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Neuropathy is a major diabetic complication. While the mechanism of this neuropathy is not well understood, it is believed to result in part from deficient nerve regeneration. Work from our laboratory established that gp130 family of cytokines are induced in animals after axonal injury and are involved in the induction of regeneration-associated genes (RAGs) and in the conditioning lesion response. Here, we examine whether a reduction of cytokine signaling occurs in diabetes. Streptozotocin (STZ) was used to destroy pancreatic β cells, leading to chronic hyperglycemia. Mice were injected with either low doses of STZ (5×60mg/kg) or a single high dose (1×200mg/kg) and examined after three or one month, respectively. Both low and high dose STZ treatment resulted in sustained hyperglycemia and functional deficits associated with the presence of both sensory and autonomic neuropathy. Diabetic mice displayed significantly reduced intraepidermal nerve fiber density and sudomotor function. Furthermore, low and high dose diabetic mice showed significantly reduced tactile touch sensation measured with Von Frey monofilaments. To look at the regenerative and injury-induced responses in diabetic mice, neurons in both superior cervical ganglia (SCG) and the 4th and 5th lumbar dorsal root ganglia (DRG) were unilaterally axotomized. Both high and low dose diabetic mice displayed significantly less axonal regeneration in the sciatic nerve, when measured in vivo, 48h after crush injury. Significantly reduced induction of two gp130 cytokines, leukemia inhibitory factor and interleukin-6, occurred in diabetic animals in SCG 6h after injury compared to controls. Injury-induced expression of interleukin-6 was also found to be significantly reduced in the DRG at 6h after injury in low and high dose diabetic mice. These effects were accompanied by reduced phosphorylation of signal transducer and activator of transcription 3 (STAT3), a downstream effector of the gp130 signaling pathway. We also found decreased induction of several gp130-dependent RAGs, including galanin and vasoactive intestinal peptide. Together, these data suggest a novel mechanism for the decreased response of diabetic sympathetic and sensory neurons to injury.

The cervical sympathetic trunks (CSTs) contain axons of preganglionic neurons that innervate the superior cervical ganglia (SCGs). Because regeneration of CST fibers can be extensive and can reestablish certain specific patterns of SCG connections, restoration of end organ function would be expected. This expectation was examined with respect to the pineal gland, an organ innervated by the two SCGs. The activity of pineal serotoninN-acetyltransferase (NAT) exhibits a large circadian rhythm that is dependent on the sympathetic input of the gland, with high activity at night. Thirty-six hours after the CSTs were crushed bilaterally, nocturnal NAT was decreased by 99%. Three months later, enzyme activity had recovered only to 15% of control values, a recovery dependent on regeneration of CST fibers. Nevertheless, a small day/night rhythm was present in lesioned animals. Neither the density of the adrenergic innervation of the gland nor the ability of an adrenergic agonist to stimulate NAT activity was reduced in rats with regenerated CSTs. In addition, stimulation of the regenerated CST at a variety of frequencies was at least as effective in increasing NAT activity as seen with control nerves. These data suggest that the failure of pineal function to recover is not attributable to a quantitative deficit in the extent of reinnervation or synaptic efficacy. Rather, we suggest that there is some loss of specificity in the synaptic connections made in the SCG during reinnervation, resulting in a loss of the central neuronal information necessary for directing a normal NAT rhythm and thus normal pineal function.

Functional noradrenergic transmission requires the coordinate expression of enzymes involved in norepinephrine (NE) synthesis, as well as the norepinephrine transporter (NET) which removes NE from the synapse. Inflammatory cytokines acting through gp130 can suppress the noradrenergic phenotype in sympathetic neurons. This occurs in a subset of sympathetic neurons during development and also occurs in adult neurons after injury. For example, cytokines suppress noradrenergic function in sympathetic neurons after axotomy and during heart failure. The molecular basis for suppression of noradrenergic genes is not well understood, but previous studies implicated a reduction of Phox2a in cytokine suppression of dopamine beta hydroxylase. We used sympathetic neurons and neuroblastoma cells to investigate the role of Phox2a in cytokine suppression of NET transcription. Chromatin immunoprecipitation experiments revealed that Phox2a did not bind the NET promoter, and overexpression of Phox2a did not prevent cytokine suppression of NET transcription. Hand2 and Gata3 are transcription factors that induce noradrenergic genes during development and are present in mature sympathetic neurons. Both Hand2 and Gata3 were decreased by cytokines in sympathetic neurons and neuroblastoma cells. Overexpression of either Hand2 or Gata3 was sufficient to rescue NET transcription following suppression by cytokines. We examined expression of these genes following axotomy to determine if their expression was altered following nerve injury. NET and Hand2 mRNAs decreased significantly in sympathetic neurons 48 hours after axotomy, but Gata3 mRNA was unchanged. These data suggest that cytokines can inhibit NET expression through downregulation of Hand2 or Gata3 in cultured sympathetic neurons, but axotomy in adult animals selectively suppresses Hand2 expression.

Adult peripheral neurons exhibit dramatic changes in gene expression after axonal injury, including changes in neuropeptide phenotype. For example, sympathetic neurons in the superior cervical ganglion (SCG) begin to express vasoactive intestinal peptide (VIP), galanin, pituitary adenylate cyclase activating polypeptide (PACAP), and cholecystokinin after axotomy. Before these changes, nonneuronal cells in the SCG begin to express leukemia inhibitory factor (LIF). When the effects of axotomy were compared in LIF-/- and wild-type mice, the increases in VIP and galanin expression were less in the former, though significant increases still occurred. LIF belongs to a family of cytokines with overlapping physiological effects and multimeric receptors containing the subunit gp130. Real-time PCR revealed large increases in the SCG after axotomy in mRNA for three members of this cytokine family, interleukin (IL)-6, IL-11, and LIF, with modest increases in oncostatin M, no changes in ciliary neurotrophic factor, and decreases in cardiotrophin-1. To explore the role of these cytokines, animals with selective elimination of the gp130 receptor in noradrenergic neurons were studied. No significant changes in mRNA levels for VIP, galanin, and PACAP were seen in axotomized ganglia from these mutant mice, while the increase in cholecystokinin was as large as that seen in wild-type mice. The data indicate that the inductions of VIP, galanin, and PACAP after axotomy are completely dependent on gp130 cytokines and that a second cytokine, in addition to LIF, is involved. The increase in cholecystokinin after axotomy, however, does not require the action of these cytokines.

Axotomy of sympathetic and sensory neurons leads to changes in their neuropeptide phenotypes. These changes are mediated in part by the induction of leukemia inhibitory factor (LIF) by nonneuronal cells. In the present study, we identified satellite/Schwann cells as a possible source of the injury-induced LIF. Using a Schwann cell line, SC-1 cells, we examined mechanisms of LIF induction. LIF mRNA levels increased rapidly when the cells were treated with 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate, phorbol 12-myristate 13-acetate (PMA), or A23187. Among these reagents, PMA was the most efficacious. Inhibition of protein kinase C (PKC) by GF-109203X significantly reduced the PMA-induced LIF mRNA levels. As PKC is known to activate the extracellular signal-regulated kinase (ERK) signaling pathway, the involvement of this pathway in the PMA-stimulated induction of LIF mRNA was examined. Phosphorylation of ERKs was increased following PMA treatment in SC-1 cells. Moreover, inhibition of ERK kinase activity by PD98059 dramatically reduced PMA-stimulated phosphorylation of ERKs and induction of LIF mRNA. These results indicate that LIF mRNA levels can be regulated by ERK activation via stimulation of PKC in Schwann cells.

