Immunohistochemical study of Pax6 and Reissner fibre expression in the prenatal development of the mouse subcommissural organ

The subcommissural organ (SCO) releases glycoproteins into the ventricular cerebrospinal fluid (CSF), where they form Reissner's fibre (RF) and also secretes a CSF-soluble material different from RF-material. Pax6 is a transcription factor important for the regulation of cell proliferation, migration and differentiation in the developing brain. In the present work, we studied wild-type, heterozygous and homozygous Sey mice to compare the expression of RF-antibody and Pax6 in the SCO and adjacent structures. In wild-type mice between E15 to E18, we observed Pax6 expression in cells surrounding the secretory cells of the SCO, and RF-immunoreactive material only in the SCO ependymal cell layer and its basal process. In the heterozygous mice, the neuroanatomical structure of the SCO was present, but RF-antibody staining and Pax6 expression was scarce or almost undetectable; in the homozygous mice neither SCO nor other epithalamic structures were found. We suggest that Pax6 expression at the periphery of the SCO is essential for the development and activity of the organ (AU)

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Organization of radial and non-radial glia in the developing rat thalamus.

The organization of glia and its relationship with migrating neurons were studied in the rat developing thalamus with immunocytochemistry by using light, confocal, and electron microscopy. Carbocyanine labeling in cultured slice of the embryonic diencephalon was also used. At embryonic day (E) 14, vimentin immunoreactivity was observed in radial fascicles spanning the neuroepithelium and extending from the ventricular zone to the lateral surface of the diencephalic vesicle. Vimentin-immunopositive fibers orthogonal to the radial ones were also detected at subsequent developmental stages. At E16, radial and non-radial processes were clearly associated with migrating neurons identified by the neuronal markers calretinin and gamma-aminobutyric acid. Non-radial glial fibers were no longer evident by E19. Radial fibers were gradually replaced by immature astrocytes at the end of embryonic development. In the perinatal period, vimentin immunoreactivity labeled immature astrocytes and then gradually decreased; vimentin-immunopositive cells were only found in the internal capsule by the second postnatal week. Glial fibrillary acidic protein immunoreactivity appeared at birth in astrocytes of the internal capsule, but was not evident in most of the adult thalamic nuclei. Confocal and immunoelectron microscopy allowed direct examination of the relationships between neurons and glial processes in the embryonic thalamus, showing the coupling of neuronal membranes with both radial and non-radial glia during migration. Peculiar ultrastructural features of radial glia processes were observed. The occurrence of non-radial migration was confirmed by carbocyanine-labeled neuroblasts in E15 cultured slices. The data provide evidence that migrating thalamic cells follow both radial and non-radial glial pathways toward their destination.

Projections from the hypothalamus to the posterior lobe in rats during ontogenesis: 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate tracing study.

The objective of this study was to determine the schedule of the arrival of the axons from the hypothalamus to the posterior lobe of the pituitary (PL) in rats during ontogenesis by using the fluorescent lipophilic carbocyanine dye 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate (DiI) as a retrograde tracer. After preliminary fixation of the brain, DiI crystals were implanted in the PL on embryonic day 15 (E15), E16, E17, and E19 as well as on postnatal day 2 (P2) and P9. This was followed by a DiI retrograde diffusion along the plasma membrane and subsequent staining of hypothalamic neuronal cell bodies. The supraoptic nucleus (SO) contained an accumulation of fluorescent cells that extended toward the diamond-like swelling of the third ventricle as early as E15. These data suggest that the magnocellular neurons of the SO send their axons to the PL at the very beginning of differentiation, perhaps even before reaching their final position. The initial axons of the neurons of the paraventricular nucleus proper (PV) appeared to reach the PL significantly later, at E17. In addition to the SO and the PV, accessory magnocellular nuclei contributed to the innervation of the PL in perinatal rats. The neurons of the retrochiasmatic accessory nucleus first sent their axons to the PL on E16-E17. Axons that originated from other accessory hypothalamic nuclei reached the PL after birth, suggesting a delay in their involvement in the regulation of visceral functions compared with other magnocellular nuclei. Thus, the axons of magnocellular neurons reach the PL unexpectedly early in embryogenesis, raising the possibility of the functional significance of vasopressin and oxytocin as fetal neurohormones.

