Regulation of acetylcholine synthesis: does cytoplasmic acetylcholine control high affinity choline uptake?

When brain synaptosomes are obtained from animals that have been injected intravenously with [2H4]choline 1 minute before being killed, their high affinity [3H] choline uptake is correlated inversely with their acetylcholine content and directly with the rate at which they synthesize [2H4]acetylcholine. The control of such choline uptake by the cytoplasmic acetylcholine concentration is proposed as a mechanism regulating acetylcholine synthesis in cholinergic nerve terminals.

Acetylcholine release from striatal slices of young adult and aged Fischer 344 rats.

The release of endogenous and newly synthesized acetylcholine (ACh) was examined in neostriatal slices prepared from young adult (10-month) and aged (28-month) Fischer 344 rats. Both spontaneous and potassium-stimulated release were tested after various in vitro incubation times (1, 3 or 5 hr). The potassium-stimulated release of ACh from slices of 28-month rats was decreased by 53% when tested after incubating the slices 1 hr. The age-related differences in ACh release lessened if the slices were incubated for longer times (3 or 5 hr) before monitoring release. The spontaneous release of ACh was similar among the slices from both age-groups and at all times points monitored. When the neostriatal slices were incubated in medium supplemented with deuterated choline, the release of both the endogenous and newly synthesized ACh from slices of 28-month rats was decreased by 33% when tested after a 1-hr incubation, but was again similar to that released from slices of 10-month rats when tested after a 3-hr incubation. Choline release from slices of the 28-month rats was similar to that released from the slices of the 10-month rats when acetylcholinesterase (AChE) was inhibited during the release incubation. In slices with intact AChE activity, however, the age-related difference in choline release was similar to that observed for ACh release when AChE was inhibited. That is, when AChE activity was intact, the potassium-stimulated choline release from slices of 28-month rats was less than that released from slices of 10-month rats when release was tested after a 1-hr incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-related differences in the phosphoinositide system in rat neostriatum.

The phosphoinositide cycle is an important second messenger system that may be involved in the therapeutic action of lithium. Elderly patients taking lithium often experience beneficial effects at lower doses and serum levels than their younger counterparts. To investigate age-related differences in lithium's effects on the phosphoinositide system, neostriatal slices from 3-, 10-, and 24- to 26-month-old Fischer 344 rats were labeled with [3H]-myoinositol and exposed for a 25-minute incubation period to oxotremorine-M and varying lithium chloride concentrations. Inositol phosphatase accumulation was significantly greater in 10- and 24- to 26-month-old than in 3-month-old rats. Further investigations may clarify the underlying mechanisms for such age-related differences.

Characterization of muscarinic receptors modulating acetylcholine release in the rat neostriatum.

Two classical and two nonclassical muscarinic agents were tested for their effects on neostriatal acetylcholine (ACh) release. The classical muscarinic antagonist, atropine (0.1-2 microM), increased ACh release in a dose-dependent fashion, whereas the 'nonclassical' antagonist, pirenzepine (2-200 microM), had no effect on ACh release. The muscarinic agonist, oxotremorine (1-100 microM), and its analog, oxotremorine-M (0.1-50 microM), both decreased ACh release in a dose-dependent fashion. The inhibitory potency of oxotremorine-M was approximately 20 times that of oxotremorine, and the inhibitory effects of both agonists were blocked by atropine. The results obtained with the muscarinic antagonists indicate that 'M-2 type' muscarinic receptors mediate ACh release in the neostriatum, but further pharmacological analyses are required to assign with certainty the receptor subtype involved in neostriatal ACh release.

Presynaptic muscarinic modulation of nicotinic excitation in the rat neostriatum.

