Rat Submandibular Ganglion Research Articles

Active Dendritic Conductances Dynamically Regulate GABA Release from Thalamic Interneurons

Inhibitory interneurons in the dorsal lateral geniculate nucleus (dLGN) process visual information by precisely controlling spike timing and by refining the receptive fields of thalamocortical (TC) neurons. Previous studies indicate that dLGN interneurons inhibit TC neurons by releasing GABA from both axons and dendrites. However, the mechanisms controlling GABA release are poorly understood. Here, using simultaneous whole-cell recordings from interneurons and TC neurons and two-photon calcium imaging, we find that synchronous activation of multiple retinal ganglion cells (RGCs) triggers sodium spikes that propagate throughout interneuron axons and dendrites, and calcium spikes that invade dendrites but not axons. These distinct modes of interneuron firing can trigger both a rapid and a sustained component of inhibition onto TC neurons. Our studies suggest that active conductances make LGN interneurons flexible circuit-elements that can shift their spatial and temporal properties of GABA release in response to coincident activation of functionally related subsets of RGCs.

ω-Conotoxin CVID Inhibits a Pharmacologically Distinct Voltage-sensitive Calcium Channel Associated with Transmitter Release from Preganglionic Nerve Terminals

Neurotransmitter release from preganglionic parasympathetic neurons is resistant to inhibition by selective antagonists of L-, N-, P/Q-, R-, and T-type calcium channels. In this study, the effects of different omega-conotoxins from genus Conus were investigated on current flow-through cloned voltage-sensitive calcium channels expressed in Xenopus oocytes and nerve-evoked transmitter release from the intact preganglionic cholinergic nerves innervating the rat submandibular ganglia. Our results indicate that omega-conotoxin CVID from Conus catus inhibits a pharmacologically distinct voltage-sensitive calcium channel involved in neurotransmitter release, whereas omega-conotoxin MVIIA had no effect. omega-Conotoxin CVID and MVIIA inhibited depolarization-activated Ba(2+) currents recorded from oocytes expressing N-type but not L- or R-type calcium channels. High affinity inhibition of the CVID-sensitive calcium channel was enhanced when position 10 of the omega-conotoxin was occupied by the smaller residue lysine as found in CVID instead of an arginine as found in MVIIA. Given that relatively small differences in the sequence of the N-type calcium channel alpha(1B) subunit can influence omega-conotoxin access (Feng, Z. P., Hamid, J., Doering, C., Bosey, G. M., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 15728-15735), it is likely that the calcium channel in preganglionic nerve terminals targeted by CVID is a N-type (Ca(v)2.2) calcium channel variant.

Pre- and postsynaptic actions of ATP on neurotransmission in rat submandibular ganglia

The pre- and postsynaptic actions of exogenously applied ATP were investigated in intact and dissociated parasympathetic neurones of rat submandibular ganglia. Nerve-evoked excitatory postsynaptic potentials (EPSPs) were not inhibited by the purinergic receptor antagonists, suramin and pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), or the desensitising agonist, α,β-methylene ATP. In contrast, EPSPs were abolished by the nicotinic acetylcholine receptor antagonists, hexamethonium and mecamylamine. Focal application of ATP (100 μM) had no effect on membrane potential of the postsynaptic neurone or on the amplitude of spontaneous EPSPs. Taken together, these results suggest the absence of functional purinergic (P2) receptors on the postganglionic neurone in situ. In contrast, focally applied ATP (100 μM) reversibly inhibited nerve-evoked EPSPs. Similarly, bath application of the non-hydrolysable analogue of ATP, ATPγS, reversibly depressed EPSPs amplitude. The inhibitory effects of ATP and ATPγS on nerve-evoked transmitter release were antagonised by bath application of either PPADS or suramin, suggesting ATP activates a presynaptic P2 purinoceptor to inhibit acetylcholine release from preganglionic nerves in the submandibular ganglia. In acutely dissociated postganglionic neurones from rat submandibular ganglia, focal application of ATP (100 μM) evoked an inward current and subsequent excitatory response and action potential firing, which was reversibly inhibited by PPADS (10 μM). The expression of P2X purinoceptors in wholemount and dissociated submandibular ganglion neurones was examined using polyclonal antibodies raised against the extracellular domain of six P2X purinoceptor subtypes (P2X 1–6). In intact wholemount preparations, only the P2X 5 purinoceptor subtype was found to be expressed in the submandibular ganglion neurones and no P2X immunoreactivity was detected in the nerve fibres innervating the ganglion. Surprisingly, in dissociated submandibular ganglion neurones, high levels of P2X 2 and P2X 4 purinoceptors immunoreactivity were found on the cell surface. This increase in expression of P2X 2 and P2X 4 purinoceptors in dissociated submandibular neurones could explain the increased responsiveness of the neurones to exogenous ATP. We conclude that disruption of ganglionic transmission in vivo by either nerve damage or synaptic blockade may up-regulate P2X expression or availability and alter neuronal excitability.

