Intermediolateral Research Articles

Electroacupuncture reduced airway inflammation by activating somatosensory-sympathetic pathways in allergic asthmatic rats.

Electroacupuncture (EA) treatment is efficacious in patients with respiratory disorders, although the mechanisms of its action in lung-function protection are poorly understood. This study aimed to explore the neuroanatomical mechanisms of EA stimulation at the BL13 acupoint (Feishu, EA-BL13) improvement in asthma. Allergic asthma was induced by intranasal 2.0% ovalbumin (OVA) instillation combined with intraperitoneal injection of the 10.0% OVA. The levels of interleukin (IL)-4 and IL-5 were detected by enzyme-linked immunosorbent assay. Hematoxylin and eosin and periodic acid-schiff stain were used to evaluate inflammatory cell infiltration and mucus secretion. Cellular oncogene fos induction in neurons after EA stimulation was detected by immunofluorescent staining. The mRNA expression levels of adrenergic receptors were quantified with real-time polymerase chain reaction. EA improved airway inflammation and mucus secretion mainly by activating somatosensory-sympathetic pathways (P<0.001). Briefly, the intermediolateral (IML) nuclei of the spinal cord received signals from somatic EA stimulation and then delivered the information via the sympathetic trunk to the lung. Excited sympathetic nerve endings in lung tissue released large amounts of catecholamines that specifically activated the β2 adrenergic receptor (β2AR) on T cells (P<0.01) and further decreased the levels of IL-4 and IL-5 (P<0.001) through the cyclic adenosine monophosphate/protein kinase A signaling pathway. This study provided a new explanation and clinical basis for the use of EA-BL13 as a treatment for allergic asthma in both the attack and remission stages and other respiratory disorders related to airway inflammation.

Differential dopamine modulation of spinal reflex amplitudes is associated with the presence or absence of the autonomic nervous system

The spinal cord contains a highly collateralized network of descending dopamine (DA) fibers that stem from the dorso-posterior hypothalamic A11 region in the brain, however, the modulatory actions of DA have generally only been assessed in lumbar segments L2-L5. In contrast to these exclusively sensorimotor segments, spinal cords segments T1-L2 and, in mouse, L6-S2, additionally contain the intermediolateral (IML) nucleus, the origin of autonomic nervous system (ANS).Here, we tested if the different spinal circuits in sensorimotor and IML-containing segments react differently to the modulation of the monosynaptic reflex (MSR) by DA. Bath-application of DA (1 μM) led to a decrease of MSR amplitude in L3-L5 segments; however, in IML-containing segments (T10-L2, and S1/2) the MSR response was facilitated. We did not observe any difference in the response between thoracic (sympathetic) and lumbosacral (parasympathetic) segments. Application of the D2-receptor agonists bromocriptine or quinpirole mimicked the effects of DA, while blocking D2 receptor pathways with raclopride or application with the D1-receptor agonist SKF 38393 led to an increase of the MSR in L3-L5 segments and a decrease of the MSR in IML-containing segments. In contrast, in the presence of the gap-junction blockers, carbenoloxone and quinine, DA modulatory actions in IML-containing segments were similar to those of sensorimotor L3-L5 segments.We suggest that DA modulates MSR amplitudes in the spinal cord in a segment-specific manner, and that the differential outcome observed in ANS segments may be a result of gap junctions in the IML.

Characterization of neuronal discharge in the thoracic spinal cord during cardiac stress in a porcine model

