Spinal Sympathetic Preganglionic Neurons Research Articles

Chronic Cervical Midline Contusion in Rats Disrupts Multiple Facets of Cardiovascular Function

Cardiovascular dysfunction is the leading cause of morbidity and mortality following spinal cord injury (SCI). There are a limited number of studies assessing cardiovascular deficits after cervical SCI. This study aimed to assess disrupted cardiovascular function following a chronic severe incomplete C8 SCI in rats, via damaging descending sympathetic projections to the spinal sympathetic preganglionic neurons. Rats were first trained on the isometric pull task before receiving a C8 midline contusion (Infinite Horizon Impact Device; 250 kilodynes; 0 s dwell time) or laminectomy and were tested one day per week from weeks 7-13 following surgery. Isometric forelimb strength and correlates of muscle endurance were monitored during pull task sessions. Cardiovascular function was evaluated 16 weeks post-surgery. Tilt table testing was used to induce an orthostatic challenge by tilting rats 90 degrees head-up from a supine position and 90 degrees head-down from supine position as a control. Following tilt table testing, dobutamine (DOB; 2 μg/kg/min and 10 μg/kg/min) and norepinephrine (NE; 1 μg/kg/min and 4 μg/kg/min) were infused into the femoral vein separately to assess cardiovascular responses to a pharmacological challenge. We monitored cardiovascular physiology during all terminal testing. A blood pressure catheter was inserted into the left common carotid artery and advanced to the aortic arch to monitor interatrial blood pressure, 2 pairs of impedance cardiography (ICG) electrodes were placed on the left side of the thorax to evaluate inotropic correlates of cardiac function, and 2 photoplethysmography (PPG) sensors were affxed over the forelimbs flexor muscles to examine peripheral hemodynamics. To verify the accuracy of ICG-derived cardiac function, rats were examined by echocardiography at week 15. Spinal cords were collected for lesion analysis and were also stained for tyrosine hydroxylase. Our preliminary results demonstrate that chronic severe incomplete C8 SCI resulted in a significant and sustained hypotension during the 3-minute period of 90 degrees head-up tilting (systolic blood pressure = ~ -22 mmHg; diastolic blood pressure = ~ -19 mmHg) compared to laminectomy. There were also significant differences in left ventricular ejection time, heart rate, and stroke volume across groups, whereas forelimb hemodynamics did not differ across groups. Additionally, correlates of forelimb muscle endurance significantly decreased in SCI rats, despite similar isometric forelimb strength across groups. Intravenous infusion of DOB and NE demonstrated significant and dose-dependent effects on cardiovascular parameters. Overall, our data demonstrate that chronic severe C8 SCI in rats leads to orthostatic hypotension and also impairs multiple additional aspects of function. This study was supported by: NIH (R01 NS131493) and Florida Department of Health (COPBC). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Interrelationships Between Spinal Sympathetic Preganglionic Neurons, Autonomic Systems and Electrical Synapses Formed by Connexin36-containing Gap Junctions

Spinal sympathetic preganglionic neurons (SPNs) are among the many neuronal populations in the mammalian central nervous system (CNS) where there is evidence for electrical coupling between cell pairs linked by gap junctions composed of connexin36 (Cx36). Understanding the organization of this coupling in relation to autonomic functions of spinal sympathetic systems requires knowledge of how these junctions are deployed among SPNs. Here, we document the distribution of immunofluorescence detection of Cx36 among SPNs identified by immunolabelling of their various markers, including choline acetyltransferase, nitric oxide and peripherin in adult and developing mouse and rat. In adult animals, labelling of Cx36 was exclusively punctate and dense concentrations of Cx36-puncta were distributed along the entire length of the spinal thoracic intermediolateral cell column (IML). These puncta were also seen in association with SPN dendritic processes in the lateral funiculus, the intercalated and central autonomic areas and those within and extending medially from the IML. All labelling for Cx36 was absent in spinal cords of Cx36 knockout mice. High densities of Cx36-puncta were already evident among clusters of SPNs in the IML of mouse and rat at postnatal days 10–12. In Cx36BAC::eGFP mice, eGFP reporter was absent in SPNs, thus representing false negative detection, but was localized to some glutamatergic and GABAergic synaptic terminals. Some eGFP+ terminals were found contacting SPN dendrites. These results indicate widespread Cx36 expression in SPNs, further supporting evidence of electrical coupling between these cells, and suggest that SPNs are innervated by neurons that themselves may be electrically coupled.

