Rat Superior Cervical Ganglion Cells Research Articles

Special AT-rich sequence-binding protein-2 (Satb2) — Independent initiation of cholinergic sweat gland innervation in rat in vivo

The transcription factor special AT-rich sequence-binding protein-2 (Satb2) is induced and critical for the establishment of the cholinergic phenotype in rat superior cervical ganglion cells in culture during switching from a noradrenergic to the cholinergic phenotype upon exposure to soluble differentiation factors. To obtain evidence for Satb2 determining the sympathetic cholinergic sudomotor phenotype also in vivo, we traced the postnatal expression profile of Satb2 in the rat stellate ganglion in comparison to the vesicular acetylcholine transporter (VAChT), choline acetyltransferase (ChAT), and the neuropeptide calcitonin gene-related peptide (CGRP), all required for sudomotor function in adult rats.

Acute isolation of rat superior cervical ganglion cells and identification of the electrophysiological properties

Objective To establish a method for acute isolation of rat superior cervical ganglion (SCG) cells and identify the electrophysiological properties.Methods Sprague-Dawley rats of both sexes,aged 5-12 days,were decapitated.The SCGs were removed quickly,and the single SCG cell was enzymatically isolated from the SCGs.When the holding potential was - 60 mV,100 μmol/L acetylcholine was applied and the nicotinic acetylcholine receptor currents were recorded by whole-cell patch-clamp technique.When the holding potential was 760 mV,65 mmol/L KCl was applied and quantal release of catecholamines was detected by using carbon fiber electrodes.Results SCG cells with normal electrophysiological properties were isolated.Typical nicotinic acetylcholine receptor currents and quantal release of catecholamines were recorded successfully.Conclusion The cells suitable for patch-clamp experiments can be obtained by using the method for acute isolation of rat SCG cells. Key words: Ganglia, sympathetic; Neurons, efferent; Cell separation; Electrophysiology

Statins prevent cholinesterase inhibitor blockade of sympathetic α7-nAChR-mediated currents in rat superior cervical ganglion neurons

Statins are reported to be beneficial in treating a multitude of disorders including dementia due to Alzheimer disease (AD) and vascular dementia (VaD) with varying, yet-to-be determined mechanisms of actions. Although cholinesterase inhibitors (ChEIs) are still recommended as the primary drug of choice for AD and related diseases, their efficacy is frequently questioned. We recently reported that alpha7-neuronal acetylcholine nicotinic receptor (alpha7-nAChR)-mediated neurogenic vasodilation of porcine cerebral arteries was blocked by ChEIs, and this blockade was prevented by statin pretreatment. The exact mechanism of interaction between ChEIs and statins remains unclear. Activation of alpha7-nAChRs located on perivascular postganglionic sympathetic nerve terminals releases norepinephrine, which then acts on presynaptic beta(2)-adrenoceptors located on neighboring nitrergic nerve terminals, resulting in nitric oxide release and vasodilation. The present study, therefore, was designed to determine whether interaction of ChEIs and statins occurs at the alpha7-nAChR level. We examined effects of concurrent application of ChEIs and statins on alpha7-nAChR-mediated inward currents in primary neuronal cultures of rat superior cervical ganglion cells, the origin of the perivascular sympathetic innervation to the cerebral arteries. The results indicated that physostigmine, neostigmine, and galantamine inhibited choline- and nicotine-induced whole cell currents in a concentration-dependent manner. This inhibition, which was noncompetitive in nature, was prevented by concurrent application of mevastatin and lovastatin in a concentration-dependent manner. These results suggest that statins protect alpha7-nAChR function directly at the receptor level. Since alpha7-nAChR is neuroprotective, having beneficial effects on memory and cerebral vascular function, its functional inhibition by ChEIs may explain in part the limitation of its effectiveness in AD and VaD therapy. Protection of alpha7-nAChR function from ChEI inhibition by concurrent administration of statins may provide an alternative strategy in improving the efficacy of AD and VaD therapy.

SERCA function declines with age in adrenergic nerves from the superior cervical ganglion.

