Brainstem Raphe Nuclei Research Articles

Transcranial sonography in obsessive–compulsive disorder

There is convergent evidence that basal ganglia structures are involved in the pathogenesis of obsessive–compulsive disorder (OCD). It has been also assumed that OCD is caused by a central serotonergic dysfunction. Transcranial sonography (TCS) has become a reliable, sensitive and non-invasive diagnostic tool concerning the evaluation of extrapyramidal movement disorders. This study used TCS to examine the alterations in different parenchymal regions, especially concerning serotonergic brainstem raphe nuclei as well as basal ganglia in OCD. Thirty-one OCD patients were compared with 31 matched healthy controls. Echogenecities were investigated according to the examination protocol for extrapyramidal disorders using a Siemens Sonoline® Elegra system. Obsessive–compulsive disorder patients showed reduced echogenity of the serotonergic brainstem raphe nuclei (32.3%) compared with healthy controls (16.1%). In nine OCD-patients (31%), but only in 2 control subjects (6.2%), a hyperechogenicity of the caudate nucleus was found. Patients with OCD significantly more often reveal a hypoechogenic brainstem raphe possibly reflecting altered serotonergic neurons there and a hyperechogenicity of caudate nucleus indicating structural or molecular cell changes. Further research is warranted to examine, whether TCS is useful in order to classify OCD and its subtypes.

HIGHER IN VIVO SEROTONIN-1A BINDING IN POSTTRAUMATIC STRESS DISORDER: A PET STUDY WITH [11C]WAY-100635

Brain serotonin-1A receptors (5-HT1A ) are implicated in anxiety. We compared regional brain 5-HT1A binding in medication-free participants with posttraumatic stress disorder (PTSD) and healthy volunteers using fully quantitative positron emission tomography (PET) methods. Twenty patients with DSM-IV PTSD (13 with comorbid major depressive disorder, [MDD]) and 49 healthy volunteers underwent PET imaging with 5-HT1A antagonist radioligand [C-11]WAY100635. Arterial blood sampling provided a metabolite-corrected input function and the concentration of free ligand in plasma (fP ) for estimation of regional binding potential, BPF ( = Bavailable / KD ). Linear mixed modeling compared BPF between groups across regions of interest (ROIs). The PTSD group had higher 5-HT1A BPF across brain ROIs (P = .0006). Post hoc comparisons showed higher 5-HT1A BPF in PTSD in all cortical ROIs (26-33%), amygdala (34%), and brainstem raphe nuclei (43%), but not hippocampus. The subgroup of seven PTSD patients without comorbid MDD had higher 5-HT1A BPF compared with healthy volunteers (P = .03). This is the first report of higher brainstem and forebrain 5-HT1A binding in vivo in PTSD. The finding is independent of MDD. PTSD and MDD have in common an upregulation of 5-HT1A binding including midbrain autoreceptors that would favor less firing and serotonin release. This abnormality may represent a common biomarker of these stress-associated brain disorders.

Lack of serotonin reuptake during brain development alters rostral raphe-prefrontal network formation

Besides its “classical” neurotransmitter function, serotonin (5-HT) has been found to also act as a neurodevelopmental signal. During development, the 5-HT projection system, besides an external placental source, represents one of the earliest neurotransmitter systems to innervate the brain. One of the targets of the 5-HT projection system, originating in the brainstem raphe nuclei, is the medial prefrontal cortex (mPFC), an area involved in higher cognitive functions and important in the etiology of many neurodevelopmental disorders. Little is known, however, about the exact role of 5-HT and its signaling molecules in the formation of the raphe-prefrontal network. Using explant essays, we here studied the role of the 5-HT transporter (5-HTT), an important modulator of the 5-HT signal, in rostral raphe-prefrontal network formation. We found that the chemotrophic nature of the interaction between the origin (rostral raphe cluster) and a target (mPFC) of the 5-HT projection system was affected in rats lacking the 5-HTT (5-HTT−/−). While 5-HTT deficiency did not affect the dorsal raphe 5-HT-positive outgrowing neurites, the median raphe 5-HT neurites switched from a strong repulsive to an attractive interaction when co-cultured with the mPFC. Furthermore, the fasciculation of the mPFC outgrowing neurites was dependent on the amount of 5-HTT. In the mPFC of 5-HTT−/− pups, we observed clear differences in 5-HT innervation and the identity of a class of projection neurons of the mPFC. In the absence of the 5-HTT, the 5-HT innervation in all subareas of the early postnatal mPFC increased dramatically and the number of Satb2-positive callosal projection neurons was decreased. Together, these results suggest a 5-HTT dependency during early development of these brain areas and in the formation of the raphe-prefrontal network. The tremendous complexity of the 5-HT projection system and its role in several neurodevelopmental disorders highlights the need for further research in this largely unexplored area.

