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Choline esters of polyenoic fatty acids block cleavage divisions of sea urchins and evoke the formation of one-cell multinuclear embryos. If the fatty acids AA-Ch or DHA-Ch are added at the mid or late blastula stage, many cells are extruded, forming extra-embryonic cell clusters near the animal pole of embryos or larvae. Both effects are prevented by dimethylaminoethyl esters of polyenoic fatty acids (AA-DMAE or DHA-DMAE) or their 5-hydroxytryptamides. Nicotinic acetylcholine receptor antagonists, imechine, d-tubocurarine or QX-222 provide partial protection against AA-Ch or DHA-Ch. The organophosphate pesticide, chlorpyrifos, or a combination of (-)-nicotine + phorbol 12-myristate 13-acetate, also evoke the mass extrusion of transformed embryonic cells at the animal pole of larvae. These effects are similarly antagonized by AA-DMAE, DHA-DMAE, or fatty acids 5-hydroxytryptamides. Taking together, these results suggest that AA-Ch and DHA-Ch act on sea urchin embryos and larvae as agonists of acetylcholine receptors, whereas AA-DMAE and DHA-DMAE act as antagonists. The ability of fatty acids 5-hydroxytryptamides to prevent the effects of AA-Ch or DHA-Ch may be due to restoration of the normal dynamic balance of cholinergic and serotonergic signaling during cleavage divisions and gastrulation.

The problem of pre-nervous neurotransmitter systems arose from studies carried out on different groups of invertebrates and vertebrates in the late 1950s to early 1960s. These investigations were motivated by an hypothesis formulated by K. S. Koshtoyants concerning the similarity between pre-nervous control processes and neuronal functions. Here, we review new data related to the embryogenetic and morphogenetic functions of serotonin (5-HT) and 5-HT-like substances in early embryos of sea urchins, mouse, and other species. Accumulating evidence across animal phyla indicates that 5-HT, together with other classical neurotransmitters, regulates basic developmental processes, including cell proliferation, migration, differentiation, and morphogenesis. Future investigations of cellular and molecular mechanisms underlying phylogenetically old functions of neurotransmitters could provide new insights into the evolutionary emergence of the vertebrate nervous system.

This paper summarizes a conference held at the National Institute of Child Health and Human Development on December 6-7, 1999, on self-injurious behavior [SIB] in developmental disabilities. Twenty-six of the top researchers in the U.S. from this field representing 13 different disciplines discussed environmental mechanisms, epidemiology, behavioral and pharmacological intervention strategies, neurochemical substrates, genetic syndromes in which SIB is a prominent behavioral phenotype, neurobiological and neurodevelopmental factors affecting SIB in humans as well as a variety of animal models of SIB. Findings over the last decade, especially new discoveries since 1995, were emphasized. SIB is a rapidly growing area of scientific interest to both basic and applied researchers. In many respects it is a model for the study of gene-brain-behavior relationships in developmental disabilities.

Results of the present study demonstrate that activation of the adenylyl cyclase/protein kinase A (PKA) pathway leads to increased levels of insulin-like growth factor I (IGF-I) in cultured embryonic mouse mandibular mesenchymal cells. Treatment of serum-free cultures with 10(-8) M 8-OH-DPAT (DPAT) or with 10(-5) M forskolin in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX; 10(-5) M) increased levels of IGF-I (but not IGF-II), as measured by [(125)I]protein A immunobinding. In a previous study, we showed that DPAT, forskolin, IBMX and the 5-HT(4) receptor agonist SC53116 all increased the synthesis of cyclic adenosine monophosphate (cAMP) in these cultures. Taken together, these results provide evidence that stimulation of the adenylyl cyclase/PKA pathway in embryonic mandibular mesenchymal cells positively regulates IGF-I. This is supported by the ability of the PKA inhibitor Rp-cAMPS to block increases in IGF-I caused by both DPAT and forskolin. Consistent with these results, DPAT and forskolin increased phosphorylation of the cAMP response element binding protein (CREB), which was also blocked by Rp-cAMPS. These results suggest that activation of 5-HT receptors positively coupled to the adenylyl cyclase/PKA pathway may promote transcription of IGF-I through a cAMP response element (CRE) in the IGF-I promoter. This may represent one mechanism whereby 5-HT positively regulates IGF-I expression in developing craniofacial mesenchymal cells., (Copyright 2001 S. Karger AG, Basel)

The GLUT4 glucose transporter is primarily expressed in skeletal muscle, heart and adipose tissue, where its expression is postnatal, coincident with the acquisition of insulin-regulated glucose transport. In muscle, contraction also regulates GLUT4 activity in the postnatal animal. Here we demonstrate that GLUT4 is expressed in the developing mouse embryo with specific tissue and spatiotemporal patterns. From embryonic day 9 (E9; E1 = day of copulation plug) to postnatal day 70 (P70), mice were analyzed for GLUT4 mRNA and protein expression by in situ hybridization, immunohistochemistry and immunoblot. Specificity was confirmed with sense riboprobe hybridization and peptide competition, respectively. At E9, GLUT4 was detected in the cranial neural folds in the outer (mantle) layer of the neuroepithelium. At E10, expression was present throughout the developing heart and was prominent in the endocardial cushions through E12. At E10-12, GLUT4 was also prominent in craniofacial mesenchyme. GLUT4 expression in cartilage and bone was evident at E12 and was maintained throughout early postnatal life. GLUT4 was apparent throughout embryonic development in the ventricular epithelium, choroid plexus and in the developing cerebellum. At birth, cardiac expression was reduced and GLUT4 was most evident in cartilage, bone and specific brain regions. In the latter, GLUT4 expression was most evident in the cerebellum, specifically in the external granular layer through P7 and in the internal granular layer thereafter. Maximal GLUT4 protein levels in the cerebellum were measured between P14 and P21 and were reduced in the adult brain. These findings suggest that GLUT4-mediated glucose transport may play important roles during development of the brain and nonneuronal tissues in the mouse embryo., (Copyright 2000 S. Karger AG, Basel.)

