Oxytocin acts at V1 receptors to excite sympathetic preganglionic neurones in neonate rat spinal cord in vitro

Intracellular recordings were made from sympathetic preganglionic neurones (SPNs) in transverse slices of thoraco-lumbar spinal cord of young rats (12–20 days old). A small group of SPNs generally having higher membrane potentials (− 70mV) compared to a remaining group (− 66mV) showed spontaneous oscillations of their membrane potential. Oxytocin superfused in concentrations of 0.1–30 μM had four effects on SPNs, inducing slow depolarisation, EPSPs, IPSPs and brief rhythmic oscillations. The slow depolarisation was unaffected by TTX whereas this abolished the other changes. The oxytocin-induced depolarisation was associated with a slow inward current and was not reversed at membrane potentials negative toE_K, it increased at more positive potentials and was still present in low Ca²⁺ and high Mg²⁺ solutions. These features of the oxytocin induced current are similar to those of the TTX resistant voltage dependent Na⁺ current described in brainstem autonomic neurones. Vasopressin superfused at concentrations of 0.1 μM to 30 μM had similar effects on SPNs to those of oxytocin. A comparison of the effects of oxytocin and vasopressin on the same neurones revealed that oxytocin was almost 10 times less potent than vasopressin. The effects of oxytocin were not mimicked by a selective oxytocin agonist but were mimicked by a selective vasopressin V_1a agonist and blocked by a selective V_1a antagonist. Therefore it is concluded that the effects of oxytocin on SPNs are mediated by the vasopressin V_1a receptor. It is suggested that oxytocin and vasopressin terminals in the lateral horn are part of a descending system controlling oscillating networks of SPNs in the spinal cord.     

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.     

Excitation and inhibition of rat sympathetic preganglionic neurones by catecholamines

The actions of microiontophoretically applied catecholamines on antidromically identified sympathetic preganglionic neurones (SPN) in the upper thoracic spinal cord of the anaesthetized rat were investigated. Noradrenaline (NA) excited the majority of neurones (50/71), however, a significant number were inhibited by the catecholamine (17/71). Adrenaline excited 4/9 SPN and inhibited 2/9. Dopamine had excitatory actions on SPN (3/3). Dual actions of NA on the same SPN were demonstrated, with the actions of the catecholamine being modulated by excitatory amino acids. NA was also shown to induce burst firing in 21% of SPN.     

The response of individual sympathetic preganglionic neurones to microelectrophoretically applied endogenous monoamines

In anaesthetized cats the effect on antidromically identified single sympathetic preganglionic neurones (SPN) in the third thoracic segment of microelectrophoretically applied monoamines, amino acids and acetyl choline was examined. 5-Hydroxytryptamine (5-HT) creainine sulphate and bimaleate excited a majority of SPN. A few cells were inhibited by 5-HT creatinine sulphate. These effects were observed on spontaneously active SPN (cardiac and non-cardiac type) and on silent SPN. Noradrenaline, adrenaline and dopamine inhibited all ‘types’ of SPN, including spontaneously active neurones silent neurones activated by glutamate ordl-homo- cysteic acid and neurones synaptically activated by electrically stimulating a brain stem excitatory region. Acetyl choline had no effect on different types of SPN.     

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.     

Effects of glutamate and aspartate on sympathetic preganglionic neurons in the upper thoracic intermediolateral nucleus of the cat

Glutamate and aspartate excited all spontaneously active sympathetic preganglionic neurons (SPNs) tested in the intermediolateral nucleus of spinal segments T1-T3. Most silent neurons could be induced to discharge but the remainder showed only a decrease in antidromic spike amplitude. These effects were typically fast (on, off less than 1 s). D,L-Homocysteic acid also produced excitation; this effect was typically slower. Glutamate and aspartate were usually equipotent; 20% were differentially sensitive to aspartate, 10% to glutamate.     

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.     

Effects of intranasal oxytocin and vasopressin on cooperative behavior and associated brain activity in men

The neural mechanisms supporting social bonds between adult men remain uncertain. In this double-blind, placebo-controlled study, we investigate the impact of intranasally administered oxytocin (OT) and vasopressin (AVP) on behavior and brain activity among men in the context of an iterated Prisoner's Dilemma game, which models a real-life social situation. fMRI results show that, relative to both AVP and placebo, OT increases the caudate nucleus response to reciprocated cooperation, which may augment the reward of reciprocated cooperation and/or facilitate learning that another person can be trusted. OT also enhances left amygdala activation in response to reciprocated cooperation. Behaviorally, OT was associated with increased rates of cooperation following unreciprocated cooperation in the previous round compared with AVP. AVP strongly increased cooperation in response to a cooperative gesture by the partner compared with both placebo and OT. In response to reciprocated cooperation, AVP increased activation in a region spanning known vasopressin circuitry implicated in affiliative behaviors in other species. Finally, both OT and AVP increase amygdala functional connectivity with the anterior insula relative to placebo, which may increase the amygdala's ability to elicit visceral somatic markers that guide decision making. These findings extend our knowledge of the neural and behavioral effects of OT and AVP to the context of genuine social interactions.     

