Sympathoadrenal Preganglionic Neurons Research Articles

The abnormalities of adrenomedullary hormonal system in genetic hypertension: Their contribution to altered regulation of blood pressure.

It is widely accepted that sympathetic nervous system plays a crucial role in the development of hypertension. On the other hand, the role of adrenal medulla (the adrenomedullary component of the sympathoadrenal system) in the development and maintenance of high blood pressure in man as well as in experimental models of hypertension is still controversial. Spontaneously hypertensive rats (SHR) are the most widely used animal model of human essential hypertension characterized by sympathetic hyperactivity. However, the persistence of moderately elevated blood pressure in SHR subjected to sympathectomy neonatally as well as the resistance of adult SHR to the treatment by sympatholytic drugs suggests that other factors (including enhanced activity of the adrenomedullary hormonal system) are involved in the pathogenesis of hypertension of SHR. This review describes abnormalities in adrenomedullary hormonal system of SHR rats starting with the hyperactivity of brain centers regulating sympathetic outflow, through the exaggerated activation of sympathoadrenal preganglionic neurons, to the local changes in chromaffin cells of adrenal medulla. All the above alterations might contribute to the enhanced release of epinephrine and/or norepinephrine from adrenal medulla. Special attention is paid to the alterations in the expression of genes involved in catecholamine biosynthesis, storage, release, reuptake, degradation and adrenergic receptors in chromaffin cells of SHR. The contribution of the adrenomedullary hormonal system to the development and maintenance of hypertension as well as its importance during stressful conditions is also discussed.

Thermogenesis mediated by a capsaicin-sensitive area in the ventrolateral medulla.

Systemic administration of capsaicin elicits heat production, which can be observed in decerebrated preparations but is blocked by spinal transection. To identify the critical locus involved in the capsaicin-induced thermogenesis in the brainstem, we studied the effect of capsaicin on rats with bilateral electrolytic lesions in the premotor areas of sympathoadrenal preganglionic neurons. Lesions in the rostral ventrolateral medulla (RVLM), but not in other regions, largely attenuated the capsaicin-induced heat production. Unilateral microinjection of 30-100 nl capsaicin (0.5%, w/v) into the RVLM elicited a heat production response, whereas capsaicin injection in neighboring areas or vehicle injection into the RVLM did not affect heat production. These results suggest that the thermogenic effect of capsaicin is mediated, at least in part, by some capsaicin-sensitive structure in the RVLM.

Analysis of a multi-mutant herpes simplex virus type 1 for gene transfer into sympathetic preganglionic neurons and a comparison to adenovirus vectors

A non-replicating triple-mutant herpes simplex virus (14H Δ3vhsZ) expressing the bacterial marker enzyme β-galactosidase, was assessed for neurotropism and cytopathic effects as a vector for gene transfer into differentiated phaeochromocytoma 12 cells in vitro and into spinal sympathetic neurons in vivo. In the in vivo study, the 14H Δ3vhsZ was injected into the adrenal gland of hamsters. For comparison, an evaluation of two adenovirus vectors, AdCA17lacZ and AdCA36lacZ, was performed. Infection of the differentiated phaeochromocytoma 12 cells by 14H Δ3vhsZ resulted in intense β-galactosidase staining in 80–90% of the cells without changes in cell morphology, detected by light microscopy, after a period of four days. No cytoskeletal disruption was detected by immunocytochemistry for the neurofilament protein and no apoptosis was demonstrated by the Hoescht stain for nuclear chromatin in virus-infected cells in comparison to mock-infected control cells. Two to three days after adrenal inoculation with 14H Δ3vhsZ, β-galactosidase was detected in 240 preganglionic neurons per hamster ( n=8), a number equal to about 25% of the population of targeted neurons. The β-galactosidase reaction product extended throughout the normal kite-shaped neuronal somata and extensive dendritic arbour. The number decreased to 120 by five days ( n=3) and to two by eight days ( n=4). This decrease was presumably due to loss of expression of the marker gene and not to cell death because, at eight days, the number of sympathetic preganglionic neurons in the nucleus intermediolateralis, pars principalis, that were immunoreactive for the neurotransmitter enzyme choline acetyltransferase, and demonstrated nicotinamide adenine dinucleotide phosphate-diaphorase activity, were the same on the infected left side of the cord as on the uninfected right side. Inflammatory cells surrounded some of the infected neurons at five days but by eight days the infiltrate was reduced. Infection of differentiated phaeochromocytoma 12 cells by AdCA17lacZ and AdCA36lacZ also resulted in marker gene expression in a large proportion of the cells (80–90%) in the absence of cytopathic effects. In contrast, four days after adrenal injection of AdCA17lacZ or AdCA36lacZ ( n=5 for each) only an average of three preganglionic neurons per hamster expressed β-galactosidase activity, despite clear adrenal infection. AdCA17lacZ and AdCA36lacZ both produced light patches of staining confined to the neuronal soma. These neurons had normal morphology but sometimes were surrounded by an inflammatory infiltrate. In conclusion, the non-replicating herpes simplex virus, 14H Δ3vhsZ, had minimal cytotoxic effects in neurons, in vitro or in vivo, and was efficiently transported from the adrenal gland to infect many sympathoadrenal preganglionic neurons. In contrast, very few neurons demonstrated β-galactosidase activity after injection into the adrenal gland of AdCA17lacZ and AdCA36lacZ. Therefore, 14H Δ3vhsZ is a more suitable vector than either of the adenovirus vectors tested for eliciting short-term changes in preganglionic neuron gene expression.

