Effect of acute administration of morphine on newly synthesized 5-hydroxytryptamine in spinal cord of the rat

The effect of morphine (10 mg/kg, s.c.) on the rate of [³H]5-HT synthesis in brain and spinal cord following intravenous injection of [³H]tryptophan was studied in the rat.

(1) In the spinal cord morphine induced an increase in [³H]TRP uptake which was significant 30 and 60 min after the injection, and a clear enhancement of the formation of [³H]5-HT which was significant 15 min after the injection and which peaked at 30 min.

In the forebrain, the increase in [³H]TRP accumulation was significant as early as 15 min after morphine. At this time, the rate of 5-HT synthesis was not modified, but it was significantly increased 30 and 60 min after the injection.

(2) Increases in [³H]5-HT synthesis rate were suppressed by naloxone (1 mg/kg, i.m.). However this narcotic antagonist did not significantly reduce the increased accumulation of [³H]TRP in brain and spinal cord due to morphine treatment.

These results demonstrate that morphine induces a fast and marked increase in 5-HT synthesis, and suggest that this increase is only partly related to an increase in the availability of tryptophan to the central nervous system.These results are in good agreement with recent investigations showing the involvement of the raphe-spinal system in morphine analgesia.     

The effect of modification of 5-hydroxytryptamine function in nucleus raphe magnus on nociceptive threshold

Thresholds to noxious heat stimulation were increased following microinjection of zimelidine, an inhibitor of 5-hydroxytryptamine (5-HT) re-uptake, into the nucleus raphe magnus (NRM) of rats. Pretreatment with intraperitoneally given cinanserin reduced this effect but pretreatment with intraperitoneally given phenoxybenzamine did not. Fenfluramine, which causes the release of 5-HT from synaptic terminals also elevated nociceptive thresholds following microinjection into NRM. Subanalgesic doses of morphine or zimelidine elevated nociceptive thresholds when microinjected together into NRM. The elevation of nociceptive threshold produced by microinjection of morphine into NRM was reduced by simultaneous microinjection of cinanserin into NRM. Cinanserin alone had no effect when microinjected into NRM. These findings suggest that inhibition of the re-uptake of 5-HT in NRM can elevate nociceptive thresholds and that there may be an interaction between the effects of morphine and 5-HT in NRM.     

Inhibition of sympathetic preganglionic discharges by epinephrine and and alpha-methylepinephrine

The present study was undertaken to determine the effect of iontophoretic applications of epinephrine (E) and its derivative alpha-methylepinephrine (mE) on the discharges of sympathetic preganglionic neurons (SPNs). Spontaneously active SPNs located in thoracic segment T2 were antidromically identified in White Carneaux pigeons anesthetized with urethane and immobilized with purified alpha-cobratoxin. All SPNs tested were inhibited by E, mE, several other catecholamines, clonidine, GABA, glycine and morphine. The inhibitory effects of E and mE but not those of GABA were antagonized by iontophoretic applications of the preferential alpha 2-antagonists piperoxane and yohimbine, but not by the alpha 1-antagonist prazosin or the beta-antagonist sotalol when similarly applied. The inhibitory effects of GABA, glycine and morphine were respectively antagonized by bicuculline methiodide, strychnine and naloxone, but these antagonists failed to alter the action of E. It is concluded that (1) epinephrine and its alpha-methyl derivative inhibit the discharges of SPNs via the activation of alpha 2-receptors and (2) the epinephrine-induced inhibition does not result from the secondary release of GABA, glycine or opioid peptides from afferent terminals or interneurons.     

Lateralisation of projections from the rostral ventrolateral medulla to sympathetic preganglionic neurons in the rat

Spinally projecting sympathoexcitatory neurons in the rostral ventrolateral medulla (RVLM), synapse with sympathetic preganglionic neurons (SPN) and regulate the activity of sympathetic nerves that control the heart, blood pressure and the adrenal medulla (AM). However, the degree of lateralization of the bulbospinal projections to SPN innervating specific targets is poorly understood. Three approaches were used in this study. Anterograde tracer was iontophoresed into a pressor site in the RVLM (left or right) and retrograde tracer injected into the superior cervical ganglion (SCG, right) and the AM (left). Close appositions between anterogradely labelled axons and retrogradely labelled SCG- or AM-SPN were counted. Projections to the SCG were bilateral. Projections to the AM were markedly ipsilateral. In the second part, retrograde tracers were injected unilaterally into the region of the intermediolateral cell column at spinal segment T2 or T8 on one side and the number of labelled neurons in the RVLM counted. The results from each level of injection were similar showing that 63–64% of the neurons were ipsilateral. Responses to glutamate microinjection into the RVLM on adrenal nerve (left) and superior cervical nerve (left) activity were measured. The ratio of the nerve responses was the same even when different sides of the RVLM were injected. The anterograde data strongly suggest that the RVLM projections to AM-SPN are predominantly ipsilateral. Although other experimental approaches also attempted to investigate lateralization, the retrograde data target different and functionally heterogeneous pools of SPN that may mask the ipsilateral projection to the AM. Similarly, chemical stimulation of the RVLM will excite not only monosynaptic projections but also polysynaptic projections that may also mask the predominant ipsilateral monosynaptic projection to AM.     

