Transmission pathways in the rat superior cervical ganglion

On isolated preparations of the superior cervical ganglion (SCG, n = 8) taken from 21-day-old rats, we studied the intraganglion pathways and mechanisms underlying generation of synaptic responses of SCG neurons to antidromic stimulation. One of the three nerves connected with the SCG was stimulated, and compound action potentials were recorded simultaneously from the other two nerves; then, the order of stimulated and recorded nerves was changed. Orthodromic stimulation of the cervical sympathetic nerve (CSN) evoked responses in the internal carotid nerve (ICN), which could be completely blocked by hexamethonium, and responses in the external carotid nerve (ECN), which contained a component that was not blocked by this of the ECN caused responses in the CSN, which were not blocked by hexamethonium. Effects of superfusion of the SCG with a Ca²⁺-free solution allowed us to conclude that the hexamethonium-insensitive component of the responses in the CSN and ECN and ECN-CSN conduction can be explained by the presence of direct fibers going from the CSN to the ECN with no synaptic relay. Possible mechanisms underlying antidromic stimulation-induced synaptic responses in SCG neurons are discussed. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 396–399, July–October, 2007.     

Chronic exposure to a 60-Hz electric field: effects on synaptic transmission and peripheral nerve function in the rat

Several reports have suggested that the nervous system can be affected by exposure to electric fields and that these effects may have detrimental health consequences for the exposed organism. The purpose of this study was to investigate the effects of chronic (30-day) exposure of rats to a 60Hz, 100-kV/m electric field on synaptic transmission and peripheral-nerve function. One hundred forty-four rats, housed in individual polycarbonate cages were exposed to uniform, vertical, 60-Hz electric fields in a system free of corona discharge and ozone formation and in which the animals did not receive spark discharges or other shocks during exposure. Following 30 days of exposure to the electric field, superior cervical sympathetic ganglia, vagus and sciatic nerves were removed from rats anesthetized with urethan, placed in a temperature-controlled chamber, and superfused with a modified mammalian Ringer's solution equilibrated with 95% O2 and 5% CO2. Several measures and tests were used to characterize synaptic transmission and peripheral-nerve function. These included amplitude, area, and configuration of the postsynaptic or whole-nerve compound-action potential; conduction velocity; accommodation; refractory period; strength-duration curves; conditioning-test (C-T) response, frequency response; post-tetanic response; and high-frequency-induced fatigue. The results of a series of neurophysiologic tests and measurements indicate that only synaptic transmission is significantly and consistently affected by chronic (30-day) exposure to a 60-Hz, 100-kV/m electric field. Specifically, and increase in synaptic excitability was detected in replicated measurements of the C-T response ratio. In addition, there are trends in other data that can be interpreted to suggest a generalized increase in neuronal excitability in exposed animals.     

Chronobiological Studies on the Hypotensive Effect of Prostaglandin E2 and Arachidonic Acid in the Rat

The present study was designed to determine whether biological rhythm variations could be detected in the hypotensive action of prostaglandin E₂ (PGE₂) and arachidonic acid (AA) in normal rats. Doses of 1.0 μg kg^-1 of PGE₂ or 0.5 mg kg^-1 of AA were administered to pentobarbital-anesthetized rats at 6 times of the day. Maximal reduction of systolic and diastolic blood pressures was obtained when PGE₂ or AA were administered to rats between 0930 and 1200. The lowest falls in blood pressure were found when the same doses of the two substances were injected between 0300 and 0500. Mechanisms to explain these circadian variations are suggested.     

