Diencephalic Regions Research Articles

Roles of Pax-6 in murine diencephalic development.

Pax-6 is one of the earliest regulatory genes to be expressed in the diencephalon. We tested whether normal Pax-6 protein is required for early diencephalic development by examining morphology, precursor proliferation and patterns of regulatory gene expression in the embryonic diencephalon of Small-eye mice (Pax-6 mutants). In Small-eye mice, diencephalic morphology was abnormal at all the embryonic ages studied (days 10.5, 12.5 and 14.5). Regional differences in diencephalic cell density were lost, the diencephalon/mesencephalon boundary was unclear and the third ventricle was enlarged. We estimated diencephalic proliferative rates after labelling with bromodeoxyuridine and found that they were abnormally low in mutants aged embryonic day 10.5. In older mutants, the diencephalon contained fewer cells than normal. In wild-type E14.5 diencephalon, Pax-6, Dlx-2 and Wnt-3 are expressed in discrete regions along the rostrocaudal and dorsoventral axes. In situ hybridizations for these genes in E14.5 Small-eye mice revealed discrete zones of diencephalic expression that had similar relative positions to those in wild-type mice. Some differences of detail in their expression were seen: Pax-6 had an expanded rostral domain of expression and an abnormally indistinct caudal boundary; Dlx-2 had a diffuse, rather than a sharp, caudal boundary of expression; the normally high dorsal midline expression of Wnt-3 was lost. We conclude that normal expression of Pax-6 is required for the correct regulation of diencephalic precursor proliferation. Pax-6 may also control some aspects of diencephalic differentiation, but its mutation in Small-eye mice does not preclude the development of a degree of diencephalic regionalization resembling that in normal mice.

Memory beyond the hippocampus

Improved neuroanatomical knowledge, technical and methodological innovations (such as PET), and more refined conceptualizations of memory have inspired a reappraisal of theoretical beliefs regarding the role of the hippocampus in memory. In the past few years, it has become apparent that the influence of the medial temporal lobe regions extends beyond memory and that memory processes (such as encoding, consolidation and retrieval) involve not only the hippocampus and the medial temporal and diencephalic regions, but also widely distributed neocortical and perhaps even cerebellar regions.

Neurobehavioral outcome in pediatric craniopharyngioma.

Neurobehavioral dysfunction occurs in children with craniopharyngioma, both before and after treatment, and its impact on outcome may not be fully appreciated. Also unclear is whether neurobehavioral outcome relates more to tumor location or surgical factors. We reviewed the records of 20 children with craniopharyngioma who were seen between 1983 and 1995. All children had subfrontal craniotomy and either partial (14 children) or gross total (6 children) resection of their tumors. In addition to traditional neuropsychological testing, we assessed social behavior and school performance using standardized ratings based on family interviews and school records. Over a mean follow-up period of 38 months, only 3 of 20 children had a good outcome in all three categories, and 12 of 20 had moderate or severe impairment in at least one category. Outcome did not differ between those who had partial and those with gross total resection. We conclude that neurobehavioral disorders are common and cause important morbidity in children after treatment for craniopharyngioma. To evaluate these impairments in future outcome studies, standard neuropsychological testing should be supplemented by specific behavioral assessments to capture the full range of neurobehavioral disability. In this series, partial versus gross total resection did not influence outcome, implying that tumor location in diencephalic and limbic regions is a more important factor.

Sexual dimorphism of arg-vasotocin gene expressing neurons in the telencephalon and dorsal diencephalon of the domestic fowl. An immunocytochemical and in situ hybridization study.

