Age-related loss of cardiac vagal preganglionic neurones in spontaneously hypertensive rats.

Despite the findings that impaired vagal control of the heart rate occurs in human hypertension, leading to greater cardiovascular risk, the mechanism of this impairment is as yet unknown. Observations in humans and experiments in the spontaneously hypertensive rat (SHR) suggested that such impairment may be related to an anomaly in central vagal neurones. We therefore set out to determine whether the numbers and distribution of cardiac-projecting vagal preganglionic neurones in the medulla of adult (12 week) hypertensive SHR are different from those in young (4 week) prehypertensive SHR and in age-matched Wistar-Kyoto (WKY) rats of two age groups. The number of vagal neurones, identified by labelling with the fluorescent tracer DiI applied to the heart, was essentially similar in the three areas of the medulla analysed (dorsal vagal nucleus, nucleus ambiguus and intermediate reticular zone) in young SHR and young or adult WKY rats. In contrast, fewer vagal neurones were labelled in adult SHR compared with young SHR or WKY rats. This difference was due to highly significant reductions in vagal neurones in the dorsal vagal nucleus and nucleus ambiguus on the right side of the medulla. These observations suggest that a loss of parasympathetic preganglionic neurones supplying the heart with axons in the right vagus nerve, or a remodelling of their cardiac projections, may explain the known impairment of the baroreceptor reflex gain controlling heart rate in hypertension.

Substance P (NK1) and somatostatin (sst2A) receptor immunoreactivity in NTS-projecting rat dorsal horn neurones activated by nociceptive afferent input.

Spinal neurones that receive inputs from primary afferent fibres and have axons projecting supraspinally to the medulla oblongata may represent a pathway through which nociceptive and non-nociceptive peripheral stimuli are able to modulate cardiorespiratory reflexes. Expression of the neurokinin-1 (NK1) receptor is believed to be an indicator of lamina I cells that receive nociceptive inputs from substance P releasing afferents, and similarly, sst2A receptor expression may be a marker for neurones receiving somatostatinergic inputs. In this study, immunoreactivity for these two receptors was investigated in rat spinal neurones retrogradely labelled by injections of cholera toxin B or Fluorogold into the nucleus of the solitary tract (NTS). In addition, nociceptive activation of these labelled cells was studied by immunodetection of Fos protein in response to cutaneous and visceral noxious chemical stimuli. NK1 and sst2A receptors in lamina I were localised to mainly separate populations of retrogradely labelled cells with fusiform, flattened and pyramidal morphologies. Examples of projection neurones expressing both receptors were, however observed. With visceral stimulation, many retrogradely labelled cells expressing c-fos were immunoreactive for the NK1 receptor, and a smaller population was sst2A positive. In contrast, with cutaneous stimulation, only NK1 positive retrogradely labelled cells showed c-fos expression. These data provide evidence that lamina I neurones receiving noxious cutaneous and visceral stimuli via NK1 receptor activation project to NTS and so may be involved in coordinating nociceptive and cardiorespiratory responses. Moreover, a subpopulation of projection neurones that respond to visceral stimuli may receive somatostatinergic inputs of peripheral, local or supraspinal origins.

Ionotropic glutamate receptor subunit immunoreactivity of vagal preganglionic neurones projecting to the rat heart.

The ionotropic glutamate receptor subunits expressed by vagal preganglionic neurones in the rat medulla oblongata were examined by using fluorescence immunolabelling combined with retrograde neuronal tracing. The general population of these neurones in the medulla was identified by intraperitoneal injections of Fluorogold and also with choline acetyltransferase antibodies. Cardiac projecting neurones were specifically identified by applying the fluorescent tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine (DiI) to the heart or by injecting cholera toxin B-subunit into the pericardium. Both tracers labelled populations of neurones lying in the dorsal vagal nucleus, intermediate reticular formation and nucleus ambiguus, and when both tracers were applied simultaneously, approximately 50% of cells were dual-labelled. Control experiments established that the labelling was specific for neurones projecting to the heart. Most vagal preganglionic neurones, including those projecting to the heart, irrespective of their location in the medulla, had a similar profile of glutamate receptor immunoreactivity. Labelling of somata for the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) subunit GluR1 was weak or absent, while labelling with antibodies directed to GluR2, a common sequence of GluR2 and GluR3, and GluR4 was moderate or intense. All neurones studied appeared to express the N-methyl-D-aspartate (NMDA) receptor subunit NR1, and while antibodies recognising the NR2A and NR2B splice variants gave strong labelling, immunoreactivity with a NR2B specific antibody was weaker. Weak to moderate labelling was seen in some neurones using antibodies to the kainate receptor subunits KA2 and GluR5-7. These results are consistent with neurophysiological data indicating the presence of AMPA, NMDA and kainate responses in cardiac vagal preganglionic neurones, and suggest that these neurones are similar to other vagal parasympathetic preganglionic neurones in expressing mainly AMPA receptor subunits GluR2-4.

Ultrastructural Relationships Between GABAergic Terminals and Cardiac Vagal Preganglionic Motoneurons and Vagal Afferents in the Cat: A Combined HRP Tracing and Immunogold Labelling Study.

The ultrastructural relationships between gamma-aminobutyric acid-immunoreactive (GABA-ir) neurons and other neurons of the nucleus tractus solitarius (NTS) and motoneurons of the nucleus ambiguus (NA) and dorsal motor vagal nucleus (DMVN), were examined by electron microscopic (EM) immunogold labelling with an anti-GABA antiserum on brain stem sections in which vagal motoneurons and vagal afferent fibres were labelled with horseradish peroxidase (HRP). HRP was applied to the cervical vagus or the cardiac vagal branch of anaesthetized cats. After 24 - 48 h survival, brains were glutaraldehyde-fixed and a stable HRP-tetramethylbenzidine reaction product compatible with EM processing was revealed on 250 microm vibratome sections. Following osmium postfixation, dehydration and resin embedding, GABA-ir was localized on ultrathin sections by an immunogold technique. GABA-ir axon terminals, heavily and specifically labelled with gold particles, were very numerous within NTS, DMVN and NA. All terminals contained small, clear, pleomorphic vesicles and a few also contained larger dense cored vesicles. The density of gold particles over clear vesicles, dense cored vesicles and mitochondria was significantly greater than over the cytoplasm of these terminals. GABA-ir synapses were found on the soma and dendrites of neurons, but rarely on other axon terminals within NTS, where GABA-ir cell bodies and dendrites were also seen. These received synaptic contacts from both GABA-ir terminals and from HRP-labelled vagal afferents. In both the DMVN and NA, similar GABA-ir synapses were present on both the soma and dendrites of HRP-labelled motoneurons. GABA synapses were also present on other cell types in DMVN. These observations provide an anatomical basis for a GABAergic inhibition of neurons forming the central pathways of cardiovascular and other autonomic reflexes.