Pregnenolone and dehydroepiandrosterone administration in neonatal rats alters the immunoreactivity of hippocampal synapsin I, neuropeptide Y and glial fibrillary acidic protein at post-puberty.

It is well documented that neurosteroids administered during the neonatal period influence the development of several brain systems. In our previous study, pregnenolone administered to rats during the neonatal period altered adenosinergic and dopaminergic functions in the striatum and cerebral cortex. The present study examined the effects of the treatment with pregnenolone and dehydroepiandrosterone (DHEA) from the postnatal day (P) 3-P7 on synapsin I (a marker for presynaptic terminals) and glial fibrillary acidic protein (GFAP: a marker for astroglia) levels in the hippocampus of Sprague-Dawley rats at 3 and 7 weeks of age. In addition, neuropeptide Y and dynorphin A immunoreactivity was measured. The administration of pregnenolone and DHEA to neonatal rats significantly altered the expression of synapsin I in the dentate gyrus and CA3 region at post-puberty but not at pre-puberty. A significantly greater expression of GFAP-immunoreactive astrocytes or processes was demonstrated in the pregnenolone- and DHEA-treated groups at both pre-puberty and post-puberty. A significant increase in the number and size of GFAP-immunoreactive astrocytes and in the extension of arborization was seen in the overall hippocampus. The number of neuropeptide Y-positive cells in the hilus region was also significantly increased in the neurosteroid-treated group at post-puberty. No differences were detected in dynorphin A immunoreactivity among the experimental groups. These results of this study suggest that pregnenolone and DHEA play an important role in the development of hippocampus.

Adenosine A2A, 5-HT1A and 5-HT7 receptor in neonatally pregnenolone-treated rats.

Steroid hormones synthesized in the brain, called 'neurosteroids', modulate neuronal activity. We treated neonatal rats with a main precursor of the neurosteroidogenesis, pregnenolone, and examined adenosine A2A receptor, 5- hydroxytryptamine (5-HT)1A and 5-HT7 receptor densities in the front-parietal cortex in juvenile and adult rats. In receptor binding assay using [3H]CGS21680 and [3H]8-OH-DPAT, it was shown that neonatal pregnenolone-treatment induced a significant decrease in the adenosine A2A receptor density with no significant effects on the 5-HT1A and 5-HT7 receptor densities.

Nicotine exposure during pregnancy is a factor which influences serotonin transporter density in the rat brain.

We examined the effects of nicotine exposure during pregnancy on serotonin transporter (SERT) expression in the brain. Nicotine (6 mg/kg/day) was administered to pregnant rats via subcutaneous injections or infusion pumps. Irrespective of the route of administration, nicotine increased SERT density in the forebrain on postnatal day 22, but not in the other brain regions. Our results suggest that nicotine use by pregnant women might be an environmental factor influencing SERT expression in their children.

Cell biology of limb patterning.

Of vertebrate organ systems, the developing limb has been especially well characterized. Morphological studies have yielded a wealth of information describing limb outgrowth and have allowed for the identification of a multitude of important factors. In terms of the latter, key signaling pathways are known to control numerous aspects of limb development, including establishment of the early limb field, determination of limb identity, elongation of the limb bud, specification of digit pattern, and sculpting of the digits. Modification of underlying signaling pathways can thus result in dramatic alterations of the limb phenotype, accounting for many of the diverse limb patterns observed in nature. Given this, it is clear that signaling pathways regulate the highly orchestrated and tightly controlled sequence of cellular events necessary for limb outgrowth; however, exactly how molecular signals interface with the cell biology of limb development remains largely a mystery. In this review we first provide an overview of a number of the morphogenetic signaling pathways that have been identified in the developing limb and then review how a subset of these signals are known to modify cell behaviors important for limb outgrowth.

Inhibition of polarizing activity in the anterior limb bud is regulated by extracellular factors.

