Peter G. Smith

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.

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)

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.

Physiological and ultrastructural observations on regenerated carotid sinus nerves after removal of the carotid bodies in cats

Abstract Carotid bodies were excised bilaterally in cats and the cut carotid sinus nerves allowed to regenerate freely into the region of the carotid bifurcation for 31 to 315 days. Electrophysiological recordings from regenerated nerves showed spontaneous activity and mechanoreceptive units in some nerves. None of the nerves were excited by physiological or chemical stimuli known to excite chemoreceptors, but electrical stimulation of the regenerated axons evoked reflex ventilatory responses. Serial section light-microscopic analysis of the regenerated nerves revealed bundles of axons branching from the carotid sinus nerve near the site of transection and coursing into the area of the carotid bifurcation. Electron-microscopic examination of the most distal region of the regenerated nerve which could be identified showed axonal bundles comprised of myelinated and unmyelinated fibers characteristic of the mature sinus nerve; axonal sprouts were rarely encountered. Carotid body type 1 or type II cells were never observed. Infrequently, neuronal specializations containing mitochondria and synaptic vesicles were seen in connective tissue in the distal regions of the regenerated nerve. The vascularity of the regenerated nerve was at least comparable to that of the normal carotid sinus nerve. The results indicate that chemoreceptivity is not a property of carotid sinus nerve sensory axons when they are allowed to regenerate freely into the area of the carotid bifurcation after removing the carotid body. Consequently, these observations do not provide support for theories which consider chemoreception to be an intrinsic property of carotid sinus neurons. It is concluded that some extrinsic factor normally provided by the carotid body is essential to the transduction process which initiates depolarization of the sensory axons terminating within this organ.

Functional plasticity in the sympathetic nervous system of the neonatal rat

The age-dependent effects of sympathetic superior cervical ganglionectomy on smooth muscle alpha-adrenoceptor contractile responses were investigated in Müllers muscle in the superior eyelid of the Sprague-Dawley rat. Ganglionectomies in juvenile (32 days) and adult (70 days) rats produced sustained sympathetic denervation, determined by absence of contractile responses to electrical stimulation of ipsilateral and contralateral sympathetic chains and reduced responses to indirect release of nerve terminal norepinephrine by tyramine. Denervation was associated with diminished contractile responses and postjunctional supersensitivity to alpha-adrenoceptor stimulation by methoxamine. In contrast, contractile responses in rats ganglionectomized as neonates (day 3) were greater than those of other groups and sensitivity to methoxamine was comparable to control values. Responses to tyramine suggested that noradrenergic reinnervation had occurred. Electrical stimulation of the ipsilateral chain did not elicit contractions; however, stimulation of the contralateral chain evoked contractions in all neonatally ganglionectomized preparations and responses were present by 20 days of age. Contralateral reinnervation was derived from postganglionic sympathetic neurons with axons in the internal carotid nerve. This pathway is probably not formed by collateral sprouting of neurons with bilateral projections at the time of surgery for contralateral stimulation did not evoke contractions in the neonate. It is concluded that Müllers muscle is reinnervated by sympathetic neurons in the contralateral chain after denervation in neonates but not in older animals, and it is suggested that the higher potential for plasticity is attributable to the immaturity of the neonatal sympathetic nervous system.

Relation between functional maturation of cervical sympathetic innervation and ontogeny of α-noradrenergic smooth muscle contraction in the rat

