subunit mRNAs.

The ratio of mRNA not selected for polyadenylation (non-poly(A)+ selected) to mRNA selected for polyadenylation (poly(A)+) for the beta 1, alpha 1 and gamma 2 subunits of the GABAA receptor complex was examined in rats as a function of age. RNA was extracted from whole brain of rats that were either 0, 1, 3, 5 or over 60 days of postnatal age. Poly(A)+ mRNA was purified by oligo(dT)-cellulose chromatography. Non-poly(A)+ selected mRNA and poly(A)+ mRNA for the GABAA receptor beta 1, alpha 1 and gamma 2 subunits were examined by Northern blot analysis using cDNA probes specific for these subunits. Levels of GABAA receptor beta 1 subunit mRNA were also examined by solution hybridization analysis with a beta 1 riboprobe. Analysis of Northern blots revealed that levels of poly(A)+ beta 1 subunit mRNA were highest at 0 days of age, but decreased and reached adult levels by 5 days of postnatal age. However, levels of the beta 1 subunit message extracted from non-poly(A)+ selected mRNA were not significantly different at any of the ages examined, suggesting the existence of a population of beta 1 subunit mRNA that is not polyadenylated. The age-related discrepancy between beta 1 subunit levels measured in non-poly(A)+ selected mRNA and poly(A)+ mRNA was also observed using solution hybridization analysis. In contrast, levels of both non-poly(A)+ selected mRNA and poly(A)+ mRNA for the alpha 1 subunit of the GABAA complex increased from 0 days of age to adulthood. Similarly, levels of both non-poly(A)+ selected mRNA and poly(A)+ mRNA for the GABAA receptor gamma 2 subunit increased with age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Region-specific expression of GABAA receptor alpha 3 and alpha 4 subunits mRNAs in the rat brain.

The expression of mRNAs encoding the alpha 3 and alpha 4 subunits of the gamma-aminobutyric acid A (GABAA) receptor in the rat brain was investigated by in situ hybridization histochemistry. Both subunits showed a wide but uneven distribution, which did not coincide with the distribution of any other subunit so far reported. The cerebral cortex, anterior olfactory nucleus, lateral septum, subiculum, lateral and medial nuclei of the amygdaloid complex, anterior nuclei of the thalamus, pars compacta of the substantia nigra, trigeminal sensory nuclei, and cochlear nucleus were some of the areas where strong expression of mRNA for both the alpha 3 and alpha 4 subunits was detected. In the mitral cell layer of the olfactory bulb, the preoptic area and locus coeruleus, strong expression of only the alpha 3 subunit was detected. In the granular cell layer of the olfactory bulb, caudate-putamen, tenia tecta, pyramidal cell layer of the CA region and granular cell layer of the dentate gyrus in the hippocampal formation, dorsomedial and ventrolateral nuclei of the thalamus, dorsal part of the lateral geniculate body, preolivary nuclei and pontine nuclei, only the alpha 4 subunit showed strong expression. The diverse distribution of these two subunits is considered to indicate that each has a different role in the central nervous system.     

Immunohistochemical studies on distribution of GABAA receptor complex in the rat brain using antibody against purified GABAA receptor complex

The distribution of the gamma-aminobutyric acid (GABA)A receptor/benzodiazepine receptor/Cl- channel complex in the rat brain was examined immunohistochemically using the specific antibody against purified GABAA receptor complex. The immunization of white albino rabbit with purified GABAA receptor complex resulted in the formation of specific antibody as indicated by the immunoprecipitation test. Immunohistochemical examinations using the antiserum on rat brain slices by the peroxidase-antiperoxidase method revealed the presence of the following immunoreactive sites which coincided with a previous report using antibody against L-glutamic acid decarboxylase; ventromedial nucleus of hypothalamus, red nucleus, globus pallidus, zona compacta and zona reticulata of substantia nigra, layers of Purkinje cells and granular cells of cerebellum, layers III-V of cerebral cortex and stratum radiatum of hippocampus. These results strongly suggest that immunohistochemical application of the antibody against the purified GABAA receptor complex is a useful tool for identifying GABAergic neurons having GABAA receptor complex-mediated synapses.     

Developmental changes in distribution of NMDA receptor channel subunit mRNAs.

In situ hybridization analyses have revealed drastic changes in expression and distribution of five subunit mRNAs of the mouse NMDA receptor channel during brain development. The epsilon 1 subunit mRNA is expressed postnatally and widely in the brain. On the other hand, the epsilon 2 subunit mRNA is found throughout the entire embryonic brain, but its expression becomes restricted to the forebrain at postnatal stages. The epsilon 3 subunit mRNA appears postnatally and predominantly in the cerebellum, whereas the epsilon 4 subunit mRNA is abundantly expressed in the diencephalon and the brainstem at embryonic and neonatal stages. In contrast, the zeta 1 subunit mRNA distributes ubiquitously in the brain throughout development. These findings suggest that changes in the subunit composition of the NMDA receptor channel take place during brain development.     

