Retrogradely transported CTB-saporin kills sympathetic preganglionic neurons

Aiming to ablate sympathetic preganglionic neurons (SPN) innervating a defined target, we injected saporin conjugated to cholera toxin B subunit (CTB) unilaterally into the superior cervical ganglion of rats. In spinal cord segments T1-T3, the numbers of cholinergic neurons in the intermediolateral cell column ipsilateral and contralateral to the injected ganglion were significantly different by 3 days post-injection. By day 14, 77% of ipsilateral cholinergic neurons had disappeared. A higher percentage of neurons were killed in T1-T2 than in T3. Comparing SPN counts from CTB-saporin injected rats and counts from rats receiving unconjugated CTB into the superior cervical ganglion indicated that 84% of SPN supplying the ganglion had died by 14 days. Retrogradely transported CTB-saporin kills sympathetic preganglionic neurons and may also eliminate other types of neurons that transport CTB.     

Expression of Fos protein in adrenal preganglionic neurons following chemical stimulation of the rostral ventrolateral medulla of the rat

The ventrolateral medulla is known to be involved in the regulation of arterial blood pressure, especially via its connections with sympathetic preganglionic neurons (SPNs) mainly located in the intermediolateral nucleus of the spinal cord. It has been shown that stimulation of the rostral part of the ventrolateral medulla (RVLM) elicits a release of catecholamines from the adrenal medulla. The aim of the present study was to demonstrate the existence of a functional pathway between the RVLM and adrenal SPNs using the combination of a retrograde tract tracing technique (cholera toxin B subunit) with the immunohistochemical detection of Fos protein following the chemical stimulation of RVLM. The data obtained showed that: (1) chemical stimulation of the RVLM induced Fos immunoreactivity in the intermediolateral nucleus and particularly in SPNs projecting to the adrenal medulla; (2) along the thoracic segments T2-T12, 26.1% of retrogradely identified adrenal SPNs were Fos-immunoreactive with the greatest percentage (30.9%) in the T8 segment. These results favored a functional control of the RVLM on adrenal SPNs which may contribute to a substantial activation of the cardiovascular system via the release of adrenal catecholamines.     

Activation of adrenal preganglionic neurons during autonomic dysreflexia in the chronic spinal cord-injured rat

Autonomic dysreflexia (AD) occurs in a majority of high paraplegic and quadriplegic patients and is particularly characterized by a paroxysmal hypertension elicited by somatic or visceral stimuli. We have previously shown that plasma adrenaline and noradrenaline levels were significantly increased during episodes of AD in the 30-day spinal cord-injured (SCI) rats, suggesting the participation of adrenal catecholamines in the cardiovascular changes associated to AD. Thus, adrenal sympathetic preganglionic neurons (SPN) could be activated by visceral afferences leading to AD. The aim of this study was then to demonstrate whether visceral stimulation that induces AD activates adrenal SPN in chronic SCI rats. To this end, a retrograde tracer, the cholera toxin B subunit (CTB), was combined with the immunocytochemical detection of Fos protein after visceral stimulation. Chronic SCI rats received a CTB injection into the adrenal gland and, 3 days later, were stimulated by repetitive distension of the colon. Results showed that this stimulation elicited typical hypertensive episodes of AD and a significant increase in the number of double-labeled neurons (CTB/Fos immunoreactive neurons) in the thoracic spinal cord below the level of injury (T4 segment) when compared to the stimulated non-SCI rats. In conclusion, visceral stimulations in the chronic SCI rats activate adrenal SPN, which could induce release of catecholamines by the adrenal medulla. The present study brings new data on the spinal mechanisms of AD cardiovascular dysfunctions.     

Calretinin and calbindin D-28k,but not parvalbumin protect against glutamate-induced delayed excitotoxicity in transfected N18-RE 105 neuroblastoma-retina hybrid cells

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 approximately 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.     

Distribution of sympathetic preganglionic neurons innervating the kidney in the rat: PRV transneuronal tracing and serial reconstruction.

