Elliott Mills

Hypoxia‐induced catecholamine secretion in isolated newborn rat adrenal chromaffin cells is mimicked by inhibition of mitochondrial respiration

1 In newborn mammals, systemic hypoxia provokes catecholamine secretion from the adrenal medulla. In contrast to adults, this release is independent of sympathetic innervation. We have studied the cellular processes involved in hypoxia‐induced catecholamine secretion, employing fluorimetric techniques to measure changes in [Ca2+]i, NADH and mitochondrial potential, and voltammetric techniques to record changes in PO2 and catecholamine secretion. 2 In adrenal chromaffin cells freshly dissociated from newborn rats, severe hypoxia increased [Ca2+]i and secretion of catecholamines, indicating that the response of the newborn adrenal medulla to hypoxia is an intrinsic property of these cells. Discrete quantal secretory events were identifiable, suggesting an exocytotic mechanism of secretion. 3 Hypoxia‐induced secretion was only seen when PO2fell below 5 mmHg, similar to the threshold arterial PO2 reported to stimulate release in vivo. Such oxygen tensions also inhibited mitochondrial metabolism, shown by an increase in NADH autofluorescence. We therefore explored the involvement of mitochondria in oxygen sensing. Inhibition of mitochondrial respiration either by CN− at complex IV or by rotenone at complex I mimicked severe hypoxia, reversibly increasing both [Ca2+]i and catecholamine secretion. The CN−‐induced depolarization of the mitochondrial inner membrane potential preceded the increase in [Ca2]i by ∼6 s. 4 The effects of severe hypoxia and CN− on [Ca2+]i and catecholamine secretion were not additive, suggesting a common mechanism. 5 Chemical anoxia failed to increase [Ca2+]i in a significant proportion of cells dissociated from 2‐ to 4‐week‐old rats. Thus, the sensitivity to hypoxia is specific to adrenal chromaffin cells dissociated from newborn rats. 6 These data indicate that hypoxia‐induced catecholamine secretion in the newborn adrenal medulla is mediated by reversible inhibition of mitochondrial respiration, leading to an increase in [Ca2+]i and catecholamine secretion.

Development of sympathetic ganglionic neurotransmission in the neonatal rat. Pre- and postganglionic nerve response to asphyxia and 2-deoxyglucose

To determine the time course of development of neurotransmission in the sympathetic ganglion of the rat, pre- and postganglionic activity was recorded from the cervical sympathetic trunk in anesthetized neonatal and mature preparations. Tonic activity and responses to two stimuli, cellular hypoglycemia induced by 2-deoxyglucose and asphyxia, which are known to evoke CNS-mediated sympathetic activation in mature rats were measured. In 2-11-day-old neonates, tonic preganglionic activity recorded from the cervical sympathetic nerve and responses to hypoglycemia and asphyxia were comparable to or greater than that in mature rats. In 17-19-day-old neonates these variables were elevated to twice the adult value. In contrast, tonic postganglionic activity recorded from the internal carotid nerve was barely detectable through 5 days of age and there was no response to hypoglycemia. During asphyxia, maximum postganglionic impulse frequency and total number of impulses discharged were 10-20% of the mature value through the 5th postnatal day and the duration of the postganglionic response was only 25% of the preganglionic response. Tonic postganglionic activity and response to stimuli were equivalent to those in mature rats by the 10th postnatal day. The compound action potential evoked in the postganglionic axons by direct electrical stimulation was comparable in 4-5 and 10-13 day-old rats. In the concluded that functional ganglionic neurotransmission is established in the neonatal rat between the 5th and 10th postnatal day. The relation between biochemical changes associated with maturation of the postganglionic neuron, ganglionic synaptogenisis and neurotransmission is discussed. It is concluded that synaptogenisis and onset of neurotransmission are causally associated with development of CNS regulation of postganglionic activity and end organ response rather than with maturation of the postganglionic neuron and that cholinergic excitation of the postganglionic neuron adequate to evoke action potentials is not essential to initiate maturation of the neuron.

Restoration of reflex ventilatory response to hypoxia after removal of carotid bodies in the cat