Inhibitory and indirect excitatory effects of dopamine on sympathetic preganglionic neurones in the neonatal rat spinal cord in vitro

Regions of the thoraco-lumbar spinal cord containing sympathetic preganglionic neurones are rich in dopamine terminals. To determine the influence of this innervation intracellular recordings were made from antidromically identified sympathetic preganglionic neurones in (400 micrometers) transverse neonatal rat spinal cord slices. Dopamine applied by superfusion caused a slow monophasic hyperpolarisation in 46% of sympathetic preganglionic neurones, a slow monophasic depolarisation in 28% of sympathetic preganglionic neurones and a biphasic effect consisting of a slow depolarisation followed by a slow hyperpolarisation or vice-versa in 23% of sympathetic preganglionic neurones. Three percent of sympathetic preganglionic neurones did not respond to the application of dopamine. Low Ca2+/high Mg2+ Krebs solution or TTX did not change the resting membrane potential but abolished the slow depolarisation elicited by dopamine, indicating this was synaptic and did not prevent the dopamine induced hyperpolarisation. The dopamine induced slow hyperpolarisation was mimicked by the selective D1 agonists SKF 38393 or SKF 81297-C and blocked by superfusion with the D1 antagonist SCH 23390. It was not prevented by superfusion of the slices with alpha1 or alpha2 or beta-adrenoceptor antagonists, whereas the inhibitory or excitatory actions of adrenaline were prevented by alpha1 or alpha2 antagonists, respectively. The dopamine induced slow depolarisation occurring in a sub-population of sympathetic preganglionic neurones was mimicked by quinpirole, a D2 agonist, and blocked by haloperidol, a D2 antagonist. Haloperidol did not block the dopamine induced hyperpolarisations. Dopamine also induced fast synaptic activity which was mimicked by a D2 agonist and blocked by haloperidol. D1 agonists did not elicit fast synaptic activity.     

Effect of preganglionic stimulation or chronic decentralization on neurotensin-like immunoreactivity in sympathetic ganglia of the cat

In pentobarbital-anesthetized cats, supramaximal stimulation (40 Hz, 2 h) of the preganglionic input to the acutely decentralized right stellate (RSG) or superior cervical (RSCG) ganglion resulted in a decrease in neurotensin (NT)-like immunoreactivity (IR), by 83% in the SG and by 46% in the SCG, as determined by radioimmunoassay. Chronic (7 days) decentralization of the ganglia resulted in a similar depletion of NT-like IR (SG: 86%; SCG: 76%). Supramaximal stimulation (40 Hz, 2 h) of the intact postganglionic outflow of either ganglion had no effect on NT-like IR. These data suggest that NT in the SG and SCG is present in preganglionic axons and is released by activation of these axons.     

The role of glutamate and vasopressin in the excitation of RVL neurones by paraventricular neurones

Neurones in the paraventricular nucleus of the hypothalamus project to rostral ventrolateral medullary spinally projecting vasomotor neurones. We studied the excitatory action and the role of glutamate and vasopressin in this pathway in anaesthetised rats. A five barrel micropipette assembly was used for extracellular recording of neuronal activity and for microiontophoresis of drugs into the vicinity of identified medullary vasomotor neurones. Iontophoresis of L-glutamate or vasopressin into the vicinity of a vasomotor neurone increased activity, effects which were blocked by simultaneous iontophoretic application of a glutamate receptor antagonist, or a vasopressin V(1a) antagonist respectively. Paraventricular neurones were activated either by microinjecting D,L-homocysteic acid or by disinhibition by microinjecting bicuculline. The excitatory effects on vasomotor neurones, of paraventricular nucleus stimulation at some sites were prevented by simultaneous microiontophoretic application of kynurenic acid or at other sites by application of V(1a) antagonist. Neither antagonist altered the ongoing activity of the vasomotor neurones. Therefore, glutamate or vasopressin may act as excitatory neurotransmitters at synapses of paraventricular neurones on rostral ventrolateral medullary vasomotor neurones.     

