Peter N. McWilliam

Glutamate, γ-aminobutyric acid and tachykin-inimmunoreactive synapses in the cat nucleus tractus solitarii

SummaryNeurophysiological and pharmacological evidence suggests that glutamate, γ-aminobutyric acid and tachykinins (substance P and neurokinin A) each have a role in cardiovascular regulation in the nucleus tractus solitarii. This study describes the ultrastructural relationships between nerve terminals immunoreactive for these substances in the nucleus tractus solitarii of the cat using post-embedding immunogold (single and double) labelling techniques on sections of tissue embedded in LR White resin. The technique combines a high specificity of labelling with good ultrastructural and antigenic preservation. Glutamate-immunoreactive terminals, recognized by their high density of gold particle labelling compared to the mean tissue level of labelling, accounted for about 40% of all synaptic terminals in the region of the nucleus tractus solitarii analysed (medial, dorsal, interstitial, gelatinosus and dorsolateral subnuclei). They appeared to comprise several morphological types, but formed mainly asymmetrical synapses, most often with dendrites of varying size, and contained spherical clear vesicles together with fewer dense-cored vesicles. Substance P- and neurokinin A-immunoreactive terminals were fewer in number (9% of all terminals) but similar in appearance, with the immunoreaction restricted to the dense-cored vesicles. Analysis of serial- and double-labelled sections showed a co-existence of substance P and neurokinin A-immunoreactivity in 21% of glutamate-immunoreactive terminals. Immunoreactivity for γ-aminobutyric acid was found in 33% of all terminals in the nucleus tractus solitarii. These predominantly contained pleomorphic vesicles and formed symmetrical synapses on dendrites and somata. Possible sites of axo-axonic contact by γ-aminobutyric acid-immunoreactive terminals onto glutamateor tachykinin-immunoreactive terminals were rare, but examples of adjacent glutamate and γ-aminobutyric acid-immunoreactive terminals synapsing on the same dendritic profile were frequent. These results provide an anatomical basis for a γ-aminobutyric acid mediated inhibition of glutamatergic excitatory inputs to the nucleus tractus solitarii at a post-synaptic level.

Glutamate‐immunoreactivity in identified vagal afferent terminals of the cat: a study combining horseradish peroxidase tracing and postembedding electron microscopic immunogold staining

Using electron microscopic immunohistochemistry we have shown that strong glutamate‐immunoreactivity (glutamate‐ir) is present in neuronal cell bodies of the nodose ganglion, axons in the tractus solitarius and afferent terminals in the nucleus tractus solitarii. Vagal afferent fibres were specifically labelled by transganglionic retrograde transport of horseradish peroxidase (HRP). Fifty‐seven per cent of the HRP‐labelled terminals in the dorsomedial medulla were found to contain a high level of glutamate‐ir, suggesting that a population of vagal afferent fibres uses glutamate as a neurotransmitter substance. There were no apparent ultrastructural differences between glutamate‐ir and non‐glutamate‐ir vagal afferent terminals, both classes mainly containing rounded vesicles and forming asymmetric synapses. However, some difference in their preference for postsynaptic target was noted. The great majority (83%) of non‐glutamate‐ir vagal afferent terminals made axodendritic synapses, but only just over half (57%) of the glutamate‐ir vagal terminals made synaptic contact with dendrites. Approximately 13% of the HRP‐labelled terminals were found to make synaptic contact with HRP‐labelled dendrites or soma of motoneurones of the dorsal vagal motor nucleus, confirming the existence of monosynaptic connections between vagal afferent fibres and vagal motoneurones.

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 γ‐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 μm 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.

Electrical stimulation of the vagus increases extracellular glutamate recovered from the nucleus tractus solitarii of the cat by in vivo microdialysis

Release of glutamate into the extracellular space of the cat nucleus tractus solitarii (NTS) was measured by in vivo microdialysis and high performance liquid chromatography. Perfusion of the probe with 100 mM potassium increased glutamate release by 211% (P < 0.001), while electrical stimulation of the cervical vagus increased release by 53% (P < 0.01). These results are compatible with the hypothesis that glutamate is a neurotransmitter released by vagal afferent nerve terminals in the NTS.

A gamma‐aminobutyric‐acid‐mediated inhibition of neurones in the nucleus tractus solitarius of the cat.