1. Intracellular calcium is a universal second messenger integrating numerous cellular pathways. An age-related breakdown in the mechanisms controlling [Ca2+]i homeostasis could contribute to neuronal degeneration. One component of neuronal calcium regulation believed to decline with age is the function of sarco/endoplasmic reticulum calcium ATPase (SERCA) pumps. 2. Therefore we investigated the impact of age on the capacity of SERCA pumps to control high (68 mM) [K+]-evoked [Ca2+]i-transients in acutely dissociated superior cervical ganglion (SCG) cells from 6- and 20-month-old Fisher-344 rats. Calcium transients were measured by fura-2 microfluorometry in the presence of vanadate (0.1 microM) to selectively block plasma membrane calcium ATPase (PMCA) pumps, dinitrophenol (100 microM) to block mitochondrial calcium uptake and extracellular sodium replaced with tetraethylammonium to block Na+/Ca2+-exchanger, thus forcing the neuronal cells to rely on SERCA uptake to control [Ca2+]i homeostasis. 3. In the presence of these calcium buffering blockers, the rate of recovery of [Ca2+]i was significantly slower and time to recover to approximately 90% of resting [Ca2+]i was significantly greater in SCG cells from old (20 months) compared with young (6 months) animals. 4. This age-related change in the recovery phase of [K+]-evoked [Ca2+]i-transients could not be explained by differences in the sensitivity of SCG cells to the calcium buffering blockers, as no age-related difference in basal [Ca2+]i was observed. 5. These studies illustrate that when rat SCG cells are forced to rely on SERCAs to buffer [K+]-evoked [Ca2+]i-transients, an age-related decline in SERCA function is revealed. Such age-related declines in calcium regulation coupled with neuronal sensitivity to calcium overload underscore the importance of understanding the components of [Ca2+]i homeostasis and the functional compensation that may occur with advancing age.

Sodium overload through voltage-dependent Na + channels induces necrosis and apoptosis of rat superior cervical ganglion cells in vitro

Using the failure to exclude trypan blue as a criterion for cell death, we found that veratridine, the voltage-dependent Na + channel activator, exerted its toxicity to cultured sympathetic neurons in a dose-dependent manner (half-maximal toxicity occurred at 2 μM). The co-presence of tetrodotoxin completely reversed the toxicity only at concentrations of veratridine <20μM. Veratridine neurotoxicity was due to the influx of Na +; a medium low in Na + (36 mM) completely abolished its neurotoxicity, whereas a Ca 2+-free medium did not attenuate its neurotoxicity. Furthermore, the buffering action of 1,2-Bis-(2-aminophenoxy)ethane-N,N,N′,N′,-tetraacetate (BAPTA) on veratridine-induced increase in intracellular Ca 2+ levels neither blocked veratridine neurotoxicity in normal medium, nor attenuated the low Na + effect. Elevated K + effectively blocked veratridine neurotoxicity in a Ca 2+-dependent manner. Cytoplasmic pH measurements using a fluorescent pH indicator demonstrated that cellular acidification (from pH 7.0 to pH 6.5) occurred upon treatment with veratridine. Both veratridine-induced acidification and cell death were ameliorated by 5-(N-ethyl-N-isopropyl)amiloride, the specific inhibitor of the Na +/H + exchanger (IC 50 = 0.5 μM). Finally, necrosis occurred predominantly in veratridine neurotoxicity, but both staining with bis-benzimide and TUNEL analysis showed nuclear features of apoptosis in sympathetic neurons undergoing cell death.

The role of ryanodine receptors in the cyclic ADP ribose modulation of the M-like current in rodent m1 muscarinic receptor-transformed NG108-15 cells.