Effect of ethanol on calcium regulation in rat fetal hypothalamic cells in culture.

The effects of acute exposure to ethanol on calcium regulation in primary cultures of rat fetal hypothalamic cells was studied with the use of the calcium indicator fura-2 and digital imaging techniques. We found that ethanol caused cytoplasmic calcium to increase in a dose-dependent and reversible manner, and these increases could be observed at pharmacologically relevant doses (34 mM). At 170 mM ethanol 65% of 1059 cells examined responded to ethanol with an increase in cytoplasmic calcium. Removing bath calcium eliminated the ethanol-induced calcium response in most cells (76% of 427 cells). In most cells exposure to thapsigargin (20 nM) had no significant effect on the ethanol-induced calcium increase (87% of 67 cells examined). The ethanol-induced calcium increase was reduced by 79+/-5% (n=110 cells) by the P/Q-type calcium channel blocker omega-agatoxin-TK (20 nM), by 51+/-10% (n=115 cells) by the N-type calcium channel blocker omega-conotoxin-GVIA (100 nM), and by 26+/-3% (n=90 cells) by the T-type calcium channel blocker flunarizine (1 microM). The L-type calcium channel blocker nifedipine (1 microM) had complex actions, sometimes inhibiting and sometimes increasing the calcium response. These results demonstrate that ethanol can directly modulate cytoplasmic calcium levels in hypothalamic cells mostly by a pathway that involves extracellular calcium and voltage-dependent calcium channels, and that this response may participate in the biological effects of acute ethanol exposure.

Effects of ethanol on basal and adenosine-induced increases in beta-endorphin release and intracellular cAMP levels in hypothalamic cells.

Recently we have shown that the cAMP system is involved in ethanol-regulated beta-endorphin (beta-EP) release from rat hypothalamic neurons in primary cultures. The cascade of events that leads to activation of cAMP following ethanol treatment in hypothalamic beta-EP neurons is not apparent. In this study the role of adenosine, a cAMP regulator, in ethanol-regulated beta-EP release was determined by measuring the cellular incorporation of [3H]adenosine, intracellular cAMP levels and media immunoreactive (IR) beta-EP levels in cultures of rat hypothalamic cells following ethanol treatments in the presence and absence of an adenosine agonist and antagonist. Acute exposure to a 50 mM dose of ethanol for a period of 1 h increased media levels of IR-beta-EP and cellular contents of cAMP, but the ethanol treatment decreased [3H]adenosine uptake. Constant exposure to a 50 mM dose of ethanol for a period of 48 h, failed to alter media levels of IR-beta-EP, cell content of cAMP and [3H]adenosine uptake. The media level of IR-beta-EP was elevated following treatment with adenosine receptor agonist phenyl-isopropyl adenosine (PIA) and was reduced following treatment with adenosine receptor antagonist isobutylmethylxanthine (IBMX) or with adenosine uptake inhibitor adenosine deaminase. The level of cellular cAMP was also increased by PIA but was decreased by IBMX and adenosine deaminase. The stimulatory actions of the adenosine agonist PIA on IR-beta-EP release and on cAMP production were potentiated by simultaneous incubation with ethanol for 1 h. However, chronic ethanol exposure reduced PIA-induced IR-beta-EP release and cAMP production. Additionally, both IBMX and adenosine deaminase reduced ethanol-induced IR-beta-EP release and cAMP levels. These results suggest that ethanol inhibits adenosine uptake in IR-beta-EP neurons in the hypothalamus, thereby increasing extracellular levels of adenosine and leading to activation of membrane adenosine receptors, cAMP production and IR-beta-EP secretion from these neurons. Chronic ethanol desensitizes the adenosine-regulated cAMP production and IR-beta-EP release from hypothalamic neurons.

Brain-derived neurotrophic factor and neurotrophin-3 enhance somatostatin gene expression through a likely direct effect on hypothalamic somatostatin neurons.