Ionic selectivity of native ATP-activated (P2X) receptor channels in dissociated neurones from rat parasympathetic ganglia.

1. The relative permeability of the native P2X receptor channel to monovalent and divalent inorganic and organic cations was determined from reversal potential measurements of ATP-evoked currents in parasympathetic neurones dissociated from rat submandibular ganglia using the dialysed whole-cell patch clamp technique. 2. The P2X receptor-channel exhibited weak selectivity among the alkali metals with a selectivity sequence of Na(+) > Li(+) > Cs(+) > Rb(+) > K(+), and permeability ratios relative to Cs(+) (P(X)/P(Cs)) ranging from 1.11 to 0.86. 3. The selectivity for the divalent alkaline earth cations was also weak with the sequence Ca(2+) > Sr(2+) > Ba(2+) > Mn(2+) > Mg(2+). ATP-evoked currents were strongly inhibited when the extracellular divalent cation concentration was increased. 4. The calculated permeability ratios of different ammonium cations are higher than those of the alkali metal cations. The permeability sequence obtained for the saturated organic cations is inversely correlated with the size of the cation. The unsaturated organic cations have a higher permeability than that predicted by molecular size. 5. Acidification to pH 6.2 increased the ATP-induced current amplitude twofold, whereas alkalization to 8.2 and 9.2 markedly reduced current amplitude. Cell dialysis with either anti-P2X(2) and/or anti-P2X(4) but not anti-P2X(1) antibodies attenuated the ATP-evoked current amplitude. Taken together, these data are consistent with homomeric and/or heteromeric P2X(2) and P2X(4) receptor subtypes expressed in rat submandibular neurones. 6. The permeability ratios for the series of monovalent organic cations, with the exception of unsaturated cations, were approximately related to the ionic size. The relative permeabilities of the monovalent inoganic and organic cations tested are similar to those reported previously for cloned rat P2X(2) receptors expressed in mammalian cells.

Calcium channels controlling acetylcholine release from preganglionic nerve terminals in rat autonomic ganglia

Little is known about the nature of the calcium channels controlling neurotransmitter release from preganglionic parasympathetic nerve fibres. In the present study, the effects of selective calcium channel antagonists and amiloride were investigated on ganglionic neurotransmission. Conventional intracellular recording and focal extracellular recording techniques were used in rat submandibular and pelvic ganglia, respectively. Excitatory postsynaptic potentials and excitatory postsynaptic currents preceded by nerve terminal impulses were recorded as a measure of acetylcholine release from parasympathetic and sympathetic preganglionic fibres following nerve stimulation. The calcium channel antagonists ω-conotoxin GVIA (N type), nifedipine and nimodipine (L type), ω-conotoxin MVIIC and ω-agatoxin IVA (P/Q type), and Ni 2+ (R type) had no functional inhibitory effects on synaptic transmission in both submandibular and pelvic ganglia. The potassium-sparing diuretic, amiloride, and its analogue, dimethyl amiloride, produced a reversible and concentration-dependent inhibition of excitatory postsynaptic potential amplitude in the rat submandibular ganglion. The amplitude and frequency of spontaneous excitatory postsynaptic potentials and the sensitivity of the postsynaptic membrane to acetylcholine were unaffected by amiloride. In the rat pelvic ganglion, amiloride produced a concentration-dependent inhibition of excitatory postsynaptic currents without causing any detectable effects on the amplitude or configuration of the nerve terminal impulse. These results indicate that neurotransmitter release from preganglionic parasympathetic and sympathetic nerve terminals is resistant to inhibition by specific calcium channel antagonists of N-, L-, P/Q- and R-type calcium channels. Amiloride acts presynaptically to inhibit evoked transmitter release, but does not prevent action potential propagation in the nerve terminals, suggesting that amiloride may block the pharmacologically distinct calcium channel type(s) on rat preganglionic nerve terminals.