1. The role of cyclic ADP ribose and ryanodine receptors in the inhibition of the M-like current (IK(M,ng)) by acetylcholine was investigated in m1 muscarinic receptor-transformed mouse neuroblastoma-rat glioma hybrid (NG108-15) cells using patch-clamp techniques and calcium microfluorimetry. 2. Acetylcholine (1-100 microM) decreased IK(M,ng) by up to 55 %. Application, via the patch pipette, of the cyclic ADP ribose antagonists 8-amino-cyclic ADP ribose (10-100 microM) and 8-bromo-cyclic ADP ribose (100-1000 microM) reduced this inhibition of IK(M,ng) in a concentration-dependent manner. The half-maximal inhibition concentrations for 8-amino- cyclic ADP ribose and 8-bromo-cyclic ADP ribose were around 40 microM and 1 mM, respectively. 3. Neither of the cyclic ADP ribose antagonists altered the amplitude of IK(M,ng) per se, or the incidence of the concurrent Ca2+-activated K+ current (IIK(Ca)) activation, also mediated by acetylcholine. 4. The ryanodine receptor modulators ryanodine (1-10 microM) and Ruthenium Red (10 microM) did not alter IK(M,ng) amplitude or IK(M,ng) inhibition mediated by acetylcholine. There was a statistically significant increase in the proportion of cells showing outward currents in the presence of Ruthenium Red. 5. Intracellular calcium levels measured with fura-2 microfluorimetry were increased with low concentrations of ryanodine (1 microM), more consistently with caffeine (10 mM), and in almost every case with both bradykinin (300 nM) and acetylcholine (100 microM). Caffeine-, but not bradykinin-evoked responses were abolished by preincubation with ryanodine (10 microM). 6. The fast 'rundown rate' of the M-current recorded in rat superior cervical ganglion cells under whole-cell conditions precluded an investigation of the effects of intracellular dialysis of cyclic ADP ribose. However, when cyclic ADP ribose (5 microM) was applied directly to the cytoplasmic face of inside-out membrane patches excised from rat superior cervical ganglion cells containing M-channels, it had no effect on the main parameters of single channel activity (conductance, mean open time or frequency of opening). 7. These results indicate that cyclic ADP ribose acts on a specific intracellular site to mediate IK(M,ng) inhibition. However, unlike previously established effects of cyclic ADP ribose, the ryanodine receptor is not required, suggesting that another molecular target may be involved. Studies at the single channel level indicate that cyclic ADP ribose may not act directly on the M-channels in inside-out patches.

On-going and reflex synaptic events in rat superior cervical ganglion cells.

1. Synaptic events evoked by brief noxious cutaneous stimuli were recorded in sympathetic neurones in the superior cervical ganglion of anaesthetized rats. 2. On-going excitatory synaptic potentials (ESPs) and/or action potentials (APs) were recorded in 69% of neurones at mean frequencies that varied from 0.01 to 6.3 Hz in different cells. From histograms of ESP amplitude during membrane hyperpolarization, it appears that most cells received one (52%), or two or more (36%), suprathreshold inputs and several subthreshold inputs with overlapping amplitudes. 3. Pinching the skin for 1-3 s evoked either a brief burst of synaptic events (lasting about 300 ms) preceding a few seconds of inhibition (burst-inhibitory (BI) neurones), or simply an excitation (excitatory (E) neurones), or no response (O neurones). In 60% of BI neurones, a second burst occurred after the end of the pinch. 4. BI neurones had a higher frequency of on-going synaptic activity (2.9 +/- 0.5 Hz, n = 15) than E neurones (0.2 +/- 0.1 Hz, n = 5) or O (0.2 +/- 0.1 Hz, n = 5) neurones. Most neurones with two or more suprathreshold inputs were BI neurones. In 20% of neurones (all BI with high rates of synaptic activity), several other inputs had ESPs with amplitudes close to threshold. 5. Subthreshold and suprathreshold inputs responded in the same way in only 45% of neurones, but suprathreshold inputs were excited in 73% of BI and all E neurones. The order of recruitment of different inputs varied from trial to trial. If classification was based only on suprathreshold responses, there were 36% BI, 32% E and 32% O neurones. 6. In the majority of neurones, postganglionic discharge was initiated exclusively by suprathreshold inputs, even during reflex excitation. 7. Qualitatively similar, but smaller, responses were evoked by a puff of air on the abdomen in 71% of cells tested. 8. The data suggest that the natural discharge of SCG neurones is largely determined by the activity of one or two preganglionic inputs with high quantal contents. BI neurones may include vasoconstrictor neurones, whereas the other types include secretomotor, pilomotor and other neurones projecting to targets in the head.

Effects of a cognition-enhancer, linopirdine (DuP 996), on M-type potassium currents (IK(M)) and some other voltage- and ligand-gated membrane currents in rat sympathetic neurons.

