Intact Superior Cervical Ganglia Research Articles

Patch-clamp analysis of nicotinic synapses whose strength straddles the firing threshold of rat sympathetic neurons.

Neurons in paravertebral sympathetic ganglia are innervated by converging nicotinic synapses of varying strength. Based upon intracellular recordings of excitatory postsynaptic potentials (EPSPs) with sharp microelectrodes these synapses were classified in the past as either primary (strong) or secondary (weak) by their ability to trigger postsynaptic action potentials. Here we present an analysis of 22 synapses whose strength straddled threshold, thereby distinguishing them from the original classification scheme for primary and secondary synapses. Recordings at 36°C were made from intact superior cervical ganglia isolated from 13 male and 3 female Sprague-Dawley rats and from 4 male spontaneously hypertensive (SHR) rats. Ganglia were pretreated with collagenase to permit patch recording. By dissecting a 1 cm length of the presynaptic cervical sympathetic nerve as part of the preparation and through use of graded presynaptic stimulation it was possible to fractionate synaptic inputs by their distinct latencies and magnitudes, and by the presynaptic stimulus threshold for each component. Comparison of cell-attached extracellular recordings with intracellular recordings of synaptic potentials and synaptic currents indicated that straddling EPSPs are not an artifact of shunting damage caused by intracellular recording. The results also showed that in cells where a single presynaptic shock elicits multiple action potentials, the response is driven by multiple synapses and not by repetitive postsynaptic firing. The conductance of straddling synapses also provides a direct estimate of the threshold synaptic conductance (9.8 nS ± 7.6 nS, n = 22, mean ± SD). The results are discussed in terms of their implications for ganglionic integration and an existing model of synaptic amplification.

HCN Channel Blockers Act Pre‐ and Post‐Synaptically to Inhibit Synaptic Transmission and Gain in the Rat Superior Cervical Sympathetic Ganglion

Hyperpolarization‐activated cation channels encoded by HCN subunits are permeable to Na+ and K+ ions and carry inward h‐currents. The h‐current (Ih) was originally discovered in the heart, where it is the principal pacemaker current. Ivabradine, a selective blocker of Ih, was approved in the US in 2015 as an adjunct to β‐adrenergic blockade for clinical management of heart failure. Recently we observed that sympathetic neurons in the rat superior cervical ganglion (SCG) express prominent Ih responses that can be blocked by ZD7288, another selective antagonist. The goal of the present study was to test the hypothesis that Ih modulates synaptic transmission in sympathetic ganglia. We did this using dissociated SCG neurons and using acutely isolated intact ganglia, treated with enzymes to permit whole‐cell recording, together with simultaneous presynaptic stimulation and extracellular recording. When synaptic transmission was elicited by submaximal 1 Hz stimulation in the isolated intact SCG, the extracellular postsynaptic compound action potential was inhibited by about 20% by 10–40 μM Ivabradine and by 10–20 μM ZD7288. In dissociated SCG neurons, we measured Ih under voltage clamp, fit the data to a Hodgkin‐Huxley model and then used the model to implement virtual Ih under dynamic clamp. When neurons were probed with virtual nicotinic fast EPSPs created with dynamic clamp, blocking Ih with 10–15 μM ZD7288 increased the threshold‐synaptic conductance (thresh‐gsyn) from 7.0 ± 0.9 nS to 12.1 ± 2.0 nS (p<0.0001) and decreased synaptic gain from 2.2 to 1.7 in response to noisy patterns of virtual synaptic activity that were designed to mimic physiological activity in vivo. This indicates that the native postsynaptic Ih normally enhances synaptic amplification to increase postsynaptic spike output by the SCG. When the native Ih was reconstituted with virtual Ih, synaptic amplification was restored. Shifting the voltage‐dependence of activation to mimic binding of cyclic AMP to HCN channels further modulated thresh‐gsyn and synaptic gain. To assess presynaptic actions of Ih, we then measured nicotinic cholinergic synaptic currents evoked by minimal presynaptic stimulation of the intact SCG. Ivabradine and ZD7288 both reduced peak EPSC amplitude and total charge by 40 to 50% while also increasing the failure rate. This indicates that native Ih normally enhances the probability of acetylcholine release and the amount of release by preganglionic terminals. Taken together the results suggest that clinical use of Ivabradine may have the heretofore unknown effect of reducing peripheral sympathetic activity through its pre‐ and postsynaptic effects on ganglionic transmission. This may have beneficial therapeutic consequences by countering the sympathetic hyperactivity that can drive human hypertension and that exacerbates the progression of heart failure.Support or Funding InformationSupported by the Great Rivers Affiliate of the American Heart Association through Grant‐in‐Aid 13GRNT1685007

