Serotonergic innervation and serotonin receptor expression of NPY-producing neurons in the rat lateral and basolateral amygdaloid nuclei

Pharmacobehavioral studies in experimental animals, and imaging studies in humans, indicate that serotonergic transmission in the amygdala plays a key role in emotional processing, especially for anxiety-related stimuli. The lateral and basolateral amygdaloid nuclei receive a dense serotonergic innervation in all species studied to date. We investigated interrelations between serotonergic afferents and neuropeptide Y (NPY)-producing neurons, which are a subpopulation of inhibitory interneurons in the rat lateral and basolateral nuclei with particularly strong anxiolytic properties. Dual light microscopic immunolabeling showed numerous appositions of serotonergic afferents on NPY-immunoreactive somata. Using electron microscopy, direct membrane appositions and synaptic contacts between serotonin-containing axon terminals and NPY-immunoreactive cellular profiles were unequivocally established. Double in situ hybridization documented that more than 50 %, and about 30–40 % of NPY mRNA-producing neurons, co-expressed inhibitory 5-HT1A and excitatory 5-HT2C mRNA receptor subtype mRNA, respectively, in both nuclei with no gender differences. Triple in situ hybridization showed that individual NPY mRNA-producing interneurons co-express both 5-HT1A and 5-HT2C mRNAs. Co-expression of NPY and 5-HT3 mRNA was not observed. The results demonstrate that serotonergic afferents provide substantial innervation of NPY-producing neurons in the rat lateral and basolateral amygdaloid nuclei. Studies of serotonin receptor subtype co-expression indicate a differential impact of the serotonergic innervation on this small, but important, population of anxiolytic interneurons, and provide the basis for future studies of the circuitry underlying serotonergic modulation of emotional stimulus processing in the amygdala.     

Neuromodulatory role of serotonin in the ferret thalamus

Serotonergic fibers broadly innervate the thalamus and may influence the sleep wake cycle, attention, and other processes through modulation of neurons in this structure. However, the actions of serotonin in the dorsal thalamus have been investigated in detail only in the dorsal lateral geniculate nucleus. In the present study, we examined the action of serotonin in several different regions of the ferret dorsal thalamus, including the associative nuclei, using the in vitro slice preparation and intracellular recording techniques. In nearly all nuclei examined, the predominant action of serotonin was one of hyperpolarization and inhibition of the tonic firing mode. The magnitude of the hyperpolarizing response decreased with age and varied greatly across and somewhat within nuclei maintaining the following relationship (in descending order of magnitude): lateral posterior, lateral dorsal, pulvinar, mediodorsal, center median, anteroventral, central lateral, ventral basal, and medial geniculate. This hyperpolarization is elicited through two mechanisms: one direct and the other via local interneurons. The direct action occurs through an increase in potassium conductance mediated through the 5-HT(1A) receptor. This conclusion is supported by the findings that it persists in the presence of tetrodotoxin and block of GABAergic synaptic transmission, the reversal potential shifts in a Nernstian fashion with changes in extracellular potassium concentration, and the response is antagonized by the 5-HT(1A) antagonist WAY100635 and mimicked by the application of the 5-HT(1A)-selective agonist 8-OH DPAT. The second mechanism by which 5-HT evoked a hyperpolarization was through the activation of local interneurons. In slices in which GABA receptors were not blocked, 5-HT application increased the frequency and amplitude of spontaneous inhibitory postsynaptic potentials (IPSPs) occurring in thalamocortical neurons. Application of 5-HT to physiologically or morphologically identified interneurons evoked a prolonged suprathreshold depolarization. Our results suggest that serotonergic inputs act differentially across the thalamus in a complex manner involving direct and indirect mechanisms. It appears that 5-HT has a greater direct postsynaptic inhibitory influence in the posterior, medial, and intralaminar nuclei than in the primary sensory nuclei.     