Spinal origin of sympathetic preganglionic neurons in the rat

The segmental distribution of sympathetic preganglionic neurons (SPNs) and dorsal root ganglion cells (DRGs) was studied after Fluoro-gold injections into the major sympathetic ganglia and adrenal gland in rats. A quantitative assessment of the segmental and nuclear locations was made. Four general patterns of innervation were apparent: (1) a large number of SPNs (1000–2000/ganglion) innervate the sympathetic ganglia which control head or thoracic organs and a relatively small number of SPNs (100–400/ganglion) innervate the sympathetic ganglia controlling the gut, kidney, and pelvic organs; this difference in density of innervation probably relates to the level of fine control that can occur in these end organs by the SPNs; (2) the reverse pattern is seen in the DRG labeling where a large number of DRGs were labeled after Fluoro-gold injections into the preaortic ganglia (celiac, superior, and inferior mesenteric) and a small number were labeled after injections into the cervical sympathetic ganglia; (3) the intermediolateral cell column is the main source of SPNs except for the inferior mesenteric ganglion which is innervated predominantly by SPNs originating in the central autonomic nucleus (75%); the lateral funiculus is a source of SPNs mainly for the cervical sympathetic ganglia; and (4) each sympathetic ganglion and the adrenal gland receives a multisegmental SPN and DRG input with one segment being the predominant source of the innervation. The adrenal gland shows an intermediate position in terms of the density of SPN input (800 cells) and dorsal root input (300 cells); it has a widespread segmental input (T₄-T₁₂) with the T₈ segment being the major source.     

Glutamate-immunoreactive synapses on retrogradely-labelled sympathetic preganglionic neurons in rat thoracic spinal cord

Retrograde tracing with cholera toxin B subunit (CTB) combined with post-embedding immunogold labelling was used to demonstrate the presence of glutamate-immunoreactive synapses on sympathetic preganglionic neurons that project to the adrenal medulla or to the superior cervical ganglion in rat thoracic spinal cord. At the electron microscope level, glutamate-immunoreactive synapses were found on retrogradely labelled nerve cell bodies and on dendrites of all sizes. Two-thirds of the vesicle-containing axon profiles that were directly apposed to, or synapsed on, CTB-immunoreactive sympathoadrenal neurons were glutamate positive. The proportion of glutamate-immunoreactive contacts and synapses on sympathoadrenal neurons decreased to zero when the anti-glutamate antiserum was absorbed with increasing concentrations of glutamate from 0.1 mM to 10 mM. Double immunogold labelling for glutamate and gamma-aminobutyric acid (GABA) showed that glutamate-immunoreactive profiles did not contain GABA and that GABA-immunoreactive profiles did not contain glutamate. These results suggest that glutamate is the major excitatory neurotransmitter to sympathoadrenal neurons and possibly to other sympathetic preganglionic neurons in the intermediolateral cell column of the spinal cord.     

Functional properties of lumbar preganglionic neurones

Lumbar preganglionic neurones projecting through WRL₂ and L₃ to lumbar ganglia caudal to L₄ were investigated for those functional properties which are typical for postganglionic vasoconstrictor neurones supplying muscle and skin and for postganglionic sudomotor neurones. The properties tested were the cardiac rhythmicity of the activity and the reactions to systemic hypoxia, to noxious stimulation of skin and (in part of the experiments) to vibrational stimuli. Furthermore, resting activity and conduction velocities of the axons were measured.426 neurones were investigated. 311 (73%) of them were silent and could — as far as tested — not be excited by the afferent stimuli used. The conduction velocities of the axons of these neurones ranged from 0.5 to about 16 m/sec.115 neurones had resting activity of 0.1–4.6 impulses/sec. The conduction velocities of their axons ranged from 0.5 to about 12 m/sec. 80 preganglionic neurones with resting activity were classified on the basis of the reflexes in these neurones to afferent stimuli.Preganglionic neurones reacting like postganglionic vasoconstrictor neurones to muscle (excited by systemic hypoxia and/or by noxious stimulation of skin; with cardic rhythmicity) were classified as type 1 neurones (26 from 80 neurones tested). The resting activity of these neurones was1.8 ± 1.3impulses/sec(mean± 1S.D.). Their axons conducted with3.9 ± 2.2m/sec.Preganglionic neurones reacting like the majority of the postganglionic vasoconstrictor neurones to hairy and hairless skin (inhibited by systemic hypoxia and/or noxious cutaneous stimuli) were classified as type 2 neurones (48 from 80 neurones investigated). In 40% of these neurones the activity had cardiac rhythmicity. The resting activity was0.9 ± 0.6impulses/sec. The distribution of the conduction velocities of the axons of these neurones was bimodal. They conducted on the average with1.3 ± 0.6m/sec and6.6 ± 2.2m/sec respectively.A neurones were found (6 fronm 80 neurones) which were activated by vibrational stimuli (activation of Pacinian corpuscles by tapping on the hindfoot). Since this type of activation is typical for postganglionic sudomotor neurobes they were classified as type 3 neurones. The activity of these neurones had no cardiac rhythmicity.Indirect measurements of the conduction velocities of preganglionic axons converging onto postganglionic neurones supplying skeletal muscle and hairy skin yielded values which were statistically not different from the conduction velocities of the axons of type 1 and type 2 neurones respectively. These measurements support the classification into type 1 and type 2 preganglionic neurones. The implications of this study are discussed.     

