The synthesis, turnover and release of surplus acetylcholine in a sympathetic ganglion

1. Surplus acetylcholine (ACh) is the extra ACh that accumulates in cholinergic nerve endings when they are exposed to an anticholinesterase agent. The synthesis and turnover of this ACh was examined in the cat's superior cervical ganglion.2. Surplus ACh did not accumulate in chronically decentralized ganglia perfused with eserine-choline-Locke solution, and this shows that it is stored in presynaptic nerve terminals.3. Surplus ACh accumulated more rapidly in ganglia perfused with eserine than in ganglia perfused with neostigmine or with ambenonium; accumulation was delayed by 45-60 min when a quaternary anticholinesterase was used. However, the release of ACh upon preganglionic nerve stimulation was the same during perfusion with eserine, neostigmine or ambenonium. It is concluded that intracellular acetylcholinesterase normally destroys surplus ACh, whereas extracellular enzyme destroys released ACh.4. When ganglia were perfused with [(3)H]choline and eserine, the surplus ACh that accumulated was labelled but its specific radioactivity was only 38% of that of the choline added to the perfusion fluid.5. Surplus ACh was not released by nerve stimulation and was not mobilized for release during, or after, prolonged nerve stimulation. It is concluded that ACh released by nerve impulses is replaced by synthesis at the site of ACh storage and not by movement of ACh from the surplus pool.6. The accumulation of surplus ACh no more than doubled the total ACh content of ganglia, but turnover of ACh continued when the total amount was constant. Surplus ACh may contribute to spontaneous ACh output from eserinized preparations.7. When ganglia were perfused with a medium containing high K(+) (56 mM), surplus ACh was released.     

The preferential release of newly synthesized transmitter by a sympathetic ganglion

1. The acetylcholine (ACh) store of cat's superior cervical ganglia was replaced with radioactive ACh by perfusion, during stimulation, with [(3)H]choline-Locke solution. Perfusion was continued with Locke containing unlabelled choline (Ch) (in physiological concentration) and the release of labelled and unlabelled ACh was measured.2. Electrical stimulation of the preganglionic sympathetic nerve (20/sec or 5/sec), or stimulation by perfusing with raised K, released ACh that had a lower specific radioactivity than ganglionic ACh. The proportion of released ACh that was labelled was slightly higher when stimulation was at lower frequency or by K.3. Preganglionic nerve stimulation released, in the first few minutes, ACh that had a specific activity 70-80% of ganglionic ACh, but after 5 min the proportion of label in the released ACh fell to 35-45% of that in the ganglion.4. It is concluded that newly synthesized ACh is released before equilibration with preformed stores, and the significance of this to the mechanism of transmitter release is discussed.     

The effect of preganglionic nerve stimulation on the accumulation of certain analogues of choline by a sympathetic ganglion.

1. Cat superior cervical ganglia were perfused with a Krebs solution containing 10(-6) M [3H]homocholine (2-hydroxypropyl-trimethylammonium) or 10(-5) M [14C]triethylcholine (2-hydroxyethyl-triethylammonium). Preganglionic nerve stimulation (20 Hz) increased the accumulation of homocholine (3-2-fold) and of triethylcholine (2-1-fold). This increased accumulation during stimulation was not the result of increased metabolism. 2. The increased accumulation of homocholine or triethylcholine induced by pregnaglionic nerve stimulation was not reduced by tubocurarine or by atropine, but it was blocked by choline and by hemicholinium. These results suggested that preganglionic nerve stimulation increased choline analogue accumulation into cholinergic nerve terminals. 3. The increased accumulation of homocholine or of triethylcholine induced by preganglionic nerve stimulation was reduced when the Ca2+ concentration was reduced and was abolished in the absence of Ca2+. However, changes in the Mg2+ concentration which depressed acetylcholine (ACh) release by amounts comparable to those induced by altered Ca2+ concentrations did not alter the uptake of homocholine or triethylcholine. It is concluded that the uptake of choline analogues is not regulated by transmitter release but that stimulation increases the uptake of the choline analogues by a Ca2+-dependent mechanism. 4. The accumulation of ACh by ganglia perfused with a Krebs solution containing choline and high MgSO4 (18 mM) was measured. The ACh content of these ganglia did not increase, although choline transport presumably exceeded that necessary for ACh synthesis to replace released ACh. It is concluded that choline transport does not limit ACh synthesis in ganglia.     

