Dopaminergic modulation of the septo-hippocampal cholinergic system activity under stress

The effects of the dopaminergic agonist apomorphine or the antagonist sulpiride on high affinity choline uptake and newly synthesized acetylcholine release by hippocampal synaptosomal preparations, were examined in rats subjected to immobilization stress. Increased dopamine uptake by septal synaptosomal preparations was taken as evidence for increased mesoseptal dopaminergic activity in response to stress. While apomorphine treatment failed to alter choline uptake or acetylcholine release in unhandled rats, it did however prevent the stress-induced increase in these cholinergic parameters. In contrast, after treatment with sulpiride both choline uptake and acetylcholine release were increased in unhandled rats, as they were after acute stress. Acute stress of sulpiride treated rats however resulted in changes similar to those produced by administration of either sulpiride or stress separately. We conclude that the mesoseptal dopaminergic system plays an important role in modulating the activity of the septo-hippocampal cholinergic system under stress.     

Synaptic Function of Cholinergic-Specific Chol-1α Ganglioside

The function of a cholinergic-specific ganglioside, Chol-1alpha, was investigated. The release of acetylcholine from synaptosomes was inhibited by anti-Chol-1alpha monoclonal antibody but not by monoclonal antibodies against other brain gangliosides tested. Chol-1alpha ganglioside stimulated the high-affinity choline uptake by synaptosomes and consequently enhanced acetylcholine synthesis, resulting in an increased release of acetylcholine from synaptosomes. The memory and learning abilities of rats given anti-Chol-1alpha antibody were remarkably suppressed. These in vitro and in vivo studies suggest that Chol-1alpha ganglioside plays a pivotal role in cholinergic synaptic transmission and participates in cognitive function.     

SODIUM-DEPENDENT HIGH AFFINITY CHOLINE UPTAKE: A REGULATORY STEP IN THE SYNTHESIS OF ACETYLCHOLINE

The sodium-dependent high affinity choline uptake into synaptosomes from rat brain has been studied after in vivo treatments which would alter the activity of cholinergic neurons. We utilized a number of treatments to reduce the activity of cholinergc neurons in the brain. Administration of pentobarbital (65 mg/kg), chloral hydrate (40 mg/kg) and γbutyrelactone (750 mg/kg) caused a 50-80% reduction in sodium-dependent high affinity choline uptake in several brain regions (30 min). This depression was not found 24 h after injection. Interruption of the cholinergic septal-hippocampal or habenuleinterpeduncular tracts by lesions (10 min-1 h) also caused a similar, large reduction in sodium-dependent high affinity choline uptake in the hippocampus and the interpeduncular nucleus respectively. We reversed the inactivity after pentobarbital administration by direct electrical stimulation of the cholinergic septal-hippocampal tract. Stimulation (40 Hz) for 10-15 min completely reversed the depression in sodium-dependent high affinity choline uptake. Stimulation at lower frequencies or for shorter times caused a partial reversal. Administration of pentylenetetrazol (75 mg/kg), a convulsant, was utilized to increase the activity of central cholinergic neurons. After drug administration, we found a large (60%) increase in sodium-de-pendent high affinity choline uptake. This increase was not found in the hippocampus when cholinergic afferents were interrupted by septal lesion prior to drug administration. We also examined the uptake after administration of cholinergic drugs. Oxotremorine (0.75 mg/kg), a muscarinic agonist which reduces acetylcholine release and turnover, caused a reduction in uptake. On the other hand, administration of scopolamine (5 mg/kg), a cholinergic antagonist which increases acetylcholine turnover, caused an increase in sodium-dependent high affinity choline uptake. Addition of any drug utilized, drectly to uptake samples, did not alter uptake. We examined the conversion of [³H]choline to [³H]acetylcholine in hippocampal synaptosomes after septal lesion, pentylenetetrazol administration and in untreated controls. In all cases, 60-70% of the total sodium-dependent tritium content was present as [³H]acetylcholine. Evidence was presented that homoexchange is not or is less involved in choline uptake than in GABA uptake. A kinetic analysis of sodium-dependent high affinity choline uptake was performed after all treatments. We found changes in V_max, after all treatments, which were consistently in the same direction as the alterations in activity. The proposal is made that the sodium-dependent high affinity choline uptake is coupled to cholinergic activity in such a way as to regulate the entry of choline for the maintenance of acetylcholine synthesis. The findings also lead us to propose that sodium-dependent high affinity choline uptake in vitro be utilized as a rapid, relative measure of the activity of cholinergic nerve terminals in vivo.     