Electrophysiological and pharmacological properties of GABAergic cells in the dorsal raphe nucleus

The dorsal raphe nucleus (DRN) is the origin of the central serotonin [5-hydroxytryptamine (5-HT)] system and plays an important role in the regulation of many physiological functions such as sleep/arousal, food intake and mood. In order to understand the regulatory mechanisms of 5-HT system, characterization of the types of neurons is necessary. We performed electrophysiological recordings in acute slices of glutamate decarboxylase 67–green fluorescent protein knock-in mice. We utilized this mouse to identify visually GABAergic cells. Especially, we examined postsynaptic responses mediated by 5-HT receptors between GABAergic and serotonergic cells in the DRN. Various current responses were elicited by 5-HT and 5-HT_1A or 5-HT_2A/2C receptor agonists in GABAergic cells. These results suggested that multiple 5-HT receptor subtypes overlap on GABAergic cells, and their combination might control 5-HT cells. Understanding the postsynaptic 5-HT feedback mechanisms may help to elucidate the 5-HT neurotransmitter system and develop novel therapeutic approaches.     

GABA(B) receptors in the median raphe nucleus: distribution and role in the serotonergic control of hippocampal activity.

Previous studies have shown that serotonergic neurons of the median raphe nucleus have a suppressive effect on theta synchronization in the hippocampus. Median raphe lesion, suppression of 5-HT neuronal activity by administration of GABA(A) receptor antagonist or by glutamate blockade or depletion produced long-lasting non-interrupted hippocampal theta in freely behaving rats independent of behavior and in rats anesthetized with urethane. Serotonergic neurons show a characteristic sleep-wake pattern of activity and there is evidence that GABAergic mechanisms play an important role in their regulation. In this study we analyzed the distribution and subcellular localization of GABA(B) receptors in the midbrain raphe complex using combined 5-HT/GABA(B) receptor immunohistochemistry at the light and electron microscopic levels and studied the effects of their pharmacological manipulation on hippocampal electroencephalographic activity in urethane-anesthetized rats. We found that sustained infusion of the GABA(B) receptor agonist baclofen into the median raphe nucleus, using the microdialysis technique, elicited lasting theta activity in the hippocampus. The effect was antagonized by selective GABA(B) receptor antagonists. The predominant localization of GABA(B) receptors in the median, as well as in dorsal raphe was found on serotonergic neurons which strongly indicates that the increase in theta occurrence after baclofen injection resulted from suppression of the serotonergic output originating from the median raphe. On the electron microscopic level, we found GABA(B) receptors located extrasynaptically indicating that these receptors are preferentially activated by strong inputs, i.e. when GABA released from the synaptic terminals is sufficient to spill over from the synaptic cleft. Such conditions might be satisfied during rapid eye movement sleep when GABAergic neurons in the raphe are firing at their highest rate and in rhythmic synchronized bursts.Our data indicate that midbrain raphe GABA(B) mechanisms play an important role in behavioral state control and in hippocampal activity, in particular.     

Nuclear organization of the serotonergic system in the brain of the rock cavy (Kerodon rupestris)

Serotonin, or 5-hydroxytryptamine (5-HT), is a substance found in many tissues of the body, including as a neurotransmitter in the nervous system, where it can exert different post-synaptic actions. Inside the neuro-axis, 5-HT neurons are almost entirely restricted to the raphe nuclei of the brainstem. As such, 5-HT-immunoreactivity has been considered a marker of the raphe nuclei, which are located in the brainstem, at or near the midline. The present study investigated distribution of serotonergic neurons in the brain of the rock cavy (Kerodon rupestris), a rodent species inhabiting the Brazilian Northeast. The cytoarchitectonic location of serotonergic neurons was established through a series of 5-HT immunostained sections, compared with diagrams obtained from adjacent coronal and sagittal sections stained by the Nissl method. The following nuclei were defined: the rostral group, consisting of rostral linear raphe, caudal linear raphe, median and paramedian raphe, dorsal raphe, and pontine raphe nuclei, and the caudal group composed of raphe magnus, raphe pallidus and raphe obscurus nuclei. Other serotonergic neuronal clusters, such as the supralemniscal group and the rostral and caudal ventrolateral medulla oblongata clusters, were found outside the midline. Rare 5-HT-producing neurons were identified in the lateral parabrachial nucleus and in the pontine reticular formation, mostly along fibers of the lateral lemniscus. Despite exhibiting some specializations, the picture outlined for serotonergic groups in the rock cavy brain is comparable to that described for other mammalian species.     

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.     