Impaired maturation of serotonergic function in the dentate gyrus associated with epilepsy

Temporal lobe epilepsy is believed to develop after an initial precipitating injury, usually suffered in childhood or adolescence, and aspects include impaired maturation of the hippocampus, and specifically the dentate gyrus. The dentate gyrus receives a major serotonergic input from the brainstem raphe nuclei, and the serotonergic system may regulate neurogenesis in the developing and mature hippocampus. The aim of this work was to investigate changes which may be associated with abnormal functioning of the serotonergic system in the pilocarpine model of epilepsy, where spontaneous seizures are induced by administration of pilocarpine at 6weeks of age. Application of serotonin (100μM) led to a transient hyperpolarization of the resting membrane potential and decrease of the input resistance mediated by the 5-HT1A receptor that was similar between control and pilocarpine-treated animals and unaffected by the age of the animal. In the younger, but not in older control animals, serotonin led to a 5-HT2 receptor-mediated long-term depression of evoked postsynaptic currents, a normal functional shift in the early adulthood of the Wistar rat. In pilocarpine-treated animals, this long-term depression persisted in older animals, indicating impaired maturation of the dentate gyrus. These data may indicate 5-HT2 receptor function to be affected by the pathology of temporal lobe epilepsy.

Study on the Property Change of Processed Rhizoma Coptidis Based on 5-HT Level and Brain Tissues Morphology of Rats

All Chinese medicinal substances have a special property (cold, hot, warm or cool) according to the theories of Traditional Chinese Medicine (TCM). Processing of Chinese Materia Medica ( pao zhi ) is always applied to modify the property according to clinical needs in TCM. The purpose of this paper was to evaluate the processing effects on the property of Rhizoma Coptidis (RC). The wine-processed RC (WPRC, with less cold property in TCM) and bile-processed RC (BPRC, with colder property in TCM) were prepared, the level of 5-hydroxytryptamine (5-HT) of rats was determined by fluorescence spectrophotometry and the morphology of rats’ brain tissue was analyzed by immunohistochemistry. Compared to crude RC treated group, the distribution of 5-HT-immunopositive neurons and cell staining in the brainstem raphe nuclei were remarkably denser and deeper in BPRC treated group, while the distribution of 5-HT-immunopositive neurons was sparse and the cell staining was lighter in WPRC group. The work provided a meaningful way in the research of processing effect on properties of Chinese medicinal substances.

OP.01 The neurobiology of sleep

It has been established in the course of the past decade that the level of arousal at any time reflects an intricate interplay between a number of distinct wakefulness-promoting and...

Role of serotonin transporter inhibition in the regulation of tryptophan hydroxylase in brainstem raphe nuclei: time course and regional specificity

