Number Of Serotonergic Neurons Research Articles

Approach/Avoidance Behavior to Novel Objects is Correlated with the Serotonergic and Dopaminergic Systems in the Brown Rat (Rattus norvegicus)

The brown rat (Rattus norvegicus) is known to show three types of behavioral responses to novel objects. Whereas some rats are indifferent to novel objects, neophobic and neophilic rats show avoidance and approach behavior, respectively. Here, we compared the dopaminergic, serotonergic, and noradrenergic systems immunohistochemically among these rats. Trapped wild rats and laboratory rats were first individually exposed to the novel objects in their home cage. Wild rats were divided into neophobic and indifferent rats depending on their behavioral responses. Similarly, laboratory rats were divided into neophilic and indifferent rats. Consistent with the behavioral differences, in the paraventricular nucleus of the hypothalamus, Fos expression in corticotropin-releasing hormone-containing neurons was higher in the neophobic rats than in the indifferent rats. In the anterior basal amygdala, the neophobic rats showed higher Fos expression than the indifferent rats. In the posterior basal amygdala, the neophobic and neophilic rats showed lower and higher Fos expressions than the indifferent rats, respectively. When we compared the neuromodulatory systems, in the dorsal raphe, the number of serotonergic neurons and Fos expression in serotonergic neurons increased linearly from neophobic to indifferent to neophilic rats. In the ventral tegmental area, Fos expression in dopaminergic neurons was higher in the neophilic rats than in the indifferent rats. These results demonstrate that approach/avoidance behavior to novel objects is correlated with the serotonergic and dopaminergic systems in the brown rat. We propose that the serotonergic system suppresses avoidance behavior while the dopaminergic system enhances approach behavior to novel objects.

Chronic exposure of female mice to selective serotonin reuptake inhibitors during lactation induces vocal behavior deficits in pre-weaned offspring

Developmental factors for autism spectrum disorders (ASDs) have been an ongoing debate despite an increasing number of reports on genetic factors. Recent studies have suggested maternal intake of selective serotonin reuptake inhibitors (SSRIs) as a possible developmental factor elevating the risk for ASD in offspring. Here, we show that maternal exposure of mice to an SSRI, Fluoxetine (FLX), induces abnormal ultrasonic vocalizations (USVs), an indicator of ASD-related behavior. We tested the effect of FLX intake during pregnancy, lactation, or both. We found that the lactation and both conditions decreased the number of USVs emitted by offspring pups. An index for assessing the syllables' frequency modulation revealed that highly modulated syllables appeared to be inhibited only in both conditions. Furthermore, we found that the number of serotonergic neurons at adulthood was reduced in the progeny of mice treated with FLX in all conditions. In addition, maternal exposure to FLX through pregnancy and lactation induced a high death rate of early post-natal pups. These suggest that the maternal exposure to SSRIs affects early development of offsprings as well as the serotonergic system. Focusing on vocal communication, our results indicate that intake of an SSRI during lactation increases the risk of abnormal USVs in pups, and provides potential insights into the development of ASD.

Intra-uterine diazepam exposure decreases the number of catecholaminergic and serotoninergic neurons of neonate rats

Benzodiazepines, such as diazepam (DZP), are used to treat anxiety disorders, and are prescribed to pregnant woman for therapeutic purposes. Concerns regarding their consequences on postnatal development rise as they cross the placenta and interact with the embryo. Occurrence of malformation and behavioral syndromes have been reported for different ages, but little is known about their effects on the brain after exposure during intrauterine life. Thus, we sought to evaluate the effects of intrauterine exposure to DZP on the number of brainstem’s catecholaminergic and serotonergic neurons, implicated in respiratory control, in male and female rats on postnatal (P) day 12–13, using immunofluorescence labeling for tyrosine–hydroxylase (TH) and serotonin (5–HT). We observed a reduction in the number of catecholaminergic neurons for males and females. Special attention is given to the reduction in the density of neurons in the A6 region, involved in ventilatory responses to CO2. Interestingly, only males showed a reduction in the number of serotonergic neurons, while females were not affected. These findings suggest that in utero exposure to DZP results in deleterious neuroanatomical effects on P12–13 rats and raises a note of concern for women clinicians to make more informed choices about the use of anxiolytic treatments during gestation.

