Glutamate circuits in selected medullo-spinal areas regulating cardiovascular function

1. The importance of the medullo-spinal neuronal pools in the regulation of cardiovascular function has been known for a long time. However, important groups of these neurons, interconnections between them and the neurotransmitters released at their projections have been identified with certainty only during the past two decades. 2. Some of the medullo-spinal neuronal pools mediating cardiovascular function include the nucleus tractus solitarius, caudal ventrolateral medullary depressor area, rostral ventrolateral medullary pressor area, nucleus ambiguus and intermediolateral cell column of the thoracolumbar spinal cord. Interactions between these selected neuronal groups and neurotransmitters in the pathways connecting them are discussed in the present short review.     

Both dorsal and ventral spinal cord pathways contribute to overground locomotion in the adult rat

Identification of long tracts responsible for spontaneous locomotion is critical for spinal cord injury (SCI) repair strategies. We recently demonstrated that extensive demyelination of adult rat thoracic ventral columns, ventromedial, and ventrolateral white matter produces persistent, significant open-field hindlimb locomotor deficits. Locomotor movements resulting from stimulation of the pontomedullary locomotor region are inhibited by dorsolateral funiculus (DLF) lesions suggesting that important pathways for locomotion may also exist in the dorsal white matter. However, dorsal hemisections that interrupt dorsal columns/dorsal corticospinal tract (DC/CST) and DLF pathways do not produce persistent, severe locomotor deficits in the adult rat. We studied the contributions of myelinated tracts in the DLF and DC/CST to overground locomotion following complete conduction blockade of axons in the ventrolateral funiculus (VLF), a region important for locomotor movements and for transcranial magnetic motor-evoked potentials (tcMMEP). Animals received ethidium bromide plus photon irradiation to produce discrete demyelinating lesions sufficient to stop axonal conduction in the VLF, combined VLF + DLF, or combined VLF + DC/CST. Open-field BBB scores and tcMMEPs were studied at 1, 2, 3, and 4 weeks postlesion. VLF lesions resulted in mean BBB scores of 17 at 4 weeks. VLF + DC/CST and VLF + DLF lesions resulted in mean BBB scores of 15.9 and 11.1, respectively. TcMMEPs were absent in all lesion types confirming VLF conduction blockade throughout the study. Our data indicate that significant contributions to locomotion from myelinated pathways within the rat DLF can be revealed when combined with simultaneous compromise of the VLF.     

Isthmic origin of neurons of the rat substantia nigra

This study provides new data on the time of origin, the generation site and the migration route of the young neurons of the substantia nigra of the rat during embryogenesis. The neurons of the substantia nigra are generated on day 12, 13, 14 and 15 of gestation. They settle following a light spatiotemporal rostrocaudal gradient from day 12 to 15. The neurons of the substantia nigra are generated at two different points of the basal plate at the level of the fovea isthmi (meso-isthmic junction) and migrate in radial pattern as two definite streams toward the ventral mesencephalon. From this point they move rostralwards along the surface towards their final site.The main findings of this work are the disclosure that the neurons of the substantia nigra are generated in the region of the isthmus rhombencephali and that its cells do not migrate between existing cells of the mesencephalic tegmentum but first migrate ventralwards in a radial pattern and then rostrally towards their definite site. Numerous neurons of the basal mesencephalon and of the midline structures of the caudal mesencephalon are apparently derived from the region of the fovea isthmi.     

Depressor responses to L-proline microinjected into the rat ventrolateral medulla are mediated by ionotropic excitatory amino acid receptors