Excessive sacral sweating caused by autonomic dysreflexia after traumatic brainstem injury

Abstract Rationale: Autonomic dysreflexia is a release phenomenon of spinal sympathetic preganglionic neurons from supraspinal inhibition, possibly of the lower brainstem origin. It is most frequently observed in patients with severe spinal lesions above the mid-thoracic cord. Theoretically, brainstem lesions may cause autonomic dysreflexia; however, such reports are rare. The author reports a case of autonomic dysreflexia restricted to the sacral area that appeared after brainstem lesions due to a traffic accident. Patient concerns: A 36-year-old man suffered from traumatic subarachnoid hemorrhage and hematomas in the lower brainstem. After he became alert and ambulant, he presented with neurological deficits suggestive of right-sided brainstem lesions, which were visualized on magnetic resonance images. He also complained of excessive sweating in the sacral area whenever he had a desire to void. Diagnoses: On the basis of neurological and radiological findings, and results of the thermal sweating test and sweating induced by bladder or rectal distension, his sacral hyperhidrosis was attributed to autonomic dysreflexia. Interventions: Baclofen (15 mg per day) was administered for more than three months, and the sacral hyperhidrosis gradually subsided. Outcomes: After three months, the patient returned home with minor gait disturbances and sacral sweating. Lessons: This may be the first report of excessive sweating due to autonomic dysreflexia caused by traumatic brainstem lesions restricted to the sacral territory. These observations suggest possible somatotopic arrangements, even in the supraspinal inhibitory pathways. Baclofen may be one of the choices for similar cases.

Spinal relay neurons for central control of autonomic pathways in a photoperiodic rodent.

Location and distribution of spinal sympathetic preganglionic neurons projecting to the superior cervical ganglion were investigated in a rodent model organism for photoperiodic regulation, the Djungarian hamster (Phodopus sungorus). Upon unilateral injection of Fluoro-Gold into the superior cervical ganglia, retrograde neuronal tracing demonstrated labeled neurons ipsilateral to the injection site. They were seen in spinal segments C8 to Th5 of which the segments Th1 to Th3 contained about 98% of the labeled cells. Neurons were found in the spinal cord predominantly in the intermediolateral nucleus pars principalis and pars funicularis. At the same time, the central autonomic area and the intercalated region contained only very few labeled cells. In the intermediolateral nucleus, cells often were arranged in clusters, of which several were seen in each spinal segment. Selected sections were exposed to antibodies directed against arginine-vasopressin, neuronal nitric oxide synthase, neuropeptide Y, neurotensin, oxytocin or substance P. It was found that about two-thirds of sympathetic preganglionic neurons produced the gaseous neuroactive substance nitric oxide and that few contained small amounts of neuropeptide Y. Fibers of putative supraspinal origin immunopositive for either arginine-vasopressin, neuronal nitric oxide synthase, neuropeptide Y, neurotensin, oxytocin or, in particular, substance P were found in the vicinity of labeled sympathetic preganglionic neurons. These results demonstrate the location of relay neurons for autonomic control of cranial and cardial structures and provide further knowledge on neurochemical properties of sympathetic preganglionic neurons and related structures.

Spinal Oxygen Sensors (SOS): A Novel Oxygen Sensing Mechanism Involved in Cardiovascular Responses to Hypoxia