An electrophysiological analysis of the synthesis of acetylcholine in preganglionic nerve terminals

1. An electrophysiological analysis has been made of the synthesis of acetylcholine (ACh) in the preganglionic nerve terminals of the isolated superior cervical ganglion of the guinea-pig. The mean amplitude of excitatory post-synaptic potentials recorded intracellularly was taken as a measure of the ACh output per impulse from the terminals of a preganglionic axon.2. Prolonged repetitive stimulation of the cervical sympathetic trunk at 10 and 20 Hz led to a decline in ACh output over the first 5-15 min and then a maintained output for periods of up to an hour. The mean level of maintained output was 0.4 of the peak initial output.3. The maintained level of output was shown to be equal to the rate of synthesis of new transmitter and was not dependent on the addition of choline (3 x 10(-5)M) to the fluid in the organ bath.4. The ACh output per minute was shown to be directly proportional to the frequency of stimulation.5. A model has been proposed of the storage and synthesis of ACh in preganglionic nerve terminals during prolonged stimulation, in which choline from the hydrolysis of released ACh is the main source of substrate for synthesis of new transmitter, and the rate at which synthesis proceeds is controlled by the rate at which transmitter is released.     

An electrophysiological analysis of the storage of acetylcholine in preganglionic nerve terminals

1. An electrophysiological analysis has been made of the storage of acetylcholine (ACh) in the preganglionic nerve terminals of the isolated superior cervical ganglion of the guinea-pig. The mean amplitude of excitatory post-synaptic potentials recorded intracellularly was taken as a measure of the ACh output per impulse from the terminals of a preganglionic axon.2. Prolonged repetitive stimulation of the cervical sympathetic trunk at 10 and 20 Hz in the presence of hemicholinium No. 3 led to an exponential decline of ACh output as the transmitter formed before the beginning of stimulation was depleted.3. The rate of decline of ACh output during stimulation at 20 Hz (tau = 0.83-1.95 min) was about twice as fast as that at 10 Hz (tau = 1.00-6.83 min).4. The results suggest that, during prolonged stimulation, ACh is released from a single store in the preganglionic nerve terminal and that each impulse releases a constant fraction of the store of the transmitter.     

Cholinergic receptors at sympathetic preganglionic nerve terminals

1. In the paravertebral sympathetic chain of bullfrogs, some part of the preganglionic nerve located within the ganglion was depolarized transiently when acetylcholine (ACh) was directly applied to the ganglion, particularly in the presence of an anticholinesterase (anti-ChE). The axonal part of the preganglionic nerve, on the other hand, showed no detectable depolarization following direct application of ACh (with anti-ChEs) to the interganglionic nerve trunk.2. The ACh depolarization was markedly depressed by nicotine, and less markedly by (+)-tubocurarine, whereas it was not affected by atropine. Nicotine, similar to ACh, transiently depolarized only the intraganglionic portion of the presynaptic fibres.3. The action potentials, recorded from the axonal as well as the terminal parts of the preganglionic nerve, showed spike potentials followed by a marked negative after-potential. The negative after-potential was followed by a positive after-potential which was markedly enhanced by repetitive nerve stimulation.4. A slow negative potential followed the positive after-potential in the repetitive responses of the terminal parts of the preganglionic nerve. The slow negative potential was enhanced by anti-ChEs, eliminated by ACh and nicotine, and unaffected by atropine.5. The amplitude of the action potentials of the terminal parts of the preganglionic nerve, and particularly that of the negative after-potential, was significantly depressed during the development of ACh depolarization as well as the slow negative potential, indicating that the two types of slow depolarization originated in the intraganglionic portion of the presynaptic fibres, presumably somewhere near or at the nerve terminals.6. ACh depolarization similar to that observed with bullfrog sympathetic ganglia was observed with rat superior cervical ganglia. The present experiments provide evidence that a certain part of the preganglionic nerve terminals is depolarized by the action of the transmitter released from their endings.     