Drugs that selectively inhibit the serotonin transporter (SERT) are widely prescribed for treatment of depression and a range of anxiety disorders. We studied the time course of changes in tryptophan hydroxylase (TPH) in four raphe nuclei after initiation of two different SERT inhibitors, citalopram and fluoxetine. In the first experiment, groups of Sprague–Dawley rats received daily meals of rice pudding either alone (n=9) or mixed with citalopram 5 mg/kg/day (n=27). Rats were sacrificed after 24 h, 7 days or 28 days of treatment. Sections of dorsal raphe nucleus (DRN), median raphe nucleus (MRN), raphe magnus nucleus (RMN) and caudal linear nucleus (CLN) were processed for TPH immunohistochemistry. Citalopram induced a significant reduction in DRN TPH-positive cell counts at 24 h (41%), 7 days (38%) and 28 days (52%). Similar reductions in TPH-positive cell counts were also observed at each timepoint in the MRN and in the RMN. In the MRN, citalopram resulted in significant reductions at 24 h (26%), 7 days (16%) and 28 days (23%). In the RMN, citalopram induced significant reductions of TPH-positive cell counts at 24 h (45%), 7 days (34%) and 28 days (43%). By contrast, no significant differences between control and treatment groups were observed in the CLN at any of the time points that we studied. To investigate whether these changes would occur with other SERT inhibitors, we conducted a second experiment, this time with a 28-day course of fluoxetine. As was observed with citalopram, fluoxetine induced significant reductions of TPH cell counts in the DRN (39%), MRN (38%) and RMN (41%), with no significant differences in the CLN. These results indicate that SERT inhibition can alter the regulation of TPH, the rate limiting enzyme for serotonin biosynthesis. This persistent and regionally specific downregulation of serotonin biosynthesis may account for some of the clinical withdrawal symptoms associated with drugs that inhibit SERT.

Role of Central 5‐Hydroxytryptamine Receptors in Micturition Control

5‐Hydroxytryptamine (5‐HT) receptors in the central nervous system have been implicated in the control of micturition. In rats and cats, spinal and supraspinal reflex pathways involved in lower urinary tract (LUT) function exhibit dense serotonergic innervation and multiple 5‐HT receptors. At present, the most investigated 5‐HT receptor that has been shown to play a role in the control of micturition is the 5‐HT1A receptor, followed by 5‐HT7, 5‐HT2, and 5‐HT3 receptors. Most studies focus on the effects of these receptors on the parasympathetic outflow to the bladder and the somatic outflow to the external urethral sphincter (EUS) in rats. Earlier studies have shown that the activation of 5‐HT‐containing brain areas (the raphe nuclei in the caudal brainstem) inhibits bladder activity. However, 5‐HT1A and 5‐HT7 receptors have recently been suggested to have an excitatory role in the control of bladder function. 5‐HT1A receptors act at both a spinal and supraspinal level, while 5‐HT7 receptors act only at a supraspinal level. The activation of 5‐HT2A receptors in rats results in an increase in EUS activity. Overall, the pharmacological manipulation of the central serotonergic system can be a promising treatment strategy for LUT dysfunction. However, more studies are mandatory to fully understand the role of multiple 5‐HT receptors in the control of LUT function.

Nonserotonergic projection neurons in the midbrain raphe nuclei contain the vesicular glutamate transporter VGLUT3

The brainstem raphe nuclei are typically assigned a role in serotonergic brain function. However, numerous studies have reported that a large proportion of raphe projection cells are nonserotonergic. The identity of these projection cells is unknown. Recent studies have reported that the vesicular glutamate transporter VGLUT3 is found in both serotonergic and nonserotonergic neurons in both the median raphe (MR) and dorsal raphe (DR) nuclei. We injected the retrograde tracer cholera toxin subunit B into either the dorsal hippocampus or the medial septum (MS) and used triple labeled immunofluorescence to determine if nonserotonergic raphe cells projecting to these structures contained VGLUT3. Consistent with previous studies, only about half of retrogradely labeled MR neurons projecting to the hippocampus contained serotonin, whereas a majority of the retrogradely labeled nonserotonergic cells contained VGLUT3. Similar patterns were observed for MR cells projecting to the MS. About half of retrogradely labeled nonserotonergic neurons in the DR contained VGLUT3. Additionally, a large number of retrogradely labeled cells in the caudal linear and interpeduncular nuclei projecting to the MS were found to contain VGLUT3. These data suggest the enigmatic nonserotonergic projection from the MR to forebrain regions may be glutamatergic. In addition, these results demonstrate a dissociation between glutamatergic and serotonergic MR afferent inputs to the MS and hippocampus suggesting divergent and/or complementary roles of these pathways in modulating cellular activity within the septohippocampal network.