BACKGROUNDIn healthy individuals, when blood oxygenation decreases, cardiorespiratory reflexes are triggered in an attempt to restore oxygen supply to vital organs. The carotid bodies are the primary respiratory oxygen chemoreceptors but cardiovascular responses to hypoxia such as increase in heart rate and blood pressure persist in their absence, suggesting an additional high‐fidelity oxygen sensor. Previously we discovered that spinal sympathetic preganglionic neurons (SPN), are exquisitely sensitive to oxygen; here we investigate the oxygen sensing mechanisms and test the role of these spinal oxygen sensors (SOS) in cardiorespiratory responses to asphyxia‐like stimuli.OBJECTIVETo study the cellular oxygen sensing mechanism and contribution of the SOS in responses to cardiorespiratory crisis.METHODSWe investigated the cellular mechanism of oxygen sensing in artificially‐perfused (in situ) and slice (in vitro) thoracic spinal cord preparations, recording sympathetic nerve root and single cell responses to hypoxia during pharmacological interrogation. To determine if the SOS are involved in cardiorespiratory responses to asphyxia, we also used an in situ rat spinal cord – carotid body ‐ brainstem preparation in which each oxygen sensitive compartment is separately perfused while recording phrenic (respiratory) and splanchnic (sympathetic) nerve activity.RESULTSOur data suggest the SOS use a novel oxygen sensing mechanism. This mechanism involves two interacting NADPH and oxygen‐dependent enzymes: Neuronal Nitric Oxide Synthase (NOS1) and NADPH oxidase (NOX2). NOS1 is expressed in surprising abundance in the SOS and is oxygen sensitive across the entire physiological range. Hence, in the presence of oxygen, NOS1 is likely to utilize most of the available NADPH in the cell. When oxygenation falls during hypoxia, NOS1 activity is reduced, increasing NADPH availability for NOX2. NOX2 produces Reactive Oxygen Species (ROS) which in turn, activate ROS‐dependent internal Ca2+ stores and/or Ca2+ channels leading to increased intracellular Ca2+, neuronal firing and, consequently, SOS responses to hypoxia. Functionally, during hypoxia, the SOS enhance sympathetic and breathing activity, while shortening apnea and gasping towards recovery, and are capable of triggering brief periods of sympathetic and respiratory‐like activity in the brainstem’s absence.CONCLUSIONSThe results provide critical new knowledge required to unlock the cellular mechanisms involved in how the body mounts emergency responses to conditions that involve chronic and acute hypoxia.Support or Funding InformationUniversity of Calgary Eyes High; Hotchkiss Brain Institute; Alberta Children’s Hospital Research Institute; Alberta Innovates Health Solutions; MITACS Globalink; Canadian Institutes of Health Research.

Chapter 3 - Adaptations in autonomic nervous system regulation in normal and hypertensive pregnancy

There is an increase in basal sympathetic nerve activity (SNA) during normal pregnancy; this counteracts profound primary vasodilation. However, pregnancy also impairs baroreflex control of heart rate and SNA, contributing to increased mortality secondary to peripartum hemorrhage. Pregnancy-induced hypertensive disorders evoke even greater elevations in SNA, which likely contribute to the hypertension. Information concerning mechanisms is limited. In normal pregnancy, increased angiotensin II acts centrally to support elevated SNA. Hypothalamic sites, including the subfornical organ, paraventricular nucleus, and arcuate nucleus, are likely (but unproven) targets. Moreover, no definitive mechanisms for exaggerated sympathoexcitation in hypertensive pregnancy have been identified. In addition, normal pregnancy increases gamma aminobutyric acid inhibition of the rostral ventrolateral medulla (RVLM), a key brainstem site that transmits excitatory inputs to spinal sympathetic preganglionic neurons. Accumulated evidence supports a major role for locally increased production and actions of the neurosteroid allopregnanolone as one mechanism. A consequence is suppression of baroreflex function, but increased basal SNA indicates that excitatory influences predominate in the RVLM. However, many questions remain regarding other sites and factors that support increased SNA during normal pregnancy and, more importantly, the mechanisms underlying excessive sympathoexcitation in life-threatening hypertensive pregnancy disorders such as preeclampsia.

Subregional differences in GABAA receptor subunit expression in the rostral ventrolateral medulla of sedentary versus physically active rats.

Neurons in the rostral ventrolateral medulla (RVLM) regulate blood pressure through direct projections to spinal sympathetic preganglionic neurons. Only some RVLM neurons are active under resting conditions due to significant, tonic inhibition by gamma-aminobutyric acid (GABA). Withdrawal of GABAA receptor-mediated inhibition of the RVLM increases sympathetic outflow and blood pressure substantially, providing a mechanism by which the RVLM could contribute chronically to cardiovascular disease (CVD). Here, we tested the hypothesis that sedentary conditions, a major risk factor for CVD, increase GABAA receptors in RVLM, including its rostral extension (RVLMRE ), both of which contain bulbospinal catecholamine (C1) and non-C1 neurons. We examined GABAA receptor subunits GABAAα1 and GABAAα2 in the RVLM/RVLMRE of sedentary or physically active (10-12 weeks of wheel running) rats. Western blot analyses indicated that sedentary rats had lower expression of GABAAα1 and GABAAα2 subunits in RVLM but only GABAAα2 was lower in the RVLMRE of sedentary rats. Sedentary rats had significantly reduced expression of the chloride transporter, KCC2, suggesting less effective GABA-mediated inhibition compared to active rats. Retrograde tracing plus triple-label immunofluorescence identified fewer bulbospinal non-C1 neurons immunoreactive for GABAAα1 but a higher percentage of bulbospinal C1 neurons immunoreactive for GABAAα1 in sedentary animals. Sedentary conditions did not significantly affect the number of bulbospinal C1 or non-C1 neurons immunoreactive for GABAAα2 . These results suggest a complex interplay between GABAA receptor expression by spinally projecting C1 and non-C1 neurons and sedentary versus physically active conditions. They also provide plausible mechanisms for both enhanced sympathoexcitatory and sympathoinhibitory responses following sedentary conditions.