A study of cholinoceptive cells in the lateral geniculate nucleus

1. The dorsal lateral geniculate nucleus (LGN) of the cat stains densely for acetylcholinesterase, which is present in intra-axonal and extracellular locations.2. Acetylcholine (ACh), cholinomimetic drugs, anticholinesterases and ACh antagonists were administered iontophoretically to neurones in the LGN.3. ACh excited eighty-six of 184 (46.7%) geniculate neurones and depressed seven (3.8%).4. The excitatory response to ACh was frequently larger than that to L-glutamate and had a comparable time course.5. There was a considerable variation in the proportion of ACh sensitive cells in different animals. ACh firing was facilitated by optic nerve or visual stimulation.6. Carbamylcholine was the most active of the choline esters tested, frequently exceeding ACh in potency. The other choline esters and nicotine were consistently less active than ACh.7. Anticholinesterases, eserine, neostigmine and edrophonium potentiated the action of ACh, and often caused excitation. Eserine caused an initial small enhancement in the amplitude of the focal potential evoked by optic nerve stimulation followed by a reduction in amplitude and prolongation of duration of the potential.8. Atropine and benzoquinonium effectively prevented the ACh excitation of many cells. Dihydro-beta-erythroidine failed to cause a significant reduction in the magnitude of the ACh response. All three ACh antagonists failed to reduce the excitant effects of optic nerve or visual stimulation.9. Stimulation of the mesencephalic reticular formation caused either an enhancement or a reduction in the excitability of ACh sensitive neurones in the LGN. Benzoquinonium abolished the excitatory effects of reticular formation stimulation.10. The findings presented in this paper suggest that ACh is the transmitter released by terminals of nerves of the reticular formation projection to the LGN.     

Changes in statistical release parameters during prolonged stimulation of preganglionic nerve terminals.

1. An analysis has been made of the release of acetylcholine (ACh) from the preganglionic nerve terminals of the isolated superior cervical ganglion of the guinea-pig during prolonged repetitive stimulation (10 Hz), using the amplitude of the excitatory post-synaptic potential (e.p.s.p.) as a measure of the amount of ACh released. 2. The decline in the mean amount of ACh released by each impulse over 20 min of continuous stimulation was accompanied by a small decrease in the mean miniature (min.) e.p.s.p. amplitude (less than 20%), both in the presence and absence of supplementary choline (3 x 10(-5) M). During stimulation in the presence of hemicholinium-3 (HC-3) (2 x 10(-5) M), the fall in min.e.p.s.p. amplitude was significantly larger. 3. Amplitude-frequency histograms of e.p.s.p.s evoked at different times after the beginning of stimulation were usually well predicted by binomial statistics, using the min.e.p.s.p.s released during each sample period as a measure of the quantal unit. In the other cases, histograms could be predicted using Poisson's Law. 4. A decline in quantal content, m, occurred in all experiments. In the presence of synthesis, with or without added choline, this was always associated with a decrease in the binomial parameter, n, and, in many cases, with a decrease in the binomial parameter, p. During stimulation in the presence of HC-3, a larger fall in p was observed in all experiments. 5. The results suggest that depletion of the ACh stored in the terminal decreases both the size and number of quanta available for release, and that the decrease in the amount of ACh in each quantum reduces the probability of its release.     