Regulation of Corticolimbic Reactivity via the 5-HT 1A Autoreceptor in the Pathophysiology and Treatment of Depression

According to the National Institute of Mental Health, major depressive disorder (MDD) is among the leading causes of mortality and disability in the USA. In a given year, nearly 7% of Americans experience at least one clinically significant episode of MDD, manifested by a combination of symptoms that interfere with the ability to work, study, sleep, eat and enjoy once pleasurable activities. While an MDD episode may occur only once, they are typically recurring and result in lifetime disability. Although there are a number of widely applied pharmacological and psychological approaches to the treatment of MDD, the efficacy of these is poor, with less than 50% of individuals experiencing a therapeutic response [1]. Combination therapies may lead to higher rates of therapeutic response for a few individuals, but many others will experience severe side effects leading to treatment discontinuation [2]. In light of the immense personal and public health burden of MDD, the existing poor efficacy of available treatments has precipitated dedicated research efforts to better understand the pathophysiology of MDD with the aim of identifying predictive markers of disease liability and generating more effective intervention. Although the neurobiological mechanisms through which disturbances in emotional behavior, mood and temperament emerge and contribute to the development of MDD are not wholly understood, a myriad of converging evidence inextricably links these phenomena with functioning of the central serotonin (5-HT) system and its inter-related subsystems. Multiple mechanisms contribute to the regulation of 5-HT neurotransmission and variability in these regulatory mechanisms is associated with individual differences in temperament and risk for MDD. Key among these is the 5-HT type 1A (5-HT1A) somatodendritic autoreceptor, which is expressed on a population of serotonergic neurons projecting from the brainstem raphe nuclei to cortical and limbic target regions. The 5-HT1A autoreceptor mediates negative-feedback regulation of 5-HT neurons through G-protein coupled signaling cascades, resulting in diminished neuronal activity and subsequent 5-HT release at postsynaptic targets. These postsynaptic targets include the amygdala and prefrontal cortex, key nodes of a corticolimbic circuitry supporting emotional reactivity and control. In the context of emotional behavior, the subcortical amygdala mediates rapid physiological and behavioral arousal in response to salient, biological stimuli (e.g., threat-related facial expressions, such as anger and fear). In addition to regulating the activity of numerous hypothalamic and brainstem autonomic centers, the amygdala can stimulate the activity of the ventromedial and orbitofrontal cortices [3]. These prefrontal regions are critical for the integration of amygdala-mediated arousal in the service of complex, adaptive behavioral responses to environmental challenges [4,5]. The subsequent stimulation of dorsomedial, ventrolateral and dorsolateral prefrontal regions, resulting from such engagement of the ventromedial and orbitofrontal cortices, then precipitates top-down inhibition of the amygdala [6–8]. The coordinated activity within this distributed corticolimbic circuitry largely governs emotional arousal and regulation, and dysfunction of circuit components are pathological markers of MDD [9,10].

Specification and differentiation of serotonergic neurons

Serotonin is an important neurotransmitter with multiple functions in the whole central nervous system. Its synthesis, however, is restricted to a very limited number of cells in the brainstem raphe nuclei with a vast axonal network. These cells express markers of the serotonin lineage such as the rate-limiting enzyme in serotonin synthesis, tryptophan hydroxylase 2, the serotonin transporter, and the transcription factor Pet1. Pet1 together with Lmx1b, Nkx2.2, Mash1, Gata2, Gata3, and Phox2b form a transcriptional network, which specifies the differentiation of serotonergic neurons around embryonic day 11 in the mouse. These cells are generated in rhombomeres r1-r3 and r5-r7 caudal to the midbrain- hindbrain organizer under the control of the fibroblast growth factors 4 and 8 and sonic hedgehog (SHH) from precursors, which have produced motoneurons before. Because serotonin is a relevant pathophysiological factor in several neurological diseases such as bipolar disorder and depression tools to generate or maintain serotonergic neurons might be of therapeutic value. Such tools can be assessed in embryonic stem cells, which can be differentiated in vitro to produce serotonergic neurons. Culture systems for these cells including embryoid bodies based and monolayer differentiation have been established, which allows the generation of up to 50% serotonergic neurons in all neurons developed.

