DNA microarray analysis and functional profile of pituitary transcriptome under core-clock protein BMAL1 control.

Although it is known to contain five cell types that synthesize and release hormones with a circadian pattern, the pituitary gland is poorly characterized as a circadian oscillator. By a differential microarray analysis, 252 genes were found to be differentially expressed in pituitaries from Bmal1(-/-) knockout versus wild-type mice. By integrative analyses of the data set with the Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources annotation analysis system, pituitary genes with altered expression in Bmal1(-/-) mice were dispatched among functional categories. Clusters of genes related to signaling and rhythmic processes as well as transcription regulators, in general, were found enriched in the data set, as were pathways such as circadian rhythm, transforming growth factor ß (TGFß) signaling, valine, leucine, and isoleucine degradation, and peroxisome proliferator-activated receptor (PPAR) signaling pathways. Gene Ontology term overrepresentation analyses revealed significant enrichment for genes involved in 10 key biological processes. To determine whether genes with altered expression in Bmal1(-/-) mice were actually circadian genes, we further characterized in the mouse pituitary gland the daily pattern of some of these genes, including core-clock genes. Core-clock genes and genes selected from three identified overrepresented biological processes, namely, hormone metabolic process, regulation of transcription from RNA polymerase II promoter, and cell adhesion, displayed a rhythmic pattern. Given the enrichment in genes dedicated to cell adhesion and their daily changes in the pituitary, it is hypothesized that cell-cell interactions could be involved in the transmission of information between endocrine cells, allowing rhythmic hormone outputs to be controlled in a temporally precise manner.

Vitamin A is a necessary factor for sympathetic-independent rhythmic activation of mitogen-activated protein kinase in the rat pineal gland.

The circadian clock in the suprachiasmatic nucleus (SCN) controls day-to-day physiology and behavior by sending timing messages to multiple peripheral oscillators. In the pineal gland, a major SCN target, circadian events are believed to be driven exclusively by the rhythmic release of norepinephrine from superior cervical ganglia (SCG) neurons relaying clock messages through a polysynaptic pathway. Here we show in rat an SCN-driven daily rhythm of pineal MAPK activation that is not dependent on the SCG and whose maintenance requires vitamin A as a blood-borne factor. This finding challenges the dogma that SCG-released norepinephrine is an exclusive mediator of SCN-pineal communication and allows the assumption that humoral mechanisms are involved in pineal integration of temporal messages.

Adrenergic inducibility of AP-1 binding in the rat pineal gland depends on prior photoperiod.

The main known function of the pineal gland in mammals is the temporal synchronization of physiological rhythms to seasonal changes of day length (photoperiod). In rat, the transcription factor activating protein-1 (AP-1) displays a circadian rhythm in its DNA binding in the pineal gland, which results from the rhythmic expression of Fra-2. We postulated that, if AP-1 is an important component of pineal gland functioning, then variations in photoperiodic conditions should lead to an adaptation of the AP-1 binding rhythm. Here we show that AP-1 binding patterns adapt to variations in lighting conditions, in the same way as the rhythm of arylalkylamine-N-acetyltransferase (AA-NAT) activity. This adaptation appeared to result from photoperiodic adaptation of the rhythmic fra-2 gene expression and was reflected by an adapted delay between the onset of night and the acrophase of the nocturnal peak. We further showed that photoperiodic adaptation of both the AP-1 binding and AA-NAT activity rhythms resulted from adapted changes in adrenergic inducibility of both variables at night onset. We finally provided evidence that AP-1 shared with the CREM gene encoding the transcriptional repressor protein inducible cAMP early repressor (ICER) the ability to be hypersensitive or subsensitive to adrenergic stimuli, depending on prior photoperiod.

Long-term variations of AP-1 composition after CRH stimulation: consequence on POMC gene regulation.

It has been shown previously that the CRH-induced POMC gene transcription in the corticotroph cell line AtT-20 involves an increase in AP-1 DNA binding activity that remained elevated for at least 24 h, while induction of c-fos was transient. We showed here that there were dramatic changes in protein components of AP-1 including an initial recruitment of the transcriptional activators c-Fos and Jun-B then of Fra-2 and Jun-D. Changes in AP-1 composition were concomitant with a decrease in POMC mRNA. Moreover, the presence of Fra-2/Jun-D dimers suppressed the CRH-induction of c-fos mRNA expression as well as c-Fos/Jun-B recruitment in AP-1 complexes, suggesting the existence of autoregulatory loops of AP-1 composition that involve complex interactions between the different members of the Jun and Fos families. It is concluded that CRH stimulation of corticotroph cells involves successive recruitment of activators and repressors, possibly contributing to prevent over expression of POMC.