The organization of spinal motor circuitry to the kidney is not well-characterized and changes in renal innervation have been associated with disease states such as hypertension found in the spontaneously hypertensive rat or renal hypertension. Here, we describe the segmental and intra-segmental organization of the spinal motor circuitry that was resolved after neurotropic viral injection into the kidney and retrograde transneuronal transport to the spinal cord. In the first experiment, the serial reconstruction of infected neurons in the thoracolumbar spinal cord from T8-L1 was performed following injection of pseudorabies virus (PRV, Bartha strain) into either the cranial pole, the caudal pole or both the cranial and caudal poles of the left kidney in male rats. In the second experiment, rats received injections of two different PRV strains that were genetically engineered to express unique reporter molecules; one of the engineered strains was injected into the cranial pole and the other was injected into the caudal pole. Either 3- or 4-day post-infection, the animals were anesthetized and sacrificed by transcardial perfusion. PRV-infected neurons were located by immunocytochemistry against either PRV itself (experiment 1) or the unique marker proteins (experiment 2). After injection of both poles of the kidney, the majority of the infected neurons were found in the ipsilateral intermediolateral cell column (IML) from T10 to T12 with the mode at T11. Infected neurons were found in discrete neuron clusters in the intermediolateral cell column along the longitudinal axis in a repeating pattern of high and low density that has been called "beading". Three observations indicated a topographic distribution of renal sympathetic preganglionic neurons (SPN). First, after injection into either the cranial or caudal poles of the kidney, the mode of infected cells was located in segments T11 and T12, respectively. The one spinal segment shift in the mode suggested a topographic distribution. Second, in spinal segments T8-L1, comparison of the distributions of the neurons innervating each pole of the left kidney revealed an overlap in the distribution, except in the T11 segment. In the T11 segment, the neurons projecting to each pole tended to segregate into separate populations. Third, in rats that received injections of two PRV strains that were genetically engineered to express unique markers into opposite poles of the kidney, a segregation of neurons projecting to the cranial and caudal poles of the kidney was noted again in the T11 spinal segment and the segregation at adjacent spinal levels was obvious. The analysis of the distribution of infected neurons within each spinal cord segment (intra-segmental distribution) revealed three different patterns along the cranial-caudal dimension. In segments T8-T10, >60% of the infected neurons were located in the caudal half of the spinal segment. In segments T12-L1, >60% of the infected neurons were located in the cranial half of the spinal segment. In segment T11, the neurons were more evenly distributed throughout the segment. These intra-segmental distribution patterns were found after both 3- or 4-day survival periods post-infection and were found in most animals. The distribution of clusters of neurons revealed a similar intra-segmental pattern. Thus, as was described previously for the sympathetic postganglionic neurons that innervate the kidney, the present work indicates a topographic organization in the second-order neurons in the renal sympathetic efferent pathway. The physiological significance of this anatomical organization remains to be determined.     

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.     

Spinocerebellar tract neurons with axons passing through the inferior or superior cerebellar peduncles. A retrograde horseradish peroxidase study in rats

Using the retrograde horseradish peroxidase (HRP) method, we determined whether axons of the spinocerebellar tract (SCT) neurons pass through the superior (SCP) or the inferior (ICP) cerebellar peduncle in rats. Following bilateral section of either the SCPs or the ICPs, HRP was injected into the cerebellar anterior lobe and lobule VI, and the resulting labeled neurons were quantitatively examined throughout the length of the spinal cord. Almost all SCT neurons in the central cervical nucleus, Clarke's column and lamina VII of the third cervical (C3) to third thoracic (T3) segments and the T11 to fifth lumbar (L5) segments, and the majority of SCT neurons in the ventrolateral part of the anterior horn of the L6 to caudal (Ca) segments and laminae V of the C8-L5 segments and VII of the L6-Ca segments project their axons through the ICPs. The majority of spinal border cells (T11-L5) and a large number of SCT neurons in lamina VII of the C3-T3, T11-L5 and L6-Ca segments project their axons through the SCPs. A nearly equal number of SCT neurons in lamina VIII (C1-L6) send axons through the SCPs or the ICPs. The proportion of SCT neurons projecting via the SCPs versus those projecting via the ICPs was approximately 1:5.     