The essential amino acid L-proline produces a depressor response when microinjected into the caudal ventrolateral medulla (CVLM) of anesthetized rats. L-proline may activate some excitatory amino acid (EAA) receptors. The present study tested this hypothesis by investigating the effects of two ionotropic excitatory amino acid receptor antagonists on the depressor response to L-proline in the CVLM: the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptor-selective antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the N-methyl-D-aspartate (NMDA) receptor-selective antagonist MK801. Urethane-anesthetized rats received arterial catheters and their ventrolateral medulla surface was exposed. Injections of the antagonists CNQX and MK801 (2 mM, 68 nl in each case) into the CVLM completely blocked depressor responses to subsequent administration of AMPA (2 pmol/34 nl) and NMDA (2 pmol/34 nl), respectively. The depressor response to L-proline (3.4 nmol/34 nl) was strongly inhibited by prior injection of CNQX (2 mM, 68 nl) and significantly attenuated by prior injection of a high dose (20 mM, 68 nl), but not a low dose (2 mM, 68 nl), of MK801. The results indicate that the depressor response to L-proline in the CVLM includes mechanisms of ionotropic excitatory amino acid receptors.     

Coexpression of vesicular glutamate transporter-3 and gamma-aminobutyric acidergic markers in rat rostral medullary raphe and intermediolateral cell column

Markers of serotonergic, gamma-aminobutyric acid (GABA)-ergic (glutamic acid decarboxylase, 67 kDa isoform; GAD-67), and glutamatergic transmission (vesicular glutamate transporter 3; VGLUT3) have been detected in presumed sympathetic premotor neurons of the medullary raphe, a region that controls sympathetic tone to brown fat, skin blood vessels, and heart. In this study, the degree of coexpression of these markers was examined in raphe neurons by simultaneous histological detection of tryptophan hydroxylase (TrpOH) immunoreactivity with GAD-67 mRNA and VGLUT3 mRNA. Over half (52%) of the VGLUT3 mRNA-positive neurons expressed one or both of the other markers. The proportion of VGLUT3 neurons containing at least one of the other two markers was even higher (89%) for VGLUT3 spinally projecting neurons. VGLUT3 neurons containing markers for both serotonin and GABA were especially numerous (50-72%, depending on rostrocaudal level) within the marginal layer of raphe pallidus and the parapyramidal region. The dual GABAergic and glutamatergic nature of some bulbospinal raphe neurons was suggested by the presence of nerve terminals immunoreactive (ir) for both VGLUT3 and GABA in the intermediolateral cell column (IML) as detected by electron microscopy. VGLUT3-ir terminals formed approximately equal numbers of symmetric and asymmetric synapses onto presumed preganglionic neurons (nitric oxide synthase-ir profiles) or GABA-ir dendrites in IML, and terminals immunoreactive for both VGLUT3 and GABA always formed symmetric synapses. These data suggest that medullary raphe VGLUT3 neurons could inhibit sympathetic outflow and that their spinal targets include both preganglionic neurons and GABAergic interneurons.     

Characteristics of the trigeminal depressor response in cats

We studied the effects of electrical stimulation of the inferior alveolar nerve (IAN) on cardiovascular responses in cats. There was statistical correlation between cardiovascular response and prestimulus mean arterial blood pressure (MABP) and heart rate (HR). A trigeminal depressor response (TDR) was induced when the prestimulus MABP and HR were above 95 mm Hg and 140 beats/min, respectively. We investigated further to identify the vasomotor regulating center and neural transmitters involved in TDR. In the medulla, electrical stimulation of the dorsomedial medulla, the infratrigeminal nucleus (IFT), and the rostral ventrolateral medulla (RVLM) induced a vasopressor response. We confirmed that neurons in the RVLM were retrogradely labeled by wheat germ agglutinin-conjugated horseradish peroxidase injection into the nucleus intermediolateralis of the spinal cord. The vasopressor response induced by IFT stimulation was similar to that induced by IAN stimulation. Vasodepressor responses were induced when the caudal ventrolateral medulla, the nucleus tractus solitarius, the lateral tegmental field, the trigeminal nucleus interpolaris, the trigeminal spinal tract, and the paramedian reticular nucleus were stimulated. These responses, however, were not similar to the vasodepressor response induced by IAN stimulation but were similar to the cardiovascular response induced by vagal afferent stimulation. After spinalization or lesion of the RVLM, MABP and HR decreased and TDR completely disappeared. Inhibitory synaptic ligands and receptors were localized using immunohistochemical techniques. Neurons immunopositive for adrenaline, noradrenaline, and gamma-aminobutyric acid (GABA), and adrenaline alpha(2A), GABA(A), GABA(B), and glycine receptors were distributed along the sympatho-reflexive route including the RVLM and IFT. These results suggest that TDR could be induced as negative feedback to sympathetic hyperactivity whenever MABP and HR are high, because of the inhibitory control of the RVLM.     