Localization of noradrenergic terminals in sympathetic preganglionic nuclei of the rat: Demonstration by immunocytochemical localization of dopamine-β-hydroxylase

The noradrenergic (NE) innervation to sympathetic preganglionic nuclei in the rat thoracic cord was studied by immunocytochemical localization of dopamine-β-hydroxylase (DBH), a specific NE antigen. DBH antisera was prepared against DBH purified from bovine adrenal medulla.The most intense immunoreaction was observed within the intermediolateral cell column (IML) of the spinal cord, the major sympathetic preganglionic nucleus in mammals. DBH was also localized in both the central autonomic and intercalated nuclei, cell groups known to contain sympathetic preganglionic visceral motor neurons. Two weeks following a midthoracic spinal transection, DBH immunoreactivity was no longer observed caudal to the lesion. Thus, the cells of origin of these noradrenergic terminals are supraspinal. Following a midthoracic hemisection DBH, immunoreactivity was similarly reduced in both the ipsilateral and contralateral IML caudal to the lesion. Therefore, bulbospinal NE neurons project bilaterally to sympathetic preganglionic nuclei.     

Neuronal responses to 5-hydroxytryptamine and dorsal raphe stimulation within the globus pallidus of the rat

The projection from the dorsal raphe nucleus (DRN) to the globus pallidus (GP) was investigated electrophysiologically, in the urethane-anesthetized rat together with the responsiveness of cells in the GP to 5-hydroxytryptamine (5-HT) and noradrenaline (NA). The majority of spontaneously active cells in the GP had high regular firing rates. They were unaffected by both DRN stimulation (69/83 cells) and iontophoretically applied 5-HT (38/63 units) or NA (30/42 units) but were inhibited by GABA. A few cells (N = 10) were recorded from, that were spontaneously active but with a much lower and less regular firing rate, which, however, seemed to be much more responsive to 5-HT. In addition, DL-homocysteic acid (DLH) was used to activate silent cells and all seven cells activated in this manner were inhibited by 5-HT. In addition 5/6 cells that had their firings maintained by DLH were inhibited by stimulation of the dorsal raphe. The results show a lack of responsiveness to both 5-HT and DRN stimulation of the typically regular spontaneously active pallidal neurons. There seems to be a small population of normally quiescent cells, however, that is sensitive to 5-HT and receives an input from the DRN.     

Segmental distribution of peptide-like immunoreactivity in cell bodies of the thoracolumbar sympathetic nuclei of the cat

The distribution of leucine-enkephalin, methionine-enkephalin, neurotensin, somatostatin, substance P, oxytocin, vasopressin, and neurophysin II in cell bodies of sympathetic autonomic nuclei of the thoracolumbar (T-L) spinal cord was studied immunohistochemically in cats after intrathecal administration of colchicine. Neurons containing only enkephalin-, neurotensin-, somatostatin-, and substance P-like immunoreactivity (ENK, NT, SS, SP, respectively) were found in the intermediolateral nucleus pars principalis (IMLp) and pars funicularis (IMLf), the nucleus intercalatus (IC), and the central autonomic area (CA). The size, shape, location, and numbers of the peptide-positive neurons in the IMLp, IMLf, and IC suggested that they were sympathetic preganglionic neurons (SPN). This was confirmed by a combined retrograde tracing/immunohistochemical study showing that most of these neurons at the levels of the T-L cord known to provide preganglionic fibers to the stellate ganglion were SPN. On the other hand, the functional identification of the neurons in the CA is uncertain as neurons were not observed which were both retrogradely labelled and contained ENK, NT, SS, or SP. Immunoreactive neurons in each area were counted in ten sections from each segment from C8 to L4. In the IMLp, the SPN with ENK were greatest in number (up to 25) in segments T4-T7 and L2-L3. The maximum number of SPN containing NT was found in segments T4-T7 (45 neurons). Of the four peptides, neurons containing SS were found in the greatest number (up to 48 in segments T2-T6); neurons containing SP were found in the smallest number (15 or fewer per segment). Few SPN containing each of the four peptides were found in the IC; CA neurons with ENK and NT were also few in number. A comparison of the numbers of immunoreactive neurons in the IML with earlier estimates for the total numbers of SPN in the IML at each level showed that the proportions of IML neurons containing each of the four peptides were fairly consistent throughout the T-L cord, with some exceptions. These results suggest that the innervation of visceral organs is not obviously peptide-specific, although some organs may be innervated by a greater proportion of SPN containing one of these peptides. Finally, the presence of ENK, NT, SS, and SP in SPN suggests that these four peptides act as neurotransmitters in preganglionic pathways to sympathetic ganglia.     

