5-Hydroxytryptamine3 Receptors Sited on Cholinergic Axon Terminals of Human Cerebral Cortex Mediate Inhibition of Acetylcholine Release

Synaptosomes prepared from freshly obtained human cerebral cortex and labeled with [3H]choline have been used to investigate the modulation of [3H]acetylcholine ([3H]ACh) release by 5-hydroxytryptamine (5-HT). The Ca(2+)-dependent release of [3H]-ACh occurring when synaptosomes were exposed in superfusion to 15 mM KCl was inhibited by 5-HT (0.01-1 microM) in a concentration-dependent manner. The effect of 5-HT was mimicked by 1-phenylbiguanide, a 5-HT3 receptor agonist, but not by 8-hydroxy-2-(di-n-propylamino)tetralin, a 5-HT1A receptor agonist. The 5-HT3 receptor antagonists tropisetron and ondansetron blocked the effect of 5-HT, whereas spiperone and ketanserin were ineffective. It is suggested that cholinergic axon terminals in the human cerebral cortex possess 5-HT receptors that mediate inhibition of ACh release and appear to belong to the 5-HT3 type.     

Characterization of N-[3H]Methylcarbamylcholine Binding Sites and Effect of N-Methylcarbamylcholine on Acetylcholine Release in Rat Brain

The present experiments show that N-[3H]-methylcarbamylcholine ([3H]MCC) binds specifically and with high affinity to rat hippocampus, frontal cortex, and striatum. The highest maximal density of binding sites was apparent in frontal cortex and the lowest in hippocampus. [3H]MCC binding was potently inhibited by nicotinic, but not muscarinic, agonists and by the nicotinic antagonist dihydro-beta-erythroidine in all three brain regions studied. The effect of unlabeled MCC on acetylcholine (ACh) release from slices of rat brain was tested. The drug significantly enhanced spontaneous ACh release from slices of hippocampus and frontal cortex, but not from striatal slices. This effect of MCC to increase ACh release from rat hippocampus and frontal cortex was antagonized by the nicotinic antagonists dihydro-beta-erythroidine and d-tubocurarine, but not by alpha-bungarotoxin or by the muscarinic antagonist atropine. The MCC-induced increase in spontaneous ACh release from hippocampal and frontal cortical slices was not affected by tetrodotoxin. The results suggest that MCC might alter cholinergic transmission in rat brain by a direct activation of presynaptic nicotinic receptors on the cholinergic terminals. That this alteration of ACh release is apparent in hippocampus and frontal cortex, but not in striatum, suggests that there may be a regional specificity in the regulation of ACh by nicotinic receptors in rat brain.     

Long-Term Survival of Intrastriatal Dopaminergic Grafts: Modulation of Acetylcholine Release by Graft-Derived Dopamine

The nigrostriatal dopaminergic system of rats was unilaterally lesioned with 6-hydroxydopamine. Part of the animals was grafted 2 weeks later with fetal dopaminergic cells on the lesioned side; untreated rats of the same strain served as controls. Both 3 and 12-14 months after surgery the striatal dopamine (DA) content and the in vivo rotational response following injection of D-amphetamine showed significant changes in grafted as compared to lesioned animals. At 12-14 months after transplantation, the electrically evoked release of tritiated DA and acetylcholine (ACh) in slices (preincubated with [3H]DA or [3H]choline, respectively) of striata of intact, lesioned, or grafted animals was also investigated. Electrical field stimulation of striatal slices of the lesioned side did not evoke any significant [3H]DA overflow, whereas a marked [3H]DA release was observed in slices of grafted and control striata. Moreover, both DL-amphetamine (3 microM) and nomifensine (10 microM) strongly enhanced basal 3H outflow in these slices. Electrically evoked [3H]ACh release was significantly reduced in slices from all striatal tissues by 0.01 microM apomorphine. In slices from denervated striata a clearcut hypersensitivity for this action of apomorphine was present, indicating supersensitivity of DA receptors on cholinergic terminals; this hypersensitivity was significantly reduced in graft-bearing striata. Furthermore, because this hypersensitivity was unchanged in slices of lesioned striata under stimulation conditions (four pulses/100 Hz) avoiding inhibition by endogenously released DA, it is concluded that lesion-induced DA receptor supersensitivity is caused by an increase in receptor density or efficacy rather than by a decreased competition between endogenous and exogenous agonists. Both reuptake blockade of DA with nomifensine (10 microM) and release of endogenous DA by DL-amphetamine (3 microM) potently reduced [3H]ACh release only in control and grafted but not in lesioned tissue. In experiments using potassium-evoked [3H]ACh release, tetrodotoxin had no effect on the inhibitory activity of amphetamine and nomifensine, indicating that the DA receptors involved in their indirect inhibitory action are located directly on the cholinergic terminals.     