Distinguishing characteristics of serotonin and non-serotonin-containing cells in the dorsal raphe nucleus: electrophysiological and immunohistochemical studies

The membrane properties and receptor-mediated responses of rat dorsal raphe nucleus neurons were measured using intracellular recording techniques in a slice preparation. After each experiment, the recorded neuron was filled with neurobiotin and immunohistochemically identified as 5-hydroxytryptamine (5-HT)-immunopositive or 5-HT-immunonegative. The cellular characteristics of all recorded neurons conformed to previously determined classic properties of serotonergic dorsal raphe nucleus neurons: slow, rhythmic activity in spontaneously active cells, broad action potential and large afterhyperpolarization potential. Two electrophysiological characteristics were identified that distinguished 5-HT from non-5-HT-containing cells in this study. In 5-HT-immunopositive cells, the initial phase of the afterhyperpolarization potential was gradual (tau=7.3+/-1.9) and in 5-HT-immunonegative cells it was abrupt (tau=1.8+/-0.6). In addition, 5-HT-immunopositive cells had a shorter membrane time constant (tau=21.4+/-4.4) than 5-HT-immunonegative cells (tau=33.5+/-4.2). Interestingly, almost all recorded neurons were hyperpolarized in response to stimulation of the inhibitory 5-HT(1A) receptor. These results suggested that 5-HT(1A) receptors are present on non-5-HT as well as 5-HT neurons. This was confirmed by immunohistochemistry showing that although the majority of 5-HT-immunopositive cells in the dorsal raphe nucleus were double-labeled for 5-HT(1A) receptor-IR, a small but significant population of 5-HT-immunonegative cells expressed the 5-HT(1A) receptor. These results underscore the heterogeneous nature of the dorsal raphe nucleus and highlight two membrane properties that may better distinguish 5-HT from non-5-HT cells than those typically reported in the literature. In addition, these results present electrophysiological and anatomical evidence for the presence of 5-HT(1A) receptors on non-5-HT neurons in the dorsal raphe nucleus.     

5-HT1A receptor mRNA and immunoreactivity in the rat medial septum/diagonal band of Broca-relationships to GABAergic and cholinergic neurons

Activation of 5-HT1A receptors results in a variety of physiological responses, depending on their localization on neurons with different phenotypes in the brain. This study investigated the localization of 5-HT1A receptor mRNA and 5-HT1A receptor immunoreactivity in cell bodies of the rat septal complex using in situ hybridization and immunohistochemistry. In adjacent sections of the medial septum/diagonal band of Broca (MSDB), the distribution of cell bodies expressing 5-HT1A receptor mRNA was closely related to cells labeled with oligonucleotide probes to GAD (glutamic acid decarboxylase), VAChT (vesicular acetylcholine transporter) or parvalbumin mRNA. Using antiserum to GAD and antibodies to GABA, 5-HT1A receptor immunoreactivity was demonstrated in a majority of GABAergic cells in the MSDB. 5-HT1A receptor-immunoreactive GABAergic cells in the MSDB were also demonstrated to contain the calcium-binding protein parvalbumin, a marker for septohippocampal projecting GABAergic neurons. In the lateral septum, 5-HT1A receptor immunoreactivity was colocalized with the calcium-binding protein calbindin D-28k, a marker for septal GABAergic somatospiny neurons. 5-HT1A receptor immunoreactivity was also detected in a subpopulation of VAChT-containing cholinergic neurons of the MSDB. In MSDB neurons, colocalization of 5-HT1A and 5-HT2A receptor immunoreactivities was demonstrated. These observations suggest that serotonin via 5-HT1A receptors may represent an important modulator of hippocampal transmission important for cognitive and emotional functions through actions on both GABAergic and cholinergic neurons of the rat septal complex. In addition, 5-HT may exert its effects in the MSDB via cells expressing both 5-HT1A and 5-HT2A receptors.     

Serotonin modulates the population activity profile of olfactory bulb external tufted cells