Background: Biogenic amine neurons are involved in a number of mental diseases including addiction and alcohol dependence. Because chronic ethanol is known to cause supersensitivity to NMDA excitotoxicity in cortical neurons, this study sought to determine the effect of ethanol treatment on biogenic amine neurons., Methods: To determine if ethanol exposure alters the vitality of biogenic amine neurons, cultures were prepared from E14 rat brain. After 24 hr in culture, cells were divided into control or ethanol (100 mM) treatment groups, cultured an additional 48 hr, and then half of them exposed to NMDA for 25 min. The NMDA was then removed and cells cultured in fresh media for an additional 24 hr to allow for excitotoxic delayed neuronal death. Cultures were then stained with antibodies to 5-hydroxytryptamine or tyrosine hydroxylase to identify serotonin and dopamine neurons, respectively. Cultures were analyzed for cell number and neuronal morphology., Results: Ethanol treatment alone had no effect on biogenic amine cell number, soma area, number of neurites, or terminal segments, although the field area of dopamine neurons was decreased. Treatment with 30 microM NMDA had no effect on controls, but significantly decreased dopamine neurons in ethanol-treated cultures as well as reduced soma area, field area, number of neurites and number of terminal segments. Treatment with higher concentrations of NMDA reduced dopamine and serotonin neurons in both controls and ethanol-treated groups, and ethanol treatment significantly enhanced NMDA excitotoxic effects. Treatment with Brain Derived Neurotrophic Factor (BDNF) prevented ethanol sensitization to NMDA excitotoxicity., Conclusions: These studies suggest that ethanol treatment sensitizes biogenic amine neurons to excitotoxic insults. Ethanol sensitization of biogenic amine neurons to insults could contribute to the development of mental disease.

Evidence from the present study suggests that activation of both 5-HT(1A) and 5-HT(4) (5-hydroxytryptamine) receptor subtypes stimulates cyclic adenosine monophosphate (cAMP) synthesis in cultured embryonic mouse mandibular mesenchymal cells (micromass cultures). When these cells were grown in serum-free medium and treated with 10(-8) M agonist selective for either the 5-HT(1A) or 5-HT(4) receptor subtype (8-OH-DPAT and SC53116, respectively), this significantly stimulated cAMP synthesis and increased insulin-like growth factor I (IGF-I), but not IGF-II, protein levels compared to vehicle-treated controls, as measured by semi-quantitative immunobinding assays. Consistent with these results, IGF-I was significantly decreased when mandibular mesenchymal cells were grown in serum-containing medium (which contains micromolar amounts of 5-HT from fetal calf serum) and treated with 10(-8) M antagonist selective for the 5-HT(1A) or 5-HT(4) receptor subtype (NAN-190 on SDZ-205,557). Forskolin also stimulated cAMP and IGF-I (but not IGF-II) in both serum-containing and serum-free cultures. These results indicate that activation of 5-HT receptors that increase cAMP promotes synthesis of IGF-I. This may occur by activation of the cAMP response element sequence present in the IGF-I promoter region. Stimulation of the adenylyl cyclase pathway by activation of 5-HT(1A) or 5-HT(4) receptors may be one mechanism by which serotonin regulates IGF-I synthesis in developing craniofacial mesenchymal cells.

Serotonin (5-HT) is a neurotransmitter that also functions as a hormone and a growth factor. 5-HT is involved in numerous physiological actions and displays complex pharmacological properties. As a growth factor, 5-HT plays a role in cell proliferation and differentiation of neuronal and nonneuronal tissue and it transduces its signals through more than fourteen subtypes of 5-HT receptors. Since determination of the expression and distribution is important for understanding the role of the 5-HT receptors, we have developed an RNase protection assay (RPA) that allows the simultaneous analysis of 5-HT2AR, 5-HT2BR, and 5-HT2CR per sample of RNA. This multiprobe set also comprises probes for two house-keeping genes, L32 and GAPDH, which control for sample-loading errors. Using this RPA probe set, we have examined the relative expression of 5-HT2AR, 5-HT2BR, and 5-HT2CR in rat embryos inclusively from embryonic day (ED) 9 to 21 of development. Our data indicate that 5-HT2AR levels gradually increased from ED11 to ED21. The expression of 5-HT2BR was decreased between ED9 to ED11 then remained relatively constant through ED21. 5-HT2CR was initially expressed at residual levels between ED9 and ED12 but dramatically increased to a peak level at ED13, then decreased by ED17. Expression of the 5-HT2 receptors in these tissues was confirmed independently by RT-PCR indicating that there is developmental regulation in the expression of these receptors. The 5-HT2R multiprobe assay will be useful for detecting relative changes in the expression of these receptors in developmental, normal and pathological tissues as well as for monitoring relative changes in expression resulting from the use of pharmaceutical agents.

GABA exerts a variety of trophic influences on developing brain cells, as reviewed in this issue. During early stages of brain development, GABAergic axons course through regions where other neurotransmitter phenotypes are being generated. This raises the question of whether GABA may influence the ontogeny of these neurotransmitter systems in the embryonic brain. The brainstem provides a good example of this relationship, since GABAergic axons pass through the anlage of the developing raphe nuclei when serotonergic (5-HT) neurons are just beginning to differentiate and migrate away from the ventricular zone. Evidence that GABA regulates development of these and adjacent noradrenergic neurons has recently been obtained using embryonic brainstem cultures, which contain differentiating 5-HT, tyrosine hydroxylase (TH), and GABA neurons. These cultures also express multiple GABAA-receptor subunits that form functional GABAA/Cl- channels. GABAA receptor ligands produce differential effects on survival and growth of monoamine (5-HT, TH) and GABA neurons, and on expression of GABAA subunits in these cultures. These findings provide evidence that GABA can selectively regulate development of neurons of different neurotransmitter phenotypes, as well as developmental expression of its own receptors, and suggest that in utero exposure to GABAA receptor ligands could produce imbalances in monoaminergic versus GABAergic neurotransmission in the developing brain. Dieldrin, an organochlorine pesticide that acts as a GABAA antagonist, has potent effects on survival, and neurite outgrowth by 5-HT neurons, and GABAA subunit expression in brainstem cultures. Thus, maternal exposure to organochlorine pesticides could pose a risk to fetal brain development, especially during the first trimester of pregnancy.

GABA acts as a trophic signal for cultured embryonic rat monoamine neurons by activating GABA(A) receptors. These effects are blocked by the organochlorine insecticide dieldrin and the classic GABA(A) antagonist bicuculline. Both dieldrin and another organochlorine insecticide, lindane, block the effects of GABA on the GABA(A) receptor by binding directly to the Cl- channel. Therefore, prenatal exposure to these chemicals could lead to disturbances in the trophic actions of GABA on monoamine neurotransmitter systems in the embryonic brain and produce alterations in GABA(A) receptor expression and function. Effects of daily prenatal exposure to organochlorine insecticide (dieldrin or lindane) or bicuculline from embryonic day (E)12-17 were determined in brains of E17 fetal rats using t-[35S]butyl-bicyclophosphorothionate ([35S]TBPS) binding. This radioligand was chosen because, like organochlorine insecticides, it binds directly to GABA(A) receptor/Cl- channels. [35S]TBPS binding was analyzed in extensively washed membranes from E17 brainstem and whole brain with the brainstem removed ('rest of brain') at a TBPS concentration that approximated the KD determined in [35S]TBPS saturation binding experiments performed on normal E17 rat brainstem. In utero exposure to dieldrin, lindane, or bicuculline from E12-E17 caused a significant reduction in the amount of [35S]TBPS binding in E17 brainstem compared to vehicle-injected controls, but had no significant effect on 'rest of brain'. These data suggest that in utero exposure to organochlorine insecticides that act as GABA(A) antagonists negatively regulate expression of GABAA receptors in fetal brainstem. If these effects persist, they could lead to disturbances in postnatal functions of the ascending GABAergic system, possibly with behavioral consequences.

