Development of the serotonergic cells in murine raphe nuclei and their relations with rhombomeric domains

The raphe nuclei represent the origin of central serotonergic projections. The literature distinguishes seven nuclei grouped into rostral and caudal clusters relative to the pons. The boundaries of these nuclei have not been defined precisely enough, particularly with regard to developmental units, notably hindbrain rhombomeres. We hold that a developmental point of view considering rhombomeres may explain observed differences in connectivity and function. There are twelve rhombomeres characterized by particular genetic profiles, and each develops between one and four distinct serotonergic populations. We have studied the distribution of the conventional seven raphe nuclei among these twelve units. To this aim, we correlated 5-HT-immunoreacted neurons with rhombomeric boundary landmarks in sagittal mouse brain sections at different developmental stages. Furthermore, we performed a partial genoarchitectonic analysis of the developing raphe nuclei, mapping all known serotonergic differentiation markers, and compared these results, jointly with others found in the literature, with our map of serotonin-containing populations, in order to examine regional variations in correspondence. Examples of regionally selective gene patterns were identified. As a result, we produced a rhombomeric classification of some 45 serotonergic populations, and suggested a corresponding modified terminology. Only a minor rostral part of the dorsal raphe nucleus lies in the midbrain. Some serotonergic neurons were found in rhombomere 4, contrary to the conventional assumption that it lacks such neurons. We expect that our reclassification of raphe nuclei may be useful for causal analysis of their differential molecular specification, as well as for studies of differential connectivity and function.     

Development and organization of the descending serotonergic brainstem-spinal projections in the sea lamprey

The organization and development of the descending spinal projections from serotonergic rhombencephalic neurons in the larval sea lamprey were investigated by double labeling, tract-tracing methods and immunocytochemistry against serotonin. The results showed that two serotonergic populations of the isthmic and vagal reticular regions present reticulospinal neurons from the beginning of the larval period. Of the three serotonergic subpopulations recognized in the isthmic reticular group [Abalo, X.M., Villar-Cheda, B., Meléndez-Ferro, M., Pérez-Costas, E., Anadón, R., Rodicio, M.C., 2007. Development of the serotonergic system in the central nervous system of the sea lamprey. J. Chem. Neuroanat. 34, 29-46], only two - the medial and ventral subpopulations - project to the spinal cord, with most of the projecting cells in the caudal part of the medial isthmic subpopulation. Occasional cells projecting to the spinal cord were observed in the ventral subpopulation. The vagal reticular serotonergic nucleus situated in the caudal rhombencephalon also presents cells with descending projections. The early development of the brainstem serotonergic projections to the spinal cord appears to be a conserved trait in all vertebrates studied. Although a serotonergic hindbrain-spinal projection system appears to have been present before the divergence of agnathans and gnathostomes, no serotonergic cells were observed in the raphe region in lamprey. Moreover, proportionally more rostral hindbrain serotonergic cells contribute to the spinal serotonergic projections in the sea lamprey than in jawed vertebrates.     

Exposure to an open-field arena increases c-Fos expression in a subpopulation of neurons in the dorsal raphe nucleus, including neurons projecting to the basolateral amygdaloid complex