Abstract The functional ontogeny of a sympathetic neuroeffector system consisting of postganglionic neuron and smooth muscle end organ was studied in vivo in the rat at selected times from birth through 75 days of age. The maximum noradrenergic contractile response of the smooth muscle (Mu¨llers) component of the levator palpebrae was assessed with the directly acting agonist, methoxamine. The functional capacity of the nerve terminals innervating the muscle to evoke contraction was determined by displacing their endogenous noradrenaline with tyramine. The integrity of the pathway from preganglionic axon to end organ was tested by stimulating the preganglionic axons electrically. The smooth muscle contractile response to methoxamine increased in a step-wise fashion through the 20th postnatal day. On day 1, the response was 2% of the mature (70–75 day) value. The response increased to 17% on day 4, and remained at this level through the 9th day. A second increase to 32% occurred between the 10th and 12th day, and there was no further increase through the 19th day. Responses increased to 64% of the mature value by the 45–50th postnatal day. Responses to tyramine were always comparable to those evoked by methoxamine. Electrical stimulation of the preganglionic nerve failed to evoke contractions on days 1 and 4, but from day 8 on, responses to preganglionic stimulation, tyramine and methoxamine were comparable. Decentralization of the superior cervical ganglion on the 1st postnatal day decreased the response to endogenously-released nerve terminal noradrenaline by 25% at 18–19 days postnatally and the deficit persisted to maturity. In contrast, the response to direct noradrenergic stimulation was attenuated at 70–75 days postnatally, but not at 18–19 and 45–50 days. Sham operation itself significantly decreased responses to methoxamine and tyramine at 18–19 and 45–50 days of age, but not at 70–75 days. The following conclusions were reached. Functional capacity of the postganglionic sympathetic nerve terminals develops at least as rapidly as the capacity of the smooth muscle to respond directly to α-noradrenergic stimulation. The connection between central nervous system and postganglionic sympathetic neuron is not required to initiate functional maturation of the neuron, postsynaptic α-noradrenergic receptors or post-receptor smooth muscle contractile elements. After ganglionic neurotransmission is established, there is a significant, though modest, transynaptically mediated contribution to overall functional maturation of postganglionic neuron and end organ. The maturational deficit (about 25%) which follows decentralization of the postganglionic neuron on the 1st postnatal day is manifest first in the sympathetic nerve terminal; this, in turn, appears to lead to a deficit in smooth muscle response to direct α-noradrenergic stimulation. Thus, decentralization in the neonate prior to establishment of ganglionic neurotransmission leads to subsensitivity of α-noradrenergic end organ response while decentralization in mature animals is known to result in supersensitivity. Since the contribution of neural, transynaptic factors to maturation was modest in this sympathetic neuroeffector system, consideration is given to the possibility that humoral factors such as thyroid and growth hormone play major roles in development of the postganglionic neuron and end organ.

Evidence for central reorganization of ventilatory chemoreflex pathways in the cat during regeneration of visceral afferents in the carotid sinus nerve

Abstract There are two sets of peripheral arterial chemoreceptors in the cat, the carotid bodies innervated by the carotid sinus nerve and the aortic bodies with afferents in the aortic depressor nerves. Reflex stimulation of ventilation in response to hypoxia is abolished acutely after interrupting the sensory pathway from the carotid body chemoreceptors in the cat even though the reflex pathway from the aortic body chemoreceptors is intact. However, in chronically maintained preparations, there is a restoration of the hypoxic response which is mediated by the aortic chemoreflex pathway. It was proposed that restoration was due to a ‘central reorganization’ of chemoreflex pathways which followed interruption of the sensory pathway from the carotid bodies and that the reorganization enhanced the efficacy of the aortic ventilation chemoreflex. This proposal was tested in the present experiments by measuring reflex ventilatory and cardiovascular responses to electrical stimulation of the sensory nerves containing aortic and carotid chemoreceptor afferents following bilateral interruption of carotid sinus nerves and carotid body resection. Responses measured acutely (1–6 h) after interruption were compared with those measured 60–80 and 110–140 days later. At 60–80 days, a chemoreflex response (increase in tidal volume of ventilation) to stimulation of the interrupted carotid sinus sensory pathway was markedly attenuated while the response to stimulation of the uninterrupted pathway in aortic depressor nerves was enhanced. At 110–140 days, the tidal volume response to carotid sinus nerve stimulation was greatly enhanced while the aortic depressor nerve response declined from the elevated level. There were significant but less pronounced changes in the response of other ventilatory and cardiovascular variables to aortic depressor nerve and carotid sinus nerve stimulation. The results support the idea that there is a ‘central reorganization’ of chemoreflex pathways which is reflected functionally by changes in the efficacy of reflexes evoked from aortic depressor nerve and carotid sinus nerve. The changes are analagous to those occurring in somatic reflexes during regeneration of sensory nerves. It is suggested that the changes in efficacy of carotid sinus nerve reflexes are due to a degenerative loss of synapses of the central projections of interrupted carotid sinus nerve sensory axons (degenerative atrophy) and subsequent regenerative like changes (regenerative proliferation) in the central projections. The changes in the efficacy of aortic depressor nerve reflexes may be attributed to formation of new synapses by converging central projections of this uninterrupted pathway (reactive synaptogenesis) and subsequent regression of the newly formed synapses.

Toxic effects of hypoxia on neonatal cardiac function in the rat: α-adrenergic mechanisms

Abstract Stimulation of peripheral α-adrenergic receptors by circulating catecholamines derived from the adrenal medulla is essential for surviving neonatal hypoxia. In 1-day-old rats, where sympathetic innervation of cardiovascular sites has not yet developed, blockade of these receptors results in failure of cardiac performance from a progressive decline in sinus rate and atrioventricular conduction deficits. These effects are absent in 8-day-old rats, where sympathetic efferent innervation has become established.