The relationship of transmitter release and storage to fine structure in a sympathetic ganglion

Summary Cat sympathetic ganglia were prepared for electron microscopy by perfusion fixation with glutaraldehyde in the presence of Mg⁺⁺. At resting boutons de passage the populations of synaptic vesicles were 6400 per bouton. The vesicle distributions displayed many of the features of spheres in close-packing. Calculations based on a vesicle close-packing hypothesis gave a figure of 8000 vesicles per bouton. In ganglia stimulated for 20 min at 20/s the vesicle populations were reduced to 25%, and to 28.5% in ganglia in which acetylcholine (ACh) synthesis was inhibited by hemicholinium (HC-3). The reduction was to 34% when stimulation was for 1 min. In ganglia stimulated for 20 min at 1/s and 4/s the vesicle populations were reduced to 44% and 46% respectively. Even following 1 min stimulation at 4/s over half the boutons showed significant loss of vesicles. ACh stores in ganglia are known not to be depleted by any of these procedures except stimulation in the presence of HC-3. The fraction of ganglionic ACh stores known to be released by stimulation for 1 min at 20/s or 4/s and by 20 min stimulation at 1/s is substantially less than the fraction of vesicles lost. The observations therefore were not readily accounted for by the vesicle theory of transmitter storage and release. They were consistent with the idea the ACh is stored in vesicles at rest, but that during maintained activity over half the bouton ACh is free in the cytoplasm. The concentration of cytoplasmic ACh was calculated to be approximately 50–150 mm 1^–1. Examination of the hypothesis that ACh may be released from this cytoplasmic pool during synaptic activation indicated an efflux of approximately 1.5–3.0 × 10^–12 M Ach cm^–2 synapsing membrane/impulse.Medical Research Associate of the Medical Research Council of Canada.     

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.     

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.     

Spontaneous and evoked release of acetylcholine and a cholinergic false transmitter from brain slices: comparison to true and false transmitter in subcellular stores

Rat cerebral cortex slices were incubated with [¹⁴Ccholine and [³Hhomocholine to allow synthesis of [¹⁴Cacetylcholine and [³Hacetylhomocholine; release of the acetylated compounds was tested in the presence of eserine and hemicholinium-3. Both [¹⁴Cacetylcholine and [³Hacetylhomocholine were released spontaneously by a Ca²⁺-independent mechanism; exposure of the tissue to a high K⁺ medium resulted in a Ca²⁺-dependent increase in [¹⁴Cacetylcholine release (30-fold) and in [³Hacetylhomocholine release (5 to 7-fold). Thus, spontaneous and K⁺-evoked transmitter release could be distinguished on the basis of their molar ratios of true to false transmitters and these ratios were compared to the molar ratios of the transmitters in subcellular fractions prepared from the incubated tissue. The molar ratio of acetylcholine to acetylhomocholine released from the tissue spontaneously differed from the ratio in subcellular fractions containing occluded transmitters. The molar ratio of acetylcholine to acetylhomocholine released by K⁺ differed from the ratio in a fraction (H) containing occluded transmitters but was similar to the ratio in the fraction (D) containing monodisperse synaptic vesicles and the fraction (0) containing the majority of a soluble cytoplasmic marker. Comparison of the transmitter contents of subcellular fractions from unstimulated and K⁺-stimulated tissue showed that the three fractions (D, H and O) lost equal proportions of both transmitters as a result of K⁺ stimulation.It is concluded that acetylhomocholine may be released from brain slices both spontaneously and in response to stimulation via mechanism similar to those that release acetylcholine, but that there must be some differences in specificity of the acetylcholine storage and/or release processes. The results also indicate that spontaneous transmitter release originates from extravesicular stores; the results are consistent with a vesicular site of origin of evoked transmitter release but do not distinguish between nerve terminal vesicles and cytoplasm as the immediate source of evoked transmitter release.     

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.     

Persistent enhancement of transmitter release accompanying long-term potentiation in the guinea pig hippocampus

In order to examine temporal changes in enhancement of transmitter release during long-term potentiation (LTP), we examined amplitude fluctuation of excitatory postsynaptic potentials (EPSPs) for longer periods than 2 h after tetanic stimulation (up to 4 h in the longest observation). The relative magnitude of excitatory postsynaptic potentiation (EPSP) fluctuation (coefficient of variation, CV) reduced throughout the observation periods in association with an increase in EPSP amplitude after tetanic stimulation. The reciprocals of squared CVs (= mean²/variance) were almost in proportion to the magnitude of LTP, and the ratio of 1/CV² and the LTP magnitude did not change significantly for up to 4 h. These findings suggest that a prolonged enhancement of transmitter release from presynaptic terminals underlies LTP, and the relative contribution of this presynaptic enhancement does not change significantly for 2 h (maybe up to 4 h, or longer) after tetanic stimulation.     