In rat neostriatal slices, cholinergic agents were tested for their effects on endogenous ACh release and on electrical activity. ACh release was evoked by 25 mM K+ during two 5-min periods between which a slice was allowed to rest for 20 min; drugs were present during the second stimulation period. In the absence of a cholinesterase inhibitor, only Ch outflow was monitored. For the recording of electrical activity, intrastriatal stimulation evoked field potentials which were monitored in the absence and presence of drugs in the perfusate. Atropine (1-100 microM) increased endogenous ACh release by 32-91% and effective doses were 10-fold lower in the presence of a cholinesterase inhibitor. Atropine also increased the amplitudes of synaptic population spikes in the field potentials. The muscarinic agonists muscarine (100 microM) and oxotremorine (25 and 100 microM) decreased endogenous ACh release. Atropine (10 microM) blocked the depressant effect of muscarine (100 microM). Muscarine (100 microM-1 mM) and oxotremorine (10-100 microM) decreased the electrically evoked excitation in the rat neostriatal slices, and their effects were reversed by atropine. Only higher concentrations of nicotine (1 and 5 mM) decreased the synaptic population spikes, but potassium-stimulated Ch outflow was not affected. It is concluded that in the neostriatum presynaptic muscarinic receptors modulate nicotinic excitation since potassium-stimulated ACh release and intrinsically evoked synaptic excitation are influenced by muscarinic drugs in the same way.

Muscarinic modulation of endogenous acetylcholine release in rat neostriatal slices.

Some classical and nonclassical muscarinic agents were tested for their effects on potassium-evoked acetylcholine (ACh) release from rat neostriatal slices. Release was monitored by measuring endogenous ACh when acetylcholinesterase (AChE) was inhibited with physostigmine (30 microM) or by measuring endogenous choline when AChE activity was left intact. The classical antagonist, atropine (0.1-2 microM), induced an increase in release whether AChE activity was inhibited or intact. The putative M-1 selective antagonist, pirenzepine, had minimal effects over a broad concentration range (2-200 microM) and induced an increase in ACh release only when AChE activity was inhibited. The classical agonist, oxotremorine (10-100 microM) decreased effectively ACh release (by 22-35%), but only when AChE activity was intact. The oxotremorine analog, oxotremorine-M, was apparently more potent than oxotremorine, but also decreased ACh release (by 24-41%) only when AChE activity was intact. Another oxotremorine analog, BM-5, behaved more like a muscarinic antagonist in its effects on neostriatal ACh release, and the highest concentration tested (100 microM) increased release (by 47%) when AChE activity was left intact. As in many other cholinergic systems, the agonists tested in this study were not selective in their action on ACh release modulation in the rat neostriatum. The antagonists, however, were more selective in their action, i.e., pirenzepine was relatively ineffective, and on the basis of this selective action, it can be concluded that modulation of endogenous ACh release in the rat neostriatum is mediated by a M-2 muscarinic receptor subtype.

Acetylcholine metabolism and choline availability at the neuromuscular junction of mature adult and aged rats.

1. The balance between acetylcholine (ACh) synthesis and degradation and the availability of choline were studied in the neuromuscular junction of rats aged 10 (mature adult) and 28 (aged) months. Endogenous and 2H4-labelled variants of ACh and choline were assayed during steady-state stimulated and resting conditions using gas chromatography-mass spectrometry. 2. Endogenous ACh levels were 34% less per nerve terminal in the older rats; in non-innervated tissue, the levels were 12-14% as large as in innervated tissue. Endogenous choline levels in innervated and non-innervated tissue were similar and were 28% higher in the older animals. 3. Accumulation of 2H4-labelled choline was significantly greater in the 28-month animals; the specific activity at equilibrium was 60% higher in the older rats. 4. Incorporation of [2H4]choline into [2H4]ACh was significantly faster in the aged rats; times to half-maximal values were 2.0 and 0.6 min in the 10- and the 28-month animals, respectively. There was no measurable synthesis of ACh in the non-innervated tissue. 5. There were no major age-related differences in the amount of ACh released during stimulation between 1 and 20 Hz. In contrast, choline efflux during stimulation was significantly greater in the aged animals; flux values (+/- S.E. of mean) were 0.48 (+/- 0.014) and 0.66 (+/- 0.038) nmol/min for the 10- and the 28-month rats, respectively. 6. Under resting conditions, endogenous ACh efflux was significantly greater in the older rats; the rates (+/- S.E. of mean) were 2.00 (+/- 0.21) and 3.05 (+/- 0.43) pmol/min for the younger and the older animals, respectively; similarly, choline efflux was greater in the aged tissue. 7. These results indicated that lower intracellular ACh levels can be attributed to enhanced leakage rather than to decreased synthesis. Greater ACh efflux is accompanied by correspondingly greater choline uptake in the 28-month rats; this is associated with greater choline efflux and higher steady-state choline levels in the aged tissue.