Virtual leak channels modulate firing dynamics and synaptic integration in rat sympathetic neurons: implications for ganglionic transmission in vivo.

The synaptic organization of paravertebral sympathetic ganglia enables them to relay activity from the spinal cord to the periphery and thereby control autonomic functions, including blood pressure and body temperature. The present experiments were done to reconcile conflicting observations in tissue culture, intact isolated ganglia and living animals. By recording intracellularly from dissociated neurons and intact ganglia, we found that when electrode damage makes cells leaky it could profoundly distort cellular excitability and the integration of synaptic potentials. The experiments relied on the dynamic clamp method, which allows the creation of virtual ion channels by injecting current into a cell based upon a mathematical model and using rapid feedback between the model and cell. The results support the hypothesis that sympathetic ganglia can produce a 2.4-fold amplification of presynaptic activity. This could aid understanding of the neural hyperactivity that is believed to drive high blood pressure in some patients. The excitability of rat sympathetic neurons and integration of nicotinic EPSPs were compared in primary cell culture and in the acutely isolated intact superior cervical ganglion using whole cell patch electrode recordings. When repetitive firing was classified by Hodgkin's criteria in cultured cells, 18% displayed tonic class 1 excitability, 36% displayed adapting class 2 excitability and 46% displayed phasic class 3 excitability. In the intact ganglion, 71% of cells were class 1 and 29% were class 2. This diverges from microelectrode reports that nearly 100% of superior cervical ganglion neurons show phasic class 3 firing. The hypothesis that the disparity between patch and microelectrode data arises from a shunt conductance was tested using the dynamic clamp in cell culture. Non-depolarizing shunts of 3-10nS converted cells from classes 1 and 2 to class 3 dynamics with current-voltage relations that replicated microelectrode data. Primary and secondary EPSPs recorded from the intact superior cervical ganglion were modelled as virtual synapses in cell culture using the dynamic clamp. Stimulating sympathetic neurons with virtual synaptic activity, designed to replicate in vivo recordings of EPSPs in muscle vasoconstrictor neurons, produced a 2.4-fold amplification of presynaptic activity. This gain in postsynaptic output did not differ between neurons displaying the three classes of excitability. Mimicry of microelectrode damage by virtual leak channels reduced and eventually obliterated synaptic gain by inhibiting summation of subthreshold EPSPs. These results provide a framework for interpreting sympathetic activity recorded from intact animals and support the hypothesis that paravertebral ganglia function as activity-dependent amplifiers of spinal output from preganglionic circuitry.

Differential Inhibition of Ca2+ Channels by α2-Adrenoceptors in Three Functional Subclasses of Rat Sympathetic Neurons

A comparison of identified sympathetic neurons in the isolated intact superior cervical ganglion revealed that secretomotor, pilomotor, and vasoconstrictor cells differ in their action potential mechanisms and in their postsynaptic alpha(2)-adrenergic responses to 10 microM norepinephrine (NE). In normal saline, the half-width of the spike afterhyperpolarization (AHP) in secretomotor neurons (103.5 +/- 6.2 ms) was twofold that recorded in vasoconstrictor neurons (47.7 +/- 2.9 ms) and 1.5-fold that in pilomotor neurons (71.4 +/- 10.3 ms). Bath-applied NE reversibly inhibited the action potential repolarization shoulder, AHP amplitude, and AHP duration in secretomotor and pilomotor neurons to a similar extent, but had no effect on vasoconstrictor neurons. The insensitivity of vasomotor neurons to NE was not an artifact produced by microelectrode recording because all three cell groups were similar in terms of resting potential and input resistance. Moreover, NE insensitivity was not a natural consequence of briefer AHP duration in vasoconstrictor cells. Adding 10 mM TEA(+) caused marked accentuation of the shoulder and AHP duration in vasoconstrictor neurons and comparable changes in the other two cell types, but did not unmask any sign of NE sensitivity in the vasoconstrictors. However, the spike shoulder and AHP in vasoconstrictors were Cd(2+) sensitive, blocked by omega-conotoxin, an N-type calcium channel antagonist, and inhibited by oxotremorine-M, a muscarinic receptor agonist. These data show that NE can differentially modulate functional subsets of mammalian sympathetic neurons and that NE insensitivity can serve as a practical experimental criterion for identification of vasomotor neurons in the isolated ganglion.