Anterior-posterior patterning of the developing limb is largely viewed as a function of polarizing activity. Recent evidence in polydactylous mutants, however, indicates that development of proper pattern also requires the involvement of inhibitory pathways in the anterior limb that prevent secondary polarizing zone formation, thus limiting the number of digits produced. We report the novel finding that grafts of extracellular matrix from the Mouse Posterior Limb Bud-4 cell line can induce supernumerary digits, including digits with posterior phenotype, from anterior chick limb mesenchyme. Unlike previously described mechanisms of pattern specification during limb development, it is shown that the extracellular matrix effect is not associated with release of an active signal. Rather, evidence is presented suggesting that heparan sulfate moieties in extracellular matrix grafts bind an endogenous, extracellular factor involved in inhibition of anterior polarizing activity, leading to derepression of the anterior limb and induction of polarizing zone marker genes including Sonic hedgehog and Bone morphogenetic protein-2.

Effects of neonatal melatonin administration on the extra-hypothalamic regions in rat brains: effects on the serotonergic system.

The effects of 100microg melatonin injection at postnatal day 5 (PD 5) on the development of the central serotonergic systems in male and female rats were investigated. The contents of serotonin (5-HT) and 5-hydroxy-3-indolacetic acid (5-HIAA) were measured in several extrahypothalamic regions at 3, 10 and 42 weeks of age. The neonatal melatonin administration increased both 5-HT and 5-HIAA levels in the striatum throughout the examined period. In the hippocampus, an increase in 5-HIAA contents by neonatal melatonin administration was found at 3 weeks but not 10 or 42 weeks of age. There were no significant differences in the effects of melatonin between male and female rats. These results indicated that exogenous melatonin administration during the early neonatal period influenced the development of the serotonergic systems in extrahypothalamic regions including the hippocampus and the striatum.

Evaluation of biologically based dose-response modeling for developmental toxicity: a workshop report.

Biologically based dose-response (BBDR) modeling represents a novel approach for quantitative assessment of health risk by incorporating pharmacokinetic and pharmacodynamic characteristics of a chemical and by relating the immediate cellular responses to a cascade of aberrant biological actions that leads to detectable adverse outcomes. The quantitative relationship of each of the intervening events can be described in mathematical forms that are amenable for adjustment and extrapolation over a range of doses and across species. A team of investigators at the Reproductive Toxicology Division of the U.S. Environmental Protection Agency has explored the feasibility of BBDR modeling by examining the developmental toxicity of a known teratogen, 5-fluorouracil. A panel of researchers from academic and industrial laboratories, biomathematical modelers, and risk assessment scientists was convened in a workshop to evaluate the approaches undertaken by the EPA team and to discuss the future prospects of BBDR modeling. This report summarizes the lessons learned from one approach to BBDR modeling and comments from the panelists: while it is possible to incorporate mechanistic information into quantitative dose-response models for the assessment of health risks, the process is enormously data-intensive and costly; in addition, the confidence of the model is directly proportional to our current understanding of basic biology and can be enhanced only through the ongoing novel discoveries. More importantly, the extent of "uncertainty" (inherent with the default assumptions associated with the NOAEL or benchmark approach) reducible by BBDR modeling requires further scrutiny and comparison.

Influence of FGF4 on digit morphogenesis during limb development in the mouse.

Much of what we currently know about digit morphogenesis during limb development is deduced from embryonic studies in the chick. In this study, we used ex utero surgical procedures to study digit morphogenesis during mouse embryogenesis. Our studies reveal some similarities; however, we have found considerable differences in how the chick and the mouse autopods respond to experimentation. First, we are not able to induce ectopic digit formation from interdigital cells as a result of wounding or TGFbeta-1 application in the mouse, in contrast to what is observed in the chick. Second, FGF4, which inhibits the formation of ectopic digits in the chick, induces a digit bifurcation response in the mouse. We demonstrate with cell marking studies that this bifurcation response results from a reorganization of the prechondrogenic tip of the digit rudiment. The FGF4 effect on digit morphogenesis correlates with changes in the expression of a number of genes, including Msx1, Igf2, and the posterior members of the HoxD cluster. In addition, the bifurcation response is digit-specific, being restricted to digit IV. We propose that FGF4 is an endogenous signal essential for skeletal branching morphogenesis in the mouse. This work stresses the existence of major differences between the chick and the mouse in how digit morphogenesis is regulated and is thus consistent with the view that vertebrate digit evolution is a relatively recent event. Finally, we discuss the relationship between the digit IV bifurcation restriction and the placement of the metapterygial axis in the evolution of the tetrapod limb.

