Excitatory action of lead on rat sympathetic preganglionic neurons in vitro and in vivo

Lead exposure elicited an increase in blood pressure and was considered to be a cardiovascular risk factor. The involvements of sympathetic nervous system and circulating catecholamines have been implicated in lead-induced hypertension. This study examined the effects of PbCl(2) on sympathetic preganglionic neurons (SPNs) in vitro and in vivo. In vitro electrophysiological study showed that superfusion of a low concentration (5 microM) of PbCl(2), which had no effects on membrane potential and spontaneous discharge rate, enhanced excitatory postsynaptic potentials (EPSPs) in some of the SPNs examined but inhibited inhibitory postsynaptic potentials (IPSPs) in other SPNs tested. A higher concentration (50 microM) of PbCl(2) inhibited both EPSPs and IPSPs in all SPNs examined. In vivo study showed that intrathecal injection of PbCl(2) (10 and 100 nmol) via an implanted cannula to the T7-T9 segments of urethane-anesthetized rats increased both the heart rate and mean arterial pressure. The pressor and tachycardic responses of intrathecal PbCl(2) (100 nmol) were attenuated by pretreatment with intravenous administration of hexamethonium (10 mg/kg) or intrathecal AP-5 (DL-2-amino-5-phosphonovaleric acid, 100 nmol), but were not significantly antagonized by prior intrathecal administration of CNQX (6-cyano-7-nitroquinoxaline-2,3-dione, 100 nmol). Taken together, these results demonstrated that lead may exert a stimulatory effect on SPNs, which may result from the enhancement of EPSPs and inhibition of IPSPs by low concentrations of lead.     

Migration patterns of sympathetic preganglionic neurons in embryonic rat spinal cord

The displacement of immature neurons from their place of origin in the germinal epithelium toward their adult positions in the nervous system appears to involve migratory pathways or guides. While the importance of radial glial fibers in this process has long been recognized, data from recent investigations have suggested that other mechanisms might also play a role in directing the movement of young neurons. We have labeled autonomic preganglionic cells by microinjections of horseradish peroxidase (HRP) into the sympathetic chain ganglia of embryonic rats in order to study the migration and differentiation of these spinal cord neurons. Our results, in conjunction with previous observations, suggest that the migration pattern of preganglionic neurons can be divided into three distinct phases. In the first phase, the autonomic motor neurons arise in the ventral ventricular zone and migrate radially into the ventral horn of the developing spinal cord, where, together with somatic motor neurons, they form a single, primitive motor column (Phelps P. E., Barber R. P., and Vaughn J. E. (1991). J. Comp. Neurol. 307:77–86). During the second phase, the autonomic motor neurons separate from the somatic motor neurons and are displaced dorsally toward the intermediate spinal cord. When the preganglionic neurons reach the intermediolateral (IML) region, they become progressively more multipolar, and many of them undergo a change in alignment, from a dorsoventral to a mediolateral orientation. In the third phase of autonomic motor neuron development, some of these cells are displaced medially, and occupy sites between the IML and central canal. The primary and tertiary movements of the preganglionic neurons are in alignment with radial glial processes in the embryonic spinal cord, an arrangement that is consistent with a hypothesis that glial elements might guide autonomic motor neurons during these periods of development. In contrast, during the second phase, the dorsal translocation of preganglionic neurons occurs in an orientation perpendicular to radial glial fibers, indicating that glial elements are not involved in the secondary migration of these cells. The results of previous investigations have provided evidence that, in addition to glial processes, axonal pathways might provide a substrate for neuronal migration. Logically, therefore, it is possible that the secondary dorsolateral translocation of autonomic preganglionic neurons could be directed along early forming circumferential axons of spinal association interneurons, and this hypothesis is supported by the fact that such fibers are appropriately arrayed in both developmental time and space to guide this movement.     