Serotonergic systems in the dorsal raphe nucleus are thought to play an important role in the regulation of anxiety states. To investigate responses of neurons in the dorsal raphe nucleus to a mild anxiety-related stimulus, we exposed rats to an open-field, under low-light or high-light conditions. Treatment effects on c-Fos expression in serotonergic and non-serotonergic cells in the midbrain raphe nuclei were determined 2 h following open-field exposure or home cage control (CO) conditions. Rats tested under both light conditions responded with increases in c-Fos expression in serotonergic neurons within subdivisions of the midbrain raphe nuclei compared with CO rats. However, the total numbers of serotonergic neurons involved were small suggesting that exposure to the open-field may affect a subpopulation of serotonergic neurons. To determine if exposure to the open-field activates a subset of neurons in the midbrain raphe complex that projects to forebrain circuits regulating anxiety states, we used cholera toxin B subunit (CTb) as a retrograde tracer to identify neurons projecting to the basolateral amygdaloid complex (BL) in combination with c-Fos immunostaining to identify cells that responded to open-field exposure. Rats received a unilateral injection of CTb into the BL. Seven to 11 days following CTb injection rats were either, 1) exposed to an open-field in low-light conditions, 2) briefly handled or 3) left undisturbed in home cages. Dual immunostaining for c-Fos and CTb revealed an increase in the percentage of c-Fos-immunoreactive BL-projecting neurons in open-field-exposed rats compared with handled and control rats. Dual immunostaining for tryptophan hydroxylase and CTb revealed that a majority (65%) of BL-projecting neurons were serotonergic, leaving open the possibility that activated neurons were serotonergic, non-serotonergic, or both. These data are consistent with the hypothesis that exposure to anxiogenic stimuli activates a subset of neurons in the midbrain raphe complex projecting to amygdala anxiety circuits.     

Distribution of serotonin-immunoreactive neurons in the brain of sockeye salmon fry

The organization of the serotonergic cell groups in the brainstem of fishes and amphibians has received relatively little attention. It has been generally assumed that they are little differentiated and constitute a median cell column throughout the brainstem, and that laterally migrated serotonergic cell groups are largely lacking. In the present study we present evidence to the contrary. By the use of a sensitive immunocytochemical technique for the visualization of serotonin-immunoreactive (5HTir) neurons, we have been able to make a detailed delineation of the putatively serotonergic neuronal groups throughout the brain. In the epithalamus, 5HTir neurons were located in the left habenular nucleus in its dorsal subdivision. 5HTir neural elements, primarily photoreceptor cells, were present throughout the pineal organ and in some cases also in the parapineal organ. In the periventricular zones of the hypothalamus and posterior tuberculum, 5THir cerebrospinal fluid-contacting neurons were located in the paraventricular organ and in the dorsal, ventral and caudal zones of the periventricular hypothalamus. In the dorsal thalamus/synencephalon, 5THir neurons surround the tractus habenulo-interpeduncularis (fasciculus retroflexus). In the brainstem, several groups of 5HTir neurons could be discerned, that for reasons of topological similarity were named according to Lidov and Molliver a raphe pallidus/obscurus-complex (B1 and B2), raphe magnus (part of B3), median raphe (B8) possibly including raphe pontis (B5), raphe dorsalis (B4, B6 and B7), and B9. 5HTir neurons were observed in the central gray of the IVth ventricle, dorsal to the noradrenergic isthmal neurons and lateral to the brachium conjunctivum, in an area topologically equivalent with the dorsal subdivision of the locus coeruleus in mammals. In addition, small numbers of 5HTir neurons were located in the lobi faciales. Thus, the presence of well-differentiated groups of migrated serotonergic neurons is not an advanced trait of amniote brains, but may be a pattern common to all vertebrates.     

Characterization of the raphe nuclei of the reptile Ctenosaura pectinata.

The brain stem of the lizard Ctenosaura pectinata was studied in 10 microns thick sections following the Nissl and eosin-hematoxilin techniques. Furthermore, the distribution of serotonin-containing neuronal somata in this encephalic region was determined by means of an indirect immunofluorescence technique using a specific antibody to serotonin. Two of the cellular groups of the brain stem were identified as the superior and inferior raphe nuclei, which show serotonergic cells of variable size (between 17 and 30 microns). The results obtained in the present study together with information coming from other authors, suggest that serotonergic neuronal systems placed at brain stem level of vertebrates are phylogenetically ancient.     