Circadian binding activity of AP-1, a regulator of the arylalkylamine N-acetyltransferase gene in the rat pineal gland, depends on circadian Fra-2, c-Jun, and Jun-D expression and is regulated by the clock's zeitgebers.

The daily rhythm in circulating melatonin is driven by a circadian rhythm in the expression of the arylalkylamine N:-acetyltransferase gene in the rat pineal gland. Turning off expression of this gene at the end of night is believed to involve inhibitory transcription factors, among which Fos-related antigen 2 (Fra-2) appears as a good candidate. Circadian rhythms in the expression of three proteins of activating protein-1 (AP-1) complexes, namely, Fra-2, c-Jun, and Jun-D, are shown here to account for circadian variations in AP-1 binding activity. Quantitative variations in the Fra-2 component over the circadian cycle were associated with qualitative variations in protein isoforms. Destruction of the suprachiasmatic nucleus resulted in decreased nocturnal AP-1 activity, showing that AP-1 circadian rhythm is driven by this nucleus. Exposure to light during subjective night and administration of a serotonin 5-HT(1A)/5-HT(7) receptor agonist during subjective day, respectively, induced a 50% decrease and a 50% increase in both AP-1 and Fra-2 expression. These effects were impaired by suprachiasmatic nucleus lesions. These data show that pineal AP-1 binding activity, which results from Fra-2 expression, can be modulated by light and serotonin through the suprachiasmatic nucleus according to a "phase dependence" that is characteristic of the rhythm of clock sensitivity to both zeitgebers.

Is light-regulated AP-1 binding in the rat suprachiasmatic nucleus gated by the circadian clock?

In mammals, photic entrainment of circadian rhythms likely involves light- and clock-dependent expression of immediate early genes, including fos-like and jun-like genes, in the rat suprachiasmatic nucleus. Using an electrophoretic mobility shift assay, we evaluated whether the photic regulation of DNA-binding activity and composition of activating protein-1 (AP-1) complexes in the suprachiasmatic nucleus is also dependent on circadian phase. Phase-dependent light inducibility in the expression of fra-2 and c-fos genes and in immunoreactive Fra-2 and c-Fos protein expression was also evaluated, by in situ hybridization and immunocytochemistry. Light's effects on AP-1 DNA-binding differed both qualitatively and quantitatively according to the circadian phase at which light was applied. This phase dependence accounted for by both compartmentalization of proteins involved in constitutive AP-1 complexes within the nucleus or cytoplasm and control of the extent to which the expression of specific complexes was induced. It was then shown that the mechanisms by which the circadian clock gates the photic induction of AP-1 components differed according to the nature of the protein.

Identification and role of serotonin 5-HT1A and 5-HT1B receptors in primary cultures of rat embryonic rostral raphe nucleus neurons.

Autoregulatory mechanisms affecting serotonin [5-hydroxytryptamine (5-HT)] release and synthesis during the early period of development were investigated in dissociated cell cultures raised from embryonic rostral rat rhombencephalon. The presence of 5-HT1A and 5-HT1B receptors in serotoninergic neurons was assessed using binding assays. The involvement of 5-HT1A and 5-HT1B receptors in the control of the synthesis and release of [3H]5-HT was studied using biochemical approaches with several serotoninergic receptor ligands. A mean decrease of 30% in [3H]5-HT synthesis and release was observed in the presence of 5-HT (10(-8) M), the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), the 5HT1B/1A agonist 5-methoxy-3-(1,2,5,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969), the 5-HT1B agonist 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one (CP-93,129), and the 5-HT(1D/1B) agonist sumatriptan. Inhibition of 5-HT synthesis and release induced by 8-OH-DPAT was blocked by chiral N-tert-butyl-3-[1-[1-(2-methoxy)phenyl]piperazinyl]-1-phenylpropionam ide dihydrochloride quaternary-hydrate (WAY 100135) (10(7) M) or methyl 4-[4-[4-(1,1,3-trioxo-2H-1,2-benzoisothiazol-2-yl)butyl]-1-p iperazinyl]-1Hindole-2-carboxylate (SDZ 216-525) (10(-7)M), and that of CP-93,129 was blocked by methiothepin (10(-7) M). Paradoxically, extracellular levels of [3H]5-HT increased in the presence of 8-OH-DPAT and RU 24969 at 10(-6) M. 5-HT uptake experiments showed that these two agonists interacted with the 5-HT transporter. 5-HT1 binding sites (620 fmol/mg of protein) and 5-HT1A (482 fmol/mg of protein) and 5-HT1B (127 fmol/mg of protein) receptors were detected in 12-day in vitro cell cultures. Experiments carried out with tetrodotoxin suggested that 5-HT1A receptors are located on nerve cell bodies, whereas 5-HT1B receptors are located on the nerve terminals. We concluded that autoregulatory mechanisms involving 5-HT1A and 5-HT1B autoreceptors are functionally mature in cells from rostral raphe nuclei during the early period of development.