Subunit- and brain region-specific reduction of GABAA receptor subunit mRNAs during chronic treatment of rats with diazepam

The mRNA levels for several GABA_A receptor subunits were measured by Northern blot analysis. Rats were treated for 3 wk by continuous release of diazepam (DZP) from subcutaneous reservoirs, and then sacrificed immediately or 48 h after removing the reservoirs. Poly(A)⁺ RNAs, isolated from cerebral cortex, cerebellum, and hippocampus, were hybridized with oligonucleotide probes for GABA_A receptor subunits and a cDNA probe for β-actin. Subunit mRNAs were expressed relative to the corresponding β-actin mRNA. DZP treatment decreased the α₁ subunit mRNA level 40% in hippocampus, but it was not changed in cortex or cerebellum. The α₅ subunit mRNA level was decreased in cerebral cortex (28%) and hippocampus (15%). The γ₂ subunit mRNA level was decreased (40%) only in cortex. DZP treatment did not affect α₂, α₃, α₄, β₂, or β₃ subunit mRNA levels. Decreases in mRNA levels had reversed within 48 h after stopping chronic treatment. Acute DZP did not change α₁, α₅, or γ₂ subunit mRNA levels. The decreases in GABA_A receptor subunit mRNA levels were specific to subunit and brain region. These results, coupled with those after chronic flurazepam treatment, also indicated that the effects on GABA_A receptor subunit mRNA levels are specific to the benzodiazepine (BZ) used for chronic treatment.     

Stoichiometries of AMPA receptor subunit mRNAs in rat brain fall into discrete categories

In situ hybridization was used to estimate the relative concentrations of mRNAs encoding different subunits (GluR1-4) of α-amino 3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors in rat brain and to test the hypothesis that within-region expression profiles reflect a limited number of recurring patterns. Fractional subunit mRNA concentrations were calculated for 33 brain regions, and cluster analysis methods were applied to test for statistically meaningful groupings in the data. Four relatively homogeneous classes were identified and designated as AMPA receptor (AR) categories, numbered according to dominant subunit mRNAs. The AR-1 class (47% GluR1 mRNA) was expressed by structures near the mesodiencephalic border, including basal ganglia-related areas. The AR-2 class (57% GluR2 mRNA) was expressed in cortex and tectum. The AR-1,2 class (31% GluR1, 45% GluR2) was found in the largest number of regions, including such dissimilar cell fields as hippocampus and substantia nigra pars compacta. The AR-2,3 grouping (33% GluR2, 31% GluR3) was associated with the sensory relay and reticular thalamic nuclei. It is suggested that AR-1,2 and AR-2, the most closely related categories in clustering space, are largely telencephalic receptors with the former predominant in the subcortex and the latter in the cortex. The AR-2,3 class is associated with ascending sensory stations, whereas AR-1 appears to include several smaller categories expressed by specialized systems. If the balance of subunit mRNAs is reflected at the protein level, then the present data suggest that forebrain AMPA-type glutamate receptors can be classified into a limited number of recurring types. J. Comp. Neurol. 385:491–502, 1997. © 1997 Wiley-Liss, Inc.     

Changes in neocortical and hippocampal GABAA receptor subunit distribution during brain maturation and aging

gamma-Aminobutyric acid type A (GABA(A)) receptors are the most important inhibitory receptors in the central nervous system, playing a pivotal role in the regulation of brain excitability. The pentameric receptor is commonly composed of different alpha, beta, and gamma subunits which mediate the function and pharmacology of the receptor and show regional- and temporal-specific expression patterns. Under varying physiological and pathophysiological conditions, this diversity allows a multitude of adaptive changes in subunit composition leading to distinct biological and pharmacological properties of the receptor. Here, we investigated the expression of five major GABA(A) receptors subunits (alpha1, alpha2, alpha3, alpha5, gamma2) in early postnatal, adult, and aged rat brains. Immunohistochemistry was performed at postnatal day 10, 30, 60, 90, 180, 360, and 540. Morphological and semi-quantitative evaluations of regional optical densities revealed specific regional and temporal expression patterns for all subunits. The study clearly demonstrated that changes in GABA(A) receptor distribution not only occur in the early postnatal cortex and hippocampal formation but also during later periods in the adolescent and aging brain. These findings contribute to a better understanding of age-related changes in brain excitability and further elucidate the distinct pharmacological effects of different GABAergic drugs in young and elderly patients.     

Topographic atlas of somatostatin-containing neurons system in the avian brain in relation to catecholamine-containing neurons system. I. Telencephalon and diencephalon

The morphological organization of the somatostatin (SRIF)-positive neurons in the forebrain (telencephalon and diencephalon) of the warbling grass parakeet (Melopsittacus undulatus) was studied using the indirect immunohistochemical technique of Coons and co-workers ('58). In the telencephalon, a number of SRIF-positive neurons was detected in the lobus paraolfactorius, hippocampus, and paleostriatum. Furthermore, scattered SRIF-labeled cells were noticed in the area corticoidea dorsolateralis and area temporoparieto-occipitalis. A moderate density of immunoreactive fibers was found in the above areas. In addition, although the septal areas was devoid of SRIF-positive neurons, this area contained a moderate occurred in the following hypothalamic areas: (1) nucleus medialis hypothalami posterior, (2) lateral hypothalamus, and (3) mammillary nucleus. The bird hypothalamus also received a strikingly massive SRIF innervation. The heaviest concentration of SRIF-labeled fibers was detected in the medial eminence. Many SRIF-labeled fibers were also observed in other hypothalamic regions. Their locations roughly corresponded in many cases to the areas in which SRIF-positive neurons were disclosed. The overall distribution of the catecholamine system (CA) of the avian forebrain is also represented by means of histofluorescent technique. A possible interaction between SRIF and CA neurons systems is briefly discussed.     

