Hormonal regulation of sympathetic neuron development. The effects of neonatal castration

The effects of neonatal castration on neuronal ontogeny were examined in peripheral sympathetic ganglia in male Sprague-Dawley rats. Tyrosine hydroxylase (T-OH) activity, the rate-limiting enzyme in catecholamine biosynthesis and a marker of noradrenergic maturation, was examined in the hypogastric (HG) and superior cervical ganglion (SCG). Initial studies characterized the normal development of T-OH activity in HG ganglia. Neonatal castration at 10-11 days of age prevented the normal ontogeny of HG T-OH activity: T-OH activity failed to develop normally and was 17% of sham-operated littermate controls when examined at 8 weeks of age, and less than 5% when studied 10 weeks after surgery. In contrast to the effects in HG, there was no change in enzyme activity in the SCG. Replacement therapy with testosterone decanoate completely reversed the developmental alteration in enzyme activity. These observations suggest that hormonal factors modulate noradrenergic ontogeny in peripheral sympathetic ganglia but these effects appear restricted to ganglia whose targets include hormonally dependent sex organs.     

Trans-synaptic regulation of the development of end organ innervation by sympathetic neurons.

To examine the regulation of development of end organ innervation the superior cervical ganglion (SCG), and two of its target organs, the iris and pineal gland, were studied using biochemical and histofluorescent approaches. During postnatal ontogeny the activity of tyrosine hydroxylase (T-OH), which is localized to adrenergic neurons, increased 50-fold in iris, and 34-fold in pineal nerve terminals of the rat. These increases paralleled the in vitro rise in iris [3H]norepinephrine ([3H]NE) uptake, a measure of the presence of functional nerve terminal membrane. These biochemical indices of end organ innervation correlated well with developmental increases in density of innervation, adrenergic ground plexus ramification and nerve fiber fluorescence intensity as determined by fluorescence microscopy. Unilateral transection of the presynaptic cholinergic nerves innervating the SCG in 2-3-day-old rats prevented the normal development of end organ innervation: T-OH activity, [3H]NE uptake, innervation density, plexus ramification and fluorescence intensity failed to develop normally in irides innervated by decentralized ganglia. It is concluded that trans-synaptic factors regulate the maturation of adrenergic nerve terminals, and the development of end organ innervation by SCG.     

Neurotrophins and target interactions in the development and regulation of sympathetic neuron electrical and synaptic properties

The electrical and synaptic properties of neurons are essential for determining the function of the nervous system. Thus, understanding the mechanisms that control the appropriate developmental acquisition and maintenance of these properties is a critical problem in neuroscience. A great deal of our understanding of these developmental mechanisms comes from studies of soluble growth factor signaling between cells in the peripheral nervous system. The sympathetic nervous system has provided a model for studying the role of these factors both in early development and in the establishment of mature properties. In particular, neurotrophins produced by the targets of sympathetic innervation regulate the synaptic and electrophysiological properties of postnatal sympathetic neurons. In this review we examine the role of neurotrophin signaling in the regulation of synaptic strength, neurotransmitter phenotype, voltage-gated currents and repetitive firing properties of sympathetic neurons. Together, these properties determine the level of sympathetic drive to target organs such as the heart. Changes in this sympathetic drive, which may be linked to dysfunctions in neurotrophin signaling, are associated with devastating diseases such as high blood pressure, arrhythmias and heart attack. Neurotrophins appear to play similar roles in modulating the synaptic and electrical properties of other peripheral and central neuronal systems, suggesting that information provided from studies in the sympathetic nervous system will be widely applicable for understanding the neurotrophic regulation of neuronal function in other systems.     

Target regulation of sympathetic sprouting in the rat hippocampal formation

Sympathetic fibers appear in the rat hippocampal formation after lesions of the medial septal nucleus. The distribution of sympathetic axons correlates most closely with the distribution of dentate granule cells and their axons. To test the hypothesis that granule cells are the targets of sympathetic fibers, neurotoxins were injected to destroy either the granule cells (colchicine) or the pyramidal cells (kainic acid) in the hippocampal formation 1 month prior to placing septal lesions. Sympathetic fibers appeared in animals with kainic acid lesions but not in regions lacking granule cells in animals treated with colchicine. These results support the hypothesis that the granule cells of the dentate gyrus are the targets of sprouting sympathetic fibers.     