Prenatal administration of nicotine results in dopaminergic alterations in the neocortex.

This study examined the effect of two high doses (3 or 6 mg/kg/day) of nicotine administrations via injections to pregnant rats on the dopaminergic, serotonergic, and noradrenergic systems in six brain regions in young adult male rats. The 3 mg/kg/day and 6 mg/kg/ day nicotine exposure resulted in significant decreases in dihydroxyphenylacetic acid (DOPAC) content in the neocortex and in both the neocortex and in the midbrain plus pons medulla, respectively, without any effects on the other brain regions such as the hypothalamus or striatum. No significant effects of prenatal nicotine were found on norepinephrine, serotonin, or 5-hydroxy-3-indolacetic acid levels. These data demonstrated that prenatal nicotine induced disturbances in the dopaminergic system in the young adult period. Furthermore, the region-specific reductions in the DOPAC content suggests that the exposure to a high dose of nicotine in utero might cause a predisposition to diseases related to a dopaminergic dysfunction in the frontal cortex.

Serotonin-induced platelet intracellular Ca2+ responses in untreated depressed patients and imipramine responders in remission.

BACKGROUND: Intracellular Ca2+ metabolism in platelets has been investigated as a peripheral marker of affective disorders. METHODS: We investigated the intracellular free Ca2+ concentration in platelets in both untreated depressed patients with no medications and patients in remission who were treated by imipramine (IMI) (IMI responders) using a Ca(2+)-sensitive fluorescent probe fura-2. RESULTS: The increases in intracellular free Ca2+ concentration in platelets induced by stimulation with serotonin (5-HT) ([Ca2+] delta) were significantly higher in both the untreated patients and the IMI responders compared with healthy controls; however, there were no significant differences in the basal Ca2+ levels in the platelets ([Ca2+]B) among the three groups. On the other hand, in the IMI responders, we observed positive correlations between the duration of the remission and [Ca2+]B, but not [Ca2+] delta. CONCLUSIONS: Our present data suggest that the enhancement of 5-HT2A-induced Ca2+ responses persisted after remission in depressed patients, and that the duration of the remission is a factor varying the intracellular basal Ca2+ levels.

Cell migration and chick limb development: chemotactic action of FGF-4 and the AER.

Experiments have been carried out to investigate the role of the apical ectodermal ridge (AER) and FGF-4 on the control of cell migration during limb bud morphogenesis. By coupling DiI cell labeling with ectopic implantation of FGF-4 microcarrier beads we have found that FGF-4 acts as a potent and specific chemoattractive agent for mesenchymal cells of the limb bud. The response to FGF-4 is dose dependent in both the number of cells stimulated to migrate and the distance migrated. The cell migration response to FGF-4 appears to be independent of the known inductive activity of FGF-4 on Shh gene expression. We investigated the role of the AER in controlling cell migration by characterizing the migration pattern of DiI-labeled subapical cells during normal limb outgrowth and following partial AER removal. Subapical cells within 75 micrometer of the AER migrate to make contact with the AER and are found intermingled with nonlabeled cells. Thus, the progress zone is dynamic with cells constantly altering their neighbor relationships during limb outgrowth. AER removal studies show that cell migration is AER dependent and that subapical cells redirect their path of migration toward a functional AER. These studies indicate that the AER has a chemoattractive function and regulates patterns of cell migration during limb outgrowth. Our results suggest that the chemoattractive activity of the AER is mediated in part by the production of FGF-4.