Peptidergic inputs to sympathetic preganglionic neurons

This paper presents data showing that the sympathetic autonomic areas of the cat thoracolumbar spinal cord contain nerve terminals and fibres with immunoreactivity for at least seven neuropeptides. The distribution in the intermediolateral cell column of the terminals and fibres which contain enkephalin-, neuropeptide Y-, neurotensin-, substance P-, and neurophysin II-like immunoreactivity (ENK, NPY, NT, SP, and NP2, respectively) suggests that these peptides are involved in more generalized functions of the autonomic nervous system. On the other hand, peaks in density of immunoreactivity at certain levels suggest that different levels of influence of sympathetic preganglionic neurons by the various peptides may occur along the length of the thoracolumbar cord. The distribution of terminals and fibres containing somatostatin- and oxytocin-like immunoreactivity (SS and OXY) suggests that these peptides may be part of specific pathways to particular sympathetic preganglionic neurons. The possible sources of the terminals and fibres containing ENK, NPY, NT, SS, and SP include the spinal cord and supraspinal areas, whereas the source of these structures with OXY and NP2 is most likely supraspinal. The data suggest that coexistence of peptides and interactions between structures containing different neuropeptides occur in the spinal autonomic areas. It is speculated that neuropeptides have an important role to play in the regulation of the cardiovascular division of the autonomic nervous system.     

Migration patterns of sympathetic preganglionic neurons in embryonic rat spinal cord.

The displacement of immature neurons from their place of origin in the germinal epithelium toward their adult positions in the nervous system appears to involve migratory pathways or guides. While the importance of radial glial fibers in this process has long been recognized, data from recent investigations have suggested that other mechanisms might also play a role in directing the movement of young neurons. We have labeled autonomic preganglionic cells by microinjections of horseradish peroxidase (HRP) into the sympathetic chain ganglia of embryonic rats in order to study the migration and differentiation of these spinal cord neurons. Our results, in conjunction with previous observations, suggest that the migration pattern of preganglionic neurons can be divided into three distinct phases. In the first phase, the autonomic motor neurons arise in the ventral ventricular zone and migrate radially into the ventral horn of the developing spinal cord, where, together with somatic motor neurons, they form a single, primitive motor column (Phelps P. E., Barber R. P., and Vaughn J. E. (1991). J. Comp. Neurol. 307:77-86). During the second phase, the autonomic motor neurons separate from the somatic motor neurons and are displaced dorsally toward the intermediate spinal cord. When the preganglionic neurons reach the intermediolateral (IML) region, they become progressively more multipolar, and many of them undergo a change in alignment, from a dorsoventral to a mediolateral orientation. In the third phase of autonomic motor neuron development, some of these cells are displaced medially, and occupy sites between the IML and central canal. The primary and tertiary movements of the preganglionic neurons are in alignment with radial glial processes in the embryonic spinal cord, an arrangement that is consistent with a hypothesis that glial elements might guide autonomic motor neurons during these periods of development. In contrast, during the second phase, the dorsal translocation of preganglionic neurons occurs in an orientation perpendicular to radial glial fibers, indicating that glial elements are not involved in the secondary migration of these cells. The results of previous investigations have provided evidence that, in addition to glial processes, axonal pathways might provide a substrate for neuronal migration. Logically, therefore, it is possible that the secondary dorsolateral translocation of autonomic preganglionic neurons could be directed along early forming circumferential axons of spinal association interneurons, and this hypothesis is supported by the fact that such fibers are appropriately arrayed in both developmental time and space to guide this movement.     

Physiological properties of newly formed synapses between sympathetic preganglionic neurons and sympathetic ganglion neurons