Antibodies directed against ubiquitin inhibit high affinity [3H]choline uptake in rat cerebral cortical synaptosomes

Sodium-dependent [3H]choline uptake and coupled [3H]acetylcholine synthesis were inhibited in rat cerebral cortical synaptosomes in a dose- (1-10 micrograms/ml) and time-dependent manner by affinity-purified antibodies directed against ubiquitin (anti-Ub). Neither sodium-independent [3H]choline uptake nor [3H]acetylcholine release was affected by up to 10 micrograms/ml anti-Ub, indicating that the cholinergic terminals were not depolarized by the anti-Ub. Binding of anti-Ub to synaptosomes, as measured with 125I-protein A, was saturable and occurred over the same concentration range (1-10 micrograms/ml) at which uptake inhibition was observed. Although preimmune IgG bound to the synaptosome preparation to a greater extent and was apparently not readily saturable, this fortuitous binding was without effect on high affinity choline uptake and conversion to acetylcholine. The results suggest the presence of a ubiquitin-protein conjugate on the synaptosomal surface and a functional relationship between this protein conjugate and the sodium-dependent choline transport system.     

THE EFFECTS OF BOTULINUM TOXIN ON ACETYLCHOLINE METABOLISM IN MOUSE BRAIN SLICES AND SYNAPTOSOMES

The effects of Type A botulinum toxin on acetylcholine metabolism were studied using mouse brain slice and synaptosome preparations. Brain slices that had been incubated with the toxin for 2h exhibited a decreased release of acetylcholine into high K⁺ media. Botulinum toxin did not affect acetylcholine efflux from slices in normal K⁺ media. When labeled choline was present during the release incubation, a‘newly-synthesized’pool of acetylcholine was formed in the tissue. In toxin-treated slices exposed to high K⁺, both the production and the release of this‘newly-synthesized’acetylcholine were depressed. A possible explanation for these actions of botulinum toxin would be via an inhibition of the high affinity uptake of choline. This hypothesis was tested by measuring the high affinity uptake of [³H]choline into synaptosomes prepared from brain slices. Previous exposure of slices to botulinum toxin caused a significant reduction in the accumulation of label by the synaptosomes. These data are discussed in terms of our current understanding of the mechanism of action of botulinum toxin and the toxin's interaction with the mechanisms regulating acetylcholine turnover.     

Elimination of the vesicular acetylcholine transporter in the striatum reveals regulation of behaviour by cholinergic-glutamatergic co-transmission

Cholinergic neurons in the striatum are thought to play major regulatory functions in motor behaviour and reward. These neurons express two vesicular transporters that can load either acetylcholine or glutamate into synaptic vesicles. Consequently cholinergic neurons can release both neurotransmitters, making it difficult to discern their individual contributions for the regulation of striatal functions. Here we have dissected the specific roles of acetylcholine release for striatal-dependent behaviour in mice by selective elimination of the vesicular acetylcholine transporter (VAChT) from striatal cholinergic neurons. Analysis of several behavioural parameters indicates that elimination of VAChT had only marginal consequences in striatum-related tasks and did not affect spontaneous locomotion, cocaine-induced hyperactivity, or its reward properties. However, dopaminergic sensitivity of medium spiny neurons (MSN) and the behavioural outputs in response to direct dopaminergic agonists were enhanced, likely due to increased expression/function of dopamine receptors in the striatum. These observations indicate that previous functions attributed to striatal cholinergic neurons in spontaneous locomotor activity and in the rewarding responses to cocaine are mediated by glutamate and not by acetylcholine release. Our experiments demonstrate how one population of neurons can use two distinct neurotransmitters to differentially regulate a given circuitry. The data also raise the possibility of using VAChT as a target to boost dopaminergic function and decrease high striatal cholinergic activity, common neurochemical alterations in individuals affected with Parkinson's disease.     