It is now well established that neurotransmitters act as growth-regulatory signals for neuronal and non-neuronal cells of both primitive and higher organisms, where they control cell proliferation, motility, survival, growth, differentiation, and gene expression. Many of these actions are reminiscent of the actions of other growth-regulatory signals such as growth factors, neurotrophins, and proto-oncogenes. How, then, do neurotransmitters exert these effects? Although some information is available concerning second messengers activated by these neurotransmitters in developing cells, little is known about subsequent steps involving signal transduction cascades leading to their final outcomes. This review attempts to provide testable hypotheses regarding possible cellular and molecular mechanisms downstream of second messengers activated by neurotransmitters, based on recent insights into signal transduction cascades activated by classical growth-regulatory signals. In many cases, there are clear points of convergence between these pathways, raising the interesting possibility that neurotransmitters and other growth-regulatory signals may cooperate to regulate developmental functions of cells and tissues.

We have previously shown that GABA acts as a trophic signal for monoamine neurons in embryonic day 14 (E14) rat brainstem cultures [Liu et al., J Neurosci 1997a; 17:2420-2428]. The organochlorine pesticide dieldrin and the classical GABA(A) receptor antagonist bicuculline interfere with the trophic actions of GABA and alter expression of several GABA(A) receptor subunit mRNA transcripts in these cultures [Liu et al., J Neurosci Res 1997b;49:645-653]. In the present study, we investigated whether prenatal exposure to dieldrin or bicuculline from E12-17 would alter mRNA expression of alpha1, beta3, gamma1, gamma2S and gamma2L GABA(A) receptor subunits in fetal (E17) rat brainstem using competitive RT-PCR to absolutely quantify these transcripts. The effects of dieldrin and bicuculline on expression of GABA(A) receptor subunit transcripts were similar across subunits. Dieldrin and bicuculline decreased expression of alpha1, beta3 and gamma1 transcripts compared to vehicle-injected controls, but did not significantly alter expression of gamma2S and gamma2L transcripts. Taken together, these studies indicate that in utero exposure to organochlorine pesticides acting as GABA(A) receptor antagonists may alter the expression and subunit composition of developing GABA(A) receptors. If these changes persist, they could have long-lasting effects on developing GABAergic neural circuitry, GABA(A) receptor function and GABA-mediated behaviors.

Cyclodiene organochlorine pesticides, such as dieldrin, inhibit gamma-aminobutyric acid (GABA)ergic neurotransmission by blocking the Cl- channel of GABA(A) receptors. This action may make the developing nervous system especially vulnerable to these neurotoxins, which could interfere with the trophic actions of GABA on developing neurons and alter expression of GABA(A) receptors. We have used an in vitro model to determine whether exposure to dieldrin alters developmental expression of GABA(A) receptor subunit mRNA transcripts. Dissociated cell cultures were prepared from embryonic day 14 (E14) brainstem and cultured in serum-containing medium for 1 day in vitro (DIV), then treated for 2 DIV with 10 microM dieldrin in serum-free medium. This dose was based on preliminary experiments and previous studies (Nagata et al.: Brain Res 645:19-26, 1994; Pomes et al.: J Pharmacol Exp Ther 271:1616-1623, 1994). Absolute amounts of alpha1, beta3, gamma1, gamma2S and gamma2L mRNA transcripts were quantified in these cultures by quantitative, competitive reverse transcription-polymerase chain reaction (RT-PCR) using subunit-selective internal standards. The most abundant GABA(A) subunit transcript was beta3, which was much more highly expressed than gamma2S, gamma1, gamma2L, or alpha1 subunit mRNAs. Dieldrin differentially regulated expression of these transcripts. Levels of beta3 subunit transcripts were significantly increased (by 300%) by dieldrin, whereas expression of gamma2S and gamma2L transcripts were decreased (by 50% and 40%, respectively). However, dieldrin did not alter the ratio of gamma2S to gamma2L transcripts, indicating that it did not affect alternative splicing of gamma2 transcripts. Dieldrin appeared to increase expression of alpha1 subunit transcripts, but this effect was not statistically significant. Dieldrin did not significantly alter expression of gamma1 subunit transcripts. These results support the hypothesis that in utero exposure to cyclodiene pesticides could pose a risk to the developing brain by virtue of their ability to alter gene expression of GABA(A) receptor subunits, which could produce GABA(A) receptors with altered functional properties.

The inhibitory neurotransmitter GABA may act as a trophic signal for developing monoamine neurons in embryonic rat brain, because GABA neurons and their receptors appear in brainstem during generation of monoamine neurons. To test this hypothesis, we used dissociated cell cultures from embryonic day 14 rat brainstem, which contains developing serotonin (5-HT), noradrenaline (tyrosine hydroxylase; TH), and GABA neurons. Immunocytochemistry and reverse transcription-PCR (RT-PCR) revealed the presence of multiple alpha, beta, gamma, and delta subunits in these cultures. Competitive RT-PCR demonstrated high levels of beta3 subunit transcripts. Expression of functional GABAA receptors was demonstrated using 36Cl- flux assays. To investigate GABAergic regulation of neuronal survival and growth, cultures were treated for 1-3 d in vitro with 10 microM GABA and/or GABAA antagonist (bicuculline or the pesticide dieldrin). The effects of treatments were quantified by analysis of immunoreactive 5-HT, TH, and GABA neurons. GABAA receptor ligands differentially regulated neuronal survival and growth depending on neurotransmitter phenotype. GABA exerted positive effects on monoamine neurons, which were countered by bicuculline (and dieldrin, 5-HT neurons only). By itself, bicuculline produced inhibitory effects on both 5-HT and TH neurons, whereas dieldrin potently inhibited 5-HT neurons only. GABA neurons responded positively to both antagonists, but more strongly to bicuculline. Taken together, these results demonstrate that the activation/inhibition of GABAA receptors produces opposite effects on the development of embryonic monoamine and GABA neurons. This suggests that these neurotransmitter phenotypes may express GABAA receptors that differ in fundamental ways, and these differences determine the developmental responses of these cells to GABAergic stimuli.