Effects of 2,4-dithiobiuret treatment in rats on cholinergic function and metabolism of the extensor digitorum longus muscle.

Effects of 2,4-dithiobiuret (DTB) treatment in rats on neuromuscular transmission and the disposition of cholinergic substances, acetylcholine (ACh) and choline (Ch), were examined in a combined electrophysiological/biochemical study using an in vitro extensor digitorum longus (EDL) muscle-peroneal nerve preparation. EDL muscle preparations isolated from rats treated with DTB (1 mg/kg/day X 5 days, ip) displayed a 49% depression in the frequency of miniature end-plate potentials (MEPPs) and a 21% depression in mean MEPP amplitude. Statistical analysis of evoked end-plate potentials (EPPs) measured in curarized preparations indicated that the mean quantal content (m) was significantly depressed in EDL muscles from DTB-treated rats. At stimulation rates of 1, 10, 20, and 50 Hz the estimated values of m in EDL preparations from DTB-treated rats were, respectively, 21, 25, 45, and 51% of that in control preparations. Biochemical determinations of ACh and Ch revealed a significant DTB-induced increase in endogenous ACh and Ch content in EDL preparations fixed for extraction of ACh and Ch immediately after dissection from the treated rats. In vitro, however, there were negligible changes in overall ACh synthesis since the total (tissue and medium) tracer ACh (2H4-ACh) synthesized from tracer Ch (2H4-Ch; 10 microM) supplied in the perfusion medium was similar in EDL preparations from DTB-treated and control rats. Also, in EDL muscles from DTB-treated rats the resting release of ACh was not affected, but when exogenous Ch (2H4-Ch) was not supplemented in the medium the evoked release (via peroneal nerve stimulation) of ACh was depressed. Thus, decreases in spontaneous quantal ACh release, as detected in the electrophysiological experiments, were not reflected by changes in the biochemically determined ACh resting release. The biochemical determination of evoked ACh release, however, correlated with the decrease in quantal content detected in the electrophysiological analysis of evoked EPPs when exogenous Ch was not supplemented in the perfusion medium. Significant and consistent increases (two to three times) in both Ch content and efflux occurred in the EDL muscles from DTB-intoxicated rats. These results indicate that DTB induces a prejunctional impairment of neuromuscular transmission that is not specifically directed at ACh synthesis. Rather those processes by which ACh is incorporated into or released from vesicles appear to be altered.

Behavioral and neurochemical changes associated with chronic exposure to low-level concentration of pesticide mixtures.

In order to assess behavioral and neurochemical changes resulting from pesticide exposure, food-restricted male weanling rats were exposed for 90 d to low doses (1 ppb-10,000 ppb range) of individual pesticides (aldicarb, metribuzin, or methomyl) or mixtures of them. During exposure, rats were trained to run a T-maze and tested for spatial discrimination reversal learning. At sacrifice, three brain regions (cortex, hippocampus, and neostriatum) were assayed for the neurotransmitters dopamine, acetylcholine, and serotonin. Animals treated with a mixture of two insecticides and one herbicide were found to have slower speeds in maze-running (motor control) and also had altered levels of choline in their neostriatums. Rats treated with one herbicide compound (metribuzin) took longer to learn on two reversals; this group also had a significantly lower acetylcholine/choline ratio in their hippocampus.