Potentiation phase of spike timing-dependent neuromodulation by a serotonergic interneuron involves an increase in the fraction of transmitter release

In the mollusk, Tritonia diomedea, the serotonergic dorsal swim interneuron (DSI) produces spike timing-dependent neuromodulation (STDN) of the synaptic output of ventral swim interneuron B (VSI) resulting in a biphasic, bidirectional change of synaptic strength characterized by a rapid heterosynaptic potentiation followed by a more prolonged heterosynaptic depression. This study examined the mechanism underlying the potentiation phase of STDN. In the presence of 4-aminopyridine, which blocks the depression phase and enhances transmitter release from VSI, rapidly stimulating VSI led to a steady-state level of transmitter depletion during which potentiation by DSI or serotonin (5-HT) was eliminated. Cumulative plots of excitatory postsynaptic currents were used to estimate changes in the size and replenishment rate of the readily releasable pool (RRP) and the fraction of release. 5-HT application increased transmitter release without altering replenishment rate. The magnitude of 5-HT-evoked potentiation correlated with the increase in the fraction of release. A phenomenological model of the synapse further supported the hypothesis that 5-HT-induced potentiation was caused by an increase in the fraction of release and correctly predicted no change in frequency facilitation. A dynamic version of the model correctly predicted the effect of DSI stimulation under a variety of conditions. Finally, depletion of internal Ca(2+) stores with cyclopiazonic acid showed that Ca(2+) from internal stores is necessary for the 5-HT-induced potentiation. The data indicate that 5-HT released from DSI increases the fraction of the RRP discharged during VSI action potentials using a mechanism that involves Ca(2+) extrusion from internal stores, resulting in time- and state-dependent neuromodulation.     

Augmentation: A process that acts to increase transmitter release at the frog neuromuscular junction.

1. End-plate potentials (e.p.p.s) were recorded from frog neuromuscular junctions bathed in Ringer solution containing increased Mg and decreased Ca to reduce transmitter release. Conditioning and testing stimulation was applied to the nerve to study a previously uncharacterized process which acts to increase e.p.p. amplitudes. We will refer to this process as augmentation. 2. Following repetitive stimulation augmentation decayed approximately exponentially over most of its time course with a mean time constant of about 7 sec (range 4-10 sec) which is intermediate in duration between the time constants for the decay of facilitation and potentiation. 3 . The magnitude of agumentation increased with the duration of the conditioning stimulation. Assuming a multiplicative relationship between augmentation and potentiation, values of the magnitude of augmentation ranged from 0-3 to 0-6 following 50 impulses at 20/sec to 0-5-7-8 following 600 impulses at 20/sec. (An augmentation of 0-3 and 7-8 would increase e.p.p. amplitudes 1-3 and 8-8 times, respectively.) 4. The time constant characterizing the decay of augmentation remained relatively constant as the duration of the conditioning stimulation was increased. 5. Augmentation as well as facilitation and potentiation resulted from an increase in the number of quanta of transmitter released from the nerve terminal. 6. Augmentation decayed faster at higher temperatures with a mean temperature coefficient, Q10, of about 3-8. The corresponding Q10 for the decay of potentiation was found to be about 2-4. 7. It is concluded that augmentation can be a significant factor in increasing transmitter release and will therefore have to be accounted for when studying the effects of repetitive stimulation on the function of the nerve terminal or when formulating models of transmitter release.     

BDNF-Induced potentiation of spontaneous twitching in innervated myocytes requires calcium release from intracellular stores

Brain-derived neurotrophic factor (BDNF) can potentiate synaptic release at newly developed frog neuromuscular junctions. Although this potentiation depends on extracellular Ca(2+) and reflects changes in acetylcholine release, little is known about the intracellular transduction or calcium signaling pathways. We have developed a video assay for neurotrophin-induced potentiation of myocyte twitching as a measure of potentiation of synaptic activity. We use this assay to show that BDNF-induced synaptic potentiation is not blocked by cadmium, indicating that Ca(2+) influx through voltage-gated Ca(2+) channels is not required. TrkB autophosphorylation is not blocked in Ca(2+)-free conditions, indicating that TrkB activity is not Ca(2+) dependent. Additionally, an inhibitor of phospholipase C interferes with BDNF-induced potentiation. These results suggest that activation of the TrkB receptor activates phospholipase C to initiate intracellular Ca(2+) release from stores which subsequently potentiates transmitter release.     