The channel‐blocking action of methonium compounds on rat submandibular ganglion cells

The channel‐blocking action of methonium compounds on rat submandibular ganglion cells

The channel‐blocking action of methonium compounds on rat submandibular ganglion cells

The effects of drugs of the polymethylene bis‐trimethylammonium (methonium) series on the characteristics of the synaptic currents (e.s.cs) recorded from voltage‐clamped rat submandibular ganglion cells have been studied. The drugs studied were from C4 to C10 (decamethonium). All of the drugs except C4 shortened the initial decay phase of the e.s.c.; C9 and C10 produced an additional slowly decaying component. These effects were interpreted in terms of an open channel block mechanism, the calculated rate constants for association with the open channel at − 80 mV being fairly similar (5.9 × 106 to 18.1 × 106M−1 s−1) for all of the compounds except C4, which had no effect on the e.s.c. decay. All of the compounds produced use‐dependent block when tested with short trains of stimuli at 10 Hz, or with trains of ionophoretic pulses of acetylcholine, consistent with their channel blocking property. Tubocurarine had a similar effect, but not trimetaphan or mecamylamine. Recovery from use‐dependent block with short chain methonium compounds, up to C8, was very slow in the absence of agonist, being incomplete even after several minutes. With C9 or C10 or tubocurarine, recovery from use‐dependent block was complete within a few seconds. With C6 recovery in the absence of agonist was unaffected by membrane potential, but could be accelerated by applying acetylcholine with the cell depolarized to − 40 mV. This persistent block was ascribed to the ability of the blocking molecule to become trapped by closure of the channel. With C9 and C10 it is assumed that their presence inhibits channel closure, so they can escape without the help of agonist. When use‐dependent block is avoided by leaving the ganglion unstimulated during equilibration with the blocking drug, the first e.s.c. elicited shows no appreciable reduction of amplitude, though with C6, C7 or C8 subsequent responses elicited at 0.1 Hz become progressively more blocked. Even at 1 Mm, C6 does not prevent acetylcholine from opening ionic channels. It is concluded that all of the effects on e.s.c. amplitude can be interpreted in terms of channel block, there being no evidence of any receptor blocking action.

Amplitude and time course of evoked and spontaneous synaptic currents in rat submandibular ganglion cells.

1. Excitatory postsynaptic currents (EPSCs) were recorded in rat submandibular ganglion cells in vitro using the two-electrode voltage clamp technique. 2. The peak amplitude of EPSCs evoked by nerve impulses in single presynaptic fibres varied between 1.2 and 9.8 nA in different cells (mean = 4.6 +/- 2.6 nA; n = 23; -80 mV membrane potential; 22-25 degrees C). 3. Experiments were performed to re-investigate a previous hypothesis that different mechanisms underlie the generation of evoked versus spontaneous quantal EPSCs in submandibular cells. This hypothesis was based on the observation of different time courses of evoked and spontaneous EPSCs. 4. In agreement with previous studies, the time course of the decay phase of evoked EPSCs was described by the sum of two exponentials, with time constants tau 1 and tau 2 of 6.9 +/- 0.7 and 34.4 +/- 7.7 ms, respectively (n = 23; -80 mV membrane potential). 5. The double-exponential decay of evoked EPSCs persisted when transmitter release was reduced by bath addition of 100 microM cadmium chloride to the level of failures, one or several quanta. 6. Spontaneous EPSCs exhibited mean amplitudes of 81 +/- 24 pA (n = 5 cells; -80 mV membrane potential), and displayed an extremely wide range of peak amplitudes in the same cell (mean coefficient of variation (c.v.) = 0.37 +/- 0.09; n = 5 cells). In contrast to a previous report (see below), the decay phase of spontaneous EPSCs was found to exhibit a double-exponential time course with time constants similar to those of the evoked EPSC recorded in the same cell. 7. These results indicate that evoked and spontaneously released quanta of transmitter most probably act on the same population of postsynaptic receptors in submandibular ganglion cells. There is a large variability in the peak amplitudes of quantal EPSCs recorded in the same cell. This large variability is not due to electrotonic effects, since these cells lack dendrites.