Inhibition of lateral vestibular nucleus neurons by 5-hydroxytryptamine derived from the dorsal raphe nucleus

Electrophysiological studies were performed to elucidate the effect of 5-hydroxytryptamine (5-HT) originating in the dorsal raphe nucleus (DR) on neuronal activity in the lateral vestibular nucleus (LVN) neurons, using cats anesthetized with alpha-chloralose. LVN neurons were classified into monosynaptic and polysynaptic neurons according to their responses to vestibular nerve stimulation. Conditioning stimuli applied to the DR inhibited orthodromic spikes elicited by vestibular nerve stimulation predominantly in polysynaptic neurons of the LVN. The iontophoretic application of 5-HT also inhibited orthodromic spikes of the LVN neurons. A close correlation was observed between the effects of DR conditioning stimulation and iontophoretically applied 5-HT in the same neurons. These inhibitions with both treatments were antagonized during the application of methysergide, a 5-HT antagonist. In the majority of LVN polysynaptic neurons that responded to antidromic stimulation of the ipsilateral or contralateral abducens nucleus, orthodromic spikes elicited by vestibular nerve stimulation were inhibited by DR conditioning stimulation and the iontophoretic application of 5-HT. In contrast, LVN neurons that responded to antidromic stimulation of the vestibulospinal tract were rarely affected by these treatments. These results indicate that 5-HT derived from the DR inhibits the synaptic transmission of LVN polysynaptic neurons ascending to the abducens nucleus, and suggest that 5-HT derived from the DR is involved in the regulation of the vestibulo-ocular reflex.     

Sigmoid-colonic motility in health and irritable bowel syndrome: a role for 5-hydroxytryptamine

Abstract Evidence suggests that sigmoid-colonic motility is increased in patients with irritable bowel syndrome (IBS). 5-Hydroxytryptamine (5-HT) plays a role in the control of motility, but its involvement in the dysmotility seen in IBS remains unclear. To investigate the relationship between platelet depleted plasma 5-HT (PDP 5-HT) concentration and sigmoid-colonic motility in patients with IBS and healthy volunteers. Pre- and postprandial PDP 5-HT concentrations were assessed while recording sigmoid-colonic motility in 35 IBS patients (aged 19-53 years, eight male) and 16 healthy volunteers (aged 18-39 years, six male). Motility was recorded using a five-channel solid-state catheter introduced to a depth of 35 cm into an unprepared bowel. 5-Hydroxytryptamine concentration was measured by reverse-phase HPLC with fluorimetric detection. Irritable bowel syndrome patients had elevated concentrations of PDP 5-HT under fasting (P < 0.004) and fed (P = 0.079) conditions compared with controls. Likewise, they exhibited increased sigmoid-colonic motility under fasting (activity index: P < 0.02) and fed (P < 0.05) conditions compared with controls. Platelet depleted plasma 5-HT concentration positively correlated with colonic activity index under both fasting (r = 0.402; P = 0.003) and fed (r = 0.439; P = 0.001) conditions. These data show a possible relationship between endogenous concentrations of 5-HT and sigmoid-colonic motility recorded in both IBS and healthy subjects.     

Effect of iontophoretically applied 5-hydroxytryptamine on the excitability of single primary afferent C- and A-fibers in the cat spinal cord

Excitability testing of single sural afferent C- and A-fibers was employed to study possible presynaptic effects of 5-hydroxytryptamine (5-HT) applied iontophoretically at the intraspinal point of lowest threshold for their antidromic activation in anesthetized or decerebrate spinalized cats. Threshold for single fibers recorded in the sural nerve was measured prior to and during iontophoretic application of 5-HT through a micropipette positioned in close proximity to the intraspinal stimulating electrode. 5-HT produced dose-related increases in threshold for antidromic activation in 21 or 30 C-fibers. Six of 9 A δ-, and 4 of 7 Aβ-fibers were similarly affected.     

