Development of neurochemical labeling in the intermediolateral nucleus of cats' spinal cord.

NeuN is a neuron-specific nuclear protein expressed in most mature neuronal cell types, with some exceptions. These exceptions are known mainly for the brain but not for the spinal cord or the spinal visceral networks for which only scarce information is available. One of the most defined visceral structures in the spinal cord is the sympathetic intermediolateral nucleus located within the thoracolumbar segments. We investigated the NeuN staining in the intermediolateral nucleus and compared it with the staining for two neurochemical markers of visceral neurons: nitric oxide synthase and calcium-binding protein calretinin in adult cats and in kittens aged 0, 14, and 35 days. A clear NeuN-immunonegativity was obtained for intermediolateral neurons labeled for nitric oxide synthase for both adult cats and kittens. In contrast, a matched immunopositivity for the NeuN and calretinin was obtained, showing an age-dependent degree of this colocalization, which was high in newborn kittens, decreased on postnatal 14 and 35 days and persisted at a moderate level up to adulthood. Perhaps our data displayed a heterogeneity of the intermediolateral neurons.

Dopamine and ghrelin receptor co-expression and interaction in the spinal defecation centers.

BACKGROUND: Dopamine receptor 2 (DRD2) and ghrelin receptor (GHSR1a) agonists both stimulate defecation by actions at the lumbosacral defecation center. Dopamine is in nerve terminals surrounding autonomic neurons of the defecation center, whereas ghrelin is not present in the spinal cord. Dopamine at D2 receptors generally inhibits neurons, but at the defecation center, its effect is excitatory. METHODS: In vivo recording of defecation and colorectal propulsion was used to investigate interaction between DRD2 and GHSR1a. Localization studies were used to determine sites of receptor expression in rat and human spinal cord. KEY RESULTS: Dopamine, and the DRD2 agonist, quinpirole, directly applied to the lumbosacral cord, caused defecation. The effect of intrathecal dopamine was inhibited by the GHSR1a antagonist, YIL781, given systemically, but YIL781 was not an antagonist at DRD2. The DRD2 agonist, pramipexole, administered systemically caused colorectal propulsion that was prevented when the pelvic nerves were cut. Drd2 and Ghsr were expressed together in autonomic preganglionic neurons at the level of the defecation centers in rat and human. Behaviorally induced defecation (caused by water avoidance stress) was reduced by the DRD2 antagonist, sulpiride. We had previously shown it is reduced by YIL781. CONCLUSIONS AND INFERENCES: Our observations imply that dopamine is a transmitter of the defecation pathways whose actions are exerted through interacting dopamine (D2) and ghrelin receptors on lumbosacral autonomic neurons that project to the colorectum. The results explain the excitation by dopamine agonists and the conservation of GHSR1a in the absence of ghrelin.

Synapse preservation and decreased glial reactions following ventral root crush (VRC) and treatment with 4-hydroxy-tempo (TEMPOL).

Astrogliosis and microglial reactions are correlated with the formation of scar tissue and synapse loss. 4-hydroxy-tempo (TEMPOL) is a reactive oxygen species scavenger with proven neuroprotective efficacy in experimental models of traumatic injury and cerebral ischemia. TEMPOL has not, however, been applied following ventral root lesions, which are particularly correlated with the degeneration of spinal motoneurons following brachial plexus injuries. Thus, the present study investigated the effects of TEMPOL on motoneurons and adjacent glial reactions, with a particular focus on the preservation of excitatory and inhibitory spinal circuits. Adult female Sprague Dawley rats were subjected to ventral root crush (VRC) at the lumbar intumescence. Animals were divided into the following experimental groups: (a) VRC-saline treatment; (b) VRC-TEMPOL treatment (12 mg/kg, n = 5), and (c) VRC-TEMPOL treatment (250 mg/kg, n = 5). The spinal cord tissue located contralateral to the lesion was used as the control. Fourteen days after lesioning, the rats were euthanized and the spinal cords were removed for motoneuron counting and immunolabeling with glial (GFAP and Iba-1) and synapse markers (synaptophysin, VGLUT-1, and GAD65). Although TEMPOL did not exert neuroprotective effects at the studied concentrations, the modulation of glial reactions was significant at higher doses. Thus, synaptophysin staining was preserved and, in particular, VGLUT-1-positive inputs were maintained, thereby indicating that TEMPOL preserved proprioceptive glutamatergic inputs without exacerbating the rate of motoneuron degeneration. Consequently, its administration with other efficient neuroprotective substances may significantly improve the outcomes following spinal cord lesioning.

