Serotonergic influence from nucleus raphe obscurus on neurones in the periaqueductal grey matter in the rat

In rats anaesthetised with urethane, iontophoretic application of 5-hydroxytryptamine (5-HT, 5–70 nA) produced changes in ongoing activity of 41/44 neurones in the periaqueductal grey matter (PAG). The majority (85%) of responsive cells were inhibited and 15% were excited. The inhibitions were mimicked in 5/7 cells by iontophoresis of the 5-HT_1A agonist8-hydroxy-2-(di-n-proplyamino) tetralin (8-OH-DPAT, 10–30 nA) whilst excitation was produced in 3/5 cells by iontophoresis of the 5-HT₂ agonist α-methyl-5-HT (10–30 nA). Selective activation of neuronal perikarya in nucleus raphe obscurus (NRO) by microinjection of 50–100 nl,D,L-homocysteic acid (DLH) inhibited ongoing activity of 25/31 neurones tested in the PAG for periods of 30–580 s, mean 183.5 s. The duration of the inhibition was potentiated by between 36 and 300% during iontophoresis of the 5-HT re-uptake blocker paroxetine (1–25 nA, 6/6 cells). The results indicate that there is an extensive inhibitory serotonergic input to the PAG which originates, at least in part, from NRO.     

GABAergic projection from the ventral tegmental area and substantia nigra to the periaqueductal gray region and the dorsal raphe nucleus

Previous studies have shown that neurons in the ventral tegmental area (VTA) and substantia nigra (SN) project to the ventrolateral periaqueductal gray (PAGvl) and dorsal raphe nucleus (DR). Research has also shown that stimulation of neurons in the VTA/SN elicits cardiovascular depressor responses that are mediated by a projection to the PAGvl/DR. Anatomic and physiological experiments were done in the present study to determine the neurochemical identity of the VTA/SN projection to the PAGvl/DR. Experiments were done to characterize the origin and chemical nature of this projection by combining cholera toxin B tracing with immunofluorescence for the 67K isoform of glutamic acid decarboxylase (GAD) and tyrosine hydroxylase. The PAGvl/DR region was found to receive a substantial input from neurons in the VTA, SN, and deep mesencephalic nucleus. The DR was preferentially innervated by neurons in the VTA, whereas the PAGvl was preferentially innervated by neurons in the SN. A proportion of neurons in the VTA and the reticular portion of the SN found to project to the PAGvl/DR were GAD positive. In addition, experiments were done in urethane-anesthetized rats to determine whether injections of a gamma-aminobutyric acid (GABA) antagonist in the region of the PAGvl/DR attenuated the cardiovascular depressor responses produced by glutamate stimulation of the VTA/SN. Injections of the GABA-blocking agent picrotoxin (2.5 nmol, 500 nl) into the PAGvl/DR eliminated the cardiovascular responses from stimulation of the VTA/SN (0.01 M, 50 nl). The results of the present investigation provide evidence for a GABAergic projection from the VTA/SN to the PAGvl/DR. This projection may be an important regulator of the PAGvl/DR, an area of the midbrain involved in the production of behavioral and physiological responses to pain and stress.     

Cellular basis for the effects of substance P in the periaqueductal gray and dorsal raphe nucleus

Substance P (SP) is known to act at supraspinal sites to influence pain sensitivity as well as to promote anxiety. The effects of SP could be mediated in part by actions in the periaqueductal gray (PAG) and the dorsal raphe nucleus (DRN), adjoining mesencephalic cell groups that are strategically positioned to influence both nociception and mood. Previous studies have indicated that SP regulates both enkephalin and serotonin neurotransmission in these brain regions. To determine the mechanism underlying the effects of SP in the PAG and DRN, the distribution of the principal receptor for SP, the neurokinin 1 (NK1) receptor, was examined with respect to other neurotransmitter markers. PAG neurons that had NK1 receptor immunolabeling were interdigitated with and received contacts from enkephalin-containing neurons. However, only a few (16/144; 11%) neurons with NK1 receptor also contained enkephalin immunoreactivity after colchicine treatment. In the DRN, dendrites containing NK1 receptor were selectively distributed in the dorsomedial subdivision. The majority (132/137; 96%) of these dendrites did not contain immunoreactivity for the serotonin-synthesizing enzyme tryptophan hydroxylase. In contrast, neuronal profiles with NK1 receptor in both the PAG and the DRN often contained immunolabeling for glutamate. Light and electron microscopic examination revealed that 48-65% of cell bodies and dendrites with NK1 receptor were dually immunolabeled for glutamate. These data suggest that SP directly acts primarily on glutamatergic neurons in the PAG and DRN. To a lesser extent, enkephalin-containing neurons may be targeted. Through these actions, it may subsequently influence activity of larger populations of neurons containing enkephalin as well as serotonin. This circuitry could contribute to, as well as coordinate, effects of SP on pain perception and mood.     