Effects of Acetylethylcholine Mustard on [3H]Quinuclidinyl Benzilate Binding and Acetylcholine Release in Rat Brain Synaptosomes

The effects of acetylethylcholine mustard and its aziridinium derivative (AMMA) on acetylcholine (ACh) release and [3H]quinuclidinyl benzilate (QNB) binding were studied in rat cortical synaptosomes. After incubation for 5 min at 37 degrees C, AMMA reduced [3H]QNB binding with an IC50 of 9 microM. Following incubation for 5 min with 50 microM AMMA and washing, there was a 62% reduction in the [3H]QNB binding capacity with no change in the KD value for the remaining receptors, a result indicating the irreversibility of the AMMA binding. AMMA and oxotremorine both reduced the basal and 30 mM K+-induced release of newly synthesized [3H]ACh in dose-dependent manners over a 2.5-min period. At identical 50 microM concentrations, AMMA produced a much longer inhibition of basal [3H]ACh release than oxotremorine did. The inhibition of basal and 30 mM K+-induced [3H]ACh release by AMMA (10-250 microM) was blocked by 2 microM atropine during a 2.5-min release incubation, but not during a 30-min release incubation. After synaptosomes were treated with 50 microM AMMA for 5 min and the unbound drug was washed out from the tissue, [3H]ACh release (basal and K+-induced) was reduced. AMMA (50 microM) reduced high-affinity choline uptake and ACh synthesis by greater than 90% in this tissue, but these effects did not account for the [3H]ACh release inhibition, because they were not atropine sensitive and hemicholinium-3 had no effect on [3H]ACh release under the conditions used in these studies, i.e., after extracellular [3H]choline was washed out. Taken together, these results suggest that AMMA may be an irreversible agonist at presynaptic muscarinic autoreceptors.     

Nicotinic autoreceptor function in rat brain during maturation and aging: possible differential sensitivity to organophosphorus anticholinesterases