Characterization of the Inhibitory Action of Botulinum Neurotoxin Type A on the Release of Several Transmitters from Rat Cerebrocortical Synaptosomes

Under optimised conditions for intoxication, botulinum neurotoxin type A was shown to inhibit approximately 90% of Ca2+-dependent K+-evoked release of [3H]acetylcholine, [3H]noradrenaline, and [3H]dopamine from rat cerebrocortical synaptosomes; cholinergic terminals were most susceptible. In each case, the dose-response curve for the neurotoxin was extended, with about 50% of evoked release being inhibited at approximately 10 nM whereas 200 nM was required for the maximal blockade. This may suggest some heterogeneity in the release process. The action of the toxin was time and temperature dependent and appeared to involve binding and sequestration steps prior to blockade of release. The neurotoxin failed to exert any effect on synaptosomal integrity or on Ca2+-independent release of the transmitters tested; it produced only minimal changes in neurotransmitter uptake although small secondary effects were detected with cholinergic terminals. Blockade by the neurotoxin of Ca2+-dependent resting release of transmitter was apparent; Sr2+, Ba2+, or high concentrations of Ca2+ restored the resting release of 3H-catecholamine but not [3H]acetylcholine. Interestingly, none of the latter conditions or 4-aminopyridine could reverse the toxin-induced blockade of evoked release. This lack of specificity in its action on synaptosomes, and other published findings, lead to the conclusion that toxin-sensitive component(s) exist in all nerve terminals that are concerned with transmitter release.     

Calcium-dependent [3H]acetylcholine release and muscarinic autoreceptors in rat cortical synaptosomes during development

A number of presynaptic cholinergic parameters (high affinity [³H]choline uptake, [³H]acetylcholine synthesis, [³H]acetylcholine release, and autoinhibition of [³H]acetylcholine release mediated by muscarinic autoreceptors) were comparatively analyzed in rat brain cortex synaptosomes during postnatal development. These various functions showed a differential time course during development. At 10 days of age the release of [³H]acetylcholine evoked by 15 mM KCl from superfused synaptosomes was Ca²⁺-dependent but insensitive to the inhibitory action of extrasynaptosomal acetylcholine. The muscarinic autoreceptors regulating acetylcholine release were clearly detectable only at 14 days, indicating that their appearance may represent a criterion of synaptic maturation more valuable than the onset of a Ca²⁺-dependent release.     

CHOLINE: SELECTIVE ACCUMULATION BY CENTRAL CHOLINERGIC NEURONS

Abstract— Most of the cholinergic input to the hippocampus was destroyed by placement of lesions in the medial septal area. In animals with such lesions we found that hippocampal ChAc activity was reduced by 85–90% and endogenous acetylcholine levels were reduced by more than 80 %. When hippocampal synaptosomes from animals with lesions were incubated with [³H]choline at concentrations of 7.5 nm, 1 μm and 10 μm there was approximately a 60 % reduction in the uptake of [³H]choline, suggesting that cholinergic nerve endings were mainly responsible for [³H]choline uptake. At 0.1 mm concentrations of [³H]choline, there was only a 25 % reduction of choline uptake, suggesting that at higher concentrations of choline there was more nonspecific uptake. The uptake of radiolabelled tryptophan, glutamate and GABA were only slightly or not at all affected by the lesions. There was a significant reduction of uptake of radiolabelled serotonin and norepinephrine, since known monoaminergic tracts were disrupted. Choline uptake was reduced only in brain regions in which cholinergic input was interrupted (i.e. the cerebral cortex and hippocampus) and remained unchanged in other regions (i.e. the cerebellum and striatum). The time course of the reduction in choline uptake was similar to that of the reductions in ChAc activity and endogenous ACh levels; there was no decrease at 1 day, a significant decrease at 2 days, and the maximal decrease at 4 days postlesion. There was a close correlation among choline uptake, ChAc activity and ACh levels in the four brain regions examined (i.e. the striatum, cerebral cortex, hippocampus and cerebellum). Our results suggest that when hippocampal synaptosomes (and perhaps synaptosomes from other brain areas as well) are incubated in the presence of choline, at concentrations of 10 μm m or lower, then cholinergic nerve endings are responsible for the bulk of the choline accumulated by the tissue.     