Fos expression after mating in noradrenergic cells of the A1 and A2 areas of the medulla is altered by adrenalectomy

In the female rat, the integrity of the ventral noradrenergic bundle (VNAB) is necessary to carry stimuli from the uterine cervix and vagina to brain areas involved in mating-induced pseudopregnancy. Because adrenal hormones are known to alter noradrenergic function, we examined whether adrenalectomy altered mating-induced Fos expression in the A1 and A2 noradrenergic cell groups that project through the VNAB. Ovariectomized females were adrenalectomized (ADX) or sham-operated (Sham) and, 2 weeks after surgery, were given oestrogen and progesterone and mated. They received 15 intromissions, five intromissions or 15 mounts-without-intromission (mounts-only) from a male. Two hours after mating, rats were perfused and brains were collected; controls were perfused after being taken directly from their home cage. After immunocytochemical staining, Fos-immunoreactive (Fos-IR) and dopamine-beta-hydroxylase-immunoreactive (DBH-IR) cells and the percentage of DBH cells that were labelled with Fos (% DBH/Fos) were counted. In the A1 area, Fos-IR and percentage DBH/Fos were not affected by adrenalectomy. Although an overall effect of mating treatment was found for both measures, no specific mating treatment increased labelled cells above home cage levels. In the caudal, middle and rostral A2, 15 intromissions induced a significant increase in Fos-IR in Sham females above all other groups and a higher percentage of DBH/Fos in the middle and rostral A2 areas. ADX females showed no rise in either Fos-IR or percentage DBH/Fos after 15 intromissions. However, in the middle and rostral A2, ADX females showed significantly increased Fos-IR and percentage DBH/Fos after mounts-only treatment above Sham mounts-only females and all other ADX groups. These results demonstrate that adrenal hormones suppress activation of A2 cells to mounts-only stimuli but contribute to A2 activation in response to intromissions from males. The latter effect may result from stress associated with receipt of vaginocervical stimulation during mating.     

Electroacupuncture induces c-Fos expression in the rostral ventrolateral medulla and periaqueductal gray in cats: relation to opioid containing neurons

Our previous studies have shown that electroacupuncture (EA) at the Neiguan-Jianshi (P5-P6) acupoints inhibits sympathetic outflow and attenuates excitatory visceral cardiovascular reflexes through enkephalin- or beta-endorphin-related opioid receptors in the rostral ventrolateral medulla (rVLM). It is not known whether EA at these acupoints activates neurons containing enkephalin or beta-endorphin in the rVLM as well as in the periaqueductal gray (PAG) that are involved in EA-mediated central neural regulation of sympathetic activity. The present study evaluated activated neurons in the rVLM and PAG by detecting c-Fos immunoreactivity, and identified the relationship between c-Fos nuclei and neuronal structures containing enkephalin or beta-endorphin in these regions. To enhance the detection of cell bodies containing enkephalin or beta-endorphin, colchicine (90-100 microg/kg) was injected into the subarachnoid space in anesthetized cats 28-30 h prior to EA or the sham-operated control for EA. Following bilateral barodenervation and cervical vagotomy, EA (1-4 mA, 2 Hz, 0.5 ms) was performed at the P5-P6 acupoints (overlying median nerve; n=7) for 30 min. Identical procedures, with the exception of electrical stimulation, were carried out in five control animals. EA decreased blood pressure (BP) in four of seven cats (5-15 mm Hg) while the sham procedure for EA produced no responses. Perikarya containing enkephalin were found in the rVLM and rarely in the PAG, while no cell bodies labeled with beta-endorphin were identified in either region. Compared to animals in the control group, more c-Fos immunoreactivity, located principally in close proximity to fibers containing enkephalin or beta-endorphin, was observed in the rVLM and ventrolateral PAG (vlPAG) in EA-treated cats. Moreover, neurons double-labeled with c-Fos and enkephalin in the rVLM were significantly increased in cats following EA stimulation (P<0.05). These data indicate that EA at the P5-P6 acupoints activates neurons in the rVLM and vlPAG. These activated neurons contain enkephalin in the rVLM, and most likely interact with nerve fibers containing enkephalin or beta-endorphin in both the rVLM and vlPAG. The results from this study provide the first anatomical evidence showing that EA at the P5-P6 acupoints has the potential to influence neuronal structures (perikarya, axons and/or dendrites) containing enkephalin or beta-endorphin in specific regions of the brain stem. These neurons likely form the substrate for EA's influence on sympathoexcitatory cardiovascular reflexes.     