Sudden cardiac death is the leading cause of death in the United States and is often caused by ventricular tachyarrhythmias (VT; Mozaffarian et al., 2015). Myocardial ischemia triggers imbalances in sympathetic and parasympathetic modulation, via the autonomic nervous system, and leads to the pathophysiology of cardiac arrhythmias. However, the spinal mechanism of this sympathetic neural control remains poorly understood. We hypothesize that cardiac stressors elicit distinct neural signatures in the dorsal horn (DH; the spinal relay point for cardiac afferents) and the intermediolateral (IML) nucleus of the thoracic spinal cord (the primary nexus for modulation of sympathetic efferent outflow). Adult, anesthetized Yorkshire pigs (n=9; 42 ± 1.6 kg) were implanted with high‐density microarrays (64–256 electrodes; Neuronexus) in the thoracic spinal cord to record extracellular potentials. Antidromic stimulation of the T2 paravertebral chain allowed identification of sympathetic preganglionic neurons located in the IML (53 of total 1,757 neurons recorded). To characterize neuronal discharge, after a baseline recording, pigs repeatedly received: 1) chest scratch (SCR), 2) heart touch (THR), 3) ligation of the left anterior descending coronary artery (LAD), 4) occlusion of the inferior vena cava (IVC), or 5) occlusion of the descending aorta (AOC). Preliminary investigation of percent change in firing rate from baseline suggests complex neural signatures in DH and IML neurons during perturbations. A subset of DH neurons increased firing rate during SCR (8.29% of DH neurons), THR (10.70%), LAD (14.40%), IVC (21.57%), and AOC (6.13%). Firing rate increased in a subset of IML neurons as well (3.84% during SCR, 26.92% during THR, 18.87% during LAD, 20.75% during IVC, and 12.12% during AOC). In contrast, firing rate decreased in 4.89% of DH neurons during SCR, 20.88% during THR, 21.63% during LAD, 16.44% during IVC, and 9.85% during AOC. Additionally, rates decreased in a small population of IML neurons (3.84% during SCR, 7.69% during THR, 28.30% during LAD, 5.66% during IVC, and 18.18% during AOC). Subsequent analysis revealed positive correlations between DH and IML neurons, indicating excitatory neuronal connectivity. Further investigation is warranted to understand the temporally related discharge patterns as well as the inhibitory or excitatory nature of these connections. These preliminary data pave a potential pathway to develop targeted neuraxial interventions in cases of intractable ventricular arrhythmia.Support or Funding InformationHL084261, T32‐HD043730This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fos immunoreactivity in the intermediolateral nucleus induced by tendon vibration of the m. triceps surae in rats pretreated with a nitric oxide blocker or precursor

We investigated neuronal activation of the rat intermediolateral (IML) nucleus of the thoracolumbar spinal cord, initiated by Achilles tendon vibration, after intramuscular (m. triceps surae) administration of 7‑nitroindazole (7‑NI) or L‑arginine (LA). The spindle afferent response to vibratory stimuli induced a distinct bilateral increase in the activation of c‑Fos immunoreactivity in the spinal neurons in three groups of rats (tendon‑vibrated, tendon‑vibrated + 7‑NI and tendon‑vibrated + LA). The T5/T13 segments in tendon‑vibrated +7‑NI animals showed the highest increase of Fos‑immunoreactive neurons. This increase was two times higher than that in tendon only‑vibrated control rats and endon‑vibrated + LA animals. The highest mean number of labelled neurons were observed in the IML nucleus and in layers 4 and 7 of the T5-L3 segments in tendon‑vibrated and tendon‑vibrated + 7‑NI animals, and in the IML nucleus and layer 4 in tendon‑vibrated + LA rats. The highest mean number of activated neurons was found ipsilaterally in the IML nucleus of the T5/T13 segment. These results indicate that decreased nitric oxide release after injection of 7‑NI was accompanied by a potentiation of the early c‑fos gene expression induced by muscle proprioceptive activity within the thoracolumbar region of the rat spinal cord. Thus, enhanced c‑Fos immunoreactivity in the IML nucleus indicated that the sympathetic nervous system can exert a direct influence on the muscle spindles.

Seizure-Induced Sympathoexcitation Is Caused by Activation of Glutamatergic Receptors in RVLM That Also Causes Proarrhythmogenic Changes Mediated by PACAP and Microglia in Rats.

Sudden unexpected death in epilepsy is a major cause of death in epilepsy. Generally, seizures are accompanied by changes in brain function leading to uncontrolled nerve activity causing high blood pressure, rapid heart rate, and abnormal heart rhythm. Nevertheless, the brain chemicals causing these cardiovascular changes are unknown. Chemicals, such as glutamate and pituitary adenylate cyclase activating polypeptide, whose expression is increased after seizures, act on specific cardiovascular nuclei in the brain and influence the activity of the heart, and blood vessels. Microglia, which manage excitation in the brain, are commonly activated after seizure and produce pro- and/or anti-inflammatory factors. Hence, we aimed to determine the effects of blocking glutamate, pituitary adenylate cyclase activating polypeptide, and microglia in the RVLM and their contribution to cardiovascular autonomic dysfunction in seizure.