Diabetes mellitus causes changes in the activity and expression of brain tryptophan-5-hydroxylases and in the number of serotonergic neurons, which do not return to normal with insulin treatment.

Diabetes mellitus (DM) inhibits brain serotonin biosynthesis through changes in tryptophan-5-hydroxylase (TPH) activity and expression. To determine whether DM-induced changes in brain TPH1 or TPH2 expression and in the number of serotonergic neurons return to normal in diabetic rats treated with insulin. Rats with streptozotocin-induced diabetes were divided in two groups: one treated with insulin and the other without treatment. On day 14, brain stems were obtained in order to quantify L-tryptophan and 5-hydroxytryptamine levels, as well as to determine TPH activity. The expression of TPH1 and TPH2 by West-ern blot, and the number of serotonergic neurons by immunohistochemistry. In diabetic rats, a decrease in the levels of L-tryptophan, 5-hydroxytryptamine, and TPH activity was confirmed, as well as lower TPH1 and TPH2 expression and lower numbers of serotonergic neurons. When diabetic rats were treated with insulin, L-tryptophan returned to normal, but not 5-hy-droxytryptamine, TPH expression, or the number of serotonergic neurons. DM chronically inhibits the synthesis of brain 5-hydroxytryptamine through changes in TPH1 and TPH2 expression and a decrease in the number of serotonergic neurons, which persist despite insulin treatment.

Inactivation of the GATA Cofactor ZFPM1 Results in Abnormal Development of Dorsal Raphe Serotonergic Neuron Subtypes and Increased Anxiety-Like Behavior.

Serotonergic neurons in the dorsal raphe (DR) nucleus are associated with several psychiatric disorders including depression and anxiety disorders, which often have a neurodevelopmental component. During embryonic development, GATA transcription factors GATA2 and GATA3 operate as serotonergic neuron fate selectors and regulate the differentiation of serotonergic neuron subtypes of DR. Here, we analyzed the requirement of GATA cofactor ZFPM1 in the development of serotonergic neurons using Zfpm1 conditional mouse mutants. Our results demonstrated that, unlike the GATA factors, ZFPM1 is not essential for the early differentiation of serotonergic precursors in the embryonic rhombomere 1. In contrast, in perinatal and adult male and female Zfpm1 mutants, a lateral subpopulation of DR neurons (ventrolateral part of the DR) was lost, whereas the number of serotonergic neurons in a medial subpopulation (dorsal region of the medial DR) had increased. Additionally, adult male and female Zfpm1 mutants had reduced serotonin concentration in rostral brain areas and displayed increased anxiety-like behavior. Interestingly, female Zfpm1 mutant mice showed elevated contextual fear memory that was abolished with chronic fluoxetine treatment. Altogether, these results demonstrate the importance of ZFPM1 for the development of DR serotonergic neuron subtypes involved in mood regulation. It also suggests that the neuronal fate selector function of GATAs is modulated by their cofactors to refine the differentiation of neuronal subtypes.SIGNIFICANCE STATEMENT Predisposition to anxiety disorders has both a neurodevelopmental and a genetic basis. One of the brainstem nuclei involved in the regulation of anxiety is the dorsal raphe, which contains different subtypes of serotonergic neurons. We show that inactivation of a transcriptional cofactor ZFPM1 in mice results in a developmental failure of laterally located dorsal raphe serotonergic neurons and changes in serotonergic innervation of rostral brain regions. This leads to elevated anxiety-like behavior and contextual fear memory, alleviated by chronic fluoxetine treatment. Our work contributes to understanding the neurodevelopmental mechanisms that may be disturbed in the anxiety disorder.

