Wu Ma

Sex differences in expression of serotonin receptors (subtypes 1A and 2A) in rat brain: a possible role of testosterone.

Sexual differences in the expression of messenger RNA and in the binding of serotonin receptors (subtypes 1A and 2A) were studied by in situ hybridization and autoradiography ¿[3H]8-hydroxy-2(di-n-propylamino)tetralin and [3H]ketanserin binding) in the rat brain. Serotonin-1A receptor messenger RNA showed distinct expression patterns for female and male rats. Expression of serotonin-1A receptor messenger RNA was greater in males in subregions of the hypothalamus and amygdala, and less in males in subregions of the hippocampus. No significant differences in the distribution of serotonin-1A receptor binding sites were found between the sexes. Serotonin-2A receptor messenger RNA expression was comparable in males and females in all brain regions except the ventromedial hypothalamic nuclei, where lower levels were seen in females. However, the binding of serotonin-2A receptor measured with [3H]ketanserin was significantly higher in females in all regions of the hippocampus. In a separate study, gonadectomy in males significantly increased serotonin-1A messenger RNA content in the cortex, hypothalamus, hippocampus, amygdala and dorsal raphe, and decreased serotonin-2A messenger RNA in ventromedial hypothalamic nuclei only. Almost all gonadectomy-induced changes were reversed by concomitant administration of testosterone. Our data provide evidence for region-specific sex differences in serotonin receptor subtype 1A and 2A transcription and concentration in the rat brain, and further suggest a modulatory role of testosterone in serotonin (particularly subtype 1A) receptor expression. Gender and gonadal steroid effects on central serotonergic systems may underlie the reported sexual dimorphisms in affective state regulation, response to psychopharmacological agonists or pituitary adrenal activation.

Neurotrophins Stimulate Chemotaxis of Embryonic Cortical Neurons

During mammalian cortical development, neuronal precursors proliferate within ventricular regions then migrate to their target destinations in the cortical plate, where they organize into layers. In the rat, most cortical neuronal migration occurs during the final week of gestation (Bayer et al., 1991; Jacobson, 1991). At this time (E15–E21), reverse transcriptase‐polymerase chain reaction demonstrated that cortical homogenates contain mRNA encoding brain derived neurotrophic factor (BDNF) and the catalytic form of its high‐affinity receptor, TrkB. lmmunocytochemistry and in situ hybridization of sections revealed that the catalytic TrkB receptors predominantly localize to regions containing migratory cells. Many TrkB+ cells exhibited the classic morphology of migrating neurons, suggesting that TrkB ligands play a role in cortical neuronal migration. We analysed whether TrkB ligands influence the motility of embryonic cortical cells (from E15–E21) using a quantitative in vitro chemotaxis assay. High‐affinity TrkB ligands (BDNF and NT4/5) stimulated chemotaxis (directed migration) of embryonic neurons at concentrations ranging from 1 to 100 ng/ml. NT‐3, a low‐affinity TrkB ligand, only stimulated significant migration at high concentrations (±100 ng/ml). Peak migration to BDNF was observed at gestational day 18 (E18). BDNF‐induced chemotaxis was blocked by either tyrosine kinase inhibitor, K252a, or the Ca2+‐chelator, BAPTA‐AM, suggesting that BDNF‐induces motility via autophosphorylation of TrkB receptor proteins and involves Ca2+‐dependent mechanisms. BDNF‐stimulation of increased cytosolic Ca2+ was confirmed with optical recordings of E18 cortical cells loaded with Ca2+ indicator dye. Thus, signal transduction through the TrkB receptor complex directs neuronal migration, suggesting that, in vivo, BDNF exerts chemotropic effects that are critical to morphogenesis of the cortex.

Analysis of the anatomical distribution of GAD67 mRNA encoding truncated glutamic acid decarboxylase proteins in the embryonic rat brain.