Sympathoadrenal preganglionic neurons in the adult rabbit send their dendrites into the contralateral hemicord

Sympathetic preganglionic neurons projecting to the adrenal medulla of the adult rabbit were retrogradely labelled with horseradish peroxidase. Preganglionic neurons were located in thoracic spinal cord segments T3-T12, peaking in number at T8, and only ipsilateral to the side of injections. However, retrogradely labelled dendritic processes of preganglionic neurons in the intercalated nucleus pars paraependimalys, the intercalated nucleus, and possibly even in the intermediolateral cell column, were observed in the contralateral hemicord. This suggests that the activity of sympathoadrenal preganglionic neurons could be modulated by both ipsilaterally and contralaterally descending pathways.     

Glial cell line-derived neurotrophic factor rescues target-deprived sympathetic spinal cord neurons but requires transforming growth factor-beta as cofactor in vivo.

Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for several populations of CNS and peripheral neurons. Synthesis and storage of GDNF by the neuron-like adrenal medullary cells suggest roles in adrenal functions and/or in the maintenance of spinal cord neurons that innervate the adrenal medulla. We show that unilateral adrenomedullectomy causes degeneration of all sympathetic preganglionic neurons within the intermediolateral column (IML) of spinal cord segments T7-T10 that project to the adrenal medulla. In situ hybridization revealed that IML neurons express the glycosylphosphatidylinositol-linked alpha receptor 1 and c-Ret receptors, which are essential for GDNF signaling. IML neurons also display immunoreactivity for transforming growth factor-beta (TGF-beta) receptor II. Administration of GDNF (recombinant human, 1 microg) in Gelfoam implanted into the medullectomized adrenal gland rescued all Fluoro-Gold-labeled preganglionic neurons projecting to the adrenal medulla after four weeks. Cytochrome c applied as a control protein was not effective. The protective effect of GDNF was prevented by co-administration to the Gelfoam of neutralizing antibodies recognizing all three TGF-beta isoforms but not GDNF. This suggests that the presence of endogenous TGF-beta was essential for permitting a neurotrophic effect of GDNF. Our data indicate that GDNF has a capacity to protect a population of autonomic spinal cord neurons from target-deprived cell death. Furthermore, our results demonstrate for the first time that the previously reported requirement of TGF-beta for permitting trophic actions of GDNF in vitro (Kreiglstein et al., 1998) also applies to the in vivo situation.     

Substance P-containing sensory neurons in the rat dorsal root ganglia innervate the adrenal medulla.

The adrenal medulla is innervated by both cholinergic and substance P (SP)-containing fibres via the splanchnic nerve. SP has been shown to modulate catecholamine (CA) secretion in isolated chromaffin cells and in the perfused rat adrenal gland, however, the origin of SP-containing fibres is not known. In the present study, we have combined the techniques of SP immunohistochemistry and retrograde tracing with Fast blue injected into the left adrenal medulla of the rat in order to study whether SP-containing sensory neurons in the dorsal root ganglia innervate the adrenal medulla. The results showed that there were on average 281 +/- 31 SP-like immunoreactive cells in each left dorsal root ganglion, T3-T13 (range, 234 +/- 19 in T4 to 372 +/- 43 in T13, n = 8). The average total number of Fast blue-labelled cells (T3-T13) in 8 experiments was 172 +/- 26, distributed normally about a peak at T8 (33.8 +/- 6.3 cells) and T9 (33.3 +/- 6.8 cells) with the least at T3 (1.5 +/- 0.8) and T13 (5.2 +/- 2.0). No Fast blue-labelled cells were found in the right DRG. In the left DRG, the average number of cells exhibiting both SP and Fast blue labelled cells were distributed from T7 to T9. These results demonstrate that SP-containing sensory neurons in the dorsal root ganglia provide an ipsilateral innervation of the adrenal medulla in rats.     