Anatomic and functional topography of the dorsal raphe nucleus.

Serotonergic systems play an important and generalized role in regulation of sleep-wake states and behavioral arousal. Recent in vivo electrophysiologic recording studies in animals suggest that several different subtypes of serotonergic neurons with unique behavioral correlates exist within the brainstem raphe nuclei, raising the possibility that topographically organized subpopulations of serotonergic neurons may have unique behavioral or physiologic correlates and unique functional properties. We have shown that the stress-related and anxiogenic neuropeptide corticotropin-releasing factor can stimulate the in vitro neuronal firing rates of topographically organized subpopulations of serotonergic neurons within the dorsal raphe nucleus (DR). These findings are consistent with a wealth of behavioral studies suggesting that serotonergic systems within the DR are involved in the modulation of ongoing anxiety-related behavior and in behavioral sensitization, a process whereby anxiety- and fear-related behavioral responses are sensitized for a period of up to 24 to 48 h. The dorsomedial subdivision of the DR, particularly its middle and caudal aspects, has attracted considerable attention as a region that may play a critical role in the regulation of acute and chronic anxiety states. Future studies aimed at characterization of the molecular and cellular properties of topographically organized subpopulations of serotonergic neurons are likely to lead to major advances in our understanding of the role of serotonergic systems in stress-related physiology and behavior.

Robust and tissue-specific expression of TPH2 versus TPH1 in rat raphe and pineal gland

Background Regulation of raphe serotonergic cells is fundamental to the prevailing hypothesis of major depression pathophysiology. Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in serotonin biosynthesis, but brainstem TPH mRNA expression has been difficult to measure and study. Recently, a novel paralog of TPH, TPH2 (or neuronal TPH), was described, but its anatomic expression is unknown. Methods In situ hybridization histochemical survey was conducted across Sprague-Dawley rat brain for TPH1 and TPH2 mRNA. Semiquantitative techniques were used to estimate relative mRNA levels in individual cells. Results Almost exclusively, TPH2 mRNA is expressed in raphe, in a pattern overlapping the histologically defined raphe nuclei. In sharp contrast, TPH1 (the previously known TPH) is expressed predominantly in pineal gland. There is no appreciable overlap in the expression of these paralogs. The level of TPH2 mRNA expression in individual raphe cells is approximately 2.5-fold greater than the level of TPH1 expression in pinealocytes. Conclusions TPH2 mRNA has an anatomic expression pattern consistent with brainstem raphe nuclei and is likely to be the gene giving rise to the majority of TPH activity in these cells. The robust expression of TPH2 in brainstem should facilitate studies on the transcriptional regulation of raphe serotonin biosynthesis.

Symptomatic migraine and sensitization of trigeminal nociception associated with contralateral pontine cavernoma

A 38-year-old woman is described with symptomatic strictly right-sided migraine associated with a pontine cavernoma affecting the contralateral (left) nucleus raphe magnus. A persistent facilitation of the right-sided trigeminal nociception was detected interictally using the ‘nociception specific’ blink reflex, which was more pronounced during the acute attack. This case shows for the first time, an impairment of the anti-nociceptive brainstem nuclei and the facilitation of the trigeminal nociception in the same subject, thus providing further evidence for the key role of the brainstem raphe nuclei in the pathophysiology of migraine.