Regulation of the subthreshold chloride conductance in the rat sympathetic neuron

The mechanisms that control chloride conductance (gCl) in the rat sympathetic neuron have been studied by the two-electrode voltage-clamp technique in mature, intact superior cervical ganglia in vitro. In addition to voltage dependence in the membrane potential range -120/-50 mV, gCl displays time- and activity-dependent regulation (sensitization). The resting membrane potential is governed by voltage-dependent gK and gCl, which determine values of cell input conductance ranging from 7 to 18 nS (full deactivation) to an upper value of about 130 nS (full activation and maximal gCl sensitization). The quiescent neuron, held at constant membrane potential, spontaneously and gradually moved from a low- to a high-conductance status. An increase (about 40 nS) in gCl accounted for this phenomenon, which could be prevented by imposing intermittent hyperpolarizing episodes. Following spike firing, gCl increased by 20-33 nS, independent of the cell conductance value preceding tetanization, and thereafter decayed to the pre-stimulus level within 5 min. Intracellular sodium depletion and its successive ionophoretic restoration moved the neuron from a stable low-conductance state to maximum gCl sensitization, pointing to a link between gCl sensitization and [Na+]i. The dependence of gCl build-up on [Na+]i and the time-course of such Na+-related modulation have been examined: gCl sensitization was absent at 0 [Na+]i, was well developed (20 nS) at 15 mM and tended towards a saturating value of 60 nS for higher [Na+]i. Sensitization was transient in response to neuron activity. In the silent neuron, sensitization of gCl shifted membrane potential over a range of about 15 mV.

Synaptic and Somatic Effects of Axotomy in the Intact, Innervated Rat Sympathetic Neuron

A biophysical description of the axotomized rat sympathetic neuron is reported, obtained by the two-electrode voltage-clamp technique in mature, intact superior cervical ganglia in vitro. Multiple aspects of neuron functioning were tested. Synaptic conductance activated by the whole presynaptic input decreased to 29% of the control value (0.92 muS per neuron) 1 day after axotomy and to 18% after 3 days. Despite the decrease in amplitude of the macroscopic current, miniature excitatory postsynaptic current (mEPSC) mean conductance, acetylcholine (ACh) equilibrium potential, and EPSC decay time constant were unaffected. Synaptic efficacy was tested during paired-pulse or maintained stimulation (5, 10, and 15 Hz, 10-s duration). Quantal release in axotomized neurons was preserved during the tetanus despite the reduction of the initial EPSC amplitude, suggesting that ACh secretion depended on the number of surviving synapses; each of them exhibited dynamic behavior during trains similar to that of normal synapses. Facilitation of EPSC amplitude was noted in 2-day axotomized neurons during the first few impulses in the train. Voltage-dependent potassium currents (the delayed I(KD) and the transient I(A)) exhibited an early drastic decrease in peak amplitude; these effects persisted 7 days after axotomy. Marked changes in I(A) kinetics occurred after injury: the steady-state inactivation curve shifted by up to +17 mV toward positive potentials and the voltage sensitivity of inactivation removal became steeper. I(A) impairment was reflected in a reduced inward threshold charge for discharge and reduced spike repolarization rate. Synaptic and somatic data were applied in a mathematical model to describe the progressive decrease in the safety factor, and the eventual failure of ganglionic transmission after axotomy.