Linopirdine is a cognition enhancer which augments depolarization-induced transmitter release in the cortex and which is under consideration for potential treatment of Alzheimer's disease. It has previously been reported to inhibit M-type K+ currents in rat hippocampal neurons. In the present experiments we have tested its effect on whole-cell M-currents and single M-channels, and on a range of other membrane currents, in dissociated rat superior cervical sympathetic ganglion cells. Linopirdine inhibited the whole-cell M-current with an IC50 of 3.4 microM and blocked M-channels recorded in excised outside-out membrane patches but not in inside-out patches. This suggests that linopirdine directly blocks M-channels from the outside. It was much less effective in inhibiting other voltage-gated potassium currents [delayed rectifier (IK(V)), IC50 63 microM; transient (IA) current, IC50 69 microM] and produced no detectable inhibition of the fast and slow Ca(2+)-activated K+ currents IC and IAHP or of a hyperpolarization-activated cation current (IQ/Ih) at 10-30 microM. However, it reduced acetylcholine-activated nicotinic currents and GABA-activated Cl- currents with IC50 values of 7.6 and 26 microM respectively. It is concluded that linopirdine shows some 20-fold selectivity for M-channels among different K+ channels but can also block some transmitter-gated channels. The relationship between M-channel block and the central actions of linopirdine are discussed.

Intracellular Calcium Directly Inhibits Potassium M Channels in Excised Membrane Patches from Rat Sympathetic Neurons

Complex effects of altering intracellular [Ca 2+] on M-type K + currents have previously been reported using whole-cell current recording. To study the direct effect of Ca 2+ on M-channel activity, we have applied Ca 2+ to the inside face of membrane patches excised from rat superior cervical sympathetic ganglion cells. Ca 2+ rapidly and reversibly inhibited M-channel activity in 28/44 patches by up to 87%, with a mean IC 50 of 100 nM. This effect persisted in the absence of ATP, implying that it was not due to phosphorylation/dephosphorylation. A similar effect was observed in 13/13 cell-attached patches when cells were transiently “Ca 2+-loaded” by adding 2 mM Ca 2+ to a 25 mM K + solution bathing the extrapatch cell membrane. These observations provide new evidence that Ca 2+ can directly inhibit M channels, so supporting the view that Ca 2+ might mediate M current inhibition following muscarinic receptor activation.

Reduction in muscarinic receptors by antisense oligodeoxynucleotide

Treatment of rat superior cervical ganglion cells in culture for 4 days with an antisense oligodeoxynucleotide corresponding to a partial sequence of the mRNA coding the m1 muscarinic receptor decreased m1 receptor mRNA by 64% without affecting m2 receptor mRNA. 3H]Pirenzepine binding to M 1 receptors was decreased by 43%. Intraventricular infusion of this antisense oligodeoxynucleotide reduced muscarinic receptor density in m 1 but not M 2 receptor-rich brain regions.

Neuropeptide Y inhibits adrenergic transmitter release in cultured rat superior cervical ganglion cells by restricting the availability of calcium through a pertussis toxin-sensitive mechanism

Neuropeptide Y has been reported to inhibit the release of the adrenergic transmitter from sympathetic nerves in many tissues. The purpose of this study was to determine the mechanism of the inhibitory effect of neuropeptide Y on the release of the adrenergic transmitter in cultured superior cervical ganglion cells prelabeled with tritiated norepinephrine. In cultured superior cervical ganglion cells superfused with a HEPES-buffered saline, electrical field stimulation (1 Hz, 30 pulses, 1 ms, 60 V) increased the fractional overflow of tritium. Neuropeptide Y (50 nM) attenuated this depolarization-induced increase in transmitter release. The nonhydrolyzable cAMP analog, 8-(4-chlorophenylthio)cyclic AMP (100 μM) and the potassium channel blockers, tetraethylammonium chloride (1 mM) and 4-aminopyridine (300 μM) potentiated the electrically stimulated increase in fractional tritium overflow but failed to alter the inhibitory effect of neuropeptide Y on fractional tritium overflow. Increasing the calcium concentration in the superfusion fluid from 1.8 to 5.4 mM potentiated the electrically stimulated increase in fractional tritium overflow and attenuated the inhibitory effect of neuropeptide Y. Reduction of superfusion fluid calcium concentration to 0.5 mM decreased electrically stimulated fractional tritium overflow and augmented the inhibitory effect of NPY on release of tritium. The fractional release of tritium in response to the calcium ionophore, ionomycin, was not significantly altered by neuropeptide Y. In Fura-2-loaded isolated sympathetic neurites obtained from superior cervical ganglia explants, the depolarization-induced (54 mM KC1) increase in cytosolic calcium was attenuated by neuropeptide Y (50 nM). Pertussis toxin (100 ng/ml for 4 h) pretreatment abolished both the neuropeptide Y-induced decrease in electrically stimulated fractional tritium overflow and the reduction in depolarization-induced increase in cytosolic calcium. These data suggest that NPY inhibits adrenergic transmitter release in cultured superior cervical ganglion cells by decreasing the availability of calcium for the release process by a pertussis toxin-sensitive mechanism.