Effects of nerve growtfactor on sympathetic neuron development in normal and limbless chick embryos

Wing bud removal in chick embryos has been shown to affect the generation of sympathetic neurons prior to the normal period of limb innervation [Saltis and Rush (1995) J. auton. nerv. Sys., 51, 117–127.]. Pyknotic activity occurred earlier within the peripherally deprived ganglion, suggesting that a precocious cell death of dividing sympathoblasts led to the reduced neuronal population. We have now sought to test whether the effect of limb bud extirpation can be overcome by the administration of nerve growth factor (NGF). Specifically, the peripherally deprived ganglion has been examined for mitotic activity and total neuronal numbers. In brachial ganglia from the operated side, neuron numbers decreased by 67% by embryonic day (E) 13, but by only 28% when NGF was administered from E9. Ganglia on the unoperated side were unaffected by the NGF treatment. In contrast, in embryos receiving NGF from E5 to E9, neuron numbers in the ganglia increased by more than 100%, on both the intact and operated side. This increase was accompanied by a greater proportion of ³H-thymidine-labelled neurons. We therefore conclude that NGF, in addition to its previously described role of preventing naturally occurring neuron death, can also affect the generation of sympathetic neurons. This ability of NGF to affect gangliogenesis is most likely achieved by increasing the survival of dividing neuroblasts, although a direct effect on mitosis has not been excluded.     

Target organ destruction enhances recovery of choline acetyltransferase activity in adult rat sympathetic ganglia after denervation

We studied the effect of destruction of the adrenergic neuronal population on the recovery of preganglionic choline acetyltransferase activity in adult rat sympathetic ganglia. To produce a partial destruction of the adrenergic system, rats were injected with guanethidine for 4 weeks; the preganglionic nerve to the superior cervical ganglion was then crushed and the guanethidine injections were continued for an additional 3 days to 6 weeks. To determine that the drug was effective, tyrosine hydroxylase activity was assessed; enzymic activity was reduced by 76% or more after guanethidine administration. In addition, electron microscopy studies showed that the number of principal cell-synaptic contacts and vesicle-containing varicosities were decreased by 90% after guanethidine administration. Those measures indicated the drug effectively destroyed the postsynaptic adrenergic neurons. In contrast, crushing the preganglionic nerve in animals not treated with guanethidine did not change tyrosine hydroxylase activity, suggesting minimal nonspecific damage to the ganglion as a result of the lesion. Choline acetyltransferase activity was measured as an index of presynaptic cholinergic integrity. After crush of the preganglionic nerve, there was a gradual recovery of ganglionic choline acetyltransferase activity in the saline-injected rats from 5% of control 3 days after the crush to 49% of control after 6 weeks. On the other hand, in the ganglia of rats administered guanethidine, there was a much enhanced recovery of choline acetyltransferase activity after the nerve crush compared with saline-injected animals; in the guanethidine-injected rats, the ganglionic choline acetyltransferase activity 3 days and 6 weeks after the nerve crush was 15 and 96%, respectively, compared with the uncrushed side. These results demonstrate after destruction of the adrenergic target tissue, recovery of presynaptic choline acetyltransferase activity in the adult rat sympathetic ganglion can still occur after denervation; however, the mechanism(s) that controls the regeneration is altered, so that enzymic activity is enhanced.     

Target regulation of neurotransmitter phenotype.

Studies of sympathetic neurons developing in cell culture revealed a surprising degree of transmitter plasticity and established the role of environmental factors in determining transmitter choice. The sympathetic neurons that innervate sweat glands undergo a change in neurotransmitter phenotype from noradrenergic to cholinergic during normal development similar to that observed in culture. Cross-innervation experiments indicate that the target sweat glands induce the switch and thereby specify the phenotype of the neurons that innervate them. Thus, both the transmitter plasticity and the role of environmental influences initially elucidated in culture are part of the developmental repertoire of sympathetic neurons in vivo. Further, these findings extend considerably our understanding of the role that targets may play during development; targets may not only determine how many neurons survive but also what their properties will be.     

Cardiac contractility and the spinal sympathetic neuron

Changes in cardiac contractility in cats were measured following electrical stimulation of the thoracolumbar spinal cord. Inotropic response sites were highly localized, and inotropic changes were greatest in the region of the intermediolateral nucleus. Cardioaugmentation was elicited from all spinal segments between T1 and L4. Inotropic responses were maximal at a frequency of 25 Hz and, at this frequency, showed strong right-sided preponderance. Similar lateralization was noted for heart rate and blood pressure. The right-sided spinal lateralization of the contractility response appeared to be frequency dependent, with reversal at 10 Hz, a relationship previously demonstrated for zona intermedia pressor responses. Neither adrenalectomy nor change of anesthetic agent affected the magnitude or lateralization of the inotropic response.     