We have examined the physiological properties of transmission at newly formed synapses between sympathetic preganglionic neurons and sympathetic ganglion neurons in vitro. Chick neurons were labeled with fluorescent carbocyanine dyes before they were placed into culture (Honig and Hume, 1986), and were studied by making intracellular recordings during the first 2 weeks of coculture. Evoked monosynaptic excitatory postsynaptic potentials (EPSPs) were not observed until 48 h of coculture. Beyond this time, the frequency with which connected pairs could be found did not vary greatly with time. With repetitive stimulation, the evoked monosynaptic EPSPs fluctuated in amplitude from trial to trial and showed depression at frequencies as low as 1 Hz. To gain further information about the quantitative properties of transmission at newly formed synapses, we analyzed the pattern of fluctuations of delayed release EPSPs. In mature systems, delayed release EPSPs are known to represent responses to single quanta, or to the synchronous release of a small number of quanta. For more than half of the connections we studied, the histograms of delayed release EPSPs were extremely broad. This result suggested that either quantal reponses are drawn from a continuous distribution that has a large coefficient of variation or that there are several distinct size classes of quantal responses. The pattern of fluctuation of monosynaptic EPSPs was consistent with both of these possibilities, and was inconsistent with the possibility that monosynaptic EPSPs are composed of quantal subunits with very little intrinsic variation. Although variation in the size of responses to single quanta might arise in a number of ways, one attractive explanation for our results is that the density and type of acetylcholine receptors varies among the different synaptic sites on the surface of developing sympathetic ganglion neurons.     

Physiological properties of newly formed synapses between sympathetic preganglionic neurons and sympathetic ganglion neurons.

We have examined the physiological properties of transmission at newly formed synapses between sympathetic preganglionic neurons and sympathetic ganglion neurons in vitro. Chick neurons were labeled with fluorescent carbocyanine dyes before they were placed into culture (Honig and Hume, 1986), and were studied by making intracellular recordings during the first 2 weeks of coculture. Evoked monosynaptic excitatory postsynaptic potentials (EPSPs) were not observed until 48 h of coculture. Beyond this time, the frequency with which connected pairs could be found did not vary greatly with time. With repetitive stimulation, the evoked monosynaptic EPSPs fluctuated in amplitude from trial to trial and showed depression at frequencies as low as 1 Hz. To gain further information about the quantitative properties of transmission at newly formed synapses, we analyzed the pattern of fluctuations of delayed release EPSPs. In mature systems, delayed release EPSPs are known to represent responses to single quanta, or to the synchronous release of a small number of quanta. For more than half of the connections we studied, the histograms of delayed release EPSPs were extremely broad. This result suggested that either quantal responses are drawn from a continuous distribution that has a large coefficient of variation or that there are several distinct size classes of quantal responses. The pattern of fluctuations of monosynaptic EPSPs was consistent with both of these possibilities, and was inconsistent with the possibility that monosynaptic EPSPs are composed of quantal subunits with very little intrinsic variation. Although variation in the size of responses to single quanta might arise in a number of ways, one attractive explanation for our results is that the density and type of acetylcholine receptors varies among the different synaptic sites on the surface of developing sympathetic ganglion neurons.     

Coexistence of neuropeptides in sympathetic preganglionic neurons of the cat

Coexistence of four neuropeptides in sympathetic preganglionic neurons (SPN) was investigated immunohistochemically in cats after intrathecal administration of colchicine. Neurons were studied for the coexistence of all combinations of enkephalin-, neurotensin-, somatostatin-, and substance P-like immunoreactivity (ENK, NT, SS, and SP, respectively) in the intermediolateral cell column (IML), nucleus intercalatus (IC), and central autonomic area (CA). The results indicate that SP coexists with all three other peptides, SS coexists with NT and SP, and ENK coexists only with SP. In all cases, SPN which contained two peptides were found in the IML in almost all levels of the thoraco-lumbar cord. Much smaller numbers of SPN which contained two peptides (in the same combinations as above) were found in the IC and not all segments contained such neurons. In the CA, only one neuron was found which contained two peptides (SP/SS). The distribution of SPN containing two peptides suggests that these neurons may participate in more general functions of the autonomic nervous system and that they are not likely involved in the innervation of specific visceral organs.     