The role of serotonin release and autoreceptors in the dorsalis raphe nucleus in the control of serotonin release in the cat caudate nucleus

Using a push-pull cannula technique and an isotopic method for estimating [3H]serotonin continuously synthesized from [3H]tryptophan, the effects of changes in the release of serotonin in the dorsalis raphe nucleus on in vivo release of [3H]serotonin in the cat caudate nucleus were investigated. The increase in the release of serotonin in the dorsalis raphe nucleus caused by local application of parachlorophenylethylamine (10(-6) M) reduced striatal [3H]serotonin release. This inhibition in serotonin release in the striatum was blocked by the prior and continuous local superfusion of the dorsal raphe with methiothepin (10(-6) M), a serotonin autoreceptor antagonist. GABA (5 x 10(-5) M) applied to the dorsalis raphe reduced both local and striatal release of [3H]serotonin. However, picrotoxin (10(-5) M), a GABA A receptor antagonist applied locally in the dorsalis raphe nucleus increased [3H]serotonin release while decreasing striatal [3H]serotonin release. This decrease in serotonin release in the striatum was again blocked by continuous superfusion of the raphe with methiothepin. Furthermore, superfusion of serotonergic cell bodies of the dorsalis raphe nucleus with methiothepin alone never altered local release or striatal release of [3H]serotonin. These data strongly suggest that the release of serotonin from the cell body in the dorsalis raphe nucleus phasically controls release of the amine at the axonal nerve ending through serotonergic autoreceptors located on serotonergic nerve cell bodies in the dorsalis raphe nucleus. The origin of the serotonin released in the dorsalis raphe nucleus and the possibility that this type of regulation could be related to changes in nerve impulse conduction of the serotonergic raphe-striatal system are discussed.     

Repeated social defeat increases reactive emotional coping behavior and alters functional responses in serotonergic neurons in the rat dorsal raphe nucleus

Chronic stress is a vulnerability factor for a number of psychiatric disorders, including anxiety and affective disorders. Social defeat in rats has proven to be a useful paradigm to investigate the neural mechanisms underlying physiologic and behavioral adaptation to acute and chronic stress. Previous studies suggest that serotonergic systems may contribute to the physiologic and behavioral adaptation to chronic stress, including social defeat in rodent models. In order to test the hypothesis that repeated social defeat alters the emotional behavior and the excitability of brainstem serotonergic systems implicated in control of emotional behavior, we exposed adult male rats either to home cage control conditions, acute social defeat, or social defeat followed 24 h later by a second social defeat encounter. We then assessed behavioral responses during social defeat as well as the excitability of serotonergic neurons within the dorsal raphe nucleus using immunohistochemical staining of tryptophan hydroxylase, a marker of serotonergic neurons, and the protein product of the immediate-early gene, c-fos. Repeated social defeat resulted in a shift away from proactive emotional coping behaviors, such as rearing (explorative escape behavior), and toward reactive emotional coping behaviors such as freezing. Both acute and repeated defeat led to widespread increases in c-Fos expression in serotonergic neurons in the dorsal raphe nucleus. Changes in behavior following a second exposure to social defeat, relative to acute defeat, were associated with decreased c-Fos expression in serotonergic neurons within the dorsal and ventral parts of the mid-rostrocaudal dorsal raphe nucleus, regions that have been implicated in 1) serotonergic modulation of fear- and anxiety-related behavior and 2) defensive behavior in conspecific aggressive encounters, respectively. These data support the hypothesis that serotonergic systems play a role in physiologic and behavioral responses to both acute and repeated social defeat.     

Prostaglandin EP3 receptor protein in serotonin and catecholamine cell groups: a double immunofluorescence study in the rat brain