Analysis of the Late Steps of Exocytosis: Biochemical and Total Internal Reflection Fluorescence Microscopy (TIRFM) Studies

1. Time with Julie in his laboratory at the NIH in the early 1970s is remembered. The experience led to a life-long interest in the regulation of catecholamine secretion. Here are summarized aspects of this work.2. The relationship between ATP-dependent priming of exocytosis and the polyphosphoinositides is reviewed. In addition, studies are summarized in which total internal reflection fluorescent microscopy (TIRFM) was used to visualize secretory granule behavior before exocytosis and individual exocytotic events.3. Quantitative optical analysis indicates that chromaffin granule motion is highly restricted but regulated. Granules can undergo significant motion in the 100 ms prior to fusion and interactions with the plasma membrane leading to fusion can occur within this time. The small motions may permit granules adjacent to the plasma membrane to repetitively sample microdomains of the plasma membrane, thereby increasing the probability of fruitful interactions that lead to fusion.     

Chronobiological Studies on the Hypotensive Effect of Prostaglandin E2 and Arachidonic Acid in the Rat

The present study was designed to determine whether biological rhythm variations could be detected in the hypotensive action of prostaglandin E₂ (PGE₂) and arachidonic acid (AA) in normal rats. Doses of 1.0 μg kg^?1 of PGE₂ or 0.5 mg kg^?1 of AA were administered to pentobarbital-anesthetized rats at 6 times of the day. Maximal reduction of systolic and diastolic blood pressures was obtained when PGE₂ or AA were administered to rats between 0930 and 1200. The lowest falls in blood pressure were found when the same doses of the two substances were injected between 0300 and 0500. Mechanisms to explain these circadian variations are suggested.     

Insights on the role of adhesion related molecules on stem cells

The development of the peripheral nervous system (PNS) is a highly dynamic process, during which motor and sensory axons innervate distal targets, such as skeletal muscles and skin. Axonal function depends critically on support from Schwann cells, the main glial cell type in the PNS. Schwann cells originate from the neural crest, migrate along outgrowing axons and associate with axons along their entire length prior to ensheathment or myelination. How axonal growth and the migration of Schwann cells is coordinated at the level of reciprocal axon-glial signaling is the fascinating subject of ongoing research. Neuregulin-1 (NRG1) type III, an axonal membrane-bound ligand for receptor tyrosine kinases of the ErbB family, acts as a “master regulator” of peripheral myelination. In addition, NRG1-ErbB signaling directs the development of the Schwann cell lineage and regulates the proliferation and survival of Schwann cells. Studies in zebrafish have identified a direct role of NRG1 type III in Schwann cell migration, but to what extend NRG1 serves a similar function in the mammalian PNS is not clear. We have employed a mouse superior cervical ganglion explant culture system, in which the migration of endogenous Schwann cells along outgrowing axons can be visualized by time-lapse imaging. Using this approach, we found that NRG1 type III-ErbB signaling regulates the colonization of distal axonal segments by Schwann cells. However, our data suggest an indirect effect of NRG1 type III-ErbB signaling via the support of Schwann cell survival in proximal axonal regions rather than a direct effect on Schwann cell motility.     

Distribution of pluripotent neural crest cells in the embryo and the role of brain-derived neurotrophic factor in the commitment to the primary sensory neuron lineage

Many early migratory neural crest cells are pluripotent in the sense that their progeny are able to generate more than one differentiated phenotype (Sieber-Blum and Cohen, 1980, Dev. Biol. 80:95–106; Baroffio, Dupin, and Le Douarin, 1988, Proc. Natl. Acad. Sci. USA 85:5325–5329; Bronner-Fraser and Fraser, 1988, Nature 335:161–164; Sieber-Blum, 1989a, Science 243:1608–1611; Ito and Sieber-Blum, 1991, Dev. Biol. 148:95–106). At trunk levels, the neural crest contains two classes (Sieber-Blum and Cohen, 1980) and at posterior rhombencephalic levels, three different classes of pluripotent cells (Ito and Sieber-Blum, 1991). We investigated cell differentiation by in vitro clonal analysis to determine when in development the pool of pluripotent neural crest cells becomes exhausted. The data suggest that different classes of pluripotent cells, precursor cells with more restricted developmental potentials, and apparently committed cells, exist at sites of advanced migration (posterior branchial arches) and even at target sites of neural crest cell differentiation [posterior branchial arches, dorsal root ganglia (DRG), sympathetic ganglia (SG), and epidermal ectoderm]. Some putative classes of pluripotent cells persist well into the second half of embryonic development. These observations have implications for our understanding of the mechanisms that control neural crest cell migration and differentiation. They support the idea that cues originating from the microenvironment affect differentiation of pluripotent neural crest cells. One such signal appears to be brain-derived neurotrophic factor (BDNF). In the presence of BDNF, but not nerve growth factor (NGF), there is a significant increase in the number of neural crest cells per colony that express a sensory neuron-specific marker. Because this increase is not accompanied by a corresponding increase in the total number of cells per colony, this suggests that BDNF plays a role in cell type specification. © 1993 John Wiley & Sons, Inc.     

