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.     

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.     

Effects of oxytocin and vasopressin on thoracic sympathetic preganglionic neurones in the cat

When applied by iontophoresis onto single sympathetic preganglionic neurones in the intermediolateral nucleus of segments T₁–T₃ in the cat, oxytocin and vasopressin each had an excitatory effect. This effect consisted of a prolonged (30–300 sec) after-discharge following termination of application. These results indicate that oxytocin and vasopressin each exert excitatory effects on sympathetic preganglionic neurones and support the possibility that they may be chemical mediators of synaptic transmission in the intermediolateral nucleus, perhaps in cardioacceleratory and/or pressor pathways descending from the paraventricular nucleus of the hypothalamus.     

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.     

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.     

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.     

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.     

Sympathetic preganglionic neurones in neonatal rat spinal cord in vitro: electrophysiological characteristics and the effects of selective excitatory amino acid receptor agonists

Intracellular recordings were made from 52 lateral horn neurones in thin slices of neonatal rat thoracolumbar spinal cord. Of these neurones 12 were spontaneously active and the remainder silent. A number of these cells could be activated antidromically by stimulation of ventral roots. The conduction velocity of the antidromic potential was estimated to be 0.9–2 m/s which is within the range reported for axons of sympathetic preganglionic neurones (SPNs). The membrane properties of antidromically identified SPNs were similar to other lateral horn neurones included in this study and comparable to those reported for SPNs by others. Spontaneous burst firing was recorded in 3 neurones and activity in a further 5 neurones was characterized by the discharge of an action potential followed by an afterhyperpolarization potential (AHP) of peak amplitude 3–13 mV and duration 0.5–4 s. The AHP had an initial fast component (fAHP) which was sensitive to the potassium channel blocker tetraethylammonium (TEA), and a second slower component (sAHP) which was both sensitive to extracellular calcium and TEA. The effects of the selective excitatory amino acid receptor agonists N-methyl-d-aspartate (NMDA), kainate and quisqualate were investigated by superfusion of the agonists, at known concentrations (100 nM to 100 μM). These agonists induced concentration-dependent depolarizations which were primarily associated with a reduction in neuronal input resistance. NMDA-induced depolarizations were potentiated in the absence of magnesium. In a number of neurones NMDA, kainate and quisqualate (1–50 μM) induced, in addition to a depolarizing response, the discharge of small (1.5–6.5 mV), brief (7–20 ms) IPSPs which were reversed just below resting membrane potential at −64 mV. These results suggest that both NMDA and non-NMDA receptors may have an important role in the excitation of SPNs and of inhibitory interneurones presynaptic to SPNs.     

Sympathetic preganglionic neurons in the isolated spinal cord of the neonatal rat

A preparation of the isolated spinal cord of the neonatal rat was developed for the study of sympathetic preganglionic neurons (PGNs). PGNs were identified for extracellular single unit recording by their location and by antidromic activation by ventral root stimulation. PGNs could be synaptically activated by stimulation of the dorsal root and spinal pathways. Spontaneous firing was observed in 18% of the PGNs. The average firing rate was 1 Hz with a range of 0.3 to 2 Hz.PGNs (and motoneurons) were visualized by incubating vental roots in horseradish peroxidase (HRP) solutions. The location and morphology of PGNs were similar to those reported in studies using adult animals. Primary afferent fibers were visualized by incubating dorsal roots in HRP solutions. Dorsal root projections appeared mature in the neonatal rat. Primary afferents did not appear to project directly to PGNs.It is concluded that PGNs are viable in this preparation and that spinal sympathetic systems are relatively mature in the neonatal rat.     

Direct synaptic contacts of catecholamine axons on the preganglionic sympathetic neurons in the rat thoracic spinal cord

Preganglionic sympathetic neurons were labelled by retrograde transport of horseradish peroxidase, while catecholamine axon varicosities were marked by the uptake of 5-hydroxydopamine in the intermediolateral nucleus of the rat. The direct synaptic contacts from the catecholamine axons to the preganglionic sympathetic neurons were demonstrated. Catecholamine axons formed symmetric synapses.     

Glutamate excitation of oxytocin neurones in vitro involves predominantly non-NMDA receptors

Experiments were undertaken to compare effects of the NMDA and non-NMDA receptor antagonists, AP5 (40 μM) and NBQX (10 μM), on glutamate-induced firing in supraoptic oxytocin (OT) and vasopressin (VP) neurones in vitro. In putative OT neurones NBQX caused a significantly greater reduction in firing than AP5, whilst in putative VP neurones both antagonists reduced activity powerfully and to a similar extent. The relatively small effect of AP5 in putative OT neurones was unaffected by the removal of extracellular magnesium. These results suggest that glutamate-induced firing in putative OT neurones is predominantly controlled by non-NMDA receptors.     