A strong sex dimorphism in the distribution of immunoreactive arginine-vasotocin (AVT) and AVT mRNA was observed in telencephalic and dorsal diencephalic areas of the domestic fowl using immunocytochemistry and in situ hybridization. Two subgroups of immunoreactive parvocellular perikarya surrounded by dense plexus of immunoreactive fibres were found within the bed nucleus of the stria terminalis and the dorsal part of the diencephalic paraventricular region of males. No signs of immunoreactivity were observed within corresponding regions of the female brain. Instead, in females a few scattered weakly stained perikarya were observed rostrally to the level of the anterior commissure, juxtapositioned to the nucleus accumbens and the floor of the lateral ventricle. The distribution of AVT mRNA containing cell profiles fully confirmed the immunocytochemical findings. Osmotic stress induced by water deprivation for 48 h had no influence on the number of immunoreactive or AVT mRNA containing parvocellular cell bodies. However, it resulted in an increase of immunoreactive cell area in the bed nucleus of the stria terminalis and dorsal diencephalon of 5. 9 and 11.7%, respectively. We suggest that the sexually dimorphic vasotocinergic circuit may be involved in the co-ordination of behavioural and autonomic functions in response to environmental stress.

Effects of in utero cocaine exposure on the expression of mRNAS encoding the dopamine transporter and the D1, D2 and D5 dopamine receptor subtypes in fetal rhesus monkey

The effects of in utero cocaine exposure on the development of the mRNAs encoding the dopamine transporter (DAT) and the D1, D2 and D5 dopamine receptor subtypes were determined in fetal monkey brains at day 45 and day 60 of gestation. Pregnant monkeys were treated with cocaine 3 mg/kg or saline i.m., four times a day from day 18 of gestation until the pregnancy was terminated at day 45 or day 60. The fetal brains were dissected, and tissue RNA extracted and quantified using ribonuclease protection assay analysis. In day 45 fetal monkeys, dopamine D1 and D2 receptor subtype mRNAs and DAT mRNA were found in low quantities both in control and cocaine-treated subjects. In day 60 fetal monkeys, D1 receptor mRNA levels were highest in the frontal cortex/striatal area, and low to moderate quantities were found in diencephalic and mesencephalic fetal brain regions. Dopamine D2 receptor mRNA levels were highest in the frontal cortex/striatal area, diencephalon and the midbrain, moderate in the brainstem and low in the caudal temporal lobe and surrounding cortical areas. Dopamine D5 receptor mRNA was expressed in low quantities throughout the day 60 fetal monkey brain, whereas DAT mRNA was found in the midbrain only. In utero cocaine exposure caused a significant increase in dopamine D1, D2 and D5 receptor subtype mRNAs in the frontal cortex/striatal area of day 60 fetal monkeys. These results support the hypothesis that dopamine synthesis and release may be reduced in cocaine-treated fetuses, which results in dopamine receptor up-regulation.

Effects of in utero cocaine exposure on the expression of mRNAS encoding the dopamine transporter and the D1, D2 and D5 dopamine receptor subtypes in fetal rhesus monkey

The effects of in utero cocaine exposure on the development of the mRNAs encoding the dopamine transporter (DAT) and the D1, D2 and D5 dopamine receptor subtypes were determined in fetal monkey brains at day 45 and day 60 of gestation. Pregnant monkeys were treated with cocaine 3 mg/kg or saline i.m., four times a day from day 18 of gestation until the pregnancy was terminated at day 45 or day 60. The fetal brains were dissected, and tissue RNA extracted and quantified using ribonuclease protection assay analysis. In day 45 fetal monkeys, dopamine D1 and D2 receptor subtype mRNAs and DAT mRNA were found in low quantities both in control and cocaine-treated subjects. In day 60 fetal monkeys, D1 receptor mRNA levels were highest in the frontal cortex/striatal area, and low to moderate quantities were found in diencephalic and mesencephalic fetal brain regions. Dopamine D2 receptor mRNA levels were highest in the frontal cortex/striatal area, diencephalon and the midbrain, moderate in the brainstem and low in the caudal temporal lobe and surrounding cortical areas. Dopamine D5 receptor mRNA was expressed in low quantities throughout the day 60 fetal monkey brain, whereas DAT mRNA was found in the midbrain only. In utero cocaine exposure caused a significant increase in dopamine D1, D2 and D5 receptor subtype mRNAs in the frontal cortex/striatal area of day 60 fetal monkeys. These results support the hypothesis that dopamine synthesis and release may be reduced in cocaine-treated fetuses, which results in dopamine receptor up-regulation.