The effect of temperature change upon transmitter release, facilitation and post-tetanic potentiation

1. End-plate potentials (e.p.p.s) and miniature end-plate potentials (m.e.p.p.s) were intracellularly recorded from rat diaphragm phrenic nerve preparations in vitro at temperatures between 7 degrees and 40 degrees C.2. The quantal content of e.p.p.s and the frequency of m.e.p.p.s showed broadly similar relationships with temperature, with maxima about 20 degrees and above 39 degrees C.3. Analysis of the change in e.p.p. quantal content showed that the maximum about 20 degrees C was accompanied by a similar maximum of p, the probability of release of quanta. The maximum above 39 degrees C was associated with a rise in n, a presynaptic store of material needed for release.4. The rate at which transmitter could be mobilized was linear in an Arrhenius plot with an apparent activation energy of 25 kcal deg(-1).5. Facilitation and post-tetanic potentiation (PTP) were shown to be entirely attributable to changes in p.6. It is suggested that facilitation and PTP have a common basis and that the (temperature-dependent) rate of Ca removal from intracellular sites at which it exerts its action is as important a determinant of the magnitude of quantal release as is the amount of Ca combining with these sites.     

A quantitative description of tetanic and post-tetanic potentiation of transmitter release at the frog neuromuscular junction.

1. End-plate potential (e.p.p.s) were recorded with a surface electrode from frog neuromuscular junctions blocked with high Mg and low Ca to study post-tetanic potentiation (potentiation). 2. The magnitude of potentiation was not directly related to the number of conditioning impulses, but was a function of the frequency and duration of the conditioning stimulation. 3. Potentiation was always greater following an equal number of impulses delivered at a higher frequency of stimulation. 4. Plots of the magnitude of potentiation against the number of conditioning impulses would sometimes show an upward inflexion depending on the parameters of stimulation. 5. These experimental observations were described by a model based on the assumption (1) that potentiation is linearly related to a residual substance, R(t), which accumulates in the nerve terminal during repetitive stimulation, and (2) that each nerve impulse adds an identical increment, r, of this residual substance. The data were not described by assuming a 4th power relationship between potentiation and R(t). 6. The upward inflexion in potentiation (see paragraph 4) is described by the model as resulting from an increase in the time constant for the decay of potentiation as the magnitude of potentiation increases. 7. The increment of residual substance r added by each impulse was independent of the amount of transmitter released during the conditioning train. This increment typically increased transmitter release by amount 1% of the control level in the absence of potentiation. 8. Suggestions are given to explain why potentiation of transmitter release, which is thought to arise from an accumulation of Ca-2+ in the nerve terminal, can be described assuming a linear relationship between potentiation and R(t), the proposed substance responsible for potentiation, under experimental conditions in which a 3rd to 4th power relationship would be expected to exist between external Ca concentration and evoked transmitter release.     

Comparison of transmitter release properties of embryonic sympathetic neurons growing in vivo and in vitro

The functional behavior of embryonic chick sympathetic neurons was determined by inducing release of [3H]norepinephrine by electrical stimulation of sympathetic neurons growing in the chick heart and in culture, with and without heart cells. A very close correspondence between the functional behavior of neurons developing with the heart cells, either in vivo or in vitro, was demonstrated. For example, the outflow of tritium from [3H]norepinephrine loaded sympathetic neurons of 15-day-old chick heart was about three times more at 10 Hz than at 1 Hz. In contrast, the outflow of tritium from 12-day-old [3H]norepinephrine loaded cultured sympathetic neurons was inversely related to the frequency of stimulation (outflow at 1 Hz was about three time more than at 10 Hz). When neurons were co-cultured with the heart cells, the frequency-outflow relationship reverted to that seen in the intact heart. Electrically-evoked outflow of tritium from the heart was reduced in a concentration-dependent manner by 3-30 nM tetrodotoxin, abolished in 0.25 mM Ca medium, and potentiated by 3 mM tetraethylammonium. In sharp contrast, the outflow evoked by stimulation of cultured neurons was neither blocked by 30-300 nM tetrodotoxin, low Ca, nor potentiated by tetraethylammonium. However, when neurons were co-cultured with heart cells, the evoked outflow was blocked by 30 nM tetrodotoxin and low Ca, and potentiated by tetraethylammonium. Veratrine (10 microM) had very little effect on the outflow from cultured neurons but induced a massive outflow from co-cultures as well as hearts. Neurons grown in a medium conditioned by the heart cells were not sensitive to tetrodotoxin and veratrine. It is implied that cultured sympathetic neurons are endowed mostly with Ca channels, and that the Na channels become functional only when neurons are grown with the target cells. This dramatic alteration in the functional behavior of neurons co-cultured with heart cells indicates that the effector organ has an important role in the development of ionic conductances of sympathetic neurons growing in the body and in culture.