Light-induced variations in AP-1 binding activity and composition in the rat suprachiasmatic nucleus.

Expression of immediate early genes, including fos-like and jun-like genes, in the suprachiasmatic nucleus is believed to be part of the mechanism for photic entrainment of circadian rhythms to the environmental light/dark cycle. However, the effects of a light stimulus on activating protein-1 (AP-1) complexes in the suprachiasmatic nucleus remain unclear. The photic regulation of AP-1 DNA-binding activity and composition in the rat suprachiasmatic nucleus was evaluated by using an electrophoretic mobility shift assay. A light pulse given during subjective night induced an increase in AP-1 binding activity when either nuclear or whole-cell extracts from suprachiasmatic nuclei were used. Under constant dark conditions, proteins that are predominant components of AP-1 complexes are Fra-2 and Jun-D. Under light stimulation, c-Fos and Jun-B consistently increased, as expected, but this was also the case for Fra-2, Jun-D, and c-Jun, although to a lesser extent. An immunocytochemical study of the Fra-2 expression pattern demonstrated the presence of the protein in the ventrolateral as well as in the dorsomedial subdivisions of the suprachiasmatic nucleus. Light regulation of Fra-2 immunoreactivity, however, appeared to be restricted to the ventrolateral subdivision. It is concluded that light may be acting both by increasing constitutive AP-1 complexes and by inducing the expression of specific complexes.

Post-lesion up-regulation of 5-HT1B binding sites in the suprachiasmatic nucleus may be reversed after spontaneous or graft-induced serotonin reinnervation.

We have previously reported that selective axotomy of serotoninergic neurons produced by an intraventricular injection of 5, 7-dihydroxytryptamine is followed by an increase in 5-HT1B binding sites in the suprachiasmatic nucleus of the hypothalamus. This post-lesion up-regulation is shown here to be spontaneously reversed after long-term survival in spite of an incomplete reinnervation of the nucleus. Recovery may be accelerated by fetal raphe transplants that produce more rapid reinnervation.

Serotonin directly stimulates luteinizing hormone-releasing hormone release from GT1 cells via 5-HT7 receptors.

Luteinizing hormone-releasing hormone (LHRH release, which serves as the primary drive to the hypothalamic-pituitary gonadal axis, is controlled by many neuromediators. Serotonin has been implicated in this regulation. However, it is unclear whether the central effect of serotonin on LHRH secretion is exerted directly on LHRH neurosecretory neurons or indirectly via multisynaptic pathways. The present studies were undertaken in order to examine whether LHRH secretion from immortalized LHRH cell lines is directly regulated by serotonin and, if so, to identify the receptor subtype involved. 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a 5-HT1A/7 receptor agonist, stimulated LHRH release from GT1-1 cells. This effect was blocked by ritanserin, a 5-HT2/7 receptor antagonist, but not by SDZ-216-525, a 5-HT1A antagonist. Basal LHRH release was not affected by the 5-HT2 agonist DOI. Reverse transcription and polymerase chain reaction technique (RT-PCR) was used in order to identify 5-HT1A and 5-HT7 receptor mRNA in immortalized LHRH cell lines. GT1-1 cells express mRNA for the 5-HT7, but not the 5-HT1A receptor subtypes. These results demonstrate a direct stimulatory effect of serotonin on LHRH release via 5-HT7 receptor.