Effect of low and high doses of nitrous oxide on preproenkephalin mRNA and its peptide methionine enkephalin levels in the hypothalamus

The effect of exposure to nitrous oxide (N 2O) on the levels of preproenkephalin mRNA in the hypothalamus of rats was examined. In the first experiment, rats were exposed to 1000 ppm N 2O for 8 h a day over 4 days. Compared with controls (which were exposed to air over the same duration), the N 2O exposed animals exhibited significant elevations in preproenkephalin mRNA levels in the hypothalamus. In a second experiment, rats were exposed to 60% N 2O or air for 12, 24 and 48 h duration, and hypothalamic levels of preproenkephalin mRNA as well as methionine enkephalin were analyzed. Compared with controls, N 2O exposed rats exhibited significant elevations in preproenkephalin mRNA levels. The levels on preproenkephalin mRNA were significantly higher after 48 h of N 2O exposure than after 12 h of N 2O exposure. Similarly, the concentration of methionine enkephalin was significantly higher after 24 and 48 h of exposure to N 2O than after exposure to 12 h of N 2O or air. These results indicate that (a) exposure to N 2O results in significant elevations in preproenkephalin mRNA levels, (b) the increased preproenkephalin mRNA levels appear to be proportional to the concentration of N 2O exposure as well as the duration of N 2O exposure, and (c) N 2O-induced elevation in preproenkephalin mRNA levels is associated with corresponding increase in tissue concentrations of methionine enkephalin. In total, these results suggest that N 2O selectively stimulates synthesis of methionine enkephalin in the diencephalic region of the brain.

Localization of galanin and its binding sites in the quail brain

Avian galanin was first isolated from the chicken intestine, and subsequently we isolated the same peptide from the quail oviduct. Galanin is known to be a multifunctional peptide in the neuroendocrine system in mammals, while little information is available for the galanin action in the central nervous system in birds. In the present study, therefore, we examined the presence and the localization of galanin and its binding sites in the quail brain. The binding of [ 125I]iodo-avian galanin to brain tissues specifically inhibited as a function of the concentrations of unlabeled avian galanin. In all of the observed brain regions, the number of galanin-binding sites when compared on the basis of unit weight was maximal in the interbrain including the preoptic and hypothalamic regions and minimal in the cerebellum. The Scatchard plot analysis of the galanin binding to the interbrain revealed that the apparent equilibrium constant of dissociation ( K d was 0.281 (95% confidence interval, 0.234-0.351) nM, suggesting the presence of a single class of high-affinity binding sites. Although galanin-like immunoreactivity was found in several restricted regions throughout the brain, the most intense immunoreaction was present in the diencephalic region. Abundant immunoreactive cell bodies and/or fibers were localized in several preoptic and hypothalamic nuclei, i.e., the nucleus preopticus medialis (PONI), the nucleus paraventricularis (PVN), the nucleus periventricularis hypothalami (PHN) and the nucleus infundibuli (IN), that are known to be involved in the control of reproductive behaviors as well as the pituitary hormone secretion. A substantial number of immunoreactive cell bodies and/or fibers was further observed within the septal nuclei in the telencephalon and several mesencephalic nuclei including the nucleus intercollicularis (ICo) involved in the control of vocalizations. These results suggest that avian galanin may act as an important factor to modulate the some reproductive behaviors and/or the pituitary hormone secretion in the bird.