Protection of Ischaemic Synaptosomes from Calcium Overload by Addition of Exogenous Lactate

In depolarised anoxic synaptosomes, in which lactate production was significantly raised compared with normoxic conditions, calcium uptake, net acetylcholine release, and the intrasynaptosomal calcium concentration were all significantly lowered. In contrast, lactate production in synaptosomes incubated under aglycaemic- and ischaemic-type conditions was significantly lower and basal calcium uptake, acetylcholine release, and intrasynaptosomal calcium concentration were elevated compared with normoxia. In addition, the increase in intrasynaptosomal calcium concentration under the ischaemic-type condition appeared to be greater than could be accounted for by the rise in calcium uptake alone. Intrasynaptosomal pH reflected the lactate production under each condition investigated. Addition of exogenous lactate to normoxic synaptosomes mimicked the effects observed in anoxia, suggesting that lactate itself may have blocked the calcium uptake, inhibiting the rise in intrasynaptosomal calcium and acetylcholine release occurring in depolarised anoxic synaptosomes. When lactate was added to ischaemic synaptosomes, the large rise in intrasynaptosomal calcium concentration, calcium uptake, and acetylcholine release were decreased, suggesting that lactate may have a protective role in preventing cell death by calcium overload under ischaemic-type conditions. Evidence is presented to suggest that the effect of L-lactate was due to the lactate moiety itself rather than the associated acidosis.     

Specific lysis of GABAergic synaptosomes by an antiserum to glutamate decarboxylase

An antiserum to pure glutamate decarboxylase (GAD) when incubated with rat cortical synaptosomes in the presence of complement caused release of 33-53% of lactate dehydrogenase (LDH) and 22-41% of total GAD. In addition most of the gamma-aminobutyrate (GABA) present was released. Anti-GAD antiserum alone, or complement alone, were without action. The antiserum plus complement had no effect on noradrenaline or choline uptake, and did not release choline acetylase (ChAT). Anti-ChAT serum plus complement released 30-37% of ChAT and 10-13% of LDH. It prevented choline uptake. This serum did not produce GAD release or prevent GABA, choline or noradrenaline uptake. When cortical synaptosomes were exposed to both antisera plus complement, their actions were strictly additive. The data indicate specific lysis of GABAergic and cholinergic synaptosomal sub-populations.     

Biochemical evidence that acetylcholine release from cholinergic nerve terminals is mostly vesicular

The nature of the intraterminal compartments from which acetylcholine (ACh) is released following presynaptic stimulation was investigated. This was pursued by examining the effects of the anticholinergic drug 2-(4-phenylpiperidino)cyclohexanol (AH5183) on the release of newly synthesized [3H]ACh and of endogenous ACh from purified cholinergic nerve terminals (synaptosomes) which were isolated from the electric organs of Torpedo. Preincubation of the synaptosomes, with AH5183 (1-10 microM), does not affect either the intraterminal synthesis of [3H]ACh or the uptake of its precursors, but results in a marked inhibition (85%) of the release of the newly synthesized [3H]ACh. However, when AH5183 is added following the accumulation of [3H]ACh in the nerve terminals, it does not affect [3H]ACh release. AH5183 also has no effect on the release of preformed endogenous ACh. These findings, together with the previous in vitro demonstrations that AH5183 is a potent inhibitor of ACh uptake into isolated cholinergic vesicles, suggest that most of the synaptosomal ACh is secreted by a vesicular mechanism.     

Cetiedil, a Drug That Inhibits Acetylcholine Release in Torpedo Electric Organ

The effects of cetiedil, a vasodilatator substance with reported anticholinergic properties, were examined on cholinergic presynaptic functions at the nerve electroplaque junction of Torpedo marmorata using either synaptosomes or slices of intact tissue. Cetiedil abolished the calcium-dependent release of acetylcholine (ACh) triggered by depolarization or by addition of A23187 ionophore, a finding localizing the site of action downstream from the calcium entry step. In addition, a direct effect on the release process itself was indicated by the observation that cetiedil blocks the release of ACh mediated by a recently isolated presynaptic membrane protein, the mediatophore, reconstituted into ACh-containing proteoliposomes. In all three preparations, ACh release was inhibited by cetiedil with a Ki of 5-8 microM. Under the conditions used in these release experiments, the synthesis of ACh and its compartmentation within the nerve terminals were not modified. However, the drug was able to reduce high-affinity choline uptake and vesicular ACh incorporation when it was given together with the radioactive precursor, a result showing that cetiedil has a broad inhibitory action on cholinergic uptake processes.     