1. Classical neurotransmitters (such as acetylcholine, biogenic amines, and GABA) are functionally active throughout ontogenesis. 2. Based on accumulated evidence, reviewed herein, we present an hypothetical scheme describing developmental changes in this functional activity, from the stage of maturing oocytes through neuronal differentiation. This scheme reflects not only the spatio-temporal sequence of these changes, but also the genesis of neurotransmitter functions, from "protosynapses" in oocytes and cleaving embryos to the development of functional neuronal synapses. 3. Thus, it appears that neurotransmitters participate in various forms of intra- and intercellular signalling throughout all stages of ontogenesis.

Serotonin (5-hydroxytryptamine; 5-HT) uptake sites are transiently expressed in craniofacial epithelia and mesenchyme, including the tooth germ, during mouse embryogenesis. Based on malformations and patterns of cell proliferation and death in cultured mouse embryos exposed to 5-HT uptake inhibitors, it has been hypothesized that 5-HT acts as a dose-dependent morphogenetic signal for craniofacial development. The present study was designed to investigate the effect of 5-HT on tooth-germ formation in serum-free mandibular explant cultures prepared from embryonic day-13 (plug day = embryonic day-1) mouse embryos. In the absence of serum or a 5-HT supplement, tooth germs develop only to the bud stage in these cultures. When explants were cultured for 8 days in a defined medium supplemented with 5-HT, late bell-stage tooth germs were stimulated to develop in a dose-dependent manner. This effect was reversed by addition of the 5-HT uptake inhibitor fluoxetine. Anti-5-HT immunocytochemistry demonstrated specific uptake of 5-HT by developing tooth germ and mandibular epithelium, which could also be blocked by fluoxetine. These results suggest that 5-HT may regulate dental differentiation, and that intracellular uptake is required for this action.

Three lines of transgenic (Tg) mice carrying a fusion gene linking the mouse metallothionein-I promoter to a cDNA encoding human insulin-like growth factor binding protein-1 (hIGFBP-1) were found to express the transgene in brain. As judged by comparing Tg brain weights to those of non-transgenic littermates, adult hemizygotic Tg mice of each line exhibited brain growth retardation (16.2%, 14.4% and 8.1% reductions in weight, respectively in each line). In two lines, total brain DNA and protein content were decreased. Further analysis indicated that the brain growth retardation was manifested in the second week of postnatal life. Given that the insulin-like growth factors (IGFs) stimulate cell proliferation and/or survival in neural cultures and that hIGFBP-1, when present in a molar excess, inhibits IGF interactions with their cell surface receptors, the brain growth retardation in hIGFBP-1 Tg mice likely results from hIGFBP-1 inhibition of IGF-stimulated growth-promoting actions. These hIGFBP-1 Tg mice should prove useful in defining IGF actions during postnatal brain maturation.

Evidence from the developing serotonergic system provides support for the working hypothesis that an important function of receptors expressed by developing glia may be to mediate the effects of neurotransmitters on growth and differentiation of the neurons that utilize them, by regulating the production of glial-derived neurotrophic factors. Regional heterogeneity of glial expression may reflect the specific roles played by these receptors within different compartments of a developing neurotransmitter system. During the construction of neural pathways, the specificity of these neurotransmitter-mediated neuronal-glial interactions would require appropriate spatiotemporal expression of glial receptors and neurotrophic factors, as well as specificity of these factors for neurons of appropriate neurotransmitter phenotypes.

In the adult nervous system, neurotransmitters act as chemical mediators of intercellular communication by the activation of specific receptors and second messengers in postsynaptic cells. This specialized role may have evolved from more primitive functions in lower organisms where these substances were used as both intra- and intercellular signalling devices. This view derives from the finding that a number of 'classical' neurotransmitters are present in primitive organisms and early embryos in the absence of a nervous system, and pharmacological evidence that these substances regulate morphogenetic activities such as proliferation, differentiation, cell motility and metamorphosis. These phylogenetically old functions may be reiterated in the developing nervous system and in the humoral functions of neurotransmitters outside the nervous system. This review will provide evidence for this hypothesis based on the commonality of signal transduction mechanisms used in primitive organisms, early embryos and non-neuronal cells, and relate these relationships to the functions of neurotransmitters in the developing nervous system. This discussion has generally been limited to neurotransmitters where non-neuronal functions have been studied and information regarding the involvement of receptors and second messenger pathways is available.

Expression of the c-fyn proto-oncogene was analysed in the developing and adult rat brain by subcellular fractionation and immunocytochemistry using polyclonal antibodies specific for its protein product (p59fyn). Immunoperoxidase staining revealed widespread localization of p59fyn in developing axonal tracts throughout the fetal (E18) rat brain. fyn immunoreactivity was not observed in most axon-rich regions of the adult brain, but continued to be expressed at elevated levels in the adult olfactory and vomeronasal systems. At other sites in the adult rat brain, fyn immunoreactivity was restricted to cell bodies of neuronal subpopulations, especially those in brain stem and hypothalamic nuclei, and to subpopulations of glial cells along axonal tracts in the medulla, optic nerve and white matter of the spinal cord. In the peripheral nervous system, fyn staining was also prominent in Schwann cells. Subcellular fractionation of fetal and adult rat brain confirmed the immunocytochemical localization, demonstrating an enrichment of p59fyn in membranes from a fetal brain fraction containing nerve growth cones, and lower levels in adult brain where there was only a small enrichment in synaptosomal membranes. The developmental regulation of p59fyn suggests that the fyn tyrosine kinase may serve separate cellular roles in axonal growth and specialized functions of mature neurons and glia.

The expression of mRNAs for two 5-HT receptors (5-HT1C, 5-HT2) has been investigated by evaluating in situ hybridization in the prenatal rat CNS. At Embryonic Day 14 (E14), the highest signal for 5-HT1C was found in the choroid plexus, while the marginal/intermediate (m/i) zones of the midbrain, brain stem (including monoaminergic groups), and spinal cord also displayed label. By E18-21 a number of more rostral regions contained transcript, including the hippocampus (CA1), in addition to more intense signal in midbrain, brain stem, and spinal cord. Expression in the choroid plexus appeared to peak between E16-E18, although considerable hybridization signal remained at E21. 5-HT2 transcripts were also detected at E14. Label was present in m/i zones of the midbrain and in a number of other areas. In comparison to 5-HT1C, 5-HT2 mRNA was distributed over a wider rostral-caudal extent at this age. As with 5-HT1C mRNA, signal increased over rostral and brain stem areas at late gestational ages with significant labeling appearing in the olfactory bulb, cerebellum, cortical plate and subplate, hippocampus (dentate gyrus), and monoaminergic nuclei. 5-HT1C and 5-HT2 receptor transcripts were also present over the meninges at E16 and may represent transient expression of these receptors. These expression patterns in the embryonic rat brain, in conjunction with previous evidence indicating that 5-HT can act as a differentiation signal for target neurons, suggests that prenatal 5-HT receptors are positioned to play a role in the prenatal development of the CNS.