Serotonergic neurons in the raphe nuclei constitute one of the most prominent neuromodulatory systems in the brain. Projections from the dorsal and median raphe nuclei provide dense serotonergic innervation of the glomeruli of olfactory bulb. Odor information is initially processed by glomeruli, thus serotonergic modulation of glomerular circuits impacts all subsequent odor coding in the olfactory system. The present study discloses that serotonin (5-HT) produces excitatory modulation of external tufted (ET) cells, a pivotal neuron in the operation of glomerular circuits. The modulation is due to a transient receptor potential (TRP) channel-mediated inward current induced by activation of 5-HT(2A) receptors. This current produces membrane depolarization and increased bursting frequency in ET cells. Interestingly, the magnitude of the inward current and increased bursting inversely correlate with ET cell spontaneous (intrinsic) bursting frequency: slower bursting ET cells are more strongly modulated than faster bursting cells. Serotonin thus differentially impacts ET cells such that the mean bursting frequency of the population is increased. This centrifugal modulation could impact odor processing by: 1) increasing ET cell excitatory drive on inhibitory neurons to increase presynaptic inhibition of olfactory sensory inputs and postsynaptic inhibition of mitral/tufted cells; and/or 2) coordinating ET cell bursting with exploratory sniffing frequencies (5-8 Hz) to facilitate odor coding.     

Functional anatomy of 5-HT2A receptors in the amygdala and hippocampal complex: relevance to memory functions

The amygdaloid complex and hippocampal region contribute to emotional activities, learning, and memory. Mounting evidence suggests a primary role for serotonin (5-HT) in the physiological basis of memory and its pathogenesis by modulating directly the activity of these two areas and their cross-talk. Indeed, both the amygdala and the hippocampus receive remarkably dense serotoninergic inputs from the dorsal and median raphe nuclei. Anatomical, behavioral and electrophysiological evidence indicates the 5-HT_2A receptor as one of the principal postsynaptic targets mediating 5-HT effects. In fact, the 5-HT_2A receptor is the most abundant 5-HT receptor expressed in these brain structures and is expressed on both amygdalar and hippocampal pyramidal glutamatergic neurons as well as on γ-aminobutyric acid (GABA)-containing interneurons. 5-HT_2A receptors on GABAergic interneurons stimulate GABA release, and thereby have an important role in regulating network activity and neural oscillations in the amygdala and hippocampal region. This review will focus on the distribution and physiological functions of the 5-HT_2A receptor in the amygdala and hippocampal region. Taken together the results discussed here suggest that 5-HT_2A receptor may be a potential therapeutic target for those disorders related to hippocampal and amygdala dysfunction.     

The organization of serotonergic projections to cerebral cortex in primates: retrograde transport studies.

Retrograde axonal transport and immunocytochemical methods were utilized to determine the origin of serotonergic afferents to selected primary projection and association areas of cerebral cortex in macaque monkeys. After injections of Fast Blue or Diamidino Yellow in primary motor, somatosensory, or visual cortex, retrogradely labeled neurons are found in both the dorsal and median raphe nuclei. The sets of dorsal raphe neurons which innervate these cortical areas differ in their spatial distributions along the rostrocaudal axis of the brainstem; a coarse rostrocaudal topographic relationship is found between these groups of dorsal raphe neurons and their cortical targets. In contrast, neurons in the median raphe which innervate these primary projection areas are not differentially distributed along the rostrocaudal axis. However, in both the median and dorsal raphe nuclei, most neurons projecting to primary visual cortex are situated lateral to the cells which project to motor and somatosensory areas; many of these visually projecting neurons lie among the fascicles of the medial longitudinal fasciculus. For comparison with the serotonergic innervation of primary projection areas, the locations of raphe cells projecting to three areas of association cortex were examined: dorsolateral prefrontal cortex, area 5 and area 7b. Neurons projecting to each of these association areas are found throughout the dorsal and median raphe nuclei. Their distributions are similar to one another; however, more cells projecting to dorsolateral prefrontal cortex are in the rostral part of the dorsal raphe. The dorsal and median raphe neurons projecting to these association areas are intermingled with neurons projecting to motor and somatosensory cortex, but are medial to most of those projecting to visual cortex. Thus, separate cortical areas are innervated by different sets of raphe neurons; these sets partially overlap, yet differ in their rostrocaudal and mediolateral distributions. Ascending serotonergic projections to cerebral cortex form a widely distributed system which exhibits a highly intricate anatomic organization. The present observations support the hypothesis that the dorsal raphe nucleus is comprised of distinct sets of neurons whose output is distributed to multiple, interconnected cortical areas; these serotonergic projections may play a role in the coordination of excitability in functionally related areas of cortex. In contrast, the serotonergic projections arising from the median raphe appear to be more divergent and are likely to have a global influence on cortical activity. Since these individual raphe nuclei have different projection patterns, they are likely to have distinct functional roles.     