Effects of secondary binding by activator and inhibitor peptides on covalent intermediates of pig pepsin.

1. Cerebral-cortex synaptosomes were shown to synthesize (14C)acetylcholine after incubation with (14C)choline, and 25mM-KCl released (14C)acetylcholine (but not (14C)choline) into the medium by a Ca2+-dependent and Mg2+-sensitive process. 2. The K+-stimulated release of (14C)acetylcholine was inhibited by more than 80% after preincubation of the synaptosomes with 10(5) mouse lethal doses of botulinum toxin/ml. (14C)choline uptake, (14C)acetylcholine synthesis, intrasynaptosomal K+ and occluded lactate dehydrogenase were unaffected by the toxin. It also failed to prevent the K+-stimulated release of (3H)noradrenaline and (14C)glycine from synaptosomes. 3. Fractionation of hypo-osmotically shocked synaptosomes revealed that more than 75% of the radioactive acetylcholine was in the cytoplasmic compartment, although the vesicle pellet contained more total acetylcholine than the cytoplasmic pool. Consequently the specific radioactivity of acetylcholine in the cytoplasmic pool was almost 5 times that of the vesicles. This distribution was unaffected by preincubation with botulinum toxin. It is concluded that the toxin acts directly on the release of acetylcholine, rather than influencing its storage. 4. After K+-stimulation, toxin-inhibited synaptosomes contained increased amounts of total acetylcholine, which suggests that its rate of synthesis is controlled by depolarization rather than release.     

Specific immunolysis of serotonergic nerve terminals using an antiserum against tryptophan hydroxylase

An antiserum to tryptophan hydroxylase purified from whole rat brain when incubated with rat striatal synaptosomes in the presence of complement caused release of 18% of LDH, 20% loss of potassium and 60% loss of tryptophan hydroxylase. Uptake of 5-HT was reduced by 60%. Anti-tryptophan hydroxylase alone, or complement alone were without action. The antiserum plus complement had no effect on DA uptake and did not release TH or GAD. These results suggest selective lysis of serotonergic nerve terminals had occurred. The antiserum plus complement reduced choline uptake by 45%. However, this did not seem due to lysis of cholinergic terminals, as ChAT was not released.     

Chronic nicotine differentially affects the function of nicotinic receptor subtypes regulating neurotransmitter release

It is known that nicotine can activate several subtypes of release-regulating presynaptic nicotinic receptors (nAChRs) including those situated on central noradrenergic, dopaminergic, cholinergic and glutamatergic axon terminals. The objective of this study was to investigate the effects of chronic administration of (-)nicotine on the function of the above autoreceptors and heteroreceptors using rat superfused synaptosomes. In hippocampal synaptosomes prelabelled with [3H]noradrenaline (NA) the nicotine-evoked overflow of [3H]NA was higher in rats treated with nicotine for 10 days (via osmotic mini-pumps) than in vehicle-treated rats. In striatal synaptosomes, prelabelled with [3H]dopamine (DA), chronic nicotine did not modify the releasing effect of nicotine. No significant change was observed in experiments with synaptosomes from nucleus accumbens prelabelled with [3H]DA. Exposure of hippocampal synaptosomes prelabelled with [3H]choline to nicotine elicited release of [3H]acetylcholine; this effect was almost abolished in synaptosomes from animals administered nicotine for 10 days, suggesting down-regulation of nicotinic autoreceptors. In hippocampal synaptosomes prelabelled with [3H]D-aspartate, the releasing effect of epibatidine following chronic nicotine treatment did not differ from that in controls. The K+-evoked exocytotic release of the neurotransmitters tested was not modified by long-term nicotine administration. The results show that chronic nicotine differentially affects the function of release-regulating nAChR subtypes.     

The Effects of Chronic Manganese Chloride Treatment Expressed as Age-Dependent, Transient Changes in Rat Brain Synaptosomal Uptake of Amines

Abstract: As a result of chronic manganese treatment of rats from conception onwards, a decrease was observed in the uptake of dopamine, but not of noradrenaline or serotonin, by synaptosomes isolated from hypothalamus, striatum, and midbrain and in choline uptake by hypothalamic synaptosomes obtained from 70–90-day-old animals. In 100–120-day-old manganese-treated rats the only difference observed was increased choline uptake by striatal synaptosomes. All comparisons were with age-matched controls. These results, which are consistent with views of a dopaminergic and cholinergic involvement in manganese encephalopathy, point out that changes in these systems are observable only at specific times during manganese intoxication.     