Prostaglandin E(2) exerts diverse physiological actions in the central nervous system with unknown mechanisms. We have reported the immunohistochemical localization of the EP3 receptor, one of the prostaglandin E receptor subtypes, in various brain regions including many monoaminergic nuclei. In the present study, a double immunofluorescence technique with an antibody to EP3 receptor and antibodies to markers for monoamine neurons was employed to examine the expression of the receptor in serotonin and catecholamine neurons, and to reveal the distribution of the receptor-expressing monoamine neurons in the rat brain. Almost all serotonergic cells in the medulla oblongata (B1-B4) exhibited EP3 receptor-like immunoreactivity, whereas mesencephalic and pontine serotonergic cell groups (B5-B9) contained relatively small populations of EP3 receptor-immunoreactive cells. In the catecholaminergic cell groups, many of the noradrenergic A7 cells in the subcoeruleus nucleus showed immunoreactivity for the receptor. The locus coeruleus exhibited EP3 receptor-like immunoreactivity densely in the neuropil and occasionally in neuronal cell bodies, all of which were immunopositive for dopamine beta-hydroxylase, as observed by confocal laser microscopy. Many of the other noradrenergic and adrenergic cell groups contained small populations of EP3 receptor-like immunoreactive cells. In contrast, no EP3 receptor-like immunoreactivity was detected in the noradrenergic A2 and A4, the adrenergic C2, and all the dopaminergic cell groups.The expression of EP3 receptor by most of the serotonergic, noradrenergic and adrenergic cell groups suggests that prostaglandin E(2) modulates many physiological processes mediated by widely distributed monoaminergic projections through activation of the EP3 receptor on the monoaminergic neurons; for instance, it may modulate nociceptive and autonomic processes by affecting the descending serotonergic pathway from the raphe magnus nucleus to the spinal cord.     

Regulation of the transmitter phenotype of rostral and caudal groups of cultured serotonergic raphe neurons

We have studied the regulation of survival and serotonergic markers by neurotrophins and several trophically active cytokines in neurons cultured from the embryonic rat raphe region under defined conditions. At embryonic day 14, saturating concentrations of brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4 and basic fibroblast growth factor elicited a two- to 2.5-fold increase in numbers of tryptophan hydroxylase- and serotonin-immunoreactive neurons over a four-day culture period. Transforming growth factor beta-1 and glial cell line-derived neurotrophic factor were less potent, while fibroblast growth factor-5 was only marginally effective. Distinct responses to different factors were noted depending on embryonic age and regional origin of serotonergic neurons. Thus, brain-derived neurotrophic factor augmented numbers of tryptophan hydroxylase-positive neurons at embryonic day 16 by a factor of seven, but only 1.5- to two-fold when cultures were established from day 13 or 14 embryos. In cultures of rostral serotonergic groups (B4-B9), numbers of tryptophan hydroxylase-positive neurons decreased in the absence of factors, whereas numbers of tryptophan hydroxylase-immunoreactive neurons in cultures from caudal serotonergic groups (B1-B3) increased during a 12-day culture period. There was no evidence that serotonergic neurons undergo apoptosis (as visualized by terminal deoxynucleotidyl transferase dUTP nick end labeling) or proliferate (as visualized by 5-bromodeoxyuridine incorporation) in culture. Numbers of serotonergic neurons also increased when cultures were treated with a brief 24-h pulse of brain-derived neurotrophic factor, supporting the notion that changes in numbers of serotonergic neurons reflected alterations of phenotype rather than cell death or proliferation. The ability of cells to specifically take up the serotonin analog 5,7-dihydroxytryptamine was also up-regulated by brain-derived neurotrophic factor in both rostral and caudal raphe cultures. Lability of the serotonergic phenotype was further suggested by the observation that ciliary neurotrophic factor fully prevented the brain-derived neurotrophic factor-mediated increase in tryptophan hydroxylase-positive neurons. The effect of ciliary neurotrophic factor was dependent on the presence of astrocytes. We conclude that serotonergic neurons show spatially and temporally distinct responses to neurotrophic factors, which seem to have a profound influence of the transmitter phenotype rather than on survival.     

Discharge properties of neurons of the median raphe nucleus during hippocampal theta rhythm in the rat