The soma and proximal dendrites of sympathetic preganglionic neurons innervating the major pelvic ganglion in female rats receive predominantly inhibitory inputs

Sympathetic preganglionic neurons (SPNs) in the intermediolateral (IML) and dorsal commissural nucleus (DCN) of the thoracolumbar segments of the spinal cord contribute to the autonomic control of the pelvic visceral organs. We examined the morphology of these neurons at the light and electron microscopic level and quantified the boutons apposing the soma and proximal dendrites of the SPNs innervating the major pelvic ganglion (MPG) in female rats. The majority of these cells resided in the DCN (61.6±6.2%) and IML (33.2±4.4%) nuclei. Measurements of cell volume and shape revealed no differences between SPNs sampled from the DCN and IML populations. Ultrastructural studies of DCN and IML SPNs revealed that coverage of SPNs by synaptic inputs is sparse, with an average of 11.60±2.41% of the soma membrane and 16.33±6.18% of proximal dendrites apposed by boutons, though some somata exhibited no synaptic coverage. Three distinct types of boutons were found to appose the SPN somata and dendrites. The putatively inhibitory F-type bouton covered a significantly greater percentage of membrane on the soma (8.48±2.12%) and dendrites (12.65±4.34%), than the S-type bouton, a putatively excitatory bouton, which only covered 2.94±0.70% of the somatic and 3.68±2.98% of the dendritic membranes. Boutons with dense-core vesicles were rare. Our results demonstrate that SPNs of the DCN and IML of female rats are similar morphologically, and that synaptic input on these cells, though sparse, is predominantly inhibitory.

Evidence for functional ghrelin receptors on parasympathetic preganglionic neurons of micturition control pathways in the rat

1. Previous work indicates that agonists of ghrelin receptors can act within the spinal cord to stimulate autonomic outputs to the colorectum and to blood vessels. Because of the close relationship between colorectal and urinary bladder control, we have investigated whether ghrelin receptor agonists also stimulate spinal centres that influence the bladder. 2. The ghrelin receptor agonist capromorelin (10 mg/kg), injected intravenously in anaesthetized male rats, disrupted the ongoing cycle of micturition reflexes and caused phasic oscillations in pressure that averaged approximately 20 mmHg. Fluid output from the bladder was diminished. The effects of capromorelin were inhibited by hexamethonium (10 mg/kg bolus followed by 4 mg/kg per h infusion, i.v.) and were further reduced by atropine (5 mg/kg bolus followed by 2.5 mg/kg per h infusion, i.v.). Capromorelin (250 microg) injected directly into the spinal cord at the lumbosacral level also increased contractile activity of the bladder. However, capromorelin, up to 0.1 mmol/L, had no effect on the tension of isolated muscle strips from the bladder. Effects of intravenous capromorelin (10 mg/kg) on bladder pressure were still observed after the descending pathways in the spinal cord were disrupted at the thoracic level. 3. In situ hybridization studies revealed ghrelin receptor gene expression in neurons of the autonomic intermediolateral (IML) cell columns. Following a series of micturition reflexes elicited by infusion of saline into the bladder, the immediate early gene product c-Fos was observed in neurons of the lumbosacral IML and approximately 20% of these also expressed ghrelin receptor gene transcripts. 4. It is concluded that ghrelin receptors are expressed by lumbosacral autonomic preganglionic neurons of the micturition reflex pathways and that ghrelin receptor agonists stimulate these neurons.