Excitatory and indirect inhibitory actions of 5-hydroxytryptamine on sympathetic preganglionic neurones in the neonate rat spinal cord in vitro

The action of 5-hydroxytryptamine (5-HT) on sympathetic preganglionic neurones (SPN) was studied by intracellular recordings in thin slices of neonatal rat spinal cord in vitro. Superfusion of 5-HT (1–270 μM) to SPN caused a concentration dependent slow depolarization or inward current and an increase in synaptic activity consisting of both EPSPs and IPSPs. The slow depolarization was still present after superfusion with TTX. Similar effects were seen during superfusion with 5-carboxamidotryptamine (5-CT) or -methyl-5-hydroxytryptamine (-me-5-HT). A comparison with the potency of 5-HT was made for 5-CT or -me-5-HT on the same neurone by determining the magnitude of the slow depolarization to different concentrations of agonist. This showed that the apparent potency of the agonists was 5-CT> 5-HT> -me-5-HT even in the presence of fluoxetine, a 5-HT uptake inhibitor. The 5-HT-induced slow depolarization was partially blocked by ketanserin but full recovery was not observed. The results suggest that the excitatory action of 5-HT on SPN is mediated via an atypical 5-HT₂ receptor or a 5-HT_1C-like receptor. The 5-HT-induced IPSPs were reversibly blocked by superfusion with strychnine, suggesting they were mediated by glycine.     

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.     

The influence of the paraventriculo-spinal pathway, and oxytocin and vasopressin on sympathetic preganglionic neurones

In anesthetized rats the effect of two procedures was studied on antidromically identified sympathetic preganglionic neurones (SPN) in the second thoracic (T) segment of the spinal cord: the application of iontophoresed oxytocin and vasopressin, and bipolar electrical stimulation of the paraventricular nucleus of the hypothalamus (PVN). In the majority of cases (16/23) oxytocin inhibited SPN firing, 1/23 being excited. Vasopressin inhibited 8/14 neurones and excited 4/14. PVN stimulation inhibited SPN apparently by an action on the membrane of SPN. The possibility that oxytocin and vasopressin act as transmitters in the paraventriculo-spinal pathway, and their possible involvement in the mediation of PVN evoked inhibition of SPN activity has been discussed.     

Pharmacological characterization of 5-hydroxytryptamine receptor types in the guinea-pig proximal colon

It was investigated which 5-hydroxytryptamine (5-HT) receptors mediate the responses to 5-HT in the longitudinal muscle layer of the guinea-pig proximal colon, using selective 5-HT receptor antagonists and the 5-HT analogues α-methyl-5-HT (2-Me-5-HT), 2-methyl-5-HT (2-Me-5-HT), and 5-methoxytryptamine (5-MeOT). 5-HT as well as its analogues induced concentration-related contractions, at low concentrations preceded by relaxations. The 5-HT concentration-contractile response curve was biphasic whilst the curves to α-Me-5-HT, 2-Me-5-HT, and 5-MeOT were monophasic. Tetrodotoxin (TTX) abolished the relaxations, and it inhibited the contractions to all agonists. In the presence of TTX, blockade of either 5-HT₂ (ketanserin) or 5-HT₃ receptors (ondansetron, tropisetron) reduced the contractions to 5-HT, whereas blockade of both 5-HT receptor types at the same time abolished them. In the absence of TTX, the contractions to 5-HT were inhibited by antagonists at 5-HT₂ (ketanserin), 5-HT₃ (granisetron, nanomolar concentration of tropisetron) and also 5-HT₄ receptors (micromolar concentration of tropisetron). Contractions to α-Me-5-HT did not seem to be sensitive to 5-HT₂ receptor blockage with ketanserin, but in the presence of TTX the contractions were abolished by the 5-HT₂ receptor antagonist. The 5-HT₃ receptor antagonist granisetron abolished contractions to 2-Me-5-HT. In the presence of TTX, the 5-HT₂ receptor antagonist ketanserin abolished contractions to 5-MeOT, and in the absence of TTX the contractions to 5-MeOT were highly sensitive blockade of 5-HT₄ receptors with tropisetron. Blockage of either 5-HT₁ (methiothepin), 5-HT₂ (ketanserin), 5-HT₃ (ondansetron, granisetron, tropisetron) or 5-HT₄ (tropisetron) receptors did not abolish the relaxations to 5-HT or 5-MeOT. In conclusion, 5-HT induces contractions of the longitudinal muscle of the guinea-pig proximal colon, through the stimulation of 5-HT₂ receptors on the smooth muscle cells and 5-HT₃ receptors and putative neuronal 5-HT₄ receptors. 5-HT evokes relaxations via an unknown neuronal receptor.     