1. Extracellular recordings were made from 123 synaptically activated neurones in the nucleus tractus solitarius of the chloralose‐anaesthetized cat. 2. Ninety‐one neurones were activated by electrical stimulation of cardiac or pulmonary vagal branches and thirty‐two by stimulation of the aortic nerve. 3. Ionophoretic application of GABA abolished or markedly reduced the evoked or spontaneous activity of each neurone tested. These inhibitory effects were antagonized by the simultaneous ionophoretic application of bicuculline. 4. Glycine inhibited the evoked activity of 60% of neurones tested. This inhibition could be antagonized by the simultaneous application of strychnine. 5. Application of bicuculline alone increased the evoked or spontaneous activity of a large proportion of the neurones; strychnine alone had no significant effect on the evoked or spontaneous activity of the neurones. 6. These results are consistent with the hypothesis that GABA acts as an inhibitory transmitter substance within the nucleus tractus solitarius.

Co-localization of neurotransmitter immunoreactivities in putative nitric oxide synthesizing neurones of the cat brain stem

The distribution of nitric oxide producing neurones in the medulla oblongata of the cat was investigated using nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry, and nitric oxide synthase (NOS) immunohistochemistry. The pattern of staining obtained with both methods was found to be similar. Strongly diaphorase and NOS reactive neurones were present in the paramedian and lateral tegmental fields, including the regions occupied by the A1/C1 catecholamine cell groups, the nucleus ambiguus and lateral reticular nucleus, and in a number of sensory nuclei including the nucleus of the tractus solitarius and the dorsal column nuclei. The extent of co-localization of NADPH-diaphorase with a number of neuropeptides and neurotransmitters was investigated by combining NADPH-diaphorase histochemistry with immunocytochemistry for neuropeptide Y, somatostatin, glutamate, cholecystokinin and tyrosine hydroxylase. NADPH-diaphorase reaction product was observed in neurones immunoreactive for glutamate and somatostatin. These double-labelled cells were found in the paramedian region, lateral reticular field, the nucleus prepositus hypoglossi and in the rostral nucleus of the tractus solitarius. In the rostral ventrolateral medulla NADPH-diaphorase/somatostatin immunoreactive cells were found in the paragigantocellular nucleus. NADPH-diaphorase/glutamate immunoreactive cells overlapped the nucleus ambiguus, the lateral reticular nucleus and the A1/C1 catecholaminergic cell groups. In addition, a few NADPH-diaphorase/glutamate immunoreactive cells were found in the paraolivary area and gigantocellular tegmental field, in the external cuneate and infratrigeminal nuclei. The functional implications of the co-localization of nitric oxide with these neurotransmitters in areas of the medulla concerned with cardiovascular regulation is discussed.

Changes in R‐R interval at the start of muscle contraction in the decerebrate cat.

1. The effect on R‐R interval of a brief hindlimb contraction, elicited by electrical stimulation of L7 ventral roots, was investigated in decerebrate cats. The first series of experiments was performed at both low and high carotid sinus pressure to vary the level of vagal tone. When carotid sinus pressure was elevated to increase vagal tone, contraction commenced 1 s later. 2. The change in R‐R interval at low carotid sinus pressure was expressed as the difference between the mean of the five R‐R intervals immediately preceding contraction and the mean of the last five R‐R intervals at the end of a 5 s contraction. At high carotid sinus pressure, the change was expressed as the difference between the mean of the last five R‐R intervals at the end of a 5 s contraction and the mean of five R‐R intervals at an equivalent time after raising pressure alone. 3. Hindlimb contraction at low carotid sinus pressure produced a significant reduction in R‐R interval from 359 +/‐ 25 (mean +/‐ S.E.M. n = 8) to 336 +/‐ 24 ms (P less than 0.005). At high carotid sinus pressure the response was enhanced with contraction producing a reduction in R‐R interval from 474 +/‐ 45 to 419 +/‐ 47 ms (P less than 0.001). 4. The shortening of R‐R interval produced by hindlimb contraction at high carotid sinus pressure, 55 +/‐ 8 ms, was significantly greater than that observed at low sinus pressure, 23 +/‐ 5 ms (P less than 0.001, n = 8, paired t test). This pattern of response was also seen at stimulation frequencies as low as 10 Hz. 5. In a second series of experiments, designed to determine the latency of the cardiac acceleration, the minimum latency between the onset of L7 ventral root stimulation and the end of the first shortened R‐R interval was 687 +/‐ 29 ms (n = 5). 6. Atropine (0.4 mg kg‐1, I.V.) prevented a 5 s contraction from producing any change in R‐R interval. 7. These results indicate that afferent information originating from receptors in contracting muscles is responsible for producing an immediate shortening of R‐R interval, which is mediated by vagal withdrawal. The possibility that the shortening of R‐R interval at the start of contraction is linked to a reduction in arterial baroreceptor reflex sensitivity, possibly via inhibitory effects on neurones forming the central pathway of the baroreceptor reflex, is discussed.