Evidence that transmitter can be released from regions of the nerve cell other than presynaptic axon terminal: axonal release of acetylcholine without modulation

Release of acetylcholine from isolated preganglionic axons of sympathetic nerve trunk (cervical preganglionic sympathetic branch) of the cat was studied. In response to depolarization (KCl, 48.4 mM) acetylcholine was released into the eserinized Krebs solution. This release was shown to be dependent on extracellular Ca2+. Electrical stimulation (1 Hz) enhanced the release of acetylcholine from the isolated axonal preparation. The release by stimulation proved to be tetrodotoxin-sensitive and Ca2+-dependent. Evidence has been obtained that the acetylcholine released from sympathetic nerve trunks originates from the axon and not from Schwann cells: 5 days after section of the nerve, there was no release in response to stimulation. The release of acetylcholine from the axon is unlike that from axon terminals in that the rate of release cannot be enhanced by the inhibition of Na, K-adenosine 5'-triphosphatase (ouabain 2 X 10(-5) M) and cannot be modulated by noradrenaline (10(-6) M) or by morphine. Furthermore, although isolated nerve trunks took up [3H]choline by a hemicholinium-sensitive process, no radioactivity could be released upon electrical stimulation. It is suggested that the release of acetylcholine is not confined to axon terminals, but that it can be non-synaptically released by depolarization from axons provided Ca2+ is present.     

The formation of synapses in mammalian sympathetic ganglia reinnervated with preganglionic or somatic nerves

1. A study has been made of the formation of synapses in the superior cervical ganglion of the guinea-pig, during reinnervation either with axons of the cervical sympathetic trunk, or with somatic axons of the nerve to the sternohyoid muscle.2. No significant changes in either the geometry or electrical parameters of sympathetic motorneurones were detected following denervation for periods of 3-6 weeks, or after reinnervation with either preganglionic or somatic axons.3. The post-ganglionic action potential reappeared about 4 weeks after preganglionic trunk section (eighteen of eighteen ganglia); its amplitude increased progressively and was almost normal by more than 10 weeks after nerve section. A very small response was detected from thirteen of eighteen ganglia after periods longer than 8 weeks after cross-reinnervation with somatic axons.4. Regenerated preganglionic or somatic nerve terminals were demonstrated around the ganglion cells using ZIO impregnation and electron-microscopy; the structure of these terminals was unchanged following regeneration into the ganglia, although many more synapses were formed by preganglionic terminals than by somatic terminals.5. The facilitation of evoked synaptic potentials which occurs during repetitive stimulation of preganglionic axons was retained following their regeneration, whereas most synapses formed on ganglion cells by regenerating somatic axons showed facilitation of transmitter release during trains of stimuli, rather than the normal depression.6. These observations suggest that the structure and electrical properties of adult mammalian autonomic motorneurones are not under neural control, but that these neurones do show some selectivity in the type of nerve which they will permit to form synapses on them.     

Storage and release of acetylcholine in a sympathetic ganglion

1. The hypotheses of preferential release of newly synthesized acetylcholine (ACh) and two compartment storage of transmitter in the cat superior cervical ganglion have been re-examined by testing, first, the assumption that ganglionic ACh stores do not alter during a 20 min rest following 60 min preganglionic nerve stimulation at 20/s, and secondly, the implication that the rate of ACh release should be high near the onset of activity and decline to a lower rate with time irrespective of the frequency of stimulation.2. The ganglionic ACh stores were found to increase by 38 +/- 8% within 20 min following 60 min preganglionic nerve stimulation at 20/s, and this extra ACh was releasable.3. The rate of ACh release from ganglia perfused with cat plasma and stimulated at 4/s increased over the first 5 min of stimulation to reach a 27% higher rate that was maintained.4. Correction of the original data to allow for the post-activation increase in ACh stores suggests that newly synthesized ACh equilibrates with most of the preformed stores. The time course of ACh release at 4/s does not support the two compartment model as currently formulated.5. These findings resolve in part a conflict between the physiological data and a recent hypothesis for ACh storage based on ganglion morphology.     