The neurotransmitter serotonin (5-HT) was localized in the ectoplacental cone (EPC) and placenta of the day 9-12 (E9-12) mouse embryo in vivo and in whole embryo cultures, using immunocytochemistry with a specific 5-HT antiserum. In uncultured conceptuses, 5-HT immunoreactivity (5-HT IR) was most intense in the EPC at E9 (2-7 somites), particularly in giant cells around the periphery. Nuclear staining was observed in lightly staining giant cells and in small cells in the core of the cone. By E10 (18-24 somites) 5-HT IR in the placenta was less intense and almost exclusively limited to giant cells, where it was localized to chromatin-like material in nuclei. The same pattern and level of 5-HT IR persisted through E12. In the placenta, 5-HT IR appeared to be most intense in giant cells located near aggregations of platelets in decidual blood vessels. 5-HT IR was enhanced in cultured conceptuses, and further increased when exogenous 5-HT was added to the culture medium. Immunoreactivity was greatly reduced by adding the 5-HT uptake inhibitor fluoxetine to the culture medium, or culturing conceptuses in medium containing 5-HT depleted rat serum. Thus, 5-HT was apparently taken up from the culture medium. In conceptuses exposed to exogenous 5-HT, immunoreactivity in the placenta appeared as a gradient from the giant cells to the inner layers, suggesting that these cells may transport 5-HT toward the embryo. No evidence of 5-HT synthesis by the EPC/placenta was found. These results suggest that 5-HT present in the EPC/placenta is due to uptake, not synthesis. Possible sources and functions of 5-HT in the developing placenta are discussed.

During craniofacial development in the mouse embryo (days 9-12 of gestation; plug day = day 1), transient expression of serotonin (5-HT) uptake in epithelial structures of this region correlates with critical morphogenetic events (Lauder et al., '88; Shuey, '91; Shuey et al., '89, '92). The purpose of the present investigation was to assess the possible functional significance of these uptake sites by examination of patterns of dysmorphology following exposure of embryos to selective 5-HT uptake inhibitors. Exposure of mouse embryos in whole embryo culture to sertraline, at a concentration (10 microM) which produced no evidence of general embryotoxicity, caused craniofacial malformations consistent with direct action at 5-HT uptake sites. Two other 5-HT uptake inhibitors, fluoxetine and amitriptyline, produced similar defects. The critical period of sertraline exposure occurred on days 10-11. The observed craniofacial defects were associated with decreased proliferation and extensive cell death in mesenchyme located 5-6 cell layers deep from the overlying epithelium. In contrast, the subepithelial mesenchymal layers showed normal or elevated levels of proliferation. From these results it appears that inhibition of 5-HT uptake into craniofacial epithelia may produce developmental defects by interference with serotonergic regulation of epithelial-mesenchymal interactions important for normal craniofacial morphogenesis.

To study the phenotypic specificity of S-100 beta and insulin-like growth factor II (IGF-II) for developing monoamine neurons, serotonin (5-HT) neurons from the embryonic day 14 (E14) rostral raphe or dopamine (TH) neurons from the substantia nigra/ventral tegmental area were cultured for 3 days in vitro (3 DIV) in the presence of these factors. Neuronotrophic effects were analyzed by computer-assisted morphometry of 5-HT and TH-immunoreactive neurons. S-100 beta and IGF-II differentially regulated the growth of 5-HT and TH neurons but did not affect their survival. S-100 beta significantly increased several parameters of neurite outgrowth by 5-HT neurons but inhibited the spatial extent (field area) of TH neurites. IGF-II promoted growth of cell bodies of both phenotype, but only stimulated neurite outgrowth by TH neurons. S-100 beta and IGF-II differentially affected the number of GFAP immunoreactive cells from raphe and substantia nigra, but these effects did not correlate with the specificity of neuronotrophic effects. S-100 beta and IGF-II immunoreactivities were expressed in glial cultures derived from the same brain regions, raising the possibility that these factors have autocrine effects on glia as well as paracrine actions on neurons. The results of this study suggest that specificity of neurotrophic factors for particular embryonic neurons may be correlated with their neurotransmitter phenotype.

To test the hypothesis that glia mediate interactions between embryonic serotonergic (5-HT) neurons and dopamine neurons, we studied the effects of 5-HT in co-cultures of E14 raphe neurons of mesencephalic dopamine neurons and radial glia/astrocytes derived from the same (homotypic) or opposite (heterotypic) brain region using a dose (10(-5) M) that would produce 5-HT uptake into glial cells as well as activate 5-HT receptors. Morphometric analysis of 5-HT and tyrosine hydroxylase (TH) immunoreactive neurons revealed regional differences in the effects of 5-HT (and nialamide) on survival, cell soma size, and dendrite-like neurite outgrowth in neuronal-glial co-cultures. In general, 5-HT had more significant effects on both types of monoamine neuron when they were cultured with mesencephalic glia (GSN). Stimulatory effects of 5-HT on growth of TH neurons in GSN cultures suggest that developing raphe axons, which reach the mesencephalon during the early differentiation of these neurons, may enhance the influence of local glial-derived trophic factors. Likewise, the promotion of 5-HT neuronal survival in these cultures suggests that glial factors in the mesencephalon may contribute to the support of 5-HT neurons in addition to the influences of raphe glia. The inhibitory effects of 5-HT on neurite outgrowth by raphe neurons in GSN co-cultures indicates enhanced sensitivity of these neurons to the inhibitory effects of 5-HT in the presence of mesencephalic glia. The region-specific effects of 5-HT and nialamide in glial co-cultures suggest that raphe and mesencephalic glia may express different capacities for 5-HT uptake, receptors, and/or monoamine oxidase (MAO) activities. These characteristics could be important for the specificity of growth-regulatory influences of glial cells on the development of brain monoamine neurons.

A prenatal atlas of the mouse brain is presently unavailable and is needed for studies of normal and abnormal development, using techniques including immunocytochemistry and in situ hybridization. This atlas will be especially useful for researchers studying transgenic and mutant mice. This collection of photomicrographs and corresponding drawings of Gestational Day (GD) 14 mouse brain sections is an excerpt from a larger atlas encompassing GD 12-18. In composing this atlas, available published studies on the developing rodent brain were consulted to aid in the detailed labeling of embryonic brain structures. C57Bl/6J mice were mated for 1 h, and the presence of a copulation plug was designated as GD 0. GD 14 embryos were perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer and embedded in paraffin. Serial sections (10 microns thickness) were cut through whole heads in sagittal and horizontal planes. They were stained with hematoxylin and eosin and photographed. Magnifications were 43X and 31X for the horizontal and sagittal sections, respectively. Photographs were traced and line drawings prepared using an Adobe Illustrator on a Macintosh computer.