Differential distribution of estrogen receptor (ER)-alpha and ER-beta in the midbrain raphe nuclei and periaqueductal gray in male mouse: Predominant role of ER-beta in midbrain serotonergic systems

We examined the distribution of estrogen receptor (ER)-alpha and ER-beta immunoreactive (ir) cells in the dorsal (DRN) and median/paramedian (MPRN) raphe nuclei in male mice. ER-alpha ir neurons were scattered across the three subdivisions (ventral, dorsal, and lateral) of the DRN and the MPRN. Robust ER-beta ir cells were observed throughout the raphe nuclei, and were particularly abundant in the ventral and dorsal subdivisions of the DRN. Using dual-label immunocytochemistry for ER-alpha or ER-beta with tryptophan hydroxylase (TPH), the rate-limiting enzyme for 5-hydroxytryptamine (5-HT) synthesis, over 90% of ER-beta ir cells exhibited TPH-ir in all DRN subdivisions, whereas only 23% of ER-alpha ir cells contained TPH. Comparisons of ER-alpha knockout (alphaERKO) as well as ER-beta knockout (betaERKO) mice with their respective wild-type (WT) littermates revealed that gene disruption of either ER-alpha or ER-beta did not affect the other ER subtype expression in the raphe nuclei. In situ hybridization histochemistry revealed that there was a small but statistically significant decrease in TPH mRNA expression in the ventral DRN subdivision in betaERKO mice compared with betaWT mice, whereas TPH mRNA levels were not affected in alphaERKO mice. These findings support a hypothesis that ER-beta activation may contribute to the estrogenic regulation of neuroendocrine and behavioral functions, in part, by acting directly on 5-HT neurons in the raphe nuclei in male mice.     

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.     

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.     

Involvement of medullary GABAergic and serotonergic raphe neurons in respiratory control: electrophysiological and immunohistochemical studies in rats

In the present study we first examined the possible involvement of the putative neurotransmitters gamma-aminobutyric acid (GABA) and serotonin (5-HT) in raphe-induced facilitatory or inhibitory effects on the respiratory activity of rats. Secondly, we investigated the possibility of spinal projections of GABAergic and serotonergic neurons from the medullary raphe nuclei to the phrenic motor nucleus (PMN). We observed that an intravenous (i.v.) injection of (+)-bicuculline, a GABA(A) receptor antagonist, significantly reduced respiratory inhibition induced by electrical stimulation of the raphe magnus (RM) or the raphe obscurus (RO). On the other hand, an i.v. injection of methysergide, a broad-spectrum 5-HT receptor antagonist, significantly reduced the respiratory facilitation induced by electrical stimulation of the raphe pallidus (RP) or RO. By using a combined method of retrograde tracing with Texas Red injected into the PMN region at segments C4 and C5 and immunohistochemical labeling, we observed that glutamic acid decarboxylase (GAD; a GABA synthesizing enzyme) immunopositive and Texas Red double labeled neurons were predominantly localized in the RM, and additionally in the RO. However 5-HT immunopositive and Texas Red double-labeled neurons were predominantly localized in the RP, and additionally in the RO and RM. These findings suggest that RM-, or RO-induced inhibitory effects, are transmitted, at least in part, to the PMN via a direct GABAergic descending pathway. The RP-, or RO-induced facilitatory effects in rats however, are transmitted via a serotonergic descending pathway.     

Non-serotonergic dorsal and median raphe projection onto parvalbumin- and calbindin-containing neurons in hippocampus and septum

The median raphe nucleus is involved in controlling and maintaining hippocampal activity through its projection to inhibitory neurons in medial septum and hippocampus. It has been shown that anterogradely axonal-traced fibers originating in the median raphe nucleus project onto calbindin-containing neurons in hippocampus and parvalbumin-containing neurons in medial septum. Parallel immunohistochemistry studies showing serotonin fibers contacting calbindin- and parvalbumin-positive neurons have led to the assumption that raphe fibers projecting on these types of neurons are mainly serotonergic. However, in both dorsal and median raphe nucleus there is a large amount of non-serotonergic neurons which also are projecting neurons, indicating that a part of the raphe fibers projecting to hippocampus and septum may be non-serotonergic. Our aim was to determine whether there is a non-serotonergic projection from the raphe nucleus onto calbindin- and parvalbumin-containing neurons in hippocampus and septum. Biotin dextran amine was used as the anterograde neuronal tracer and injected into either dorsal or median raphe nucleus. By use of triple immunofluorescence-labeling we analyzed the serotonergic content of the biotin dextran amine-labeled fibers contacting parvalbumin- and calbindin-positive neurons. Surprisingly, we found a significant non-serotonergic projection from both dorsal and median raphe nuclei onto calbindin- and parvalbumin-containing interneurons in septum and hippocampus, with a preference in hippocampus for projecting onto calbindin-positive neurons. These results indicate that the raphe nuclei may exert their control on hippocampal and septal activity not only through a serotonergic projection, but also through a significant non-serotonergic pathway.     