The action of extracellular cations on the release of the sympathetic transmitter from peripheral nerves

1. The noradrenaline stores in the sympathetic nerve endings of the cat colon were labelled in vivo with (+/-)-[7-(3)H]noradrenaline (500 muc, 11.3 mug) injected 3 hr before killing.2. The colon was removed and immersed in an organ bath containing Krebs solution. The vascular bed was perfused from the inferior mesenteric artery to the colic vein.3. The effects of Ca(2+), Ba(2+) and Mg(2+) on the output of [(3)H]noradrenaline and [(3)H]metabolites in the venous effluent were measured before and after electrical stimulation of the post-ganglionic inferior mesenteric nerves at 10 impulses/sec.4. Nerve stimulation increased the efflux of [(3)H]noradrenaline when the perfusion fluid contained Ca(2+). Variations in Ca(2+) concentration (1.5-10 mM) did not affect this response.5. Removal of Ca(2+) from the fluid passing through the vascular bed (and therefore from the region of the sympathetic nerve terminals), abolished the output of [(3)H]noradrenaline in response to nerve stimulation. There was no change when phenoxybenzamine was added to prevent the binding of transmitter on to post-synaptic receptors on the effector organ.6. The output of transmitter was not changed when Ca(2+) was present in the blood vessels of the colon even though it was removed from the solution in contact with the remainder of the tissue.7. Nerve stimulation released [(3)H]noradrenaline when Ba(2+) was used as a substitute for Ca(2+); Mg(2+) was not an effective substitute for Ca(2+) as then nerve stimulation did not increase the output of radioactive noradrenaline or metabolites.8. Ba(2+) also increased the resting output of [(3)H]noradrenaline in the absence of nerve stimulation. Addition of Ca(2+) or Mg(2+) did not change this spontaneous release but it was augmented by removal of Ca(2+).9. It is concluded that Ca(2+) is essential for release of the sympathetic transmitter by nerve stimulation but not for the spontaneous output that occurs in the absence of nervous activity. The site of action of Ca(2+) is considered to be the terminals of adrenergic fibres.     

Synthesis,storage and release of [14C]acetylcholine in isolated rat diaphragm muscles

1. Segments of rat diaphragms were kept in choline-free media for 4 hr and were then exposed to a physiological concentration of [¹⁴C]-choline (30 μM) at 37° C. The synthesis, storage and subsequent release of [¹⁴C]acetylcholine by the muscles was assessed by isotopic- and bio-assays after isolation of the transmitter by paper electrophoresis.2. Replacement of endogenous acetylcholine (0·92 μ-mole/kg) with labelled acetylcholine proceeded slowly at rest, but rapidly during nerve stimulation. [¹⁴C]Acetylcholine accumulated most rapidly when hydrolysis of the released transmitter, and thus the re-use of endogenous choline, was prevented by an esterase inhibitor. Fully replaced stores were maintained during nerve stimulation by synthesis rates sufficient to replenish at least 35% of the store size in 5 min.3 In the presence of hemicholinium-3, which inhibits choline uptake, acetylcholine stores declined rapidly during stimulation, and residual synthesis was slight, indicating little intraneural choline. Net choline uptake into nerve terminals was estimated from the highest observed synthesis rate and from previous measurements of the number and size of terminals, as 3-6 p-mole/cm² sec.4. Transmitter synthesis was localized in the region of end-plates, and was reduced to a few per cent of normal 6 weeks after phrenic nerve section. Release experiments suggested that at least half of the acetylcholine in phrenic nerves is in their terminals; from this content and the morphology of the terminals, the average concentration of transmitter in the whole endings would appear to be about 50 m-mole/l. Homogenization of the muscles freed choline acetyltransferase into solution, but left some [¹⁴C]acetylcholine associated with small particles, presumably synaptic vesicles.5. Resting transmitter release was about 0·013% of stores/sec. With 360 nerve impulses at 1-20/sec, release increased up to 0·43% of stores/sec, and amounted to 3·5-7 × 10^-18 moles per end-plate per impulse. The release rate was unaffected by the doubling of store size which occurred with eserine, but the extra transmitter did help to maintain releasable stores during prolonged stimulation. Experiments with fractional store labelling indicated that newly synthesized acetylcholine was preferentially released.6. Preformed [³H]acetylcholine was not taken up and retained by muscle or nerve cells in the absence of an esterase inhibitor. With eserine present, labelled acetylcholine was taken up uniformly by muscle segments; when eserine was then removed, radioactive acetylcholine remained only near neuromuscular junctions.     