Acetylcholine (ACh) release is modulated pre-synaptically by both muscarinic and nicotinic receptor-mediated processes. While muscarinic autoreceptors inhibit ACh release, nicotinic autoreceptors enhance ACh release and thus disruption of these processes could potentially affect cholinergic toxicity following exposure to anticholinesterases. Marked age-related differences in sensitivity to some organophosphorus (OP) anticholinesterases have been reported. We compared nicotinic autoreceptor function (NAF) during maturation and aging and evaluated its potential modulation by the common OP insecticide, chlorpyrifos (CPF). Cortical synaptosomes were pre-loaded with [3H]choline, superfused (0.6 ml/min) with physiological buffer and [3H]ACh release was evoked with potassium (KCl, 9 mM), with or without co-addition of exogenous ACh to stimulate nicotinic autoreceptors. Fractions of perfusate were subsequently collected and area under the curve (AUC) for [3H] was analyzed by scintillation counting. The difference in evoked release due to co-addition of exogenous ACh was defined as NAF. Under these conditions, atropine (ATR, 0.1 microM) appeared requisite for NAF; thus this muscarinic antagonist was subsequently added to all perfusion buffers. In synaptosomes from adult tissues, exogenous ACh (3-100 microM) significantly increased release in a concentration-dependent manner. The nicotinic antagonist mecamylamine (MEC, 100 microM) substantially reduced the potassium-evoked release elicited by co-addition of ACh (10 microM). Interestingly, the nicotinic agonists nicotine (NIC) and dimethylphenylpiperazinium (DMPP; 0.1-10 microM) had no effect on release. The active metabolite of CPF (i.e. chlorpyrifos oxon (CPO), 1-10 microM) inhibited NAF in vitro. Maturation-related expression of NAF was noted (AUC with co-addition of 10 microM ACh: 7-day rats, 7+/-6; 21-day rats, 44+/-6; 90-day rats, 196+/-37; 24-month rats, 173+/-52). NAF was substantially reduced (67-91%) 96 h after maximum tolerated dosages of CPF in adult and aged rats (279 mg/kg, sc) but not in juveniles (127 mg/kg, sc), even though AChE inhibition was similar among the age groups (>80%). Together these data suggest that NAF is differentially expressed during maturation and that this neuromodulatory process may be selectively altered by some OP insecticides, potentially contributing to age-related differences in response to AChE inhibitors. As NAF has been postulated to be activated under conditions of 'impaired' cholinergic function, selective alteration of this pre-synaptic process by OP anticholinesterases may be also important in age-related conditions associated with cholinergic hypofunction.     

Differential Alteration of Various Cholinergic Markers in Cortical and Subcortical Regions of Human Brain in Alzheimer''s Disease

The main objective of the present study was to determine whether cholinergic markers (choline acetyltransferase activity and nicotinic and muscarinic receptors) are altered in Alzheimer's disease. Choline acetyltransferase activity in Alzheimer's brains was markedly reduced in various cortical areas, in the hippocampus, and in the nucleus basalis of Meynert. The maximal density of nicotinic sites, measured using the novel nicotinic radioligand N-[3H]methylcarbamylcholine, was decreased in cortical areas and hippocampus but not in subcortical regions. M1 muscarinic cholinergic receptor sites were assessed using [3H]pirenzepine as a selective ligand; [3H]pirenzepine binding parameters were not altered in most cortical and subcortical structures, although the density of sites was modestly increased in the hippocampus and striatum. Finally, M2-like muscarinic sites were studied using [3H]-acetylcholine, under muscarinic conditions. In contrast to M1 muscarinic sites, the maximal density of M2-like muscarinic sites was markedly reduced in all cortical areas and hippocampus but was not altered in subcortical structures. These findings reveal an apparently selective alteration in the densities of putative nicotinic and muscarinic M2, but not M1, receptor sites in cortical areas and in the hippocampus in Alzheimer's disease.     

Effect of Chronic Nicotine Treatment on Nicotinic Autoreceptor Function and N-[3H]Methylcarbamylcholine Binding Sites in the Rat Brain

It has been reported that N-methylcarbamylcholine (MCC), a nicotinic agonist, binds to central nicotinic receptors and causes an increase of acetylcholine (ACh) release from certain central cholinergic nerve terminals. The present experiments determine whether these two phenomena change in response to the chronic administration of nicotine, a procedure known to result in an increase in nicotinic binding sites. Chronic nicotine caused a brain region-specific up-regulation of [3H]MCC sites; binding increased in the frontal cortex, parietal cortex, striatum, and hippocampus, but not in the occipital cortex or cerebellum. The effect of nicotine was selective to nicotinic binding sites, because muscarinic sites, both M1 ([ 3H]pirenzepine) and M2 ([3H]ACh), were unaffected by chronic nicotine treatment. MCC increased the release of ACh from the frontal cortex and hippocampus by a calcium-dependent mechanism; MCC did not alter ACh release from striatum or occipital cortex of control animals. The MCC-induced increase in ACh release was not apparent in those animals which had been treated with nicotine. There was a partial recovery of nicotinic autoreceptor function when animals were allowed to recover (4 days) following chronic nicotine treatment, but the density of binding sites remained increased compared to control. Chronic nicotine did not change the potassium-evoked release of ACh from the frontal cortex or hippocampus, but decreased this measure from striatum. It also decreased the ACh content of the striatum, but not that of the cortex or the hippocampus; the activity of choline acetyltransferase was not altered in any of the regions tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct evidence that release-stimulating alpha7* nicotinic cholinergic receptors are localized on human and rat brain glutamatergic axon terminals