Different adrenal sympathetic preganglionic neurons regulate epinephrine and norepinephrine secretion

Brain stimulation or activation of certain reflexes can result in differential activation of the two populations of adrenal medullary chromaffin cells: those secreting either epinephrine or norepinephrine, suggesting that they are controlled by different central sympathetic networks. In urethan-chloralose-anesthetized rats, we found that antidromically identified adrenal sympathetic preganglionic neurons (SPNs) were excited by stimulation of the rostral ventrolateral medulla (RVLM) with either a short (mean: 29 ms) or a long (mean: 129 ms) latency. The latter group of adrenal SPNs were remarkably insensitive to baroreceptor reflex activation but strongly activated by the glucopenic agent 2-deoxyglucose (2-DG), indicating their role in regulation of adrenal epinephrine release. In contrast, adrenal SPNs activated by RVLM stimulation at a short latency were completely inhibited by increases in arterial pressure or stimulation of the aortic depressor nerve, were unaffected by 2-DG administration, and are presumed to govern the discharge of adrenal norepinephrine-secreting chromaffin cells. These findings of a functionally distinct preganglionic innervation of epinephrine- and norepinephrine-releasing adrenal chromaffin cells provide a foundation for identifying the different sympathetic networks underlying the differential regulation of epinephrine and norepinephrine secretion from the adrenal medulla in response to physiological challenges and experimental stimuli.     

G protein activation inhibits gating charge movement in rat sympathetic neurons

G protein-coupled receptors (GPCRs) control neuronal functions via ion channel modulation. For voltage-gated ion channels, gating charge movement precedes and underlies channel opening. Therefore, we sought to investigate the effects of G protein activation on gating charge movement. Nonlinear capacitive currents were recorded using the whole cell patch-clamp technique in cultured rat sympathetic neurons. Our results show that gating charge movement depends on voltage with average Boltzmann parameters: maximum charge per unit of linear capacitance (Q_max) = 6.1 ± 0.6 nC/µF, midpoint (V_h) = –29.2 ± 0.5 mV, and measure of steepness (k) = 8.4 ± 0.4 mV. Intracellular dialysis with GTPS produces a nonreversible 34% decrease in Q_max, a 10 mV shift in V_h, and a 63% increase in k with respect to the control. Norepinephrine induces a 7 mV shift in V_h and 40% increase in k. Overexpression of G protein ₁₄ subunits produces a 13% decrease in Q_max, a 9 mV shift in V_h, and a 28% increase in k. We correlate charge movement modulation with the modulated behavior of voltage-gated channels. Concurrently, G protein activation by transmitters and GTPS also inhibit both Na⁺ and N-type Ca²⁺ channels. These results reveal an inhibition of gating charge movement by G protein activation that parallels the inhibition of both Na⁺ and N-type Ca²⁺ currents. We propose that gating charge movement decrement may precede or accompany some forms of GPCR-mediated channel current inhibition or downregulation. This may be a common step in the GPCR-mediated inhibition of distinct populations of voltage-gated ion channels. ion channel modulation; G protein-coupled receptors; charge movement     

Arachidonic acid both inhibits and enhances whole cell calcium currents in rat sympathetic neurons

We recently reported thatarachidonic acid (AA) inhibits L- and N-type Ca²⁺ currentsat positive test potentials in the presence of the dihydropyridine L-type Ca²⁺ channel agonist (+)-202-791 in dissociatedneonatal rat superior cervical ganglion neurons [Liu L and RittenhouseAR. J Physiol (Lond) 525: 291-404, 2000]. In thisfirst of two companion papers, we characterized the mechanism ofinhibition by AA at the whole cell level. In the presence of either-conotoxin GVIA or nimodipine, AA decreased current amplitude,confirming that L- and N-type currents, respectively, were inhibited.AA-induced inhibition was concentration dependent and reversible withan albumin-containing wash solution, but appears independent of AAmetabolism and G protein activity. In characterizing inhibition, anAA-induced enhancement of current amplitude was revealed that occurredprimarily at negative test potentials. Cell dialysis with albuminminimized inhibition but had little effect on enhancement, suggestingthat AA has distinct sites of action. We examined AA's actions oncurrent kinetics and found that AA increased holdingpotential-dependent inactivation. AA also enhanced the rate of N-typecurrent activation. These findings indicate that AA causes multiplechanges in sympathetic Ca²⁺ currents.