Intrathecal Intermittent Orexin-A Causes Sympathetic Long-Term Facilitation and Sensitizes the Peripheral Chemoreceptor Response to Hypoxia in Rats

Intermittent hypoxia causes a persistent increase in sympathetic nerve activity (SNA), which progresses to hypertension in conditions such as obstructive sleep apnea. Orexins (A and B) are hypothalamic neurotransmitters with arousal-promoting and sympathoexcitatory effects. We investigated whether the sustained elevation of SNA, termed sympathetic long-term facilitation, after acute intermittent hypoxia (AIH) is caused by endogenous orexin acting on spinal sympathetic preganglionic neurons. The role of orexin in the increased SNA response to AIH was investigated in urethane-anesthetized, vagotomized, and artificially ventilated Sprague-Dawley rats (n = 58). A spinally infused subthreshold dose of orexin-A (intermittent; 0.1 nmol × 10) produced long-term enhancement in SNA (41.4% ± 6.9%) from baseline. This phenomenon was not produced by the same dose of orexin-A administered as a bolus intrathecal infusion (1 nmol; 7.3% ± 2.3%). The dual orexin receptor blocker, Almorexant, attenuated the effect of sympathetic long-term facilitation generated by intermittent orexin-A (20.7% ± 4.5% for Almorexant at 30 mg∙kg(-1) and 18.5% ± 1.2% for 75 mg∙kg(-1)), but not in AIH. The peripheral chemoreflex sympathoexcitatory response to hypoxia was greatly enhanced by intermittent orexin-A and AIH. In both cases, the sympathetic chemoreflex sensitization was reduced by Almorexant. Taken together, spinally acting orexin-A is mechanistically sufficient to evoke sympathetic long-term facilitation. However, AIH-induced sympathetic long-term facilitation appears to rely on mechanisms that are independent of orexin neurotransmission. Our findings further reveal that the activation of spinal orexin receptors is critical to enhance peripheral chemoreceptor responses to hypoxia after AIH.

Changes in the Gene c-fos Expression in the Rat Spinal Cord after Suppression of Activity of the Cerebral Monoaminergic Systems

We detected changes in the gene c-fos expression induced by activation of muscle afferents in the rat spinal cord after systemic introduction of reserpine (irreversible suppressor of vesicular transporter of monoamines, 1.5 mg/kg, intraperitoneally) and with no action of this agent. Numbers of Fosimmunoreactive (Fos-ir) neurons were calculated in the gray matter of the lumbar spinal segments after unilateral vibrational stimulation (VS) of the Achilles tendon of the mm. gastrocnemius–soleus. In the spinal cord of rats preliminarily injected with reserpine, greater numbers of Fos-ir neurons were observed; these units were localized mostly in layers 4–7 and in the nucl. intermediolateralis (35.4 ± 1.6 and 16.7 ± 0.9 positive neurons per 40 μm-thick slice) of segments L1–L2, and also in layers 4–7 and layer 9 (Fos-ir motoneurons) of segments L4–L5 (51.7 ± 3.4 and 11.4 ± 1.5 labeled units, respectively). The numbers of activated cells in the above structures of the spinal cord after VS but without preliminary injections of reserpine were, on average, 25.6 ± 1.4 and 3.5 ± 0.5, 27.8 ± ± 0.9 and 6.9 ± 0.3 units, respectively. Most ipsilateral Fos-ir motoneurons (86%) were localized in the lateral pool of layer 9, and only 14% of labeled motoneurons were localized in its medial regions. The results obtained show that weakening of monoaminergic influences resulting from administration of reserpine is accompanied by increase in the activity of intraspinal neuronal networks activated by proprioceptive afferent volleys, and the effects of the above inputs on spinal motoneurons and sympathetic preganglionic neurons are intensified. Weakening of inhibitory control, realized by inhibitory interneurons in the pathways of transmission of excitatory influences from muscle afferents to motoneurons, which was observed in our experiments after suppression of monoaminergic modulatory systems, can be considered a significant factor responsible for the development of rigidity/spasticity of the limb muscles manifested in neurodegenerative diseases and after traumas of the spinal cord.

Novel Disabled-1-expressing neurons identified in adult brain and spinal cord.