The existence on glutamatergic nerve endings of nicotinic acetylcholine receptors (nAChRs) mediating enhancement of glutamate release has often been suggested but not demonstrated directly. Here, we study the effects of nAChR agonists on [3 H]-d-aspartate ([3 H]-d-ASP) release from synaptosomes superfused in conditions known to prevent indirect effects. Nicotinic receptor agonists, while unable to modify the basal [3 H]-d-ASP release from human neocortex or rat striatal synaptosomes, enhanced the Ca2+ -dependent exocytotic release evoked by K+ (12 mm) depolarization. Their rank order of potency were anatoxin-a > epibatidine > nicotine > ACh (+ atropine). The anatoxin-a effect, both in human and rat synaptosomes, was antagonized by mecamylamine, alpha-bungarotoxin or methyllycaconitine. The basal release of [3 H]ACh from human cortical synaptosomes was increased by (-)-nicotine (EC50 = 1.16 +/- 0.33 microm) or by ACh plus atropine (EC50 = 2.0 +/- 0.04 microm). The effect of ACh plus atropine was insensitive to alpha-bungarotoxin, methyllycaconitine or alpha-conotoxin MII, whereas it was totally antagonized by mecamylamine or dihydro-beta-erythroidine. To conclude, glutamatergic axon terminals in human neocortex and in rat striatum possess alpha7* nicotinic heteroreceptors mediating enhancement of glutamate release. Release-enhancing cholinergic autoreceptors in human neocortex are nAChRs with a pharmacological profile compatible with the alpha4beta2 subunit combination.     

Nicotinic Autoreceptors Mediating Enhancement of Acetylcholine Release Become Operative in Conditions of "Impaired" Cholinergic Presynaptic Function

Abstract: The existence in the mammalian CNS of release-inhibiting muscarinic autoreceptors is well established. In contrast, few reports have focused on nicotinic autoreceptors mediating enhancement of acetylcholine (ACh) release. Moreover, it is unclear under what conditions the function of one type of autoreceptor prevails over that of the other. Rat cerebrocortex slices, prelabeled with [³H]choline, were stimulated electrically at 3 or 0.1 Hz. The release of [³H]ACh evoked at both frequencies was inhibited by oxotremorine, a muscarinic receptor agonist, and stimulated by atropine, a muscarinic antagonist. Nicotine, ineffective at 3 Hz, enhanced [³H]ACh release at 0.1 Hz; mecamylamine, a nicotinic antagonist, had no effect at 3 Hz but inhibited [³H]ACh release at 0.1 Hz. The cholinesterase inhibitor neostigmine decreased [³H]ACh release at 3 Hz but not at 0.1 Hz; in the presence of atropine, neostigmine potentiated [³H]ACh release, an effect blocked by mecamylamine. In synaptosomes depolarized with 15 mM KCI, ACh inhibited [³H]ACh release; this inhibition was reversed to an enhancement when the external [Ca²⁺] was lowered. The same occurred when, at 1.2 mM Ca²⁺, external [K⁺] was decreased. Oxotremorine still inhibited [³H]ACh release at 0.1 mM Ca²⁺. When muscarinic receptors were inactivated with atropine, the K⁺ (15 mM)-evoked release of [³H]ACh (at 0.1 mM Ca²⁺) was potently enhanced by ACh acting at nicotinic receptors (EC₅₀? 0.6 µM). In conclusion, synaptic ACh concentration does not seem to determine whether muscarinic or nicotinic autoreceptors are activated. Although muscarinic autoreceptors prevail under normal conditions, nicotinic autoreceptors appear to become responsive to endogenous ACh and to exogenous nicotinic agents under conditions mimicking impairment of ACh release. Our data may explain in part the reported efficacy of cholinesterase inhibitors (and nicotinic agonists) in Alzheimer's disease.     