Immunohistochemical Localization of [Met 5]enkephalin and [Met 5]enkephalin-Arg 6-Gly 7-Leu 8 in sympathetic and parasympathetic neurons and nerve fibers projecting to the rat submandibular gland

The localization of [Met 5]enkephalin, [Met 5]enkephalin-Arg 6-Gly 7-Leu 8, vasoactive intestinal polypeptide and tyrosine hydroxylase immunoreactivities was studied in the submandibular gland of adult Sprague-Dawley and Wistar rats using the indirect immunofluorescence technique. Immunoreactivities for [Met 5]enkephalin and [Met 5]enkephalin-Arg 6-Gly 7-Leu 8, a proenkephalin A-derived octapeptide, showed identical distributions. A large number of enkephalin-immunoreactive nerve fibers were detected around secretory acini, along intercalated ducts, convoluted granular tubules, intra- and interlobular ducts, as well as in close contact with blood vessels. The submandibular ganglia contained several enkephalin-immunoreactive neurons and nerve fibers. In the superior cervical ganglion numerous enkephalin-immunoreactive neurons and nerve fibers were also detected. Immunohistochemical co-localization studies indicated that [Met 5]enkephalin and [Met 5]enkephalin-Arg 6-Gly 7-Leu 8 immunoreactivities co-exist with vasoactive intestinal polypeptide in a subpopulation of neurons of the rat submandibular ganglia, in nerve trunks along the salivary ducts of the gland, and in nerve fibers around the acini. Uni- or bilateral superior cervical ganglionectomies for 1–4 weeks resulted in a complete disappearance of tyrosine hydroxylase immunoreactivity in the glandular parenchyma, while moderate tyrosine hydroxylase immunoreactivity was seen in some neurons of the submandibular ganglia. Abundant [Met 5]enkephalin-Arg 6-Gly 7-Leu 8-immunoreactive nerve fibers were still seen around the acini and blood vessels, as well as close to salivary ducts. These operations did not affect the [Met 5]enkephalin-Arg 6-Gly 7-Leu 8-immunoreactive neurons in the submandibular ganglia. Many principal neurons in the superior cervical ganglion contained both [Met 5]enkephalin-Arg 6-Gly 7-Leu 8 and tyrosine hydroxylase immunoreactivity. Nerve ligation experiments indicated that [Met 5]enkephalin-Arg 6-Gly 7-Leu 8-immunoreactive sympathetic fibers project along the external carotid nerve. Accordingly, nerve fibers were found around the acini and blood vessels as well as in nerve trunks along the salivary ducts of the submandibular gland, showing co-localization of [Met 5]enkephalin-Arg 6-Gly 7-Leu 8 and tyrosine hydroxylase. Taken together, these observations suggest that the nerve fibers of the rat submandibular gland containing proenkephalin A-derived peptides are of both sympathetic and parasympathetic origin.

Nicotinic receptors of frog ganglia resemble pharmacologically those of skeletal muscle