Direct evidence for the link between monoaminergic descending pathways and motor activity. I. A study with microdialysis probes implanted in the ventral funiculus of the spinal cord.

Monoaminergic projections to the spinal cord are involved in the modulation of motor, autonomic, and sensory functions. More specifically, the increase of electrical activity of serotonergic neurons in raphe obscurus has been correlated with locomotion in treadmill-trained cats [Jacobs, B.L. and Fornal, C., Trends Neurosci., 9 (1993) 346-352]. In order to test the direct correlation between locomotion and the release of monoamines, microdialysis probes were permanently implanted for 45 days into the ventral funiculus of the spinal cord (white matter) of adult rats. Eight days after implantation, these rats were subjected to an endurant exercise on a treadmill, and dialysis sessions were organized in such a way that microdialysate samples of 15 min duration were collected during pre-, per- and post-exercise periods. Measurements of serotonin, 5-hydroxyindoleacetic acid, dopamine and 3-methoxy-4-hydroxyphenylethylglycol concentration in the extracellular space showed significant increases during locomotion when compared with both pre- and post-exercise values. Histological analysis shows that serotonergic axons were present close to the dialysis probe. These results demonstrate that the implantation of a microdialysis probe in the ventral funiculus, close to a potential target of monoaminergic projections, is a suitable technique for the collection of neuromediators released during spontaneous running.

Stimulatory effects of 5HT1A receptor agonists on luteinizing hormone-releasing hormone release from cultured fetal rat hypothalamic cells: interactions with progesterone.

Previous works have suggested an interactive stimulatory effect of progesterone (P) and serotonin (5-HT) on luteinizing hormone release. The purpose of the present study was to determine whether 5-HT via 5-HT1A receptors interacts with P in the process of luteinizing hormone-releasing hormone (LHRH) release. Using fetal hypothalamic neurons in primary cell cultures the first goal of this study was to determine the effects of 5-HT1A receptor agonists on LHRH secretion. 8-Hydroxy-2 (di-n-propylamino) tetralin (8-OH-DPAT) or ipsapirone (10(-5) M) significantly stimulated LHRH release. Pharmacological studies have allowed to rule out the possible involvement of alpha 2- or beta-adrenoreceptors, or 5-HT uptake sites, in the stimulatory effect of 8-OH-DPAT on LHRH release, thus demonstrating the specific involvement of 5-HT1A receptors in the stimulation of LHRH release. The second goal was to test the ability of P to stimulate LHRH release from fetal hypothalamic neurons. P (10(-6) M) applied for 30 or 120 min significantly stimulated LHRH secretion. The maintenance of the stimulation of LHRH release by P after a cycloheximide treatment or by an impermeable analog of P, P-3-BSA, has suggested a nongenomic effect of P on LHRH release. The effects of a pretreatment of cells by P on 8-OH-DPAT-induced LHRH release were tested. While 10(-7) M P alone did not stimulate LHRH release, this concentration of steroid potentiated the LHRH response to 10(-5) M 8-OH-DPAT. These findings led to the conclusion that P acting at the level of the plasma membrane potentiates the stimulatory effect of 5-HT1A receptor agonists on LHRH release.

Impairment of serotoninergic transmission is followed by adaptive changes in 5HT1B binding sites in the rat suprachiasmatic nucleus.

Serotonin1B (5-HT1B) receptor binding in the suprachiasmatic nucleus (SCN) following impairment of serotoninergic transmission was studied by quantitative autoradiography. Serotonin (5-HT) denervation with 5,7-dihydroxytryptamine (5,7-DHT) caused a significant increase in the density of 5-HT1B receptors in both the ventral (62%) and dorsal (53%) parts of the SCN as early as 3 days after axotomy. The magnitude of this increase did not differ 3, 15 or 21 days post-lesion. An up-regulation of 5-HT1B receptors with similar magnitude was obtained in the two parts of the SCN after inhibition of 5-HT synthesis by chronic parachlorophenylalanine treatment. In this case, up-regulation was shown to be reversible after restoration of 5-HT synthesis with L-5-hydroxytryptophan. These results indicate that 5-HT1B receptor density in the SCN was inversely correlated with 5-HT levels. These plastic properties exhibited by 5-HT1B receptors in the SCN are discussed in relation to the mode of 5-HT transmission and possible localization of the receptors onto the main chemically defined cell populations of the nucleus.