Vasoactive intestinal peptide suppresses neuronal cell death induced by nerve growth factor deprivation in rat sympathetic ganglion cells in vitro

Neuropeptides were examined for their effects on the survival of cultured rat superior cervical ganglion cells after acute deprivation of nerve growth factor (NGF). Vasoactive intestinal peptide (VIP, 3 μM) delayed the neuronal death about 6 h alone. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.2 mM) greatly potentiated its effect, reducing EC 50 from 2.5 μM to 8 nM. The neuronal death was completely suppressed under this condition. On the other hand, substance P (1–100 μM) or enkephalin (1–100 μM) alone did not modify the death, whereas the latter (100 μM) enhanced the survival-promoting effect of membrane depolarization with elevated K +. These results suggest strongly that neuropeptides regulate the NGF-independent survival of sympathetic neurons through a cAMP-dependent mechanism.

L-type calcium channel regulates depolarization-induced survival of rat superior cervical ganglion cells in vitro.

L-type calcium channel regulates depolarization-induced survival of rat superior cervical ganglion cells in vitro.

Effects of axotomy or target atrophy on membrane properties of rat sympathetic ganglion cells.

1. The electrical properties of rat superior cervical ganglion cells were examined in vitro with intracellular microelectrodes after axotomy or atrophy of the submandibular salivary gland. 2. Membrane time constant, input resistance and excitatory synaptic potentials (EPSPs) were decreased to about 50% of their control values 7-10 days after axotomy. 3. Axotomized ganglion cells also showed reduced action potentials and after-hyperpolarizations (AHPs). The AHP duration was reduced to 40% of the control value. 4. In 25% of the axotomized cells, the action potential was followed by an after-depolarization (ADP) instead of the AHP that was always present in control cells. In eleven out of seventeen axotomized cells with ADP, preganglionic stimulation failed to evoke an EPSP, whereas the failure of the synaptic response was never observed in control cells and occurred only in two of fifty-three axotomized cells with AHP. 5. In some axotomized cells with AHP, a depolarizing potential developed after a train of action potentials. This was never observed in control cells. 6. Sympathetic neurones innervating the submandibular gland in control animals had membrane properties similar to those observed in other ganglion cells. 7. The properties of neurones innervating the submandibular gland remained unaltered after the experimentally induced atrophy of the gland. 8. It is concluded that the marked effects of short-term axotomy on membrane properties of sympathetic ganglion cells are not reproduced by long-term atrophy of the target tissue.

Calcium entry through nicotinic receptor channels and calcium channels in cultured rat superior cervical ganglion cells.