Biophysical properties of the silent and activated rat sympathetic neuron following denervation

A biophysical description of the denervated rat sympathetic neuron is reported, obtained by the two-electrode voltage-clamp technique in mature intact superior cervical ganglia in vitro. At membrane potential values negative to −50 mV, the normal, quiescent neuron displays voltage-dependent K and Cl conductances; following direct or synaptic stimulation (15Hz for 10 s), the neuron moves to a new resting state characterized by increased amplitude and voltage dependence of Cl conductance. Denervation produces two main effects: 1) resting Cl conductance gradually increases while its voltage-dependence decreases; by 30 days a high-conductance resting state prevails, almost independent of membrane potential in the −50/−110 mV range; 2) the increase in amplitude and voltage-dependence of Cl conductance, produced by direct stimulation in control neurons, is less marked in denervated neurons, and is observed over an increasingly small range of membrane potentials. Thirty days after denervation, the prevailing high-conductance resting state appears virtually insensitive to changes in membrane potential and stimulation. Voltage-dependent potassium currents involved in spike electrogenesis (the delayed compound potassium current and the fast transient potassium current) exhibit an early drastic decrease in peak amplitude in the denervated neuron; the effect is largely reversed after 6 days. Remarkable changes in fast transient potassium current kinetics occur following denervation: the steady-state inactivation curve shifts by up to +15 mV toward positive potential and voltage sensitivity of inactivation removal becomes more steep. A comprehensive mathematical model of the denervated neuron is presented that fits the neuron behavior under current-clamp conditions. It confirms that neuronal excitability is tuned by the conductances (mostly chloride conductance) that control the resting membrane potential level, and by fast transient potassium current. Impairment of the latter reduces both inward threshold charge for firing and spike repolarization rate, and fast transient potassium current failure cancels the voltage dependence of both processes.

Differential expression and regulation of the high-affinity choline transporter CHT1 and choline acetyltransferase in neurons of superior cervical ganglia

Previous studies revealed that leukemia inhibitory factor (LIF) and retinoic acid (RA) induce a noradrenergic to cholinergic switch in cultured sympathetic neurons of superior cervical ganglia (SCG) by up-regulating the coordinate expression of choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter. Here, we examined the effect of both factors on high-affinity choline uptake (HACU) and on expression of the high-affinity choline transporter CHT1. We found that HACU and CHT1-mRNA levels are up-regulated by LIF and down-regulated by RA in these neurons. Thus, in contrast to LIF, RA differentially regulates the expression of the presynaptic cholinergic proteins. Moreover, we showed that untreated SCG neurons express HACU and CHT1-mRNAs at much higher levels than ChAT activity and transcripts. In intact SCG, CHT1-mRNAs are abundant and synthesized by the noradrenergic neurons themselves. This study provides the first example of CHT1 expression in neurons which do not use acetylcholine as neurotransmitter.

Calcium current components in intact and dissociated adult mouse sympathetic neurons

We examined which types of high threshold Ca 2+ channels are activated by depolarization in intact and dissociated sympathetic neurons from adult mouse superior cervical ganglia (SCG). Ba 2+ currents were recorded with microelectrodes and discontinuous voltage clamp from neurons in intact ganglia, and using the perforated patch clamp technique in dissociated cells. Peak current was larger in intact neurons, although the voltage dependence was similar. Successive application of ω-conotoxin GVIA, ω-conotoxin MVIIC and nifedipine revealed that the total current in intact cells was composed by 29% N-type, 13% P/Q-type, 32% L-type and 26% resistant to blockade (R-type). In dissociated cells, the N component was larger and the L component smaller, whereas P/Q-type and R-type were similar. Peak currents evoked with an action potential waveform instead of a square pulse were larger in both preparations but the proportions of each component were similar. We conclude that dissociating and culturing somata results in data that only partially reflect the situation in intact neurons. Assuming that the main effect of dissociation is the removal of mature dendritic membrane, the data suggest that L channels are more abundant on dendrites and N channels on the soma of intact sympathetic neurons, whereas P/Q and R channels may be uniformly distributed over the cell surface. Finally, in intact SCG neurons from rats, the proportions of current evoked by a pulse were: 49% N-type, 11% P/Q-type, 21% L-type and 20% R-type when nifedipine was applied last, suggesting that there are species differences in the expression of L and N channels.