Origins of GABA B receptor-like immunoreactive terminals in the rat spinal dorsal horn

By means of immunohistochemistry for γ-aminobutyric acid receptor B subtype (GABA BR), the origins of GABA BR-like immunoreactive (GABA BR-LI) terminals in the rat spinal dorsal horn were investigated. After dorsal root rhizotomy and/or spinal cord hemisection, the densities of GABA BR-LI terminals were remarkably depleted in the ipsilateral superficial dorsal horn of relevant segments, whereas GABA BR-LI neurons and sparsely distributed GABA BR-LI terminals remained. After injection of Fluoro-Gold (FG) into the left side of superficial lumbar dorsal horn, FG retrograde-labeled neurons were mainly observed in the ipsilateral rostral ventromedial medulla (RVM) and brainstem raphe nuclei. Some of the FG-labeled neurons, especially in the RVM, exhibited GABA BR-like immunoreactivity. Additionally, immunofluorescence histochemical double-staining revealed that the majority of GABA BR-LI neurons in the periaqueductal gray (PAG), RVM and brainstem raphe nuclei showed 5-hydroxytryptamine (5-HT)-like immunoreactivity. The present study morphologically proves that GABA BR-LI terminals in the spinal dorsal horn originate from peripheral afferents, intrinsic neurons and supraspinal structures; GABA BR and 5-HT co-exist in many neurons in the PAG, RVM and brainstem raphe nuclei. Considering that PAG, RVM, brainstem raphe nuclei and spinal dorsal horn are important structures involved in the pain modulation, we suggest that the descending pain modulation system might be mediated, at least in part, by GABA BR.

5-HT3Receptors Mediate Serotonergic Fast Synaptic Excitation of Neocortical Vasoactive Intestinal Peptide/Cholecystokinin Interneurons

Neocortical neurons expressing the serotonin 5-HT3 receptor (5-HT3R) were characterized in rat acute slices by using patch-clamp recordings combined with single-cell RT-PCR and histochemical labeling. The 5-HT3A receptor subunit was expressed selectively in a subset of GABAergic interneurons coexpressing cholecystokinin (CCK) and vasoactive intestinal peptide (VIP). The 5-HT3B subunit was never detected, indicating that 5-HT3Rs expressed by neocortical interneurons did not contain this subunit. In 5-HT3A-expressing VIP/CCK interneurons, serotonin induced fast membrane potential depolarizations by activating an inward current that was blocked by the selective 5-HT3R antagonist tropisetron. Furthermore, we observed close appositions between serotonergic fibers and the dendrites and somata of 5-HT3R-expressing neurons, suggestive of possible synaptic contacts. Indeed, in interneurons exhibiting rapid excitation by serotonin, local electrical stimulations evoked fast EPSCs of large amplitude that were blocked by tropisetron. Finally, 5-HT3R-expressing neurons were also excited by a nicotinic agonist, indicating that serotonergic and cholinergic fast synaptic transmission could converge onto VIP/CCK interneurons. Our results establish a clear correlation between the presence of the 5-HT3A receptor subunit in neocortical VIP/CCK GABAergic interneurons, its functional expression, and its synaptic activation by serotonergic afferent fibers from the brainstem raphe nuclei.

Olfactory ensheathing cells promote locomotor recovery after delayed transplantation into transected spinal cord.

We demonstrated recently that transplantation of olfactory ensheathing cells from the nasal olfactory mucosa can promote axonal regeneration after complete transection of the spinal cord in adult rat. Ten weeks after transection and transplantation there was significant recovery of locomotor behaviour and restoration of descending inhibition of spinal cord reflexes, accompanied by growth of axons across the transection site, including serotonergic axons arising from the brainstem raphe nuclei. The present experiment was undertaken to determine whether olfactory ensheathing cells from the olfactory mucosa are capable of promoting regeneration when transplanted into the spinal cord 4 weeks after transection. Under general anaesthesia, thoracic spinal cord at the T10 level was transected completely in adult rats. Four weeks later, the scar tissue and cavities at the transection site were removed to create a 3-4 mm gap. Into this gap, between the cut surfaces of the spinal cord, pieces of olfactory lamina propria were placed. Ten weeks later, the locomotor activity of these animals was significantly improved compared with control animals, which received implants of either pieces of nasal respiratory lamina propria or collagen (Basso, Beattie, Bresnahan Locomotor Rating Scale scores 4.3 + 0.8, n = 6 versus 1.0 + 0.2, n = 10, respectively; P < 0.001). Ten weeks after transplantation the behavioural recovery was still improving. Regrowth of brainstem raphe axons across the transplant site was shown by the presence of serotonergic axons in the spinal cord caudal to the transection site, and by retrograde labelling of cells in the nucleus raphe magnus after injections of fluorogold into the caudal spinal cord. Neither serotonergic axons nor labelled brainstem cells were observed in the control animals. These results indicate that olfactory ensheathing cells from the nasal olfactory lamina propria have the ability to promote spinal cord regeneration when transplanted 4 weeks after complete transection. Olfactory ensheathing cells are accessible and available in the human nose; the present study further supports clinical use of these cells in repairing the human spinal cord via autologous transplantation.