Medullary and spinal cord projections from cardiovascular responsive sites in the rostral ventromedial medulla

The rostral ventromedial medulla (RVMM) is a sympathoexcitatory area. However, little is known about its efferent projections. In this study, biotinylated dextran amine (BDA) or Phaseolus vulgaris leucoagglutinin (PHA-L) were used to investigate the medullary and spinal cord projections from pressor sites in RVMM. Initially, RVMM was systematically explored in urethane-anesthetized rats using microinjection of L-glutamate for sites that elicited increases in arterial pressure. A pressor area was identified that included the rostral magnocellular reticular and rostral lateral paragigantocellular reticular nuclei. In the second series of experiments, BDA or PHA-L was iontophoretically injected into RVMM pressor sites. Anterograde labeling was observed throughout the brainstem and spinal cord, bilaterally, but with an ipsilateral predominance. Dense labeling was observed within the nucleus of the solitary tract (NTS); the greatest density of labeling was observed in the caudal dorsolateral, medial, and ventrolateral subnuclei. Additionally, light to moderately dense labeling was found within the nucleus substantia gelatinosus and commissural nucleus. In the nucleus ambiguus/ventrolateral medullary (Amb/VLM) region, the density of labeling was greatest in caudal regions. Within Amb, most of the labeling was localized to its external formation. Anterograde labeling was also found throughout the spinal cord. In the thoracolumbar segments, dense axonal labeling was observed within the dorsolateral funiculus. These labeled axons innervated the intermediolateral nucleus and the central autonomic area. Taken together, these data suggest that RVMM neurons elicit increases in sympathetic activity by likely providing a direct excitatory input to spinal sympathetic preganglionic neurons, and by a direct inhibitory input to medullary cardioinhibitory and depressor areas.     

Motor cortical control of cardiovascular bulbar neurones projecting to spinal autonomic areas

There is evidence that the motor cortex is involved in cardiovascular adjustments associated with somatic motor activity, as it has functional connections with the ventrolateral medulla, a brainstem region critically involved in the control of blood pressure and the regulation of plasma catecholamine levels. The ventrolateral medulla sends projections to the spinal intermediolateral nucleus, where preganglionic neurones controlling heart and blood vessels (T2 segment) and adrenal medulla (T8 segment) are found. The aim of the present study was to determine whether electrical stimulation of the rat motor cortex induces cardiovascular responses and Fos expression in ventrolateral medulla neurones projecting to the T2 and T8 segments. After a set of experiments designed to record cardiovascular parameters (blood pressure and plasma catecholamine levels), injections of retrograde tracer (Fluorogold) were performed in the intermediolateral nucleus of two groups of rats, at the T2 or at the T8 segmental levels. Five days later, the motor cortex was stimulated in order to induce Fos expression in the ventrolateral medulla. Stimulation of the motor cortex induced: (1). hypotension and a significant decrease in plasma noradrenaline levels, and (2). a significant increase in the number of the double-labelled neurones in the rostral ventrolateral medulla projecting to T2. These data demonstrate that cardiovascular adjustments, preparatory to, or concomitant with, motor activity may be initiated in the motor cortex and transmitted to cardiac and vasomotor spinal preganglionic neurones, via the ventrolateral medulla.