Components of the Reelin-signaling pathway are highly expressed in embryos and regulate neuronal positioning, whereas these molecules are expressed at low levels in adults and modulate synaptic plasticity. Reelin binds to Apolipoprotein E receptor 2 and Very-low-density lipoprotein receptors, triggers the phosphorylation of Disabled-1 (Dab1), and initiates downstream signaling. The expression of Dab1 marks neurons that potentially respond to Reelin, yet phosphorylated Dab1 is difficult to detect due to its rapid ubiquitination and degradation. Here we used adult mice with a lacZ gene inserted into the dab1 locus to first verify the coexpression of β-galactosidase (β-gal) in established Dab1-immunoreactive neurons and then identify novel Dab1-expressing neurons. Both cerebellar Purkinje cells and spinal sympathetic preganglionic neurons have coincident Dab1 protein and β-gal expression in dab1(lacZ/+) mice. Adult pyramidal neurons in cortical layers II-III and V are labeled with Dab1 and/or β-gal and are inverted in the dab1(lacZ/lacZ) neocortex, but not in the somatosensory barrel fields. Novel Dab1 expression was identified in GABAergic medial septum/diagonal band projection neurons, cerebellar Golgi interneurons, and small neurons in the deep cerebellar nuclei. Adult somatic motor neurons also express Dab1 and show ventromedial positioning errors in dab1-null mice. These findings suggest that: (i) Reelin regulates the somatosensory barrel cortex differently than other neocortical areas, (ii) most Dab1 medial septum/diagonal band neurons are probably GABAergic projection neurons, and (iii) positioning errors in adult mutant Dab1-labeled neurons vary from subtle to extensive.

Identification of the spinal pathways involved in the recovery of baroreflex control after spinal lesion in the rat using pseudorabies virus.

Neurons in the rostroventrolateral medulla (RVLM) mediate baroreflex regulation (BR) of spinal sympathetic preganglionic neurons. Previously, our laboratory has shown that recovery of BR occurs in the rat after spinal hemisection. (Zahner MR, Kulikowicz E, and Schramm LP. Am J Physiol Regul Integr Comp Physiol 301: R1584-R1590, 2011). The goal of these experiments was to determine whether the observed recovery of BR is mediated by the reorganization of ipsilateral pathways or by compensation by spared contralateral pathways. To determine this, we infected the left kidney in rats with the retrograde transynaptic tracer, pseudorabies virus (PRV), either 1 or 8 wk after left spinal hemisection at either T(3) or T(8), or after a sham lesion. In sham-lesioned rats, PRV infection of RVLM neurons was bilateral. In all rats with a left hemisection, regardless of the location of the lesion (T(3) or T(8)) or postlesion recovery time (1 or 8 wk), PRV infection of left RVLM neurons was significantly reduced compared with sham-lesioned rats (P < 0.05). In a separate group of rats, we performed BR tests by measuring responses of left renal sympathetic nerve activity to pharmacologically induced decreases and increases in arterial pressure. In rats with T(8) left hemisection and 8-wk recovery, BR was robust, and acute right upper thoracic hemisection abolished all BR of left renal sympathetic nerve activity. Collectively, these data suggest that the recovery of BR is not mediated by reorganization of ipsilateral bulbospinal connections, but instead by improved efficacy of existing contralateral pathways.

Reelin induces a common signal for spinal cord and cerebral cortical migration (Commentary on Krüger et al.)