Phase shifts in circadian peripheral clocks caused by exercise are dependent on the feeding schedule in PER2::LUC mice

Circadian rhythms are regulated by the suprachiasmatic nucleus (SCN) clock, which is the main oscillator and peripheral clock. SCN clock can be entrained by both photic and non-photic stimuli, and an interaction exists between photic and non-photic entrainment. Moreover, peripheral circadian clocks can be entrained not only by scheduled restricted feeding, but also by scheduled exercise. Thus, the entrainment of peripheral circadian clocks may be the result of an interaction between the entrainment caused by feeding and exercise. In this study, we examined the effect of wheel-running exercise on the phase of the peripheral clocks (kidney, liver and submandibular gland) in PER2::LUC mice under various feeding schedules. Phase and waveforms of the peripheral clocks were not affected by voluntary wheel-running exercise. Exercise for a period of 4 h during the early dark period (morning) delayed the peripheral clocks, while exercise for the same duration during the late dark period (evening) advanced the peripheral clocks. The feeding phase was advanced and delayed by evening and morning exercise, respectively, suggesting that the feeding pattern elicited by the scheduled exercise may entrain the peripheral clocks. Exercise did not affect the phase of the peripheral clock under the 1 meal per day schedule. When the phase of the peripheral clocks was advanced by the feeding schedule of 2 or 4 meals per day during light and/or dark periods, wheel-running exercise during the morning period significantly and equally shifted the phase of all organs back to the original positions observed in mice maintained under free-feeding conditions and with no exercise. When the schedule of 2 meals per day during the dark period failed to affect the phase of peripheral clock, morning exercise did not affect the phase. Wheel-running exercise increased the levels of serum corticosterone, and the injection of dexamethasone/corticosterone instead of exercise shifted a phase that had advanced under the feeding schedule of 2 meals per day, back to the normal position. The liver and submandibular glands exhibit higher sensitivity to dexamethasone than the kidneys. In adrenalectomized mice, treadmill-induced normalization of the advanced phase under a feeding schedule of 2 meals per day was not observed. In summary, scheduled exercise-induced phase shifts were weaker compared to scheduled feeding-induced phase shifts. The phase advance caused by the feeding schedule of 2 or 4 meals per day was suppressed by wheel-running, treadmill exercise or dexamethasone/corticosterone injection in the early dark period (morning). Corticosterone release may be involved in exercise-induced phase shift of peripheral clocks. These results suggest that there is an interaction between the phase shifts caused by feeding and exercise schedules in peripheral clocks.     

Expression of vitamin D receptors in the superior cervical ganglia of rats

We investigated the role of vitamin D in the sympathetic nervous system including the distribution of vitamin D receptors (VDR), 1α-hydroxylase and 24-hydroxylase (CYP24) in neuronal subpopulations and satellite glia in the superior cervical ganglia (SCGs) of rats using immunohistochemistry. VDR immunoreactivity was observed in the cytoplasm and nucleus of nearly all neurons in the SCG. Intensity of VDR fluorescence was significantly greater in the cytoplasm of neuropeptide Y (NPY) negative somata compared to NPY positive neurons. Immunoreactivity for 1α-hydroxylase also was observed in the cytoplasm of all neurons of the SCG, but the intensity of fluorescence was less in the nuclei. To the contrary, the immunoreactivity for CYP24 was stronger in the nuclei, although it was present at lower intensity also in the cytoplasm of neurons. VDR and 1α-hydroxylase immunofluorescence was observed in many non-neuron cells, except satellite glial cells, which exhibited weak CYP24 immunofluorescence. Expression of VDRs and key metabolizing enzymes indicated the importance of vitamin D in the autonomic nervous system and the ability of sympathetic neurons to activate and deactivate vitamin D for its autocrine and paracrine roles.     

