Rabbit Superior Cervical Ganglion Research Articles

Substance P: A Putative Sensory Transmitter in Mammalian Autonomic Ganglia

Repetitive presynaptic stimulation elicited slow membrane depolarization in neurons of inferior mesenteric ganglia from guinea pigs. This response was not blocked by cholinergic antagonists but was specifically and reversibly inhibited by a substance P analog, (D-Pro2, D-Phe7, D-Trp9)-substance P, which also depressed the depolarization induced by exogenously applied substance P. The atropine-sensitive slow excitatory and slow inhibitory postsynaptic potentials evoked in neurons of rabbit superior cervical ganglia were not affected by the substance P analog. These and previous results provide strong support for the hypothesis that substance P or a closely related peptide is the transmitter mediating the slow depolarization. The latter may represent a sensory input from the gastrointestinal tract to neurons of the prevertebral ganglia.

Distribution of protein I and regulation of its state of phosphorylation in the rabbit superior cervical ganglion

The distribution and regulation of the state of phosphorylation of Protein I have been studied in the rabbit superior cervical sympathetic ganglion. The data indicate that the ganglion contains two pools of Protein I: a presynaptic pool that represents 60% of the total ganglion Protein I and a postsynaptic pool that represents 40% of the total ganglion Protein I. The state of phosphorylation of presynaptic Protein I, but not that of postsynaptic Protein I, is regulated by nerve impulse conduction, by dopamine, and by a high K+ concentration. Studies of the extracellular calcium requirements for Protein I phosphorylation, as well as peptide-mapping analyses of Protein I, suggest that the effects of nerve impulse conduction and of a high K+ concentration are mediated through the activation of calcium-dependent protein kinases and that the effect of dopamine is mediated through the activation of cyclic AMP-dependent protein kinase. The total amount of postsynaptic Protein I, but not that of presynaptic Protein I, is decreased by short periods of exposure to cycloheximide, a protein synthesis inhibitor. It is proposed that Protein I located in presynaptic nerve terminals plays a functional role in those terminals and that the Protein I located in cell bodies is newly synthesized and en route to nerve terminals.

Distribution of muscarinic receptors in mammalian sympathetic ganglion: autoradiographic and electrophysiological studies

Localization of muscarinic receptors in rabbit superior cervical ganglion (SCG) neurons was investigated by means of light microscopy autoradiographic study of binding sites for the labeled selective muscarinic antagonist atropine, and intracellular recordings were made from single ganglionic neurons to determine their response to local iontophoretic application of the selective muscarinic agonist—5-methylfurmethide (MF). Autoradiography showed that muscarinic receptors are located predominantly on the dendrites of ganglion neurons and on their soma near large processes. The remaining soma areas are usually devoid of muscarinic receptors or their density is much lower than in dendritic areas. MF-elicited depolarization was seen in 34% of neurons studied; an initial hyperpolarization was followed by depolarization in 23% of the neurons and no responses occurred in the remaining 43%. Lowering Ca 2+ and increasing Mg 2+ content in the external medium abolished MF-hyperpolarization but not MF-depolarization. The following properties of MF-depolarization support the concept of dendritic localization of muscarinic receptors: (i) the time-course of MF-depolarization varies widely in different cells; (ii) its rise time increases with the increase of the MF dose; (iii) MF is more effective in inducing a MF-depolarization if it is applied to cell processes than if it is applied to cell soma. The origin of MF-hyperpolarization is discussed.

Voltage-dependent actions of short-chain polymethylene bis-trimethylammonium compounds on sympathetic ganglion neurons

Effects of polymethylene bis-trimethylammonium compounds (with 4–7 carbons in the polymethylene chain, C4–C7) on voltage-dependence of fast excitatory postsynaptic current (EPSC) were studied in voltage-clamped neurons of the isolated rabbit superior cervical ganglion. All these compounds shortened the EPSC decay (which remained single-exponential) and decreased (or reversed) the dependence of the EPSC decay on membrane hyperpolarization. All drugs slightly decreased the EPSC amplitude; in addition, C6 and C7 decreased their dependence on membrane hyperpolarization. It is suggested that shortening of the EPSC decay produced by ganglion-blocking agents results from their binding to the open ionic channel (channel-blocking effect). The ratio of channel-blocking activities of these drugs correlates with the well-known ratio of their ganglion-blocking activities. It is suggested that the channel-blocking activities of polymethylene bis-trimethylammonium compounds determine their ganglion-blocking activities. The model of channel-blocking action is discussed.