Following axonal damage, sympathetic neurons are capable of regenerating and reinnervating their target tissues. Some years ago exogenous administration of polyamines was shown to enhance this regeneration. Recently, it was found that axonal injury leads to a dramatic up-regulation of the expression of arginase I in sympathetic neurons. This enzyme catalyzes the conversion of arginine to ornithine, which can subsequently be converted to the diamine putrescine and, ultimately, to the polyamines spermidine and spermine. In the present study, using an antiserum that reacts with both spermidine and spermine, we have found an increase in polyamine levels in both neurons and non-neuronal cells in the superior cervical ganglion 2 and 5 days following transection of the ganglion's postganglionic trunks. Using PC12 cells primed with nerve growth factor and then stripped off the culture dish and replated as a model system for axotomized sympathetic neurons, we found that spermidine treatment, with or without nerve growth factor, resulted in an increased percentage of cells with a neurite whose length was at least twice the diameter of the neuron's cell body. These increases could be seen within 48 h and were still evident after 8 days. Together, these data support the possibility that endogenous polyamines are involved in the normal regeneration which occurs following sympathetic axonal damage.

Synaptic transmission in the superior cervical ganglion (SCG) is mediated by nicotinic acetylcholine receptors (nAChR). After transection of the postganglionic nerves of the SCG in the adult rat, the transcript levels of four of the five nAChR subunits present in the ganglion, alpha3, alpha5, alpha7, and beta4, decrease dramatically. In the present study, the effect of axotomy on nAChR subunit expression was examined at the protein level, focusing on the alpha7 and beta4 subunits. Immunohistochemistry with monoclonal antibody mAb306 (for the alpha7 subunit) and polyclonal antibody 4886 (for the beta4 subunit) showed that immunoreactivities for both alpha7 and beta4 subunits were concentrated in neurons in the intact ganglion. Results from double staining with antibodies to these subunits and to tyrosine hydroxylase, the enzyme that catalyzes the rate-limiting step in the biosynthesis of the sympathetic neurotransmitter norepinephrine, demonstrated that most neurons in the SCG express both the alpha7 and beta4 subunits. Three days after axotomy, the number of immunolabeled neurons and the intensity of the immunostaining per labeled neuron were decreased for both subunits. Decreases in subunit levels were also observed by Western blot analysis. Observing changes in these subunits over time after surgery revealed that, while the protein level of the alpha7 subunit recovered substantially within 2 weeks after the lesion, that of the beta4 subunit stayed low. These data demonstrate that decreases in nicotinic receptor subunits are among the changes in proteins that occur in axotomized sympathetic neurons, and suggest that these decreases may contribute to the depression in ganglionic synaptic transmission observed in axotomized ganglia.

Axotomy of adult peripheral neurons produces decreases in the levels of transcripts for a number of proteins involved in synaptic transmission. For example, tyrosine hydroxylase and neuropeptide Y mRNA decrease in axotomized sympathetic neurons in the superior cervical ganglion (SCG). In the present study, the effects of axotomy on the expression of nicotinic receptor subunit transcripts were examined in the SCG and the results were compared to those produced by deafferentation and explantation. Normally, neurons in the SCG express five different nicotinic subunits: alpha3, alpha5, alpha7, beta2, and beta4. Forty-eight hours after axotomy in vivo or explantation, dramatic decreases in these transcripts were seen, except for beta2, which increased. In contrast, deafferentation of the SCG had negligible effects on any of these transcripts. Both leukemia inhibitory factor (LIF) and nerve growth factor (NGF) have been shown to play a role in the decrease in neuropeptide Y mRNA expression after axotomy. In the cases of these nicotinic receptor transcripts, however, similar decreases were seen in wild-type and LIF knockout animals. Furthermore, administration of an antiserum to NGF in intact animals produced no changes in transcript levels. On the other hand, providing exogenous NGF to axotomized SCG in vivo or in explant cultures partially prevented the decreases in the transcripts for alpha3, alpha5, alpha7, and beta4. These data indicate that axotomy produces dramatic decreases in the expression of several nicotinic receptor subunit transcripts, and that the molecular signals underlying these changes differ from those previously shown to mediate the decrease in neuropeptide Y expression.

Large changes in neuronal gene expression occur in adult peripheral neurons after axonal transection. In the rat superior cervical ganglion, for example, neurons that do not normally express vasoactive intestinal peptide (VIP) or galanin do so after postganglionic nerve transection. These effects of axotomy could result from a number of aspects of the surgical procedure. To test the idea that the important variable might be the disconnection of axotomized neuronal cell bodies from their target tissues, we examined the effects of producing such a disconnection by means of the compound 6-hydroxydopamine (6-OHDA), a neurotoxin that causes degeneration of sympathetic varicosities and avoids many of the complications of surgery. Two days after 6-OHDA treatment, VIP and galanin immunoreactivities had increased two- and 40-fold, respectively. Nevertheless, these increases were substantially smaller than the 30- and 300-fold changes seen after surgical axotomy. When expression of VIP and galanin was examined at the mRNA level, however, comparable increases were found after either procedure. The results indicate that chemical destruction of sympathetic varicosities produces an equivalent signal for increasing VIP and galanin mRNA as does axonal transection. The differences in the neuropeptide levels achieved suggests that peptide expression after nerve transection is regulated both at the mRNA and protein levels.

The neuropeptide phenotype of adult sympathetic neurons changes dramatically after postganglionic nerve transection. Studies, thus far, have been done on the superior cervical ganglion; however, one limitation of this preparation is that it is necessary to transect the postganglionic axons quite close to the ganglion. In the present study, we examined the effects of axonal damage on galanin-like immunoreactivity in the middle and inferior cervical ganglion complex. With these ganglia, it is possible to transect postganglionic axons at a considerable distance from their cell bodies and, therefore, to examine the extent to which local tissue damage, rather than specific axonal transection, is required for these changes in neuropeptide phenotype to occur. The anatomy of this system also allowed us to determine the extent to which the changes in galanin expression are restricted to those neurons that have been axotomized. The axons of a small population of the neurons in the middle and inferior cervical ganglia complex project into the cervical sympathetic trunk. Within two days after this trunk was transected, there was an increase in the level of galanin-like immunoreactivity in the complex and in the number of immunostained principal neurons. These neurons were concentrated primarily in the most rostral part of the complex. An increase in galanin-like immunoreactivity also occurred in response to the systemic administration of the sympathetic neurotoxin 6-hydroxydopamine. In that case, many more neurons were affected than after transection of the cervical sympathetic trunk, and the neurons were distributed evenly throughout the ganglion complex. After the cervical sympathetic trunk was cut, the cell bodies of the axotomized neurons were identified by retrograde labeling from the point of transection with Fast Blue. The vast majority (86%) of neurons that exhibited galanin-like immunoreactivity were labeled with Fast Blue, indicating that the changes in peptide expression occur primarily, and perhaps exclusively, in axotomized neurons. Forty-two per cent of the Fast Blue-labeled neurons exhibited galanin-like immunoreactivity, suggesting that sympathetic neurons within a ganglion do not behave as a homogenous population in response to axotomy. The results also indicate that tissue damage in the vicinity of a ganglion is not a necessary triggering stimulus for this change in neuropeptide phenotype. The data are consistent with the hypothesis that a specific lesion, namely axonal transection, either produces a stimulatory factor or removes an inhibitory factor that regulates the expression of galanin.

Vasoactive intestinal peptide (VIP) expression increases in sympathetic neurons when they are grown in dissociated cell or explant cultures and when they are axotomized in vivo. In dissociated cell culture, the magnitude of the VIP increase was reduced when nonneuronal cells were removed and medium conditioned by ganglionic nonneuronal cells increased VIP in neuron-enriched cultures. Antiserum Against cholinergic differentiation factor (also leukemia inhibitory factor; CDF/LIF), but not against ciliary neurotrophic factor, immunoprecipitated this activity. Medium conditioned by sympathetic ganglion explants also contained a VIP-stimulatory molecule that was immunoprecipitated by CDF/LIF antiserum, and CDF/LIF antiserum partially blocked VIP induction in explants. CDF/LIF mRNA was increased in dissociated cell cultures, in ganglion explants and in vivo after axotomy. Our results suggest that CDF/LIF released from ganglionic nonneuronal cells plays an important role in regulating VIP after axotomy. 1994 John Wiley & Sons, Inc.