Localization of galanin and its binding sites in the quail brain

Avian galanin was first isolated from the chicken intestine, and subsequently we isolated the same peptide from the quail oviduct. Galanin is known to be a multifunctional peptide in the neuroendocrine system in mammals, while little information is available for the galanin action in the central nervous system in birds. In the present study, therefore, we examined the presence and the localization of galanin and its binding sites in the quail brain. The binding of [ 125 I]iodo-avian galanin to brain tissues specifically inhibited as a function of the concentrations of unlabeled avian galanin. In all of the observed brain regions, the number of galanin-binding sites when compared on the basis of unit weight was maximal in the interbrain including the preoptic and hypothalamic regions and minimal in the cerebellum. The Scatchard plot analysis of the galanin binding to the interbrain revealed that the apparent equilibrium constant of dissociation ( K d was 0.281 (95% confidence interval, 0.234-0.351) nM, suggesting the presence of a single class of high-affinity binding sites. Although galanin-like immunoreactivity was found in several restricted regions throughout the brain, the most intense immunoreaction was present in the diencephalic region. Abundant immunoreactive cell bodies and/or fibers were localized in several preoptic and hypothalamic nuclei, i.e., the nucleus preopticus medialis (PONI), the nucleus paraventricularis (PVN), the nucleus periventricularis hypothalami (PHN) and the nucleus infundibuli (IN), that are known to be involved in the control of reproductive behaviors as well as the pituitary hormone secretion. A substantial number of immunoreactive cell bodies and/or fibers was further observed within the septal nuclei in the telencephalon and several mesencephalic nuclei including the nucleus intercollicularis (ICo) involved in the control of vocalizations. These results suggest that avian galanin may act as an important factor to modulate the some reproductive behaviors and/or the pituitary hormone secretion in the bird.

GAP-43 mRNA expression in early development of human nervous system.

The temporal and spatial distribution of GAP-43 mRNA in early human development, from 6 to 23 gestational weeks (g.w.), was examined by in situ hybridization histochemistry. GAP-43 mRNA was expressed as early as 6 g.w. in all regions of developing nervous system, the spinal cord, brainstem, cerebellum, diencephalic and telencephalic regions. Although the pronounced level of expression persisted during the entire examined period, the intensity of expression varied along the spatial axis over time. Analysis at the cellular level revealed that early on in development (6 g.w.) GAP-43 mRNA was expressed in the entire neuroblast population. With the onset of differentiation, at 13-23 g.w., GAP-43 mRNA expression had switched to the neurons that are in the process outgrowth. The highest level of GAP-43 mRNA expression was localized in the regions consisting of differentiating neurons, such as the cortical plate and intermediate zone of the telencephalic wall, and several delineated subcortical and thalamic nuclei. The spatial and temporal pattern of GAP-43 mRNA expression obtained suggests a possible dual role of GAP-43 in the development of the human nervous system: in the embryonic brain it could be involved in fundamental processes underlying cell proliferation; in the fetal brain its expression is specifically correlated with differentiation and the outgrowth of axons.

Mechanisms of the effects of fenfluramine on type II diabetes mellitus in combination with obesity

With obesity-related diabetes, it is possible to use biguanides, which have a slight anorectic effect. The presence of side effects in biguanides often complicates their long-term use, especially in elderly patients who have a history of chronic diseases of the lungs, heart, kidneys, and liver. In such cases, it is advisable to prescribe anorectic drugs that simultaneously affect carbohydrate metabolism.One of these drugs is p-ethyl-a-methyl-3-fluoromethylphenethylamine hydrochloride (minifage, ponderal), which has a stimulating effect on the central nervous system. Fenfluramine reduces the supply and synthesis of monoamines (5-hydroxytryptamine) in the diencephalic regions of the brain, due to which there is a decrease in food requirements and a decrease in body weight.A number of researchers have shown that the hypoglycemic effect of the drug occurs due to peripheral action. Fenfluramine affects the extraneuronal oxidation of monoamins and reduces the level of tryptophan in the blood. T. Pssquire in experiments on rats confirmed that this effect of the drug is achieved by improving the sensitivity of peripheral tissues to insulin, especially muscles.Some researchers observed a decrease in plasma fatty acids and cholesterol levels during treatment with ferfluramine.However, the issue of the effect of the drug on the residual secretion of the C-peptide of b-cells of the pancreas during treatment with fenfluramine is not covered in the literature available to us.In our study, 26 patients with obesity (the control group consisted of 18 healthy subjects who had previously impaired glucose tolerance) showed that fenfluramine restores phase I of insulin secretion on the background of an intravenous load with glucagon or glucose, and compensation for diabetes mellitus during treatment fenfluramine is apparently associated not only with a direct stimulating effect on the -cells of the islets of Langerhans but also with its peripheral effect.

Neurophysiology of converging synaptic inputs from the rat prefrontal cortex, amygdala, midline thalamus, and hippocampal formation onto single neurons of the caudate/putamen and nucleus accumbens.