The actions of ACh antagonists were studied on synaptic currents of autonomic ganglia of the frog. Fast excitatory synaptic currents (ESCs) were recorded from cardiac and paravertebral neurons with the use of the 2-microelectrode voltage-clamp method. The actions of 4 ACh antagonists, tubocurarine, hexamethonium, trimetaphan, and decamethonium were studied. Tubocurarine was effective at reducing the peak amplitude of ESCs (50% inhibition at 3 microM). In contrast, tubocurarine (1-30 microM) reduced the time constant of ESC decay by only 9% compared with controls. Both of these effects of tubocurarine were independent of membrane potential. Hexamethonium was a weak inhibitor of ESCs; at 600 microM peak amplitude was reduced only to about 60% of controls and decay time constants were unaffected at concentrations between 10 and 600 microM. These effects of tubocurarine and hexamethonium are consistent with these drugs being receptor antagonists with no evidence of ion channel block. Trimetaphan (3-100 microM) and decamethonium (100 microM) reduced the peak amplitude of ESCs. In the presence of 100 microM trimetaphan or 10 microM decamethonium, ESC decays were biexponential. The 2 exponential components induced by the presence of these drugs were faster and slower, respectively, than the single-exponential component of control ESC decays. The effects of these 2 drugs were more pronounced at hyperpolarized potentials and are consistent with a channel-blocking action. The actions of the 4 ACh antagonists on frog autonomic ganglia are similar to their effects at the neuromuscular junction but dissimilar to their effects on the rat submandibular ganglion.(ABSTRACT TRUNCATED AT 250 WORDS)

Neonatal synapse elimination in the rat submandibular ganglion: effect of retarded target growth.

1. We have studied synapse elimination in the submandibular ganglion of neonatal rats to determine the effects of retarded target growth on synaptic development. Neurons of this ganglion provide parasympathetic innervation to the submandibular and sublingual salivary glands. 2. Ligating the main salivary ducts 2-4 days after birth at a point where nerve fibers were not damaged reduces gland weight by 55% during the 2nd wk after birth and 80% by adulthood. 3. In control animals, the average number of preganglionic inputs/neuron normally declines steadily during the first few weeks after birth, before stabilizing during the 5th wk at the control adult level. Between birth and adulthood, the number of ganglionic neurons increases by 150%. 4. Ganglia from duct-ligated animals showed an acceleration in the process of synapse elimination. Input number in experimental ganglia reached the control adult level during the 3rd wk after birth. This acceleration is confined solely to ganglia that innervate the underdeveloped glands. 5. The loss of inputs was not further enhanced by prolonged target atrophy. Thus average input numbers to neurons of 5th wk or adult experimental ganglia were not different from age-matched control values. 6. No differences from control values were seen in most cases for resting potentials, input resistances, or cell size. However, the increase in neuron number was retarded in experimental animals, and the number of synapses/neuronal profile was reduced in the adult animals. 7. Thus subnormal target growth leads to an acceleration in the process of synaptic elimination in neonatal rats. This acceleration may be mediated by alterations in the level of trophic factors emanating from the target.

The channel-blocking action of methonium compounds on rat submandibular ganglion cells.

The effects of drugs of the polymethylene bis-trimethylammonium (methonium) series on the characteristics of the synaptic currents (e.s.cs) recorded from voltage-clamped rat submandibular ganglion cells have been studied. The drugs studied were from C4 to C10 (decamethonium). All of the drugs except C4 shortened the initial decay phase of the e.s.c.; C9 and C10 produced an additional slowly decaying component. These effects were interpreted in terms of an open channel block mechanism, the calculated rate constants for association with the open channel at -80 mV being fairly similar (5.9 X 10(6) to 18.1 X 10(6)M-1S-1) for all of the compounds except C4, which had no effect on the e.s.c. decay. All of the compounds produced use-dependent block when tested with short trains of stimuli at 10 Hz, or with trains of ionophoretic pulses of acetylcholine, consistent with their channel blocking property. Tubocurarine had a similar effect, but not trimetaphan or mecamylamine. Recovery from use-dependent block with short chain methonium compounds, up to C8, was very slow in the absence of agonist, being incomplete even after several minutes. With C9 or C10 or tubocurarine, recovery from use-dependent block was complete within a few seconds. With C6 recovery in the absence of agonist was unaffected by membrane potential, but could be accelerated by applying acetylcholine with the cell depolarized to -40 mV. This persistent block was ascribed to the ability of the blocking molecule to become trapped by closure of the channel. With C9 and C10 it is assumed that their presence inhibits channel closure, so they can escape without the help of agonist. When use-dependent block is avoided by leaving the ganglion unstimulated during equilibration with the blocking drug, the first e.s.c. elicited shows no appreciable reduction of amplitude, though with C6, C7 or C8 subsequent responses elicited at 0.1 Hz become progressively more blocked. Even at 1 mM, C6 does not prevent acetylcholine from opening ionic channels. It is concluded that all of the effects on e.s.c. amplitude can be interpreted in terms of channel block, there being no evidence of any receptor blocking action.