Although neurotrophins (NTs) have been extensively studied as neuronal survival factors in some areas of the central nervous system, little is known about their function or cellular targets in the hypothalamus. To understand their functional significance and sites of action on hypothalamic neurons, we examined the effects of their cognate ligands on neuropeptide content and messenger RNA (mRNA) expression in somatostatin neurons present in fetal rat hypothalamic cultures. Treatments were performed in defined insulin-free medium between days 6 and 8 of culture, since the maximal effects of NTs on somatostatin content and mRNA expression were observed after 48-h incubations. Brain-derived neurotrophic factor and NT-3, but not nerve growth factor, induced a dose-dependent increase in somatostatin content, which was influenced by plating density. The same treatment increased somatostatin mRNA and immunostaining intensity of somatostatin neurons, but had no effect on the number of these labeled neurons. The increased levels of somatostatin (peptide and mRNA) induced by NTs were not blocked by tetrodotoxin or by glutamate receptor antagonists, suggesting that endogenous neurotransmitters (e.g. glutamate) were not involved in these effects. In contrast, the stimulatory effects were completely blocked by K-252a, an inhibitor of tyrosine kinase (Trk) receptors, whereas the less active analog K-252b was ineffective. Double-labeling studies demonstrated that both TrkB or TrkC receptors were located on somatostatin neurons. Our results show that, in rat hypothalamic cultures, brain-derived neurotrophic factor, and NT-3 have a potent stimulatory effect on peptide synthesis in somatostatinergic neurons, likely through direct activation of TrkB and TrkC receptors.

Energy dependency of glucocorticoid exacerbation of gp120 neurotoxicity.

The HIV envelope glycoprotein, gp120, a well documented neurotoxin, may be involved in AIDS-related dementia complex. gp120 works through an NMDA receptor- and calcium-dependent mechanism to damage neurons. We have previously demonstrated that both natural and synthetic glucocorticoids (GCs) exacerbate gp120-induced neurotoxicity and calcium mobilization in hippocampal mixed cultures. GCs, steroid hormones secreted during stress, are now shown to work in conjunction with gp120 to decrease ATP levels and to work synergistically with gp120 to decrease the mitochondrial potential in hippocampal cultures. Furthermore, energy supplementation blocked the ability of GCs to worsen gp120's effects on neuronal survival and calcium mobilization. A GC-induced reduction in glucose transport in hippocampal neurons, as previously documented, may contribute to this energetic dependency. These results may have clinical significance, considering the common treatment of severe cases of Pneumocystis carinii pneumonia, typical of HIV infection, with large doses of synthetic GCs.

Methylazoxymethanol acetate-induced abnormalities in the entorhinal cortex of the rat; parallels with morphological findings in schizophrenia.

It has been suggested repeatedly that the non-heritable factors in the pathogenesis of schizophrenia involve abnormalities of prenatal neurodevelopment. Furthermore, post-mortem studies show neuropathology of apparently developmental origin in the entorhinal cortex and other brain regions of schizophrenic subjects. In an attempt to model a developmental defect of the entorhinal region in the rat, cerebrocortical proliferation was briefly interrupted during its earliest stages, when the entorhinal area is thought to undergo major cell division. Specifically, the experimental set-up involved the administration of methylazoxymethanol acetate (MAM) on 1 of 4 consecutive days of embryonal development, from E9 to E12. Analysis of the forebrain in adult animals shows reduction of the entorhinal cortex in rats treated on each of these days. This effect shifts from lateral to medial divisions of the entorhinal cortex with later administration of MAM, following a known developmental gradient. Morphological consequences of MAM administration appear to be largely confined to the entorhinal cortex in the groups treated on E9 to E11, although slight reductions of the frontal and occipital neocortex were also observed in these animals. MAM treatment on E12 produces relatively more widespread damage, as reflected among other in a small reduction of brain weight. The described brain abnormalities are not accompanied by obvious phenotypical changes in any, but the E12-treated group. They, moreover, involve cortical thinning, disorganised cortical layering, and abnormal temporal asymmetries. These finding bare some similarity to observations in brains of schizophrenic subjects. The possible relevance of this approach in modeling neurodevelopmental aspects of schizophrenia is discussed.

Inorganic Pi increases neuronal survival in the acute early phase following excitotoxic/oxidative insults.