Neurophysiological responses mediated by projections from five telencephalic and diencephalic regions (the infra- and prelimbic portions of the prefrontal cortex, amygdala, midline and intralaminar thalamic nuclei, entorhinal cortex and subiculum/CA1) to the caudate/putamen (CPu) and nucleus accumbens (Acb) of the dorsal and ventral striatum were studied in chloral-hydrate-anesthetized rats. Both extra- and intracellular in vivo recording techniques were used. A retrograde tracer (wheatgerm agglutinin-apo-horseradish peroxidase-5 nm colloidal Gold) was deposited in some animals in the vicinity of recording sites to confirm that stimulating electrodes were located near cells that projected to the striatum. Electrical stimulation of these five regions, respectively, evoked excitatory responses in 60%, 22%, 51%, 25%, and 17% of striatal neurons. Some responses, particularly with thalamic stimulation, showed short-term frequency potentiation in which 5/s stimulation increased the probability of spike firing. About half of responsive cells showed convergent excitation to more than one stimulating site. It was possible with convergent excitatory responses to show synaptic interactions: simultaneous activation of more than one site produced spatial and temporal summation to increase the probability of spike firing. Up to 5-way convergence onto single striatal neurons and up to 3-way interactions could be shown. These results indicate that functional influences from the hippocampal formation can converge with other excitatory input onto single striatal neurons to effect synaptic integration.

Neurophysiology of converging synaptic inputs from the rat prefrontal cortex, amygdala, midline thalamus, and hippocampal formation onto single neurons of the caudate/putamen and nucleus accumbens

Neurophysiological responses mediated by projections from five telencephalic and diencephalic regions (the infra- and prelimbic portions of the prefrontal cortex, amygdala, midline and intralaminar thalamic nuclei, entorhinal cortex and subiculum/CA1) to the caudate/putamen (CPu) and nucleus accumbens (Acb) of the dorsal and ventral striatum were studied in chloral-hydrate-anesthetized rats. Both extra- and intracellular in vivo recording techniques were used. A retrograde tracer (wheatgerm agglutinin-apo-horseradish peroxidase-5 nm colloidal Gold) was deposited in some animals in the vicinity of recording sites to confirm that stimulating electrodes were located near cells that projected to the striatum. Electrical stimulation of these five regions, respectively, evoked excitatory responses in 60%, 22%, 51%, 25%, and 17% of striatal neurons. Some responses, particularly with thalamic stimulation, showed short-term frequency potentiation in which 5/s stimulation increased the probability of spike firing. About half of responsive cells showed convergent excitation to more than one stimulating site. It was possible with convergent excitatory responses to show synaptic interactions: simultaneous activation of more than one site produced spatial and temporal summation to increase the probability of spike firing. Up to 5-way convergence onto single striatal neurons and up to 3-way interactions could be shown. These results indicate that functional influences from the hippocampal formation can converge with other excitatory input onto single striatal neurons to effect synaptic integration. © 1996 Wiley-Liss, Inc.

Diencephalic Asymmetries

HARRIS, J. A., V. GUGLIELMOTTI AND M. BENTIVOGLIO. Diencephalic asymmetries. NEUROSCI BIOBEHAV REV 20(4) 637–643, 1996.—Structural asymmetry in diencephalic regions has been reported in a number of studies since the pioneering observations by Kemali and Braitenberg, Atlas of the frog's brain. Springer Verlag: 1969. Anatomical differences between the left and right habenulae have been identified in many lower vertebrate species. While there are few reports of structural asymmetry in the dorsal thalamus, there is evidence that asymmetrical thalamofugal projections can be induced in the visual system of chicks by lateralized sensory stimulation prior to hatching. Finally, there have been consistent reports of differences between left and right sides of the hypothalamus in their sensitivity to the effects of circulating gonadal hormones in rats. In most cases, these asymmetries are sex-linked and correspond to a lateralization of function. Although the significance of many of these diencephalic asymmetries is still enigmatic, their existence indicates that asymmetry is not a phylogenetically recent feature of the brain, and that left-right differences in the brain may be mediated by a common ontogenetic mechanism and may underlie the development of highly specialized functions. Copyright © 1996 Elsevier Science Ltd.

