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.     

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.     

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.     

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.     

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.     

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.     

Noradrenaline modifies sympathetic preganglionic neuron spike and afterpotential

Sympathetic preganglionic neurons were antidromically identified in the slice of the upper thoracic spinal cord of the adult cat, maintained in vitro. In normal Krebs solution, spikes evoked by intracellular stimulation had a marked ‘hump’ on the repolarization phase and were followed by an afterhyperpolarization of 2.8 s duration and 16.6 mV peak amplitude. Superfusion with Krebs solution containing noradrenaline 10–50 μm reversibly abolished the ‘hump’ of the spike and the late component of the afterhyperpolarization. In addition, it caused the appearance of a depolarizing afterpotential of 100–600 ms duration. This depolarization could result in repetitive firing of the neuron in response to a single intracellular current pulse.     

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.     

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.     

C1 neurons in the rat rostral ventrolateral medulla differentially express vesicular monoamine transporter 2 in soma and axonal compartments

Vesicular monoamine transporter 2 (VMAT2) packages biogenic amines into large dense core and synaptic vesicles for either somatodendritic or synaptic release from neurons of the CNS. Whilst the distribution of VMAT2 has been well characterized in many catecholaminergic cell groups, its localization amongst C1 adrenergic neurons in the medulla has not been examined in detail. Within the rostral ventrolateral medulla (RVLM), C1 neurons are a group of barosensitive, adrenergic neurons. Rostral C1 cells project to the thoracic spinal cord and are considered sympathetic premotor neurons. The majority of caudal C1 cells project rostrally to regions such as the hypothalamus. The present study sought to quantitate the somatodendritic expression of VMAT2 in C1 neurons, and to assess the subcellular distribution of the transporter. Immunoreactivity for VMAT2 occurred in 31% of C1 soma, with a high proportion of these in the caudal part of the RVLM. Retrograde tracing studies revealed that only two of 43 bulbospinal C1 neurons contained faint VMAT2-immunoreactivity, whilst 88 +/- 5% of rostrally projecting neurons were VMAT2-positive. A lentivirus, designed to express green fluorescent protein exclusively in noradrenergic and adrenergic neurons, was injected into the RVLM to label C1 neurons. Eighty-three percent of C1 efferents that occurred in close proximity to sympathetic preganglionic neurons within the T(3) intermediolateral cell column contained VMAT2-immunoreactivity. These data demonstrate differential distribution of VMAT2 within different subpopulations of C1 neurons and suggest that this might reflect differences in somatodendritic vs. synaptic release of catecholamines.     

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.     

Segmental distribution of corticotropin-releasing factor-like and vasoactive intestinal peptide-like immunoreactivities in presumptive sympathetic preganglionic neurons of the cat

The distribution of corticotropin-releasing factor (CRF), vasoactive intestinal peptide (VIP), and luteinizing hormone-releasing hormone (LH-RH) in cell bodies of sympathetic autonomic nuclei of the thoracolumbar spinal cord was studied immunohistochemically in cats after intrathecal administration of colchicine. Neurons containing CRF-like immunoreactivity (CRFir) and VIP-like immunoreactivity (VIPir), but not LH-RH-like immunoreactivity, were found in the intermediolateral nucleus pars principalis (IMLp) and pars funicularis (IMLf). On the basis of identification in previous studies and the size, shape, and location of the immunoreactive cells, it is suggested that the neurons are sympathetic preganglionic neurons. Most of the neurons with CRFir (85.5%) were found in the IMLp in segments T2-T7 and L2-L3 and the remaining 14.5% were found in the IMLf in segments T2-T5. The largest proportion of neurons with VIPir (93.7%) was found in the IMLp in segments T2, T4-T7, and T9-T13. Only 6.3% of the neurons containing VIPir were found in the IMLf in segments T2, T4, T5, and T10. These findings suggest that CRF and VIP may participate in peptide-specific pathways to peripheral organs.     

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.     

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.     

Inhibition of sympathetic activity by stimulating in the raphe nuclei and the role of 5-hydroxytryptamine in this effect

The possibility that the putative transmitter 5-hydroxytryptamine (5-HT) is involved in the mediation of long latency to onset raphe-spinal inhibition of sympathetic preganglionic neurones was investigated in anaesthetized cats by stimulating sites located in nucleus raphe pallidus and obscurus and recording sympathetic discharge in T₃ or T₁₀ white rami evoked either reflexively or by intraspinal stimulation at cervical level. Several putative 5-HT anttagonists were administered intravenously (i.V.) or topically to the spinal cord. In 7 cats lysergic acid diethylamide (LSD) in a dose range 25–50 μg/kg i.v. or 0.6 μg topically, reversibly reduced the raphe spinal inhibition by 40–100%. Topical application was more effective than i.v. administration. In 5 cats stimulating within the ventromedial reticular formation at sites unlikely to involved 5-HT neurons produced a short latency to onset inhibition which was unaffected by LSD. Methysergide, cinanserin and cyproheptadine depressed sympathetic discharge in the absence of brain stimulation in cats with CNS intact and in unanaesthetized decerebrate spinal cats. The results are discussed in the light of the known actions of the putative 5-HT antagonists.     