Inorganic phosphate (Pi) plays a vital role in intracellular energy metabolism. Its many effects include stimulation of glucose use, enhancement of high-energy phosphate concentrations, and modulation of cytosolic free [Ca2+]. Cultured fetal rat cortical neurons constitutively import Pi, and cytosolic levels positively correlate with [ATP], [NADPH], and energy charge. In the present study, we demonstrate that the concentration of intracellular Pi is an important determinant of acute neuronal survival after an excitotoxic or oxidative insult to cultured fetal rat cortical neurons. Extracellular Pi dose-dependently enhanced survival of cortical neurons after exposure to NMDA at early (< or = 6 h) time points after termination of the insult. Pi similarly increased neuronal survival after exposure to kainic acid or H2O2. Pi-exposed neurons had higher basal intracellular [Pi], [ATP], and [GSH], and slightly lower cytosolic free [Ca2+], compared with Pi-deprived neurons. Pi-exposed neurons maintained increased [ATP] after exposure to NMDA and displayed reduced formation of reactive oxygen species after exposure to kainic acid or H2O2, compared with Pi-deprived neurons. These findings demonstrate that changes in extracellular and intracellular Pi can affect neuronal survival after excitotoxic or oxidative insults.

Leptin inhibits in vitro hypothalamic somatostatin secretion and somatostatin mRNA levels.

Leptin, the product of the ob gene, is a recently discovered hormone secreted by adipocytes that regulates food intake and energy expenditure. The site of action of leptin is likely to be the hypothalamus, since this area is important in the control of food intake and leptin receptor mRNA is particularly abundant in this area. In order to further unravel the mechanisms by which leptin acts, we have studied the effect of leptin on in vitro somatostatin synthesis and secretion. Leptin administration to fetal rat neurones in monolayer culture led to a time dependent decrease in basal somatostatin secretion and somatostatin mRNA levels, the maximal effect being observed with 6x10(-8) M leptin after 24 h incubation. Furthermore, leptin completely blunted 10(-7) M Neuropeptide Y-induced increase in somatostatin secretion and somatostatin mRNA levels as well as 10(-3) M (Bu)2-cAMP and 10(-6) M A23187-induced somatostatin secretion. Finally, leptin (3x10(-8) M M) also inhibited low glucose (1.1 mM) induced-somatostatin secretion in perifused adult hypothalami. This data indicates that leptin can influence the neuroendocrine system by regulating hypothalamic somatostatin gene expression.

Evidence for neurotensin autoreceptors and relationship of neurotensin and its receptors with tyrosine hydroxylase-positive neurons in rat primary hypothalamic cultures.

Neurotensin is present in high quantity in the hypothalamus, where it regulates pituitary hormone secretion. A relationship between dopaminergic and neurotensinergic systems has been suggested in the hypothalamus in studies showing an effect of neurotensin on tuberoinfundibular dopaminergic neurons. In order to determine the anatomical basis of such interactions, primary cultures of rat hypothalamic neurons were used. Tyrosine hydroxylase and neurotensin containing cells were identified by immunocytochemistry and neurotensin binding sites by [125I]Tyr3-neurotensin autoradiography. Colocalization studies showed that neurotensin immunoreactivity was present in 16% of tyrosine hydroxylase-positive cells, and that these neurotensin/tyrosine hydroxylase neurons represented more than half (58%) of the neurotensinergic population. Five percent of the tyrosine hydroxylase-positive cells had neurotensin binding sites, suggesting that only a restricted number of hypothalamic dopaminergic neurons is responsive to neurotensin. Neurotensin binding sites were also found on some neurotensin-positive cells, demonstrating for the first time the presence of autoreceptors for this peptide on neurons. These results in primary cultures provide a cellular basis for direct effects of neurotensin on a subpopulation of hypothalamic dopaminergic cells, and support the possibility of an autocrine action of neurotensin in the hypothalamus.

Dietary vitamin E prophylaxis and diabetic embryopathy: morphologic and biochemical analysis.