Effects of ATP on intracellular calcium dynamics of neurons and satellite cells in rat superior cervical ganglia.

Adenosine 5'-triphosphate (ATP) which is released from neuronal and non-neuronal tissues interacts with cell surface receptors to produce a broad range of physiological responses. The present study addressed the issue of whether the cells of the superior cervical ganglia (SCG) respond to ATP. To this end, the dynamics of the intracellular calcium ion concentration ([Ca2+]i) of neurons and satellite cells in intact SCG was analyzed by laser scanning confocal microscopy. ATP produced an increase of [Ca2+]i in both neurons and satellite cells; initially, ATP elicited [Ca2+]i increase in satellite cells and, subsequently, a [Ca2+]i change in neurons was observed. P1 purinoceptor agonists had no effect on this process, but P2 purinoceptor agonists induced [Ca2+]i increase and suramin totally inhibited ATP-induced [Ca2+]i dynamics in both neurons and satellite cells. In satellite cells, Ca2+ channel blockers and the removal of extracellular Ca2+, but not thapsigargin pretreatment, abolished ATP-induced [Ca2+]i dynamics. In contrast, thapsigargin pretreatment abolished ATP-induced [Ca2+]i dynamics in neurons. Reactive blue-2 inhibited the ATP-induced reaction on neurons alone. Uridine 5'-triphosphate caused a [Ca2+]i increase in neurons and alpha,beta-methylene ATP caused a [Ca2+]i increase in satellite cells. We concluded that neurons respond to extracellular ATP mainly via P2Y purinoceptors and that satellite cells respond via P2X purinoceptors.

Plasticity in the phenotypic expression of catecholamines and vasoactive intestinal peptide in adult rat superior cervical and stellate ganglia after long-term hypoxia in vivo

Sympathetic ganglia in the adult rat contain various populations of nerve cells which demonstrate plasticity with respect to their transmitter phenotype. The plasticity of the neuronal cell bodies and of the small intensely fluorescent cells in the superior cervical and stellate ganglia in response to hypoxia in vivo (10% O 2 for seven days) was assessed by studying the expression of catecholamines and vasoactive intestinal peptide. The levels of norepinephrine, dopamine, 3,4-dihydroxyphenylacetic acid and vasoactive intestinal peptide immunoreactivity were determined. In addition, the density of the immunohistochemical staining of cells for tyrosine hydroxylase and vasoactive intestinal peptide was evaluated. In the intact superior cervical ganglion, hypoxia increased the dopamine level as well as the density of small intensely fluorescent cells immunolabelled for tyrosine hydroxylase and vasoactive intestinal peptide. In the axotomized ganglion, hypoxia elicited a twofold rise in the level of the vasoactive intestinal peptide as well as enhancing the density of neuronal cell bodies immunostained for this peptide. Thus, the effect of hypoxia on the expression of vasoactive intestinal peptide expression in neurons was dependent on neural interactions. In the intact stellate ganglion, hypoxia alone induced a 1.5-fold increase in the density of neuronal cell bodies immunostained for vasoactive intestinal peptide. Thus, ganglia-specific factors appeared to play a role in determining changes in neuronal phenotype in response to hypoxia. The present study provides evidence for the involvement of dopamine and vasoactive intestinal peptide in ganglionic responses to long-term hypoxia as well as for differential responses by the two ganglionic cell populations, i.e. neuronal cell bodies and small intensely fluorescent cells. Changes in the expression of the vasoactive intestinal peptide during long-term hypoxia may be of energetic, trophic and/or synaptic significance. Hypoxia may be considered to be a vasoactive intestinal peptide-inducing factor in sympathetic ganglia.

Cytochrome b558 and hydrogen peroxide production in small intensely fluorescent cells of sympathetic ganglia.