Fos expression in serotonergic neurons in the rat brainstem following noxious stimuli: an immunohistochemical double-labelling study

In order to detect whether there were different expression patterns of Fos protein induced by somatic or visceral noxious stimulation in the serotonergic neurons in the rat brainstem, an immunohistochemical double-labelling technique for serotonin (5-HT) and Fos was employed after subcutaneous or stomach injection of formalin. The two stimuli were matched in pilot experiments to produce maximum Fos expression. The expression of Fos protein in 5-HT-containing neurons (5-HT/Fos co-localized neurons) could be observed in the ventrolateral subdivision of the midbrain periaqueductal grey, interpeduncular nucleus, paramedian raphe nucleus, all of the brainstem raphe nuclei, the alpha part of the gigantocellular reticular nucleus and the lateral paragigantocellular reticular nucleus. The locations of the 5-HT/Fos co-localized neurons in the brainstem of animals subjected to somatic noxious stimulation were similar to those subjected to visceral noxious stimulation. However, the number and proportion of the 5-HT/Fos co-localized neurons in the median raphe nucleus and nucleus raphe obscurus of the rat subjected to visceral noxious stimulation were statistically greater than those in rats subjected to somatic noxious stimulation. These results suggest that serotonergic neurons in median raphe nucleus and nucleus raphe obscurus have a tendency to higher neuronal activity after visceral noxious stimulation.     

Origin of projections from the midbrain raphe nuclei to the hypothalamic paraventricular nucleus in the rat: A combined retrograde and anterograde tracing study

A number of neuronal functions governed by the hypothalamic paraventricular nucleus are influenced by serotonin, and it is generally believed that the moderate density of serotonin-immunoreactive fibres and terminals within the paraventricular nucleus originates from the midbrain dorsal and median raphe nuclei. To further evaluate the intricate anatomy of projections from brain stem raphe nuclei of the rat, a combination of retrograde and anterograde tracing experiments were conducted to determine the medullary raphe nuclei projection to the paraventricular nucleus. Rhodamine-labelled latex microspheres, Cholera toxin subunit B and FluoroGold we used as retrograde tracers. Intracerebroventricular injections into the third ventricle of all retrograde tracers labelled a distinct population of neurons in the dorsal raphe situated in the subependymal stratum adjacent to the cerebral aqueduct indicating that these cells take up the tracer from the cerebrospinal fluid. Very few retrogradely labelled neurons were seen in the median raphe after i.c.v. administration of the tracers. Retrograde tracers delivered into the medial part of the paraventricular nucleus labelled no further cells in the midbrain dorsal and median raphe nuclei, whereas a substantial number of retrogradely labelled cells emerged in the pontine raphe magnus. However, when the retrograde tracers were delivered into the lateral part of the paraventricular nucleus, avoiding leakage of the tracer into the ventricle, very few labelled neurons were seen in the dorsal and median raphe, whereas the prominent labelling of raphe magnus neurons persisted. The anatomical organization of nerve fibres terminating in the area of paraventricular nucleus originating from midbrain raphe nuclei was studied in a series of anterograde tracing experiments using the plant lectin Phaseolus vulgaris leucoagglutinin. Injections delivered into the dorsal raphe or median raphe labelled but a few fibres in the paraventricular nucleus proper. A high number of fine calibered nerve fibres overlying the ependyma adjacent to the paraventricular nucleus was, however, seen after the injections into the subependymal rostral part of the dorsal raphe. Injections delivered into the raphe magnus gave rise to a dense plexus of terminating fibres in the parvicellular parts of the paraventricular nucleus and moderately innervated the posterior magnocellular part of the paraventricular nucleus as well as the magnocellular supraoptic nucleus. Concomitant visualization of serotonin-immunoreactive neurons and retrograde FluoroGold-tracing from the paraventricular nucleus revealed that none of the serotonergic neurons of the raphe magnus projects to this nucleus, while a few of the neurons putatively projecting to the paraventricular nucleus from the median raphe are serotonergic.