Compensatory neuritogenesis of serotonergic afferents within the striatum of a transgenic rat model of Parkinson’s disease

The majority of patients with Parkinson’s disease (PD) suffer from L-DOPA-induced dyskinesia (LID). Besides a dysfunctional dopaminergic system, changes of the serotonergic network may be linked to this severe and adverse symptom. Particularly, serotonergic neurons have the potential to synthesize dopamine, likely associated with a disproportional dopamine release within the striatum. We hypothesized that the serotonergic system is adaptively altered in the striatum due to the reduced dopaminergic input. To answer this question, we analyzed a transgenic rat PD model ubiquitously expressing human α-synuclein using a bacterial artificial chromosome. Neurite analysis showed a profound loss of dopaminergic fibers by ~30–40% within the dorsal striatum paralleled by a ~50% reduction of dopaminergic neurons in the substantia nigra pars compacta. In contrast, serotonergic fibers showed an increased fiber density in the dorsal striatum by ~100%, while the number of serotonergic neurons within the raphe nuclei (RN) and its proximal neuritic processes were unaffected. Furthermore, both the dopaminergic and serotonergic fiber density remained unchanged in the neighboring motor cortex M1/M2. Interestingly, essential enzymes required for L-DOPA turnover and dopamine release were expressed in serotonergic neurons of the RN. In parallel, the serotonergic autoreceptor levels involved in a serotonergic negative feedback loop were reduced within the striatum, suggesting a dysfunctional neurotransmitter release. Overall, the increased serotonergic fiber density with its capacity for dopamine release within the striatum suggests a compensatory, site-specific serotonergic neuritogenesis. This maladaptive serotonergic plasticity may be linked to adverse symptoms such as LIDs in PD.

Damage to Arousal-Promoting Brainstem Neurons with Traumatic Brain Injury.

Coma and chronic sleepiness are common after traumatic brain injury (TBI). Here, we explored whether injury to arousal-promoting brainstem neurons occurs in patients with fatal TBI. Postmortem examination of 8 TBI patients and 10 controls. Compared to controls, TBI patients had 17% fewer serotonergic neurons in the dorsal raphe nucleus (effect size: 1.25), but the number of serotonergic neurons did not differ in the median raphe nucleus. TBI patients also had 29% fewer noradrenergic neurons in the locus coeruleus (effect size: 0.96). The number of cholinergic neurons in the pedunculopontine and laterodorsal tegmental nuclei (PPT/LDT) was similar in TBI patients and controls. TBI injures arousal-promoting neurons of the mesopontine tegmentum, but this injury is less severe than previously observed in hypothalamic arousal-promoting neurons. Most likely, posttraumatic arousal disturbances are not primarily caused by damage to these brainstem neurons, but arise from an aggregate of injuries, including damage to hypothalamic arousal nuclei and disruption of other arousal-related circuitries.

Severely impaired hippocampal neurogenesis associates with an early serotonergic deficit in a BAC α-synuclein transgenic rat model of Parkinson's disease