The role of circadian rhythm on the pharmacokinetic of methotrexate in streptozotocin-induced diabetes mellitus rats

Chronopharmacokinetic studies have been conducted both in animals and humans. Anticancer agents are of great interest due to their narrow therapeutic range and large pharmacokinetic variability. It was reported that the pharmacokinetics of MTX showed a circadian rhythm in rats and humans. Since diabetes-induced physiological changes can affect pharmacokinetics of drugs, it was reported that MTX blood concentration in diabetic rats was higher than that of the control groups. The present study was designed to elucidate whether these diabetes-induced changes in pharmacokinetics occurred during the day and thus administered MTX at four different times in streptozotocin-induced diabetes mellitus (SIDM) rats. Blood samples were drawn at 5, 15, 30, and 60 min after IV infusion of MTX in both the SIDM and control groups. Control and SIDM Area under the concentration - time curve (AUC) values showed a significant circadian rhythm with a peak located in mid-dark phase at 14:00. Clearance values were significantly low at 14:00 in the diabetic group when compared to other periods and the control group. The MTX AUC was increased when treatment with dexamethasone was given to suppress the endogenous production of corticosterone in both control and SIDM rats. These results suggest that the extent of MTX pharmacokinetics varies with the time of day in the SIDM rats and these variations might be related to changes in corticosterone concentrations.     

Bone morphogenetic protein down-regulation of neuronal pituitary adenylate cyclase-activating polypeptide and reciprocal effects on vasoactive intestinal peptide expression

Among bone morphogenetic proteins (BMPs), the decapentaplegic (Dpp; BMP2, BMP4) and glass bottom boat (Gbb/60A; BMP5, BMP6, BMP7) subgroups have well-described functions guiding autonomic and sensory neuronal development, fiber formation and neurophenotypic identities. Evaluation of rat superior cervical ganglia (SCG) post-ganglionic sympathetic neuron developmental regulators identified that selected BMPs of the transforming growth factor beta superfamily have reciprocal effects on neuronal pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) expression. Dpp and Gbb/60A BMPs rapidly down-regulated PACAP expression, while up-regulating other sympathetic neuropeptides, including PACAP-related VIP. The suppressive effects of BMP on PACAP mRNA and peptide expression were potent, efficacious and phosphorylated mothers against decapentaplegic homolog (Smad) signaling-dependent. Axotomy of SCG dramatically increases PACAP expression, and the possibility that abrogation of inhibitory retrograde target tissue BMP signaling may contribute to this up-regulation of sympathetic neuron PACAP was investigated. Replacement of BMP6 to SCG explant preparations significantly blunted the injury-induced elevated PACAP expression, with a concomitant decrease in sympathetic PACAP-immunoreactive neuron numbers. These studies suggested that BMPs modulate neuropeptide identity and diversity by stimulating or restricting the expression of specific peptidergic systems. Furthermore, the liberation of SCG neurons from target-derived BMP inhibition following axotomy may be one participating mechanism associated with injury-induced neuropeptidergic plasticity.     