Developmental distribution of reelin-positive cells and their secreted product in the rodent spinal cord

To date, only sympathetic and parasympathetic preganglionic neurons are known to migrate abnormally in reeler mutant spinal cord. Reelin, the large extracellular matrix protein absent in reeler, is found in wild-type neurons bordering both groups of preganglionic neurons. To understand better Reelin's function in the spinal cord, we studied its developmental expression in both mice and rats. A remarkable conservation was found in the spatiotemporal pattern of Reelin in both species. Numerous Reelin-expressing cells were found in the intermediate zone, except for regions containing somatic and autonomic motor neurons. A band of Reelin-positive cells filled the superficial dorsal horn, whereas only a few immunoreactive cells populated the deep dorsal horn and dorsal commissure. High levels of diffuse Reelin product were detected in the lateral marginal and ventral ventricular zones in both rodent species. This expression pattern was detected at all segmental spinal cord levels during embryonic development and remained detectable at lower levels throughout the first postnatal month. To discriminate between the cellular and secreted forms of Reelin, brefeldin A was used to block secretion in organotypic cultures. Such perturbations revealed that the high levels of secreted Reelin in the lateral marginal zone were derived from varicose axons of more medially located Reelin-positive cells. Thus, the laterally located secreted Reelin product may normally prevent the preganglionic neurons from migrating too far medially. Based on the strong evolutionary conservation of Reelin expression and its postnatal detection, Reelin may have other important functions in addition to its role in neuronal migration.     

Efferent projections of C3 adrenergic neurons in the rat central nervous system

C3 neurons constitute one of three known adrenergic nuclei in the rat central nervous system (CNS). While the adrenergic C1 cell group has been extensively characterized both physiologically and anatomically, the C3 nucleus has remained relatively obscure. This study employed a lentiviral tracing technique that expresses green fluorescent protein behind a promoter selective to noradrenergic and adrenergic neurons. Microinjection of this virus into the C3 nucleus enabled the selective tracing of C3 efferents throughout the rat CNS, thus revealing the anatomical framework of C3 projections. C3 terminal fields were observed in over 40 different CNS nuclei, spanning all levels of the spinal cord, as well as various medullary, mesencephalic, hypothalamic, thalamic, and telencephalic nuclei. The highest densities of C3 axon varicosities were observed in Lamina X and the intermediolateral cell column of the thoracic spinal cord, as well as the dorsomedial medulla (both commissural and medial nuclei of the solitary tract, area postrema, and the dorsal motor nucleus of the vagus), ventrolateral periaqueductal gray, dorsal parabrachial nucleus, periventricular and rhomboid nuclei of the thalamus, and paraventricular and periventricular nuclei of the hypothalamus. In addition, moderate and sparse projections were observed in many catecholaminergic and serotonergic nuclei, as well as the area anterior and ventral to the third ventricle, Lamina X of the cervical, lumbar, and sacral spinal cord, and various hypothalamic and telencephalic nuclei. The anatomical map of C3 projections detailed in this survey hopes to lay the first steps toward developing a functional framework for this nucleus.     

Regulation of cardiac sympathetic afferent reflex by GABA(A) and GABA(B) receptors in paraventricular nucleus in rats

The aim of the present study was to determine the role of GABA_A and GABA_B receptors in paraventricular nucleus (PVN) in regulating cardiac sympathetic afferent reflex (CSAR). Under urethane (800 mg/kg) and α-chloralose (40 mg/kg) anesthesia, renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and heart rate (HR) were recorded in sinoaortic-denervated and cervical-vagotomized rats. CSAR was evaluated based in the response of RSNA to epicardial application of capsaicin (0.3 nmol) or bradykinin (1 nmol). Bilateral PVN microinjection of the GABA_A receptor agonist isoguvacine (10 nmol) attenuated CSAR, while the GABA_B receptor agonist baclofen (1 nmol) abolished CSAR. Both isoguvacine and baclofen greatly decreased baseline RSNA and MAP. The GABA_A receptor antagonist gabazine (0.1 nmol) had no significant effect on CSAR, but the GABA_B receptor antagonist CGP-35348 (10 nmol) enhanced CSAR. Gabazine caused greater increases in baseline RSNA and MAP than CGP-35348. Vigabatrin (10 nmol), a selective GABA-transaminase inhibitor which increases endogenous GABA level, abolished CSAR, and decreased baseline RSNA, MAP and HR. The effects of vigabatrin were antagonized by combined gabazine (0.1 nmol) and CGP-35348 (10 nmol). The results indicate that activation of either GABA_A or GABA_B receptors in the PVN inhibits CSAR, while blockage of GABA_B receptors in the PVN enhances CSAR. Endogenous GABA in the PVN could have an important role in regulating CSAR.     

Neuropeptide Y innervation of nitric oxide-synthesizing sympathetic preganglionic neurons in humans

We sought to determine whether neuropeptide Y (NPY) terminals are concentrated in the intermediolateral (IML) cell column and innervate human NADPH-diaphorase (NADPH-d)-reactive sympathetic preganglionic neurons (SPns). Spinal cords were obtained at autopsy from one man and two women, cut into segments, and immersion fixed in 2% paraformaldehyde for 24 h. The T1, T3, T6 and T8 spinal cord segments were cut serially at 50 m in the coronal, sagittal and horizontal planes. Alternating consecutive sections were double stained for NADPH-d and NPY or NPY alone. NPY-immunoreactive fibers were identified at all levels analyzed and varicosities appeared to run and cover the NADPH-d processes for long distances. NPY-immunoreactive varicosities were heavily concentrated around the soma and proximal dendrites of NADPH-d SPNs. NPY may exert many possible actions at the level of the IML cell column. Depletion of NPY-containing bulbospinal neurons may contribute to sympathetic failure in disorders such as multiple system atrophy.