Parkinson's disease (PD) is a multisystem disorder, involving several monoaminergic neurotransmitter systems resulting in a broad range of motor and non-motor symptoms. Pathological hallmarks of PD are the loss of dopaminergic neurons and the accumulation of alpha-synuclein, however also being present in the serotonergic raphe nuclei early in the disease course. The dysfunction of the serotonergic system projecting to the hippocampus may contribute to early non-motor symptoms such as anxiety and depression. The adult hippocampal dentate gyrus (DG), a unique niche of the forebrain continuously generating new neurons, may particularly present enhanced susceptibility towards accumulating alpha-synuclein levels. The underlying molecular mechanisms in the context of neuronal maturation and survival of new-born neurons are yet not well understood. To characterize the effects of overexpression of human full-length alpha-synuclein on hippocampal cellular and synaptic plasticity, we used a recently generated BAC alpha-synuclein transgenic rat model showing important features of PD such as widespread and progressive alpha-synuclein aggregation pathology, dopamine loss and age-dependent motor decline. At the age of four months, thus prior to the occurrence of the motor phenotype, we observed a profoundly impaired dendritogenesis of neuroblasts in the hippocampal DG resulting in severely reduced survival of adult new-born neurons. Diminished neurogenesis concurred with a serotonergic deficit in the hippocampus as defined by reduced levels of serotonin (5-HT) 1B receptor, decreased 5-HT neurotransmitter levels, and a loss of serotonergic nerve terminals innervating the DG/CA3 subfield, while the number of serotonergic neurons in the raphe nuclei remained unchanged. Moreover, alpha-synuclein overexpression reduced proteins involved in vesicle release, in particular synapsin-1 and Rab3 interacting molecule (RIM3), in conjunction with an altered ultrastructural architecture of hippocampal synapses. Importantly, BAC alpha-synuclein rats showed an early anxiety-like phenotype consisting of reduced exploratory behavior and feeding. Taken together, these findings imply that accumulating alpha-synuclein severely affects hippocampal neurogenesis paralleled by impaired 5-HT neurotransmission prior to the onset of aggregation pathology and overt motor deficits in this transgenic rat model of PD.

Maternal viral infection during pregnancy impairs development of fetal serotonergic neurons.

Maternal viral infection during pregnancy induces morphological abnormalities in the fetus and may cause emotional and psychological problems in offspring through unknown mechanisms. We have previously shown that prenatal exposure of rats to chemicals such as thalidomide causes an autistic-like phenotype in offspring, indicating that prenatal events affecting serotonergic development may cause developmental disorder. We investigated whether prenatal viral infection altered the expression of neurotransmitters involved in the emotional or psychological status of offspring. We here took advantage of the polyriboinosinic:polyribocytidylic acid (poly I:C) system, the synthetic double-stranded RNA, which is often used in animal models of viral infection. Ten mg/kg of poly I:C was intraperitoneally injected on gestational day (GD) 9 and counted the numbers of serotonin-immunopositive cells on GD15 using flat whole-mount preparation method, resulting 11.1% of increase in the number of serotonergic neurons in poly I:C group. Furthermore, there was a significant decrease in hippocampal serotonin content in offspring by postnatal day 50 following poly I:C administration by high-performance liquid chromatography. Since serotonin is known to link with behavior and emotion after birth, these results suggest that maternal viral infection might cause, in addition to morphological abnormalities, serotonin-related pathogenesis such as neurodevelopmental disorders including autism spectrum disorders.

Acute noxious stimulation modifies morphine effect in serotonergic but not dopaminergic midbrain areas

It is poorly understood if and how pain may modify the effect of opioids on neural systems that contribute to reward and addictive behavior. We hypothesized that the activation of ascending dopaminergic and serotonergic nuclei by morphine is modified by the presence of noxious stimulation. Immunohistochemical double-labeling technique with Fos was used to examine if an intraplantar formalin injection, an acute noxious input, changed the effect of morphine on dopaminergic neurons of the ventral tegmental area (VTA), and serotonergic neurons of the dorsal raphe nucleus (DR). Four groups of rats were analyzed: (1) control injected with normal saline s.c., (2) rats treated with formalin into the hind paw 30 min after normal saline injection, (3) rats injected with morphine sulfate s.c., and (4) rats treated with formalin into the hind paw 30 min after morphine injection (morphine/formalin). Following morphine injection, there was an increase in the number of dopaminergic neurons in the VTA with Fos immunolabeling. However, noxious stimulation did not detectably change morphine's effect on Fos expression in VTA dopamine neurons. In contrast, the number of serotonergic neurons containing Fos was increased in the morphine/formalin group compared to all other groups and this effect was topographically selective for the dorsal area of the DR at mid rostro–caudal levels. Therefore, morphine's activation of the VTA, which is associated with motivated behavior and reward seeking, appears similar in the context of pain. However, activation of the ascending serotonin system, which influences mood and has the capacity to modify reward pathways, appears different. In addition, these findings reveal interactions between nociceptive signaling and opioids that contrasts with the notion that opioids simply block access of nociceptive signaling to supraspinal structures.