The adult rat superior cervical ganglion (SSG) contains low levels of galanin- and vasoactive intestinal peptide-(VIP) like immunoreactivity, with very few immuno-stained principal neurons. Immunoreactivity for both neuropeptides increases in these neurons after explanation or postganglionic axotomy in vivo. Northern blot analysis had demonstrated concomitant increases in mRNAs encoding these peptides. To localize cells in axotomized ganglia which increase their expression of these mRNAs, we performed in situ hybridization studies. In control SCG, only a few principal neurons contained mRNA for either galanin or VIP. After 48 h in organ culture, galanin mRNA was expressed in the majority of principal neurons. At 48 h after in vivo axotomy of both postganglionic trunks of the SCG, the internal and external carotid nerves, the distribution and number of neurons expressing galanin mRNA increased similarly to that seen in culture. Lesioning either trunk alone produced increases in galanin mRNA localized to those regions of the ganglion containing neurons that project into the lesioned trunk. Transection of the predominantly preganglionic cervical sympathetic trunk increased galanin mRNA expression in a small population of neurons that nerve trunk. The distributions of these labeled neurons, together with previous neuroanatomical studies, suggests that they had been axotomised by the lesions. Similar studies examining VIP mRNA expression demonstrated that although considerably fewer VIP mRNA expressing neurons than galanin mRNA expressing neurons were present after axotomy, the distribution of neuropeptide mRNA-positive cells were similar in both cases. These observations suggest that increases in the peptide galanin and VIP after nerve transection result from changes in the levels of their mRNAs in those neurons that have been axotomized. 1994 John Wiley & Sons, Inc.

The regulation of the expression of substance P (SP) in the rat superior cervical ganglion was compared to that of vasoactive intestinal peptide (VIP) in vivo after axotomy and in vitro after explantation. Previous studies have demonstrated that both neuropeptides increase after explantation, depolarization, and decentralization; however, whereas VIP expression increases after postganglionic axotomy, SP expression reportedly does not. To compare the effect of axotomy on these two peptides directly, the content of both was determined in individual ganglia at various times after surgery. The level of VIP-like immunoreactivity (IR) is increased at 2 days, reaches a peak at 6 days, and then declines by 14 days to approximately half its peak value. The level of SP-IR also increases 2 days after axotomy, but returns to control values by day 6. The increase in SP-IR is accompanied by an increase in beta-preprotachykinin mRNA, suggesting that the elevation in SP content is due, at least in part, to enhanced peptide synthesis. Immunocytochemical localization of SP-IR revealed the presence of immunoreactive principal neurons in axotomized, but not in sham-operated ganglia. Similarities in the regulation of these two neuropeptides were also investigated in organ culture by examining the effects of dexamethasone and interleukin-1 beta on VIP content, since the former has been shown to prevent the increase in SP in culture, while the latter has been found to enhance this increase (Kessler, Adler, Bell, et al., 1983, Neuroscience 9:309-321; Freidin and Kessler, 1991, Proc. Natl. Acad. Sci. USA 88:3200-3203; Hart, Shadiack, and Jonakait, 1991, J. Neurosci. Res. 29:282-291). As with SP expression, dexamethasone reduces the increase in VIP expression, while interleukin-1 beta increases it. Thus, both in vivo and in vitro, similar changes in VIP and SP expression are observed following a number of experimental manipulations, suggesting that expression of the two peptides is regulated by qualitatively similar mechanisms in sympathetic neurons.

Electrical stimulation of the preganglionic fibers innervating the rat superior cervical ganglion (SCG) produces both short-term and long-term increases in tyrosine hydroxylase (TH) activity that are not completely blocked by nicotinic antagonists. Vasoactive intestinal peptide (VIP) and secretin, two neuropeptides known to produce short-term increases in TH activity, were examined for their ability to produce long-term increases in this enzyme activity. Culturing the SCG in the presence of either peptide produced a 30–50% increase in TH activity measured 2 days later. The results raise he possibility that one of these peptides or a related molecule participates in the transsynaptic induction of ganglionic TH.

We have utilized the favorable signal-to-noise ratios provided by whole- cell recording, combined with variance analysis, to determine the pre- or postsynaptic actions of a variety of manipulations on unitary EPSPs evoked by low-intensity stimulation of afferents to CA1 pyramidal neurons in slices of hippocampus. Estimates of quantal content (mcv) were determined by calculating the ratio of the squared average unitary EPSP amplitude (determined from 150–275 responses) to the variance of these responses (M2/sigma 2), while quantal amplitudes (qcv) were estimated by calculating the ratio of the response variance to average EPSP size (sigma 2/M). Estimates of mcv were highly correlated with those determined using the method of failures (mf). With paired stimulation (50 msec interpulse interval) there was a significant facilitation of the second unitary EPSP, accompanied by an increase in mcv, but not qcv, suggesting that this facilitation was of presynaptic origin. Superfusion of hippocampal slices with various concentrations of adenosine, the A1-selective adenosine receptor agonist cyclohexyladenosine, or the Ca2+ channel blocker cadmium significantly reduced average unitary EPSP amplitudes and mcv, without significantly altering qcv, suggesting a presynaptic locus for this inhibition. The 50% effective concentration for the apparent presynaptic action of adenosine on mcv in the present study (5.7 microM; 95% confidence limits = 4.2–7.7 microM) was significantly lower than its EC50 for reducing conventional, large EPSPs (33 microM; recorded with high- resistance microelectrodes), or extracellular field EPSPs (29 microM), as previously reported by this laboratory. The glutamate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) reduced average unitary EPSP amplitudes; in contrast to the above manipulations, it had no effect on mcv, but significantly altered qcv, which is consistent with its presumed postsynaptic mechanism of action. We conclude from these data that adenosine presynaptically reduces synaptic strength at Schaffer collateral-commissural synapses in the hippocampus by diminishing the number of quanta released, not by reducing the size of these individual quanta or postsynaptic sensitivity to excitatory neurotransmitter. These results suggest that the mechanism by which adenosine inhibits synaptic transmission in the hippocampus is similar, if not identical, to the mechanism by which it inhibits synaptic transmission at the neuromuscular junction.

The distributions of vasoactive intestinal peptide (VIP)- and peptide histidine isoleucine amide (PHI)-immunoreactivities (IR) in the thoracic spinal cord of the rat are described. VIP- and PHI-IR were present in cells and fibers in the lateral spinal nucleus, lamina VII, nucleus proprius, substantia gelatinosa, intermediolateral cell column, and the area around the central canal. The functions of these peptides in the thoracic spinal cord are not known; however, their locations suggest that they are involved in sensory and autonomic functions, among others.

Vasoactive intestinal peptide (VIP) increased catecholamine biosynthesis in bovine adrenal chromaffin cells by 50-200%. Six related peptides produced no effects. In addition, VIP increased tyrosine hydroxylase (TH) activity measured in gel-filtered supernatants prepared from homogenates of treated cells. The hypothesis that cyclic AMP is the second messenger involved in these effects of VIP was also evaluated. VIP led to an elevation of cyclic AMP levels, and this increase occurred over a similar concentration range and time course as the activation of TH and the increase in catecholamine biosynthesis. Each measure reached maximal levels at 10-20 microM VIP within 1 min and remained elevated for at least 16 min. These changes produced by VIP were paralleled by enhanced phosphorylation of TH, and this phosphorylation occurred on a single tryptic peptide that was the same peptide whose phosphorylation has been previously shown to be stimulated by forskolin. In contrast to VIP and forskolin, 12-O-tetradecanoylphorbol 13-acetate, a phorbol ester known to activate protein kinase C, increased the phosphorylation on a total of three tryptic peptides of TH. Our results indicate that VIP stimulates catecholamine biosynthesis in chromaffin cells through the phosphorylation and activation of TH and support the conclusion that a cyclic AMP-dependent phosphorylation of TH is responsible for these effects.