Noradrenaline modifies sympathetic preganglionic neuron spike and afterpotential

Sympathetic preganglionic neurons were antidromically identified in the slice of the upper thoracic spinal cord of the adult cat, maintained in vitro. In normal Krebs solution, spikes evoked by intracellular stimulation had a marked ‘hump’ on the repolarization phase and were followed by an afterhyperpolarization of 2.8 s duration and 16.6 mV peak amplitude. Superfusion with Krebs solution containing noradrenaline 10–50 μm reversibly abolished the ‘hump’ of the spike and the late component of the afterhyperpolarization. In addition, it caused the appearance of a depolarizing afterpotential of 100–600 ms duration. This depolarization could result in repetitive firing of the neuron in response to a single intracellular current pulse.     

Modulation of parasympathetic neuron phenotype and function by sympathetic innervation

Selective sympathetic nerve dysfunction occurs during aging and in certain disease states. Here, we review findings concerning the effects of chronic sympathetic denervation on parasympathetic innervation to orbital target tissues in the adult rat. Long-term sympathetic denervation was induced by excising the ipsilateral superior cervical ganglion for 5-6 weeks prior to analyses. Following sympathectomy, pterygopalatine ganglion parasympathetic neurons show reduced nitric oxide synthase protein in their somata and projections to vascular targets. Laser Doppler measurements of ocular blood flow indicate that sympathectomy is also accompanied by reduced nitrergic vasodilatation. In the superior tarsal muscle of the eyelid, parasympathetic varicosities, normally, are distant to smooth muscle cells but make axo-axonal contacts with sympathetic nerves, consistent with physiological evidence showing only prejunctional inhibitory effects on sympathetically mediated smooth muscle contraction. Following sympathectomy, parasympathetic varicosities proliferate and closely appose smooth muscle cells, and this is accompanied by establishment of parasympathetic-smooth muscle excitatory neurotransmission. Many pterygopalatine parasympathetic neurons normally contain nerve growth factor (NGF) protein and express NGF mRNA. However, following chronic sympathectomy or elimination of sympathetic impulse activity, NGF mRNA and protein are markedly reduced, indicating that sympathetic neurotransmission enhances NGF expression in parasympathetic neurons. Together, these findings portray a striking dependency of parasympathetic neurons on sympathetic nerves to maintain normal phenotype and function. Sympathetic influences on parasympathetic neurons may be mediated, in part, through axo-axonal synapses. NGF synthesis and release by parasympathetic neurons may represent a molecular basis underlying the formation of these synapses, and up-regulation of NGF synthesis by sympathetic nerve activity may act to reinforce these associations.     

Lymphocyte-mediated regulation of neurotransmitter gene expression in rat sympathetic ganglia.

It has been previously shown that sympathetic noradrenergic nerve fibers, in addition to supplying the smooth muscle of the splenic capsule, trabeculae and blood vessels, also form very tight appositions with lymphocytes of the periarteriolar lymphatic sheath. To determine whether there is a direct communication between the sympathetic neurons and the immune cells we have grown dissociated superior cervical ganglion (SCG) neurons together with splenic lymphocytes. Sympathetic neurons were grown both as mixed preparations (neurons and non-neuronal ganglion cells) and neuron-enriched preparations. These systems were used to investigate whether coculture with splenocytes alters neurotransmitter gene expression in SCG cultures. Northern blot analysis was used to measure changes in neurotransmitter mRNA expression. The results showed that expression of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, was significantly decreased when SCG cultures were grown in the presence of spleen cells compared to control SCGs grown either alone or in the presence of erythrocytes. When the mitogen concanavalin A (ConA) was used to stimulate the spleen cells in the cocultures the decrease in TH was more pronounced. In contrast, preprotachykinin-A (PPT-A) mRNA expression in cultured SCGs increased in the cocultures. Another neuropeptide, neuropeptide Y (NPY), showed different responses in the presence of stimulated vs. unstimulated splenocytes. NPY mRNA was slightly increased in the presence of resting spleen cells, but showed a 70% decrease when ConA was added to the cocultures. Thus, our results suggest that lymphocytes can differentially regulate neurotransmitter gene expression in sympathetic ganglia.     

Interaction between medullary and cervical regulation of renal sympathetic activity.