Age-related differences in the effects of some muscarinic agents on acetylcholine release from rat neostriatal slices.

The purpose of this study was to investigate whether the muscarinic modulation of neostriatal acetylcholine release changes with senescence. Neostriatal slices from Fischer 344 rats aged 3, 10 and 28 months were prepared and incubated in Krebs-Ringer bicarbonate buffer oxygenated with 95% O2/5% CO2. Acetylcholine release from slices of each age group was monitored in the presence or absence of muscarinic agents, and the release in the presence of the drug was compared to the release from slices of age-matched controls in the absence of drug. The muscarinic agonist, oxotremorine, and two muscarinic antagonists, atropine and pirenzepine, were tested for their effects on acetylcholine release. Pirenzepine is selective in its interaction with the M1 muscarinic receptor subtype; atropine and oxotremorine are nonselective in their actions. Of the three drugs tested, pirenzepine displayed a significant age-related difference in its effects on acetylcholine release. Whereas the effects of pirenzepine (50 microM) on acetylcholine release modulation in slices from the 3-month rats were negligible, the M1-selective antagonist increased the release of acetylcholine from slices of 10- and 28-month rats by another 42 and 192% (P less than .05), respectively. Atropine (1 microM) was also tested, and an increase in acetylcholine release by another 64, 104 and 218% (all P less than .05) was observed in slices from the 3-, 10- and 28-month rats, respectively. In the presence of oxotremorine (50 microM), acetylcholine release decreased in slices from the 3-month rats by 35% (P less than .1), but changed by only 7 and 15% in the 10- and 28-month slices, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of various experimental manipulations on neostriatal acetylcholine and dopamine release.

The release of endogenous acetylcholine and dopamine and the appearance of their metabolites, choline and dihydroxyphenylacetic acid (DOPAC), from neostriatal slices prepared from Fischer 344 rats was examined under various experimental conditions. There was a dose-dependent increase in the amount of neurotransmitter or metabolite as the medium potassium concentration was increased from 5 to 50 mM. Over an eight minute period in Krebs Ringer bicarbonate buffer containing 25 mM potassium, the rate of release of acetylcholine was 6 to 13 times greater than that of dopamine. The dopamine endogenous to the slice preparation appeared to have little effect on the release of endogenous acetylcholine since manipulations that significantly altered dopamine release (depletion with 6-hydroxydopamine or uptake inhibition with nomifensine) had minimal effects on the cholinergic neurons. In contrast, increasing the endogenous acetylcholine in the preparation by inhibiting acetylcholinesterase resulted in a 1.2 to 12 fold increase in dopamine release depending upon the incubation time and the potassium concentration. These studies indicate that within the neostriatal slices there is minimal influence of the endogenous dopamine on the cholinergic neurons, whereas the extracellular acetylcholine can influence dopamine release when its concentration is increased by inhibition of acetylcholinesterase.

Choline uptake and acetylcholine synthesis in synaptosomes: investigations using two different labeled variants of choline.

