Differential long-term effect of AF64A on [3H]ACh synthesis and release in rat hippocampal synaptosomes.

The activities of various presynaptic cholinergic parameters were determined in hippocampal synaptosomes of rats 29 weeks after intracerebroventricular injection of ethylcholine aziridinium (AF64A) (3 nmol/2 microliters/side) or vehicle (saline). Synaptosomes were preloaded with [3H]choline ([3H]Ch), treated with diisopropyl fluorophosphate to inhibit cholinesterase activity and then were assayed for their content of [3H]Ch and [3H]acetylcholine ([3H]ACh) and for their ability to synthesize and release [3H]ACh. In synaptosomes from AF64A-treated rats compared with synaptosomes from vehicle-treated rats we observed that: (i) specific uptake of [3H]Ch was reduced to 60% of control; (ii) residing [3H]ACh levels were 43% of control while residing [3H]Ch levels were 72% of control; (iii) basal and K(+)-induced [3H]ACh release were 77% and 73% of control, respectively; (iv) high K(+)-induced synthesis of [3H]ACh was only 9% of control; (v) but, choline acetyltransferase activity remained relatively high, being 80% of control. These results suggest that AF64A-induced cholinergic hypofunction is expressed by both loss of some cholinergic neurons and impairment in the functioning of the spared neurons.     

Storage and release of acetylcholine in rat cortical synaptosomes: Effects ofd,l-2-(4-phenylpiperidino)cyclohexanol (AH5183)

A post-stimulation synthesis of acetylcholine (ACh), its incorporation into a 'stable-bound' (vesicular) compartment and subsequent release, were compared in K+-stimulated synaptosomes, in the absence and presence of 10 microM AH5183. The drug depressed by 16% the net intrasynaptosomal formation of ACh from 1 microM [3H]choline (Ch) in the medium, by competitively inhibiting (Ki approximately equal to 20 microM) the high-affinity Ch transport, but it had no direct effect on the intraterminal synthesis of ACh per se. The drug reduced incorporation of newly synthesized [3H]ACh into synaptic vesicles by 55% and subsequent K+-depolarization-induced release of [3H]ACh by 83%, although it had no effect on Ca2+ influx into synaptosomes. These results are consistent with the hypothesis that AH5183 blocks cholinergic neurotransmission presynaptically by interfering with recharging of synaptic vesicles with ACh. Since the reduction of ACh release in the presence of AH5183 had no direct effect on ACh synthesis, these results also suggest that the transmitter release is not prerequisite for enhancement of Ch uptake and ACh synthesis in stimulated nerve terminals.     

Downregulation of muscarinic- and 5-HT1B-mediated modulation of [H]acetylcholine release in hippocampal slices of rats with fimbria-fornix lesions and intrahippocampal grafts of septal origin

Adult Long-Evans female rats sustained electrolytic fimbria-fornix lesions and, two weeks later, received intrahippocampal suspension grafts of fetal septal tissue. Sham-operated and lesion-only rats served as controls. Between 6.5 and 8 months after grafting, both the [³H]choline accumulation and the electrically evoked [³H]acetylcholine ([³H]ACh) release were assessed in hippocampal slices. The release of [³H]ACh was measured in presence of atropine (muscarinic antagonist, 1 μM), physostigmine (acetylcholinesterase inhibitor, 0.1 μM), oxotremorine (muscarinic agonist, 0.01 μM–10 μM), mecamylamine (nicotinic antagonist, 10 μM), methiothepin (mixed 5-HT₁/5-HT₂ antagonist, 10 μM), 8-OH-DPAT (5-HT_1A agonist, 1 μM), 2-methyl-serotonin (5-HT₃ agonist, 1 μM) and CP 93129 (5-HT_1B agonist, 0.1 μM–100 μM), or without any drug application as a control. In lesion-only rats, the specific accumulation of [³H]choline was reduced to 46% of normal and the release of [³H]ACh to 32% (nCi) and 43% (% of tissue tritium content). In the grafted rats, these parameters were significantly increased to 63%, 98% and 116% of control, respectively. Physostigmine reduced the evoked [³H]ACh release and was significantly more effective in grafted (−70%) than in sham-operated (−56%) or lesion-only (−54%) rats. When physostigmine was superfused throughout, mecamylamine had no effect. Conversely, atropine induced a significant increase of [³H]ACh release in all groups, but this increase was significantly larger in sham-operated rats (+209%) than in the other groups (lesioned: +80%; grafted: +117%). Oxotremorine dose-dependently decreased the ([³H]ACh) release, but in lesion-only rats, this effect was significantly lower than in sham-operated rats. Whatever group was considered, 8-OH-DPAT, methiothepin and 2-methyl-serotonin failed to induce any significant effect on [³H]ACh release. In contrast, CP 93129 dose-dependently decreased [³H]ACh release. This effect was significantly weaker in grafted rats than in the rats of the two other groups. Our data confirm that cholinergic terminals in the intact hippocampus possess inhibitory muscarinic autoreceptors and serotonin heteroreceptors of the 5-HT_1B subtype. They also show that both types of receptors are still operative in the cholinergic terminals which survived the lesions and in the grafted cholinergic neurons. However, the muscarinic receptors in both lesioned and grafted rats, as well as the 5-HT_1B receptors in grafted rats show a sensitivity which seems to be downregulated in comparison to that found in sham-operated rats. In the grafted rats, both types of downregulations might contribute to (or reflect) an increased cholinergic function that results from a reduction of the inhibitory tonus which ACh and serotonin exert at the level of the cholinergic terminal.     