Small intensely fluorescent (SIF) cells are paraganglionic cells derived from sympathicoblasts which may serve as interneurons, endo-/paracrine cells or arterial chemoreceptors within sympathetic ganglia. Like paraganglionic cells of other locations, e.g., carotid body glomus cells, they are responsive to hypoxia. Recent studies on glomus cells and other hypoxia-sensing cells suggested the involvement of a b558-type cytochrome and intracellular generation of H2O2 in the process of oxygen sensing. In the present study, we demonstrate the occurrence of the small subunit of cytochrome b558, p22phox, in SIF cells of guinea-pig sympathetic ganglia by immunohistochemistry using two different antisera. H2O2 production was monitored in explanted intact superior cervical ganglia of 2-day-old rats by confocal laser scanning analysis of rhodamine 123 fluorescence generated due to oxidation of dihydrorhodamine 123 by H2O2. Using this technique, SIF cell clusters appeared as sites of highest H2O2 production within the ganglia. Thus, SIF cells exhibit two key features of an oxidase system generating reactive oxygen species. This may be involved in the proposed chain of events in oxygen sensing, but alternative cellular functions of this system have also to be considered.

Myosin II is involved in transmitter release at synapses formed between rat sympathetic neurons in culture

The presynaptic function of myosin II was studied at cholinergic synapses formed between rat superior cervical ganglion neurons in culture. Immunofluorescent staining showed that myosin 11 was colocalized with synaptophysin at the presynaptic nerve terminals. Antimyosin II antibody introduced into presynaptic neurons inhibited synaptic transmission. Transmission was also inhibited in a dose-dependent manner by two inhibitors of myosin light chain kinase: a peptide, SM-1, and an organic inhibitor, wortmannin. The inhibition produced by these agents was dependent on presynaptic activity. Extracellularly applied wortmannin also blocked synaptic transmission, but its effects were slower in onset. Wortmannin also decreased postsynaptic potentials and post-tetanic potentiation in intact superior cervical ganglia. These results suggest a model in which myosin light chain kinase phosphorylates myosin, and the resultant change in actin-myosin interactions is involved in neurotransmitter release.

An mRNA homologous to interleukin-1 receptor type I is expressed in cultured rat sympathetic ganglia

Interleukin-1 (IL-1) induces substance P (SP) gene expression in cultured rat superior cervical ganglion (SCG) explants. In order to study the molecular mechanism of this action of IL-1, the presence of an interleukin-1 receptor (IL-1R) activity and the identity of an mRNA homologous to known IL-1R sequence was determined in SCG. The SP increase is blocked by recombinant IL-1 receptor antagonist protein, so IL-1 must be interacting with a specific receptor. We have cloned cDNA homologous to IL-1R Type I from rat SCG using a reverse transcription-polymerase chain reaction (RT-PCR). The resulting cDNA sequence is strongly homologous with mouse and human IL-1R cDNA of the T cell and fibroblast type (type I; encoding an 80-kDa protein). mRNA specific for IL-1R can be readily detected in intact SCG by quantitative RT-PCR and S1 hybridization. However, the level of IL-1R mRNA increases 3–6-fold by 2 days in culture. This increase is independent of the presence of dexamethasone, IL-1β or IL-1 receptor antagonist protein ligands. The increase of IL-1R following explantation, a model of nerve injury, may provide a mechanism linking inflammatory signalling to neuronal phenotypic changes.

Selective alterations in transglutaminase activity of rat superior cervical ganglia in response to neurotransmitters, high potassium and sialic acid-containing compounds

We examined the in vitro effects of neurotransmitters, high KCl as well as sialic acid-containing compounds (GM1; SC) on transglutaminase (TG) activity in isolated superior cervical ganglia (SCG) one week after denervation or axotomy. Following denervation, TG activity in SCG decreased to 83% of the unoperated control value, whereas that of axotomized ganglia was 28% of control. Thus, TG activity was relatively unaffected when sympathetic ganglionic neurons were preserved, but was markedly reduced under conditions where neurons were degenerating. Addition of ACh (0.1 mM) to the medium during aerobic incubation stimulated TG activity more than 3-fold in denervated ganglia but had no effect on TG activity in axotomized ganglia. Similarly, the NE (0.05 mM)-induced decreased of TG activity observed in intact SCG was also seen following denervation (−4%) but not following axotomy. In denervated SCG, the stimulatory effects of ACh were virtually abolished by co-addition of the cholinergic antagonists, atropine or hexamethnium, while the suppressant effects of NE were blocked by the adrenergic antagonists, propranolol prozosin or yohimbine. These results imply that transmitter-induced rapid changes in TG activity occur predominantly in ganglionic neurons. When the ganglia were depolarized by high KCl (50 mM), a significant increase in TG activity in each intact, denervated and axotomized SCG was seen with qualitatively similar manner, suggesting that high KCl-induced depolarization effects both neuronal and glial components in the SCG. The marked increase in ganglionic TG activity in response to GM1 (5 nM) and synthetic SC (0.02 mM) were lost in denervated SCG but only partially reduced in axotomized SCG. These findings indicate that the target cells responding to sialyl compounds are preganglionic nerve terminals and/or glial cells.