Two muscarinic depolarizing mechanisms in mammalian sympathetic neurons

A voltage-sensitive outward membrane current (‘M’) and a consequent change in conductance (ΔG) appear with a slow time-constant, in principal neurons of rabbit superior cervical ganglion (SCG), only when membrane potentials (V m) are depolarized to <—60 mV. Effects of muscarine on the voltage-current curves indicate that, in this depolarized range of <—60 mV, suppression of M-current could contribute a muscarinic depolarization accompanied by a decrease in G; but that, at all V ms tested (about —90 to —40 mV), there is an additional larger muscarinic depolarization with no ΔG. Thus, the muscarinic depolarizing response and the equivalent slow excitatory postsynaptic potential in the rabbit SCG may consist of two different components: one is due to the suppression of M-current and is substantial only in the depolarized range; the other is probably mediated via an intracellular increase in cyclic GMP and can account for most or all of the response at V ms more negative than —55 mV.

Nerve impulses increase the phosphorylation state of protein I in rabbit superior cervical ganglion.

Protein I, a neurone-specific protein concentrated at synapses, is present in most, and probably all, presynaptic nerve terminals where it is at least partially associated with neurotransmitter vesicles (refs 1–5 and P. De Camilli, W. B. Huttner, R. Cameron, M. Harris and P.G., in preparation). Protein I is a major endogenous substrate in nervous tissue for both cyclic AMP-dependent1 and calcium/calmodulin-dependent6–8 protein kinases. Neurotransmitters9,10, apparently acting through cyclic AMP, and depolarizing agents9–11, apparently acting through calcium, have been shown to increase the state of phosphorylation of protein I in intact preparations of the central and peripheral nervous systems. To determine whether impulse conduction regulates the state of phosphorylation of protein I, we have now studied this process in the rabbit superior cervical ganglion, a well characterized neuronal preparation that is suitable for both physiological and biochemical studies. Using a technique developed to quantify the state of phosphorylation of the small amounts of protein I contained in ganglion tissue, we show here that in physiological conditions brief periods of impulse conduction increase the state of phosphorylation of protein I in this ganglion.

Two modes of activity of nicotinic acetylcholine receptor channels in sympathetic neurons

Spectral analysis of acetylcholine (ACh) noise was performed in voltage-clamped neurons of the isolated rabbit superior cervical ganglion at 34–37°C and at membrane potential −80 mV. Two modes of activity were found in the ionic channels of nicotinic ACh receptors, with mean channel life-times of τ f = 1.1 ± 0.1 ms for fast-operating channels and τ s = 5.6 ± 0.6 ms for slow-operating channels. Excitatory postsynaptic current (EPSC) decays exponentially with a time constant which is very close to τ s, indicating that the slow-operating channel activity determines the duration of EPSC. The mean value of conductance of single nicotinic ACh-receptor channel is 36 ± 3 pS.

Electrophysiological and cyclic AMP responses to beta-adrenergic receptor stimulation and catecholamine levels in cat and rabbit sympathetic ganglion

The effects of beta 1- and beta 2-selective and nonselective adrenergic agonists and antagonists were studied in the cat and rabbit superior cervical ganglion (SCG) in situ and in vitro. In experiments on the cat SCG in situ beta-adrenergic agonists induced an increase in positive afterpotential (PAP) of compound ganglionic action potential evoked by preganglionic stimulation in the following order of potency: isoprenaline (ISO) > salbutamol ⪢ tazolol. Beta-adrenergic antagonists inhibited the ISO response in the following order of potency: propranolol > exaprolol ⪢ H 35/25. Practolol did not exhibit beta-adrenergic blocking action; on the contrary, in higher doses it enhanced the response to ISO. In higher doses all beta-adrenergic antagonists tested depressed ganglionic transmission in an approximately similar range of doses. In experiments in vitro, using sucrose-gap technique, ISO induced depolarization of the cat SCG and this effect was inhibited in a dose-dependent manner by propranolol. Beta-adrenergic agonists did not induce depolarization of the rabbit SCG in experiments in situ and in vitro. The level of noradrenaline and dopamine in the cat and rabbit SCG did not differ significantly. Low level of adrenaline was found in the cat SCG; however, no adrenaline was detected in the rabbit SCG. No statistically significant change of cyclic AMP content was observed in the cat and rabbit SCG or in the incubation medium under conditions similar to those in which ISO induced depolarization of the cat SCG. It is concluded that the beta-adrenergic receptors present in the cat SCG resemble the beta 2-subtype and that the presence of an electrophysiological response to beta-adrenergic agonists appears to be species-dependent. The depolarization of the cat SCG due to beta-adrenergic receptor stimulation is not coupled with cyclic AMP increase.