Pirenzepine-insensitive muscarinic autoreceptors regulate acetylcholine release in human neocortex

The release of [³H]acetylcholine ([³H]ACh) and its modulation mediated by autoreceptors were investigated in synaptosomes prepared from fresh human cerebral cortex prelabelled with [³H]choline ([³H]Ch) and depolarized in superfusion with 15 mM KCl. The K⁺-evoked release of tritium was almost totally accounted for by unmetabolized [³H]ACh and was largely calcium-dependent. Exogenous ACh decreased the depolarization-evoked release of [³H]ACh in a concentration-dependent manner (EC₅₀ = 1.5 μM). The inhibitory effect of ACh on [³H]ACh release was counteracted by the non-selective muscarinic antagonist atropine. In contrast, the selective M₁ receptor antagonist pirenzepine was ineffective. It is concluded that muscarinic autoreceptors regulating the release of ACh are present on cholinergic nerve terminals of human cerebral cortex and appear to belong to a pirenzepine-insensitive subtype.     

Effects of ovariectomy and estradiol benzoate on high affinity choline uptake, ACh synthesis, and release from rat cerebral cortical synaptosomes

Several presynaptic processes were studied in cerebral cortical synaptosomes prepared from intact adult female rats or from ovariectomized animals that received 3 subcutaneous injections of either estradiol benzoate (10 μg/kg) or vehicle. Injections were given 1/day, every other day, and animals were sacrificed 1 h after the last injection. High affinity choline uptake and coupled acetylcholine (ACh) synthesis were reduced by ovariectomy, and restored to control levels by the estradiol benzoate injections. In contrast, low affinity choline uptake and depolarization-induced [³H]ACh release were unaffected by either ovariectomy or estradiol benzoate injections. These results suggest that changes in estradiol levels may alter the high affinity transport process regulating ACh synthesis in this tissue.     

Choline's phosphorylation in rat striatal slices is regulated by the activity of cholinergic neurons

The mechanism by which populations of brain cells regulate the flux of choline (Ch) into membrane or neurotransmitter biosynthesis was investigated using electrically stimulated superfused slices of rat corpus striatum. [Me-¹⁴C]Ch placed in the superfusion medium for 30 min during a 1-h stimulation period was incorporated into tissue [¹⁴C]phosphorylcholine (PCh) and [¹⁴C]phosphatidylcholine (PtdCh). Stimulation also caused a profound inhibition of PCh synthesis and a 10-fold increase in [¹⁴C]ACh release into the medium; it failed to affect tissue [¹⁴C]ACh levels. This effect was not explained by changes in ATP levels nor in the kinetic properties of Ch kinase (E.C. 2.7.1.32) or Ch acetyltransferase (ChAT) (E.C. 2.3.1.7). To investigate the mechanism of these effects, Ch uptake studies were performed with and without hemicholinium-3 (HC3), a selective inhibitor of high affinity Ch uptake. A two-compartment model accurately fit the observed data and yielded aK_m for Ch uptake of 5 μM into cholinergic structures and 72 μM into all other cells. Using this model it was estimated that cholinergic neurons account for 60% of observed uptake of Ch at physiologic Ch concentrations, even though they represent fewer than 1 % of the total cells in the slice. The model also predicts that an increase in Ch uptake within cholinergic neurons, reported to be associated with depolarization [4,27,32], would significantly inhibit Ch uptake into all other cells, and would account for the observed decrease in PCh synthesis.     