In mammals there are two sets of peripheral arterial chemoreceptors, the carotid bodies innervated by the sinus branch of the glossopharyngeal nerve and the aortic bodies innervated by the vagus nerves. The afferent impulse discharge from both receptors increases during hypoxia and there is a reflexly mediated increase in ventilation (hypoxic hyperventilation). In the present study we tested this response by exposing anesthetized cats to decreased inspired O2 concentration before and up to 315 days after bilateral resection of the carotid bodies. Acutely after removing the carotid bodies, hypoxic hyperventilation was abolished. This observation supports the view that the reflex pathway from the aortic body receptors normally contributes minimally to hypoxic hyperventilation. Subsequently, there was a restoration of hypoxic hyperventilation. Restoration was significant 30–43 days after removing the carotid bodies, it reached 70% of the preoperative value at 93–111 days and was essentially complete in terminal experiments 260–315 days after carotid body resection. In terminal experiments, hypoxic hyperventilation was not affected by recutting the regenerated carotid sinus nerves but was abolished completely by bilateral transection of the cervical vagosympathetic trunks. The restored ventilatory response was due predominantly to an increase in rate of breathing while an increase in tidal volume was predominant before carotid body resection. Resting ventilation breathing room air was not consistently decreased after carotid body resection while expired CO2 was elevated from day 20 to day 111 and at the preoperative level in terminal experiments. n nIt is concluded that restoration of hypoxic hyperventilation in the cat after carotid body resection is mediated by the reflex pathway from aortic body chemoreceptors. The possible contribution of chemo-receptive regenerated carotid sinus nerve axons was excluded. It is suggested that restoration may be a consequence of the central reorganization of chemoreceptor afferent pathways consequent to interruption of the carotid body reflex pathway and that as a result the ‘gain’ of the aortic body ventilatory chemoreflex is enhanced.

Ontogeny of neural and non-neural contributions to arterial blood pressure in spontaneously hypertensive rats.

Arterial blood pressure was measured directly by cannulation in anesthetized spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats on postnatal Days 1, 5, 9, 20, 42, and 82-86. The time course for development of the following variables was established: resting diastolic and basal (after ganglionic blockade) pressure, the neural contribution to resting pressure (resting minus basal pressure), vascular reactivity to a noradrenergic agonist, methoxamine, and to endogenous sympathetic nerve terminal norepinephrine released by tyramine (maximum pressor response and ED50) and resting and basal heart rate. Resting diastolic pressure was higher in SHR compared to WKY by 24 hours after birth. In both strains, the increase in resting diastolic pressure with age was interrupted by a plateau period (Days 5-9 in SHR; Days 9-20 in WKY). Juxtaposition of the development curves was such that the interstrain differences in pressure were statistically significant in all periods studied except Days 5 and 9. Both basal and neurally mediated components of resting diastolic blood pressure were elevated in SHR compared to WKY. The magnitude of the interstrain difference in basal pressure remained constant during development while the magnitude of the neurally mediated component showed accelerated development through 42 days of age. Reactivity to methoxamine and tyramine was higher in SHR, but the magnitude of the difference did not change with age. Cardiac sympathetic tone was higher in SHR than WKY, but did not account for the increased resting diastolic pressure in SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Catecholamine content of the carotid body in cats ventilated with 8 - 40% oxygen.

Abstract The content of catecholamines in the carotid body varies directly with inspired oxygen concentration in the range between 8 and 40% oxygen. The decrease in content during hypoxia is mediated mainly through an efferent neural pathway in the carotid sinus nerve.

Degenerative and regenerative changes in central projections of glossopharyngeal and vagal sensory neurons after peripheral axotomy in cats: a structural basis for central reorganization of arterial chemoreflex pathways.

Hypoxic hyperventilation in cats is a reflex normally initiated by afferent impulses originating in the carotid body and conducted to the brain stem by the carotid sinus nerves. The reflex response is abolished acutely after section of carotid sinus nerves and excision of the carotid bodies; but, chronically, there is a chemoreflex restoration which is mediated by the aortic body via the aortic depressor nerves. The restoration is associated temporally with changes in efficacy of ventilatory reflexes elicited by electrically stimulating carotid sinus and aortic depressor nerves, and these changes are postulated to reflect a central reorganization of the reflex pathways. In the present study, histological and ultrastructural techniques were used to investigate the neuroanatomical basis of the reorganization. The brain stem of the cat was examined using the Fink-Heimer silver stain to determine if degenerating axons were present following section of the carotid sinus nerve peripheral to its sensory ganglion. Degeneration was found 4-15 days postoperatively and the distribution of the axons corresponded with that reported for central projections of carotid sinus nerves labeled by transganglionic transport of horseradish peroxidase. The fine structure of nerve terminals in nucleus tractus solitarius was then examined with electron microscopy after cutting the vagus and glossopharyngeal nerves unilaterally peripheral to the sensory ganglia. Structural changes consistent with nerve terminal degeneration were observed 4-91 days postoperatively, and presumptive axonal sprouts were seen at 56-91 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Time course for development of vagal inhibition of the heart in neonatal rats

Abstract Heart rate was recorded in anesthetized rats 1, 3 and 7 days after birth, and the cut peripheral end of the right vagus nerve stimulated electrically at frequencies of 5, 10, 20 and 40 Hz. Control heart rate increased from 344 ± 8 beats/min on eay one to 490 ± 10 beats/min on day seven. Stimulation 6–24 hours after birth slowed the heart rate. At 3 days, slowing was more marked at all frequencies and greatly enhanced at 20 and 40 Hz. It is concluded that efferent cardiac vagal fibers conduct action potentials and that ganglionic and post-ganglionic neurotransmission is established in rats within 6 hours after birth.