Dorsal raphe and external electrical stimulation modulate noxious input to single neurons in nucleus parafascicularis thalami

Spontaneous discharges and nociceptive responses of 47 parafascicularis thalami (PF) neurons were recorded extracellularly and comparisons were made between the effects of these discharges following focal dorsal raphe stimulation (DRS) and bilateral pinnal electrical stimulation (PES). Eighty-three percent of PF neurons (N = 39) responded to noxious stimulus, about 69% of the PF responsive cells (N = 27) were excited during noxious stimuli and thus categorized as "nociceptive-on" cells. The remaining 31% (N = 12) were suppressed by the noxious stimuli, and were categorized as "nociceptive-off" cells. DRS and PES attenuated the spontaneous activity of the "nociceptive-on" neurons as well as the noxious input to these cells, while the spontaneous activity of the "nociceptive-off" cells was suppressed only following DRS and not following PES. Moreover, PES displayed disinhibiting properties, namely, it reduced the suppression effects elicited by noxious input. In conclusion, it was demonstrated that both focal DRS and noninvasive PES were effective in modulating pain input to single neurons in the PF.     

Changes of reactions of neurones in dorsal raphe nucleus and locus coeruleus to electroacupuncture by hypothalamic arcuate nucleus stimulation

In this experiment the role of the hypothalamic arcuate nucleus (ARC) in acupuncture analgesia and its mechanisms were studied with behavioural and electrophysiological methods. After ARC stimulation the analgesic effect of acupuncture was enhanced significantly and the responses of neurones to electroacupuncture were increased in the dorsal raphe nucleus (DR) and reduced in the locus coeruleus (LC), which could be reversed by intraperitoneal injection of naloxone. The results indicate that ARC might participate in acupuncture analgesia via changing the responses of DR and LC neurones to electroacupuncture, a process in which opiate-like substances (probably beta-endorphin) are involved.     

Responses of cat C1 spinal cord dorsal and ventral horn neurons to noxious and non-noxious stimulation of the head and face

Previous anatomical studies have shown that trigeminal and cervical afferent nerve fibers project to the upper cervical segments of the spinal cord. To determine the response properties of neurons in the upper cervical spinal cord, we studied the response of C1 dorsal and ventral horn cells to electrical and graded mechanical stimulation of the face, head and neck in anesthetized cats. Neurons were classified as low-threshold-mechanoreceptive (LTM), wide-dynamic-range (WDR), nociceptive-specific (NS) or unresponsive, based on their responsiveness to graded mechanical stimulation. Extracellular single unit recordings were obtained from 118 neurons excited by cervical (24), trigeminal (39) or both cervical and trigeminal (55) stimulation and from 24 neurons unresponsive to peripheral stimulation. Based on neuronal mechanical response properties, 52.2% of the responsive neurons were classified as LTM, 35.9% as WDR and 11.9% as NS. WDR neurons exhibited more convergence and had larger receptive fields than either NS or LTM neurons. WDR and NS neurons had longer first spike latencies than LTM neurons at all tested sites. Only WDR neurons were found to project to the contralateral caudal thalamus. Within C1, LTM neurons were located primarily in laminae III and IV, WDR neurons in lamina V and NS neurons in laminae VII and VIII. These data suggest that some neurons in the first cervical segment of the spinal cord receive convergent input from trigeminal and cervical pathways and may be involved in mediating orofacial and cranial pain.     