Differential synaptic inputs to the cell body and proximal dendrites of preganglionic parasympathetic neurons in the rat conus medullaris

Preganglionic parasympathetic neurons (PPNs) reside in the intermediolateral (IML) nucleus of the rat lumbosacral spinal cord and contribute to the autonomic control of visceral pelvic organs. PPNs provide the final common pathway for efferent parasympathetic information originating in the spinal cord.We examined the detailed ultrastructure of the type and organization of synaptic inputs to the cell body and proximal dendrites of PPNs in the rat conus medullaris. The PPNs were retrogradely labeled by a systemic administration of the B subunit of cholera toxin conjugated to horseradish peroxidase.We demonstrate four distinct types of synaptic boutons in apposition with PPN somata and proximal dendrites: S-type boutons show clear, spheroid vesicles; F-type boutons show flattened vesicles; dense-cored vesicle-type (DCV-type) boutons show a mixture of clear and dense-cored vesicles; L-type boutons were rare, but large, exhibited clear spheroid vesicles, and were only encountered in apposition with the PPN dendrites in our sample. The membrane surface covered by apposed boutons was markedly higher for the proximal dendrites of PPNs, compared with their somata. The inhibitory synaptic influence was markedly higher over the PPN somata compared with their proximal dendrites, as suggested by the higher proportion of putative inhibitory F-type boutons in apposition with the soma and a higher frequency of S-type boutons per membrane length for the proximal dendrites. Our studies suggest that the synaptic input to PPNs originates from multiple distinct sources and is differentially distributed and integrated over the cell membrane surface.

Location of parotid preganglionic neurons in the inferior salivatory nucleus and their relation to the superior salivatory nucleus of rat

In major brain maps the location of the salivatory nuclei of the rat is depicted from the level of the root of the facial nerve to the level of the rostral tip of the nucleus of the solitary tract. Most published data deal with the superior salivatory nucleus (SSN). In the present study the topography of the parasympathetic preganglionic neurons of the inferior salivatory nucleus (ISN) that innervate the parotid gland through the otic ganglion was determined by means of a retrograde transneuronal labeling technique. Parasympathetic, sympathetic and sensory neurons were labeled following injection of the virus into the parotid gland. The majority of the ISN neurons were found dorsal to the facial motor nucleus, embedded in the parvocellular reticular formation. In addition to the ISN neurons, virus-labelled cells were present in the intermediolateral (IML) cell column of the thoracic spinal cord, in the brainstem catecholamine groups, and in medullary raphe neurons. The removal of the ipsilateral superior cervical ganglion prior to the virus injection into the parotid gland did not influence the labeling of the ISN neurons but labeled neurons were not observed in the IML and A5 catecholamine cell group. In our previous study we had defined the relationship between the lacrimal and submandibular subdivison of the SSN, while in the present study we defined the relationship between the ISN and the lacrimal subdivision of SSN: the later located ventrolaterally to the caudal portion of the ISN. On the basis of these data a three-dimensional topography is given suggesting the relationship between the ISN and SSN.

Localization of 5‐HT1A/B receptors in the rat brainstem, their distribution in IML‐projecting cells and activation during acute hypoxia

Serotonin (5‐HT) type‐1 receptors contribute to cardiorespiratory (CR) function. Yet, distribution of these receptors in CR nuclei, and specifically in cells projecting to sympathetic preganglionic neurons in the intermediolateral (IML) cell column, is lacking. Whether 5‐HT1 containing cells are activated during the chemoreflex is also not known. We hypothesized acute hypoxia (10% O2, 3 hr) would stimulate IML‐projecting cells containing 5‐HT1 receptors. Immunohistochemistry in rat brainstem slices retrogradely‐labeled with fluorogold (FG, 1%) in the IML (T2 level) was used to test this notion. Fos‐like reactivity was used as a marker of cell stimulation. Fos, FG, 5‐HT1A and 5‐HT1B distribution was examined. 5‐HT1A and 1B labeling was similarly distributed in the following nuclei: nucleus of the solitary tract (NTS), facial, lateral paragigantocellularis, hypoglossal, ambiguus, rostral and caudal ventral lateral medulla (RVLM, CVLM), and occasionally, the Botzinger and pre‐Botzinger. In the RVLM, ~26% of 5‐HT1A and 18% of 5‐HT1B co‐labeled with FG, and a greater percent of 5‐HT1B than 1A co‐labeled with PNMT. In the NTS, 5‐HT1A/B did not co‐label with FG. Hypoxia increased Fos in the NTS, RVLM and CVLM compared to room air. In the NTS, a small percentage of Fos cells contained 5‐HT1A and in RVLM, some co‐labeled with FG and 5‐HT1B. These data suggest 5‐HT1 receptors may play a role in the CR response to hypoxia.

