David E. Millhorn

Stimulation by central command of locomotion, respiration and circulation during exercise

We studied the relationships between exercise (locomotion) and respiratory and circulatory responses in 19 cats that walked or ran normally on a treadmill, and in 16 paralyzed animals during fictive locomotion, i.e., locomotory activity in motor nerves to the legs. Preparations included anesthetized cats with intact brains and unanesthetized decorticate (hypothalamic) and decerebrate (mesencephalic) animals. Spontaneous actual locomotion and fictive locomotion occurred in all preparations except the mesencephalic cats. Electrical stimulation or injection of a GABA antagonist (picrotoxin) into the hypothalamic locomotor region caused locomotion to develop. In all cases when locomotion occurred, respiration and arterial pressure increased in proportion to the level of locomotor activity despite control or ablation of respiratory feedback mechanisms. Respiration and arterial pressure increased similarly during fictive locomotion despite the absence of muscular contraction or limb movement and the lack of change of metabolic rate. We conclude that the study provides experimental support for the feed-forward, or command signal, hypothesis for the genesis of proportional changes of respiration and circulation that occur during exercise. Feedback mechanisms are not required for its operation. We suggest that command signals emanating from the hypothalamus provide the primary drive for changes of respiration and circulation during exercise.

Prolonged stimulation of respiration by a new central neural mechanism

The inspiratory responses to stimulation by peripheral chemoreceptor, central chemoreceptor and calf muscle afferents were studied in anesthetized, or decerebrate, paralyzed cats whose end-tidal PCO2 was servo-controlled and kept constant. All stimuli were associated with immediate increases of inspiratory (phrenic) activity and at offset were followed by a respiratory afterdischarge lasting approximately five minutes. The level of inspiratory activity following decay of the afterdischarge was the same as the prestimulation control after central chemoreceptor and calf muscle stimulation. However, after peripheral chemoreceptor afferent stimulation the stable level of inspiratory activity following the afterdischarge had increased over the prestimulation level and remained elevated for as long as it was followed, up to 90 min. Decerebration, vagotomy, and section of the spinal cord at C--T1 did not prevent this long-lasting increase in respiration. We conclude that we have demonstrated a new ponto-medullary neural mechanism which is uniquely activated by peripheral chemoreceptor afferent input; once activated, this mechanism sustains respiration at an increased level for a long period of time.

Prolonged stimulation for respiration by endogenous central serotonin

We have recently reported a new neural brainstem mechanism which is uniquely activated by stimulation of carotid body afferent input to the brain and which facilitates respiration for hours after the immediate affects of the stimulation have dissipated (Millhorn, Eldridge and Waldrop, 1980). In the present study respiratory responses to carotid body or carotid sinus nerve stimulation were measured in vagotomized, anesthetized, and paralyzed cats whose end-tidal PCO2 and temperature were servo-controlled and kept constant. The responses of animals pretreated with various serotonin antagonists and a dopamine-norepinephrine antagonist were compared to the responses of untreated control animals. All three differently acting serotonin antagonists (methysergide, parachlorophenylalanine, and 5, 7-dihydroxytryptamine) either prevented or significantly reduced the magnitude of the long-lasting respiratory response whereas the dopamine-norepinephrine antagonist (alpha-methyltyrosine) failed to alter it. We conclude that the long-lasting increase of respiratory activity following stimulation of carotid body afferents is due to activation of an endogenous central serotoninergic mechanism which facilitates respiration.

Regulation of Tyrosine Hydroxylase Gene Expression in the Rat Carotid Body by Hypoxia

Abstract: The activity (Vmax) of tyrosine hydroxylase (TH; EC 1.14.16.2), the rate limiting enzyme in the synthesis of catecholamines, is increased in carotid body, superior cervical ganglion, and the adrenal medulla during hypoxia (i.e., reduced Pao2). The present study was undertaken to determine if the increase in TH activity in these tissues during hypoxia is regulated at the level of TH mRNA. Adult rats were exposed to hypoxia (10% O2) or room air for periods lasting from 1 to 48 h. The carotid bodies, superior cervical ganglia, and adrenals were removed and processed for in situ hybridization using 35S‐labeled oligonucleotide probes. The concentration of TH mRNA was increased by hypoxia at all time points in carotid body type I cells, but not in cells of either superior cervical ganglion or adrenal medulla. The increase in TH mRNA in carotid body during hypoxia did not require innervation of the carotid body or intact adrenal glands. In addition, hypercapnia, another physiological stimulus of carotid body activity, failed to induce an increase in TH mRNA in type I cells. Our findings suggest that hypoxia stimulates TH gene expression in the carotid body by a mechanism that is intrinsic to type I cells.

Depolarization and stimulation of neurons in nucleus tractus solitarii by carbon dioxide does not require chemical synaptic input.

The effects of elevated CO2 (i.e. hypercapnia) on neurons in the nucleus tractus solitarii were studied using extracellular (n = 82) and intracellular (n = 33) recording techniques in transverse brain slices prepared from rat. Synaptic connections from putative chemosensitive neurons in the ventrolateral medulla were removed by bisecting each transverse slice and discarding the ventral half. In addition, the response to hypercapnia in 20 neurons was studied during high magnesium-low calcium synaptic blockade. Sixty-five per cent of the neurons (n = 75) tested were either insensitive or inhibited by hypercapnia. However, 35% (n = 40) were depolarized and/or increased their firing rate during hypercapnia. Nine out of 10 CO2-excited neurons retained their chemosensitivity to CO2 in the presence of high magnesium-low calcium synaptic blockade medium. Our findings demonstrate that many neurons in the nucleus tractus solitarii were depolarized and/or increased their firing rate during hypercapnia. These neurons were not driven synaptically by putative chemosensitive neurons of the ventrolateral medulla since this region was removed from the slice. Furthermore, because chemosensitivity persisted in most neurons tested during synaptic blockade, we conclude that some neurons in the nucleus tractus solitarii are inherently CO2-chemosensitive. Although the function of dorsal medullary chemosensitive neurons cannot be determined in vitro, their location and their inherent chemosensitivity suggest a role in cardiorespiratory central chemoreception.