Differential expression of neurotensin receptor mRNA in the dopaminergic cell groups of the rat diencephalon and mesencephalon

Several lines of evidence support interactions between neurotensin (NT) and dopaminergic (DAergic) neurons in the brain. In order to obtain further knowledge about the anatomical substrate for such interactions, the distribution of cells expressing the cloned neurotensin receptor (NTR) mRNA was examined in relation to tyrosine hydroxylase (TH) mRNA-expressing cells within different subnuclei of the diencephalon and ventral mesencephalon of the male rat. In situ hybridization was performed on consecutive sections labeled with ³³P-labeled oligonucleotide probes. In the hypothalamus, NTR mRNA signals were mostly found in the suprachiasmatic, dorsomedial, dorsal premammillary, and supramammillary nuclei. On the other hand, DAergic cells of the periventricular nucleus of the hypothalamus and dorsal aspect of the arcuate nucleus, revealed by TH in situ hybridization, did not exhibit NTR mRNA even though dense NT binding sites have been previously described in this nuclei. In the zona incerta, TH mRNA-containing cells were concentrated in the medial part, with little overlap with NTR mRNA-expressing cells located mainly in its mediolateral extent. In contrast, the distribution of both markers was superimposable within the different subdivisions of the ventral tegmental area and substantia nigra, as previously suggested, but also in the retrorubral field. These anatomical data further support the NT-dopamine interactions on both mesocorticolimbic and nigrostriatal DAergic systems. Moreover, the results suggest that diencephalic DAergic neurons do not synthesize the cloned NTR mRNA or express it at considerably lower levels than DAergic mesencephalic cells. © 1995 Wiley-Liss, Inc.     

Transient expression of GABAA receptor subunit mRNAs in the cellular processes of cultured cortical neurons and glia.

In this study, we have studied by in situ hybridisation histochemistry the expression and intracellular distribution of the GABAA receptor subunit mRNAs in cultured neurons obtained from postnatal day 1-3 rats in order to determine how neurotransmitter receptor expression may be regulated during development of the nervous system. In postnatal cortical cells, we found that GABAA receptor subunit mRNAs coding for alpha2, alpha5, beta2, beta3 and gamma2 subunits were transiently expressed in the cellular processes and growth cones after 1-3 days in culture. These observations indicate that GABAA receptor subunit mRNAs are transported (or trafficked) into the cellular processes of early postnatal cortical cells. These selective localisations were rarely observed after 5 days in culture and only in cells which had not made cell-to-cell contact. The localisation of subunit mRNAs in the processes was more effectively maintained up to 5 days or even longer if cell-to-cell contact was avoided by culturing the cells at low density or by inhibiting neurite sprouting pharmacologically with the GABA receptor channel antagonist TBPS. Finally, immunocytochemistry revealed the expression of GABAA receptors in the growth cones of pyramidal neurons in culture. Thus, the expression of mRNA correlates to the expression of protein. These results suggest that the selective trafficking of GABAA receptor subunit mRNAs during synaptogenesis may be regulated by synapse formation and/or glial-neural communication.     

Differential and transient expression of GABAA receptor alpha-subunit mRNAs in the developing rat CNS.

The expression of mRNAs coding for alpha 1, alpha 2, alpha 3, alpha 5, and alpha 6 subunits of the GABAA neurotransmitter receptor was followed during the development of the rat CNS by in situ hybridization histochemistry. Expression of these subunit mRNAs in tissue sections of embryonic day 15 and 17 (E15, E17) whole rat and in brain at ages greater than E17 to adult were varied, transient, and region specific. Subunit mRNAs first detected at E15 were those coding for the alpha 2 and alpha 3 subunits. At E17, alpha 2, alpha 3, and alpha 5 mRNAs were present in abundance in numerous areas in the CNS, with lower but significant amounts of alpha 6 being present in the cortical neuroepithelial layers. However, alpha 6 subunit mRNA expression in the cortex declined until little or no alpha 6 mRNA was detected at E19. alpha 1 subunit mRNA first appeared at E19 in the cortex, followed by expression in the hippocampus by postnatal 5 (PN5). Particularly high expression of alpha 2 and alpha 5 subunit mRNAs was detected throughout the developing CNS, but they were most abundant in the olfactory bulb neurons. The high levels of alpha 2 and alpha 5 subunit mRNAs began to decline around PN5 to the amounts observed in adult. These results demonstrate that numerous GABAA receptor alpha-subunits are expressed before birth in a region- and age-specific manner. This complex and varied expression supports the hypothesis that GABA may play a role in cellular and synaptic differentiation.