The anti-inflammatory effect of peripheral bee venom stimulation is mediated by central muscarinic type 2 receptors and activation of sympathetic preganglionic neurons

The anti-inflammatory effect (AI) induced by peripheral injection of diluted bee venom (dBV) involves activation of spinal cord circuits and is mediated by catecholamine release from adrenal medulla, but the precise neuronal mechanisms involved are not fully understood. In a recent study, we demonstrated that an increase in spinal acetylcholine is involved in mediating the anti-inflammatory effect of dBV and that this mediation also involves adrenomedullary activation. The present study utilized the mouse air pouch inflammation model to evaluate the involvement of spinal acetylcholine receptors and sympathetic preganglionic neurons (SPNs) in dBV's anti-inflammatory effect (dBVAI). Intrathecal (IT) pretreatment with atropine (muscarinic cholinergic antagonist) but not hexamethonium (nicotinic cholinergic antagonist) significantly suppressed dBVAI on zymosan-evoked leukocyte migration. Subsequent experiments showed that IT pretreatment with methoctramine (a muscarinic receptor type 2; M(2) antagonist), but not pirenzepine (an M(1) antagonist) or 4-DAMP (an M(3) antagonist), suppressed the dBVAI. In addition, dBV stimulation specifically increased Fos expression in SPNs of the T7-T11, but not the T1-T6 or T12-L2 spinal cord segments, in animals with zymosan-induced inflammation. Moreover, IT methoctramine pretreatment suppressed this dBV-induced Fos expression specifically in SPNs of T7-T11 level. Peripheral sympathetic denervation using 6-hydroxydopamine (6-OHDA) treatment (which spares sympathetic adrenal medullary innervation) did not alter dBVAI. Collectively these results indicate that dBV stimulation leads to spinal cord acetylcholine release that in turn acts on spinal M(2) receptors, which via a hypothesized disinhibition mechanism activates SPNs that project to the adrenal medulla. This activation ultimately leads to the release of adrenal catecholamines that contribute to dBVAI.     

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.     

Segmental distribution of peptide- and 5HT-like immunoreactivity in nerve terminals and fibers of the thoracolumbar sympathetic nuclei of the cat

The distribution of leucine-enkephalin, methionine-enkephalin, neurotensin, somatostatin, substance P, oxytocin, vasopressin, neurophysin II, and serotonin in nerve terminals and fibers of sympathetic autonomic areas of the thoracolumbar (T-L) spinal cord was studied immunohistochemically in cats. Densities of these immunoreactive terminals and fibers were estimated in the intermediolateral nucleus pars principalis (IMLp) and pars funicularis (IMLf), the nucleus intercalatus (IC), and the central autonomic area (CA). Results for leucine- and methionine-enkephalin-like immunoreactivity (ENK) were similar and immunoreactivity for vasopressin was not observed. The greatest numbers of terminals and fibers in the IMLp region contained ENK, neurotensin-(NT), and serotonin-like immunoreactivity (5HT); terminals and fibers containing substance P-(SP) and neurophysin II-like immunoreactivity (NP2) were intermediate in number, and those containing somatostatin-(SS) and oxytocin-like immunoreactivity (OXY) were generally sparse. In the IC and CA, terminals and fibers containing ENK and NT were dense, those containing SP were moderate, and those containing OXY, NP2, and 5HT were sparsely represented. In the IMLp, where the largest proportion of sympathetic preganglionic neurons (SPN) is found, the greatest concentration of terminals and fibers containing ENK was found in segments T1-T8; for NT these segments were T1-T5 and T11-L1, for SP-C8-T2 and T11-L1, for NP2-T4-T7 and L2 to L3, and for 5HT-T1-T5. Terminals and fibers containing SS and OXY were present in segments C8-T10 and segments C8, T2-T8, T13, and L2 to L3, respectively. These results indicate that while ENK, NT, SP, NP2, and 5HT fibers and terminals are widely distributed throughout the T-L cord, they may influence to a greater degree the SPN in segments where they are present in greater numbers. As SS and OXY were not found at all levels of the IMLp, their functions may be more organ specific.     