Tolerance effects of non-steroidal anti-inflammatory drugs microinjected into central amygdala, periaqueductal grey, and nucleus raphe Possible cellular mechanism

Pain is a sensation related to potential or actual damage in some tissue of the body.The mainstay of medical pain therapy remains drugs that have been around for decades,like non-steroidal anti-inflammatory drugs (NSAIDs),or opiates.However,adverse effects of opiates,particularly tolerance,limit their clinical use.Several lines of investigations have shown that systemic (intraperitoneal) administration of NSAIDs induces antinociception with some effects of tolerance.In this review,we report that repeated microinjection of NSAIDs analgin,clodifen,ketorolac and xefocam into the central nucleus of amygdala,the midbrain periaqueductal grey matter and nucleus raphe magnus in the following 4 days result in progressively less antinociception compared to the saline control testing in the tail-flick reflex and hot plate latency tests.Hence,tolerance develops to these drugs and cross-tolerance to morphine in male rats.These findings strongly support the suggestion of endogenous opioid involvement in NSAIDs antinociception and tolerance in the descending pain-control system.Moreover,the periaqueductal grey-rostral ventro-medial part of medulla circuit should be viewed as a pain-modulation system.These data are important for human medicine.In particular,cross-tolerance between non-opioid and opioid analgesics should be important in the clinical setting.     

Cellular effects of swim stress in the dorsal raphe nucleus

Swim stress regulates forebrain 5-hydroxytryptamine (5-HT) release in a complex manner and its effects are initiated in the serotonergic dorsal raphe nucleus (DRN). The purpose of this study was to examine the effects of swim stress on the physiology of DRN neurons in conjunction with 5-HT immunohistochemistry. Basic membrane properties, 5-HT(1A) and 5-HT(1B) receptor-mediated responses and glutamatergic excitatory postsynaptic currents (EPSCs) were measured using whole-cell patch clamp techniques. Rats were forced to swim for 15min and 24h later DRN brain slices were prepared for electrophysiology. Swim stress altered the resting membrane potential, input resistance and action potential duration of DRN neurons in a neurochemical-specific manner. Swim stress selectively elevated glutamate EPSC frequency in 5-HT DRN neurons. Swim stress non-selectively reduced EPSC amplitude in all DRN cells. Swim stress elevated the 5-HT(1B) receptor-mediated inhibition of glutamatergic synaptic activity that selectively targeted 5-HT cells. Non-5-HT DRN neurons appeared to be particularly responsive to the effects of a milder handling stress. Handling elevated EPSC frequency, reduced EPSC decay time and enhanced a 5-HT(1B) receptor-mediated inhibition of mEPSC frequency selectively in non-5-HT DRN cells. These results indicate that swim stress has both direct, i.e., changes in membrane characteristics, and indirect effects, i.e., via glutamatergic afferents, on DRN neurons. These results also indicate that there are distinct local glutamatergic afferents to neurochemically specific populations of DRN neurons, and furthermore that these distinct afferents are differentially regulated by swim stress. These cellular changes may contribute to the complex effects of swim stress on 5-HT neurotransmission and/or the behavioral changes underlying the forced swimming test model of depression.     

Differential effects of exposure to low-light or high-light open-field on anxiety-related behaviors: relationship to c-Fos expression in serotonergic and non-serotonergic neurons in the dorsal raphe nucleus

Serotonergic systems arising from the mid-rostrocaudal and caudal dorsal raphe nucleus (DR) have been implicated in the facilitation of anxiety-related behavioral responses to anxiogenic drugs or aversive stimuli. In this study we attempted to determine a threshold to engage serotonergic neurons in the DR following exposure to aversive conditions in an anxiety-related behavioral test. We manipulated the intensity of anxiogenic stimuli in studies of male Wistar rats by leaving them undisturbed (CO), briefly handling them (HA), or exposing them to an open-field arena for 15-min under low-light (LL: 8-13 lx) or high-light (HL: 400-500 lx) conditions. Rats exposed to HL conditions responded with reduced locomotor activity, reduced time spent exploring the center of the arena, a lower frequency of rearing and grooming, and an increased frequency of facing the corner of the arena compared to LL rats. Rats exposed to HL conditions had small but significant increases in c-Fos expression within serotonergic neurons in subdivisions of the rostral DR. Exposure to HL conditions did not alter c-Fos responses in serotonergic neurons in any other DR subdivision. In contrast, rats exposed to the open-field arena had increased c-Fos expression in non-serotonergic cells throughout the DR compared to CO rats, and this effect was particularly apparent in the dorsolateral part of the DR. We conclude that exposure to HL conditions, compared to LL conditions, increased anxiety-related behavioral responses in an open-field arena but this stimulus was at or below the threshold required to increase c-Fos expression in serotonergic neurons.     