The serotonin (5-HT)-containing median raphe nucleus has been shown to be critically involved in the control of desynchronized (non theta) states of the hippocampal electroencephalogram (EEG). We examined the activity of 181 cells of the median raphe nucleus in the urethane-anesthetized rat and found that approximately 80% (145/181) of them showed changes in activity associated with changes in the hippocampal EEG. These cells were subdivided into theta-on (68%) and theta-off (32%) based on increased or decreased rates of activity with theta, respectively. They were further classified as slow-firing (~1 Hz), moderate-firing (5-11 Hz), or fast-firing (>12 Hz) theta-on or theta-off cells. The slow-firing cells as well as a subset of moderate-firing theta-off cells displayed characteristics of "classic" serotonin-containing raphe neurons. All fast-firing neurons were theta-on cells and showed either tonic or phasic (rhythmical) increases in activity with theta. We propose that: (1) the slow-firing cells (on and off) as well as a subset of moderate-firing theta-off cells are serotonergic neurons; (2) the phasic and tonic fast-firing theta-on cells are GABAergic cells; and (3) these populations of cells mutually interact in the modulation of the hippocampal EEG. An activation of local serotonergic and GABAergic theta-on cells would inhibit 5-HT slow- or moderate-firing theta-off projection cells to release or generate theta, whereas the suppression of serotonergic- or GABAergic theta-on cells would disinhibit 5-HT theta-off cells, resulting in a blockade of theta or a desynchronization of the hippocampal EEG. A role for the median raphe nucleus in memory-associated functions of the hippocampus is discussed.     

Acidosis-stimulated neurons of the medullary raphe are serotonergic

Neurons of the medullary raphe project widely to respiratory and autonomic nuclei and contain co-localized serotonin, thyrotropin-releasing hormone (TRH), and substance P, three neurotransmitters known to stimulate ventilation. Some medullary raphe neurons are highly sensitive to pH and CO(2) and have been proposed to be central chemoreceptors. Here it was determined whether these chemosensitive neurons are serotonergic. Cells were microdissected from the rat medullary raphe and maintained in primary cell culture for 13-70 days. Immunoreactivity for serotonin, substance P, and TRH was present in these cultures. All acidosis-stimulated neurons (n = 22) were immunoreactive for tryptophan hydroxylase (TpOH-IR), the rate-limiting enzyme for serotonin biosynthesis, whereas all acidosis-inhibited neurons (n = 16) were TpOH-immunonegative. The majority of TpOH-IR medullary raphe neurons (73%) were stimulated by acidosis. The electrophysiological properties of TpOH-IR neurons in culture were similar to those previously reported for serotonergic neurons in vivo and in brain slices. These properties included wide action potentials (4.55 +/- 0.5 ms) with a low variability of the interspike interval, a postspike afterhyperpolarization (AHP) that reversed 25 mV more positive than the Nernst potential for K(+), prominent A current, spike frequency adaptation and a prolonged AHP after a depolarizing pulse. Thus the intrinsic cellular properties of serotonergic neurons were preserved in cell culture, indicating that the results obtained using this in vitro approach are relevant to serotonergic neurons in vivo. These results demonstrate that acidosis-stimulated neurons of the medullary raphe contain serotonin. We propose that serotonergic neurons initiate a homeostatic response to changes in blood CO(2) that includes increased ventilation and modulation of autonomic function.     

大鼠脑干至脊髓5-羟色胺投射纤维的起源——免疫细胞化学和酶组织化学双重标记法

本文应用 HRP 逆行追踪与免疫细胞化学相结合的双重标记技术,研究了大鼠脑干至脊髓5-羟色胺(5-HT)投射纤维的起源,并对中缝核群和网状结构内5-HT 阳性细胞、HRP 标记细胞和5-HT-HRP 双重标记细胞进行了数量分析。结果表明,1.在延髓、脑桥尾侧部,5-HT-HRP双重标记细胞约占5-HT 阳性细胞的55%;占 HRP 标记细胞的45%。中脑仅有极少数5-HT-HRP双重标记细胞。2.脑干至脊髓5-HT 下行投射,主要起源于中缝苍白核、中缝隐核、中缝大核和延髓浅表弓状纤维区,所含双重标记细胞约占双重标记细胞总数的68%。3.浅表弓状纤维区、舌下神经根间核、中缝苍白核和中缝隐核主要为5-HT 下行投射;中缝大核及周围网状结构主要为非5-HT 下行投射。     

Colocalization of serotonin and vesicular glutamate transporter 3-like immunoreactivity in the midbrain raphe of Syrian hamsters (Mesocricetus auratus)