Localization of neurons in the rat spinal cord which project to the superior cervical ganglion

Horseradish peroxidase (HRP) was used to determine the location in the spinal cord of neurons projecting to the superior cervical ganglion of the rat. HRP was applied to the proximal cut end of the cervical sympathetic trunk, close to its entry into the superior cervical ganglion. After survival times of 3, 6, or 9 days, the animals were sacrificed and their spinal cords were processed to visualize HRP using diaminobenzidine, benzidine dihydrochloride, or tetramethylbenzidine. Labeled neurons were found only ipsilateral to the site of HRP application and were restricted to spinal segments C8-T5. Ninety percent of these neurons were located in segments T1-T3. Similar numbers of labeled neurons were found at survival times of 3 and 6 days and the mean number ± S.E.M. for 11 experiments at these two survival times was 1575 ± 89. Nine days after application of HRP the mean number of labeled cells and the density of label per cell were reduced. Labeled neurons were found in four regions of the spinal cord: the intermediolateral nucleus (75%), the lateral funiculus (23%), the central autonomic area (1%), and the intercalated region (1%). The cells of the intermediolateral nucleus did not form a continuous column along the rostrocaudal axis of the spinal cord, but instead were often found in clusters, several clusters being present per spinal segment.     

Localization of efferent sympathetic neurons and afferent neurons in dorsal root ganglia innervating the heart

The retrograde transport of horseradish peroxidase (HRP) has been used to study the localization and the number of neurons innervating the heart in the right stellate ganglion and accessory cervical ganglion, spinal cord and dorsal root ganglia of the cat. HRP was applied to the central cuts of anastomose of the stellate ganglion with the vagal nerve, of the vagosympathetic trunk caudal to anastomose and of the inferior cardiac nerve. HRP-labelled neurons were detected in the stellate ganglion in the regions which give off nerves, whereas in the accessory cervical ganglion labelled neurons were distributed throughout the whole ganglion. HRP-stained cells were found in the anastomose. In the spinal cord labelled neurons were detected in the lateral horn of T1-T5 segments. In the dorsal root ganglion the greatest number of neurons was observed in T2-T4 segments.     

Calbindin-immunoreactive neurons in the reticular formation of the rat brainstem: catecholamine content and spinal projections

Calbindin-D28k (calbindin) is a calcium-binding protein that is distributed widely in the rat brain. The localisation of calbindin immunoreactivity in the medulla oblongata and its colocalisation with adrenaline-synthesising neurons [phenylethanolamine-N-methyltransferase-immunoreactive (PNMT-IR)] was examined (Granata and Chang [1994] Brain Res. 645:265-277). However, detailed information about the distribution of calbindin-IR neurons in the reticular formation of the medulla oblongata in particular is lacking. In this report, the authors address this issue with an emphasis on the quantitation of calbindin-IR neurons, catecholamine neurons [tyrosine hydroxylase (TH)-IR, or PNMT-IR], and spinally projecting neurons in the ventral brainstem. Rats received injections of the retrograde tracing agent cholera toxin B (CTB) into the thoracic spinal cord or into the superior cervical ganglion. Immunocytochemistry was used to reveal calbindin, TH, PNMT, and CTB immunoreactivity. Ten calbindin-IR cell groups were identified within the pontomedullary reticular formation. Seven previously undescribed but distinct clusters of calbindin-IR neurons were found. Within the ventral pons, a population of calbindin-IR neurons occurred dorsal but adjacent to the A5 cell group. These calbindin-IR neurons did not contain either TH or PNMT immunoreactivity, and few if any of these neurons projected to the spinal cord. A distinct group of calbindin-IR neurons was present in the ventral medulla. Seventy-five percent of these calbindin-IR neurons contained TH immunoreactivity, 45% contained PNMT immunoreactivity, and 21% were spinally projecting neurons. Spinally projecting, calbindin-IR neurons were a subpopulation of PNMT-IR cells. In the caudal ventral medulla, no TH-IR or PNMT-IR cells were calbindin-IR. In the intermediolateral cell column, close appositions of calbindin-IR terminals on identified sympathetic preganglionic neurons as well as calbindin-IR synapses indicated that these neurons may affect directly the sympathetic outflow. The results demonstrate for the first time the existence of a new subpopulation of spinally projecting, PNMT-IR neurons in the rostral ventrolateral medulla.     