Fos-like protein is induced in neurons of the medulla oblongata after stimulation of the carotid sinus nerve in awake and anesthetized rats

The protooncogene c-fos is expressed rapidly, transiently and polysynaptically within neurons in response to synaptic activation and voltage-gated calcium entry into the cell. The nuclear protein product of this gene (Fos) is detectable immunohistochemically 20-90 min after cell activation and remains within the nucleus for hours after expression. The present study was undertaken to identify cells within the rat medulla oblongata that express Fos-like protein in response to stimulation of afferent fibers of the carotid sinus nerve (CSN). Direct electrical stimulation of the CSN in anesthetized animals or hypoxic stimulation in either anesthetized or awake animals resulted in a consistent and discrete distribution of Fos-like immunoreactivity (Fos-LI). Fos-LI was observed bilaterally within nucleus tractus solitarius (NTS) and the ventrolateral medulla (VLM), within area postrema and nucleus raphe pallidus, and bilaterally along the ventral medullary surface. Unstimulated animals were devoid of Fos-LI within the medulla oblongata. Furthermore, neither the surgical preparations alone nor the effects of anesthesia could account for the extent of Fos-LI observed. We believe these cells represent second- and higher-order neurons within the baroreceptor and chemoreceptor reflex pathways.

CO2 decreases membrane conductance and depolarizes neurons in the nucleus tractus solitarii

SummaryTo identify central sites of potential CO2/H+-chemoreceptive neurons, and the mechanism responsible for neuronal chemosensitivity, intracellular recordings were made in rat tissue slices in two cardiopulmonary-related regions (i.e., nucleus tractus solitarii, NTS; nucleus ambiguus, AMBc) during exposure to high CO2. When the NTS was explored slices were bisected and the ventral half discarded. Utilizing such “dorsal” medullary slices removed any impinging synaptic input from putative chemoreceptors in the ventrolateral medulla. In the NTS, CO2-induced changes in firing rate were associated with membrane depolarizations ranging from 2–25 mV (n = 15). In some cases increased e.p.s.p. activity was observed during CO2 exposure. The CO2-induced depolarization occurred concomitantly with an increased input resistance ranging from 19–23 MΩ (n = 5). The lower membrane conductance during hypercapnia suggests that CO2-induced depolarization is due to a decreased outward potassium conductance. Unlike neurons in the NTS, AMBc neurons were not spontaneously active and were rarely depolarized by hypercapnia. Eleven of 12 cells tested were either hyperpolarized by or insensitive to CO2. Only 1 neuron in the AMBc was depolarized and it also showed an increased input resistance during CO2 exposure. Our findings suggest that CO2/H+-related stimuli decrease potassium conductance which depolarizes the cell and increases firing rate. Although our in vitro studies cannot guarantee the specific function of these cells, we believe they may be involved with brain pH homeostasis and cardiopulmonary regulation.

Input‐output relationships of central neural circuits involved in respiration in cats

1. Inspiratory output responses, measured as integrated phrenic activity, to hypercapnia, to unilateral and bilateral carotid sinus nerve stimulation and to combinations of these stimuli were determined in paralysed, vagotomized and glomectomized cats whose end‐tidal PCO2 was kept constant by means of a servo‐controlled ventilator. In addition, the effect on these responses of the mechanism that causes the respiratory after‐discharge was determined.

Extent of colocalization of serotonin and GABA in neurons of the ventral medulla oblongata in rat

The colocalization of serotonin (5-hydroxytryptamine; 5-HT) and gamma-aminobutyric acid (GABA) in the ventral aspect of the rat medulla oblongata was studied using antibodies directed against 5-HT and GABA. Although 5-HT- and GABA-immunoreactive cell bodies were observed over the entire rostral-caudal extent of the ventral medulla, the colocalization of these two classical neurotransmitters in single cells was, for the most part, limited to a region that corresponds anatomically to nucleus raphe magnus/nucleus paragigantocellularis. Schematic drawings showing the distribution of 5-HT/GABA cell bodies in the ventral medulla are provided.

Mechanism of respiratory effects of methylxanthines

Neural respiratory responses to theophylline, aminophylline and ethylenediamine were determined in paralyzed, vagotomized and glomectomized cats whose end-tidal PCO2 and brain temperature were kept constant. Intravenous theophylline and aminophylline similarly stimulated respiration, but ethylenediamine had no effect. The following did not cause the response: muscular and mechanical factors, carotid body and vagal reflexes, spinally mediated mechanisms arising below C7, changes of arterial PCO2 or medullary ECF pH, changes of whole body metabolic rate or release of substances from the adrenal glands. Absence of suprapontine brain did not prevent the response. Pretreatment with a serotonin antagonist did not affect the response but two different dopamine antagonists caused its attenuation. When administered into the third ventricle, theophylline did not stimulate respiration, but both aminophylline and ethylenediamine, due to the latters ability to mimic the inhibitory effects on neurons of gamma-aminobutyric acid (GABA), caused significant depression of respiration. We conclude that the neural respiratory response to systemically administered theophylline is mediated at the level of the brainstem, and somehow involves the action of the neurochemical dopamine. The failure of cerebroventricularly administered theophylline to stimulate respiration must be related to its inability to reach the appropriate neurons from the cerebrospinal fluid.