The reeler mouse is a well-known neurological mutant, yet it remains unclear how the absence of Reelin leads to incorrect neuronal positioning during development. Reelin binds to neurons that express the lipoprotein receptors Vldlr and Apoer2, and induces tyrosine phosphorylation of the adaptor protein Disabled1 by Src-family kinases. Thus, the loss of Reelin, both Reelin receptors (Vldlr/Apoer2) or Disabled1 causes similar neuronal positioning errors. Multiple downstream pathways are activated by Disabled1 tyrosine phosphorylation and contribute to functions as diverse as dendritic maturation (Matsuki et al., 2008; Niu et al., 2004) and modulation of synaptic plasticity in adults (Beffert et al., 2005). In this issue of European Journal of Neuroscience, Krüger et al., together with a previous study (Chai et al., 2009), report a novel downstream Reelin pathway that induces the phosphorylation of n-cofilin (p-cofilin) that probably acts upon the actin cytoskeleton of migrating neurons to reduce further movement. During development, neurons migrate in the direction of highly motile filopodia on their leading processes. Cofilin acts to depolymerize F-actin and promote its disassembly, whereas p-cofilin inhibits process extension. What is intriguing about this (Krüger et al., 2010) and the previous study (Chai et al., 2009) is that, despite radically different migratory patterns, spinal cord and cerebral cortical neurons both use Reelin induction of p-cofilin to mediate neuronal positioning. Spinal sympathetic preganglionic and somatic motor neurons initially migrate together from the ventricular zone to form a primitive motor column (Fig. 1A, arrow 1). Somatic motor neurons remain ventrally, whereas sympathetic preganglionic neurons translocate dorsally in a secondary, nonradial migration to the intermediolateral horn (Fig. 1A, arrow 2). Finally, some sympathetic preganglionic neurons migrate medially along radial glia toward the midline (Phelps et al., 1993) (Fig. 1a, arrow 3). In reeler mutants, most sympathetic preganglionic neurons fail to stop in the intermediolateral horn and continue into the midline (Fig. 1B) (Yip et al., 2000; Phelps et al., 2002). Krüger et al. (2010) show that Reelin induces p-cofilin in wild-type and Vldlr mutant sympathetic preganglionic neurons that migrate normally, whereas sympathetic preganglionic neurons in reeler, Apoer2 and Disabled1 mutants do not contain p-cofilin and migrate past their correct positions. The more complex migration in the cerebral cortex is initiated by preplate formation, followed by waves of newly generated neurons that split the preplate and migrate past earlier-born neurons along radial glia, to generate ‘inside-out’ layering (Rakic & Caviness, 1995). Cortical neurons in reeler mutant mice do not split the preplate or migrate past earlier-born neurons. Reelin stabilizes leading processes in vitro (Chai et al., 2009), and yet reeler cortical neurons cease migration too soon, whereas mutant sympathetic preganglionic neurons migrate too far. These distinct positioning errors are probably due to additional factors or cues involved in correct neuronal positioning. Migration of sympathetic preganglionic neurons (SPNs) in wild-type (A) and reeler mutant (B) mice. Initially, SPNs and somatic motor neurons (SMNs) migrate out of the ventricular zone (VZ) together (arrow 1). SPNs then undergo a secondary, nonradial migration toward the intermediolateral horn (arrow 2) and finally a few SPNs migrate back toward the VZ (arrow 3). Reelin-secreting cells, illustrated in red, maintain most wild-type SPNs laterally but, in reeler mutants, the majority of SPNs fail to stop and instead migrate into the midline (arrow 3). IZ, Intermediate Zone; MZ, Marginal Zone. Drawing revised from Kubasak et al. (2004). Future studies will test if other Reelin-sensitive neurons also require p-cofilin for neuronal positioning, or if, for example, only neurons that migrate along radial glia are affected. Do neurons that only express Vldlr receptors use different positioning mechanisms? Based on past experience, additional complexity in how downstream Reelin signaling controls the critical function of neuronal migration may emerge.

Cross over of forebrain and brainstem neuronal projections to spinal cord sympathetic preganglionic neurons in the rat

Neuronal inputs from the forebrain and the brainstem to sympathetic preganglionic neurons in the spinal cord were investigated by the transneuronal retrograde tracing technique using pseudorabies virus in intact and brainstem-lesioned rats. After unilateral subcutaneous viral inoculations into the hind limb of intact rats, infected neurons were then visualized by immunostaining. At 3.5 days after inoculation, infected neurons appeared in the thoracic (T10) intermediolateral (IML) cell column. On the 4th day, infected neurons were present in the C1, A5, A6, A7 catecholamine cell groups and the rostral ventromedial medulla (RVMM). On the 5th day, viral labeling was seen in the hypothalamic paraventricular and arcuate nuclei and the lateral hypothalamic area. In all of these nuclei, the infected cells appeared bilaterally. However, the appearance of virus-labeled cells in these nuclei was unilateral following unilateral coronal sections between the medulla and the spinal cord (depending on the side of hemisection, but not on the site of virus inoculation). Midsagittal sections throughout the entire medulla oblongata did not alter the topographical pattern of virus-infected neurons in the forebrain or the brainstem. These findings indicate that descending fibers to the spinal neurons may not cross over in the lower brainstem but that they decussate within the spinal cord.