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.

Plateau potentials and membrane oscillations in parasympathetic preganglionic neurones and intermediolateral neurones in the rat lumbosacral spinal cord.

Whole-cell patch recordings were made from parasympathetic preganglionic neurones (P-PGNs) and unidentified intermediolateral (IML) neurones in thick slices of the lower lumbar and sacral spinal cord of 14- to 21-day-old rats. The P-PGNs and IML neurones examined were similar in terms of soma sizes, input resistance and capacitance, and displayed a sag conductance as well as rebound firing. In the absence of drugs, the neurones responded with either tonic or adapting firing to depolarizing current steps. However, in the presence of the group I metabotropic glutamate receptor agonist (RS)-3,5-dihydroxyphenylglycine (DHPG), almost half of the neurones displayed accelerating firing rates during the constant current injection, followed by a sustained after-discharge. In the presence of TTX, plateau potentials were observed. The firing changes and plateaux were blocked by nifedipine, an L-type Ca2+ channel blocker, and (S)-(-)-Bay K8644 was able to produce these firing changes and plateaux in the absence of DHPG, demonstrating the involvement of an L-type Ca2+ conductance. Ca2+-activated nonspecific cationic conductances also appear to contribute to the firing changes. A few neurones displayed membrane oscillations and burst firing in the presence of DHPG. The results suggest that the firing characteristics of both P-PGNs and other neurones likely to be involved in caudal spinal reflex control are not static but, rather, quite dynamic and under metabotropic glutamate receptor modulatory control. Such changes in firing patterns may be involved in normal pelvic parasympathetic reflex function during micturition, defaecation and sexual reflexes, and may contribute to the abnormal output patterns seen with loss of descending brainstem input and visceral or perineal sensory disturbances.

Death of Preganglionic Sympathetic Neurons after Surgical or Immunologic Lesion of Peripheral Processes

Three months after systemic injection of antibody to acetylcholinesterase (AChE), there is a 60% decrease in the population of preganglionic sympathetic neurons expressing choline acetyltransferase (ChAT) in the intermediolateral (IML) nucleus of the rat spinal cord. In principle, the disappearance of identifiable cholinergic neurons might reflect either outright cell death or severe atrophy with downregulation of cholinergic markers. To distinguish between these possibilities, preganglionic neurons were labeled with the retrograde tracer dye, Fast Blue, 1 week before antibody injection or surgical transection of the cervical sympathetic trunk. Three months after either treatment, the thoracic IML contained 40–60% fewer Fast Blue-labeled neurons than in controls. Therefore, preganglionic sympathetic neurons do degenerate after antibody injection or axotomy. To clarify the role of axonal damage in this process, the effects of three different mechanical lesions were examined. A lumbar ganglionectomy designed to interrupt most sympathetic axons emanating from L2 IML caused 92% loss of ChAT-positive cells observed 10 weeks later at that site. In comparison, transection of the cervical sympathetic trunk, which spared some distally directed axonal branches from the thoracic IML, caused only a 46% loss of ChAT-positive neurons at T1. Still smaller effects were seen after the same nerve was crushed, a lesion that is less destructive. Thus, the ability of central sympathetic neurons to survive a peripheral lesion may be related to the degree of axonal damage and to the opportunity for axonal regrowth.

Role of inferior olive and thoracic IML neurons in nonshivering thermogenesis in rats.