OBJECTIVE: In this study we sought to determine whether dietary supplementation with vitamin E, a known antioxidant, would reduce the incidence of diabetic embryopathy in an in vivo rat model. STUDY DESIGN: Eighty-day-old Sprague-Dawley rats were assigned to one of five groups: two control groups (groups 1 and 2) and three diabetic groups (groups 3, 4, and 5). One group of controls (group 2) and one group of diabetic rats (group 4) received dietary supplements of vitamin E (440 mg/day). The other three groups (groups 1, 3, and 5) received a normal diet only. Group 5 received insulin therapy to control glucose levels. On day 6 of gestation diabetes was induced in groups 3, 4, and 5 with streptozotocin (65 mg/kg). Animals were killed on day 12; embryos were examined for size, protein content, evidence of malformations, and superoxide dismutase activity. RESULTS: In both groups (groups 3 and 4) of diabetic rats the mean blood glucose level than was significantly higher in controls. Insulin-treated animals (group 5) had glucose levels that were comparable to those of controls. The unsupplemented diabetic group had a neural tube defect rate of 21.48% +/- 9.6% (percentage of neural tube defects per rat) and a resorption rate of 21.37% +/- 20.39% (percentage of resorptions per rat) as compared with rates in the supplemented diabetic group of 6.92% +/- 4.08% and 2.17% +/- 3.74%, respectively (p < 0.01). Groups 1, 2, and 5 had similar neural tube defect rates (6.63% +/- 5.0%, 5.01% +/- 4.87%, and 3.55% +/- 5.92%, respectively. Vitamin E levels, measured by high-performance liquid chromatography, were significantly higher in maternal serum and embryos in the supplemented groups (p < 0.001) than in controls. Superoxide dismutase activity was reduced in the diabetes groups and was not affected by vitamin E therapy. CONCLUSIONS: Supplementation with the antioxidant vitamin E confers a significant protective effect against diabetic embryopathy and may potentially serve as a dietary prophylaxis in the future. We postulate that this protective effect is mediated by a reduction in the oxidative load induced by hyperglycemia.

Excitatory actions of GABA after neuronal trauma.

GABA is the dominant inhibitory neurotransmitter in the CNS. By opening Cl- channels, GABA generally hyperpolarizes the membrane potential, decreases neuronal activity, and reduces intracellular Ca2+ of mature neurons. In the present experiment, we show that after neuronal trauma, GABA, both synaptically released and exogenously applied, exerted a novel and opposite effect, depolarizing neurons and increasing intracellular Ca2+. Different types of trauma that were effective included neurite transection, replating, osmotic imbalance, and excess heat. The depolarizing actions of GABA after trauma increased Ca2+ levels up to fourfold in some neurons, occurred in more than half of the severely injured neurons, and was long lasting (>1 week). The mechanism for the reversed action of GABA appears to be a depolarized Cl- reversal potential that results in outward rather than inward movement of Cl-, as revealed by gramicidin-perforated whole-cell patch-clamp recording. The consequent depolarization and resultant activation of the nimodipine sensitive L- and conotoxin-sensitive N-type voltage-activated Ca2+ channel allows extracellular Ca2+ to enter the neuron. The long-lasting capacity to raise Ca2+ may give GABA a greater role during recovery from trauma in modulating gene expression, and directing and enhancing outgrowth of regenerating neurites. On the negative side, by its depolarizing actions, GABA could increase neuronal damage by raising cytosolic Ca2+ levels in injured cells. Furthermore, the excitatory actions of GABA after neuronal injury may contribute to maladaptive signal transmission in affected GABAergic brain circuits.

Neurophysin expression is stimulated by dopamine D1 agonist in dispersed hypothalamic cultures.

We have exposed primary dispersed hypothalamic cultures from 14-day-old fetal Sprague-Dawley rats to substances known to either elevate adenosine 3',5'-cyclic monophosphate (cAMP) levels or increase vasopressin (VP) secretion. The levels of VP in the medium collected from the cultures were determined by radioimmunoassay, and the number of neurophysin (NP)-positive cells after immunohistochemistry was counted. cAMP-elevating agents, 3-isobutyl-1-methylxanthine (200 microM) and forskolin (25 microM), in combination (I-F) maintained NP synthesis and VP secretion in 19-day cultures. I-F replacement by K+ (28 mM), isoproterenol (10 microM), glutamate (10 microM), or bicuculline (10 microM) during the last week of culture resulted in maintenance of NP expression and transient stimulation of VP secretion, but these agents did not induce NP expression independently of I-F treatment. In contrast, exposure to the dopamine D1 agonist SKF-38393 (10 microM) significantly increased NP expression independently and after replacement of I-F. Dopamine D1A receptors were detected by immunofluorescence on NP-expressing cells, providing a morphological basis for this response. These results suggest a role for D1A receptors in the regulation of VP gene expression.