Transynaptic regulation of low-affinity p75 nerve growth factor receptor mRNA precedes and accompanies lesion-induced collateral neuronal sprouting

The bilateral sympathetic innervation of the rat pineal gland from the two superior cervical ganglia (SCG) is a useful model system to investigate the mechanisms by which intact neurons compensate for neuronal losses. Cutting of the internal carotid nerve (ICN) on one side has been shown to result in the removal of approximately one-half of the innervation to the pineal gland within 2 days. This denervation is followed by the development of collateral neuronal sprouting from the contralateral “intact” SCG, most of which takes place during the next 2 days. Using a solution hybridization protection assay, levels of low-affinity NGF receptor p75 NGFR mRNA (pg/μg total RNA) were found to be increased 25%, with no change in cyclophilin mRNA, in the SCG contralateral to the lesion performed 1 or 3 days earlier. In situ hybridization with a 35S riboprobe complementary to p75 NGFR mRNA demonstrated a large increase in this mRNA in some cells of this intact SCG at both 1 and 3 days after a contralateral ICN cut lesion. The clustering of these cells toward the rostral portion of the SCG suggests that they may overlap with the population of sympathetic neurons which provides innervation to bilaterally innervated structures such as the pineal gland. The nature of the signals involved in the regulation of NGF receptor mRNA levels and their role in initiating and maintaining collateral sprouting remain to be fully established. Nevertheless, the time course of the changes in mRNA levels suggests that regulation of the low-affinity NGF receptor gene may be involved in the sequence of events associated with the collateral sprouting response by intact sympathetic nerve cells following partial denervation.

Decentralization induces long-term c-fos protein-like immunoreactivity in non-neuronal cells in the rat superior cervical ganglion

The expression of c-fos protein(s) was studied immunohistochemically in the intact and decentralized superior cervical ganglia (SCG) of adult rats. C-fos protein(s)-like immunoreactivity (c-fos-LI) was detected in 1–2% of the principal ganglion cells but not in the satellite or Schwann cells of the intact SCG. After transection of the cervical sympathetic trunk, satellite and Schwann cells in the ganglion expressed c-fos-LI from one hour through 8 days following the operation. The maximal staining was seen 1 day postransection (d.p.t.), when the c-fos-LI was intense throughout the ganglion. No change was observed in the c-fos-LI of the principal neurons. At 8 d.p.t. the number of immunoreactive cells and their staining intensity had decreased, and at 14 d.p.t. they were back to the control level. These results suggest that a long-term expression of c-fos protein(s) is induced in the SCG satellite and Schwann cells after preganglionic denervation possibly related to their activation during nerve degeneration and regeneration.

Protein Phosphorylation in Intact Superior Cervical Ganglion During Regeneration

The incorporation of radioactive phosphate into proteins of both normal and regenerating superior cervical ganglion nerve of the rat is reported. Incorporation studies carried out by in vitro and in vivo methods are compared. In the in vitro method, excised intact ganglia or their homogenates were incubated in the presence of inorganic phosphate or ATP, respectively, under various conditions. Proteins were analyzed by gel electrophoresis followed by autoradiography, in which quantitative but not qualitative differences between regenerating and control cases were apparent. In the in vivo procedure, inorganic phosphate was injected into the living animal 4 h before removal of ganglia. At least fivefold more proteins became labeled in vivo than in vitro, whereas no similarity in the pattern of labeling between the two methods was observed. For example, the most heavily labeled protein in the in vivo method, tentatively identified as microtubule-associated protein-2, was not detected on autoradiograms of proteins labeled by the in vitro method. In this latter method, an 85-kDa species and growth-associated protein-43 were always labeled, and the extent of their phosphorylation was enhanced by the additional presence of phosphatidylserine and Ca2+, a result indicating that these labeled species are substrates of protein kinase C. The in vitro conditions also led to the labeling of proteins identified as alpha- and beta-tubulin. Comparison of the methods suggests that removal of the ganglion interferes with the function of protein phosphorylation systems and that this effect involves elements of the cytoskeleton.