Suckling-induced activation of neural c-fos expression at lower thoracic rat spinal cord segments

Suckling stimulation is essential for neuroendocrine and sympathetic reflex activation during lactation. In the present study, the induction of c-fos gene expression was used to identify neuronal populations in the spinal cord activated by acute 5 min suckling or by electrical stimulation of the central stump of the first abdominal mammary nerve in lactating rats previously separated from their litters for 6 or 18 h. In addition, to investigate whether spinal sympathetic preganglionic neurons are activated by suckling, dual immunostaining (Fos and choline acetyltransferase) was performed. Fos was expressed at low levels in continuously suckled and 6 h nonsuckled mothers, but no expression was found after 18 h of nonsuckling. On the other hand, in 6 h nonsuckled rats, significant increments in Fos expression occurred in several regions after acute suckling and after electrical stimulation. Also, the pattern of Fos expression in each spinal laminae was different for the two stimuli, i.e. more intense effects of suckling in deep laminae V–X and more intense effects in laminae I–IV with electrical stimulation. Double-labeling after suckling was found only in sympathetic preganglionic neurons from the intermedio-medial cell column, whereas after electrical stimulation, double label was observed only in neurons from the intermedio-lateral cell column. On the other hand, no effect upon Fos protein expression was observed after suckling and only a minor effect after electrical stimulation of mammary nerve in 18 h nonsuckled rats. These results are consistent with previous findings on the sympathetic reflex regulation of the mammary gland, as well as on the importance of the nonsuckling interval for optimal functioning of lactation.

Hypothalamic pathways underlying the endocrine, autonomic, and behavioral effects of leptin

Leptin affects feeding, metabolism, and neuroendocrine status. It is now clearly established that the hypothalamus coordinates these responses, though the specific brain regions engaged by leptin remain unclear. We have used combinations of neuroanatomic techniques to identify candidate pathways in the central nervous system underlying leptin action. Leptin decreases body weight in part by activating the sympathetic nervous system, resulting in increased thermogenesis and energy expenditure. We investigated hypothalamic pathways underlying leptin's effects on stimulating the sympathetic nervous system. We found that leptin activates neurons in the retrochiasmatic area (RCA) and lateral arcuate nucleus (Arc) that innervate the sympathetic preganglionic neurons in the thoracic spinal cord and also contain cocaine- and amphetamine-regulated transcript (CART). We also found that CART neurons in the RCA and the Arc coexpress pro-opiomelanocortin (POMC) mRNA. Recent studies have reinforced the view that the lateral hypothalamic area (LHA) regulates food intake and body weight. Using retrograde tracing with leptin administration, we found retrogradely labeled cells in the Arc contained neuropeptide Y (NPY) mRNA or POMC mRNA. Following leptin administration, NPY cells in the Arc did not express Fos but expressed suppressor of cytokine signaling-3 (SOCS-3) mRNA. In contrast, leptin induced both Fos and SOCS-3 expression in POMC neurons, many of which also innervated the LHA. We suggest that leptin directly activates POMC/CART neurons that project to the LHA, the paraventricular hypothalamic nucleus (PVH), and spinal sympathetic preganglionic neurons. These projections link circulating leptin and neurons that regulate feeding behavior, energy expenditure, and body weight homeostasis.

Neurokinin-1 receptor immunoreactivity in hypotension sensitive sympathetic preganglionic neurons

Substance P activation of neurokinin-1 (NK1) receptors on spinal sympathetic preganglionic neurons (SPN) influences blood pressure. We identified SPN likely to subserve the baroreceptor reflex and established if these neurons showed NK1 receptor-immunoreactivity. Nitroprusside (NP) infusion or inferior vena cava (IVC) constriction activated similar numbers of SPN. Of these, about 40% were NK1 receptor-immunoreactive after NP infusion, but only about 20% were NK1 receptor-immunoreactive after IVC constriction. The distribution of Fos/NK1 receptor SPN suggested that substance P may preferentially target sympathoadrenal SPN.

Vasopressin acting at V1-type receptors produces membrane depolarization in neonatal rat spinal lateral column neurons