GABA receptors precede glutamate receptors in hypothalamic development; differential regulation by astrocytes.

1. The developmental changes in gamma-aminobutyrate (GABA)-, glutamate-, and glycine-mediated currents in cultured embryonic neurons (n = 134) from rat hypothalamus were studied with the use of whole cell voltage-clamp recording. 2. GABA-evoked currents were detected in neurons cultured from 15-day embryos (E15) a few hours after plating. Every neuron studied from the time of plating at E15 to 2 wk later responded to GABA (30 microM). The peak and steady-state currents evoked by GABA increased by four- to fivefold within 2 wk in culture. The time constants of the desensitization of GABA currents did not change during this period. The properties of the responses to GABA were not altered by different culture densities or substrates. 3. Glycine activated receptors that were pharmacologically distinct from GABA receptors on hypothalamic neurons. The glycine responses increased by > 50-fold within 2 wk in culture. The percentage of cells responding to glycine (500 microM) was 20% at 0 days in vitro (DIV), and increased to 100% at 6 DIV. Astrocytes increased both the amplitude of glycine-mediated currents and the percentage of cells responding to glycine. 4. Glutamate-mediated currents developed later than GABA-mediated currents. The percentage of cells responding to glutamate (500 microM) increased within the 1st wk, from 20% on the day of plating to 100% after 6 DIV. Both the peak currents and the steady-state currents mediated by glutamate increased by 20-fold during the 2 wk in culture. Both the amplitude of the responses to glutamate and the percentage of cells responding to glutamate were increased by growing neurons either on an astrocyte substrate or in high-density cultures. 5. The currents and conductance changes elicited by GABA were greater than those generated by glutamate or glycine throughout the period examined. This difference was particularly evident in younger cells. After 3 days in vitro, GABA (30 microM) elicited a mean current of 1,648 pA, whereas glutamate (500 microM) only elicited a 266-pA current, and glycine (500 microM) elicited a 278-pA current from neurons growing on an astrocyte layer. 6. The expression of amino acid receptors was heterogeneous among hypothalamic neurons in younger cultures. Whereas all neurons expressed GABA receptors, some developing neurons did not express detectable glutamate receptors or glycine receptors. 7. Each of the three amino acid-evoked currents increased from E15 (1 DIV) to E20 (1 DIV), indicating an intrinsic development in the expression of the amino acid receptors in vivo. The GABA, glutamate, and glycine currents at E15, 10 DIV were similar to the currents at E20, 5 DIV (both 25 days after conception), suggesting parallel developmental patterns for amino acid receptor expression in vitro and in vivo. 8. Together, these data suggest that GABA may play a major role in early development because hypothalamic neurons are more sensitive to GABA than to either glutamate or glycine. However, glutamate and glycine receptors appear more sensitive to regulation by the local environment than GABA receptors because culture density and the astrocyte substrate have greater inductive effects on glutamate and glycine receptors than on GABA receptors.

GABA neurotransmission in the hypothalamus: developmental reversal from Ca2+ elevating to depressing.