Evidence for increased release of prostaglandins of E-type in response to orthodromic stimulation in the guinea-pig superior cervical ganglion

Prostaglandins of the E type (PGEs) stimulate cyclic adenosine-3′,5′-monophosphate (cAMP) biosynthesis both in isolated preparations of rat, guinea-pig and rabbit superior cervical ganglia (SCG) and in calf SCG slices. Electrical stimulation of preganglionic nerve fibers of the guinea-pig SCG remarkably increased PGE release and cAMP biosynthesis. These effects were blocked by reducing the Ca2+ to Mg2+ ratio in the incubation medium. Atropine (1 μM) and phentolamine (10 μM) inhibited PGE biosynthesis and significantly reduced cAMP levels.

Post-tetanic depolarization in sympathetic neurones of the guinea-pig.

1. Repetitive intracellular stimulation at a frequency of 5-30 Hz for 1-10 s evoked in neurones of the isolated inferior mesenteric and superior cervical ganglia of the guinea-pig three types of post-spike membrane potential changes: (i) hyperpolarization, (ii) hyperpolarization followed by a slow depolarization, and (iii) a second hyperpolarization following the initial two responses.2. The initial post-spike hyperpolarization had a mean duration of 2.0 s and was often associated with a fall in membrane resistance; it could be elicited in every sympathetic neurone studied. This response was termed the post-tetanic hyperpolarization (PTH).3. The slow depolarization which could be induced only in a portion of neurones had a mean amplitude and duration of 2.2 mV and 27.5 s, respectively; it was termed the post-tetanic depolarization (PTD).4. PTD was associated with a fall in membrane resistance, augmented by membrane hyperpolarization, and reduced by depolarization; its mean extrapolated equilibrium potential was -38 mV.5. PTD was not blocked by nicotinic and muscarinic antagonists, or alpha-and beta-adrenergic receptor antagonists, whereas it was suppressed by adrenaline, noradrenaline, Co(2+) and a low Ca(2+) solution.6. The amplitude of the single spike after-hyperpolarization in normal Krebs solution as well as in high K(+) solution was increased during PTD; furthermore, conditioning hyperpolarization to the level of E(K) increased the amplitude of PTD in normal Krebs as well as in high K(+) solution.7. PTD with similar amplitude, time course and membrane characteristics could be evoked in a portion of neurones of the rabbit superior cervical ganglia; however, PTD was not detected in neurones of the rat superior cervical ganglia.8. Decentralization of the guinea-pig and rabbit superior cervical ganglia for 14 d did not alter the number of neurones in which PTD could be elicited, its amplitude, or its time course.9. Our results suggest that a chemical substance(s) is responsible for the generation of PTD; it may be released from the soma and/or dendrites and acts in an auto-receptive manner on the cells in question. The nature and origin of the second hyperpolarization remain to be clarified.

Roles of cyclic nucleotides in the synaptic transmission in sympathetic ganglia of rabbits

1. Two kinds of slow postsynaptic potentials can be elicitable in the rabbit superior cervical ganglion: a dopaminergic hyperpolarizing (slow IPSP) one and a muscarinic depolarizing (slow EPSP) one, respectively. 2. Cyclic AMP may not be involved in the generation of slow IPSP, whereas it certainly mediates the dopamine-induced enduring modulatory enhancement of the slow EPSP response. 3. Cyclic GMP appears to mediate at least one of the components in the mechanism underlying the slow EPSP by generating a depolarization with no change in membrane conductance. 4. Cyclic GMP does additionally regulate the dopamine (or cyclic AMP)-induced modulatory enhancement of the slow EPSP by antagonizing it in a uniquely time-dependent fashion.