Changes in immunoreactivity for growth associated protein-43 suggest reorganization of synapses on spinal sympathetic neurons after cord transection

Cervical or high thoracic spinal cord injury often results in autonomic dysreflexia, a condition characterized by exaggerated spinal reflexes and episodic hypertension, that may be caused by reorganization of synapses on sympathetic preganglionic neurons after loss of supraspinal input. To assess remodelling of synaptic input to identified preganglionic neurons, immunoreactivity for growth associated protein-43 was examined by fluorescent and electron microscopy in control rats with intact spinal cords and in rats seven to 30 days after midthoracic cord transection. This protein is found in mature bulbospinal axons that supply spinal sympathetic nuclei and it is also known to be up-regulated in growing or sprouting axons. In the thoracic cord of control rats, fibres containing growth associated protein-43 surrounded histochemically- or retrogradely-labelled preganglionic neurons and formed a ladder-like pattern in the gray matter. Fibres travelled rostrocaudally along the lateral horn and, at approximately regular intervals, they coursed mediolaterally to form the “rungs” of a ladder. Electron microscopy revealed concentrated growth associated protein-43 in many intervaricose axon segments in the intermediolateral cell column. Less frequently, faint immunoreactivity for this protein was found in varicosities, some of which synapsed on retrogradely-labelled sympathoadrenal preganglionic neurons. Electron microscopy of conventionally processed tissue was used to determine the time-course of degeneration of severed axon terminals in the intermediolateral cell column. In spinal rats, terminals with ultrastructural signs of degeneration were numerous in the intermediolateral cell column three days after transection, but were rare at seven days and absent at 14 days. Degenerating terminals were never found in this region in control rats. Thus virtually all supraspinal inputs to preganglionic neurons had been eliminated by seven days after transection. At longer times after injury, terminals containing immunoreactivity for growth associated protein-43 must therefore arise from intraspinal neurons. The distribution of fibres immunoreactive for growth associated protein-43 changed markedly in the first 30 days after cord transection. By 14 days, the ladder-like pattern was distorted rostral to the transection by enlarged masses of immunoreactive fibres surrounding preganglionic neurons, suggesting sprouting of bulbospinal or intraspinal axons or accumulation of this protein in their terminals after the parent axon had been severed. Caudal to the transection, the ladder-like arrangement of fibres was completely replaced by a reticular network of immunoreactive fibres that extended throughout the intermediate gray matter and increased in density between 14 and 30 days. In the intermediolateral cell column, at fourteen days after transection, axons with the ultrastructural features of growth cones contained intense growth associated protein-43 immunoreactivity. Although varicosities of bulbospinal axons containing this protein had degenerated by 14 days, weak immunoreactivity was still found in varicosities that synapsed on labelled sympathoadrenal neurons. Furthermore, immunoreactivity appeared in numerous somata of presumed interneurons throughout the intermediate gray matter by 14 days and the number of somata increased by 30 days. These interneurons may be the source of this protein in the reticular network, and in growth cones and synapses. The loss of supraspinal inputs by seven days after cord transection, and the new intraspinal network of immunoreactive fibres, synapses and cells are consistent with new synapse formation on preganglionic neurons. New synapses on preganglionic neurons may be crucial for the development of autonomic dysreflexia.