Both synaptic and antidromic stimulation of neurons in the rat superior cervical ganglion acutely increase tyrosine hydroxylase activity

Electrical stimulation of the preganglionic cervical sympathetic trunk produces an acute increase in the rate of DOPA synthesis in the rat superior cervical ganglion. The present study was designed to test the possibility that this acute transsynaptic stimulation of catechol biosynthesis could be, at least in part, a consequence of an increase in the firing rate of the postganglionic sympathetic neurons. For this purpose, the effect of stimulation in vitro of the preganglionic cervical sympathetic trunk was compared to that of stimulation of the predominantly postganglionic internal and external carotid nerves. Stimulation of the cervical sympathetic trunk at 10 Hz for 30 min produced a 4.6-fold increase in DOPA synthesis, while simultaneous stimulation of the two postganglionic trunks produced a 3.1-fold increase. The internal carotid nerve is known to contain a small population of preganglionic fibers that synapse on principal neurons in the ganglion before entering this nerve trunk. To eliminate the possibility that the effect of stimulation of the internal carotid nerve is mediated by synaptic stimulation via these preganglionic "through fibers", the effect of stimulation of previously decentralized ganglia was examined. While decentralization reduced the magnitude of the effect of stimulation of the internal and external carotid nerves, a 2.0-fold increase in DOPA synthesis was still seen. In addition, when these nerve trunks were stimulated in control ganglia that had been maintained in organ culture for 48 h to allow time for the degeneration of afferent nerve terminals, DOPA synthesis increased 4.1-fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential expression of calcium channels in sympathetic and parasympathetic preganglionic inputs to neurons in paracervical ganglia of guinea-pigs

Neurons in pelvic ganglia receive nicotinic excitatory post-synaptic potentials (EPSPs) from sacral preganglionic neurons via the pelvic nerve, lumbar preganglionic neurons via the hypogastric nerve or both. We tested the effect of a range of calcium channel antagonists on EPSPs evoked in paracervical ganglia of female guinea-pigs after pelvic or hypogastric nerve stimulation. omega-Conotoxin GVIA (CTX GVIA, 100 nM) or the novel N-type calcium channel antagonist, CTX CVID (100 nM) reduced the amplitude of EPSPs evoked after pelvic nerve stimulation by 50-75% but had no effect on EPSPs evoked by hypogastric nerve stimulation. Combined addition of CTX GVIA and CTX CVID was no more effective than either antagonist alone. EPSPs evoked by stimulating either nerve trunk were not inhibited by the P/Q calcium channel antagonist, omega-agatoxin IVA (100 nM), nor the L-type calcium channel antagonist, nifedipine (30 microM). SNX 482 (300 nM), an antagonist at some R-type calcium channels, inhibited EPSPs after hypogastric nerve stimulation by 20% but had little effect on EPSPs after pelvic nerve stimulation. Amiloride (100 microM) inhibited EPSPs after stimulation of either trunk by 40%, while nickel (100 microM) was ineffective. CTX GVIA or CTX CVID (100 nM) also slowed the rate of action potential repolarization and reduced afterhyperpolarization amplitude in paracervical neurons. Thus, release of transmitter from the terminals of sacral preganglionic neurons is largely dependent on calcium influx through N-type calcium channels, although an unknown calcium channel which is resistant to selective antagonists also contributes to release. Release of transmitter from lumbar preganglionic neurons does not require calcium entry through either conventional N-type calcium channels or the variant CTX CVID-sensitive N-type calcium channel and seems to be mediated largely by a novel calcium channel.     

Impulse conduction in sympathetic nerve terminals in the guinea-pig vas deferens and the role of the pelvic ganglia.