Orthodromic production of non-cholinergic slow depolarizing response in the superior cervical ganglion of the rabbit.

1. A late slow depolarization in the rabbit superior cervical ganglion, recorded extracellularly as ;late late negative' (l.l.n.) response, can be elicited by suitably repetitive stimulation of cervical sympathetic nerve. The l.l.n. response is not blocked by strong nicotinic, muscarinic or adrenergic antagonists; it appears with latencies in seconds, rise times in minutes, durations of up to 20 min or more, and extracellular amplitudes that can exceed 1 mV when recorded in an air-gap chamber.2. The l.l.n. component is a graded post-synaptic response that decreases with a length constant similar to those of the known p.s.p.s (fast e.p.s.p., slow i.p.s.p., and slow e.p.s.p.). This and its other characteristics indicate that the l.l.n. response is neuronally generated and represents a non-cholinergic late slow depolarization. The term ;slow slow e.p.s.p.' is suggested for this response, to replace both ;slow depolarization' and ;late slow e.p.s.p.'.3. The amplitudes, evaluated relative to the compound action potentials, and the durations of l.l.n. responses recorded from intact neurones of rabbit superior cervical ganglion were considerably greater and more consistently producible than the non-cholinergic slow depolarizations recorded by others from impaled neurones of guinea-pig inferior mesenteric ganglion.4. The l.l.n. response does not exhibit the special sensitivity to sodium azide previously found for the muscarinic ;late negative' or slow e.p.s.p. response.5. The total number of orthodromic volleys is the chief determinant of the amplitude and duration of the l.l.n. response. Increases in pulse frequency, with no change in pulse number, exert only a minor influence on amplitude and duration of the l.l.n. response but can markedly decrease latency and rise time.6. Even very low pulse frequencies (e.g. 1/sec) are almost as effective as higher frequencies if a sufficiently large number of stimulus pulses is applied.7. The features of orthodromic production of the l.l.n., slow slow e.p.s.p. response, as well as the amplitudes and durations of this depolarization, indicate that this non-cholinergic post-synaptic response could, like the muscarinic slow e.p.s.p., play a significant role in mediating physiological activities of sympathetic ganglia.

Identification of a nerve growth factor receptor protein in sympathetic ganglia membranes by affinity labeling.

Membranes from adult rabbit superior cervical ganglia, cross-linked to membrane-bound 125I-labeled nerve growth factor (NGF) by the photoreactive agent hydroxysuccinimidyl-p-azidobenzoate, were found to contain two labeled components with apparent Mr = 143,000 and Mr = 112,000. At high concentrations of the cross-linker, minor amounts of a Mr = 300,000 affinity labeled product were also observed. The affinity labeled species exhibit the characteristics expected of membrane receptors for NGF. The inhibition of specific 125I-NGF binding to membranes by increasing concentrations of unlabeled NGF parallels the inhibiton of the affinity labeling of these components. Insulin, insulin-like growth factor I, multiplication stimulating activity, and epidermal growth factor do not inhibit the affinity labeling reaction. Membrane preparations of various non-neuronal tissues do not show any detectable specific cross-linking to 125I-NGF. The affinity labeled species of superior cervical ganglia are proteins and contain intrapeptide disulfide bridges compacting their molecular structure. Peptide mapping experiments indicate a close structural relationship between the Mr = 143,000- and the Mr = 112,000-labeled proteins, suggesting a transformation of the former into the latter by limited proteolysis. The results suggest that the Mr = 143,000 affinity labeled protein represents a native NGF receptor component.