Development of serotonergic neurons of dorsal raphe nuclei in mice with knockout of monoamine oxidase a and 5-HT1A and 5-HT1B autoreceptor

The morphological changes in the development of serotonergic neurons of the dorsal raphe nuclei in the medulla oblongata was studied by immunocytochemistry in mice with knockout of 1A and 1B serotonin autoreceptors as well as monoamine oxidase A. Serotonin autoreceptors regulate electric activity of serotonergic neurons as well as the synthesis and release of the neurotransmitter, while monoamine oxidase A catalyzes its degradation. These genetic modifications proved to have no effect on the number of serotonergic neurons in the medulla oblongata but induced morphofunctional changes. Decreased cell size and increased intracellular serotonin level were observed in the case of monoamine oxidase A deficiency, while excessive cell size and decreased intracellular serotonin level were observed in the case of autoreceptor deficiency. The data obtained confirm the hypothesis of autoregulation of serotonergic neurons in development.

Genesis of gasping is independent of levels of serotonin in the Pet-1 knockout mouse

Eupnea is normal breathing. If eupnea fails, as in severe hypoxia or ischemia, gasping is recruited. Gasping can serve as a powerful mechanism for autoresuscitation. A failure of autoresuscitation has been proposed as a basis of the sudden infant death syndrome. In an in vitro preparation, endogenous serotonin is reported to be essential for expression of gasping. Using an in situ preparation of the Pet-1 knockout mouse, we evaluated such a critical role for serotonin. In this mouse, the number of serotonergic neurons is reduced by 85-90% compared with animals without this homozygous genetic defect. Despite this reduction in the number of serotonergic neurons, phrenic discharge in eupnea and gasping of Pet-1 knockout mice was not different from that of wild-type mice. Indeed, gasping continued unabated, even after administration of methysergide, a blocker of many types of receptors for serotonin, to Pet-1 knockout mice. We conclude that serotonin is not critical for expression of gasping. The proposal for such a critical role, on the basis of observations in the in vitro slice preparation, may reflect the minimal functional neuronal tissue and neurotransmitters in this preparation, such that the role of any remaining neurotransmitters is magnified. Also, rhythmic activity of the in vitro slice preparation has been characterized as eupnea or gasping solely on the basis of activity of the hypoglossal nerve or massed neuronal activities of the ventrolateral medulla. The accuracy of this method of classification has not been established.

Reduced number of serotonergic neurons and behavioral changes in rats prenatally exposed to ethanol

Reduced number of serotonergic neurons and behavioral changes in rats prenatally exposed to ethanol

A global view of gene expression in lithium and zinc treated sea urchin embryos: new components of gene regulatory networks

Novel territory-specific markers from the sea urchin Strongylocentrotus purpuratus have been identified using screens for genes that are differentially expressed in lithium-treated embryos, which form an excess of endomesoderm, and in zinc-treated embryos, in which endomesoderm specification is blocked.