The rat pineal gland was chosen as a model system to study how aging affects the capacity of surviving neurons to compensate for partial destruction of a neural pathway. The pineal gland receives bilateral overlapping sympathetic innervation from the two internal carotid nerves, whose activity regulates several aspects of pineal metabolism in a circadian fashion. The most dramatic of these is the marked nighttime increase in the activity of N-acetyltransferase, the rate-limiting enzyme in melatonin synthesis. These features allow for the pineal gland to be used as a model system for studies on neuronal plasticity, since it is possible to create specific partial neural lesions and to evaluate functional recovery subsequently at the cellular level. We examined the activity of N-acetyltransferase and the content of melatonin in the pineal gland as indices of pineal function at various time points after unilateral surgical denervation (lesion of one of the two internal carotid nerves) in 4-month-(young) and 25-month-old (aged) rats. At both ages, the nighttime levels of the two parameters were significantly lower 8 h after this lesion than in sham-operated animals of the same age, indicating impaired function. When examined at later time points (i.e., 1.5 and 10 days after this lesion), both young and aged animals exhibited full recovery in these two parameters. Measurement of specific neuronal uptake of [3H]norepinephrine was utilized as an index of the number of sympathetic varicosities innervating the pineal gland. One and a half days after unilateral denervation. [3H]norepinephrine uptake in 4-, 10-, 16-, and 25-month-old animals was approximately 50% of that obtained in sham-operated animals of the same age, as expected if half of the innervation of the gland was lesioned. Ten days after surgery, [3H]norepinephrine uptake in 4- and 10-month-old animals was 78% of the value found in sham-operated animals of the same age, suggesting collateral sprouting of the remaining sympathetic nerve fibers. However, in 16- and 25-month-old animals, uptake remained at 50%. These data indicate that recovery of pineal function after unilateral denervation occurs to a similar extent and with a similar time course in young and aged animals. In contrast, the data suggest that collateral sprouting is impaired in aged animals. The fact that functional recovery occurs in young animals sooner after the lesions than does collateral sprouting and that recovery occurs in the aged animals in the apparent absence of collateral sprouting suggests that the relationship between this anatomical form of plasticity and functional recovery may be complex. Among other possibilities, collateral sprouting may play a role in the maintenance of recovered function rather than in the initial recovery process or sprouting may be functionally important after nerve losses greater than those represented by a 50% lesion.

Previous studies have shown that certain peptides of the secretin-glucagon family stimulate tyrosine hydroxylase activity in sympathetic neurons of the superior cervical ganglion and three of its end organs, i.e., the iris, pineal gland, and submaxillary gland. To determine whether a similar regulation occurs in other sympathetic neurons, the effects of two of these peptides, secretin and vasoactive intestinal peptide, were examined in the right cardiac ventricle of the rat, a tissue innervated primarily by the middle and inferior cervical ganglia. Both peptides stimulated tyrosine hydroxylase activity, measured in situ, in this tissue. In addition, several second messenger systems were investigated as possible mediators of this peptidergic stimulation of tyrosine hydroxylase activity in autonomic end organs. 8-Bromoadenosine 3′,5′-cyclic monophosphate and forskolin elevated tyrosine hydroxylase activity in slices of both the right ventricle and the submaxillary gland. 8-Bromoguanosine 3′,5′-cyclic monophosphate also stimulated tyrosine hydroxylase activity in both tissues, whereas nitroprusside stimulated activity only in the submaxillary slices. Furthermore, the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine and/or Ro 20–1724 potentiated the stimulation by secretin, as well as the stimulations by forskolin and nitroprusside. Phorbol 12,13-dibutyrate also stimulated tyrosine hydroxylase activity in cardiac and submaxillary slices; however, no potentiation of these effects was seen following addition of either phosphodiesterase inhibitor. These data, taken together with those of previous studies, suggest a role for a cyclic nucleotide, probably adenosine 3′,5′-cyclic monophosphate, in the peptidergic stimulation of tyrosine hydroxylase activity in sympathetic nerve terminals.

Previous studies have shown that preganglionic nerve stimulation in vitro increases acutely the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, in sympathetic neuronal cell bodies in the rat superior cervical ganglion (SCG). In the present study, we have examined whether a similar increase occurs after nerve stimulation in vivo, and whether this enzyme activity also increases in sympathetic nerve terminals in autonomic end organs. Immediately following stimulation at 10 Hz for 15 min in vivo, TH activity was found to have increased 4- to 8-fold in the SCG and in 3 of its end organs: the iris, the pineal gland and the submaxillary gland. These results indicate that increases in sympathetic nerve activity in vivo can lead to increases in TH activity both in adrenergic cell bodies/dendrites in the SCG and in adrenergic nerve terminals/fibers in various autonomic end organs.

The rat pineal gland is an attractive system for studies on the capacity of neural systems to recover following partial injury, allowing both for the creation of precise subtotal lesions and for the measurement of recovery of function at the cellular level. The pineal gland receives overlapping sympathetic innervation from the right and left internal carotid nerves from neurons whose cell bodies are located in the two superior cervical ganglia. This innervation regulates several aspects of pineal metabolism in a circadian fashion, with the most dramatic being a marked increase in the night-time activity of N-acetyltransferase, a key enzyme regulating the rate of melatonin synthesis. We have previously shown that a highly divergent pattern takes place in the night-time activity of this enzyme following two different unilateral lesions of the sympathetic innervation to the gland. Thus, following a unilateral lesion of the internal carotid nerve (unilateral denervation), there is an initial decline in N-acetyltransferase activity; however, normal activity is again seen during the second and subsequent nights. In contrast, a unilateral lesion of the cervical sympathetic trunk, the nerve that innervates the superior cervical ganglion (unilateral decentralization), results in "permanent" impairment of N-acetyltransferase activity. In the present study, we report that the functional capacity of the entire pathway for melatonin synthesis is similarly affected following these lesions, as reflected by the levels of melatonin and of its precursor N-acetylserotonin in the pineal gland, as well as the levels of the main melatonin metabolite 6-hydroxy-melatonin in the urine.(ABSTRACT TRUNCATED AT 250 WORDS)

Activating transcription factor 3 (ATF3) is induced in a high proportion of axotomized sensory and motor neurons after sciatic nerve transection. In the present study, we looked at the expression of this factor in the superior cervical ganglion (SCG) after axotomy and after other manipulations that induce certain aspects of the cell body response to axotomy. Sympathetic ganglia from intact rats and mice exhibit only a very occasional neuronal nucleus with activating transcription factor 3-like immunoreactivity (ATF3-IR); however, as early as 6 h and as late as 3 weeks postaxotomy, many of the neurons showed intense ATF3-IR. A second population of cells had smaller and generally less intensely stained nuclei, and at least some of these cells were satellite cells. Lesions distal to the SCG induced by administration of 6-hydroxydopamine or unilateral removal of the salivary glands produced increases in ATF3-IR similar to those seen after proximal axotomy, indicating that this response is not strictly dependent on the distance of the lesion from the cell body. Two proposed signals for triggering ATF3 expression were examined: reduction in nerve growth factor (NGF) availability and induction of the cytokine leukemia inhibitory factor (LIF). While administration of an antiserum raised against NGF to intact animals induced ATF3-IR, induction of ATF3-IR after axotomy was not reduced in LIF null mutant mice. Since axotomy, 6-hydroxydopamine, and sialectomy are known to decrease the concentration of NGF in the SCG, our data suggest that these decreases in NGF lead to increases in ATF3-IR. Furthermore, since the number of neurons in the SCG expressing ATF3-IR was greater after axotomy than after antiserum against NGF treatment, this raises the possibility that decreased NGF is not the only process regulating ATF3 expression after axotomy.