Modulation of slow postsynaptic potentials by dopamine, in rabbit sympathetic ganglion

(1) Temporary exposure of rabbit's superior cervical ganglion (SCG) to dopamine (DA), in the presence of an inhibitor of catechol-o-methyltransferase (COMT) is consistently followed by a potentiation of the slow (s)-EPSP and s-IPSP, lasting for some hours. The fast (f)-EPSP is not significantly increased, but it is better maintained than in control ganglia. (2) Exposure to the COMT-inhibitor U-0521 alone induces less but substantial potentiations of both s-PSPs. This effect is explained as due to protection of DA released intraganglionically at rest. (3) This evidence suggests that COMT may significantly limit the access of catecholamines to postsynaptic receptors, for at least certain types of neuron-to-neuron synaptic actions. (4) The potentiation of both s-PSPs, whether induced by DA in the presence of U-0521 or by U-0521 alone, is depressed by DA-1 antagonists that have been found to depress DA-stimulation of adenyl cyclase in rabbit SCG; these are spiroperidol, butaclamol and, to a lesser extent, bromocriptine. The specific ‘DA-2’ antagonists metaclopramide and sulpiride, and the α-adrenergic antagonist dihydroergotamine, did not depress potentiation. (5) Potentiation of s-EPSP is viewed as identical in nature to the previously discovered DA-modulatory enhancement of direct muscarinic depolarizing actions (by acetylcholine or its agonists). Potentiation of s-IPSP may be due to a similar DA-modulation of other muscarinic response(s) involved in mediating the s-IPSP. The consistency and comparative ease with which these DA-modulatory effects can be induced, under presently described experimental conditions, should facilitate future study of this mode of synaptic action.

Lectin-induced inhibition of nerve growth factor binding by receptors of sympathetic ganglia.

Nerve growth factor (NGF) initiates a pleiotypic response in numerous tissues derived from the neural crest by binding to specific plasma membrane receptors. In sympathetic ganglia this receptor has been characterized as a highly asymmetric, minimally hydrophobic, intrinsic membrane protein with a molecular weight of 135,000 (Costrini et al., 1979b). To further characterize this moiety we assessed the effects of lectins on 125I-NGF specific binding to preparations of particulate and nonionic detergent-extracted microsomal receptors of rabbit superior cervical ganglia (SCG). Concanavalin A (Con A) and wheat germ agglutinin (WGA), but not soybean agglutinin or Ulex europaeus 1, induced a concentration-related, carbohydrate-specific decrease in 125I-NGF binding. Following Con A exposure, 125I-NGF specific decrease in 125I-NGF binding. Following Con A exposure, 125I-NGF specific binding to particulate SCG receptors was maximally reduced to 23% of control values. WGA similarly reduced NGF binding to particulate microsomal receptors to 37% of control values. Scatchard analysis of growth factor binding following Con A exposure indicated that this lectin effect was principally due to a sixfold reduction in maximum receptor affinity. Lectin-associated impairment of NGF binding was also demonstrated by using a Triton X-100 solubilized receptor preparation. These results provide evidence that the high-affinity-state NGF receptor of SCG is a glycoprotein containing N-acetylglucosamine and alpha-D-mannopyranoside residues. These residues are probably located in close proximity to the growth factor binding region of the NGF receptor.

Inhibition of ACh release by prostaglandin E 1 in the rabbit superior cervical ganglion

Superfusion of prostaglandin E 1 (PGE 1) in low concentrations (0.01–0.5 μM) to the neurons of isolated rabbit superior cervical ganglia reversibly depressed the amplitude of the fast excitatory postsynaptic potential (f-epsp) evoked by preganglionic nerve stimulation without changing the resting membrane potential or the membrane resistance. In concentrations higher than 1 μM, PGE 1 elicited a slow membrane depolarization accompanied by a reduction in membrane resistance. PGE 1 in concentrations that did not alter the membrane potential reduced significantly the f-epsp without affecting the acetylcholine (ACh) potential induced by iontophoretic application of ACh to the ganglion cells. The results suggest that the primary action of PGE 1 in the sympathetic ganglia is to depress synaptic transmission by reducing the amount of ACh liberated from preganglionic fibers.

Cyclic AMP synthesis in guinea pig superior cervical ganglia: response to pharmacological and preganglionic physiological stimulation