Gene transfer into sympathetic preganglionic neurons in vivo using a non-replicating thymidine kinase-deficient herpes simplex virus type 1

The suitability of non-replicating thymidine kinase deficient herpes simplex virus type 1 expressing bacterial β-galactosidase ( tk -lacZ HSV-1) as a transfer vehicle into sympathetic preganglionic neurons in vivo was assessed. Many sympathoadrenal preganglionic neurons (451±105) with normal morphology were identified using β-galactosidase histochemistry two days after inoculation of tk -lacZ HSV-1 into the adrenal gland of hamsters. β-Galactosidase activity co-localized with nicotinamide adenine dinucleotide phosphate-diaphorase-positive sympathetic preganglionic neurons in the nucleus intermediolateralus, pars principalis. The maximal number of β-galactosidase expressing neurons was found two days post-inoculation but this number dropped dramatically after this time. An inflammatory infiltrate was abundant around infected neurons and in the white matter at five days and infected neurons appeared morphologically abnormal. At 26 days, the infiltrate was still present but no infected sympathoadrenal preganglionic neurons were detected. Approximately 25% fewer nicotinamide adenine dinucleotide phosphate-diaphorase-positive neurons in the nucleus intermediolateralis, pars principalis were counted ipsilaterally than contralaterally in animals infected for 14, 21 or 26 days with tk -lacZ HSV-1, compared to the 3% difference in animals mock-infected for 26 days. Approximately 33% of the estimated number of sympathoadrenal preganglionic neurons infected with tk -lacZ HSV-1 at five days were apoptotic or necrotic. About 60% of neurons infected with tk -lacZ HSV-1 at two days no longer expressed nicotinamide adenine dinucleotide phosphate-diaphorase at 14–26 days. In conclusion, the non-replicating thymidine kinase deficient HSV-1 was efficiently retrogradely transported from the adrenal gland to infect sympathoadrenal preganglionic neurons. These gene transfer experiments using tk -lacZ HSV-1 suggest that foreign gene expression in sympathetic preganglionic neurons in vivo may be maximal two days after inoculation when β-galactosidase was expressed in the greatest number of sympathetic preganglionic neurons. After two days, fewer neurons expressed β-galactosidase and the presence of tk -lacZ HSV-1 appeared to be altering protein expression in sympathetic preganglionic neurons and/or leading to the demise of the infected neuron.

Monosynaptic projections from the medullary gigantocellular reticular formation to sympathetic preganglionic neurons in the thoracic spinal cord.

Microinjection of L-glutamate into a restricted area of the medullary gigantocellular reticular formation, the gigantocellular depressor area (GiDA), lowers arterial pressure. Unlike the nuclei tractus solitarii and the caudal ventrolateral medulla, the two principle medullary vasodepressor areas, the GiDA projects directly to the spinal cord and not to the rostral ventrolateral medulla (Aicher et al. [1994] Neuroscience 60:761-779). We investigated whether neurons within GiDA directly innervate autonomic areas of the thoracic spinal cord. Fluoro-Gold injected into the thoracic spinal cord labeled neurons within functionally defined vasodepressor sites in the GiDA in the same animal. To examine the morphology of GiDA efferents to the spinal cord, the anterograde tracer Phaseolus vulgaris-leucoagglutinin was iontophoresed into the GiDA, and efferent processes in the intermediolateral cell column and nucleus intercalatus spinalis were examined by electron microscopy. Labeling was confined to axons and axon terminals (n = 144) that usually contained primarily small clear vesicles, contacted large and small dendrites, and formed symmetric (inhibitory) synapses. To determine whether some of the postsynaptic targets of GiDA efferent terminals in the thoracic spinal cord were sympathoadrenal preganglionic neurons, these neurons were retrogradely labeled from the adrenal gland with Fluoro-Gold in rats that had deposits of the anterograde tracer, biotinylated dextran amine (BDA), in the GiDA. Some BDA-containing terminals formed symmetric synapses with dendrites containing Fluoro-Gold. We conclude that a population of neurons in the GiDA monosynaptically innervates some sympathetic preganglionic neurons. The findings suggest the presence of a novel reticulospinal sympathoinhibitory projection originating in the GiDA.