Immunohistochemical profiles of spinal lamina I neurones retrogradely labelled from the nucleus tractus solitarii in rat suggest excitatory projections.

Three morphologically distinct types of lamina I neurones, fusiform, flattened and pyramidal, project from the spinal cord to the caudal part of the nucleus tractus solitarii in the rat, and may represent a pathway whereby peripheral stimuli can modify autonomic functions. The neurochemistry of these three types of projection neurones was investigated using retrograde neuronal tracing with cholera toxin B-subunit combined with dual and triple immunofluorescence labelling for different neuroactive substances. None of the lamina I neurones with immunoreactivity for GABA or glycine were found to project to the nucleus tractus solitarii, whereas high levels of glutamate immunoreactivity, which may indicate a glutamatergic phenotype, were found in 18.4% of fusiform, 9.6% of pyramidal and 2.1% of flattened projection neurones. Immunoreactivity for calbindin-D28K was present in 34.9% of fusiform cells, 18.3% of pyramidal cells and 10.5% of flattened cells, and nitric oxide synthase immunoreactivity was detected in 13.8% of fusiform cells, 1.1% of pyramidal cells and 4.2% of flattened cells that had projections to the nucleus tractus solitarii. Calbindin immunoreactivity was co-localised in major subpopulations of projection neurones of each morphological type that contained glutamate immunoreactivity, whereas co-localisation of nitric oxide synthase immunoreactivity in these neurones was relatively uncommon. The pyramidal cell was the only retrogradely labelled cell type found to be immunoreactive for substance P, but few (<5%) of these neurones were immunolabelled. These data are consistent with the hypothesis that lamina I neurones projecting to the dorsal vagal complex are not inhibitory, and that some of them, belonging mostly to the fusiform and pyramidal types, may exert excitatory, glutamate- or substance P-mediated effects upon inhibitory interneurones in the nucleus tractus solitarii. Such excitatory pathways could be involved in the attenuation of the reflex control of blood pressure by both painful and innocuous peripheral stimuli, such as those arising in injury and exercise.

Changes in the baroreceptor reflex at the start of muscle contraction in the decerebrate cat.

1. The action of muscle contraction on the sensitivity of the cardiac vagal component of the baroreceptor reflex was examined in decerebrate cats. 2. The sensitivity of the baroreceptor reflex was expressed as the difference between the maximum prolongation of the R‐R interval in response to carotid sinus baroreceptor stimulation and the mean of ten R‐R intervals immediately before carotid sinus pressure elevation. 3. Muscle contraction elicited by electrical stimulation of L7 ventral roots (50 Hz) significantly reduced the sensitivity of the baroreceptor reflex by reducing the prolongation of the R‐R interval from 269 +/‐ 31 to 159 +/‐ 22 ms. 4. Inhibition of the cardiac vagal component of the baroreceptor reflex was seen just 1 s after the onset of contraction and with stimulation frequencies as low as 10 Hz. 5. These results show for the first time that changes in the sensitivity of the baroreceptor reflex during exercise result in part from afferent information originating in the contracting muscles.

Projection sites of superficial and deep spinal dorsal horn cells in the nucleus tractus solitarii of the rat.

By using anterograde transport of biotin dextran amine injected into the cervical spinal dorsal horn, we have shown that fibres from superficial and deep dorsal horn project to the nucleus tractus solitarii via two distinct pathways. Afferent fibres from the superficial lamina (I-III) were found to course in the dorsal funiculus and terminate bilaterally in the caudal zone of the nucleus tractus solitarii (NTS), mainly within the commissural subnucleus. In contrast, afferents from the deeper dorsal horn laminae (IV-V) were found to course in the dorsolateral fasciculus and terminate ipsilaterally, mostly in the lateral areas of the caudal nucleus tractus solitarii. Similar, but more extensive patterns of labelled fibres were produced by injections into the white matter of the dorsal funiculus and dorsolateral fasciculus, respectively. These observations suggest that the caudal NTS not only serves as a location of visceral afferent convergence and integration, but may also be a receptive area for monosynaptic projections from dorsal horn neurons receiving sensory afferent inputs. Such projections may represent pathways through which NTS neurons are influenced by nociceptive and non-nociceptive information from the dorsal horn and thereby can co-ordinate the appropriate autonomic response, including adjustments in cardiorespiratory reflex output.