A segmental map of architectonic subdivisions in the diencephalon of the frog Rana perezi: acetylcholinesterase-histochemical observations.

The work examines frog diencephalic subdivisions from a segmental viewpoint and adds a number of details to the atlas of the bullfrog diencephalon reported by Neary and Northcutt [1983]. Acetylcholinesterase histochemistry was performed on brains of Rana perezi, sectioned either sagittally or in a plane roughly parallel to the optic tract to optimize detection of segmented landmarks. This material provided a very detailed picture of individual neurons, neuropils and some fiber tracts expressing the enzyme in diverse patterns characteristic for each diencephalic region. The main diencephalic areas previously recognized in the bullfrog appeared subdivided into smaller AChE-chemoarchitectonic units. Modified subdivisions are proposed for several entities: preoptic, suprachiasmatic, entopeduncular, ventral thalamic, anterior thalamic, pretectal, hypothalamic and tuberculum posterior regions. A number of cell groups are described for the first time in frogs. This mapping is expected to be useful for the interpretation of immunocytochemical and experimental hodologic results in the diencephalon of frogs and opens new possibilities for comparative analysis.

In vitro increase of aromatase mRNA in diencephalic neurons.

This study was designed to (1) examine the ability of fetal diencephalic neurons cultured in vitro to express aromatase mRNA and (2) evaluate the involvement of several environmental factors which may regulate the development and differentiation of the neurons in the central nervous system. Brain cells from fetal mice at various developmental stages were cultured as tissue slices and as primary monolayer cells, and the expression levels of aromatase mRNA in the cultured cells were measured by a quantitative reverse transcription-polymerase chain reaction method using an internal standard. On cultured slices of the diencephalic region from fetal mice on embryonic day 12 (E12), E13, or E15 on collagen-coated membranes, the expression level of the mRNA continued to increase for the initial 2-3 days as that in vivo. Time-dependent increase of aromatase mRNA was also observed for 3 days in E13 neuronal cells dissociated with papain and cultured on poly L-Lys-coated dishes in serum-free medium. However, no significant time-dependent increase of the aromatase mRNA level was observed in E10 or E11 brain cells cultured by either method. These findings suggest that the developmental increase of aromatase mRNA in diencephalic neurons is an endogenous characteristic and probably genetically determined after E12.

Host-guided migration allows targeted introduction of neurons into the embryonic brain

The stereotyped positions occupied by individual classes of neurons are a fundamental characteristic of CNS cytoarchitecture. To study the regulation of neuronal positioning, we injected genetically labeled neural precursors derived from dorsal and ventral mouse forebrain into the telencephalic vesicles of embryonic rats. Cells from both areas were found to participate in the generation of telencephalic, diencephalic, and mesencephalic brain regions. Donorderived neurons populated the host brain in distinct patterns and acquired phenotypic features appropriate for their final location. These observations indicate that neuronal migration and differentiation are predominantly regulated by non-cell-autonomous signals. Exploiting this phenomenon, intrauterine transplantation allows generation of controlled chimerism in the mammalian brain.

Resting cerebral glucose metabolism in first-episode and previously treated patients with schizophrenia relates to clinical features.

Functional neuroimaging can elucidate brain dysfunction in schizophrenia. The frontal, temporolimbic, and diencephalic regions have been implicated. There is a lack of prospective samples of first-episode and previously treated patients followed up longitudinally. Patients and controls (42 per group) were studied. Positron emission tomography with flurodeoxyglucose, cross-registered with magnetic resonance imaging, measured metabolism. Scales assessed clinical features, premorbid adjustment, and outcome. There were no differences between groups in whole-brain metabolism or regional ratios or in anterior-posterior gradients, but left midtemporal metabolism was relatively higher in patients. This was pronounced in the negative and Schneiderian and absent in the paranoid subtypes. Higher metabolism and lower relative left hemispheric values were associated with better premorbid adjustment and outcome. A higher subcortical-cortical gradient was noted in first-episode patients. There are no resting metabolic abnormalities in any brain region, but abnormal gradients are evident. These vary in subtypes, and laterality is associated with functioning. The results support the hypothesis of temporolimbic disturbance in schizophrenia that is all ready present at the onset of illness.