Excitatory action of pituitary adenylate cyclase activating polypeptide on rat sympathetic preganglionic neurons in vivo and in vitro

In vivo and in vitro experiments were undertaken to evaluate the effects of pituitary adenylate cyclase activating polypeptide-38 (PACAP-38) on rat sympathetic preganglionic neurons (SPNs). Intrathecal injection of PACAP-38 (0.1–1 nmol) via an implanted cannula to the T2-T3 segments of urethane-anesthetized adult rats caused a dose-dependent increase of mean arterial blood pressure from minutes to over 1 h. The pressor response was not antagonized by prior injection of the PACAP type II receptor antagonist PACAP_6-38 (0.5 nmol), but was significantly attenuated by prior intravenous administration of phentolamine (1 mg/kg). As a positive control, intrathecal injection of glutamate (1 μmol) and substance P (SP, 5 nmol) caused a short- and long-lasting pressor response. Vasoactive intestinal polypeptide (VIP, 1 nmol) had no significant pressor effect. In the second series of experiments, whole-cell patch recordings were made from antidromically identified SPNs of immature (12–16-day-old) rat thoracolumbar spinal cord slices. Applied to the spinal cord slices by superfusion, PACAP-38 (10–30 nM) caused intense neuronal discharges with or without a long-lasting membrane depolarization. The depolarization was not prevented by superfusing the slices with tetrodotoxin (0.3 μM) or low Ca²⁺ (0.25 mM) solution, indicating that PACAP-38 directly depolarized the SPNs. The depolarization was insensitive to the type II PACAP receptor antagonist PACAP_6-38. Collectively, these results provide evidence that PACAP-38 exerts a potent and long-lasting excitatory effect on SPNs, leading to an increase of spinal sympathetic outflow and one of the consequences of which is an elevation of blood pressure.     

An immunohistochemical study of methionine-enkephalin-Arg-Gly-Leu-like immunoreactivity-containing neurons in the parasympathetic preganglionic regions of the rat spinal cord

The localization of the methionine-enkephalin-Arg⁶-Gly⁷-Leu⁸ (Met-Enk-Arg-Gly-Leu)-like immunoreactivity-containing neurons in the rat lumbosacral spinal cord was immunohistochemically examined by an antiserum very specific to Met-Enk-Arg-Gly-Leu. The immunoreactive neurons occupied the positions corresponding to the parasympathetic preganglionic nuclei determined by the previous horseradish peroxidase (HRP)-tracing experiments. The present study suggests that the parasympathetic preganglionic neurons in the rat lumbosacral spinal cord produce preproenkephalin A and its related peptides.     

Neurokinin-1 receptors and spinal cord control of blood pressure in spontaneously hypertensive rats

In this study we examined blood pressure and heart rate responses to intrathecal administration of a synthetic NK1-receptor agonist, H₂N–(CH₂)₄–CO–Phe–Phe–Pro–NmeLeu–Met–NH2 (GR 73,632), in spontaneously hypertensive rats (SHR) and their progenitor strain, the Wistar–Kyoto rat (WKY). Sodium pentobarbitone anaesthetised rats with implanted intrathecal catheters were paralysed (pancuronium dibromide) and artificially ventilated. Injection of GR 73,632 at the T9 spinal level evoked dose-dependent increases in mean arterial pressure (MAP) in WKY and SHR. SHR had a lower MAP response threshold than WKY but increase in response with increasing dose was less in SHR than WKY. Biphasic blood pressure responses at high doses were observed in both strains. Prior administration of the NK1-receptor antagonist (3aR,7aR)-7,7-diphenyl-2-[1-imino-2(methoxyphenyl)ethyl] perhydroisoindol-4-one (RP 67,580) significantly reduced the pressor response in WKY but not SHR. The depressor response was not attenuated in either strain.     

Monoamine oxidase in the intermediolateral nucleus of the thoracic spinal cord of the rat. A histochemical study

We examined monoamine oxidase (MAO) activity in the intermediolateral nucleus (IML) of the rat thoracic spinal cord by histochemistry with tyramine as a common substrate for both MAO types A and B. Light microscopy showed MAO activity in neuronal cell bodies, processes, and varicosities. Electron microscopic examination showed both MAO-positive and -negative neuronal cell bodies. In the stained cell bodies, histochemical reaction products were localized in the cytoplasm showing a selective association with mitochondrial outer membranes. MAO-positive axon terminals were often found in contact with MAO-negative neurons but only occasionally with MAO-positive neurons. MAO histochemistry in the IML was also performed using serotonin (a MAO type A preferential substrate) and beta-phenylethylamine (a MAO type B preferential substrate). Light microscopy identified MAO activity for serotonin in a plexus of varicosities but not in any neuronal cell bodies. The activity for beta-phenylethylamine was detected frequently in neuronal cell bodies but rarely in varicosities. Our findings indicate that two groups of IML neurons can be chemically distinguished, one contains MAO type B while the other lacks both MAO types A and B. In addition, many axon terminals contain MAO type A but only a few fibers include MAO type B in the IML.     