Focal extracellular recording techniques were used to study nerve impulse propagation and the intermittent transmitter release mechanism in sympathetic nerve terminals of the guinea-pig vas deferens in vitro. In particular, the nature of impulse propagation in postganglionic nerve fibres was characterized following pre- or postganglionic stimulation. Conventional intracellular recording techniques were also used to study directly ganglionic transmission in cell bodies in the anterior pelvic ganglia. When brief electrical stimuli were applied to the hypogastric nerve trunk close to the prostatic end of the vas deferens, the nerve terminal impulses recorded extracellularly could be evoked either directly by stimulation of the parent axon (i.e. postganglionically) or indirectly by stimulation of the preganglionic nerve fibre. In 364 separate recordings, nerve terminal impulse conduction failure was not observed during trains of stimuli at 1 Hz. However, apparent "intermittent conduction" of nerve impulses was noted on 16 occasions. In these fibres, the degree of intermittent conduction decreased as the frequency of stimulation was increased. Conduction in these intermittent fibres was reversibly interrupted by removing calcium from the Krebs' solution or by the addition of the ganglion blocker, hexamethonium (30-100 microM). Thus, the cause of intermittent conduction is failure of the transmission of excitation in the sympathetic ganglia. Impulses evoked by postganglionic stimulation never failed to propagate into the nerve terminals, and changes in the shape or amplitude of the nerve terminal impulse during trains of stimuli were not detected. One effect of stimulation was a frequency-dependent increase in the latency of the nerve terminal impulse which developed during the train of stimuli. Thus, intermittence of transmitter release from individual varicosities cannot be attributed to failure of impulse propagation in sympathetic nerve terminals. Transmission in the anterior pelvic ganglia was investigated directly by making intracellular recordings from cell bodies whose terminals projected to the vas deferens. Many cell bodies received a strong synaptic input which generated an action potential in the postganglionic cell body on a one-to-one basis. However, in some cell bodies there was a low safety factor for the generation of the action potential by the excitatory postsynaptic potential. The safety factor for generating an action potential in the postganglionic cell body was raised by increasing the frequency of stimulation. These findings suggest that peripheral ganglia are not simple one-to-one relay stations, but may well play an important role in controlling the patterns of nerve impulse traffic in postganglionic sympathetic neurons.     

A long-lasting potentiation of transmitter release related to an increase in transmitter stores in a sympathetic ganglion

1. High frequency preganglionic nerve stimulation increases the acetylcholine (ACh) stores of the cat superior cervical ganglion. The increase reaches a maximum 20 min following 60 min conditioning stimulation at 20/s. The effect of this conditioning on ACh release in ganglia perfused with plasma and test stimulated at 4 or 5/s has been studied, and the relationship of ACh stores to ACh release in conditioned ganglia determined.

2. The rate of ACh release in response to test stimulation at 5/s for 15 min, starting 20 min following conditioning, was 174% of the rate found in unconditioned ganglia. The ACh stores of the conditioned ganglia at the end of the test were calculated to be 173% of the control ganglion stores.

3. When test stimulation at 4/s was started 5 min following conditioning, the rate of ACh release showed a variable pattern of increase and decrease over a 75 min period. The mean peak rate of release was about 150% of the control rate, and the duration of potentiation was about 75 min.

4. When conditioned, unperfused ganglia were tested by stimulation at 4/s the ACh stores were found to increase and decrease in parallel with the increase and decrease in ACh release rates found in the perfusion experiments.

5. It was found also that the magnitude of the increase in ACh stores and ACh release was related to the amount of ACh in the ganglionic stores at the onset of conditioning, being greater for the ganglia with the smaller initial stores.

6. It is concluded that the potentiation of ACh release in ganglia conditioned in this way is directly related to the accompanying increase in ACh stores.

7. The possible significance of alterations of ACh stores and ACh release as a mechanism of modulatory control of ganglionic transmission is discussed.