1. Patch-clamp techniques in conjunction with indo-1 fluorescent measurements were used to measure increases in intracellular free calcium concentration and membrane conductance induced by the activation of nicotinic and calcium channels in cultured rat sympathetic neurons. 2. Under voltage-clamp conditions, pressure application of the nicotinic agonist DMPP (1,1-dimethyl-4-phenylpiperazinium iodide, 100 microM, 100 ms) increased [Ca2+]i by 193 +/- 26 nM at a clamp potential of -60 mV. This was accompanied by an inward current of -4.53 +/- 0.89 nA, giving a mean ratio of the delta (Ca2+]i to the total inward charge transfer of 42.7 nmoles per litre of free calcium per nanocoulomb of charge (M/q ratio). 3. The DMPP-induced current and associated delta [Ca2+]i were reduced by mecamylamine (100 nM-10 microM) but were unaffected by alpha-bungarotoxin (100 nM) or cadmium (100 microM). 4. The M/q ratio was not affected by the holding potential (from -80 to -40 mV) but was a function of the external calcium concentration. 5. The M/q ratio was reduced by increasing the intracellular calcium buffering capacity and increased by heparin but not affected by ryanodine or by depletion of the caffeine-sensitive calcium store. 6. Under the same recording conditions, we quantified the increase in [Ca2+]i associated with activation of the voltage-dependent calcium current. On average at -60 mV, the M/q ratio of this highly calcium-selective permeability was 1961 mM nC-1, which is 46 times that obtained for the nicotinic channel. 7. Assuming constant-field theory, ion-substitution experiments suggest that in 2.5 mM external calcium, the permeability sequence for the nicotinic conductance was Cs+ < Li+ < Na+ < K+ < Ca2+. 8. We conclude that the nicotinic channels in rat sympathetic neurones are significantly permeant to Ca2+ and that the influx of Ca2+ through these channels is the principal cause of the rise in [Ca2+]i seen under voltage clamp.

Cyclic AMP modulates but does not mediate the inhibition of [ 3H]norepinephrine release by activation of alpha-2 adrenergic receptors in cultured rat ganglion cells

The purpose of this study was to determine whether a decrease in cyclic AMP accumulation mediates the inhibition of norepinephrine release in response to alpha-2 adrenergic receptor activation in cultured rat superior cervical ganglion cells. Superior cervical ganglia from neonatal rats were dissociated and cultured on collagen-coated plastic strips. Neurotransmitter release was assessed by measuring the fractional overflow of tritium in superfused cells prelabeled with [ 3H]norepinephrine. Intracellular cyclic AMP accumulation was measured using radioimmunoassay. Electrical field stimulation at 1 Hz, 30 pulses, 1 ms duration at 20 min intervals produced an increase in the fractional overflow of tritium that was composed predominantly of intact [ 3H]norepinephrine. The alpha-2 adrenergic receptor agonist UK-14,304 dose-dependently attenuated the increase in fractional tritium overflow elicited by electrical field stimulation. The adenylyl cyclase activator, forskolin, increased cyclic AMP accumulation in superior cervical ganglion cells and UK-14,304 dose-dependently inhibited forskolin-stimulated cyclic AMP accumulation. UK-14,304 had no effect on basal cyclic AMP accumulation or cyclic AMP accumulation during electrical field stimulation. Forskolin (1–10 μM) or the non-hydrolysable cAMP analog, 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (1–100 μM), slightly increased basal and dose-dependently potentiated the increase in fractional tritium overflow in response to electrical stimulation. Despite enhancement by forskolin and 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate of fractional tritium overflow caused by electrical field stimulation, UK-14304 (1–10 μM) reduced release to a similar degree as that observed in the absence of forskolin or 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate. These data suggest that in cultured rat superior cervical ganglion cells, stimulation of alpha-2 adrenergic receptors decreases cyclic AMP accumulation when adenyl cyclase is activated. A decrease in cyclic AMP does not mediate the inhibition of [ 3H]norepinephrine release in response to alpha-2 adrenergic receptor activation, although elevations in cyclic AMP can enhance electrically-stimulated release of [ 3H]norepinephrine.

Mechanism of Neurotransmitter Release Elicited by the Preferential ?1-Adrenergic Receptor Agonist Phenylephrine in Superfused Superior Cervical Ganglion Cells in Culture