Ontogeny of central serotonergic neurons in the directly developing frog, Eleutherodactylus coqui

Embryonic development of the central serotonergic neurons in the directly developing frog, Eleutherodactylus coqui, was determined by using immunocytochemistry. The majority of anuran amphibians (frogs) possess a larval stage (tadpole) that undergoes metamorphosis, a dramatic post-embryonic event, whereby the tadpole transforms into the adult phenotype. Directly developing frogs have evolved a derived life-history mode where the tadpole stage has been deleted and embryos develop directly into the adult bauplan. Embryonic development in E. coqui is classified into 15 stages (TS 1-15; 1 = oviposition/15 = hatching). Serotonergic immunoreactivity was initially detected at TS 6 in the raphe nuclei in the developing rhombencephalon. At TS 7, immunopositive perikarya were observed in the paraventricular organ in the hypothalamus and reticular nuclei in the hindbrain. Development of the serotonergic system was steady and gradual during mid-embryogenesis. However, starting at TS 13 there was a substantial increase in the number of serotonergic neurons in the paraventricular, raphe, and reticular nuclei, a large increase in the number of varicose fibers, and a differentiation of the reticular nuclei in the hindbrain. Consequentially, E. coqui displayed a well-developed central serotonergic system prior to hatching (TS 15). In comparison, the serotonergic system in metamorphic frogs typically starts to develop earlier but the surge of development that transpires in this system occurs post-embryonically, during metamorphosis, and not in the latter stages of embryogenesis, as it does in E. coqui. Overall, the serotonergic development in E. coqui is similar to the other vertebrates.

Corn feeding during development induces changes in the number of serotonergic neurons in the raphe nuclei

Serotonin (5-HT) plays a trophic role during brain development; chronic changes in cerebral concentration of this neurotransmitter during the critical stage of development can produce severe damage in the formation of the neural circuits. For the present work a hypoproteic (HYP) diet based on corn (CORN) meal which is deficient in tryptophan (TRY) was given to rats before and during pregnancy, which continued to the offspring until they reached 60 days of age. An isocaloric but hypoproteic diet containing normal amount of TRY, and normal chow (Ch) Purina were given with the same scheme to two groups of rats considered as controls. 5-HT immunohistochemistry was revealed by avidin–biotin complex (ABC) method to quantify serotonergic nerve cells in the nine raphe nuclei. The number of cells immunoreactive to 5-HT immunoreactive (5-HTir) were quantified by means of stereological analysis. Results demonstrated a significant variation in 5-HT expression in the raphe nuclei. Thus, a significant reduction in the number of 5-HTir cells in the rostral raphe nuclei was seen at all ages studied in the animals fed the corn diet, compared to data obtained from the control groups. This decrease was more evident between the postnatal ages of 30 and 60 days. It is concluded that the variations in the available TRY affect the brain cells producing 5-HT and the innervation of their target areas.

Development and distribution of serotonin in the central nervous system of Manduca sexta during embryogenesis I. The brain and frontal ganglion

Development of the serotonergic system in the brain and frontal ganglion of the Manduca embryo between 35 and 100% of development was studied immunocytochemically with an antiserum to serotonin (5-HT). Serotonin immunoreactivity was initially detectable at 40–45% development in short fibers in the head region, prior to differentiation of the brain. Immunoreactive cell bodies were first seen in the brain at 60% development, located in the protocerebrum and tritocerebrum. Thick fiber tracts crossing the midline (commissures) could also be observed at this early stage. As development of the embryo progressed, eight immunoreactive cell groups, containing a total of about 38–40 cells and four commissures with terminal arborizations appeared successively in the brain. From 75 to 100% development, no obvious changes occurred in the number or distribution of cells and the brain exhibited the same pattern of 5-HT immunoreactive cells, fiber tracts and arborizations as in last instar larvae of Manduca. However, an increase in the size of the cells in both the brain and frontal ganglion was noted between 75 and 80% development, followed by a decrease by 100% development. The frontal ganglion was found to contain three 5-HT immunoreactive cells, which appeared to send bilateral projections into the frontal connectives and the recurrent nerve. During embryonic development, the dendritic arborizations of these frontal ganglion cells increased, while the amount of 5-HT immunoreactivity in the cell bodies decreased. Thus, the serotonergic system first appears in the Manduca embryo at an early stage of development, similar to the situation in other insects as well as vertebrates. By the end of the embryonic period, the same number of serotonergic neurons are present in the brain as in larval and adult Manduca, suggesting that once formed, these cells persist through postembryonic development and metamorphosis.