Effects of Denervation and Axotomy on Nervous System-Specific Protein, Ornithine Decarboxylase, and Other Enzyme Activities in the Superior Cervical Sympathetic Ganglion of the Rat

The time courses of changes of three enolase isozymes (alpha alpha, alpha gamma, and gamma gamma), S-100 protein, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), ornithine decarboxylase (ODC), beta-galactosidase, and glucose-6-phosphate dehydrogenase (G6PDH) were examined from 1 to 14 days after cutting of the preganglionic nerve (denervation) or the postganglionic nerve (axotomy) of the superior cervical sympathetic ganglion (SCG) of the rat. The wet weight and protein content in the axotomized SCG increased continuously, to nearly twice those of the denervated SCG for 1-2 weeks after the operations. Among enolase isozymes in the SCG, neuron-specific gamma gamma-enolase decreased rapidly after denervation and stayed at a low level for 2 weeks, whereas the isozyme remained almost unchanged after axotomy. On the contrary, ganglionic alpha alpha-enolase and the alpha gamma-hybrid form increased remarkably to reach a maximum at the second day after axotomy, and remained above control for 1 to 2 weeks; these two enolase isozymes showed little change after denervation. Denervation caused a much larger increase than did axotomy in the ganglionic S-100 protein, an astrocyte-specific protein, during the first week after the operation, while the protein content decreased after 2 weeks of either denervation or axotomy. CNPase, a myelin-associated enzyme, rose suddenly 2 days after axotomy, and remained at a rather high level compared with the denervated ganglion, which showed little variation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Extracellular Choline Concentration and K+ Depolarization on Choline Kinase and Choline Acetyltransferase Activities in Superior Cervical Sympathetic Ganglia Excised from Rats

The activities of choline kinase (CK) and choline acetyltransferase (ChAT) were examined in vitro in superior cervical sympathetic ganglia (SCG) excised from rats following aerobic incubation for 1 h in a medium containing various choline concentrations, with and without application of a high KCl level (70 mM). Ganglionic CK activity was strongly inhibited (by approximately 75%) at low extracellular choline concentrations (1-5 microM) but rose as the choline concentration was raised to 10-50 microM in the incubation medium, then fell and rose again with further increases in choline concentration. A similar but moderate accelerative effect on ganglionic CK activity was also observed after addition of acetylcholine (ACh; 1 mM) without eserine. Whereas specific CK activity did not change significantly in axotomized SCG, in which the ratio of glial cells to neurons is greatly increased for a week after the operation., it was remarkably increased after denervation, in which the preganglionic cholinergic nerve terminals had degenerated. When either a high KCl level or hemicholinium-3 (HC-3; 50 microM) was added to the medium in the presence or absence of choline, ganglionic CK activity was markedly inhibited. On the other hand, ChAT activity in the SCG remained at a significantly high level during incubation with low choline concentrations (1-10 microM), but the enhanced enzyme activity became inhibited as the extracellular choline concentration was raised to 50-100 microM in the medium. Addition of HC-3 to the medium did not alter ganglionic ChAT activity at low choline concentrations. However, application of quinacrine (10 microM) considerably reduced ganglionic CK activity and also suppressed ChAT activity induced by high KCl levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of noradrenaline on the GABA response in rat isolated spiral ganglion neurons in culture

In the present study, the modulatory effects of noradrenaline (NA) on the GABA response were investigated in the isolated cultured spiral ganglion neurons of rat by using nystatin perforated patch recording configuration under voltage-clamp conditions. NA reversibly depressed GABA response in a concentration-dependent manner and neither changed the reversal potential of the GABA response nor affected the apparent affinity of GABA to its receptor. alpha2-adrenoceptor agonist and antagonist, clonidine and yohimbine mimicked and blocked the NA action on the GABA response, respectively. N-[2(methylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride (H-89), a protein kinase A inhibitor, mimicked the effect of NA on the GABA response. NA failed to affect the GABA response in the presence of both cAMP and protein kinase A modulator. However, NA still depressed the GABA response even in the presence of both phorbol-12-myristate-13-acetate, a protein kinase C activator and chelerythrine, a protein kinase C inhibitor. These results suggest that the NA suppression of the GABA response is mediated by alpha2-adrenoceptor which reduces intracellular cAMP formation through the inhibition of adenylyl cyclase. Therefore, NA input to the spiral ganglion neurons may modulate the auditory transmission by affecting the GABA response.     