Chronic imipramine administration amplifies the serotonin2A receptor-induced intracellular Ca2+ mobilization in C6 glioma cells through a calmodulin-dependent pathway.

In the present study, we examined whether chronic exposure of C6BU-1 cells to 100 nM of several different types of antidepressants directly influences serotonin2A (5-HT2A) receptor-stimulated intracellular Ca2+ mobilization. Imipramine, desipramine, clomipramine, and maprotiline amplified the 5-HT response at 48, but not at 2, h. Imipramine increased the maximum response to 5-HT without altering the EC50 of the dose-response curve. This effect was time dependent and cycloheximide blocked the maximal induction, suggesting an essential role for protein synthesis in this process. Previous exposure of the cells to thrombin or isoproterenol did not influence 5-HT2A receptor function and pretreatment with imipramine did not alter the thrombin- or bradykinin-induced Ca2+ mobilization, which indicates that the effects of imipramine appear to be specific to the 5-HT2A receptor. The effect of imipramine was potently suppressed by a calmodulin antagonist, W-13, in a dose-dependent manner. Furthermore, this amplified 5-HT response was blocked by KN-93, but not by H-7. Taken together, these results suggest that imipramine has a modulatory effect on the 5-HT2A receptor-coupled intracellular Ca2+ in C6 cells through a calmodulin-dependent pathway, possibly involving Ca2+/calmodulin kinase activation.

Electrolytes in erythrocytes of patients with depressive disorders.

The concentrations of calcium, sodium, potassium and magnesium in the erythrocytes of patients were measured in the active and remission phases of depressive disorders. Twelve patients in the active phase and 19 patients in remission with major depression were studied and compared with 20 age-matched healthy controls. Patients with major depression in both active and remission phases showed significantly lower calcium concentrations in the erythrocytes compared with controls, although no significant differences in sodium, potassium or magnesium concentrations were found among the three groups. In addition, no differences were found in the electrolyte concentrations between the active and remission phases in the same patients. This calcium concentration had no relationship to the age, gender, or medication drugs of the subjects. Low calcium concentrations were found in the erythrocytes of depressed patients, which may be a relevant marker for depression.

Prenatal dexamethasone exposure alters brain monoamine metabolism and adrenocortical response in rat offspring.

In this study, it has been clearly demonstrated that prenatal dexamethasone treatment (Dex; 0.05 mg/kg on gestational days 17, 18, and 19) resulted in the significant reductions of 5-hydroxytryptamine (5-HT) turnover in four brain regions, including the neocortex, hippocampus, hypothalamus, and midbrain + pons-medulla (M + P-M) but not in the striatum in the offspring at 3 and 14 wk of life, as well as dopamine turnover in the hypothalamus. [3H]paroxetine binding densities were increased in the hypothalamus and M + P-M at 14 wk of life, which corresponded to increased 5-HT contents in both regions. On the other hand, significantly lower norepinephrine contents in the neocortex and hippocampus were observed in the Dex group compared with the control group at 14 wk of life. In addition, the exposure to new environmental condition elevated blood corticosterone levels and enhanced behavioral activities to a greater extent in the Dex group than in controls at 7 wk of life, suggesting that elevated glucocorticoid levels during the pregnancy mimicked prenatal mild stress, producing developmental alterations in brain monoamine metabolism, endocrine response, and behavior in adult offspring.

Prenatal nicotine exposure affects the development of the central serotonergic system as well as the dopaminergic system in rat offspring: involvement of route of drug administrations.