During development of the central nervous system (CNS) the gene that encodes the 67 kDa form of glutamic acid decarboxylase (GAD) undergoes alternative splicing. The alternatively spliced variants include an exon (referred to as ES, for embryonic stop) that contains a premature stop codon. The detection of mRNA containing the ES exon in embryonic rat brain has been previously reported (Proc. Natl. Acad. Sci., 87 (1990) 8771-8775). We have used in situ hybridization to identify the anatomical distribution of ES mRNA in the embryonic rat brain during two stages of development, embryonic day 17 (E17) and E20. At E17, GAD67 mRNA was expressed in several CNS regions that were destined to contain GABAergic neurons when mature. ES transcripts were predominantly localized to ventricular zones and other regions associated with populations of proliferative cells at E17 and E20. At both ages, however, the alternatively spliced variants were also detected in regions of brain associated with migratory or post-mitotic neurons. GAD67 transcripts that did not include the ES exon were localized to anatomical areas that contained post-mitotic, and often post-migratory neurons. The temporal and spatial disappearance of mRNA containing the ES exon generally followed a caudal-to-rostral gradient which paralleled neuronal terminal mitosis and differentiation.

Anatomical Gradients in Proliferation and Differentiation of Embryonic Rat CNS Accessed by Buoyant Density Fractionation: α3, β3 and γ2 GABAA Receptor Subunit Co-expression by Post-mitotic Neocortical Neurons Correlates Directly with Cell Buoyancy

Development of the CNS occurs as complex cascade of pre‐programmed events involving distinct phases of cell proliferation and differentiation. Here we show these phases correlate with cells of specific buoyant densities which can be readily accessed by density gradient fractionation. Sprague‐Dawley dams were pulse‐labelled with bromodeoxyuridine (BrdU) and selected regions of embryonic (E) CNS tissues at El1–22 dissociated with papain into single‐cell suspensions. Proliferative cell populations were assessed by anti‐BrdU and propidium iodide staining using flow cytometry. Cell differentiation was evaluated using molecular and immunocytochemical probes against mRNAs and antigens differentiating the neuroepithelial, neuronal and glial cell lineages. The results show the emergence of distinctive spatiotemporal changes in BrdU+ populations throughout the CNS during embryonic development, which were followed by corresponding changes in the cellular distributions of antigens distinguishing specific cell types. Fractionation of neocortical cells using discontinuous Percoll gradients revealed that an increasing number of cells increase their buoyancy during corticogenesis. Immunocytochemical and molecular characterization showed that the proliferative and progenitor cell populations are for the most part associated with lower buoyancy or higher specific buoyant densities (> 1.056 g/ml) whereas the post‐mitotic, differentiated neurons generally separated into fractions of higher buoyancy or lower specific buoyant densities (<1.043 g/ml). Immunostaining with antibodies against several GABAA receptor subunits (α3, β3, γ2) revealed that the highest percent (70–90%) of immunopositive cells could be identified in the most buoyant, differentiating neurons found in the cortical plate/subplate regions, with the lowest percent of the immunopositive cells found in the least buoyant, proliferative and progenitor cell populations originating from the ventricular/subventricular zones. Taken together, these results indicate that buoyant density is distinguishing characteristic of embryonic CNS cells transforming from primarily proliferative to mainly differentiating, and that fractionation of these cells according to their buoyant densities provides rapid access to the properties of specific cell lineages during the prenatal period of CNS development.

INITIALLY EXPRESSED EARLY RAT EMBRYONIC GABAA RECEPTOR CL- ION CHANNELS EXHIBIT HETEROGENEOUS CHANNEL PROPERTIES

We have studied the earliest expression of GABA‐induced Cl– channels in the rat embryonic dorsal spinal cord (DSC) using in situ hybridization, immunocytochemistry, flow cytometry and electrophysiology. At embryonic day 13 (E13) cells in the dorsal region are still proliferating. In situ hybridization consistently showed transcripts encoding only three GABAa receptor subunits (α4, β1 and γ1); immunocytochemistry both in tissue sections and in acutely isolated cells in suspension demonstrated the expression of the corresponding proteins and also revealed staining for other subunits (α2, α3, β3, γ2). In patch‐recordings performed in cells acutely isolated from the dorsal cord, responses to GABA were detected in 356 out of 889 cells. GABA‐evoked responses, which often displayed the opening of a few channels, were mediated by Cl– ions, were inhibited by bicuculline and picrotoxin, and potentiated by benzodiazepines. Taken together, these observations indicate that Cl– channels likely involve GABAa type receptors. Fluctuation analysis revealed channel kinetics consisting of three exponential components (τs: ≈ 1, 9 and 90 ms) and a wide variety of inferred unitary conductance values, ranging between 4 and 40 pS. A comparison of these results with observations in other, later embryonic cell types and recombinant receptors suggests that most of the earliest E13 DSC GABAa receptors may include α3 subunit. These GABAa receptor Cl– channels may be activated physiologically as both GABA synthesizing enzymes and GABA are present in the E13 dorsal cord.

