Wesley P. Norman

Medullary ventral surface GABA receptors affect respiratory and cardiovascular function

We previously demonstrated that GABA and muscimol administered either into the cisterna magna or the fourth ventricle to chloralose-anesthetized cats cause respiratory depression, hypotension, and bradycardia. Injection of these substances into the lateral and third ventricles had no effect. In order to localize the site of action, muscimol and GABA were applied by Perspex rings to the ventral surface of the medulla. Application of muscimol (0.25-2.66 micrograms) to Schlaefkes area in 6 cats reduced minute ventilation from 443 +/- 38 to 291 +/- 52 ml/min by reducing tidal volume from 31.8 +/- 2.3 to 17.6 +/- 1.4 ml, without changing respiratory rate and duration of inspiration. Hypotension and bradycardia were also observed. Application of GABA (0.14-4.86 mg) produced similar effects on respiratory activity and arterial blood pressure. No significant effects occurred when high doses of these agents were applied to Loeschckes and Mitchells areas. Application of bicuculline (5-25 micrograms) to Schlaefkes area had the opposite effect of muscimol and GABA on respiratory activity and blood pressure, and reversed the respiratory and cardiovascular depressant effects of both agents. We conclude that GABA receptors are present at Schlaefkes area, and that activation of these receptors results in respiratory depression, hypotension, and bradycardia. Our results suggest that GABA may be an important inhibitory neurotransmitter in the modulation of respiratory and cardiovascular control.

Effect of electric and chemical stimulation of the raphe obscurus on phrenic nerve activity in the cat

The effect of electrical and chemical (L-glutamate) stimulation of the raphe obscurus on phrenic nerve activity was examined in the cat. Phrenic nerve activity was recorded from a C5 nerve root in anesthetized, paralyzed and artificially ventilated cats. Neural discharge was quantitated by integrating the phrenic nerve activity. The respiratory frequency was determined from the integrated nerve signal. Focal electrical stimulation (18-144 microA; 5-40 Hz; 100 microseconds pulse duration) resulted in significant (P less than 0.05) increases in both integrated phrenic nerve (IPN) amplitude and respiratory frequency. These changes were dependent upon current intensity and frequency of stimulation. The largest increases in IPN amplitude and respiratory frequency were 47 +/- 17% and 146 +/- 8%, respectively. To insure that the changes in integrated phrenic nerve activity (IPNA) were the result of stimulation of cell bodies and not axons of passage, L-glutamate (100, 200 nmol) was microinjected (100 nl) into the raphe obscurus. Significant (P less than 0.05) dose-related changes occurred in integrated phrenic nerve amplitude with an increase of 44 +/- 13% at 100 nmol and 80 +/- 13% at 200 nmol L-glutamate. No significant increase in respiratory frequency was observed with L-glutamate microinjection. The results suggest that the raphe obscurus may be involved in respiratory control.

Projections from the raphe nuclei to the phrenic motor nucleus in the cat

The purpose of this study was to identify the projections from the brain to the phrenic motor nucleus in the cat by employing a retrogradely-transported fluorescent dye. Propidium iodide was iontophoresed into the phrenic motor nucleus which is located in the fourth, fifth and sixth segments of the cervical spinal cord. Retrogradely-labeled cell bodies were found in the brainstem within recognized respiratory areas. In addition, retrogradely-labeled cell bodies were found within the raphe nuclei, nuclear areas not previously associated with respiratory control. The results of this study suggest that the raphe nuclei may play a role in the central regulation of breathing.

GABA and specific GABA binding sites in brain nuclei associated with vagal outflow