Axotomized peripheral neurons are capable of regeneration, and the rate of regeneration can be enhanced by a conditioning lesion (i.e., a lesion prior to the lesion after which neurite outgrowth is measured). A possible signal that could trigger the conditioning lesion effect is the reduction in availability of a target-derived factor resulting from the disconnection of a neuron from its target tissue. We tested this hypothesis with respect to nerve growth factor (NGF) and sympathetic neurons by administering an antiserum to NGF to adult mice for 7 days prior to explantation or dissociation of the superior cervical ganglion (SCG) and subsequently measuring neurite outgrowth. The antiserum treatment dramatically lowered the concentration of NGF in the SCG and increased the rate of neurite outgrowth in both explants and cell cultures. The increase in neurite outgrowth was similar in magnitude to that seen after a conditioning lesion. To determine if exogenous NGF could block the effect of a conditioning lesion, mice were injected with NGF or cytochrome C immediately prior to unilateral axotomy of the SCG, and for 7 days thereafter. A conditioning lesion effect of similar magnitude was seen in NGF-treated and control animals. While NGF treatment increased NGF levels in the contralateral control ganglion, it did not significantly elevate levels in the axotomized ganglion. The results suggest that the decreased availability of NGF after axotomy is a sufficient stimulus to induce the conditioning lesion effect in sympathetic neurons. While NGF administration did not prevent the conditioning lesion effect, this may be due to the markedly decreased ability of sympathetic neurons to accumulate the growth factor after axotomy.

Protein-protein interactions and calcium entry through the N -methyl- d -aspartate (NMDA)–type glutamate receptor regulate synaptic development and plasticity in the central nervous system. The EphB receptor tyrosine kinases are localized at excitatory synapses where they cluster and associate with NMDA receptors. We identified a mechanism whereby EphBs modulate NMDA receptor function. EphrinB2 activation of EphB in primary cortical neurons potentiates NMDA receptor–dependent influx of calcium. Treatment of cells with ephrinB2 led to NMDA receptor tyrosine phosphorylation through activation of the Src family of tyrosine kinases. These ephrinB2-dependent events result in enhanced NMDA receptor–dependent gene expression. Our findings indicate that ephrinB2 stimulation of EphB modulates the functional consequences of NMDA receptor activation and suggest a mechanism whereby activity-independent and activity-dependent signals converge to regulate the development and remodeling of synaptic connections.

A recent study has shown that mice containing a loss-of-function mutation in the gene encoding galanin exhibit decreased peripheral nerve regeneration after a lesion. This major advance indicates, for the first time, that the large increases in galanin expression that occur in axotomized peripheral neurons have functional consequences for regeneration. Hopefully, similar functional consequences will soon be found for other peptides induced in these neurons after axotomy, such as vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide.

Axonal transection of adult sympathetic and sensory neurons leads to a decrease in their content of target-derived nerve growth factor (NGF) and to dramatic changes in the expression of several neuropeptides and enzymes involved in transmitter biosynthesis. For example, axotomy of sympathetic neurons in the superior cervical ganglion (SCG) dramatically increases levels of galanin, vasoactive intestinal peptide (VIP), and substance P and their respective mRNAs and decreases mRNA levels for neuropeptide Y (NPY) and tyrosine hydroxylase (TH). Axotomy of sensory neurons in lumbar dorsal root ganglia (DRG) increases protein and mRNA levels for galanin and VIP and decreases levels for substance P and calcitonin gene-related peptide (CGRP). To assess whether reduction in the availability of endogenous NGF might play an important role in triggering these changes, we injected nonoperated animals with an antiserum against NGF (αNGF). αNGF increased levels of peptide and mRNA for galanin and VIP in neurons in both the SCG and DRG. NPY protein and mRNA were decreased in the SCG, but levels of TH protein and mRNA remained unchanged. In sensory neurons the levels of SP and CGRP protein decreased after αNGF treatment. These data suggest that the reduction in levels of NGF in sympathetic and sensory neurons after axotomy is partly responsible for the subsequent changes in neuropeptide expression. Thus, the peptide phenotype of these axotomized neurons is regulated both by the induction of an “injury factor,” leukemia inhibitory factor, as shown previously, and by the reduction in a target-derived growth factor.

Galanin expression is co-regulated in peripheral neurons with that of vasoactive intestinal peptide (VIP) under a variety of conditions. For example, the expression of both increase after explantation of adult rat superior cervical ganglia (SCG). Because VIP participates in a positive feedback loop regulating its own expression, we examined whether VIP also increases galanin expression. Galanin mRNA and peptide are nearly undetectable in the SCG in vivo, but increase dramatically after 24-48 h in organ culture. Addition of VIP or forskolin to the culture medium reduced galanin mRNA expression by 75% and 77%, respectively, and reduced galanin peptide expression by 76% and 82%, respectively, compared with ganglia cultured in control medium. In contrast, isoproterenol stimulation did not significantly alter levels of galanin mRNA or peptide, consistent with previous observations that isoproterenol exerts its effect on SCG non-neuronal cells, but not on neurons. The results indicate that galanin and VIP are differentially regulated in sympathetic neurons by cAMP- elevating agents.

Peripheral neurons begin to express galanin after axotomy. When neurons in the superior cervical ganglion were axotomized near (about 2 mm) from the ganglion, galanin-like immunoreactivity (IR) was maximal within 72 h. Axotomy of neurons in the middle and inferior cervical ganglion complex (MICG), which could be performed 2 cm from the ganglia, led to an additional galanin increase 7 and 14 days later. This second increase was not accompanied by changes in galanin mRNA or the number of galanin-immunostained neurons. Galanin-IR was detectable in a postganglionic trunk of the MICG 2 days after axotomy. At this time, immunoreactive fibers were only seen near the lesion site, while later they were found throughout the trunk. The data suggest that galanin is actively transported toward the site of nerve crush/transection and that the second increase in galanin-IR found in the MICG may be due to a saturation of the axonal transport system.

Neurons in the adult rat superior cervical sympathetic ganglion (SCG) dramatically increase their content of vasoactive intestinal peptide (VIP) and its mRNA after axotomyin vivoand after explantation. Because the VIP gene contains a functional cAMP response element, the effects of cAMP-elevating agents on VIP expression were examined. VIP, forskolin, or isoproterenol increased cAMP accumulation in explanted ganglia. Secretin, a peptide chemically related to VIP, or forskolin increased VIP levels above those seen in ganglia cultured in control medium, whereas treatment with VIP or secretin increased the level of peptide histidine isoleucine (PHI), a peptide coded for by the same mRNA that encodes VIP. VIP or forskolin also increased VIP–PHI mRNA. In contrast, isoproterenol did not alter levels of VIP, PHI, or VIP–PHI mRNA. Although VIP or forskolin increased cAMP levels in both dissociated neurons and in non-neuronal cells, isoproterenol significantly stimulated cAMP accumulation only in the latter. VIP6–28was an effective antagonist of the actions of exogenous VIP on cAMP and VIP–PHI mRNA in neuron-enriched cultures. When adult SCG explants were cultured in defined medium, endogenous VIP immunoreactivity was released. When VIP6–28was added to such cultures, it significantly inhibited the increase in VIP–PHI mRNA that normally occurs. These data indicate that VIP, or a closely related molecule, produced by adult neurons after injury can enhance the expression of VIP. Such a mechanism may prolong the period during which VIP is elevated after axonal damage. The possibility is also discussed that, because VIP is present in preganglionic neurons in normal animals, its release during periods of increased sympathetic nerve activity could alter VIP expression in the SCG.