Pituitary adenylate cyclase activating polypeptide-immunoreactive sensory neurons innervate rat adrenal medulla

Rat adrenal chromaffin cells were invested by a dense network of nerve fibers immunoreactive to pituitary adenylate cyclase activating polypeptide-38 (PACAP-IR). Immunohistochemical studies demonstrated the presence of PACAP-IR in nodose and dorsal root ganglion cells, but not in neurons of the intermediolateral cell column and other autonomic nuclei of the thoracic and upper lumbar spinal cord. Somata of the T7 to T12 paravertebral ganglia were PACAP-negative. A few lightly labeled neurons were occasionally noted in the dorsal motor nucleus of the vagus. Injection of the retrograde tracer Fluorogold into the left adrenal medulla 3 days prior to sacrifice resulted in the labeling of a population of neurons in the ipsilateral spinal cord intermediolateral cell column (T1 to L1), ipsilateral and contralateral nodose ganglia and ipsilateral dorsal root ganglia from T7 to T10 inclusive. A small number of lightly labeled somata was occasionally noted in the dorsal motor nucleus of the vagus. Combined retrograde tracing and PACAP immunohistochemistry showed that a population of Fluorogold-containing nodose and dorsal root ganglion cells were also PACAP-positive. Pre-treatment of the rats with capsaicin caused a marked reduction of the PACAP-IR in the adrenal gland as well as in the superficial layers of the dorsal horn and caudal spinal trigeminal nucleus. These findings, in conjunction with the apparent absence of PACAP-IR in spinal sympathetic preganglionic neurons, sympathetic postganglionic neurons, and dorsal motor nucleus of the vagus, raise the possibility that PACAP-IR fibers observed in the adrenal medulla are primarily sensory in origin. As a corollary, catecholamine secretion from chromaffin cells may be modulated by the peptidergic sensory afferents in addition to the cholinergic sympathetic preganglionic nerve fibers.     

Preganglionic innervation of the adrenal gland of the rat: a study using horseradish peroxidase.

Adrenal medullae of 15-day-old and adult rats were injected with a solution of horseradish peroxidase. Preganglionic neurons in the 15-day-old rats were readily identified in the intermediate zone of the spinal cord after staining with diaminobenzidine tetrahydrochloride; horseradish peroxidase uptake could not be demonstrated in the adult rats. Adrenal preganglionic neurons were distributed between spinal segments T1 and L1. Approximately 50% of the neurons were located within segments T7 to T10. Only slight differences were observed between the distributions of neurons projecting to the right and left adrenal. The maximum velocity of transport of horseradish peroxidase was calculated to be approximately 4 mm/hr. Horseradish peroxidase filled neurons so that it was possible to observe the major orientation of perikarya and dendrites. The perikarya and dendrites exhibited a predominately transverse orientation.     

Neuronal organization and cell interactions of the cat stellate ganglion.

The functional structure of the cat stellate ganglion (SG) and, in particular, its extra- and intraganglionic connections and neuronal organization, were investigated using histochemical, immunohistochemical, morphological and histological methods. Retrograde axonal transport of horseradish peroxidase was used to determine most of the extraganglionic interactions. Of the targets tested, the most extensive efferent connections of the SG were with the stemocleidomastoid muscle, trachea, esophagus and heart. Neurons of the SG also send a small number of postganglionic efferents to the thyroid and stomach. Furthermore, ganglion cells send axons to the spinal ganglia. Several afferent connections of the SG were determined. Sympathetic preganglionic neurons of segments C8-T10 of the spinal cord, sensory neurons of C8-T9 spinal ganglia, intramural ganglia of the thoracic viscera and the reticular formation of the medulla oblongata send their axons to the SG. Intraganglionic interactions of intemeurons with principal ganglionic cells were assumed to occur, based on the presence of interneurons immunoreactive to GABA and substance P. GABA- and substance P-immunoreactive fibers located around a small number of postganglionic neurons were also identified. Morphological study revealed asymmetry between the left and right ganglia: the right ganglion is larger than the left and contains more cells. This asymmetry was also reflected in basic structural parameters of neurons, such as average neuronal area and average diameter of cell somata. The present data has been used to develop a scheme for the basic inter- and intraneuronal connections of the cat SG. This ganglion is a true nervous center, with postganglionic neurons, some of which might be performing sensory functions, and interneurons. The ganglion is connected not only with the spinal cord and spinal ganglia, but also with neurons of the intramural ganglia and, by direct links, with efferent neurons of the medulla oblongata. Thus, the SG may play an essential role in viscera-visceral reflexes.