The current observations suggest that the raphe magnus constitute by far the largest raphe input to the paraventricular nucleus and strongly questions the earlier held view that most raphe fibres innervating the paraventricular nucleus are derived from the midbrain dorsal and median raphe. However, the source of serotonergic innervation of the paraventricular nucleus remains elusive.     

Pattern of distribution of serotonergic fibers to the thalamus of the rat

It is well established that serotonergic (5-hydroxytryptamine, 5-HT) fibers, mainly originating from the dorsal and median raphe nuclei of the brainstem, distribute throughout the forebrain, most heavily to ‘limbic’ forebrain structures. Few reports have examined the distribution of 5-HT fibers to the thalamus and none to our knowledge using immunoprocedures for the detection of the serotonin transporter (SERT)—a very sensitive marker for 5-HT fibers. Using immunohistochemical methods for SERT, we examined the pattern of distribution of 5-HT fibers to the thalamus in the rat. We show that serotonergic fibers are heavily concentrated in midline, intralaminar and association nuclei of the thalamus, and with the exception of the lateral geniculate complex, weakly distributed to principal nuclei of thalamus. Specifically, we demonstrate that 5-HT fibers are densely concentrated in the anteroventral, anteromedial and interanteromedial nuclei of the anterior thalamus, the paraventricular, rhomboid and reuniens nuclei of the midline thalamus, the central medial and central lateral nuclei of the intralaminar thalamus, the intermediodorsal nucleus, the lateral dorsal nucleus, and the dorsal and ventral lateral geniculate nuclei and intergeniculate leaflet of the LGN complex. Less densely innervated sites include the mediodorsal, paracentral, parafascicular, lateral posterior and submedial nuclei of thalamus. Remaining regions of the thalamus, largely consisting of principal nuclei, contained few 5-HT fibers. This pattern of 5-HT innervation indicates that serotonin/serotonergic fibers mainly affect thalamic nuclei with connections to ‘non-principal’ or limbic regions of the cortex (or forebrain). This suggests that serotonergic fibers to the thalamus may exert a significant influence on affective and cognitive functions, possibly complementing the actions of 5-HT fibers to other parts of the brain involved in emotional and cognitive behaviors.     

In vivo assessment of the midbrain raphe nuclear regulation of serotonin release in the hamster suprachiasmatic nucleus

Serotonin (5-HT) plays important regulatory roles in mammalian circadian timekeeping; however, little is known concerning the regulation of serotonergic activity in the circadian clock located in the suprachiasmatic nuclei (SCN). By using in vivo microdialysis to measure 5-HT release we demonstrated that electrical or pharmacological stimulations of the dorsal or median raphe nuclei (DRN and MRN, respectively) can alter basal release of 5-HT in the hamster SCN. There were similar increases in SCN 5-HT release after electrical stimulation of either the MRN or DRN, indicating that both could contribute to the serotonergic activity in the SCN. Systemic pretreatment with the 5-HT antagonist metergoline abolished DRN-induced SCN 5-HT release but had little effect on MRN-induced SCN 5-HT release, suggesting different pathways for these nuclei in regulating 5-HT output in the SCN. Microinjections of the 5-HT1A autoreceptor agonist 8-OH-DPAT or antagonist WAY 100635 into the MRN caused significant inhibition and stimulation of SCN 5-HT release, respectively. Both drugs had substantially less effect in the DRN. These differential drug actions indicate that somatodendritic 5-HT1A autoreceptors on MRN neurons provide the prominent raphe autoregulation of 5-HT output in the SCN. Collectively the current results are evidence that DRN as well as MRN neurons can contribute to the regulation of 5-HT release in the hamster SCN. On the basis of the current observations and those from recent anatomic tracing studies of serotonergic projections to SCN it is hypothesized that DRN input to the SCN could be mediated by a DRN --> MRN --> SCN pathway involving a 5-HT-sensitive multisynaptic interaction between the DRN and MRN neurons.