Axonal damage to adult peripheral neurons causes changes in neuronal gene expression. For example, axotomized sympathetic, sensory, and motor neurons begin to express galanin mRNA and protein, and recent evidence suggests that galanin plays a role in peripheral nerve regeneration. Previous studies in sympathetic and sensory neurons have established that galanin expression is triggered by two consequences of nerve transection: the induction of leukemia inhibitory factor (LIF) and the reduction in the availability of the target-derived factor, nerve growth factor. It is shown in the present study that no stimulation of galanin expression occurs following direct application of LIF to intact neurons in the superior cervical sympathetic ganglion. Injection of animals with an antiserum to nerve growth factor concomitant with the application of LIF, on the other hand, does stimulate galanin expression. The data suggest that the response of neurons to an injury factor, LIF, is affected by whether the neurons still receive trophic signals from their targets.

Publisher Summary The rate-limiting step in catecholamine biosynthesis, the hydroxylation of tyrosine (TH), is regulated primarily by two processes, one involving activation of preexisting TH molecules and the other involving the synthesis of new enzyme molecules. Activation of TH occurs rapidly after a stimulus, is quickly reversible, and involves post-translational modification of preexisting TH molecules. Increasing both cholinergic antagonists by an order of magnitude does not further diminish the increase in TH activity. This unexpected finding eventually led to the discovery that certain neuropeptides cause the activation of TH. Of the large number of neuropeptides that were tested as possible mediators of the noncholinergic transsynaptic regulation of TH, only a subgroup of the peptides of the secretin-glucagon family were found to be candidates. Five peptides from this family, namely secretin, vasoactive intestinal peptide (VIP), peptide histidine isoleucineamide (PHI), rat growth hormone-releasing hormone, and helodermin, increase TH activity acutely. With regard to the mechanisms underlying this peptidergic activation of TH, an important observation was the finding that those peptides that caused TH activation also cause increased levels of cyclic adenosine monophosphate (cAMP) in the SCG, whereas those peptides that have no effect on TH activity have no effect on cAMP levels. To determine whether neuropeptides of the secretin-glucagon family might influence catecholamine biosynthesis not only in adrenergic cells but also in adrenergic nerve terminals, tissues innervated by the SCG were examined, namely, the iris, pineal gland, and submaxillary gland. Incubation of these target tissues with secretin and VIP leads to TH activation.

In response to axonal injury, noradrenergic sympathetic neurons of the adult superior cervical ganglion (SCG) alter their neurotransmitter phenotype. These alterations include increases in the levels of the neuropeptides, galanin, vasoactive intestinal peptide (VIP), and substance P (SP) and a decrease in the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH). Previous studies have indicated that after axotomy in vivo, leukemia inhibitory factor (LIF) plays an important role in increasing the contents of galanin and VIP in the SCG. In the present study, by examining the time courses of the changes in LIF and neuropeptide mRNA and by using LIF null mutant mice, we have determined that LIF alters neuropeptide content in part by increasing levels of peptide mRNA. In addition, LIF also makes a small contribution to the axotomy-induced down-regulation of mRNA encoding TH and neuropeptide Y, both of which are normally expressed at high levels in the SCG. Finally, by using a LIF-blocking antiserum, this cytokine was found to regulate SP expression in an in vitro axonal injury model. Thus, after axotomy, a single factor, LIF, participates in the down-regulation of peptides/proteins involved in normal neurotransmission and the up-regulation of a group of neuropeptides normally not present in the SCG that may be involved in regeneration.

Changes in neuropeptide expression occur in sensory, motor, and sympathetic neurons following axotomy. The particular pattern of peptide changes that occurs varies among the three cell types. We have studied the regulation in the rat superior cervical ganglion of the expression of galanin, a peptide previously shown to increase in axotomized sensory and motor neurons. While normally only an occasional neuron exhibiting galanin-like immunoreactivity is found in this ganglion, at two days after transection of the postganglionic internal and external carotid nerves, immunostaining can be observed in many neurons throughout the ganglion. Similar changes are found when ganglia are placed in organ culture for two days. The distribution of immunostained neurons after section of only one of the postganglionic trunks suggests that changes in galanin-like immunoreactivity occur only within neurons whose axons are transected. None the less, even when both nerve trunks are transected, only about half of the neurons in the ganglion exhibit galanin-like immunoreactivity, indicating that only a proportion of the axotomized neurons exhibit a detectable response. The few immunostained neurons seen after section of the cervical sympathetic trunk may also represent axotomized neurons. Galanin-like immunoreactivity extracted from the ganglion co-chromatographs with authentic galanin, and the level of this immunoreactivity increases dramatically after axotomy and explantation, and modestly after decentralization. These same manipulations produce parallel increases in the level of galanin messenger RNA. Together, the findings indicate that the expression of galanin increases in sympathetic neurons after axotomy. Galanin is thus the first neuropeptide whose expression has been shown to increase after transection of all three types of peripheral axons that have been studied.

Specific and reproducible changes involving the cholinergic and dopaminergic systems have been described in both the aging rodent and the human nervous system. Nevertheless, relatively little information is available on changes in nicotinic cholinergic receptors occurring in normal aging, and there have been few attempts to correlate alterations in receptor densities with changes in nicotinic actions. We have utilized the nicotine-mediated stimulation of endogenous dopamine efflux in a striatal slice preparation as a functional index of responsiveness to nicotine in aging. Following incubation with nicotine, this efflux was significantly lower in 25-month-old (aged) as opposed to 4-month-old (young) rats. In contrast, the release of striatal dopamine following a high-potassium stimulus was similar at both ages. Binding studies in young and aged animals did not reveal any significant change with age in the total number of striatal nicotinic receptors recognized by either [3H]nicotine or the neuronal nicotinic antagonist 125I-neuronal bungarotoxin. However, there was a nearly 80% decline in the subpopulation of striatal nicotinic receptors jointly recognized by both nicotine and neuronal bungarotoxin, but not by alpha-bungarotoxin. Quantitative autoradiography demonstrated declines with age in this receptor subtype in several brain regions examined. Decrements in this specific subpopulation of nicotinic receptors or in the nerve cells expressing these receptors may contribute to the functional declines that take place in the aging motor and visual systems.

Serotonin N-acetyltransferase is regulated in the rat pineal gland by the gland's innervation from the superior cervical ganglion. Norepinephrine has been viewed as the sole transmitter involved in this trans-synaptic regulation; however, a possible role for vasoactive intestinal peptide (VIP) has recently emerged. VIP-like immunoreactivity was extracted from rat pineal glands and shown to co-elute on reverse-phase liquid chromatography with authentic VIP. The level of VIP-like immunoreactivity in the gland was unaffected by prior sympathetic denervation, though its level of neuropeptide Y-like immunoreactivity decreased by 85%. The results indicate that VIP and norepinephrine are not colocalized in sympathetic neurons in the pineal gland and raise questions as to the physiological role of VIP in regulation of pineal function in vivo.