Vesicular glutamate transporter 3 (VGLUT3) expression has been specifically localized to brain regions rich in serotonergic cells. It has been suggested that this transporter may contribute to the regulation of extracellular glutamate concentrations via a nonsynaptic mechanism. In this study, we examine the colocalization of vesicular glutamate transporter 3 immunoreactivity with serotonin immunoreactivity in the dorsal and median raphe nuclei of Syrian hamsters. Brain sections from adult hamsters were fluorescently labeled for serotonin-ir and VGLUT3-ir and examined using confocal microscopy. The results indicate that most serotonergic cells of the midbrain raphe also expressed vesicular glutamate transporter 3. In addition, nonserotonergic cells in these brain regions also show immunoreactivity for the transporter. These data confirm previous findings of vesicular glutamate transporter 3 expression in serotonergic and nonserotonergic neurons in rats. These findings suggest that the location of vesicular glutamate transporter 3 may be as much a function of neuroanatomical location as of the neurochemical identity of the expressing neurons.     

Serotonergic and nonserotonergic dorsal raphe neurons are pharmacologically and electrophysiologically heterogeneous

The dorsal raphe nucleus (DRN) projects serotonergic axons throughout the brain and is involved in a variety of physiological functions. However, it also includes a large population of cells that contain other neurotransmitters. To clarify the physiological and pharmacological differences between the serotonergic and nonserotonergic neurons of the DRN, their postsynaptic responses to 5-hydroxytryptamine (5-HT, serotonin) and to selective activation of 5-HT1A or 5-HT2A/C receptors and their action potential characteristics were determined using in vitro patch-clamp recordings. The slices containing these neurons were then immunostained for tryptophan hydroxylase (TPH), a marker of serotonergic neurons. It was found that subpopulations of both serotonergic and nonserotonergic neurons responded to 5-HT with outward (i.e., inhibitory) and inward (i.e., excitatory) currents, responded to both 5-HT1A and 5-HT2A/C receptor activation with outward and inward currents, respectively, and displayed overlapping action potential characteristics. These findings suggest that serotonergic and nonserotonergic neurons in the DRN are both heterogeneous with respect to their individual pharmacological and electrophysiological characteristics. The findings also suggest that the activity of the different populations of DRN neurons will display heterogeneous changes when the serotonergic tone in the DRN is altered by neurological disorders or by drug treatment.     

Serotonin and GABA are colocalized in restricted groups of neurons in the larval sea lamprey brain: insights into the early evolution of neurotransmitter colocalization in vertebrates

Colocalization of the classic neurotransmitters serotonin (5-HT) and γ-aminobutyric acid (GABA) (or the enzyme that synthesizes the latter, glutamate decarboxylase) has been reported in a few neurons of the rat raphe magnus-obscurus nuclei. However, there are no data on the presence of neurochemically similar neurons in the brain of non-mammalian vertebrates. Lampreys are the oldest extant vertebrates and may provide important data on the phylogeny of neurochemical systems. The colocalization of 5-HT and GABA in neurons of the sea lamprey brain was studied using antibodies directed against 5-HT and GABA and confocal microscopy. Colocalization of the neurotransmitters was observed in the diencephalon and the isthmus. In the diencephalon, about 87% of the serotonergic cells of the rostral tier of the dorsal thalamus (close to the zona limitans) exhibited GABA immunoreactivity. In addition, occasional cells double-labelled for GABA and 5-HT were observed in the hypothalamic tuberal nucleus and the pretectum. Of the three serotonergic isthmic subgroups already recognized in the sea lamprey isthmus (dorsal, medial and ventral), such double-labelled cells were only observed in the ventral subgroup (about 61% of the serotonergic cells in the ventral subgroup exhibited GABA immunoreactivity). An equivalence between these lamprey isthmic cells and the serotonergic/GABAergic raphe cells of mammals is suggested. Present findings suggest that serotonergic/GABAergic neurons are more extensive in lampreys than in the rat and probably appeared before the separation of agnathans and gnathostomes. Cotransmission by release of 5-HT and GABA by the here-described lamprey brain neurons is proposed.