Using sequential incubations in media of different K+ composition, we investigated the dynamics of choline (Ch) uptake and acetylcholine (ACh) synthesis in rat brain synaptosomal preparations, using two different deuterated variants of choline and a gas chromatographic-mass spectrometric (GC-MS) assay for ACh and Ch. Synaptosomes were preincubated for 10 min in a Krebs medium with or without high K+ and with 2 micrometer-[(2)H(9)]Ch. At the end of the preincubation al variants of ACh and Ch were measured in samples of the pellet and medium. In the second incubation (4 min) samples of synaptosomes were resuspended in normal or high K+ solutions containing [(2)H(4)]Ch (2 micrometer) and all variants of ACh and Ch were measured in the pellet and medium at the end of this period. This protocol allowed us to compare the effects of preincubation in normal or high K+ solution on the metabolism during a second low or high K+ incubation of a [(2)H(9)]Ch pool accumulated during the preincubation period. Moreover, we were able to compare and contrast the effects of this protocol on [(2)H(9)]Ch metabolism versus [(2)H(4)]Ch metabolism. The most striking result we obtained was that [(2)H(9)]Ch that had been retained by the synaptosomes after the preincubation was not acetylated during a subsequent incubation in normal or high K+ media. This result suggests that if an intraterminal pool of Ch is involved in ACh synthesis, the size of this pool is below the limits of detection of our assay. We have confirmed the observation that a prior depolarizing incubation results in an enhanced uptake of Ch during a second incubation in normal K+ Krebs. Moreover, Ch uptake is stimulated by prior incubation under depolarizing conditions relative to normal preincubation when the second incubation is in a high K+ solution. These results are discussed in terms of current models of the regulation of ACh synthesis in brain.

Intrinsic cholinergic excitation in the rat neostriatum: nicotinic and muscarinic receptors.

An in vitro slice technique was employed to study the receptors involved in intrinsic cholinergic excitation in the rat neostriatum. The locally evoked synaptic potentials were suppressed by antinicotinic agents, mecamylamine (10 muM), d-tubocurarine (3 muM) or hexamethonium (100 muM), but not by the antimuscarinic agent atropine (100 muM). If the slices were exposed to an acetylcholinesterase (AChE)-inhibitor (paraoxon 1--20 muM, physostigmine 0.1--0.5 muM), the synaptic potentials were potentiated. The amplitude of the orthodromic population spike increased, and it was further facilitated when the stimulus frequencies were raised from 1--3 Hz to 10--30 Hz. The frequency facilitation following exposure to an AChE-inhibitor was blocked by atropine (1--100 muM). Intracellular recording indicated that a slow depolarizing potential caused the frequency potentiation of the orthodromic discharges. Apparently rat neostriatum is similar to cholinergic systems in sympathetic ganglia and spinal Renshaw cells, in that nicotinic receptors mediate fast excitation and muscarinic receptors mediate slow excitation.

Choline and acetylcholine metabolism in rat neostriatal slices.

Choline (Ch) uptake and release and acetylcholine (ACh) synthesis and release have been studied by gas chromatography mass spectrometry (GCMS) in slices of rat neostriatum in vitro to assess the effects of depolarization by 25 mM K+ and the influence of elevated concentrations of Ch in the incubation medium. During the first 60 min after preparation, 25 mM K+ increased ACh release by 182% and reduced ACh levels by 40%. The rate of ACh synthesis was unchanged. After a 1-h equilibration period, the rate of ACh synthesis was considerably less (2.41 nmol mg-1 h-1, compared to 9.78 nmol mg-1 h-1). Exposure to 25 mM K+ during the second hour increased the rate to 6.47 nmol mg-1 h-1. During the first 10 min of exposure to 25 mM K+, ACh synthesis was reduced, regardless of incubation. Increasing concentrations of external [2H4]Ch apparently favored initial rates of net ACh synthesis, since the rank order of initial net ACh synthesis rates is the same as the rank order of external [2H4] Ch concentration under both normal and depolarized conditions. However, the only significant effect of external [2H4]Ch on ACh metabolism was that it increased ACh release during the initial 10 min, when the preparation was depolarized with K+. The efflux of endogenous [2H0]Ch was increased initially (10 min) and slowed over a 60-min period by 25 mM K+, and increased when [2H4]Ch in the medium was increased. Changes in ACh synthesis and release were dependent upon the time exposure of slices to high K+, and the results suggest that Ch favors initial rates of ACh synthesis, but that Ch influences ACh release primarily under conditions of stress (i.e., depolarization).