Vasoactive intestinal peptide (VIP)-like immunoreactivity is present at low levels in the superior cervical ganglion of the adult rat, where immunostained neural processes, but only an occasional immunostained cell body, are found. However, when ganglia are maintained for 24 or 48 hr in organ culture, their content of VIP-like immunoreactivity increases 6- or 31-fold, respectively. When examined at 24 hr, the increase in VIP-like immunoreactivity is totally blocked by an inhibitor of RNA or protein synthesis. Many neuronal cell bodies and processes with immunoreactivity for VIP and the related peptide histidine isoleucine amide (PHI) are seen in cultured ganglia. In addition, VIP/PHI mRNA is abundant in cultured ganglia but only barely detectable in ganglia prior to culture. Under the same culture conditions, neuropeptide Y-like immunoreactivity increases to a small extent, and tyrosine hydroxylase activity and total ganglion protein remain unchanged. These results support the idea that adult sympathetic neurons exhibit plasticity in neuropeptide expression and that this plasticity, in the case of VIP, depends on changes in gene expression.

Increased Ca2+ influx serves as a signal that initiates multiple biochemical and physiological events in neurons following depolarization. The most widely studied of these phenomena is the release of neurotransmitters. In sympathetic neurons, depolarization also increases the rate of synthesis of the transmitter norepinephrine (NE), via an activation of the enzyme tyrosine hydroxylase (TH), and this effect also seems to involve Ca2+ entry. We have examined whether the mechanism of Ca2+ entry relevant to TH activation is via voltage-sensitive Ca2+ channels and, if so, whether the type of Ca2+ channel involved is the same as that involved in the stimulation of NE release. We have investigated the isolated rat iris, allowing us to examine transmitter biosynthesis and release in sympathetic nerve terminals in the absence of sympathetic cell bodies and dendrites. Potassium depolarization produced a three- to fivefold increase in TH activity and an approximately 100-fold increase in NE release. Both effects were dependent on Ca2+ being present in the extracellular medium, and both were inhibited by omega-conotoxin (1 microM), which inhibits N-type voltage-sensitive Ca2+ channels. In contrast, the dihydropyridine nimodipine (1-3 microM), which blocks L-type Ca2+ channels, had no effect on either measure. These data support the hypothesis that increases in NE biosynthesis and release in sympathetic nerve terminals during periods of depolarization are both initiated by an influx of Ca2+ through voltage-sensitive Ca2+ channels and that a similar type of Ca2+ channel is involved in both processes.

The distribution of labeled cells in the chaffinch brain has been examined by autoradiography after an intramuscular injection of [ 3 H]testosterone. In the telencephalon labeled cells were particularly concentrated in nucleus magnocellularis neostriatalis anterioris, nucleus septalis lateralis, and hyperstriatum ventrale, pars caudale. In the diencephalon the nucleus periventricularis magnocellularis, the nucleus magnocellularis posterioris and the area infundibularis contained many heavily labeled cells. In the mesencephalon and medulla, labeled cells were found in the nucleus intercollicularis and in the nucleus nervi hypoglossi, respectively. Measurements of the density of grains per cell revealed (1) clear quantitative differences in the extent of labeling in different regions of the brain and (2) a wide variation in the density of labeling of individual cells within a region. The relationship between these sites of hormone binding and known sites of androgen action in the avian brain is discussed.

Electrical stimulation of the preganglionic cervical sympathetic trunk causes an increase in dopa synthesis in the postganglionic neurons in the superior cervical ganglion (SCG). This transsynaptic biochemical effect can be blocked only partially by cholinergic antagonists, suggesting the involvement of a noncholinergic preganglionic sympathetic neurotransmitter(s). A survey of a large number of possible candidates for this neurotransmitter revealed that, in addition to cholinergic agonists, only a small group of peptides (all members of the secretin-glucagon family) stimulated dopa synthesis in the SCG. The effective peptides included vasoactive intestinal peptide (VIP), peptide histidine isoleucine amide (PHI), and secretin. Consequently we looked for the presence of immunoreactivities for these three peptides in the SCG. VIP- and PHI-like immunoreactive fibers were found in the SCG and in its major pre- and postganglionic nerve trunks. The distributions of the two immunoreactivities were very similar. Immunoreactive fibers were seen both singly and in bundles. In some instances, fibers were found apposed to neuronal cell bodies in the ganglion, and occasionally dense plexuses of fibers were found surrounding the neurons. In addition, punctate immunoreactive profiles were found apposed to the neurons in what appeared to be terminal fields. A small number of immunoreactive neuronal cell bodies were also seen in the ganglion. In a few instances, it was possible to establish, in serial sections, that the same cell body was immunostained with both VIP and PHI antisera. No secretin like-immunoreactive fibers or cells were observed. The presence of VIP-like and PHI-like-immunoreactive fibers in the cervical sympathetic trunk and in the SCG strengthens the possibility that these peptides, or a related molecule(s), serve as preganglionic neurotransmitters in this ganglion.

—The period during which trans-synaptic stimulation is required by the rat superior cervical ganglion for induction of tyrosine hydroxylase by reserpine has been studied. Ganglia were decentralized on one side at various times before or after an injection of reserpine. The tyrosine hydroxylase activity of the denervated and control ganglia was assayed 72 h after drug treatment. When decentralization was performed 8 h after an injection of reserpine the increase in tyrosine hydroxylase activity was blocked in the denervated ganglia. Decentralization 12 h after reserpine treatment or later had no effect on the enzyme induction. The actual increase in tyrosine hydroxylase activity occurred between 24 and 48 h after injection of reserpine.

Secretin and vasoactive intestinal peptide (VIP) are known to stimulate tyrosine hydroxylase (TH) activity acutely in the rat superior cervical ganglion (SCG). Because TH-containing neurons in the SCG innervate the iris, submaxillary gland, and pineal gland, we examined the effects of secretin and VIP in these 3 autonomic end organs in vitro. Both peptides stimulated TH activity in each tissue. These stimulations resembled those in the SCG in that (1) secretin displayed a higher potency than VIP in all 3 end organs, (2) the peptide effects were unchanged when calcium was excluded from the incubation medium, and (3) they were mimicked by activators of the cyclic adenosine monophosphate (cAMP) pathway. These findings indicate that secretin and VIP can regulate transmitter metabolism in both the cell bodies and axon terminals of neurons originating in the SCG. Furthermore, the data raise the possibility that catecholamine synthesis in sympathetic nerve terminals is modulated by peptides released by other, nearby nerve endings.

The pineal gland is innervated by sympathetic neurons whose cell bodies are located in the superior cervical ganglia. This pathway, which carries information concerning environmental lighting to the gland, is important for maintaining a circadian rhythm in the activity of the pineal enzyme serotonin N-acetyltransferase (NAT). Lesioning this pathway blocks the normal nighttime increase in NAT activity. Following transplantation of the pineal gland to the anterior chamber of the eye, the gland becomes reinnervated by collateral sprouts of sympathetic neurons that innervate the iris. In addition, a day-night rhythm in NAT activity is restored to the transplanted glands (Moore, 1975; Backstrom et al., 1976). These findings raise the possibility that the neural regulation of pineal function may be restored to normal by collateral sprouts of foreign neurons. To determine whether this is the case, other aspects of the neural regulation of NAT activity were examined in transplanted and in situ pineal glands. When the dark period was extended into the normal light period, NAT activity decreased to daytime values in both groups, suggesting that, in both cases, darkness is only effective in maintaining high levels of NAT activity at certain times during the day-night cycle. In contrast to their similar responses to extended darkness, the 2 groups of pineal glands responded differently to a brief pulse of light during the dark period. While the light exposure caused a dramatic decrease in nighttime NAT activity in in situ pineal glands, it produced no change in this enzyme activity in transplanted glands.(ABSTRACT TRUNCATED AT 250 WORDS)

The activity of serotonin N-acetyltransferase (NATase) in the rat pineal gland exhibits a large (approximately 100-fold) circadian variation, with peak activity occurring in the dark part of the light/dark cycle. Surgical removal of both superior cervical ganglia abolishes this rhythm in enzyme activity. Unilateral ganglionectomy caused a 75% decrease in NATase activity during the dark period immediately following the operation; however, by the subsequent dark period (32 hr after operation) the rhythm in NATase activity had returned to normal. Similar results were found after the internal carotid nerve was cut, and data are presented indicating that this is the postganglionic trunk by which sympathetic neurons reach the pineal gland. Denervation of one superior cervical ganglion (unilateral "decentralization") also produced a 75% decrease in NATase activity during the dark period immediately following the operation; however, after decentralization, enzyme activity did not return to normal in subsequent cycles. It is hypothesized that this recovery is due to loss of norepinephrine uptake sites in the degenerating sympathetic nerve terminals. As a result of decreased norepinephrine uptake, the effectiveness of the norepinephrine released by surviving neurons may be enhanced. This hypothesis is supported by experiments in which pharmacological blockade of norepinephrine uptake in unilaterally decentralized animals increased NATase activity to control levels. We propose that neural systems which use transmitter uptake as the mechanism of transmitter inactivation have a built-in "reserve stimulatory capacity."