GABA receptor subunit mRNA expression in brain of conflict, yoked control and control rats

Animal conflict models have been used for years as a preclinical screen for predicting anxiolytic therapeutic efficacy. Anxiolytics, including benzodiazepines, increase punished responding. This suggests that the punished behavior may be mediated by the GABA receptor. To evaluate this hypothesis, we performed in situ hybridization histochemistry studies of GABA receptor subunits (alpha1-alpha4) and synthetic enzymes glutamic acid decarboxylase (GAD65 and GAD67) in four groups of rats: conflict (punishment), yoked controls (rats shocked without conflict training history), fixed interval only controls (rats that worked for food but were not shocked) and untreated controls. With conflict behavioral training, bilateral reduction of mRNA for the GABAA alpha1 subunit was seen relative to controls in the cortex, thalamus and hippocampus. In contrast, alteration of alpha2 mRNA levels appeared only in the yoked control group, with increased levels seen in the thalamus and cortex and decreased levels in the hippocampus. There were no differences in the alpha2 mRNA level between the control and the conflict behavioral trained animals. Further, no significant differences were found between groups in the mRNA levels for the alpha3 subunit, alpha4 subunit, GAD65, and GAD67. These results suggest that the behaviors related to conflict and uncontrollable aversive stimuli (yoked control group) are accompanied and perhaps mediated by selective changes in the GABAA alpha1 or alpha2 subunits, respectively. These findings highlight the potential usefulness of the conflict model as a means of elucidating the biological underpinnings of anxiety disorder. Published by Elsevier Science B.V. All rights reserved.

5-HT1A receptor mRNA expressions differ in the embryonic spinal cord of male and female rats.

During critical periods of development, the effects of testosterone (T) on promoting androgenization of the central nervous system (CNS) are reflected not only by behavior, morphology, and hormone secretion but also by gene expression. The mechanisms involved in sexual differentiation of the CNS, however, remain incompletely defined. The current set of experiments examined with in situ hybridization the dimorphism in 5-HT1A receptor mRNA expression in the embryonic rat spinal cord and the possible role of T in the dimorphism. We found sex-related differences in expression of 5-HT1A mRNA in the spinal cord, which were altered by a single injection of T. The results suggest that this gonadal steroid is responsible for the sexual dimorphism in 5-HT1A mRNA expression occurring during the critical period.

GABA induces GABAergic MSCs in cultured embryonic rat thalamic neurons

Application of 0.1–10 μM GABA in the vicinity of cultured embryonic rat thalamic neurons recorded with patch pipettes in the presence of 2 μM TTX induced or increased the frequency of miniature synaptic currents (MSCs) that reversed polarity at the Cl− equilibrium potential. These MSCs were blocked by the GABAA receptor antagonist bicuculline and exhibited exponential decay kinetics that closely paralleled those estimated from fluctuation analysis of Cl− channels activated pharmacologically by applying 1–10 μM GABA to the same cells. We conclude that the MSCs are mediated by GABA. Application of the GABAA receptor agonist muscimol activated Cl− current but failed to induce GABAergic MSCs while submicromolar concentrations of GABA evoked GABAergic MSCs but did not activate Cl− channels. The GABAB receptor agonist (‐)baclofen did not mimic GABA in inducing MSCs. Induction of GABAergic MSCs by GABA required extracellular Ca2+. Verapamil and Co2+, which block voltage‐dependent calcium channels, completely blocked GABA‐induced MSCs independent of their effects on the direct activation of a Cl− current response. The results indicate that GABA can trigger GABAergic Cl−‐dependent MSCs in a Cao2+‐dependent manner. The mechanism may involve a novel receptor and/or signal transduction pathway. Synapse 25:15–23, 1997.