Vasopressin depolarizes lateral horn cells of the neonatal rat spinal cord in vitro

The effects of vasopressin (VP) on lateral horn cells including a number of sympathetic preganglionic neurons contained in thin in vitro slices of neonatal rat spinal cord were investigated by means of intracellular recording techniques. Superfusion of (Arg⁸)-vasopressin (AVP, 0.01–1 μM) caused a depolarization leading, in the majority of lateral horn cells, to repetitive discharges. The AVP depolarization which could be partially reduced by low Ca/high Mg solution or tetrodotoxin, was accompanied by an increase in membrane resistance and the response was nullified near the membrane potential at which the spike afterhyperpolarization was abolished. A clear reversal of the response was not observed upon further hyperpolarization. The AVP response was blocked by the VP₁ antagonist,d-(CH₂)₅ Tyr (Me)-AVP, whereas, deamino (d-Arg⁸-vasopressin), a VP₂ agonist, at high concentrations ( 50 μM) was e ineffective or produced a small depolarization. The results indicate that AVP, acting mainly on VP₁ receptors, excited lateral horn cells by a direct depolarization and an indirect effect via the release of an excitatory transmitter(s). A reduction of a voltage-dependent K conductance may underlie the depolarizing effect of AVP.     

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.     

Role of neuron soma firing in the restoration of neurotensin store in sympathetic preganglionic neuron terminals after stimulus-evoked depletion

We have previously shown that prolonged preganglionic stimulation (e.g., 40 Hz 20 min) depletes the neurotensin (NT) store of preganglionic axon terminals in the stellate ganglion (SG) of the cat and that replenishment of the store requires several days. The present study investigates the mechanisms which control turnover of the NT store in sympathetic preganglionic axon terminals. NT content of the SG and of the preganglionic axons which innervate it was determined by radioimmunoassay in the anesthetized cat. This study shows that, during the 24 h after 40-Hz 20-min stimulation of the preganglionic input to the SG, the rate of NT accumulation proximal to a ligature on the stimulated input is three times that observed in the control. The poststimulus increase in NT accumulation rate is prevented by treatment with protein-synthesis inhibitors. When the centripetal propagation of action potentials from the stimulus site on the preganglionic axons is prevented by a tetrodotoxin bloxk applied locally during the stimulation period, the poststimulus increase in NT accumulation rate and the replenishment of the store are both prevented. These data suggest that the level of activity of the neuron regulates NT supply to the axon terminals, presumably by regulating NT synthesis. Thus, in the sympathetic preganglionic neuron, the action potential provides a mechanism for matching peptide synthesis to release.     

VGLUT1 and VGLUT2 innervation in autonomic regions of intact and transected rat spinal cord

Fast excitatory neurotransmission to sympathetic and parasympathetic preganglionic neurons (SPN and PPN) is glutamatergic. To characterize this innervation in spinal autonomic regions, we localized immunoreactivity for vesicular glutamate transporters (VGLUTs) 1 and 2 in intact cords and after upper thoracic complete transections. Preganglionic neurons were retrogradely labeled by intraperitoneal Fluoro-Gold or with cholera toxin B (CTB) from superior cervical, celiac, or major pelvic ganglia or adrenal medulla. Glutamatergic somata were localized with in situ hybridization for VGLUT mRNA. In intact cords, all autonomic areas contained abundant VGLUT2-immunoreactive axons and synapses. CTB-immunoreactive SPN and PPN received many close appositions from VGLUT2-immunoreactive axons. VGLUT2-immunoreactive synapses occurred on Fluoro-Gold-labeled SPN. Somata with VGLUT2 mRNA occurred throughout the spinal gray matter. VGLUT2 immunoreactivity was not noticeably affected caudal to a transection. In contrast, in intact cords, VGLUT1-immunoreactive axons were sparse in the intermediolateral cell column (IML) and lumbosacral parasympathetic nucleus but moderately dense above the central canal. VGLUT1-immunoreactive close appositions were rare on SPN in the IML and the central autonomic area and on PPN. Transection reduced the density of VGLUT1-immunoreactive axons in sympathetic subnuclei but increased their density in the parasympathetic nucleus. Neuronal cell bodies with VGLUT1 mRNA occurred only in Clarke's column. These data indicate that SPN and PPN are densely innervated by VGLUT2-immunoreactive axons, some of which arise from spinal neurons. In contrast, the VGLUT1-immunoreactive innervation of spinal preganglionic neurons is sparse, and some may arise from supraspinal sources. Increased VGLUT1 immunoreactivity after transection may correlate with increased glutamatergic transmission to PPN.