Delivery of a foreign gene to sympathetic preganglionic neurons using recombinant herpes simplex virus.

Two recombinant herpes simplex type 1 viruses expressing beta-galactosidase (encoded by the Escherichia coli lacZ gene) inserted into the unique long 41 (encoding virus host shutoff) or unique short 5 (encoding glycoprotein J) open reading frames were generated. Purified recombinants or wild-type herpes simplex type 1 were injected into the left adrenal gland of hamsters. Three days later, virus-infected neurons were detected in spinal cord sections from all infected hamsters. Neurons were visualized with beta-galactosidase histochemistry in spinal cord sections from hamsters infected with either of the recombinants but not with the wild-type virus. Wild-type virus could only be detected with immunocytochemistry. Insertional mutagenesis into the unique long 41 or unique short 5 regions of the herpes simplex genome by lacZ did not disrupt the neurotropic properties of the virus. Both recombinant viruses labelled the central nervous system sympathoadrenal preganglionic neurons as well as brainstem neurons. Because the virus host shutoff recombinant more readily crossed synapses to reach the brainstem compared to the glycoprotein J recombinant, the presence of glycoprotein J may facilitate cell to cell transmission in vivo. Both recombinants may be useful for the study of synaptic organization of neural circuits. Our recombinant viruses were less lytic yet neurovirulent after mutation of either glycoprotein J or virus host shutoff of herpes simplex virus type 1 wild-type. These recombinant viruses express the bacterial beta-galactosidase which is readily detectable using simple histochemistry. Inoculation of the adrenal gland or kidney with these viruses led to clear labelling of spinal cord cells. These viruses may be useful markers of specific neural circuits.

Segmental restriction and target specificity of bullfrog preganglionic neurons that exhibit galanin-like immunoreactivity

These experiments were designed to examine the distribution of galanin-like peptide immunoreactivity (GAL-IR) in bullfrog sympathetic preganglionic neurons and to identify the peripheral target organs affected by these neurons. Cells expressing GAL-IR were observed in the intermediolateral column of segments 7 and 8 only. Apparent GAL-IR innervation is present, but rare, in sympathetic chain ganglia. Double-labelling with retrogradely transported fast blue and galanin antiserum demonstrated that most GAL-IR neurons project via splanchnic nerves to innervate the adrenal gland, which receives a dense plexus of GAL-IR fibers surrounding chromaffin cells. The adrenal gland is also innervated by preganglionic neurons in segments 5 and 6 that do not express GAL-IR. Because nitric oxide is expressed in sympathoadrenal preganglionic neurons in mammals (Anderson, C.R., Neurosci. Lett., 139 (1992) 280), we examined whether it is expressed in bullfrog preganglionic neurons. Nicotinamide adenine dinucleotide phosphate-diaphorase positive neurons are present in bullfrog spinal grey at segments 5 through 8. These neurons were not double-labelled with fast blue retrogradely transported from the sympathetic chain, celiac ganglion, or adrenal gland; nor were they double-labelled with GAL-antiserum. Thus nitric oxide is apparently not expressed in bullfrog sympathetic preganglionic neurons.