The present study was undertaken to examine the effects of prenatal nicotine exposure by two different routes of drug administration, injection and infusion, on the development of monoaminergic systems and open field behavior in the neonatal and juvenile rat. The nicotine administration to pregnant Sprague-Dawley rats was carried out by subcutaneous injection (3 mg/kg twice daily) or infusion via implanted osmotic minipumps (6 mg/kg/day) from gestational day 4 (GD4) until GD20. At postnatal day 7 (PD7), 15 and 22, the contents of the neurotransmitters and their metabolites including noradrenaline (NA), dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanilic acid (HVA), serotonin (5-HT) and 5-hydroxy-3-indolacetic acid (5-HIAA) were measured in the midbrain+pons - medulla (M + P - M), forebrain and cerebellum. Prenatal nicotine exposure caused a persistent reduction of DA turnover in the forebrain at PD15 and PD22. In addition, the 5-HT system was also affected by prenatal nicotine, and reductions of 5-HT turnover in the M + P - M at PD15 and in the forebrain and the cerebellum at PD22 were found. Although there was no effect of prenatal nicotine on NE contents, the involvement of this system remains uncertain since we measured only NE contents without metabolites. In the present study, we also found significant route-related changes in the contents of the monoamines and metabolites in the NA, DA and 5-HT systems in all brain regions in rat offspring besides the effects of prenatal nicotine. In addition, the difference in administration route reflected the results of the open field test and the number of ambulations in the injection-group was less than that in the infusion-groups with no relation to nicotine administration. Therefore, such effects of "prenatal stress" accompanied by drug administration are not negligible in considering the risk assessment of prenatal nicotine exposure.

FGF-2 influences cell movements and gene expression during limb development.

FGF-2 is proposed to be an important ectodermal signal directing limb outgrowth and patterning. Consistent with this hypothesis we show that ectopic application of FGF-2 can maintain the apical ectodermal ridge (AER)-dependent expression of Sonic hedgehog (Shh), and AER-dependent zone of polarizing activity (ZPA) signaling. We also find that ectopic FGF-2 applied to the posterior wing bud caused a dramatic change in the morphology of the limb bud, and results in limbs that display a reduction in the length of individual skeletal elements and loss of digits. Associated with these morphological changes was an FGF-2-stimulated expansion and bifurcation of the expression domains of two posteriorly expressed genes, Shh and HoxD13. Applying FGF-2 at a central or anterior location in the limb bud did not alter the Shh expression domain or cause digit loss. To test whether ectopic application of FGF-2 into the posterior limb bud was influencing the movement of limb bud cells, we used the lipophilic dye DiI to map the behavior of posterior cells in response to FGF-2. In response to FGF-2 posterior limb bud cells move in both a proximal and a distal direction, causing the initially labeled cell population to bifurcate into two distinct domains. Our data suggest that FGF-2 is influencing limb outgrowth by modifying cell movements and subsequent position-specific cell-cell interactions that are important for limb morphogenesis.

Increased 5-HT2 receptor-mediated behavior 11 days after shock in learned helplessness rats.

In the learned helplessness procedure, rats can be differentiated into two distinct groups. Learned helplessness (LH) rats do not learn to escape a controllable shock while non-learned helplessness (NLH) rats learn this response. This deficit in performance in LH rats lasted for 11 days. In LH rats, pretreatment with acute desipramine (15 mg/kg i.p.) or chronic diazepam (0.95 mg/kg/day p.o. for 7 days) did not produce recovery from this deficit of performance, but pretreatment with chronic desipramine (17.7 mg/kg/day p.o. for 7 days) or chronic mianserin (6.1 mg/kg/day p.o. for 7 days) led to recovery. Before presentation of uncontrollable shock, there was no difference between LH and NLH rats, but 11 days after the shock, head shakes induced by (+/-)-1-(2,5-demethoxy-4-iodophenyl)-2-aminopropane (DOI) in LH rats was significantly more frequent than those in NLH and naive rats without change of [3H]ketanserin binding. The basal corticosterone level was higher in LH rats than in NLH rats. These findings suggest that the learned helplessness model is a reliable animal model of depression accompanied by 5-HT2 receptor hypersensitivity.