Abstract GABA levels and specific (3H) GABA binding were determined in several nuclei of cat brain. Since previous pharmacological studies (DiMicco et al. [3]) suggested that nucleus ambiguus (NA) may be the site of a GABA-receptor mediated inhibition of vagal outflow to the heart, we were interested in comparing the GABA content and density of (3H) GABA binding in NA with that of other nuclei known to contain GABAergic synapses. The GABA content of NA was 21.2 ± 2.4 nmol/mg protein, similar to that found in the caudate nucleus (28.4 ± 2.9 nmol/mg protein) and 2.5 fold higher than the GABA content of the surrounding reticular nuclei. In frozen-thawed and Triton X-100 treated membranes prepared from NA, specific GABA binding was 98 ± 28 fmol/mg protein when measured using 30 nM (3H) GABA. This was more than 3 fold higher than binding obtained in surrounding reticular tissue and approximately half the value obtained in substantia nigra. GABA content of paired right and left NA was nearly equal; however specific GABA binding of paired right and left NA differed markedly, with the right NA usually exhibiting greater specific binding than the left NA. Retrograde degeneration of vagal fibers of NA by intracranial sectioning of the right vagal trunk decreased the asymmetry in GABA binding of paired right and left NA. Asymmetry was also noted in the percent of the reflex-induced bradycardic response mediated by each vagus nerve. These results suggest that GABAergic synapses may be present in NA, and that some of the postsynaptic receptors for GABA may be associated with vagal efferents. The bilateral asymmetry in the physiological reflex-induced response coupled with the bilateral asymmetry in GABA binding in NA suggests that the degree of vagal activity emanating from NA may be determined by the density of GABA receptors.

NMDA receptors are involved at the ventrolateral nucleus tractus solitarii for termination of inspiration

The purpose of the present study was to determine whether blockade of excitatory amino acid receptors at the ventrolateral nucleus of the tractus solitarius would influence respiratory activity. This was done by microinjecting excitatory amino acid receptor antagonists into the ventrolateral nucleus of the tractus solitarius of alpha-chloralose-anesthetized animals while monitoring respiratory activity using a Fleisch pneumotachograph and arterial blood pressure and heart rate. Bilateral microinjection of the NMDA receptor antagonist, 3-[(R)-carboxypiperazin-4-yl]-propyl-1- phosphomic acid (CPP), 5.62 nmol per side, produced an increase in inspiratory duration (+4 +/- 1.6 s, n = 8) which progressed to an apneustic pattern of breathing. Similar results were obtained with CPP microinjected into the ventrolateral nucleus of the tractus solitarius of three vagotomized animals. Bilateral microinjection of a second NMDA receptor antagonist, 2-amino-7-phosphono-heptanoic acid (AP7), 562 nmol per side, produced qualitatively similar effects on respiration as seen with CPP. In contrast, blockade of non-NMDA receptors with 6-cyano-7-nitroquinoxaline-2,3-dione (CNXQ), 0.125 nmol per side, had very little effect on respiration. Activation of NMDA receptors at the ventrolateral nucleus of the tractus solitarius with bilateral microinjection of NMDA, 39 pmol, produced a large increase in expiratory duration (+11 +/- 3 s, n = 8), and apnea during the expiratory phase of the respiratory cycle in half of the animals studied. Similar results were obtained with D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazol-proprionate (AMPA). These results indicate that an endogenous excitatory amino acid released at the ventrolateral nucleus of the tractus solitarius and acting at the NMDA receptor, plays a significant role in respiratory timing.

Medullary parasympathetic projections innervate specific sites in the feline stomach

The purpose of our study was to determine the site of origin of vagal neurons that innervate specific parts of the stomach (the fundus, corpus, and antrum/pylorus). This was done by injecting the retrograde fluorescent tracer Fast Blue into these parts of the cat stomach and examining the hindbrain for cells labeled with retrograde tracer. We found that vagal preganglionic innervation to the stomach originates from two medullary nuclei, namely, the dorsal motor nucleus of the vagus (bilateral) and the nucleus retroambiguus (left). All parts of the stomach receive innervation from the dorsal motor nucleus of the vagus (primarily from the area ranging from 0.5 to 1.8 mm rostral to the obex), but only the fundus and corpus receive innervation from the nucleus retroambiguus. Injection of tracer into the fundus labeled cells within the lateral half of the dorsal motor nucleus of the vagus and injection of tracer into the antrum/pylorus labeled cells within the medial portion. Finally, injection of tracer into the corpus labeled cells throughout the mediolateral axis of the dorsal motor nucleus of the vagus. The finding of a columnar organization of the dorsal motor nucleus of the vagus implies some type of functional organization of gastrointestinal control. The fact that vagal inputs to the stomach arise from the dorsal motor nucleus of the vagus and nucleus retroambiguus suggests a separation of vagal pathways controlling different gastric functions (e.g., pacemaker activity, motility, and secretion).