The effects of stimulation of guinea pig superior cervical ganglia (SCG) in vitro with 50 μM concentrations of dopamine, L-norepinephrine or L-isoproterenol in Eagle's Medium containing 5 mM theophylline were determined. Dopamine (50 μM) produced no stimulation of cyclic AMP levels, 50 μM norepinephrine produced a doubling of cyclic AMP levels, while 50 μM isoproterenol produced a 6-fold increase in cyclic AMP levels over control values. The increases were blocked by propranolol, indicating that they were due to stimulation of a β-adrenergic receptor-adenylate cyclase complex. In the rabbit SCG, by contrast, 50 μM dopamine produced an increase in cyclic AMP levels of 60% over control values. The possible involvement of the β-receptor complexin the modulation of ganglionic transmission was tested by subjecting ganglia to supramaximal stimulation for 8 min. No elevation of cyclic AMP levels occured in guinea pig SCG, but a doubling of cyclic AMP levels in rabbit SCG was noted. Current concepts of the function of cyclic AMP in neural transmission in the SCG involve a dopaminergic SIF cell, dopamine receptor-adenylate cyclase complex, and the generation of a slow inhibitory postsynaptic potential (s-IPSP). Previous research has indicated that the guinea pig SCG lacks a s-IPSP, and its SIF cells contain norepinephrine rather than dopamine. Since preganglionic stimulation of the guinea pig SCG has now been shown not to cause increased cyclic AMP levels, and since the cyclic AMP content of the guinea pig SCG is not increased following incubation with dopamine, we conclude that a dopamine receptor-adenylate cyclase complex is not involved in the modulation of neural transmission in the guinea pig SCG.

Physical properties of the detergent-extracted nerve growth factor receptor of sympathetic ganglia.

The nerve growth factor (NGF) receptor from microsomes of adult rabbit superior cervical ganglia has been solubilized with Triton X-100 and sodium deoxycholate. The physical properties of the detergent-extracted NGF receptor were assessed by Sepharose 6B chromatography and sucrose density gradient ultracentrifugation studies in H2O and D2O. The predominant form of the NGF receptor has a Stokes radius of 71 A, a partial specific volume of 0.74 ml/g, a sedimentation coefficient of 4.3 S, and a frictional ratio of 1.8. From these parameters, it can be calculated that the NGF receptor in Triton X-100 is a minimally hydrophobic, highly asymmetric, intrinsic membrane protein with a molecular weight of approximately 135,000. A form of the receptor with a sedimentation coefficient of 10.4 S was occasionally seen which appears to represent an aggregated form of the 4.3 S moiety.

Binding characteristics and apparent molecular size of detergent solubilized nerve growth factor receptor of sympathetic ganglia.

Nerve growth factor (NGF), a hormone-like regulator of sympathetic neuron ontogeny and metabolism affects its target cells initially by associating with specific plasma membrane receptors. We have solubilized the NGF receptor of adult rabbit superior cervical ganglia (SCG) with the nonionic detergent Triton X-100. The high-affinity equilibrium binding constant of the detergent-extracted receptor is 2-8 x 10(-10) M. Gel chromatography of the receptor or the 125I-labeled NGF receptor complex on a column of Sepharose 6B indicated, in both cases, a single component of an apparent hydrodynamic radius of 71 +/- 5 A. In parallel investigations, we have confirmed the similarity between the hydrodynamic size of the NGF receptor of rabbit SCG and that of the insulin receptor of IM-9 lymphocytes evaluated by similar methods.

Activation of Acetylcholine Receptors in Mammalian Sympathetic Ganglion Neurones

Publisher Summary This chapter discusses the action of the synaptically released transmitter and exogenous ACh on the superior cervical ganglion of the rabbit and compares this action of ACh with that produced by some cholinomimetic drugs. The experiments were performed on superior cervical ganglia rapidly isolated from rabbits killed by air embolism. Judging by the latency and time course of potentials evoked by the iontophoretical application of ACh to the soma membrane, nicotinic receptors only were activated. It appears that the soma membrane of the neurones in rabbit superior cervical ganglion does not have muscarinic receptors. Sometimes, spontaneous excitatory postsynaptic potentials (EPSPs) appeared as a result of ACh application indicating excitation of presynaptic terminals. The Hill number found from dose-response relationship was 2.4 k 0.6. The receptors of soma membrane were 7.8 times less sensitive to CCh and 4.6 times less sensitive to TMA than to ACh. Depolarizations evoked by CCh and TMA had much slower time courses than similar depolarizations evoked by ACh. The results indicate that at least two ACh molecules combine with one nicotinic receptor and that the excitatory transmitter and exogenous ACh trigger identical ionic mechanisms in the membrane resulting in an increase in sodium and potassium permeabilities, while chloride permeability remains unchanged.