Effect of preganglionic stimulation on neuropeptide-like immunoreactivity in the stellate ganglion of the cat

In pentobarbital-anesthetized cats, treated with hexamethonium and atropine, 40 Hz stimulation of the preganglionic input to the decentralized right stellate ganglion caused cardioacceleration. When the 40-Hz stimulation is maintained for 2 h, this cardioacceleration was progressively attenuated and eventually irreversibly lost. At this time, neurotensin-like and leucine-enkephalin-like immunoreactivity associated with intraganglionic fibers and presumptive axon terminals was also lost. Preganglionic 40 Hz stimulation for 2 h did not change substance P-like, somatostatin-like vasoactive intestinal peptide-like and corticotropin-releasing factor-like immunoreactivity in the stellate ganglion. A 40-Hz 2-h stimulation of the intact stellate ganglion output caused no change of the neuropeptide immunoreactivity pattern. These findings suggest that neurotensin and leucine-enkephalin are released by sympathetic preganglionic axon terminals and that releasable pool of these peptides is depleted by prolonged preganglionic stimulation. The association of peptide depletion with loss of the cardioacceleration, evoked by stimulation of the input to the stellate ganglion in the presence of cholinergic antagonist, suggests the possibility that peptides are involved in the non-cholinergic mechanism of ganglion transmission mediating the cardioacceleration.     

Melatonin inhibits the substance P-induced secretion of vasopressin and oxytocin from the rat hypothalamo-neurohypophysial system: in vitro studies

The aim of the present investigations was to study the influence of substance P (a member of a family of peptides known as tachykinins) on basal and K(+)-evoked vasopressin (AVP) and oxytocin (OT) release from rat hypothalamo-neurohypophysial system in vitro as well as to determine whether this effect of substance P is sensitive to melatonin.The present results show that substance P stimulates basal AVP and OT release from isolated hypothalamo-neurohypophysial system, when used at the concentrations of 10(-6) and 10(-7)M/l. At the concentration of 10(-9)M/l, however, substance P was found to stimulate the in vitro secretion of AVP, but not that of OT. Melatonin diminished basal release of AVP; it also significantly inhibited the substance P-stimulated secretion of AVP and OT. K(+)-evoked release of the neurohypophysial hormones was not further modified by either substance P or melatonin.The present results show that the stimulatory effect of substance P on basal release of AVP and OT from rat hypothalamo-neurohypophysial system in vitro is sensitive to inhibitory influence of melatonin.     

Differential release of vasopressin and oxytocin in response to abdominal vagal afferent stimulation or apomorphine in the ferret

The aim of this study was to investigate whether direct afferent stimulation of the abdominal vagus promote release of the neurohypophyseal hormones. The nucleus of the solitary tract is the major recipient of vagal afferent information, and this region of the brainstem may also be activated by stimulation of the area postrema. For this reason apomorphine, a D₂ dopaminergic agonist which acts on the area postrema, and can evoke vasopressin secretion in man, was also investigated for its effect on vasopressin and oxytocin release. Our results show that vasopressin, but not oxytocin is released in vast amounts in response to electrical afferent stimulation of the abdominal vagus. Administration of apomorphine also evoked a massive vasopressin release with less marked effects on oxytocin. The possible functional implications of these results are discussed especially in the context of nausea and vomiting.     

Effects of androgens and estrogens on the vasopressin and oxytocin innervation of the adult rat brain

Recently we reported that castration of rats eliminates vasopressin immunoreactivity in the lateral septum and other areas that appear to receive vasopressin innervation from the bed nucleus of the stria terminalis. Testosterone treatment counteracts this effect of castration. In the present study, we investigated whether this action of testosterone depends on its androgenic or estrogenic metabolites by treating long-term castrated rats with estradiol (E) and/or 5 alpha-dihydrotestosterone (DHT) or testosterone. The brains were then processed for immunocytochemistry or radioimmunoassay. DHT did not increase vasopressin staining in the lateral septum, although it fully restored the size of the seminal vesicles. E did restore the original fiber density, but individual fibers stained more weakly than in sham-operated males. Only treatment with both E and DHT fully restored the vasopressin innervation. This pattern was also reflected in the radioimmunoassay data. The vasopressin content of the lateral septum decreased about 90% after castration but was fully restored by either testosterone or E + DHT treatment. E alone, however, was only half as effective as E + DHT. The treatments had no effect on the oxytocin content of the septum, or on the vasopressin or oxytocin content of the dorsal vagal complex. The results suggest that E mediates most of the effects of testosterone on the vasopressin innervation of the lateral septum. DHT enhances the response to E but has little effect on its own.