Orexin A-like immunoreactivity in the hypothalamus and thalamus of the Syrian hamster (Mesocricetus auratus) and Siberian hamster ( Phodopus sungorus), with special reference to circadian structures

The orexins are recently discovered neuropeptides that reportedly play a role in energy homeostasis, in addition to various other physiological processes. The synthesis of orexin A undergoes diurnal variation in certain areas of the brain, while the mutation of the orexin receptor 2 gene has been implicated in canine narcolepsy. Since the circadian pacemaker in the suprachiasmatic nucleus modulates the sleep/wake cycle, there is a putative role for orexins in the mammalian circadian system. In this study, immunohistochemical techniques were used to determine the distribution of orexin A in the structures of the hypothalamus and thalamus of Syrian and Siberian hamsters. In both species, the pattern of immunoreactivity was similar. Cells immunoreactive for orexin A were noted in the lateral hypothalamic area. Immunoreactive varicose orexin A fibres were found throughout the hypothalamus. The suprachiasmatic nucleus possessed little or no immunoreactive orexin A fibres in its core, but had fibres at its periphery. The thalamus of both species contained comparatively few immunoreactive fibres, which were mainly localised around the midline. The thalamic intergeniculate leaflet contained a plexus of immunoreactive orexin A fibres throughout its rostro-caudal extent. Three areas of the brainstem, the dorsal and median raphe nuclei and the locus coeruleus, were also investigated owing to their relevance to the circadian system and all were found to contain immunoreactive orexin A fibres. The presence of orexin A-immunoreactive fibres in the neural architecture of the mammalian circadian system suggests an important role for orexin A in circadian timekeeping processes.

Evidence of the synthesis of opioid receptor like 1 receptor in nociceptinergic neurons in rat brain suggests the existence of autoreceptor: a confocal double staining study

Opioid receptor like 1 (ORL1) receptor is a novel member of the opioid receptor family, which was not bound by any of the typical opioid receptor ligands but bound by the recently discovered nociceptin (also termed orphanin FQ) with high affinity. By using double staining of fluorescent in situ hybridization and immunohistochemistry, we observed the expression of ORL1 receptor mRNA in nociceptin-like immunoreactive neurons in multiple areas in rat brain including the hippocampus, arcuate nucleus in the hypothalamus, ventralateral periaquiductal gray (PAG) and raphe nuclei in brain stem. The expression of ORL1 mRNA in nociceptinergic neurons suggests that these receptors mediate, at least in part, the presynaptic autoreceptor functions. Further anatomical and functional significance of the autoreceptor of nociceptinergic neurons remain to be elucidated.

Serotonin-immunoreactive axons in the cell column of sympathetic preganglionic neurons in the spinal cord of the filefish Stephanolepis cirrhifer

Serotonin-immunoreactive axonal components were observed in the central autonomic nucleus (CAN), a cell column of sympathetic preganglionic neurons in the rostral spinal cord of the filefish Stephanolepis cirrhifer. Serotonin-positive axonal varicosities were seen around neuronal perikarya through the whole rostrocaudal extent of the CAN, although their distribution pattern in the rostral CAN was different from that in the caudal CAN. Electron microscopically, serotonin-positive axonal varicosities were found to make axodendritic and axosomatic synapses on CAN neurons. Many serotonin-positive neuronal cell bodies were seen in the raphe nuclei in the lower brainstem, whereas only a few were found in the spinal cord. Thus most of serotoninergic axons within the CAN were considered to originate from the raphe nuclei in the lower brainstem.