GABA is the primary inhibitory transmitter of the adult hypothalamus, synthesized by many neurons and found in 50% of the presynaptic boutons. GABA causes a decrease in Ca2+ in mature hypothalamic neurons in vitro by depressing cellular activity through opening Cl- channels. Despite the early expression of GABAA receptors in the embryonic hypothalamus (E15), the cellular function of GABA in the developing hypothalamus has received little attention. In the present study the role of GABA in modulating intracellular Ca2+ in developing hypothalamic neurons was studied with fura-2 digital imaging. GABA (0.5-500 microM) applied to embryonic hypothalamic neurons elicited a dramatic and rapid increase in intracellular Ca2+ This Ca2+ rise could be completely blocked by the GABAA antagonist bicuculline (20 microM) and persisted in the presence of tetrodotoxin (1 microM). The Ca2+ elevation induced by GABA was greater than that of equimolar concentrations of the excitatory transmitter glutamate in early development. The number of E15 neurons that responded to GABA with a Ca2+ rise increased during the first few days of culture, reaching 78% after 4 d in vitro. The Ca2+ rise was 87% blocked by cadmium (100 microM) and 85% blocked by nimodipine (1 microM), indicating that the mechanism of Ca2+ increase was primarily via L-type voltage operated Ca2+ channels. Addition of bicuculline to synaptically coupled cultures caused a significant decrease in Ca2+ 4-10 d after culturing, indicating hypothalamic neurons were secreting GABA at an early age of development, and that sufficient GABA was released to elicit an increase in Ca2+. This effect was seen even after blocking all glutamatergic activity with glutamate receptor antagonists. In contrast, GABA elicited no Ca2+ rise in older neurons (> 18 d in vitro), and the action of bicuculline reversed and caused a large increase in Ca2+ in spontaneously active neurons. Similar findings were obtained in cultures enriched in GABAergic neurons from the suprachiasmatic nucleus. To determine if the Ca2+ stimulating role of GABA on developing neurons was restricted to the hypothalamus and a few other regions, or whether it might exist throughout the brain, we examined the Ca2+ responses in cultured olfactory bulb, cortex, medulla, striatum, thalamus, hippocampus, and colliculus. The majority (75%) of developing neurons from each region showed a Ca2+ rise in response to GABA. Together these data suggest that GABA elevates Ca2+ in developing, but not mature, neurons from the hypothalamus and all other brain regions examined.(ABSTRACT TRUNCATED AT 400 WORDS)

Developmental expression of protein C and protein S in the rat.

In order to better understand the expression of the Protein C/Protein S anticoagulant system, we have isolated and characterized cDNAs coding for rat Protein C and Protein S. These cDNAs were used in Northern analysis to determine tissue-specificity and developmental expression patterns for mRNAs coding for Proteins C and S. In rats, Protein C mRNA is expressed almost exclusively in liver with a small amount of expression in kidney, diaphragm, stomach, intestine, uterus and placenta. Protein C mRNA was not expressed in brain, heart, lung, spleen, small intestine, large intestine, ovary, or urinary bladder. In liver, Protein C mRNA is expressed at very low levels at prenatal day 18 and these levels increased to maximal levels by postnatal day 13. The size of the mRNA coding for rat Protein C is approximately 1.9 kb. Rat Protein S mRNA was expressed in all tissues examined: brain, heart, lung, diaphragm, liver, spleen, stomach, small intestine, large intestine, kidney, adrenal ovary, uterus, placenta, and urinary bladder. Interestingly, there were 4 bands hybridizing with the rat protein S cDNA that were evident in many of the tissues examined, corresponding to mRNA sizes of approximately 3.5, 2.6, 1.8, and 0.3 kb. There was a difference in tissue-specificity of each mRNA. The 1.8 kb band is generally the most prominent autoradiographic band in any tissue. From these results, it is evident that the expression of Protein C mRNA is similar to that of other vitamin K-dependent proteins. The expression of Protein S mRNA, however, is surprisingly complex and may include alternative splicing of mRNA to generate the various sizes evident on Northern analysis.

Comparison of the effects of alcohol and acetaldehyde on proopiomelanocortin mRNA levels and beta-endorphin secretion from hypothalamic neurons in primary cultures.

The effects of acute and chronic treatments with ethanol and acute treatments with an ethanol metabolite, acetaldehyde, on proopiomelanocortin (POMC) mRNA expression were compared with those of these agents on the secretion of a POMC gene product, beta-endorphin (beta-EP) peptide. The level of POMC mRNA in cultured cells was determined using an RNase protection assay, and the accumulation of immunoreactive beta-EP (IR-beta-EP) peptide in the culture medium was measured by radioimmunoassay. Treatment of hypothalamic cells with 25-, 50-, and 100-mM doses of ethanol or 12.5 and 25 microM acetaldehyde for 3 h increased POMC mRNA levels. The stimulatory effect of ethanol on POMC mRNA levels lasted for a period of 12 h, although the percentage increase of the ethanol-stimulated mRNA level was gradually reduced over time. Acute treatments with ethanol and acetaldehyde also elevated IR-beta-EP secretion from the cultured neurons for a period of 12 h, and the IR-beta-EP secretory response developed desensitization after 24 h of ethanol incubation. The close association between the ethanol-induced IR-beta-EP secretion and ethanol-regulated POMC mRNA expression suggests that ethanol regulates both secretion and production of beta-EP peptide in the hypothalamic neurons.