Development of nNOS-positive preganglionic sympathetic neurons in the rat thoracic spinal cord.

To gain a better understanding of the neuroplasticity of sympathetic neurons during postnatal ontogenesis, the distribution of neuronal nitric oxide synthase (nNOS) immunoreactivity was studied in sympathetic preganglionic neurons (SPN) in the spinal cord (Th2 segment) of female Wistar rats at different ages (newborn, 10-, 20-, 30-day-old; 2-, 6-month-old; 3-year-old). In all age groups, the majority of nNOS-immunoreactive (IR) neurons was observed in the nucleus intermediolateralis thoracolumbalis pars principalis. In the first month, the proportion of nNOS-IR neurons decreased significantly from 92 ± 3.4% in newborn to 55 ± 4.6% in 1-month-old, while the number of choline acetyltransferase (ChAT)-IR neurons increased from 74 ± 4.2% to 99 ± 0.3% respectively. Decreasing nNOS expression in the first 10 days of life was also confirmed by western blot analysis. Some nNOS-IR SPN also colocalized calbindin (CB) and cocaine and amphetamine-regulated transcript (CART). The percentage of NOS(+)/CB(-) SPN increased from 23 ± 3.6% in 10-day-old to 36 ± 4.2% in 2-month-old rats. Meanwhile, the proportion of NOS(+)/CART(-) neurons decreased from 82 ± 4.7% in newborn to 53 ± 6.1% in 1-month-old rats. The information provided here will also serve as a basis for future studies investigating the mechanisms of autonomic neuron development.

Sympathetic nervous remodeling is induced in the intermediolateral nucleus after myocardial infarction - Role of BDNF-TrkB axis.

Several studies have shown that neural remodeling in stellate ganglia (SG) is induced by myocardial infarction (MI). It remains unclear whether neural remodeling after MI is limited in SG within the sympathetic nervous system (SNS). MI was induced in a rat model by ligation of the left anterior descending artery. Neural remodeling in the intermediolateral nucleus (IML) and SG was assessed by immunohistochemistry 2 weeks after MI. The mRNA and protein expressions of brain-derived neurotrophic factor (BDNF), tropomyosin-related kinase receptor (TrkB) and extracellular signal-regulated kinase (ERK) were measured by quantitative RT-PCR, immunohistochemistry and Western blotting 1 week after MI. The neuronal size and axonal density of IML were increased after MI compared to sham. The density of growth-associated protein-43, a protein upregulated in axons undergoing nerve sprouting, was increased after MI compared to sham. The fluorescence intensity of BDNF and TrkB in IML were significantly higher in the MI group than in the sham group. In addition, mRNA expressions of BDNF and TrkB in the spinal cord at the Th2 level was increased after MI. Finally, the percentage of phospho-ERK-immunoreactive cells in IML was significantly higher in the MI group than in the sham group. In conclusion, neural remodeling after MI in IML is associated with the activation of BDNF-TrkB axis. Morphological remodeling throughout the SNS may be involved in sustained activation of sympathetic tone after MI.