Analysis of current noise evoked by nicotinic agonists in rat submandibular ganglion neurones.

The kinetics of the responses of rat submandibular ganglion neurones to nicotinic agonists were investigated by the analysis of agonist-induced membrane current noise. Two kinetic components are found in the responses of these cells to acetylcholine, carbachol, suberyldicholine, dimethyl-4-phenylpiperazinium and nicotine, in agreement with previous findings of two components in evoked synaptic currents in these cells. Small, though significant, differences in the relative amplitudes of the two kinetic components were observed in the spectra of noise evoked by the different agonists. In particular, the fast component in the response to carbachol was relatively larger with respect to the slow component than with any other agonist tested. No measurable dependence of the relative sizes of the two components on agonist concentration or membrane potential was observed. The evidence supports the hypothesis that the two kinetic components are the product of two independent channel populations for which the agonists tested show some slight differences in selectivity.

The action of ganglionic blocking drugs on the synaptic responses of rat submandibular ganglion cells.

1 The effects of tubocurarine, hexamethonium and trimetaphan on the synaptic currents of rat submandibular ganglion cells have been measured at 20 degrees C by means of a two-microelectrode voltage-clamp system. The aim was to distinguish between the receptor-blocking and channel-blocking actions of those drugs, and to test for possible selectivity of action on the ;fast' and ;slow' acetylcholine-operated channels.2 Tubocurarine had no effect on the amplitude of evoked synaptic currents (e.s.cs) or miniature synaptic currents (m.s.cs), except at concentrations exceeding 20 muM. The slow component of the e.s.c. was shortened by tubocurarine, this effect becoming more marked as the cell was hyperpolarized. The timecourse of m.s.cs, which have no slow component, was unaffected.3 Hexamethonium (2-30 muM) caused a voltage-dependent reduction of e.s.c. amplitude, and voltage-dependent shortening of both fast and slow components of the e.s.c. M.s.cs were also shortened.4 Trimetaphan (2-10 muM) reduced the amplitude of e.s.cs and m.s.cs. Neither component of the e.s.c. was shortened by trimetaphan; however, the slow component was reduced in amplitude more than the fast component, so that the overall duration of the e.s.c. appeared to be reduced. At higher concentrations (15-25 muM) trimetaphan clearly shortened the fast component.5 It is concluded that tubocurarine acts selectively on the slow ionic channels, the association rate constant being 2.8 x 10(6) M(-1) s(-1) at -80 mV. Hexamethonium acts on both fast and slow channels, the association rate constants, at -80 mV, being respectively 5.3 x 10(6) M(-1) s(-1) and 1.3 x 10(7) M(-1) s(-1). With both drugs, the association rate constant increases if the cell is hyperpolarized, this effect being more pronounced with hexamethonium than with tubocurarine.6 The marked voltage-dependent reduction of e.s.c. amplitude by hexamethonium cannot be accounted for by open channel block, and requires an additional mechanism, the nature of which is discussed.7 Trimetaphan, at low concentrations, acts in a way consistent with receptor block, and shows a degree of selectivity for the slow component of the e.s.c.8 In an appendix, the effect of temporal dispersion of the time of opening of ionic channels on the amplitude and time-course of the composite synaptic response is analysed. It is concluded that the shortening of the time-constant of the e.s.c. decay by hexamethonium cannot, by itself, account for the drug's effect on e.s.c. amplitude.

The characteristics of synaptic currents and responses to acetylcholine of rat submandibular ganglion cells.