Removal of the midbrain tonic inhibitory mechanism on nonshivering thermogenesis (NST) results in increased temperatures of the interscapular brown adipose tissue (IBAT) and rectum (T(IBAT) and T(rec), respectively) via an enhanced central sympathetic output. Because it is unlikely that neurons (primary) of the midbrain inhibitory mechanism tonically inhibit the IBAT monosynaptically, there must be secondary or tertiary neurons posterior to the midbrain. Such neurons, therefore, may increase their activity during enhanced NST after removal of the midbrain tonic inhibition. The aim of the present experiments was to localize these secondary or tertiary neurons and establish descending neuronal pathway(s) that may project to the major NST effector IBAT. T(IBAT) and T(rec) increases induced by removal of the tonic inhibition by midbrain procaine microinjections were accompanied with appearance of c-Fos-positive neurons in the inferior olive (IO) and the intermediolateral (IML) cell column of the thoracic spinal cord. Electrical stimulation of and L-glutamate microinjections into the IO increased T(IBAT) and T(rec). Midbrain procaine-induced T(IBAT) and T(rec) increases were blocked by electrolytic IO lesions. These results suggest that central thermal signals produced from the lower midbrain are transmitted to IBAT through the IO and IML and that the IO has a role in the central sympathetic functions.

Effect of sympathectomy on the expression of NMDA receptors in the spinal cord.

The expression of NMDA receptors in the intermediolateral (IML) region of the upper thoracic spinal cord, was studied in 3 week old rats. The effect of section of the cervical sympathetic nerve on neuronal cell number and receptor expression was examined up to two weeks after the operation. Age-matched sham-operated and unoperated animals were used as controls. It was shown using quantitative autoradiography with the NMDA receptor antagonist [(3)H]MK-801 (dizocilpine maleate), that there was a marked downregulation of receptors in all groups of animals, beginning at approximately 4 weeks of age. However after sympathectomy, which resulted in the death of 44% of neurones in the IML by 7 days, there was a significant increase in receptor density per neurone compared to sham-operated controls. In the control animals there was a significant increase in the Kd value of the binding between 21 and 24 days after birth indicating an increased expression of a low affinity receptor, but no such increase was seen after axotomy. The results are consistent with two populations of NMDA receptors being transiently expressed in the IML in developing animals, and the higher affinity receptor being down-regulated between 4 and 5 weeks of age. The presence of the high affinity receptor subtype may predispose neurones to die after axotomy.

TrkB and Neurotrophin-4 Are Important for Development and Maintenance of Sympathetic Preganglionic Neurons Innervating the Adrenal Medulla

The adrenal medulla receives its major presynaptic input from sympathetic preganglionic neurons that are located in the intermediolateral (IML) column of the thoracic spinal cord. The neurotrophic factor concept would predict that these IML neurons receive trophic support from chromaffin cells in the adrenal medulla. We show here that adrenal chromaffin cells in the adult rat store neurotrophin (NT)-4, but do not synthesize or store detectable levels of BDNF or NT-3, respectively. Preganglionic neurons to the adrenal medulla identified by retrograde tracing with fast blue or Fluoro-Gold (FG) express TrkB mRNA. After unilateral destruction of the adrenal medulla, 24% of IML neurons, i.e., all neurons that are preganglionic to the adrenal medulla in spinal cord segments T7-T10, disappear. Administration of NT-4 in gelfoams (6 microgram) implanted into the medullectomized adrenal gland rescued all preganglionic neurons as evidenced by their presence after 4 weeks. NT-3 and cytochrome C were not effective. The action of NT-4 is accompanied by massive sprouting of axons in the vicinity of the NT-4 source as monitored by staining for acetylcholinesterase and synaptophysin immunoreactivity, suggesting that NT-4 may enlarge the terminal field of preganglionic nerves and enhance their access to trophic factors. Analysis of TrkB-deficient mice revealed degenerative changes in axon terminals on chromaffin cells. Furthermore, numbers of FG-labeled IML neurons in spinal cord segments T7-T10 of NT-4-deficient adult mice were significantly reduced. These data are consistent with the notion that NT-4 from chromaffin cells operates through TrkB receptors to regulate development and maintenance of the preganglionic innervation of the adrenal medulla.