In this morphometric analysis of immunoreactive serotonin (5-HT) and tyrosine hydroxylase (TH) neurons in culture, 5-HT and the MAO inhibitor nialamide influenced the survival, cell body size and neurite outgrowth of embryonic day 14 (E14) 5-HT neurons after treatment from 1-3 days in vitro (DIV), but did not significantly affect E14 or E15 TH neurons of either the noradrenergic or dopaminergic phenotype. These treatments had minimal effects on 5-HT neurons derived from E15 embryos. The stimulatory effects of 5-HT on survival and somal growth of E14 5-HT neurons was in contrast to its inhibitory effects on neurite outgrowth, suggesting trophic and inhibitory autoregulation of different cellular compartments of developing 5-HT neurons. The decreased sensitivity of E15 5-HT neurons to these treatments, despite similar viability and growth of these neurons in control cultures, suggests the existence of a critical period for this regulation during the initial period of serotonergic neurogenesis when these neurons are forming the bilateral B4-9 raphe complex. The lack of significant effects of 5-HT on TH neurons suggests differential sensitivities of 5-HT and TH neurons to developmental regulation by this neurotransmitter.

Acute maternal ethanol administration to C57B1/6J mice on gestational day 7 (GD7) results in facial and brain abnormalities similar to those reported in human fetal alcohol syndrome (FAS). Using this model, we assessed the damage to brain structures using histological methods and changes in developing neurotransmitter systems with immunocytochemistry. Cholinergic neurons in the forebrain were stained with a monoclonal antibody to choline acetyltransferase (ChAT). Catecholaminergic neurons in the midbrain and serotoninergic neurons in the hindbrain were stained with polyclonal antisera to tyrosine hydroxylase (TH) and serotonin (5-HT), respectively. Forebrain deficiencies, including loss of midline structures (olfactory bulbs, midline septation, medial septal area) and deficits in lateral and dorsal regions (neostriatum and cerebral cortex) were found in both GD14 embryos and GD18 fetuses. In severely affected offspring, complete loss of the septal region resulted in conjoined lateral ventricles and a reduction in the thickness of the ventricular zone surrounding the single ventricle, as well as a severe loss of ChAT neurons which would normally be located in this territory. However, no consistent changes were seen in the distribution or size of TH or 5-HT neuronal cell groups in the midbrain and hindbrain. These differences in effects on specific neurotransmitter systems reflect the fact that the forebrain is most severely affected by early ethanol administration, whereas the hindbrain is relatively spared. Such differential effects could produce an imbalance in developing neurotransmitter systems in the embryonic and fetal brain, which could explain some of the functional deficits observed in children with FAS.

To study the interactions between neurons of known transmitter phenotype and non-neuronal cells of glial or fibroblastic origin, serotonergic (5-HT) neurons were tested for their strength of adhesion and neurite outgrowth patterns on substrates of astrocytes or fibroblasts using a cell adhesion assay for transmitter-identified neurons, and morphometry of immunocytochemically stained neurons in dissociated cell cultures. Both the strength of adhesion and the rate and complexity of neurite outgrowth by 5-HT neurons were significantly greater on substrates of astrocytes compared to fibroblasts. These results provide evidence that 5-HT neurons can interact selectively with glia via cell surface determinants, and that this process may be important for the development of complex (dendrite-like) neuritic arbors. The methods developed in this study will be useful for future studies of interactions between transmitter-identified neurons and glial cells during ontogeny of the embryonic brain.

The serotonergic system is an early forming component of the CNS circuitry, beginning its development on gestational days 11-12 in the rat. Owing to its early presence in the embryonic nervous system, 5-HT has been proposed to act as a developmental signal for receptive cells. In vivo and in vitro evidence that 5-HT can influence both biochemical and morphological differentiation of raphe neurons and receptive target cells suggests that this neurotransmitter may have an organizing function in the developing nervous system which involves effects on neurite outgrowth and other aspects of neuronal differentiation, including synaptogenesis. Such functions may be mediated by a variety of 5-HT receptors located on both neuronal and non-neuronal cells. The apparent function of 5-HT as a differentiation signal in the developing nervous system raises important issues regarding the use of psychoactive serotonergic drugs by pregnant women, since these drugs may act as neural teratogens in the unborn child.

Previous studies from this laboratory have suggested that serotonergic (5-HT) neurons may influence the differentiation of their embryonic target cells in the developing rat brain. The present study was designed to determine whether or not maternal p-chlorophenylalanine (pCPA) administration could deplete serotonin (5-HT) in developing 5-HT neurons during embryonic days 13-15, when the effects of pCPA on neuronal genesis have been observed previously. For this study, pCPA was administered to timed-pregnant rats and embryos were sacrificed at two different gestational ages, embryonic days 13-14 (E13-14) and 14-15 (E14-15). Immunotitration experiments were carried out on tissue sections, using an antiserum to 5-HT-hemocyanin conjugates to obtain a relative estimate of the amount of 5-HT contained within individual 5-HT neurons of embryos from pCPA-treated and control mothers. Diminished immunoreactivity as a consequence of addition of increasing amounts of antigen was then quantitated on a relative scale by comparison with the amount of immunoreactivity present when no antigen was added to the primary antiserum. Two major findings resulted from this study: maternal pCPA treatment depleted 5-HT by approximately 50% in developing 5-HT neurons at embryonic ages E13-14 and E14-15, but depletion appeared to be greatest in the youngest embryos; developing 5-HT neurons increased their content of neurotransmitter by approximately 10-fold during this one day of embryonic development, an effect which could be observed in both pCPA-treated and control animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute maternal ethanol administration (two i.p. injections of 2.9 g ethanol/kg maternal body wt) to C57B1/6J mice during gastrulation stages of embryogenesis (gestational day 7) induces a spectrum of brain and facial malformations characteristic of those seen in the human Fetal Alcohol Syndrome. Scanning electron microscopic and light microscopic analyses of the brains of embryos of gestational days 11-14 demonstrate ventro-medial forebrain deficiencies of varying degrees of severity in affected specimens. Even at the mild end of the spectrum, reductions in the size of the septal nuclei and the shape of the third ventricle are observed. As the severity of the effect increases, the septal nuclei disappear altogether, resulting in midline fusion of the corpora striata (basal ganglia). In such cases, the third ventricle is totally absent anteriorly (preoptic area) and significantly narrowed at more posterior levels, adjacent to the ventromedial nuclei. In addition, the hippocampal primordium is absent at levels which include the corpora striata, and septation of the cerebral cortex is incomplete. More posteriorly, at the level of the posterior commissure, the hippocampal primordium is present, but greatly reduced in size, and the entire brain is distinctly narrower in width. Still further posteriorly, at levels of the metencephalon which include the tectum and cerebellar plate, the cerebral aqueduct is significantly expanded, fusion of midline (raphe) structures is incomplete and the cerebellar plate does not extend as far medially as it does normally. Interestingly, these abnormalities are analogous to those observed in the holoprosencephaly series of malformations. The results of the present study support our hypothesis that severe forms of the Fetal Alcohol Syndrome mimic certain aspects of the holoprosencephaly spectrum, and indicate that special attention should be paid to possible deficiencies in the septal nuclei and basal ganglia of children born to women who abuse alcohol. The fact that gross brain malformations can be induced in this animal model at a time corresponding to the third week of human gestation (a time when most women remain unaware of pregnancy) is of significance in terms of the possible prevention of alcohol-induced birth defects and mental deficiency in man., (Copyright © 1984. Published by Elsevier Ltd.)