Phenylephrine increased [3H]norepinephrine efflux and accumulation of cyclic AMP in cultured rat superior cervical ganglion cells superfused with Tyrode's solution. The purpose of this study was to determine the mechanism and relationship between these two events. Electrical stimulation (1-2 Hz), potassium chloride (50 mM), and the preferential alpha 1-adrenergic receptor agonist phenylephrine (1-100 microM) increased fractional tritium efflux, whereas methoxamine, cirazoline, and amidephrine were relatively ineffective. Phenylephrine, but not methoxamine and cirazoline, also increased cyclic AMP accumulation. Phenylephrine-induced tritium efflux was not altered by alpha- and beta-adrenergic receptor antagonists or by removal of extracellular calcium. Phenylephrine-induced cyclic AMP accumulation was blocked by the beta-adrenergic receptor antagonists propranolol and atenolol. Forskolin (10 microM) and the nonhydrolyzable cyclic AMP analogue 8-(4-chlorophenylthio)cyclic AMP (100 microM) had minimal effect on tritium efflux. However, phenylephrine-evoked increase in tritium efflux was dose dependently attenuated by the neuronal uptake blocker cocaine, and phenylephrine dose-dependently inhibited the incorporation of [3H]norepinephrine into neuronal stores. We conclude that the increase in tritium efflux induced by phenylephrine is independent of cyclic AMP accumulation and appears to be mediated by uptake of phenylephrine via the neuronal carrier-mediated amine transport process, which in turn promotes efflux of the adrenergic transmitter from its storage sites.

Early commitment of precursor cells from the rat superior cervical ganglion to neuronal or nonneuronal fates

To determine whether postmigratory neural crest cells retain the capacity to give rise to multiple cell types, the clonal progeny of embryonic rat superior cervical ganglion (SCG) cells were examined in culture. Double labeling with BrdU and neurofilament antibodies demonstrated that neuron precursors from the E14.5 SCG continued to proliferate for several days in culture. Using the BAG retrovirus to examine the progeny of single cells, we obtained several kinds of distinct clones from SCG cultures after 3 days. At E14.5, during peak neuro-genesis in vivo, neuron-containing clones composed of one to seven cells were common. At El 7.5, after neurons have been born in vivo, most clones in vitro contained flat cells, primarily reflecting glial cell division. Even in cultures from E13.5 ganglia, mixed clones containing neurons and flat cells were rarely observed. These observations suggest that neuronal and nonneuronal cell precursors are specified during or before early gangliogenesis.

Detection of sialic acid residues in the axonal reticulum of rat superior cervical ganglion cells by lectin-gold cytochemistry.

Highly glycosylated compounds have been demonstrated in the axonal reticulum elements of the superior cervical ganglion cells of the rat, and this is considered to suggest a connection of the reticulum with the trans Golgi side. In the present study, the axonal reticulum and the Golgi elements were further characterized by post-embedding methods of lectin-gold cytochemistry to determine their carbohydrate residues and to see, more specifically, if sialic acid residues could be detected in the axonal reticulum elements. Therefore, the affinity of neuronal cell structures for Limax flavus agglutinin (LFA), wheat germ agglutinin (WGA), and Ricinus communis agglutinin I (RCA-I) was tested in ultra-thin sections of glycolmethacrylate-embedded material, counterstained with phosphotungstic acid (PTA) at low pH. The trans Golgi network, the Golgi-associated axonal reticulum, the reticulum within axons, the large dense-cored vesicles, and the plasma membranes were reactive for all three lectins used. We conclude that the axonal reticulum elements carry sialic acid residues, relating them to the trans Golgi network. The present results support the concept that the axonal reticulum is an extension of the trans network of the Golgi apparatus specialized for neurosecretion.

Purification of the growth-associated protein GAP-43 by reversed phase chromatography: amino acid sequence analysis and cDNA identification

GAP-43 is a neuronal phosphoprotein. Increased synthesis and axonal transport of GAP-43 has been associated with axon growth, and altered phosphorylation of GAP-43 has been associated with changes in synaptic efficacy. Here we report a rapid and effective procedure employing reverse-phase HPLC for the purification of GAP-43 from rat brain. To characterize the protein purified by this procedure, we generated proteolytic fragments and determined their amino acid sequences. These directly determined sequences, corresponding to 56% of the GAP-43 amino acids, confirm recently reported sequences deduced from the nucleotide sequences of cDNAs. Using oligonucleotide probes constructed according to these amino acid sequences, we identified GAP-43 cDNAs in a library prepared from neonatal rat superior cervical ganglion cells. One of these cDNAs was 1.1 kB in size; it hybridized specifically with a 1.5 kB RNA from brain, but not from liver, and contained the entire coding sequence for GAP-43. This cDNA differed from recently reported cDNAs in its 3′ untranslated region.