Vasopressin-immunoreactive fibers have been visualized in the area of spinal lateral horn cells, including spinal sympathetic preganglionic neurons. The presence and nature of vasopressin receptors on neurons in this area were addressed using whole-cell patch-clamp techniques in transverse spinal cord slice preparations from neonatal rat. Bath applications of Arg8-vasopressin (VP) induced a slow-onset membrane depolarization accompanied by spike discharges and membrane oscillations. In voltage-clamp, applications of VP induced a reversible, tetrodotoxin-resistant and dose-dependent inward current in 90% of tested cells. This effect was blocked by a V1 receptor antagonist [D-(CH2)5 Tyr (Me)-VP], whereas a V2 receptor agonist [desamino-(D-Arg8)-vasopressin] was ineffective. Furthermore the applications of oxytocin produced significantly smaller depolarizations when compared with VP suggesting that, at least in the neonatal lateral horn cells, vasopressin rather than oxytocin is more effective ligand. Both the amplitude and duration of the VP effect were enhanced after intracellular dialysis with GTP-gamma-S, a non-hydrolyzable GTP analogue, whereas the inward current was significantly reduced after intracellular dialysis with GDP-beta-S, a stable analogue of GDP that competitively inhibits G-proteins. The observation that the VP-induced net inward current reversed at a potential close to the equilibrium for potassium ions and was associated with a decrease in membrane conductance in a majority of tested cells suggest mediation through closure of a leak potassium conductance. These data indicate that SPNs and other lateral horn cells possess functional G-protein-coupled V1-type vasopressin receptors that, in adult spinal cord, may contribute to CNS regulation of autonomic nervous system function.

Vasopressin's depolarizing action on neonatal rat spinal lateral horn neurons may involve multiple conductances.

Vasopressin-immunoreactive fibers have been visualized in the area of spinal lateral horn cells, including spinal sympathetic preganglionic neurons (SPNs). The presence and nature of vasopressin receptors on 125 neurons in this area were addressed using whole-cell patch-clamp techniques in transverse spinal cord slice preparations from neonatal rat (11-21 days). Local pressure applications of Arg-vasopressin (AVP, 1 microM) induced a slow-onset membrane depolarization accompanied by spike discharges and membrane oscillations. In voltage-clamp, applications of AVP (10 nM-1 microM) induced a reversible, tetrodotoxin-resistant and dose-dependent inward current in 90% of tested cells. This effect was blocked by a V1 receptor antagonist [D-(CH2)5 Tyr (Me)-AVP], whereas a V2 receptor agonist [desamino-(D-Arg8)-vasopressin] was ineffective. Both the amplitude and duration of the AVP effect were significantly modified after intracellular dialysis of non-hydrolysable G-protein modulators. I-V relationships, examined in 75 cells, suggested two conductances. In 36 cells the net AVP current reversed approximately -102 mV, was associated with a decrease in membrane conductance and yielded linear I-V plots, suggesting mediation through closure of a resting potassium conductance. In a further 26 cells the I-V lines remained almost parallel in the voltage range used in this study (-130 to -40 mV), while the membrane conductance was decreased in a majority of these cells. In the remaining 13 cells the net AVP current was estimated to reverse approximately -30 mV and was associated with a small increase in membrane conductance, suggesting mediation through opening of a nonselective cationic conductance. These data indicate that the majority of SPNs and other lateral horn cells possess functional G-protein-coupled V1-type vasopressin receptors in the neonatal spinal cord. In the adult spinal cord, some of these receptors are likely to participate in CNS regulation of autonomic nervous system function.

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.

Antisense to Thyrotropin Releasing Hormone Receptor Reduces Arterial Blood Pressure in Spontaneously Hypertensive Rats

We report in the present study the effect of intrathecal treatment with antisense oligonucleotides complementary to thyrotropin releasing hormone (TRH) receptor mRNA on the pressor response to intrathecal administration of TRH and on resting arterial blood pressure in Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). In 16-week-old male WKY rats, 18-base phosphodiester antisense or mismatch oligonucleotides to TRH receptor mRNA (100 micrograms per day) were injected intrathecally for 3 days. Twenty-four hours after the last injection, the magnitude of the pressor response to intrathecal TRH (10 micrograms) was significantly smaller in the antisense-treated group (n = 7) compared with mismatch-treated controls (n = 7) (change in mean arterial pressure, +20.3 +/- 3.0 versus +32.6 +/- 2.5 mm Hg, P < .01). No differences were observed in the pressor responses to injection of N-methyl-D-aspartic acid. Resting arterial blood pressure was unaffected by antisense treatment in WKY rats. In separate experiments, 16-week-old male SHR were treated with antisense (n = 7) or mismatch (n = 6) oligonucleotides for 3 days. Mean resting arterial blood pressure was significantly reduced by treatment with antisense oligonucleotides (from 157 +/- 4.8 to 119 +/- 8.8 mm Hg, P < .01), but no significant changes were observed in mismatch-treated animals. Our results suggest that the expression of TRH receptors in spinal sympathetic preganglionic neurons can be selectively reduced by intrathecal treatment with antisense oligonucleotides and that TRH projections to sympathetic preganglionic neurons play an important role in the elevation of arterial blood pressure in SHR.