Differential effect of zinc on the vertebrate GABAA‐receptor complex

1. gamma-Aminobutyric acid (GABA) responses were recorded from rat superior cervical ganglia (SCG) in culture using the whole cell recording technique. 2. Zinc (50-300 microM) reversibly antagonized the GABA response in embryonic and young post-natal neurones, while neurones cultured from adult animals were far less sensitive and occasionally resistant to zinc blockade. Cadmium (100-300 microM) also antagonised the GABA response, while barium (100 microM-2 mM) was ineffective. 3. The differential blocking effect of zinc on cultured neurones of different ages also occurred in intact SCG tissue. 4. The GABA log dose-response curve constructed with foetal or adult cultured neurones was reduced in a non-competitive manner by zinc. This inhibition was minimally affected by the membrane potential. 5. The GABA response recorded intracellularly from guinea-pig pyriform cortical slices was enhanced by zinc (300-500 microM), which occurred concurrently with a decrease in the input conductance of the cell. The enhancement was unaffected by prior blockade of the GABA uptake carrier by 1 mM nipecotic acid. This phenomenon could be reproduced by barium (300 microM) and cadmium (300 microM). 6. We conclude that the vertebrate neuronal GABAA-receptor becomes less sensitive to zinc with neural (GABAA-receptor?) development, and the enhanced GABA response recorded in the CNS is a consequence of the reduction in the input conductance and not due to a direct effect on the receptor complex.

Cooperation and competition during development: neonatal lesioning of the superior cervical ganglion induces cell death of trigeminal neurons innervating the cerebral blood vessels but prevents the loss of axon collaterals from the neurons that survive

In the adult rodent trigeminal ganglion there is a period of postnatal cell death in the population of cells with axons innervating the middle cerebral artery (O'Connor and van der Kooy, 1986b). The superior cervical ganglion (SCG) also has a projection to the middle cerebral artery (MCA; Mayberg et al., 1984; Cowen et al., 1986, 1987; present report). We hypothesized that the trigeminal ganglion cells innervating the MCA may be competing with the superior cervical projection for target area or for a target factor for survival, and thus the removal of the superior cervical projection at birth (sympathectomy) may promote the survival of some of the trigeminal-artery innervating cells that normally would die. Multiple fluorescent retrograde tracing was employed to analyze the postnatal development of the trigeminal projection to the MCA in sympathectomized and sibling control rats. We found that the SCG projection to the MCA exhibits a period of postnatal cell death. The trigeminal ganglion projection exhibits axon degeneration as well as postnatal cell death. Postnatal day 0 (P0) lesioning of the SCG did not prevent cell death or axon loss in the trigeminal projection to the cerebral artery. In fact, increased cell death of the trigeminal-artery projecting neurons was observed in the lesioned animals when compared to nonlesioned sibling controls. By P55, we found that 80% of these trigeminal neurons had died in lesioned animals, compared with 50% in controls. In both control and sympathectomized rats, close to 90% of the trigeminal neurons innervating the artery in the neonate can no longer be retrogradely labeled from the MCA by tracer applications at P25-90. Thus, although the presence of an intact SCG may protect some trigeminal-artery projecting neurons from cell death, it does not prevent axon retraction and does not permit a larger absolute number of trigeminal axons to innervate the arteries in the adult. Thus, separate mechanisms are responsible for the survival of perikarya versus the retraction of their axons from the MCA. Surprisingly, in the neonatally sympathectomized rats almost 20% of those trigeminal cells that maintained a projection to the MCA at P90 also had a projection to the forehead. In contrast, less than 3% of the artery innervating trigeminal cells in the P90 control rats had an axon collateral to the forehead.(ABSTRACT TRUNCATED AT 400 WORDS)