Monosynaptic projections from the rostral ventrolateral medulla oblongata to identified sympathetic preganglionic neurons

The rostral ventrolateral medulla oblongata plays an important role in the control of arterial blood pressure and it has strong descending projections into the intermediolateral nucleus of the thoracic spinal cord, where the majority of sympathetic preganglionic neurons are located. The purpose of this study was to see whether these projections form synaptic contacts with sympathetic preganglionic neurons in the rat. Projections from both the lateral part of the rostral ventrolateral medulla (rostroventrolateral reticular nucleus) and from the more medial region (lateral paragigantocellular nucleus) were investigated separately in view of their different functional roles in sympatho-regulation and their different chemical composition. Using anterograde tract-tracing of descending medullary pathways with Phaseolus vulgaris leucoagglutinin and retrograde labelling of sympatho-adrenal preganglionic neurons with cholera B chain conjugated to horseradish peroxidase, the existence of monosynaptic connections was sought by electron microscopy. Synaptic inputs from both the lateral and medial aspects of the rostral ventrolateral medulla oblongata were found on identified sympathetic preganglionic neurons. Synaptic specializations were of both the symmetrical and asymmetrical type. The targets of boutons forming asymmetrical synaptic contacts differed according to their origin: boutons originating from neurons in the rostroventrolateral reticular nucleus were mainly in contact with dendrites of sympathetic preganglionic neurons, while those originating from the lateral paragigantocellular nucleus mainly innervated the cell bodies. Our observations provide anatomical support for the view that there are two distinct classes of sympatho-regulatory cells in the rostral ventrolateral medulla, each of which can directly influence the activity of sympathetic preganglionic neurons; they also emphasize the importance of detailed investigation of the subrogions of the ventrolateral medulla with respect to their sympatho-regulatory functions.

Basic fibroblast growth factor prevents neuronal death and atrophy of retrogradely labeled preganglionic neurons in vivo

We have used retrograde fluorescent tracing (fast blue) both before (prelabeling) and at time points after selective unilateral adrenomedullectomy in vivo (postlabeling) in order to investigate the survival and morphology of sympathoadrenal preganglionic (SAP) neurons located in the lower thoracic intermediolateral (IML) cell column of the adult rat spinal cord. By prelabeling with fast blue it was found that the majority (i.e., more than 85%) of the SAP neurons underwent degeneration and were lost from the IML cell column within 4 weeks after peripheral target lesion and administration of intramedullary gelfoam implants containing a non-trophic control protein cytochrome c. By contrast, atrophy and loss of SAP neurons was largely prevented by local treatment (intramedullary implants) with recombinant basic fibroblast growth factor (bFGF) as determined from counts of large and “healthy-looking” prelabeled neurons within the IML column ipsilateral to the lesioned (i.e., operated) side 4 weeks postimplantation. The time course of withdrawal of preganglionic axons from their lesioned target area was investigated by fast blue injections into intramedullary (control or bFGF) implants at weekly intervals postimplantation (postlabeling) and was documented by counting the number of healthy SAP neurons that retained the label. Without bFGF treatment, progressive numerical loss of SAP neurons was evident within 1 to 4 weeks postlesioning, indicative of pronounced retrograde cell degeneration. Retrograde cell degeneration was insignificant during the first 2 weeks postimplantation after early postlesion treatment with exogenous bFGF; it was apparently postponed to occur after 1 month. Implantation of gelfoam containing neutralizing anti-bFGF-anti-bodies resulted in accelerated retrograde axon degeneration implying that bFGF is an endogenous trophic factor for SAP neurons. The results are consistent with the idea that SAP neurons actually die following peripheral target lesion and are not supported from other trophic sources. However, these neurons can be prevented from disconnection-induced death by providing exogenous bFGF. Limited amounts of endogenous FGF may also become available to SAP axons by disintegration of nerve terminal-surrounding cells delaying the process of retrograde SAP neuron death.

β-galactosidase expressing recombinant pseudorabies virus for light and electron microscopic study of transneuronally labeled CNS neurons

A β-galactosidase expressing pseudorabies virus (Bartha strain) was constructed, injected into the adrenal gland of rats, and subsequently shown to transneuronally label the CNS autonomic neurons that project to sye sympathoadrenal preganglionic neurons. Virally infected neurons were visualized with a one-step histochemical reaction using the Bluo-Gal substrate (halogenated indolyl-β- d-galactoside) for the localization of β-galactosidase activity. In some infected neurons, a Golgi-like staining of the primary and sometimes secondary dendrites could be obtained. For electron microscopic studies, the Bluo-Gal substrate produces an electron-dense reaction product that is easily identified at both low and high magnification. This virus may be useful for the study of the cell architecture and synaptic organization of transneuronally labeled neurons of functionally defined neural circuits. These results also demonstrate that it is possible to deliver foreign genes into specific chains of neurons in the mammalian CNS by means of the retrograde transneuronal viral labeling method.