Inhibition of sympathetic preganglionic discharges by epinephrine and α-methylepinephrine

The present study was undertaken to determine the effect of iontophoretic applications of epinephrine (E) and its derivative α-methylepinephrine (mE) on the discharges of sympathetic preganglionic neurons (SPNs).Spontaneously active SPNs located in thoracic segment T2 were antidromically identified in White Carneaux pigeons anesthetized with urethane and immobilized with purified α-cobratoxin.All SPNs tested were inhibited by E, mE, several other catecholamines, clonidine, GABA, glycine and morphine.The inhibitory effects of E and mE but not those of GABA were antagonized by iontophoretic applications of the preferential α₂-antagonists piperoxane and yohimbine, but not by the α₁-antogonist praxosin or the β-antagonist sotalol when similarly applied.The inhibitory effects of GABA, glycine and morphine were respectively antagonized by bicuculline methiodide, strychnine and naloxone, but these antagonists failed to alter the action of E.It is concluded that (1) epinephrine and its α-methyl derivative inhibit the discharges of SPNs via the activation of α₂-receptors and(2) the epinephrine-induced inhibition does not result from the secondary release of GABA, glycine or opioid peptides from afferent terminals or interneurons.     

In vitro experiments on the metabolism, uptake and release of 5-hydroxytryptamine in bovine retina

The accumulation, metabolism and potassium-induced release of 5-hydroxytryptamine (5-HT) in bovine retinae was studied. When isolated retina pieces were incubated at 37°C in a medium containing [¹⁴C]5-HT, tissue: medium ratios of about 5 were obtained after only 10 min. Within this time the accumulated amine is hardly metabolized and even after a 60 min period, only approximately 12% of the amine is metabolized to form 5-hydroxyindoleacetic acid. The process responsible for this accumulation showed properties of an active transport system: it was temperature-sensitive, sodium-dependent and inhibited in particular by Lilly 110140, chlorimipramine and desimipramine. The uptake of [¹⁴C]5-HT was saturated with increasing 5-HT concentrations and could be accounted for two saturable processes; a high-affinity system with aK_m1of1.3 × 10⁻⁷M and V_max1of0.65pmol/mg/10min, and a low affinity system with aK_m2of1.2 × 10⁻⁵and V_max2of29pmol/mg/10min.A rapid efflux of [¹⁴C]5-HT from tissue loaded with [¹⁴C]amine was observed when exposed to 70 mM KCl. This release was inhibited when the calcium content in the medium was replaced by sucrose. Cobalt ions added to the incubation medium also counteracted the effect of KCl, while chlorimipramine added to the medium enhanced the release of 5-HT caused by KCl. Bovine retinae also possess the enzymes necessary to produce 5-HT from trytophan. These data generally sypport the idea that 5-HT is a transmitter in the retina, although as this study shows, the amine is present only in minute quantities.     

Some characteristics of sympathetic preganglionic Neurones in the rat

In anaesthetized rats sympathetic preganglionic neurones (SPN) were identified by their antidromic response to stimulation of the ipsilateral cervical sympathetic trunk (CST). Units were recorded at a depth of 0.75-1.1 mm from the dorsal surface of the spinal cord. The majority of SPN had axonal conduction velocities less than 1 m/s. Units could be routinely held for periods of up to 1 h. Spontaneously active SPN had discharge rates within the range 0.3-8.0 Hz. Application of horseradish peroxidase (HRP) to the central cut end of the CST resulted in the labelling of neurones only on the ipsilateral side, mainly in the intermediolateral cell column (IML) and the lateral funiculus and between the first and third thoracic (T) segments.     

Evidence for the coexistence of acetylcholine and enkephalin in the sympathetic preganglionic neurons of rats

The localization of the cholinergic neurons in the lower thoracic segments of the spinal cord of rats was examined by a monoclonal antibody against choline acetyltransferase (ChAT). The ChAT-immunoreactive neurons were located in the intermediate as well as anterior gray matters. In the intermediate gray the highest incidence of the immunoreactive neurons was in the nucleus intermediolateralis, followed by the nucleus intercalatus pars paraependymalis and a few immunoreactive neurons were seen in the nucleus intercalatus proprius. In the sequential immunostaining of one and the same section of the spinal cord pretreated with colchicine using the ChAT antibody and a polyclonal antibody against methionine-enkephalin-argynine-glycine-leucine (Met-Enk-Arg-Gly-Leu), substantial numbers of neurons were immunostained simultaneously by the two antibodies in the intermediate gray matter. The present finding gives strong evidence for the coexistence of acetylcholine and enkephalins in, at least, some of the preganglionic neurons projecting their axons to the periphery.