Comparative Studies on the Orcadian Rhythm of Corticosterone Lipid and Cholesterol Levels in Adrenals and Blood of Rats

The time-dependent relationship of corticosterone, lipids and cholesterol over a 48-hr period was studied in the adrenals and blood of rats. In addition an attempt was made to determine whether there was a reciprocal dependence among these compounds and also a correlation between corticosterone and cholesterol in the adrenals and blood.

Corticosterone and cholesterol exhibit a circadian rhythm in the adrenals and blood. The same is true for lipids in the serum. A reciprocal dependence between the compounds in the adrenals and blood could not be demonstrated. Only the time-dependent processes of the corticosterone content in the adrenals and plasma are well correlated with each other. High levels of these steroids in the adrenals are associated with high levels of these steroids in the plasma and vice versa.

An inverse correlation between corticosterone and cholesterol exists in the adrenals and in blood. Maximal levels of corticosterone correspond to minimal levels of cholesterol and vice versa.     

Comparative Studies on the Orcadian Rhythm of Corticosterone Lipid and Cholesterol Levels in Adrenals and Blood of Rats

The time-dependent relationship of corticosterone, lipids and cholesterol over a 48-hr period was studied in the adrenals and blood of rats. In addition an attempt was made to determine whether there was a reciprocal dependence among these compounds and also a correlation between corticosterone and cholesterol in the adrenals and blood.

Corticosterone and cholesterol exhibit a circadian rhythm in the adrenals and blood. The same is true for lipids in the serum. A reciprocal dependence between the compounds in the adrenals and blood could not be demonstrated. Only the time-dependent processes of the corticosterone content in the adrenals and plasma are well correlated with each other. High levels of these steroids in the adrenals are associated with high levels of these steroids in the plasma and vice versa.

An inverse correlation between corticosterone and cholesterol exists in the adrenals and in blood. Maximal levels of corticosterone correspond to minimal levels of cholesterol and vice versa.     

Effects of Colchicine Application to Preganglionic Axons on Choline Acetyltransferase Activity and Acetylcholine Content and Release in the Superior Cervical Ganglion

Abstract: These experiments investigate the effect of block, by colchicine, of fast axonal transport in the cat's cervical sympathetic trunk (CST) on the superior cervical ganglion's choline acetyltransferase (ChAT) enzyme activity, acetylcholine (ACh) content, and ACh release. Electron microscopy on the segment of the CST exposed to colchicine 1 or 4 days earlier showed disappearance of microtubules and accumulation of vesicles and smooth membrane tubules but no disruption of the axonal cytomatrix. At 4 days following colchicine treatment, the number and size of synaptic boutons per grid square in the ganglion ipsilateral to the colchicine-treated CST were similar to those in the control ganglion. At 2 and 4 days following exposure of the CST to colchicine, ChAT activity in the ipsilateral ganglion was reduced to 76 ± 8 and 54 ± 6% of control values, respectively. ACh stores in the ganglia were also reduced (to 81 ± 6% of control values at 2 days and to 51 ± 5% of control values at 4 days). Ganglionic transmission and its sensitivity to blockade by hexamethonium during 2-Hz CST stimulation were not impaired at day 4 postcolchicine. ACh release evoked by 2-Hz stimulation of colchicine-treated axons was similar to release from untreated axons, despite the decrease in the ganglionic ACh content. In contrast, ACh release evoked by 20-Hz stimulation was depressed. The amount of ACh released during 5-Hz stimulation in the presence of vesamicol by the terminals of colchicine-treated axons was similar to that released by the terminals of untreated axons. These results suggest the following conclusions: (a) Colchicine-sensitive fast axonal transport contributes significantly to maintaining ChAT stores in preganglionic axon terminals. (b) The half-life of ChAT in sympathetic preganglionic terminals is ～4 days. (c) One consequence of colchicine-induced block of axonal transport is a reduced ACh content of preganglionic nerve terminals. (d) This decrease in ACh content appears to be the result of a loss in a reserve transmitter pool, whereas the size of the readily releasable compartment is maintained.