Bilateral projections of the pontine micturition center to the sacral parasympathetic nucleus in the rat

Previous work has revealed that pontine micturition center (PMC) neurons send projections to the sacral parasympathetic nucleus (SPN) of the intermediolateral (IML) regions of L6-S1 spinal cord segments in rats. Although unilateral SPN injections will retrogradely label PMC neurons bilaterally, it is not known whether single PMC neurons project bilaterally to the SPN. There may be two different populations of PMC neurons on each side of the brainstem, with both groups independently connecting to the SPNs on opposite sides of the spinal cord. To verify one of these alternatives, a small injection of either rhodamine-labeled latex microspheres or a red fluorescent emulsion was made into the SPN on one side of the cord; a similar injection of either fluorescein-tagged microspheres or a green fluorescent emulsion was made into the other. After at least seven days, the rats were perfused. Inspection of 40 μm cord sections confirmed the similar placement of these injections along the rostrocaudal axis of the cord and that no tracer had spread across midline. Thirty-micron brain sections were examined for filled neurons. Red, green and double labeled neurons were found bilaterally in the PMC, subcoeruleus, and A5 regions. Although some red nucleus cells were also filled, they were only singly labeled and always located contralateral to the injection. Finally, immunohistochemical staining of dopamine- β-hydroxylase (DBH) containing cells confirmed that some labeled cells were also noradrenergic. We therefore conclude that some PMC, subcoeruleus, and A5 neurons send axons to the SPN on both sides of the lumbosacral cord.

Basic and acidic fibroblast growth factors protect spinal motor neurones in vivo after experimental spinal cord injury.

We studied the effect of a single focal injection of recombinant basic (FGF2) or acidic (FGF1) fibroblast growth factor on the survival of spinal motor neurones at 24 h after a standardized spinal cord contusion injury (SCI) in the rat. Both FGF2 and FGF1 (3 microg), microinjected into the injury site at 5 min post-injury (p.i.), protected at least two functionally important classes of spinal motor neurones, autonomic preganglionic neurones in the intermediolateral (IML) column and somatic motor neurones in the ventral horn (VH). Moreover, there was enhanced choline acetyltransferase (ChAT) immunoreactivity in surviving VH and IML neurones, suggesting an improved functional status. Thus, neurotrophic factors such as FGF2 and FGF1 may contribute to an overall strategy to treat acute SCI and improve recovery of function.

NOS type-1 mRNA expression and protein localization in spinal autonomic neurons.

Autonomic neurons of the rat spinal cord show strong NADPH diaphorase activity and immunoreactivity for nitric oxide synthase (NOS). Here we show mRNA expression of NOS type-1 (neuronal or brain NOS) transcripts in cell bodies of sympathetic preganglionic neurons (SPNs) of the intermediolateral (IML) cell column by non-radioactive in situ hybridization using NOS-I riboprobes. Hybridization signals occurred only in neuronal cell bodies and not outside, in what appeared to be fibers and/or terminals. In preganglionic fibers of SPNs, however, dense axoplasmic immunogold labeling was detected with a monoclonal anti-NOS-I antibody. Expression of NOS-I mRNA in SPN cell bodies and axoplasmic immunolocalization of NOS-I protein suggest that protein translocation is involved in NO-mediated preganglionic control of peripheral targets.

Nucleus raphe obscurus (nRO) regulation of anorectal motility in rats

Previous research has demonstrated that anorectal contractions in the rat are modulated by activation of spinal autonomic circuits. In the present study, anterograde tracing of descending pathways originating from the caudal nucleus raphe obscurus (nRO) revealed that this nucleus projects to cells within the intermediolateral (IML) cell column of the thoracic cord and the sacral parasympathetic nucleus (SPN). These anatomical studies suggested that the nRO may influence the regulation of spinal reflexes of the pelvic floor. In a second set of experiments, acute rat preparations were used to investigate changes in anorectal motility during electrical stimulation of the nRO. Anorectal contractions were measured by a fluid-filled manometer. Electrical stimulation of the nRO significantly reduced spontaneous anorectal activity when compared to baseline contractions recorded for 1 min prior to stimulation. Stimulation sites outside the nRO did not affect anorectal contractions when compared to either (a) the 1-min pre-stimulation baseline for that site or (b) the 1-min stimulation period for sites within the nRO. Stimulation of caudal portions of the nRO were more likely than the rostral nRO to reduce anorectal contractions. Given that the SPN contains preganglionic neurons which may be involved in control of anorectal contractions (mediated via the pelvic nerve), the studies presented here suggest a functional role for nRO regulation of preganglionic motoneurons innervating the distal gut of the rat.