     

Convergence of preganglionic fibers on vasomotor neurons of the sympathetic ganglia in cats

Convergence of different preganglionic fibers on antidromically identified vasomotor neurons was studied by intracellular recording from neurons of ganglia L3 and L4 of the sympathetic chain, isolated from their rostral and caudal commissures, white ramus communicans, and muscular and cutaneous (mixed) twigs of the ventral branch and dorsal branch of the mixed nerve, in cats. Neurons activated antidromically by stimulation of these twigs were confidently considered to be vasomotor. Preganglionic fibers of only the B₂ and C groups were shown to converge on the vasomotor neurons, by contrast with the rest. Discharges of neurons were evoked only by excitation of preganglionic fibers of the B₂-group, arising mainly from higher segments of the spinal cord and entering through the rostral commissure. Vasomotor neurons also differ from the remaining ganglion cells in the properties of their axons, which conduct excitation at a significantly slower velocity (0.95±0.05 m/sec) than axons of other neurons (1.30±0.15 m/sec).I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 9, No. 6, pp. 592–597, November–December, 1977.     

Properties of axons of sympathetic preganglionic neurons in the cat lumbar spinal cord

Responses arising in ventral root filaments and antidromic discharges of single sympathetic preganglionic neurons in the lateral horn of gray matter in segment L₂ of the cat spinal cord were recorded during stimulation of the white rami communicantes in the same segment. Conduction velocities, thresholds, and refractory periods were determined for individual groups of sympathetic preganglionic fibers. Excitation was conducted more slowly along the intramedullary part of the axons of some sympathetic neurons than along the extramedullary part. In a third group of neurons studied the second antidromic discharge appeared in response to paired stimulation if the interstimulus interval was appreciably longer than their refractory period. It is postulated that axons of sympathetic preganglionic neurons in the lumber spinal cord have a thin intramedullary part and are supplied with recurrent collaterals.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 6, No. 2, pp. 143–151, March–April, 1974.     

Ceramide selectively inhibits apoptosis-associated events in NGF-deprived sympathetic neurons

Ceramide manifests both neurotoxic and neuroprotective properties depending on the experimental system. Ito and Horigome previously reported that ceramide delays apoptosis in a classic model of developmental programmed cell death, i.e. sympathetic neurons undergoing NGF deprivation.1 Here, we investigated the actions of ceramide upon the biochemical and genetic changes that occur in NGF deprived neurons. We correlate ceramide's neuroprotective actions with the ability of ceramide to antagonize NGF deprivation-induced oxidative stress and c-jun induction, both of which contribute to apoptosis in this model. However, ceramide did not block NGF deprivation-induced declines in RNA and protein synthesis, suggesting that ceramide does not slow all apoptosis-related events. Overall, these results are significant in that they show that ceramide acts early in the death cascade to antagonize two events necessary for NGF-deprivation induced neuronal apoptosis. Moreover, these results dissociate declines in neuronal function, i.e. macromolecular synthesis, from the neuronal death cascade.     

Horseradish peroxidase localization of sympathetic postganglionic and parasympathetic preganglionic neurons innervating the monkey heart

The localization of the sympathetic postganglionic and parasympathetic preganglionic neurons innervating the monkey heart were investigated through retrograde axonal transport with horseradish peroxidase (HRP). HRP (4 mg or 30 mg) was injected into the subepicardial and myocardial layers in four different cardiac regions. The animals were euthanized 84-96 hours later and fixed by paraformaldehyde perfusion via the left ventricle. The brain stem and the paravertebral sympathetic ganglia from the superior cervical, middle cervical, and stellate ganglia down to the T9 ganglia were removed and processed for HRP identification. Following injection of HRP into the apex of the heart, the sinoatrial nodal region, or the right ventricle, HRP-labeled sympathetic neurons were found exclusively in the right superior cervical ganglion (64.8%) or in the left superior cervical ganglion (35%). Fewer labeled cells were found in the right stellate ganglia. After HRP injection into the left ventricle, labeled sympathetic cells were found chiefly in the left superior cervical ganglion (51%) or in the right superior cervical ganglion (38.6%); a few labeled cells were seen in the stellate ganglion bilaterally and in the left middle cervical ganglion. Also, in response to administration of HRP into the anterior part of the apex, anterior middle part of the right ventricle, posterior upper part of the left ventricle, or sinoatrial nodal region, HRP-labeled parasympathetic neurons were found in the nucleus ambiguus on both the right (74.8%) and left (25.2%) sides. No HRP-labeled cells were found in the dorsal motor nucleus of the vagus on either side.     