Use of horseradish peroxidase to identify hindbrain sites that influence gastric motility in the cat

To identify hindbrain sites that influence gastric motility, we administered multiple injections of horseradish peroxidase into the anterior surface of the antrum near the lesser curvature in 3 cats, and used light microscopy to identify horseradish peroxidase-positive neurons in the hindbrain. Retrogradely labeled neurons were found evenly distributed on both sides in the dorsal motor nucleus of the vagus. Labeling extended from 2.5 mm rostral to 2.0 mm caudal to the obex. Labeled neurons were not localized to a specific region of the dorsal motor nucleus of the vagus: no labeling was observed in the nucleus ambiguus or in the nuclei of the solitary tract. Electrical stimulation of the dorsal motor nucleus of the vagus in the area with the greatest number of labeled cell bodies was performed in 4 cats while monitoring antral motility, arterial pressure, and heart rate. Stimulation elicited pronounced antral contractions but no changes in arterial pressure or heart rate. These data demonstrate that the retrograde neuronal tracing technique permits localization of central nervous system sites that specifically influence gastric function.

An ultrastructural study of nerve terminal degeneration in muscle spindles of the tenuissimus muscle of the cat

SummaryMuscle spindles in the tenuissimus muscle of the cat were studied between 12 and 168 h after cutting or freezing the nerve to this muscle. Degenerative changes in sensory and motor nerve terminals on intrafusal muscle fibres were observed using the electron microscope. Comparisons were made with spindles from unoperated or sham-operated cats.The earliest degenerative changes were seen in sensory and motor terminals at 20–24 h after the lesion. No nerve endings were seen by 114 h after denervation. The most consistent initial signs of degeneration were: (1) the presence of abnormal mitochondria and dense bodies in sensory terminals, and (2) a decrease in the number and clumping of synaptic vesicles combined with an increase in glycogen and neurofilaments in motor endings. Intrafusal fibres participate in the removal of degenerating sensory endings. Schwann cells phagocytose degenerating motor terminals. The disappearance of nerve terminals precedes the complete degeneration of preterminal myelinated fibres within the muscle spindle.

Cardiovascular neurons in cat caudal ventrolateral medulla: location and characterization of GABAergic input

The purposes of the present study were to: (1) characterize the GABAergic input to vasodepressor neurons in the caudal ventrolateral medulla of the cat, and (2) define more precisely the anatomical localization of these neurons in this species. This was done by microinjecting GABA receptor antagonists and agonists, and a negative allosteric modulator of the GABA receptor, namely, ethyl-beta-carboline-3-carboxylate, into the caudal ventrolateral medulla of alpha-chloralose-anesthetized animals while monitoring arterial blood pressure and heart rate. Localization studies where performed relating injection sites in the caudal ventrolateral medulla where cardiovascular responses were elicited, to neurons exhibiting immunoreactivity to tyrosine hydroxylase (TH) and phenethyl-N-methyl-transferase (PNMT). Microinjection of 1 and 10 ng of bicuculline into the caudal ventrolateral medulla produced decreases in mean blood pressure and heart rate of -34 +/- 6.4 and -49 +/- 9.2 mmHg, and -22 +/- 4.3 and -35 +/- 8.2 beats/min, respectively. Hypotension and bradycardia were also observed with picrotoxin microinjection (120 ng). Microinjection of muscimol (100-200 ng) and GABA (12 microgram) had no effect on mean blood pressure and heart rate. Microinjection of ethyl-beta-carboline-3-carboxylate also decreased mean blood pressure (-39 +/- 7.0 mmHg). The location of the micropipette tip after bicuculline microinjection in relation to TH and PNMT immunoreactive cells was as follows: (1) TH-immunoreactive cells of the A1 cell group were visible in the same relative location as the micropipette tip, and (2) no PNMT-positive cells were noted at the sites where bicuculline elicited hypotension. These results indicate that there is a tonic GABAergic input to neurons in the caudal ventrolateral medulla. The location of these neurons overlaps with the A1 cells.