Regional Selectivity of a γ-Aminobutyric Acid-Induced [3H]Acetylcholine Release Sensitive to Inhibitors of γ-Aminobutyric Acid Uptake

The effects of gamma-aminobutyric acid (GABA) on the release of [3H]acetylcholine ([3H]ACh) were studied in synaptosomes prepared from rat hippocampus, cerebral cortex, hypothalamus, and striatum and prelabelled with [3H]choline. When synaptosomes were exposed in superfusion to exogenous GABA (0.01-0.3 mM) the basal release of newly synthesized [3H]ACh was increased in a concentration-dependent way in hippocampus, cortex, and hypothalamus nerve endings. In contrast, the release of [3H]ACh was not significantly affected by GABA in striatal synaptosomes. The effect of GABA was not antagonized significantly by bicuculline or picrotoxin. Muscimol caused only a slight not significant increase of [3H]ACh release when tested at 0.3 mM whereas, at this concentration, (-)-baclofen was totally inactive. The GABA-induced release of [3H]ACh was counteracted by SKF 89976A, SKF 100561, and SKF 100330A, three strong and selective GABA uptake inhibitors. The data suggest that, in selective areas of the rat brain, GABA causes release of [3H]ACh following penetration into cholinergic nerve terminals through a GABA transport system.     

Ca2+-dependent protein phosphorylation of purely cholinergic Torpedo synaptosomes.

Preincubation of intact, purely cholinergic Torpedo synaptosomes with [32P]Pi results in the incorporation of 32P into about 10 specific proteins. Depolarizing the Torpedo synaptosomes by a high K+ buffer or treatment with the Ca2+ ionophore A23187 result in Ca2+ uptake, in acetylcholine (ACh) release, and in a marked increase of 32P incorporation into a specific protein band with an apparent subunit molecular weight of 100,000 (band alpha). The kinetics of synaptosomal 45Ca2+ uptake, of 32P incorporation into band alpha, and of ACh release is similar and reach maximal values about 45 s after the synaptosomes have been treated. Sr2+ and Ba2+ can replace Ca2+ in evoking both K+ depolarization-dependent ACh release and 32P incorporation into band alpha. The effectiveness of these ions (Ca2+ greater than Sr2+ greater than Ba2+) is similar in both cases. The data presented suggest that Ca2+ accumulation by Torpedo synaptosomes leads to an increase in the phosphorylation of a specific protein and to ACh release. This phosphoprotein may be involved in the regulation of presynaptic processes which underly ACh release.     

Interactions of lead and calcium on the synaptosomal uptake of dopamine and choline

Exposure of rodents to lead $\text{in vivo}$ has been associated with alterations in cholinergic and dopaminergic neurotransmission in the CNS. These effects have been hypothesized to result from competitive interactions between lead and calcium at sites involved in uptake and release of neurotransmitters and their precursors. These experiments reproduced the $\text{in vivo}$ observation by $\text{in vitro}$ exposure of crude synaptosomal suspensions to lead. Lead-induced inhibition of high affinity choline uptake was mimicked by reduced in vitro calcium concentrations, which suggests that lead's effects on cholinergic function are explainable by the lead-calcium hypothesis. However, inhibition of dopamine uptake was produced only by lead and not by reduced calcium; further additions of calcium did not reverse lead-induced effects on dopamine uptake. Increased calcium concentrations were shown to increase the release of dopamine; lead in the presence of normal calcium concentration did not affect dopamine release. However, more dopamine was released when increased calcium was combined with exposure to 1 × 10^?4 lead. This effect may have resulted from lead's ability to increase the uptake of calcium by synaptosomes. Thus, the interactions between lead and calcium appear to differ in terms of effects on cholinergic and dopaminergic function; in the former, the results suggest a competitive interaction similar to that shown functionally at peripheral cholinergic sites; in the latter, a different role for calcium is hypothesized which may account for the different effects of lead.