N-methyl-D-aspartic acid/glycine interactions on the control of 5-hydroxytryptamine release in raphe primary cultures.

Glutamic acid and glycine were quantified in cells and medium of cultured rostral rhombencephalic neurons derived from fetal rats. In the presence of 1 mM Mg2+, NMDA (50 microM) significantly stimulated (by 69%) release of newly synthesized 5-[3H]hydroxytryptamine ([3H]5-HT). D-2-Amino-5-phosphonopentanoate (AP-5; 50 microM) blocked the stimulatory effect of NMDA. AP-5 by itself inhibited [3H]5-HT release (by 25%), suggesting a tonic control of 5-HT by glutamate. In the absence of Mg2+, basal [3H]5-HT release was 60% higher as compared with release with Mg2+. AP-5 blocked the increased [3H]5-HT release observed without Mg2+, suggesting that this effect was due to the stimulation of NMDA receptors by endogenous glutamate. Glycine (100 microM) inhibited [3H]5-HT release in the absence of Mg2+. Strychnine (50 microM) blocked the inhibitory effect of glycine, indicating an action through strychnine-sensitive inhibitory glycine receptors. The [3H]5-HT release stimulated by NMDA was unaffected by glycine. In contrast, when tested in the presence of strychnine, glycine increased NMDA-evoked [3H]5-HT release (by 22%), and this effect was prevented by a selective antagonist of the NMDA-associated glycine receptor, 7-chlorokynurenate (100 microM). 7-Chlorokynurenate by itself induced a drastic decrease in [3H]5-HT release, indicating that under basal conditions these sites were stimulated by endogenous glycine. These results indicate that NMDA stimulated [3H]5-HT release in both the presence or absence of Mg2+. Use of selective antagonists allowed differentiation of a strychnine-sensitive glycine response (inhibition of [3H]5-HT release) from a 7-chlorokynurenate-sensitive response (potentiation of NMDA-evoked [3H]5-HT release).

Glutamate, GABA, glycine and taurine modulate serotonin synthesis and release in rostral and caudal rhombencephalic raphe cells in primary cultures.

Control of serotonin release and synthesis by amino acid neurotransmitters was investigated in rat rostral and caudal rhombencephalic raphe cells in primary cultures respectively. Endogenous amounts of taurine, glycine, GABA and glutamate were measured in both types of cultures. These amino acids were spontaneously released to the incubating medium. Exogenous taurine (10(-4) M) inhibited release and synthesis of newly formed [3H]serotonin [3H]5-HT from [3H]-tryptophan only in rostral raphe cells. Glycine (10(-3) M) decreased [3H]5-HT release in both types of cells, synthesis being diminished only in rostral raphe cells. Glycine inhibitory effect was totally blocked by strychnine (5 x 10(-5) M). GABA (10(-4) M) reduced [3H]5-HT metabolism in rostral as well as caudal raphe cells. This effect was totally antagonized in caudal and partially in rostral raphe cells by bicuculline (5 x 10(-5) M) a GABAA receptor antagonist. Baclofen (5 x 10(-5) M), a GABAB receptor agonist, induced a decrease of 5-HT release in rostral raphe cells. These observations suggest that monoamine release was entirely mediated by GABAA receptors in caudal raphe cells although GABAA and GABAB receptors were involved in control of 5-HT metabolism in rostral raphe cells. L-glutamate (10(-4) M) stimulated 5-HT metabolism in both types of cells, effect totally blocked by PK26124 (10(-6) M). N-methyl-D-aspartate (10(-4) M) enhanced 5-HT metabolism and the induced-effect was antagonized by the selective N-methyl-D-aspartate receptor antagonist D,L-2 amino-5-phosphonovaleric acid. Quisqualate (10(-5) M) stimulated [3H]5-HT release only in caudal raphe cells. This effect was mimicked by (RS)-a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, a quisqualate "ionotropic" receptor agonist, this increase being blocked by 6,7-dinitroquinoxaline 2,3-dione. These observations suggest that the glutamate stimulating-induced effect on serotonin metabolism is entirely mediated by N-methyl-D-aspartate receptor-type in rostral raphe cells and that quisqualate "ionotropic" receptors are also involved in caudal raphe cells. Taken together these results show that [3H]5-HT metabolism is controlled by taurine, glycine, GABA and glutamate in rhombencephalic raphe cells in primary cultures. However, some difference in amino acid receptor-types involved in the control of serotonin metabolism are observed according to the rostral or caudal origin of raphe cells.