Early development of the somatotopic map and barrel patterning in rat somatosensory cortex.

Several lines of evidence implicate a crucial role for thalamic afferents from the ventroposterior nucleus (VP) in the development of barrels and their characteristic pattern in the primary somatosensory cortex (S1) of rodents. We sought to determine the stage in development when VP thalamocortical afferents are first distributed in a periphery-related pattern and the sequence of events that culminate in a mature pattern. Using acetylcholinesterase (AChE) histochemistry, an early marker for VP thalamocortical afferents, and the anterograde axon tracer DiI, we show that VP thalamocortical afferents become distributed into a periphery-related pattern earlier than was previously reported, including their parcellation into a barrel-related pattern that mirrors the distribution of sensory hairs on the face. The earliest periphery-related patterning observed is transiently present in the deep cortical layers prior to the emergence of layer 4, the layer in which barrels later develop. AChE histochemistry reveals a clear sequence of maturation of the barrel pattern in the distribution of VP afferents: An initially patternless distribution of AChE-reactive afferents is followed by their distribution in a nascent trigeminal representation, from which rows subsequently emerge; barrel-related clusters of afferents then emerge from the rows. This process begins before birth, and the transition from row-related to barrel-related distributions of VP afferents is evident during the first postnatal day (P0). This demonstration of a periphery-related pattern in developing rat S1 precedes by about 2 days that revealed by any other marker reported to delineate barrels. These findings confirm that VP thalamocortical afferents are the first barrel component to have a periphery-related pattern and support the hypothesis that thalamocortical afferents provide to immature S1 the patterning information that initiates the formation of barrels and their characteristic array. Furthermore because these findings show an earlier onset for barrel formation than was previously realized, they necessitate a reevaluation of conclusions drawn from experiments examining developmental plasticity in barrel patterning.

Tetrahydrobiopterin synthesis rate and turnover time in neuronal cultures from embryonic rat mesencephalon and hypothalamus.

6-(R)-(L-erythro-1',2'-Dihydroxypropyl)-2-amino- 4-hydroxy-5,6,7,8-tetrahydropteridine (tetrahydrobiopterin, BH4) synthesis rate and turnover time were estimated in cultures derived from the embryonic rat mesencephalon (MES) and hypothalamus (HYP) by following the decline in BH4 levels after blockade of BH4 biosynthesis by N-acetylserotonin (NAS) or 2,4-diamino-6-hydroxypyrimidine (DAHP). BH4 content of both culture systems decreased by 75% following an 8-h incubation with maximally effective concentrations of NAS (200 microM) or DAHP (10 mM). Parameters describing BH4 metabolism were calculated from steady-state levels of BH4 and first-order rate constants determined by a nonlinear regression analysis of the exponential BH4 decline. These parameters were confirmed using an alternative procedure that examined the first-order rate of recovery of BH4 following termination of BH4 synthesis inhibition. Steady-state levels of BH4 in HYP cultures (70.3 +/- 9.4 pg/culture) were significantly greater than that for MES (46.5 +/- 2.8 pg/culture). The average fractional rate constants of BH4 loss for MES (0.153 +/- 0.015/h) and HYP (0.159 +/- 0.014/h) were equivalent. The calculated rate of BH4 synthesis was significantly greater for HYP (11.29 +/- 2.13 pg/culture/h) than for MES (7.11 +/- 0.85 pg/culture/h), owing to the greater steady-state concentration of BH4. BH4 turnover time for MES (6.68 +/- 0.67 h) and HYP (6.40 +/- 0.62 h) and half-life for MES (4.63 +/- 0.46 h) and HYP (4.44 +/- 0.43 h) did not differ. The turnover of the cofactor is thus rapid enough that alterations in its synthesis or degradation could acutely modify the rate of monoamine biosynthesis.(ABSTRACT TRUNCATED AT 250 WORDS)