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.

Cytoarchitecture and dendroarchitecture of sympathoadrenal preganglionic neurons in rats--a study with CB-HRP.

The cytoarchitecture and dendroarchitecture of sympathoadrenal preganglionic neurons (SAPN) that innervate the adrenal medulla have been studied in adult rats. Retrograde transport of choleragen subunit B horseradish peroxidase conjugate (CB-HRP) injected into the left adrenal medulla resulted in the labelling of SAPN in the ipsilateral intermediate zone from T1 to L1 segments. Four subnuclei of SAPN were distinguished and located in ILP, ILF, IC and ICPe of the spinal cord respectively. The dendritic trees of neurons in ILP exhibited dendritic patterns with extension oriented longitudinally and mediolaterally. The present findings provide detailed descriptions of the somatic structure and accompanying dendritic trees of SAPN. The observation also include anatomical evidence that two systems of circuits may exist in regulating the activities of SAPN; one is the classic core circuits system which mainly received the inputs from fibers in parallel direction with longitudinal dendrites in ILP and the other is the paralateral circuits system which chiefly received the inputs from fibers in perpendicular direction with radial dendrites in white matter.

CNS cell groups regulating the sympathetic outflow to adrenal gland as revealed by transneuronal cell body labelling with pseudorabies virus

The CNS cell groups that innervate the sympathoadrenal preganglionic neurons of rats were identified by a transneuronal viral cell body labelling technique combined with neurotransmitter immunohistochemistry. Pseudorabies virus was injected into the adrenal gland. This resulted in retrograde viral infections of the ipsilateral sympathetic preganglionic neurons (T 4−T 13) and caused retrograde transneuronal cell body infections in 5 areas of the brain: the caudal raphe nuclei, ventromedial medulla, rostral ventrolateral medulla, A5 cell group, and paraventricular hypothalamic nucleus (PVH). In the spinal cord, the segmental distribution of virally infected neurons was the same as the retrograde cell body labeling observed following Fluoro-gold injections in the adrenal gland except there was almost a 300% increase in the number of cells labeled and a shift in cell group distribution. These results imply there are local interneurons that regulate the sympathoadrenal preganglionic neurons. In the medulla oblongata, serotonin (5-HT)-, substance P (SP)-, thyrotropin-releasing hormone-, Met-enkephalin-, and somatostatin-immunoreactive neurons of the raphe pallidus and raphe obscurus nuclei and the ventromedial medulla were infected. In the ventromedial and rostral ventrolateral medulla, immunoreactivephenylethanolamine- N-methyltransferase, SP, neuropeptide Y, somatostatin, and enkephalin neurons were infected. The A5 noradrenergic cells were labeled, as were some somatostatin-immunoreactive neurons in this area. In the hypothalamus, tyrosine hydroxylase- and SP-immunoreactive neurons of the dorsal parvocellular PVH were infected. Only a few immunoreactive vasopressin, oxytocin, Met-enkephalin, neurotensin, and somatostatin PVH neurons were labeled.

The intermediolateral cell column of the thoracic spinal cord is comprised of target-specific subnuclei: evidence from retrograde transport studies and immunohistochemistry