We have reported that electrical or glutamate stimulation of the dorsolateral surface of the cervical spinal cord elicits a 40-60% decrease in renal sympathetic activity (RSA) in anesthetized rats. Because evoked sympatho-inhibition was observed, however, only after transection of the cervical spinal cord at C1, we suggested that unidentified supraspinal neurons affect the regulation of RSA by cervical neurons. In the present experiments, we tested the hypothesis that the modulatory supraspinal neurons are located in the ventrolateral medulla by observing the effects of rostroventral, lateral, medullary (RVLM) injections of the GABAergic agonist, muscimol, on baseline RSA and on our ability to inhibit that activity by cervical stimulation. GABAergic inhibition in the RVLM of chlorolose anesthetized rats elicited changes in RSA that were similar to those observed after transection of the spinal cord, including a 41% decrease in mean arterial pressure and a 44% increase in RSA. Moreover, after muscimol inhibition of RVLM neurons, electrical or glutamate stimulation of the dorsolateral cervical spinal cord elicited a decrease in RSA in otherwise intact rats. These results suggest that neurons in the RVLM interact with neurons in the cervical spinal cord in the regulation of RSA.     

Herbimycin A induces sympathetic neuron survival and protects against hypoxia

We have examined how herbimycin affects the survival and neuritogenesis of avian sympathetic neurons. Herbimycin promoted sympathetic neuron survival and neuritogenesis. At higher concentrations (> or = 100 ng/ml), herbimycin still enhanced neuron survival but blocked neuritogenesis. Addition of herbimycin (10-30 ng/ml) to neurons cultured in the presence of NGF or retinal conditioned medium altered neuronal morphology, with an increase in the number of neurites. Addition of NGF during hypoxia rescued 52% of the neurons compared to 14% survival in control conditions. Herbimycin alone rescued about 50% of the neurons. In the presence of NGF and 100 ng/ml herbimycin, 81% of the neurons survived hypoxia. Our results show that herbimycin promotes survival of chick sympathetic neurons and potentiates the effects of NGF.     

Target cells promote the development and functional maturation of neurons derived from a sympathetic precursor cell line

The role of target interactions in the development and functional maturation of peripheral neurons was investigated using an immortalized sympathetic precursor cell line. bMAH cells underwent neuronal differentiation in response to neurotrophic factors, but maintained an immature neuronal phenotype characterized by small cell bodies and continued cell division. Co-culture with cardiac myocytes, a target of sympathetic innervation, promoted the appearance of large-diameter postmitotic bMAH neurons. Analysis of bMAH maturation in the presence and absence of co-cultured myocytes indicated that myocyte-derived factors promoted the survival of maturing bMAH neurons prior to their acquisition of nerve growth factor dependence. Myocyte interactions also promoted the functional maturation of bMAH neurons, leading to an increase in the localization of synaptic vesicle proteins into neuritic varicosities and the acquisition of sympathetic-like intrinsic electrical properties. Like primary sympathetic neurons, mature bMAH neurons formed functional connections to cardiac myocytes as measured by evoked postsynaptic responses in connected myocytes. The effects of myocyte co-culture on developing bMAH neurons could be mimicked by myocyte conditioned medium, indicating that cardiac myocytes produce soluble factors that promote the appearance of mature neurons. These experiments indicate that targets of innervation play a role in directing the development and final maturation of peripheral neurons.     

Regulation of the subthreshold chloride conductance in the rat sympathetic neuron

The mechanisms that control chloride conductance (gCl) in the rat sympathetic neuron have been studied by the two-electrode voltage-clamp technique in mature, intact superior cervical ganglia in vitro. In addition to voltage dependence in the membrane potential range -120/-50 mV, gCl displays time- and activity-dependent regulation (sensitization). The resting membrane potential is governed by voltage-dependent gK and gCl, which determine values of cell input conductance ranging from 7 to 18 nS (full deactivation) to an upper value of about 130 nS (full activation and maximal gCl sensitization). The quiescent neuron, held at constant membrane potential, spontaneously and gradually moved from a low- to a high-conductance status. An increase (about 40 nS) in gCl accounted for this phenomenon, which could be prevented by imposing intermittent hyperpolarizing episodes. Following spike firing, gCl increased by 20-33 nS, independent of the cell conductance value preceding tetanization, and thereafter decayed to the pre-stimulus level within 5 min. Intracellular sodium depletion and its successive ionophoretic restoration moved the neuron from a stable low-conductance state to maximum gCl sensitization, pointing to a link between gCl sensitization and [Na+]i. The dependence of gCl build-up on [Na+]i and the time-course of such Na+-related modulation have been examined: gCl sensitization was absent at 0 [Na+]i, was well developed (20 nS) at 15 mM and tended towards a saturating value of 60 nS for higher [Na+]i. Sensitization was transient in response to neuron activity. In the silent neuron, sensitization of gCl shifted membrane potential over a range of about 15 mV.