Synaptogenesis was studied in the monoamine (MA) cell groups locus coeruleus (LC), dorsal and medial raphe nuclei (RN) and substantia nigra, zona compacta (SN) between day 18 of gestation and postnatal day 60 using ethanolic phosphotungstic acid (E-PTA) to visualize synaptic profiles. Nuclear area, and cellular packing density (inversely proportional to area of neuropil) were also determined. As determined using the E-PTA method, synaptogenesis begins in the neuropil of the SN first, on or before 18 days of gestation, and in the LC and RN at 19 days. Synaptogenesis on MA cell perikarya is first observed in the SN, on or before 18 days, and in the LC and RN at 20 days. The onset of somatic synaptogenesis coincides with the beginning of nuclear growth and development of the neuropil (decrease in cellular packing density) in all MA cell areas, raising the possibility of common factors in the initiation of these processes. Nonsynaptic contacts precede the appearance of synaptic profiles both in the neuropil and on the somata of the MA cells of the LC, RN and SN, and may represent precursors of mature synapses or desmosome-like contacts. Somatosomatic nonsynaptic contacts occur only prenatally between adjacent MA neurons in the LC, RN and SN. Although some synaptogenesis occurs prenatally in these MA cell groups (indiciating that these parts of the MA circuitry may be functional before birth), most of this synaptogenesis occurs postnatally and continues into adulthood. Since such synaptogenesis does not begin until 2-4 days prior to birth, whereas these neurons and their processes exhibit MA fluorescence as early as 12-14 days of gestation, they apparently are capable of synthesizing transmitter and proliferating terminals before they themselves are innervated.

Mesencephalic raphe nuclei were transplanted via the cisterna magna in the IVth ventricle of adult rats having received either a neonatal or an adult age destruction of the serotonin system. In neonatally treated rats both serotonin (5-HT) and glial fibrillary acidic protein (GFA) immunocytochemistry showed an absence of survival of 5-HT neurons in the transplants. However, many 5-HT immunoreactive cell bodies and processes were detected in 10-month-old transplants in adult treated animals. These results suggest that recognition processes between the target tissue and the implant may be a prerequisite for the survival of grafted neurons.

Serotonergic neurons throughout the brain were destroyed by early postnatal treatment of rats with an intracisternal injection of 5,7-dihydroxytryptamine (5,7-DHT), as demonstrated with biochemical measurements of serotonin and immunocytochemical localization of serotonin-containing neurons. Using these methods, it was shown that approximately 75-98% of serotonergic neurons underwent cell death in rats which were treated on day 3. In contrast, intracisternal administration of 5,7-DHT in adult rats led to the loss of distal serotonergic terminals without apparent loss of the cell bodies. Desipramine pretreatment prevented significant effects of 5,7-DHT on noradrenergic neurons.

The effects of early hypo- and hyperthyroidism on the rates of cell acquisition and proliferation have been studied in the external granular layer (EGL) of the developing rat cerebellar cortex at 10 days of age using quantitative autoradiographic methods. Both altered thyroid states reduce the rate of cell acquisition in the EGL, but appear to do so for different reasons. Hyperthyroidism shortens the average length of the cell cycle by decreasing the duration of the pre-DNA synthetic phase (G1), indicating that excess thyroxine may exert a direct effect on the EGL. This action involves the early onset of neuronal differentiation (cessation of proliferation)46 which presumably leads to the observed decrease in the rate of cell acquisition (increased doubling time). Such differentiating cells do not, however, leave the proliferative zone or the EGL prematurely, resulting in a reduced labeling index, mitotic index, and growth fraction as non-dividing cells dilute the proliferating cell population. Hypothyroidism, on the other hand, leads to no significant change in the length of the cell cycle or in the mitotic index, but causes a decreased labeling index and growth fraction, as well as a reduced rate of cell acquisition (increased doubling time). No significant change in the amount of cell death in the EGL could be found to explain this apparent discrepancy between the rate of cell proliferation (cell cycle length) and cell acqusiition. The answer to this puzzle appears to lie in the mitotic index, which is not affected to the same extent as the labeling index, although it is also slightly reduced. If cells were to remain longer in mitosis, this could result in a decreased labeling index and growth fraction but nearly normal mitotic index and cell cycle length (as measured using the % labeled mitoses method), since those cells dropping out of the cycling population would be counted as mitoses...

The development of central serotonergic neurons has been examined immunocytochemically utilizing an antiserum to serotonin (5-HT). Cells of the B4-B9 complex are first detected early on embryonic day 13 (E13; 7 mm crown rump length, CRL) and increase rapidly in number through E15 when they appear as bilateral columns situated from just caudal to the mesencephalic flexure to the pontine flexure. Aggregation of cells into subgroups is apparent soon after 5-HT neurons leave the ventricular zone, allowing the identification of certain subdivisions of the B4-B9 complex long before they assume their adult locations. The initial detection of 5-HT immunoreactive cells in the medulla occurs 1-2 days after the appearance of cells in the B4-B9 complex, although it has been reported that the time of origin of medullary raphe neurons (B1-B3) occurs before that of raphe neurons in the midbrain and pons (B4-B9). The first medullary 5-HT neurons, comprising the B3 subdivision occur ventro-laterally on E14 (10-11 mm CRL) at least 1-2 days before midline 5-HT neurons are visualized in the B1 and B2 groups. Thus, in contrast to cells in the B4-B9 complex, medullary 5-HT neurons complete much of their migration before they can be detected immunocytochemically, indicating that the time of onset of transmitter synthesis and storage may differ during differentiation of cells sharing a common neurotransmitter phenotype. The formation of ascending 5-HT fiber projections occurs rapidly from cells of the B4-B9 complex. Within 24 hours after the initial detection of 5-HT fiber immunoreactivity in such cells at E13, their axons are seen entering the caudal diencephalon (E14). These fibers have traversed the diencephalon and floor of the telencephalon by E15-E16 and reach the frontal neocortical pole by E17. The main ascending bundle of 5-HT axons courses through the diencephalon in the vicinity of the medial forebrain bundle, although some fibers also diverge and travel along certain pre-existing non-5HT pathways. However, examples are also found of acute directional changes in 5-HT fiber growth which do not appear to be associated with pre-formed non-5HT pathways. The pattern of ascending fiber outgrowth suggests a priority routing system which provides certain regions with 5-HT axons in a preferential sequence irrespective of the distance of these areas from 5-HT cell groups or from major bundles of ascending 5-HT fibers.