Analysis of the change in number of serotonergic neurons in the chick spinal cord during embryonic development

The existence of serotonin (5-HT)-containing neurons in the spinal cord of the chick embryo was examined by anti-5-HT immunocytochemistry. The first immunoreactive cells were observed in embryos at 7 days of incubation (E7) and were initially located within the floor plate of the early spinal cord. By E9, immunostained cells occurred throughout the length of the spinal cord and were frequently encountered in most transverse sections of the cord. When examined at later embryonic ages of E12, 17 and at hatching (E21 or 22), the 5-HT cells became progressively more difficult to find with the advancing age of the embryos. To determine if this population of spinal cord 5-HT neurons actually diminished during development, a detailed quantitative analysis was undertaken to estimate the number of 5-HT cells in the cord of chick embryos at different ages. The results of this investigation demonstrated that the size of the 5-HT neuronal population rose rapidly from E7 and plateaued (at approximately 3500 neurons) between E9 and E12. As anticipated, the number of 5-HT cells at E17 decreased at all cord levels. Surprisingly, however, the number of spinal cord 5-HT neurons at hatching increased (depending on the cord level) either back to, or above, the counts estimated for the earlier ages of E9 and E12. Therefore, cells expressing the 5-HT phenotype in the spinal cord of the chick embryo persist throughout the period of embryonic development, rather than appear transiently. Furthermore, the fluctuations in 5-HT cell numbers during chick embryogenesis may indicate some regulation in the expression of the 5-HT phenotype in this population of spinal cord neurons.

The development of serotonergic raphespinal projections in Xenopus laevis

The development of serotonin-immunoreactive neurons in the central nervous system of Xenopus laevis larvae has been studied with special emphasis on the development of the raphe nuclei and raphespinal projections. The first serotonergic neurons were observed in the rostral part of the brain stem at stage 25, only 28 hr after fertilization. By stage 28 some 20 serotonin-immunoreactive neurons were found in the rostral part of the brain stem, bearing small protrusions on the ventromedial side of the soma. These initial axonal outgrowths reach the rostral part of the spinal cord at stage 32. By stage 35/36 the growth cones of the descending serotonergic axons in the spinal cord have reached the level of the anus (10th to 15th myotome). Up to stage 45 the majority of the descending serotonergic axons was found in the dorsolateral part of the marginal zone of the spinal cord. After stage 45 some serotonergic axons were also found scattered over other parts of the spinal marginal zone. Collateral oranches were first observed in the caudal part of the brain stem at stage 35/36. Later they occurred also in the rostral (stage 43) and caudal (stage 50) spinal cord, usually on fibers in the ventral half of the spinal cord. The number of serotonergic neurons in the central nervous system (brain stem and hypothalamus) increased steadily throughout development until stage 45. After that the total number of serotonergic neurons in the central nervous system increased about two times faster than the number of serotonergic neurons in the raphe nuclei, due to a massive increase of serotonergic neurons in the hypothalamus.The present study shows that young, just differentiated raphe neurons already contain serotonin. The generation of these neurons appears to take place in the ventricular zone (matrix) of the brain stem between the caudal border of the mesencephalon and the entrance of the nervus octavus. From here these neurons seem to migrate to their final destination. The distribution of serotonin-immunoreactive neurons in the brain stem suggests that a superior (not described so far in Anura) and an inferior raphe nucleus can be distinguished in Xenopus. A rostrocaudal gradient seems to be present in the production of serotonergic neurons which project to the spinal cord. Spinal projections from the raphe nuclei are particularly extensive from the nucleus raphes inferior and gradually decrease rostralwards. In the rostral part of the nucleus raphes superior almost no neurons projecting to the spinal cord are found.