Effect of Adenosine, Adenosine Derivatives, and Caffeine on Acetylcholine Release from Brain Synaptosomes: Interaction with Muscarinic Autoregulatory Mechanisms

Synaptosomes, prepared from rat cerebral cortex and hippocampus, were preincubated with [methyl-3H]choline. The effect of adenosine, cyclohexyladenosine, N-ethylcarboxamide adenosine, 2'-deoxyadenosine, and oxotremorine on K+-evoked 3H efflux was investigated. High-voltage electrophoretic separation showed that in the presence of physostigmine, the K+-evoked 3H efflux from hippocampal synaptosomes was 90% [3H]acetylcholine and 10% [3H]choline. Adenosine (30 microM) and oxotremorine (100 microM) both decreased [3H]acetylcholine release from hippocampal synaptosomes. The effect was inversely proportional to the KCl concentration and disappeared at a KCl concentration of 50 mM. Cyclohexyladenosine was approximately 3,000 times more active than adenosine, whereas N-ethylcarboxamide adenosine and 2'-deoxyadenosine were inactive. This indicates that A1 adenosine receptors were involved in the inhibitory effect. Caffeine antagonized the adenosine effect, and at a concentration of 100 microM, it stimulated [3H]acetylcholine efflux. The inhibitory effect of oxotremorine was as great in cortical as in hippocampal synaptosomes. In contrast, adenosine was much less active in cortical than in hippocampal synaptosomes. When inhibitory concentrations of adenosine and oxotremorine were added together into the incubation medium, the effect of adenosine on [3H]acetylcholine release was consistently reduced. An interaction between muscarinic and A1 adenosine presynaptic receptors at a common site modulating acetylcholine release can be assumed.     

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.     

Nicotinic Stimulation of [3H]Acetylcholine Release from Mouse Cerebral Cortical Synaptosomes

The effects of nicotine and 1,1-dimethyl-4-phenylpiperazinium (DMPP) on the release of newly synthesized [3H]acetylcholine in mouse cerebral cortical synaptosomes were examined. Nicotine and DMPP produced increases in [3H]acetylcholine release, over the level of spontaneous release, of 24% and 30%, respectively, of a maximum depolarization-induced release produced by 50 mM potassium. The maximum effect was achieved at a concentration of 1 X 10(-4) M for both agents. The time course of release indicated a slow onset of action, reaching a maximum effect at 15 min of incubation. Both nicotine and DMPP also produced a slightly greater release of total tritium, measured in the absence of cholinesterase inhibition, than of [3H]acetylcholine. The release induced by nicotine was completely antagonized by hexamethonium and was largely (58%) calcium-dependent. Nicotine also produced an increase in [3H]choline accumulation into synaptosomes. These results indicate that the nicotinic agonists nicotine and DMPP can produce a moderate enhancement of acetylcholine release by a receptor-mediated action on cholinergic nerve terminals in the central nervous system.     