Effect of diabetes on in vivo and in vitro hypothalamic somatostatin release.

In order to better understand the mechanisms underlying the reduction in growth hormone (GH) secretion in diabetic rats, we studied hypothalamic somatostatin secretion both in vivo (into hypophysial portal blood) and in vitro (from hypothalamic fragments) 5, 9 and 30 days after induction of diabetes. Experimental diabetes was induced by an intraperitoneal injection of streptozotocin (STZ) at a dose of 65 mg/kg. Basal plasma GH was significantly reduced in diabetic rats at all stages. Somatostatin levels in hypophysial portal blood was unaffected in 5-day STZ-diabetic rats and significantly increased 9 days after STZ administration. Chronic insulin replacement therapy in diabetic animals partly normalized somatostatin levels as well as plasma GH and glucose levels. A good correlation was observed between in vivo and in vitro experiments. Indeed, somatostatin release from hypothalamic fragments did not change 5 days after STZ-induced diabetes and significantly increased 9 and 30 days after STZ administration. The in vitro increase in hypothalamic somatostatin secretion was observed in 10 as well as in 33 mM glucose concentration in the incubation medium. In the same experiment, the in vitro hypothalamic corticotropin-releasing factor secretion was lowered 5 and 9 days after diabetes induction. We conclude that hypothalamic somatostatin release increases in diabetic rats. These changes may contribute to the reduction in GH secretion in these animals. However, since these changes occur after the onset of plasma GH decrease, a factor(s) other(s) than somatostatin may play a causal role in the reduction in GH secretion.

Serotonin synthesis in adrenochromaffin cells.

The inter-renal (adrenal) gland of amphibians is composed of chromaffin and steroidogenic cells which can interact through a paracrine mode of communication. We have previously shown that serotonin is present in secretory granules of frog adrenochromaffin cells; concurrently, we have demonstrated that serotonin is a potent stimulator of corticosterone and aldosterone secretion by adrenocortical cells. The aim of the present study was to determine the origin of the amine contained in frog chromaffin cells. Using 3H-labelled tryptophan as a precursor, we observed the formation of substantial amounts of serotonin and its metabolite 5-hydroxyindoleacetic acid by frog inter-renal slices. Newly synthesized serotonin was secreted into the incubation medium and the release process was enhanced by depolarizing concentrations of KCl. Fluoxetine, and inhibitor of serotonin uptake, caused an increase of 3H-labelled serotonin in the incubation medium, suggesting that the indoleamine was taken up again by adrenal chromaffin cells. The capacity of the frog inter-renal gland to synthesize serotonin was also demonstrated by incubating inter-renal slices with non-labelled tryptophan or 5-hydroxytryptophan. In these conditions, we observed that the rate of synthesis was higher when 5-hydroxytryptophan was used as a a precursor, rather than tryptophan. Taken together, these results indicate that chromaffin cells, which have the capacity for synthesizing and releasing serotonin, behave like authentic serotonergic paraneurons. As far as is known, these data provide the first evidence for the occurrence of tryptophan-5-hydroxylase activity within the adrenal gland.

Population-specific modulation of 5-HT expression in cultures of embryonic rat rhombencephalon.

This study aimed at analyzing the regulation of in vitro serotonin expression by neurons taken from different regions of the embryonic rat rhombencephalon. We studied the influence of co-culture with alarplate tissue using immunocytochemical and biochemical methods. Computer-assisted densitometry was used to estimate the co-culture effects on the serotonin content of the cell bodies. The more dynamic aspects of serotonin expression, such as synthesis and release, were studied by measuring (3H)serotonin newly synthesized from (3H)tryptophan. The density of the immunostaining was significantly decreased in B1,B2 cells by co-culture with both caudal and rostral alar-plate tissue. For B4-B9 cells, only co-culture with rostral alar-plate tissue produced a significant decrease. The de novo synthesis of serotonin was significantly decreased in B1,B2 neurons co-cultured with caudal alar-plate tissue only. Once again, the B4-B9 cells proved to be less influenced by the experimental conditions, as co-culture with both types of alar-plate tissue produced no significant effect. We concluded that the in vitro expression of serotonin can be modulated by environmental factors, but the relative influence of these factors is very different in rostral versus caudal serotonin expressing cell populations.