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 7­nitroindazole (7­NI) or L­arginine (LA). The spindle afferent response to vibratory stimuli induced a distinct bilateral increase in the activation of c­Fos immunoreactivity in the spinal neurons in three groups of rats (tendon­vibrated, tendon­vibrated + 7­NI and tendon­vibrated + LA). The T5/T13 segments in tendon­vibrated +7­NI animals showed the highest increase of Fos­immunoreactive neurons. This increase was two times higher than that in tendon only­vibrated control rats and endon­vibrated + 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 tendon­vibrated and tendon­vibrated + 7­NI animals, and in the IML nucleus and layer 4 in tendon­vibrated + 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 7­NI was accompanied by a potentiation of the early c­fos gene expression induced by muscle proprioceptive activity within the thoracolumbar region of the rat spinal cord. Thus, enhanced c­Fos immunoreactivity in the IML nucleus indicated that the sympathetic nervous system can exert a direct influence on the muscle spindles.

Selective Pharmacogenetic Activation of Catecholamine Subgroups in the Ventrolateral Medulla Elicits Key Glucoregulatory Responses.

Catecholamine (CA) neurons in the ventrolateral medulla (VLM) contribute importantly to glucoregulation during glucose deficit. However, it is not known which CA neurons elicit different glucoregulatory responses or whether selective activation of CA neurons is sufficient to elicit these responses. Therefore, to selectively activate CA subpopulations, we injected male or female Th-Cre+ transgenic rats with the Cre-dependent DREADD construct, AAV2-DIO-hSyn-hM3D(Gq)-mCherry, at one of four rostrocaudal levels of the VLM: rostral C1 (C1r), middle C1 (C1m), the area of A1 and C1 overlap (A1/C1), and A1. Transfection was highly selective for CA neurons at each site. Systemic injection of the Designer Receptor Exclusively Activated by Designer Drugs (DREADD) receptor agonist, clozapine-N-oxide (CNO), stimulated feeding in rats transfected at C1r, C1m, or A1/C1 but not A1. CNO increased corticosterone secretion in rats transfected at C1m or A1/C1 but not A1. In contrast, CNO did not increase blood glucose or induce c-Fos expression in the spinal cord or adrenal medulla after transfection of any single VLM site but required dual transfection of both C1m and C1r, possibly indicating that CA neurons mediating blood glucose responses are more sparsely distributed in C1r and C1m than those mediating feeding and corticosterone secretion. These results show that selective activation of C1 CA neurons is sufficient to increase feeding, blood glucose levels, and corticosterone secretion and suggest that each of these responses is mediated by CA neurons concentrated at different levels of the C1 cell group.

Attenuation of angiotensin type 2 receptor function in the rostral ventrolateral medullary pressor area of the spontaneously hypertensive rat.

We hypothesized that blockade of angiotensin II type 2 receptors (AT2Rs) in the rostral ventrolateral medullary pressor area (RVLM) may elicit sympathoexcitatory responses which are smaller in hypertensive rats compared to normotensive rats. This hypothesis was tested in urethane-anesthetized, artificially ventilated male 14-week-old spontaneously hypertensive rats (SHR). Age-matched male Wistar-Kyoto rats (WKY) and Wistar rats were used as controls. PD123319 (AT2R antagonist) was microinjected into the RVLM and mean arterial pressure (MAP), heart rate (HR) and greater splanchnic nerve activity (GSNA) were recorded. Increases in MAP, HR and GSNA elicited by unilateral microinjections of PD123319 into the RVLM were significantly smaller in SHR when compared with those in WKY and Wistar rats. Unilateral microinjections of l-glutamate (l-Glu) into the RVLM elicited greater increases in MAP and GSNA in SHR compared to those in WKY. AT2R immunoreactivity was demonstrated in the RVLM neurons which were retrogradely labeled from the intermediolateral cell column (IML) of the spinal cord. These results indicate that AT2Rs are present on the RVLM neurons projecting to the IML and their blockade results in sympathoexcitatory responses. Activation of AT2Rs has an inhibitory influence in the RVLM and these receptors are tonically active. Attenuation of the function of AT2Rs in the RVLM may play a role in genesis and/or maintenance of hypertension in SHR.