Ca2+-Surrogate Action of Pb2+ on Acetylcholine Release from Rat Brain Synaptosomes

The effect of lead ions on the release of acetylcholine (ACh) was investigated in intact and digitonin-permeabilized rat cerebrocortical synaptosomes that had been prelabeled with [3H]choline. Release of ACh was inferred from the release of total 3H label or by determination of [3H]ACh. Application of 1 microM Pb2+ to intact synaptosomes in Ca2(+)-deficient medium induced 3H release, which was enhanced by K+ depolarization. This suggests that entry of Pb2+ into synaptosomes and Pb2(+)-induced ACh release can be augmented by activation of the voltage-gated Ca2+ channels in nerve terminals. The lead-induced release of [3H]ACh was blocked by treatment of synaptosomes with vesamicol, which prevents uptake of ACh into synaptic vesicles without affecting its synthesis in the synaptoplasm. This indicates that Pb2+ selectively activates the release of a vesicular fraction of the transmitter with little or no effect on the leakage of cytoplasmic ACh. Application of 1-50 nM (EC50 congruent to 4 nM) free Pb2+ to digitonin-permeabilized synaptosomes elicited release of 3H label that was comparable with the release induced by 0.2-5 microM (EC50 congruent to 0.5 microM) free Ca2+. This suggests that Pb2+ triggers transmitter exocytosis directly and that it is a some 100 times more effective activator of exocytosis than is the natural agonist Ca2+.     

Altered Muscarinic and Nicotinic Receptor Densities in Cortical and Subcortical Brain Regions in Parkinson's Disease

Abstract: Muscarinic and nicotinic cholinergic receptors and choline acetyltransferase activity were studied in postmortem brain tissue from patients with histopathologically confirmed Parkinson's disease and matched control subjects. Using washed membrane homogenates from the frontal cortex, hippocampus, caudate nucleus, and putamen, saturation analysis of specific receptor binding was performed for the total number of muscarinic receptors with [³H]quinuclidinyl benzilate, for muscarinic M₁ receptors with [³H]pirenzepine, for muscarinic M₂ receptors with [³H]oxotremorine-M, and for nicotinic receptors with (–)-[³H]nicotine. In comparison with control tissues, choline acetyltransferase activity was reduced in the frontal cortex and hippocampus and unchanged in the caudate nucleus and putamen of parkinsonian patients. In Parkinson's disease the maximal binding site density for [³H]quinuclidinyl benzilate was increased in the frontal cortex and unaltered in the hippocampus, caudate nucleus, and putamen. Specific [³H]pirenzepine binding was increased in the frontal cortex, unaltered in the hippocampus, and decreased in the caudate nucleus and putamen. In parkinsonian patients B_max values for specific [³H]oxotremorine-M binding were reduced in the cortex and unchanged in the hippocampus and striatum compared with controls. Maximal (–)-[³H]nicotine binding was reduced in both the cortex and hippocampus and unaltered in both the caudate nucleus and putamen. Alterations of the equilibrium dissociation constant were not observed for any ligand in any of the brain areas examined. The present results suggest that both the innominatocortical and the septohippocampal cholinergic systems degenerate in Parkinson's disease. The reduction of cortical [³H]oxotremorine-M and (–)-[³H]nicotine binding is compatible with the concept that significant numbers of the binding sites labelled by these ligands are located on presynaptic cholinergic nerve terminals, whereas the increased [³H]pirenzepine binding in the cortex may reflect postsynaptic denervation supersensitivity.     

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.     

Cholinergic receptor alterations in the cerebral cortex of spinal cord injured rat

Many areas of the cerebral cortex process sensory information or coordinate motor output necessary for control of movement. Disturbances in cortical cholinergic system can affect locomotor coordination. Spinal cord injury causes severe motor impairment and disturbances in cholinergic signalling can aggravate the situation. Considering the impact of cortical cholinergic firing in locomotion, we focussed the study in understanding the cholinergic alterations in cerebral cortex during spinal cord injury. The gene expression of key enzymes in cholinergic pathway - acetylcholine esterase and choline acetyl transferase showed significant upregulation in the cerebral cortex of spinal cord injured group compared to control with the fold increase in expression of acetylcholine esterase prominently higher than cholineacetyl transferase. The decreased muscarinic receptor density and reduced immunostaining of muscarinic receptor subtypes along with down regulated gene expression of muscarinic M1 and M3 receptor subtypes accounts for dysfunction of metabotropic acetylcholine receptors in spinal cord injury group. Ionotropic acetylcholine receptor alterations were evident from the decreased gene expression of alpha 7 nicotinic receptors and reduced immunostaining of alpha 7 nicotinic receptors in confocal imaging. Our data pin points the disturbances in cortical cholinergic function due to spinal cord injury; which can augment the locomotor deficits. This can be taken into account while devising a proper therapeutic approach to manage spinal cord injury.     