In this study we examined the hypothesis that the intermediolateral cell column (IML) of the thoracic spinal cord, the nucleus from which preganglionic sympathetic neurons originate, provides an anatomical substrate through which selective regulation of sympathetic nervous system targets is accomplished. Preganglionic sympathetic neurons of rats were retrogradely labeled by the simultaneous exposure of the cervical sympathetic trunk (CST) and the adrenal medulla to Fluoro-Gold and True blue, contrasting fluorescent dyes. Retrograde labeling from these sites revealed 2 populations of sympathetic preganglionic neurons in IML whose distribution overlapped between segments T1 and T4. In regions where these 2 groups of retrogradely labeled neurons overlapped, sympathoadrenal preganglionic (SAP) neurons occupied the most lateral aspect of the nucleus. It was also determined whether individual retrogradely labeled neurons within these two groups sent axon collaterals to both the CST and adrenal medulla. Diamidino yellow, a fluorescent retrograde tracer dye that labels only nuclei, was substituted for Fluoro-Gold and used in combination with True blue to simultaneously label preganglionic sympathetic neurons projecting to either the CST or adrenal medulla. No double-labeled cell bodies were observed in spinal cords of rats treated in this manner. Thus it appeared that the efferent projections of these 2 cell populations in IML were target-specific. Immunohistochemical analysis of the relationship between nerve fibers in the IML and preganglionic sympathetic neurons was also undertaken in an attempt to classify further these 2 populations of sympathetic preganglionic neurons. Equal proportions of identified CST and SAP neurons appeared to be apposed by varicosities immunoreactive for either somatostatin or serotonin. On the other hand, when the comparison was based on whether oxytocin-immunoreactive varicosities appeared to appose these 2 populations of retrogradely labeled sympathetic neurons, a highly significant difference was revealed. That is, oxytocin-immunoreactive fibers and terminals appeared to avoid SAP neurons. Thus these data support the hypothesis that an anatomical substrate exists in spinal cord IML whereby selective regulation of sympathetic nervous system targets may be mediated. Moreover, the lack of oxytocin-immunoreactive varicosities apposing SAP neurons in IML suggests that if the paraventricular nucleus innervates SAP neurons in IML, it does so via a population of neurons that do not use oxytocin as a neurotransmitter.

Coexistence of serotonin- and substance P-like immunoreactivity in nerve fibers apposing identified sympathoadrenal preganglionic neurons in rat intermediolateral cell column

In this study we examined the possibility that serotonin (5-HT) and substance P (SP) coexist in fibers and terminals afferent to sympathoadrenal preganglionic (SAP) neurons in the intermediolateral cell column (IML) of the spinal cord. SAP neurons in the IML were identified by retrograde labeling with either Fast Blue or True Blue injected into the adrenal medulla of rats. A simultaneous immunofluorescent double labeling technique was used to identify both 5-HT- and SP-like immunoreactivity in single tissue sections. Labeled SAP neurons were observed which were apposed by fibers immunoreactive for either neurotransmitter, as well as SAP neurons apposed by neither 5-HT- nor SP-like immunoreactive structures. In addition, 5-HT- and SP-like immunoreactivity were observed in separate fibers apposing the same labeled neuron and coexisting in fibers and terminal appearing in apposition to labeled SAP neurons. These data suggest a complex interaction by these neurotransmitters in regulating sympathetic outflow and may provide a model for interpreting conflicting observations concerning the effects of local 5-HT administration on sympathetic nerve activity.

Differential effects of transection of the spinal cord and splanchnic nerve on adrenal tyrosine hydroxylase and catecholamines

The role of spinal pathways in the regulation of adrenal medullary tyrosine hydroxylase and catecholamines was studied in adult rats subjected to spinal cord transection at the third thoracic level. In these animals the sympathoadrenal preganglionic neurons were isolated from their supraspinal afferents. This treatment led after three days to a progressive reduction of tyrosine hydroxylase activity and dopamine content (as compared to unoperated controls) until at least the 10th day. The results of the administration of dexamethasone or adrenocorticotropic hormone to spinalized rats suggest that in these animals glucocorticoid hypersecretion is not involved in the decline of adrenal tyrosine hydroxylase and that in fact adrenocorticotropic hormone supplementation can prevent it. A neurogenic origin for the depression of adrenomedullary function is favoured because unilateral splanchnicotomy (which by itself does not affect adrenal tyrosine hydroxylase), prior to cord section, prevented the diminution of tyrosine hydroxylase activity in the denervated gland. The decline of adrenal tyrosine hydroxylase and dopamine after spinal section may result from a decrease of modulatory impulses to the adrenal from decentralized sympathoadrenal preganglionic neurons in the isolated cord, following the loss of a descending facilitation of these neurons and/or the release of a segmental interneuronal inhibition of these neurons from a descending inhibitory influence. Such descending pathways may decussate partially below the low cervical level because rats with hemisection of the cord at C 6-C 7 exhibited no decline of adrenal tyrosine hydroxylase or of dopamine measured on either side seven days postoperatively.