5-Hydroxytryptamine inhibits the tachycardia induced by selective preganglionic sympathetic stimulation in pithed rats.

It has been shown in several species that serotonin (5-hydroxytryptamine; 5-HT) is able to inhibit the responses produced by sympathetic stimulation in a wide variety of blood vessels and other organs, including the heart. However, in pithed rats, the analysis of potential sympatho-inhibitory actions of 5-HT is hampered by the fact that 5-HT (given as i.v. bolus injections) produces tachycardia per se. Moreover, most studies have investigated 5-HT-induced sympatho-inhibition at only one frequency of stimulation. Thus, the present study set out to find the experimental conditions to overcome these problems. In this regard, we analyzed the potential ability of 5-HT, administered as i.v. continuous infusions, to inhibit the tachycardia caused by stimulation of the preganglionic (C7-T1) sympathetic outflow in pithed rats. Sympathetic cardiostimulation (0.01-3 Hz) resulted in frequency-dependent increases in heart rate; these responses were potentiated after desipramine (50 microg/kg, i.v.). During continuous infusions of 5-HT (3.1-10 microg/kg.min, i.v.), but not saline, the sympathetically-induced tachycardia was dose-dependently inhibited in both control and desipramine-pretreated rats. This inhibitory effect of 5-HT was significantly more pronounced at lower frequencies of stimulation. In contrast, the above infusions of 5-HT did not inhibit the tachycardia induced by i.v. bolus injections of noradrenaline in both control and desipramine-pretreated rats. Taken together, the above findings confirm that 5-HT induces inhibition of the sympathetic chronotropic outflow in the rat by acting at receptors located prejunctionally, without evoking tachycardia, over a wide range of stimulation frequencies.     

Transynaptic stimulation of phosphatidylinositol metabolism in sympathetic neurons in situ

An increase in 32P labelling of phosphatidylinositol, associated with synaptic activity, has previously been reported in excised sympathetic ganglia excited by preganglionic nerve impulses. In the present experiments a similar effect occurred in naturally-stimulated ganglia. It occurred when the preganglionic impulses were either evoked by electrical stimulation of the preganglionic nerve or were discharged spontaneously from the central nervous system. Thus increased turnover of phosphatidylinositol appears to be a normal accompaniment of synaptic transmission in these ganglia.     

Recurrent inhibition of sympathetic preganglionic neurons of the lateral horns of the spinal cord

Experiments on anesthetized and immobilized cats showed that repeated antidromic discharges can be evoked in 32.5% of sympathetic preganglionic neurons of the lateral horns in segments T3, T8–9, and L2 of the spinal cord, with intervals of 16 msec or more between them, which is much greater than the refractory period of these neurons. This feature was shown not to be connected with the properties of axons of that group of neurons and, in particular, with their after-subnormality. After orthodromic discharges in neurons of this group, for a much longer period of time than could be accounted for by possible collision, no antidromic discharges likewise were evoked. As a result of antidromic activation of some of these neurons in one segment, definite inhibition of the orthodromic response of other neurons of the same segment appeared, etiher by a reflex mechanism or through stimulation of descending pathways. The results point definitely to the existence of a mechanism of recurrent inhibition in some sympathetic preganglionic neurons of the lateral horns.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 9, No. 4, pp. 382–389, July–August, 1977.