1. Synaptic currents and responses to acetylcholine (ACh) have been recorded at 20 degrees C from rat submandibular ganglion cells by a two micro-electrode volatage-clamp technique.2. The peak amplitude (a(p)) of excitatory synaptic currents (e.s.c.s) was linearly related to membrane potential (E(m)), with a reversal potential close to - 10 mV. E.s.c.s decayed with a bi-exponential time course, the fast phase comprising just over half the total amplitude. The time constant (tau(f)) of the fast phase was 5-9 msec, while that of the slow phase (tau(s)) was 27-45 msec. The relative amplitudes of the two components remained constant at different membrane potentials, showing that the reversal potential was the same for both.3. Both tau(f) and tau(s) increased as the cell was hyperpolarized, the ratio tau(-80)/tau(-40) being about 1.6 for both fast and slow components.4. Increasing the calcium concentration from 2.5 to 7.5 mm increased the amplitude of both components by about 40% and also prolonged the synaptic currents 30-50%, its effect being slightly greater on tau(s) than on tau(f).5. In contrast to e.s.c.s, spontaneous or potassium-evoked miniature synaptic currents (m.s.c.s) showed a simple exponential decay with a time constant (tau(m.s.c.)) very similar to tau(f). tau(m.s.c.) showed the same sensitivity to membrane potential and calcium concentration as tau(f).6. In the presence of neostigmine (10 mum) e.s.c.s were prolonged, tau(f) about 3.5-fold and tau(s) about 2.5-fold. The decay remained bi-exponential, with little change in the relative amplitude or voltage-dependence of the two components. M.s.c.s were prolonged to a lesser extent (1.5-2-fold) and the voltage dependence of tau(m.s.c.) was unaffected by neostigmine.7. Reduction of the quantal content of the e.s.c. by low calcium-high magnesium solution did not affect the time course. The relative amplitudes, and the time constants of the two components were unchanged even with a 90% reduction of a(p).8. Voltage-jump studies, in which the cell was abruptly hyperpolarized by 20-40 mV during a response to ionophoretically applied ACh, showed a relaxation pattern consisting of two distinct exponential components, whose relative amplitudes varied considerably in different cells. The two rate constants tau(f.rel) and tau(s.rel) were somewhat shorter than tau(f) and tau(s) for e.s.c.s, the difference being generally less than two-fold.9. Measurements of ACh noise also revealed two kinetic components, the time constants of which corresponded closely to tau(f) and tau(s) for e.s.c.s. On the assumption that the two components represent channels of equal conductance, the single channel conductance, gamma, was calculated to be 31+/-3 pS, similar to that of endplate channels.10. It is concluded that the two kinetic components of e.s.c.s and ACh responses probably represent two distinct classes of ACh-operated ionic channels, whose mean lifetime differs about fivefold. The two types of channel show the same ionic selectivity and their mean lifetime varies in the same way with the membrane potential. The absence of a slow component in m.s.c.s suggests that the two types of channel are spatially separate in the membrane.

Dopamine-β-hydroxylase in rat submandibular ganglion cells which lack norepinephrine

Dopamine-β-hydroxylase in rat submandibular ganglion cells which lack norepinephrine

The reorganization of synaptic connexions in the rat submandibular ganglion during post-natal development.

1. The innervation of neurones in the submandibular ganglion of neonatal and adult rats has been studied with intracellular recording, and light and electron microscopy. 2. Intracellular recordings from neurones in isolated ganglia from adult animals showed that about 75% of the ganglion cells are innervated by a single preganglionic fibre. 3. However multiple steps in the post-synaptic potential (about five on average) were elicited in ganglion cells from neonatal animals by graded stimulation of the preganglionic nerve. The same result was obtained when the preganglionic fibres were stimulated at their emergence from the brainstem, indicating that neonatal neurones are innervated by several different preganglionic nerve cells. 4. The number of preganglionic fibres innervating individual ganglion cells gradually decreased during the first few weeks of life, and by about 5 weeks each ganglion cell was generally contacted by a single preganglionic axon. 5. Synapses were made on short protuberances in the immediate vicinity of the neuronal cell bodies in both neonatal and adult ganglia as shown by staining presynaptic boutons with the zinc-iodide osmium method, injection of horseradish peroxidase into ganglion cells, and electron microscopical examination. 6. Electron microscopical counts of synaptic profiles per ganglion cell perimeter showed that the number of synaptic contacts made on ganglion cells actually increased during the first few post-natal weeks, when the number of axons innervating each neurone was decreasing. 7. These results show that in the rat submandibular ganglion there is a reorganization of neuronal connexions during the first few weeks of life which results in a transition from multiple to generally single innervation of ganglion cells.