Electrical stimulation of the preganglionic cervical sympathetic trunk produces an acute increase in the rate of DOPA synthesis in the rat superior cervical ganglion. The present study was designed to test the possibility that this acute transsynaptic stimulation of catechol biosynthesis could be, at least in part, a consequence of an increase in the firing rate of the postganglionic sympathetic neurons. For this purpose, the effect of stimulation in vitro of the preganglionic cervical sympathetic trunk was compared to that of stimulation of the predominantly postganglionic internal and external carotid nerves. Stimulation of the cervical sympathetic trunk at 10 Hz for 30 min produced a 4.6-fold increase in DOPA synthesis, while simultaneous stimulation of the two postganglionic trunks produced a 3.1-fold increase. The internal carotid nerve is known to contain a small population of preganglionic fibers that synapse on principal neurons in the ganglion before entering this nerve trunk. To eliminate the possibility that the effect of stimulation of the internal carotid nerve is mediated by synaptic stimulation via these preganglionic “through fibers”, the effect of stimulation of previously decentralized ganglia was examined. While decentralization reduced the magnitude of the effect of stimulation of the internal and external carotid nerves, a 2.0-fold increase in DOPA synthesis was still seen. In addition, when these nerve trunks were stimulated in control ganglia that had been maintained in organ culture for 48 h to allow time for the degeneration of afferent nerve terminals, DOPA synthesis increased 4.1-fold. The increases in DOPA synthesis in ganglia in situ produced by both orthodromic and antidromic stimulation were accompanied by increases in the activation of the enzyme tyrosine hydroxylase measured in ganglion homogcnates. In addition, increases in norepinephrine and dopamine levels were seen. As a first step in an examination of the possibility that the increase in catechol synthesis produced by antidromic stimulation results from an influx of Ca2+ ions, decentralized ganglia and ganglia maintained in organ culture were stimulated antidromically in a low Ca2+/high Mg2+ medium. This change in the ionic composition of the medium completely abolished the effects of antidromic stimulation in decentralized ganglia and reduced the effect of antidromic stimulation of organ-cultured ganglia by about 90%. These data indicate that an increase in the firing rate of sympathetic neurons per se, in the absence of synaptic stimulation, can lead to an increase in tyrosine hydroxylase activity, and they raise the possibility that this biochemical change is mediated by an influx of Ca2+ ions.

Incubation of rat superior cervical ganglia with dimethylphenylpiperazinium (30μM) for 30 min resulted in a two-fold increase in tyrosine hydroxylase activity. This effect was completely inhibited by substance P (30 μM) but not by substance P-free acid, kassinin or physalaemin. Neither of these four peptides alone produced any change in the activity of tyrosine hydroxylase. The IC 50 for the inhibitory effect of substance P was approximately 3μM. Substance P did not inhibit the stimulatory effects of bethanechol or vasoactive intestinal peptide on this enzyme activity. Thus substance P, acting at a site which has a different pharmacology than previously characterized substance P receptors, selectively inhibits nicotinic stimulation of tyrosine hydroxylase activity. These data raise the possibility that substance P may modulate the nicotinic regulation of catecholamine synthesis in sympathetic ganglia in vivo .

The pineal gland is innervated by sympathetic neurons whose cell bodies are located in the two superior cervical ganglia and whose axons reach the gland via the two internal carotid nerves (ICNs). Bilateral decentralization of the superior cervical ganglia, produced by lesioning both cervical sympathetic trunks (CSTs), abolishes the circadian rhythm in the activity of the pineal enzyme serotonin N- acetyltransferase (NAT). We have examined the effects on NAT activity of unilaterally cutting the ICN or the CST. During the first night after either operation, nocturnal NAT activity was reduced by 75% compared to controls. However, during the second night after unilaterally cutting the ICN, NAT activity was restored to control values, and normal enzyme activity was seen in these lesioned animals for up to 1 month after this operation. On the other hand, following unilateral decentralization of one superior cervical ganglion, enzyme activity was reduced for at least 5 months. The high enzyme activity in animals with one ICN cut was abolished by cutting the contralateral CST, indicating that the recovery of NAT activity depended on the remaining intact sympathetic neurons. Electrical stimulation of the intact ICN during the daytime in animals in which the contralateral ICN was cut produced an increase in pineal NAT activity which was greater than the increase seen when similar stimulation was performed in sham- operated animals or in animals in which the contralateral superior cervical ganglion had been decentralized. The time course of the recovery of nocturnal NAT activity after unilateral denervation of the pineal gland was similar to the time course of the decrease in norepinephrine uptake sites in the gland.(ABSTRACT TRUNCATED AT 250 WORDS)

The mammalian superior cervical ganglion has been the classical preparation for studying cholinergic transmission between neurones1–3. Recently, however, evidence has been presented showing that, in addition to the postsynaptic changes mediated via nicotinic and muscarinic receptors, there is a non-cholinergic component to transmission in this ganglion4,5, as in frog paravertebral ganglia6,7. In the rabbit superior cervical ganglion, Ashe and Libet recorded a late, slow excitatory postsynaptic potential in response to preganglionic nerve stimulation in the presence of nicotinic and muscarinic antagonists4. We have found, in the rat superior cervical ganglion, that a postsynaptic biochemical consequence of preganglionic nerve stimulation, namely the acute activation of tyrosine 3-monooxygenase (tyrosine hydroxylase, TH; EC 1.14.16.2), is mediated in part by acetylcholine and in part by a non-cholinergic neurotransmitter5. The regulation of this enzyme activity is of particular interest because it catalyses the rate-limiting step in the biosynthesis of the postganglionic neurotransmitter, noradrenaline. In the present paper, we report that the relative importance of cholinergic and non-cholinergic transmission in the regulation of TH activity varies with the pattern of electrical stimulation of the preganglionic nerve trunk.

The effect of synaptic stimulation on tyrosine hydroxylase [tyrosine 3-monooxygenase: L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] activity in the rat superior cervical ganglion was studied. The preganglionic cervical sympathetic trunk was stimulated unilaterally at 10 Hz for 30 min. Forty-eight hours later tyrosine hydroxylase activity was 33% higher on the stimulated than on the control side. The enzyme activity restimulated than on the control side. The enzyme activity remained elevated in the stimulated ganglia for 2 days. No change was observed in total ganglion protein. Comparable increases in tyrosine hydroxylase activity were observed in anesthetized and conscious animals.

Androgen-concentrating cells were found in the midbrain of the chaffinch Fringilla coelebs by autoradiography using tritiated testosterone. Labeled cells were localized primarily in the nucleus intercollicularis, an area from which vocalizations can be electrically stimulated in birds. These autoradiographic results suggest that the nucleus intercollicularis is a site in the action of androgens on avian vocal behavior.