Response of serotonin-containing neurons in nucleus raphe magnus to morphine, noxious stimuli, and periaqueductal gray stimulation in freely moving cats

Extracellular single-unit recordings were made in nucleus raphe magnus in unanesthetized, unrestrained cats. Discharge of serotonergic neurons in this region was increased when animals were aroused by noxious stimuli such as pinch and radiant heating of the tail, but these cells were not specifically nociceptive. Peristimulus time histograms indicated that stimulation in the periaqueductal gray was excitatory but alveolar nerve stimulation at a noxious current intensity was no more effective than nonnoxious nerve stimulation in activating serotonergic unit discharge: Similarly, stressful treatments such as physical restraint increased the discharge of some serotonergic neurons, but these cells were activated during any period of behavioral arousal whether or not arousal was the result of aversive treatment. Injection of Formalin into the paw produced pain lasting about 30 min without increasing serotonergic unit discharge above rates observed during undisturbed active waking behavior. The activity of serotonergic neurons was not increased by an analgesic dose of morphine (2 mg/kg, i.p.). These results then are not consistent with the hypothesis that morphine analgesia depends on activation of serotonergic neurons in nucleus raphe magnus or that these cells are specifically involved in modulation of nociception. These neurons may, however, be involved in nociceptive control within the context of a general modulation of sensorimotor processes by serotonin in the central nervous system. We did observe neurochemically unidentified neurons in the medulla whose discharge was more specifically activated by aversive stimuli and also by morphine. It is possible that these neurons are more directly involved in the mediation of opiate and/or stress-induced analgesia.     

The dorsal raphe nucleus in schizophrenia: a post mortem study of 5-hydroxytryptamine neurones

The 5-hydroxytryptamine (5-HT, serotonin) system has been implicated in the pathophysiology and treatment of schizophrenia. In this study, we addressed the hypothesis that a deficit of 5-HT neurones, either inherited or acquired, is central to the developmental pathology of the disorder. We examined putative 5-HT neurones of the dorsal raphe nucleus (DRN) in post mortem , formalin-fixed tissue from 15 schizophrenic patients and 20 control subjects matched for age and gender. No significant difference was detected between these groups in the number or size (cross-sectional area or diameter) of tryptophan-hydroxylase-immunoreactive cell profiles viewed in transverse sections collected from the level of the trochlear decussation to the emergence of the trigeminal nerve. Profile number was not affected by age, gender, side of the brainstem (left or right) or post mortem interval; however, time in formalin correlated negatively with the number of neurones counted. Moreover, a significant negative correlation was detected between time in formalin and the levels of immunoreaction product (optical density), which in turn correlated positively with our profile counts. A positive correlation was found between the age of subjects and our estimates of cell size. Our results do not support the proposal that an abnormality in the number and/or size of DRN 5-HT neurones is central to the aetiopathology of schizophrenia.     

Functional properties of dopamine neurons and co‐expression of vasoactive intestinal polypeptide in the dorsal raphe nucleus and ventro‐lateral periaqueductal grey

The dorsal raphe nucleus (DRN) and ventrolateral periaqueductal grey (vlPAG) regions contain populations of dopamine neurons, often considered to be a dorsal caudal extension of the A10 group [mostly found in the ventral tegmental area (VTA)]. Recent studies suggest they are involved in promoting wakefulness and mediate some of the antinociceptive and rewarding properties of opiates. However, little is known about their electrophysiological properties. To address this, we used Pitx3‐GFP and tyrosine hydroxylase (TH)‐GFP mice to carry out targeted whole‐cell recordings from this population in acute brain slices. We found that DRN/vlPAG dopamine neurons have characteristics similar to most VTA dopamine neurons, but distinct from dorsal raphe serotonin neurons. They fire broad action potentials at a relatively slow, regular rate, exhibit a hyperpolarization‐activated inward current and delayed repolarization, and show spike‐frequency adaptation in response to prolonged depolarization. In addition, they receive fast excitatory and inhibitory synaptic inputs. Moreover, we found co‐expression of vasoactive intestinal polypeptide in small, periaqueductal dopamine neurons, but generally not in larger, more ventral dopamine neurons.     