The distribution of serotonin (5-HT) immunoreactive cells and their projections was mapped in the ganglia of the ventral nerve cord of the tobacco hornworm, Manduca sexta, during embryonic development, using an antiserum to 5-HT. Immunoreactive cells were first seen at 60% development. By 75% of embryonic development, a total of 94 immunoreactive cells were found in the ventral ganglia, including the suboesophageal ganglion. This number decreased to 80 neurons by 100% of development. About 50% of these cells were arranged in the abdominal ganglia and the rest were located in both the suboesophageal and thoracic ganglia. The suboesophageal ganglion exhibited immunoreactive segmental interneurons in areas corresponding to the mandibular, maxillary and labial neuromeres. Two pairs of immunoreactive interneurons were also observed to occur bilaterally in each of the thoracic and abdominal ganglia, with the exception of the prothoracic ganglion. This ganglion contained three pairs of bilaterally arranged immunoreactive neurons as early as 60% of embryonic development. Serotonin immunoreactivity was also found in a number of efferent neurons in the mandibular and labial neuromeres of the suboesophageal ganglion and in the prothoracic, mesothoracic and posterior abdominal ganglia. The occurrence of 5-HT in these efferent neurons suggests an involvement of serotonin in fore- and hindgut function via its effect on the visceral muscles. Immunoreactive lateral longitudinal fibers extended along the entire length of the ventral nerve cord together with dense segmental arborizations. The latter had regressed by the time the embryo was fully developed. This regression of the arborizations in the ganglia at the end of embryonic development indicates that a reorganization of 5-HT innervation occurs to support new larval functions. The time of appearance of 5-HT immunoreactive cells and fibers suggests that serotonin may play a role in the development of the ventral nerve cord.

The neuroanatomical distribution of bombesin-like immunoreactivity (BLI) in the rat central nervous system was investigated using radioimmunoassay and immunohistochemistry. Whereas cross-reactivity of the bombesin antiserum with substance P was problematic in the immunohistochemical experiments, no significant cross-reactivity with substance P was apparent in the radioimmunoassay. Results from the radioimmunoassay studies reveal particularly high concentrations of BLI in the hypothalamus, thalamus, medulla and spinal cord. Adult rats treated neonatally with capsaicin displayed significant depletions of somatostatin-like and substance P-like immunoreactivity and a small, statistically significant, reduction of BLI in the cervical spinal cord. Capsaicin treatment significantly reduced substance P-like immunoreactivity, but not somatostatin-like immunoreactivity, in the medulla and resulted in a small BLI depletion of borderline statistical significance in this brain region. Neonatally administered capsaicin treatment had no effect on the thalamic concentration of any of these three neuropeptides and neurotensin-like immunoreactivity was unchanged in all brain regions studied. These results suggest that the source of some of the BLI found in the spinal cord may be capsaicin-sensitive dorsal root ganglion cells.

Contrary to previous assumptions, we have found that a single dose of 3H-thymidine (25 muCi), injected into the yolk sac of White Leghorn chick eggs on 2 days of incubation (d.i.) only remains available for DNA-synthesizing (proliferating) cells for 48 hours following the time of injection. This finding now makes it possible to date the time of neuronal origin in the avian embryo using a single injection of isotope and a long survival time (30 days posthatch) as in mammalian studies where 3H-thymidine is only available as a short "pulse." Using this method, we have determined that neurons in the chick locus coeruleus (LC) cease proliferation on 2-6 d.i. with a peak of neuronal genesis on 3-5 d.i. In addition, neuronal genesis is not homogeneous throughout the LC cell population, but occurs in a predominantly caudorostral gradient. Conversely, the cerebellar Purkinje cells cease division on 3-8 d.i. with a peak of heavy labeling on 4-6 d.i., 1 day later than that observed in the LC.

With the method of whole mouse embryo culture, together with immunocytochemistry with an antiserum to serotonin (5-HT), sites of 5-HT uptake were found to be transiently expressed in the epithelia of the developing palate, tongue, nasal septum, and maxillary and mandibular prominences during the period of active morphogenesis (embryonic days 12-14; or E12-14). These sites had the ability to take up 5-HT when added to the culture medium in the presence of the MAO inhibitor nialamide and an antioxiant, L-cysteine (NC), and could also be seen after exposure of embryos to the 5-HT precursor L-tryptophan (L-TRP) + NC. These sites were also visible after culturing embryos without any additives, which may have been due to the presence of L-TRP in one component of the culture medium (DMEM) or to 5-HT itself, which is present in relatively high amounts in fetal calf serum. At E12-13, the appearance of 5-HT immunoreactivity (IR) at these sites after treatment with 5-HT + NC was blocked by the 5-HT uptake inhibitor fluoxetine, providing further evidence that these are true sites of 5-HT uptake. However, fluoxetine did not completely block the appearance of these sites in E14 embryos after 5-HT + NC or L-TRP + NC although it was effective with NC alone. This finding could mean that at E14 5-HT uptake into these sites occurs by mechanisms not completely blocked by fluoxetine or that there is some limited capacity for 5-HT synthesis. Taken together with results from previous studies where 1) 5-HT has been reported to stimulate palatal shelf reorientation and palatal mesenchyme cell motility in vitro [Wee et al., J Embryol Exp Morphol 53:75-90, 1979; Zimmerman et al., J Craniofac Genet Dev Biol 3:371-385, 1983] and 2) long-term culturing of mouse embryos in the presence of 5-HT or fluoxetine has been shown to cause malformations of the craniofacial region (Lauder, Thomas, and Sadler, in preparation), the results of the present study suggest that 5-HT could act as a developmental signal in the palate, oral cavity, and face during the period of active morphogenesis.