     

The effect of chronic nicotine and withdrawal on intra-neuronal dynamics of acetylcholine and related enzymes in a preganglionic neuron system of the rat

The effect of chronic nicotine treatment, given in the drinking water for 8–10 weeks in a dose equivalent to that of a heavy cigarette smoker, and of withdrawal 2 days on acetylcholine (ACh), choline acetyltransferase (CAT) and ACh-esterase (AChE) activities in the preganglionic cervical nerve and the superior cervical ganglia (SCG) of rats, were studied. Control rats were housed and handled as the nicotine rats. After cutting the preganglionic nerve 7–19 h before dissection, ACh, CAT and AChE accumulated in the nerve part proximal to the cut (relative to the nerve cell bodies in the spinal cord). A clearance of these substances was observed in the nerve distal to the cut. This indicates that all 3 substances are transported proximo-distally in this preganglionic cholinergic nerve. In the SCG, ACh was decreased already by 7 h (to about 60%), while CAT and AChE-activities were lowered to 60% and 80%, respectively, at 19 h after cutting the nerve. Chronic nicotine treatment caused an increased ACh accumulation (by about 35 %) and a decreased CAT accumulation (by about 20%) in the cut nerve, while the ganglionic levels of all 3 substances were essentially unchanged. Withdrawal of nicotine for 2 days prior to the final experiments caused a reduced ACh-accumulation (by about 30%) in the nerve and normalized the CAT accumulation. The AChE-activity of intact nerve was markedly increased to about 175% of control, and the transportable fraction of AChE (clearance distal to the cut) was about twice as large as in control. In the SCG withdrawal caused marked changes in the ACh content, which was decreased to 62% of control. CAT-activity was increased to 117% of control while AChE was unchanged. Our hypothesis to explain the results is that chronic nicotine treatment and in particular withdrawal of nicotine may cause marked alterations in the activity of the preganglionic neuron. This may induce changes in the intra-axonal transport of the 3 substances and an increased turnover of ACh in the nerve terminals of the SCG after withdrawal of nicotine.     

The pathway for the slow inhibitory postsynaptic potential in bullfrog sympathetic ganglia

Intracellular and sucrose gap recording techniques were used to examine synaptically evoked potentials and the response of neurons in bullfrog paravertebral sympathetic ganglia to muscarinic agonists. These neurons were defined as either B or C cells on the basis of the conduction velocity of antidromically evoked action potentials. Following stimulation of preganglionic C-fibers in the rostral portion of the VIIIth spinal nerve, a fast nicotinic excitatory postsynaptic potential (EPSP) and a slow atropine-sensitive inhibitory postsynaptic potential (IPSP) could be recorded intracellularly in C cells of the IXth and Xth paravertebral ganglia treated with 70 microM d-tubocurarine chloride (dTC). Under these conditions, local iontophoretic application of acetylcholine (ACh) could produce a slow hyperpolarization of C cell membrane potential. ACh hyperpolarizations or slow IPSPs were not detected in ganglionic B cells. Stimulation of the preganglionic B-fibers in the sympathetic chain produced a fast nicotinic EPSP and a slow muscarinic EPSP in ganglionic B cells. A small population of C cells also received cholinergic B-fiber innervation from the sympathetic chain and exhibited a slow IPSP upon tetanic stimulation of this pathway. When curarized ganglia were examined by means of sucrose gap recording, superfusion of the muscarinic agonist, methacholine (MCh), produced an initial hyperpolarization (MChH) followed by a depolarization (MChD). Both responses were blocked by atropine and therefore presumably reflect the activation of muscarinic receptors involved in the generation of the slow IPSP and the slow EPSP, respectively. Although synaptic transmission was blocked by Ringer solution containing 4 mM Co2+, neither this solution nor 10 microM tetrodotoxin reduced the amplitude of the MChH. The MChH was slightly reduced by Ringer solution containing 0.1 mM Ca2+, however, the response could be restored by the addition of 6 mM Mg2+. These results indicate that the MChH in curarized bullfrog sympathetic ganglia results from a direct muscarinic action on ganglionic cells. This suggests that the slow IPSP is mediated by ACh released from cholinergic preganglionic fibers that make synaptic contact with ganglionic C 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 omega-conotoxin GVIA (N type), nifedipine and nimodipine (L type), omega-conotoxin MVIIC and omega-agatoxin IVA (P/Q type), and Ni2+ (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.