Differential behavioral and neurochemical effects following lesions of the dorsal or median raphe nuclei in rats

Locomotor activity and regional forebrain levels of serotonin (5-HT) were measured following the placement of electrolytic lesions in either the dorsal or median raphe nucleus of adult male rats. In the first two experiments, control lesions were placed in the brachium conjunctivum, and in the third experiment, a sham lesion group served as control. Median lesions significantly increased locomotor actiivity as measured in tilt cages, by 250–300% on the second day post-lesion, and this elevation stabilized at approximately 100% above control levels on day 9 post-lesion. There were no statistically significant differences in the amount of locomotor activity in the dorsal, brachium or sham lesioned groups on any post-lesion day. When the amount of 5-HT depletion was measured 5 days post-lesion, it was found that the dorsal (D) and median (M) lesions produced similar reductions in cerebral cortex (D — 40%; M — 31%); hypothalamus (D — 54%; M — 58%) and striatum (D — 50%; M — 29%). However, the effects of the two lesions were markedly different in the hippocampus. The dorsal lesion produced a non-significant 10% reduction in hippocampal 5-HT level, while the median lesion caused an 82% reduction. On the basis of these data it is hypothesized that a reduction in hippocampal 5-HT may account for the increased activity in the median lesioned group.     

The effects of electrical stimulation of nucleus raphe magnus and nucleus reticularis gigantocellularis on medial thalamic neurons--special reference to noxious neurons

Many previous studies revealed that electrical stimulation of brainstem inhibits activities of spinal dorsal horn cells, and that the inhibitory fibers, especially raphe-spinal system, descend in the dorsolateral funiculus (DLF) of the spinal cord. But the effect of such stimulation upon thalamic neurons are still unknown. The author tried to reveal how the stimulation of the nucleus raphe magnus (NRM) and the nucleus reticularis gigantocellularis (NGC) affect the medial thalamic neurons. Thirty-two adult cats (2.0-5.0 kg) were anesthetized with pentobarbital and immobilized by succinil choline infusion, and were maintained with 0.3-0.5% of halothane during experiments. The sural nerve was exposed and electrically stimulated with an intensity strong enough to activate C-fibers. To record single unit responses from the medial thalamus, a tungsten microelectrode (1.2-5 M omega at 1000 Hz) was inserted through a burr hole near the vertex contralateral to the sural nerve stimulation. Posterior fossa craniectomy was performed to insert 3 stimulation electrodes into NRM and bilateral sides NGC. Total of 183 single units were recorded from the medial thalamic region. They were classified into 45 noxious (N), 29 tap (T), 105 spontaneous (S) and 4 inhibitory (I) types according to the response patterns to contralateral sural nerve stimulation. N type neurons were mainly in the parafascicular region (Pf) and subparafascicular region (Spf). NRM stimulation (333Hz, 100-200 microA) inhibited 84% of N type, 57% of T type and 46% of S type neurons. The inhibitory ratio of N type neurons is significantly (p less than 0.01) higher than those of T and S type neurons, but there is no significant difference between T and S type.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory effects evoked from both the lateral and ventrolateral periaqueductal grey are selective for the nociceptive responses of rat dorsal horn neurones

In rats anaesthetized with alphaxalone/alphadolone a comparative study was made of the inhibitory effects on dorsal horn neurones evoked by chemical stimulation at identified pressor and depressor sites in the lateral and ventrolateral periaqueductal grey (PAG). Stimulating micropipettes were inserted stereotaxically into the lateral or ventrolateral PAG at sites where microinjection of -homocysteic acid (DLH) evoked increases or decreases respectively in mean arterial blood pressure. The effects of DLH microinjection at these sites were tested against the responses of dorsal horn neurones to noxious and innocuous stimuli applied to their cutaneous receptive fields. Single unit extracellular recordings were made from 15 Class 1 (low-threshold) and 37 Class 2 (wide dynamic range) dorsal horn neurones in laminae II–VI of the lower lumbar spinal cord. The responses of Class 1 neurones to innocuous prodding of their receptive fields were unaffected by neuronal activation in either the lateral or ventrolateral PAG. The nociceptive (noxious pinch/heat) responses of most Class 2 neurones were strongly inhibited by chemical stimulation in either sector of the PAG. The low threshold (prod) responses of the same neurones were generally unaffected or only weakly inhibited by identical stimulation, regardless of stimulation site. No significant differences were found between the effects of lateral vs. ventrolateral PAG stimulation on the responses of dorsal horn neurones. These results do not support the view that dorsal horn neurones may be inhibited with different selectivities by hyper- and hypotensive regions of the PAG.     