Differential distribution of N-acetylaspartylglutamate and N-acetylaspartate immunoreactivities in rat forebrain

Contradictory immunohistochemical data have been reported on the localization of N-acetylaspartylglutamate in the rat forebrain, using different carbodiimide fixation protocols and antibody purification methods. In one case, N-acetylaspartylglutamate immunoreactivity was observed in apparent interneurons throughout all allocortical and isocortical regions, suggesting possible colocalization with GABA. In another case, strong immunoreactivity was observed in numerous pyramidal cells in neocortex and hippocampus, suggesting colocalization with glutamate or aspartate. Reconciling these disparate findings is crucial to understanding the role of N-acetylaspartylglutamate in nervous system function. Antibodies to N-acetylaspartylglutamate and a structurally related molecule, N-acetylaspartate, were purified in stages, and their cross-reactivities with protein conjugates of N-acetylaspartylglutamate and N-acetylaspartate were monitored at each stage by solid-phase immunoassay. Reduction of the cross-reactivity of the anti-N-acetylaspartylglutamate antibodies of N-acetylaspartate-protein conjugates to about 1% eliminated significant staining of most pyramidal neurons in the rat forebrain. Utilizing highly purified antibodies, the distributions of N-acetylaspartylglutamate and N-acetylaspartate were examined in several major telencephalic and diencephalic regions of the rat, and were found to be distinct. N-acetylaspartylglutamate-immunoreactivity was observed in specific neuronal populations, including many groups thought to use GABA as a neurotransmitter. Among these were the globus pallidus, ventral pallidum, entopeducular nucleus, thalamic reticular nucleus, and scattered non-pyramidal neurons in all layers of isocortex and allocortex. N-acetylaspartate-immunoreactivity was more broadly distributed than N-acetylaspartylglutamate-immunoreactivity in the rat forebrain, appearing strongest in many pyramidal neurons. Although N-acetylaspartate-immunoreactivity was found in most neurons, it exhibited a great range of intensities between different neuronal types.

Avian neurosteroids. II. Localization of a cytochrome P450scc-like substance in the quail brain

We have recently demonstrated that the avian brain as well as the peripheral steroidogenic glands produces pregnenolone, the main precursor of steroid hormones, on the basis of biochemical studies. Therefore, the immunohistochemical and Western immunoblotting analyses with a polyclonal antibody directed against the purified bovine adrenal cytochrome P450scc, which is involved in pregnenolone formation, was undertaken to investigate the localization of a cytochrome P450scc-like substance in the brain of adult male Japanese quails. P450scc-like immunoreactive cells were distributed in several telencephalic, diencephalic and mesencephalic regions. An intense immunoreaction was observed in somata of Purkinje cells and in the dendrites extending through the cerebellar molecular layer. Clusters of immunoreactive cell bodies were also detected in the hyperstriatum accessorium (HA), the ventral portions of both the archistriatum and the corticoid area, the preoptic area (POA), the anterior hypothalamus (AHy) and the dorsolateral thalamus (DL). Preadsorbing the antibody with cytochrome P450scc resulted in a complete absence of P450scc-like immunoreactivity in all of positively stained cells. On Western immunoblotting with the P450scc antibody, a P450scc-like substance was present in homogenates of the several brain regions that contain immunohistochemically stained cells. Immunohistochemical experiments using both antibodies against P450scc and the glial fibrillary acidic protein (GFA), a specific marker of glial cells, indicated that many HA cells contained both P450scc-like and GFA-like immunoreactivities. However, no immunoreactivity for GFA was observed in Purkinje cells and the cells localized in the ventral portion of the corticoid area, despite the presence of P450scc-like immunoreactivity. These results confirm our previous findings of pregnenolone biosynthesis and suggest the presence of a P450scc-like substance in both neuronal and glial cells of the quail brain.