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.     

Regulation of Histamine Release and Synthesis in the Brain by Muscarinic Receptors

The cholinergic modulation of histamine release and synthesis was studied in rat brain slices or synaptosomes labeled with L-[3H]histidine. Carbachol in increasing concentrations progressively reduced the K+-induced [3H]histamine release from cortical slices. Pirenzepine, a preferential M1-receptor antagonist, reversed the carbachol effect in an apparently competitive manner and with Ki values of 1-6 X 10(-8) M. 11-[(2-[(Diethylamino)methyl]-1-piperidinyl)acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116), considered a preferential M2-receptor antagonist, reversed the carbachol effect with a mean Ki of approximately 2 X 10(-7) M. Oxotremorine behaved as a partial agonist in the modulation of histamine release. Neostigmine, an acetylcholinesterase inhibitor, inhibited the K+-induced release of [3H]histamine from cortical slices, and the effect was largely reversed by pirenzepine, an observation suggesting a modulation by endogenous acetylcholine. The effects of carbachol and pirenzepine were observed with slices of other brain regions known to contain histaminergic nerve terminals or perikarya, as well as with cortical synaptosomes. The two drugs also modified, in opposite directions, [3H]histamine formation in depolarized cortical slices. In vivo oxotremorine inhibited [3H]histamine formation in cerebral cortex, and this effect was reversed by scopolamine. When administered alone, scopolamine failed to enhance significantly the 3H- labeled amine formation, a finding suggesting that muscarinic receptors are not activated by endogenous acetylcholine released under basal conditions. It is concluded that muscarinic heteroreceptors, directly located on histaminergic nerve terminals, control release and synthesis of histamine in the brain. These receptors apparently belong to the broad M1-receptor category and may correspond to a receptor subclass displaying a rather high affinity for AF-DX 116.     

Distinct Muscarinic Receptor Subtypes Differentially Modulate Acetylcholine Release from Corticocerebral Synaptosomes

The effect of McN-A-343 and oxotremorine on acetylcholine (ACh) release and choline (Ch) transport was studied in corticocerebral synaptosomes of the guinea pig. The synaptosomes were preloaded with [3H]Ch after treatment with the irreversible cholinesterase inhibitor, diisopropyl fluorophosphate, and then tested for their ability to release isotope-labeled ACh and Ch in the presence and absence of these agents. The kinetics of release were determined at the resting state (basal release) and in the presence of 50 mM K+. Under either condition, McN-A-343 enhanced the release of isotope-labeled ACh, whereas oxotremorine inhibited the K(+)-evoked release but had no effect on the basal release. The enhancing effect of McN-A-343 on basal ACh release was fully blocked by the selective M1 muscarinic antagonist, pirenzepine (100 nM). In contrast to its enhancing effect on ACh release, McN-A-343 potently inhibited Ch efflux as well as Ch influx. These effects were not blocked by atropine, a nonselective muscarinic antagonist. Oxotremorine had no effect on Ch transport. Binding studies showed that McN-A-343 was 3.6-fold more potent in displacing radiolabeled quinuclidinyl benzilate from cerebral cortex muscarinic receptors (mostly M1 subtype) than from cerebellar receptors (mostly M2 subtype), whereas oxotremorine was 2.6-fold more potent in the cerebellum. The displacements of radio-labeled pirenzepine and cis-dioxolane confirmed the M1 subtype preference of McN-A-343 and the M2 subtype preference of oxotremorine.(ABSTRACT TRUNCATED AT 250 WORDS)