Effects of periaqueductal gray and raphe magnus stimulation on the responses of spinocervical and other ascending projection neurons to non-noxious inputs

Experiments were performed in cats anesthetized with α-chloralose to examine the effects of stimulating in the periaqueductal gray (PAG) and nucleus raphe magnus (NRM) on the responses of spinocervical cells and unidentified ascending projection neurons to non-noxious peripheral stimuli. Peripheral stimuli consisted of low amplitude sinusoidal displacements applied to either the glabrous skin or the hairy skin of the neuron's receptive field. Stimulating in either the periaqueductal gray or nucleus raphe magnus reduced the impulse activity of most neurons in both groups. By applying brainstem stimuli at various phases of the sinusoidal peripheral stimulus, it was demonstrated that the effects of stimulating either the PAG or NRM on the responses of both types of neurons was dependent on the timing of the electrical stimuli relative to the peripheral input. The effects of stimulating in the PAG and NRM on the responses of these cells to non-noxious stimuli were reversibly blocked by naloxone. It was concluded that stimulating in the nucleus raphe magnus and in the periaqueductal gray can produce dramatic modifications in the responses of spinocervical cells and unidentified ascending projection neurons to non-noxious peripheral stimuli, suggesting a role for these descending systems in non-noxious information processing.     

Differential in vivo clearance of serotonin in rat dorsal raphe nucleus and CA3 region

In vivo chronoamperometric recordings were used to determine if the majority of serotonin transporters (SERTs) in the dorsal raphe nucleus (DRN) are functionally active. This was achieved by comparing the clearance of exogenously applied serotonin (5-HT) from the extracellular fluid (ECF) of the DRN to that in the CA3 region of the hippocampus, an area with lower SERT density. Serotonin was pressure ejected into these regions in anesthetized rats and reproducible electrochemical signals measured by carbon fiber microelectrodes were recorded. Consistent with SERT density as measured by [3H]cyanoimipramine binding in these brain regions (DRN>CA3), clearance of 5-HT was significantly faster in DRN compared to that in the CA3 region. The selective serotonin reuptake inhibitor, fluvoxamine, prolonged 5-HT clearance in both CA3 and DRN. It is known that the norepinephrine transporter (NET) contributes to clearance of 5-HT in the dentate gyrus (DG) but not in CA3. Given that the DRN receives noradrenergic innervation, it was also determined if the NET contributes to 5-HT clearance in the DRN. Destruction of the NET with the neurotoxin 6-hydroxydopamine failed to alter 5-HT clearance parameters in the DRN. These data support the hypothesis that serotonin transporters are functionally active in the DRN, that serotonin clearance is mediated primarily by the SERT in the DRN and that the faster clearance of 5-HT from this region is related to its greater density of functional SERTs.     

Differential autoinhibition of 5-hydroxytryptamine neurons by 5-hydroxytryptamine in the dorsal raphe nucleus

5-Hydroxytryptamine neurons in the dorsal raphe nucleus are under autoinhibitory control by endogenous 5-hydroxytryptamine. Tonic activation of 5-hydroxytryptamine 1A autoreceptors was demonstrated in awake animals, but was inconsistently observed in anaesthetized animals and slice preparations, leading to questioning of its physiological significance. We re-evaluated autoinhibition in single-unit recordings from deeply seated 5-hydroxytryptamine neurons in slices in which endogenous 5-hydroxytryptamine bioavailability was restored by supplementing its precursor L-tryptophan. In these conditions, the application of the neutral 5-hydroxytryptamine 1A receptor antagonist WAY-100635 markedly increased 5-hydroxytryptamine neuron firing. Responses to WAY-100635 in single experiments ranged from a lack of effect to a several-fold increase in firing rate, suggesting that 5-hydroxytryptamine neurons in the dorsal raphe nucleus represent a heterogeneous population regarding their susceptibility to autoinhibition by endogenous 5-hydroxytryptamine.