Role of carotid body catecholamines in chemoreceptor function

Publisher Summary This chapter presents the results of measurements of the noradrenaline and the dopamine content of the cat carotid body. The strongest support for a dopaminergic mechanism inhibiting chemoreceptor discharge in the carotid body comes from pharmacological studies in the cat. These studies still leave open the possibility that there is also an α-adrenergic mechanism that inhibits chemoreceptor discharge. Systemic hypoxia, a stimulus that is considered to activate the inhibitory mechanism as well as chemoreceptor afferents, depletes carotid body noradrenaline in the cat, a result compatible with an α-adrenergic mechanism. The effect of this stimulus on dopamine in the cat carotid body has not been determined. Indirect evidence suggests that hypoxia also activates catecholamine biosynthesis in the cat carotid body.

Maturation of sympathetic neurotransmission in the efferent pathway to the rat heart: Ultrastructural analysis of ganglionic synaptogenesis in euthyroid and hyperthyroid neonates

Abstract Synapses and ganglion cell nuclear profiles were counted in the ganglion which gives rise to the postganglionic sympathetic innervation of the neonatal rat heart. Synapse counts were low in 2-day-old rats in which the heart is not responsive to central stimulation of the sympathetic nervous system. At 8 days of age, when pre- and postganglionic elements of the efferent sympathetic system are functional, the number of synapses per unit area is increased five-fold, while the packing density of ganglion cells is decreased, as reflected in fewer nuclear profiles counted per unit area. Administration of tri-iodothyronine to 1-day-old rats is known to render the cardiac sympathetic nervous system functional within 24 h. In ganglia from 2-day-old tri-iodothyronine-treated rats, the relative number of synapses per unit area was comparable to that of 8-day-old controls, while the packing density of ganglion cell nuclear profiles was not affected as compared with 2-day-old controls. Cytochemical reactions for catecholamines were not qualitatively different in the three groups. This study demonstrates that ganglionic synaptogenesis is temporally associated with functional maturation of the sympathetic control of the neonatal rat heart, and that the effect of tri-iodothyronine in producing precocious functional maturation of the cardiac sympathetic nerves is selective for synaptogenesis as opposed to a generalized cytological effect.

Reinnervation of Müller's smooth muscle by atypical sympathetic pathways following neonatal ganglionectomy in the rat: Structural and functional investigations of enhanced neuroplasticity

Müllers extraocular smooth muscle is reinnervated by sympathetic nerves following denervation by ipsilateral superior cervical ganglionectomy in neonates but not in older animals. Experiments were performed to determine: (1) the source and extent of reinnervation, (2) the role of impulse activity in sympathetic outgrowth and (3) the effects of reinnervation on smooth muscle maturation. Müllers muscles were evaluated structurally (muscle volume, catecholamine histochemistry, retrograde labeling of sympathetic neurons) and functionally (contractile responses to electrical stimulation of postganglionic innervation and adrenoceptor agonist) in control preparations and in muscles following neonatal ipsilateral superior ganglionectomy, ipsilateral decentralization, ipsilateral superior ganglionectomy combined with contralateral decentralization of chemical (guanethidine) sympathectomy. Fluorescent tracer injections of muscles in adult control rats labeled cells in the ipsilateral superior (98%) and middle cervical ganglia. Acute ipsilateral superior ganglionectomy produced complete degeneration of sympathetic innervation of Müllers muscle in neonatal and adult rats. In preparations denervated neonatally and maintained chronically, muscles were reinnervated by neurons in both the contralateral superior and ipsilateral middle cervical ganglia. The total number of neurons reinnervating the muscle was one half that of controls. Sectional density of innervation was 45% of control. Electrical stimulation of postganglionic axons in the contralateral pathway produced muscle contractions with a prolonged time course. Reinnervation alleviated, in part, deficits in muscle volume and contraction which occurred following sustained denervation by chemical sympathectomy. Decentralization decreased ipsilateral muscle volume but did not affect numbers of neurons projecting to or nerve density within the muscle. Stimulation frequencies required to produce a 50% maximum contraction were reduced in these preparations. Decentralization of the contralateral ganglion did not impede sprouting into the denervated muscle, as nerve density and number of labeled cells were comparable to muscles reinnervated by contralateral ganglia with intact preganglionic innervation. However, maximum contraction to electrical stimulation was reduced. Comparisons with ipsilaterally decentralized muscles revealed that increased stimulation frequencies were required for 50% maximum contraction.(ABSTRACT TRUNCATED AT 400 WORDS)