Receptor autoradiography in thoracic spinal cord: correlation of neurotransmitter binding sites with sympathoadrenal neurons

Receptor autoradiography was combined with the retrograde labeling of sympathoadrenal neurons by fast blue to determine whether opiate, serotonin, catecholamine, or cholinergic binding sites could be spatially correlated with preganglionic neurons in the rat intermediolateral cell column (IML) that project to the adrenal gland. [3H]Dihydromorphine (DHM) was used for the visualization of mu opiate receptors, [3H]lysergic acid diethylamide (LSD) for serotonin receptors, [3H]para-aminoclonidine (pAC) for alpha 2-adrenergic receptors, and [3H]quinuclidinyl benzilate (QNB) for muscarinic cholinergic receptors. While qualitative assessment of autoradiograms indicated that alpha 2-adrenergic and muscarinic receptors were concentrated in the intermediate zone of the spinal cord, quantitation of grains in specific regions of the intermediate gray revealed that alpha 2-adrenergic and serotonergic receptors were more highly concentrated over sympathoadrenal preganglionic neurons than over other regions in IML or the adjacent intermediate gray matter. Information concerning the distribution of neurotransmitter-binding sites in other regions of thoracic spinal cord was also obtained. All ligands showed relatively dense binding sites in the superficial laminae of the dorsal horn, and all but [3H]DHM revealed similar densities of binding sites in the region adjacent to the central canal. Only [3H]QNB revealed a high density of binding sites in the ventral horn of the spinal cord.

Sympathoadrenal preganglionic neurons: their distribution and relationship to chemically-coded fibers in the kitten intermediolateral cell column

The location of those sympathetic preganglionic neurons in the spinal cord that project to the adrenal medulla — the sympathoadrenal preganglionic (SAP) neurons — was studied by the method of retrograde axonal transport of the fluorescent dye Fast Blue. The distribution of chemically-coded fibers and their relationship to the SAP neurons was also investigated using the indirect immunofluorescence technique. In kittens, 5 of a 1% solution of Fast Blue was injected into the medulla of the left adrenal gland. After a survival period of 5 days, the spinal cords from C8 to L5 were sectioned and processed for the localization of enkephalin-, neurophysin-, oxytocin-, serotonin-, substance P- and somatostatin-like immunoreactivity. Retrogradely labeled neurons were found in the ipsilateral intermediolateral cell column (IML) (89.8% of all retrogradely labeled neurons) from T1 to L4, and in the contralateral IML (10.2%) from T1 to L4. The enkephalin, serotonin and substance P immunoreactive fibers appeared to surround both the retrogradely labeled and unlabeled IML neurons. The somatostatin immunoreactive fibers were observed only in proximity to the retrogradely labeled neurons. Only a sparse population of neurophysin and oxytocin immunoreactive fibers were observed in IML, and were not seen to be in apposition to the retrogradely labeled neurons.

Participation of spinal monoaminergic and cholinergic systems in the regulation of adrenal tyrosine hydroxylase

The role of spinal pathways in the regulation of tyrosine hydroxylase activity (ATHA) in the actual medulla was characterized in adult rats with surgical transection of the upper thoracic cord. The animals received injections of centrally active agonists, which could then act on the sympathoadrenal preganglionic neurons isolated from their supraspinal afferents. All agonists tested produced distinct increases of tyrosine hydroxylase activity in rats with a surgical sham operation. The injection of quipazine or oxotremorine into spinalized rats led to decreased tyrosine hydroxylase activity. Clonidine and several dopamine agonists did not affect tyrosine hydroxylase activity. Considering the mechanism of the observed changes in tyrosine hydroxylase activity it is concluded that the changes are mediated by neural pathways and that variations in the secretion of adrenocorticotrophic hormone (ACTH) do not play a role. The results suggest (a) that there are serotonergic and cholinergic (muscarinic) receptors in the lower thoracic cord generating a net inhibitory action on tyrosine hydroxylase activity; and (b) that increases of tyrosine hydroxylase activity mediated by dopaminergic drugs probably originate from areas rostral to the thoracic cord. It is possible that an α-adrenergic system participates in the regulation of tyrosine hydroxylase activity in the adrenal medulla.