Modulation of hippocampal norepinephrine release by cholinergic agonists is altered by AF64A lesion

The effect of lesioning hippocampal cholinergic neurons with the neurotoxin AF64A on the ability of cholinergic agonists to modulate stimulation-induced release of ³H-norepinephrine (NE) from rat hippocampal slices was studied. Rats received intracerebroventricular injections of either AF64A (ethylcholine mustard aziridinium, 2 nmol) or vehicle (sham operated). Six weeks after treatment, release of ³H-NE evoked by electrical stimulation (2 Hz, 2 min) in the presence or absence of cholinergic agonists and/or antagonists was measured. Activation of M₂ receptors with oxotremorine (in the presence of the M₁ antagonist pirenzepine) caused a small inhibition of NE release, which was abolished in hippocampi from AF64A-treated rats. The K_d for high-affinity binding of the selective M₂ ligand [³H] AF-DX 384 was increased 10-fold in lesioned tissues. The M₁ selective agonist McN-A-343 produced a significant enhancement of NE release, which was unchanged by AF64A lesion. Binding studies with [³H] pirenzepine showed no change in the affinity or number of M₁ receptors. Nicotine also caused a significant enhancement of evoked NE release, but this effect was markedly reduced in tissues from AF64A-treated rats. AF64A treatment caused a twofold decrease in the number of [³H] nicotine binding sites. This study suggests that long-term lesion of hippocampal cholinergic neurons with AF64A alters the function of postsynaptic muscarinic M₂ and nicotinic cholinergic receptors that modulate the release of NE in the hippocampus.     

Differential alterations of cortical cholinergic and neurotensin markers following ibotenic acid lesions of the nucleus basalis magnocellularis

The present study determined whether cortical cholinergic neurons recover functionally following the loss of afferent projections from the nucleus basalis magnocellularis (nbm). At various points following ibotenic acid lesions of the nbm, choline acetyltransferase (ChAT) activity or the capacity of cortical cholinergic neurons to synthesize [³H]acetylcholine (ACh) from the precursor molecule [³H]choline were measured in the frontoparietal cortex. First, cortical ChAT activity was decreased by 21% and 35% on the side ipsilateral to the lesion at 1 and 2 weeks following the nbm lesion, respectively. By 6 weeks following nbm lesions, cortical ChAT activity returned to control levels and remained at control levels at 10 weeks following nbm lesions. However, by 13 weeks following nbm lesions, we observed a 21% increase in ChAT activity on the side ipsilateral to the lesion. ChAT activity in the nbm remained unchanged over the time course studied. Secondly, there was a parallel reduction (by 43%) in the capacity of frontoparietal cortex slices from the side ipsilateral to the lesion to synthesize [³H]ACh by 2 weeks following nbm lesions. By 13 weeks following the lesion there was a significant increase (29%) in the synthetic capacity of cortical cholinergic neurons compared to the 2 week time point. Third, the content of neurotensin in the frontoparietal cortex was significantly decreased by 25% and 36%, at 2 weeks and 13 weeks following nbm lesions, respectively. Neurotensin levels in the nbm were not affected by ibotenic acid lesions. In contrast, [¹²⁵I]neurotensin binding sites in the frontal or parietal cortex were not altered at 2 weeks following nbm lesions. In summary, the results of the present study provide evidence for functional recovery by cortical cholinergic neurons following selective lesions of the nbm. However, the reduction of cortical cholinergic terminals resulting from the lesion appears to cause irreversible trans-synaptic decreases in cortical neurotensin levels.     

Neurotransmitter release and its presynaptic modulation in the rat hippocampus after selective damage to cholinergic or/and serotonergic afferents

Male Long-Evans rats sustained injections of 5,7-dihydroxytryptamine (5,7-DHT) into the fimbria-fornix and the cingular bundle or/and intraseptal injections of 192 IgG-saporin to induce serotonergic or/and cholinergic hippocampal denervations; Sham-operated rats served as controls. Four to ten weeks after lesioning, we measured (i). the electrically evoked release of acetylcholine ([3H]ACh), noradrenaline ([3H]NA) and serotonin ([3H]5-HT) in hippocampal slices in the presence of drugs acting on auto- or heteroreceptors, (ii). the nicotine-evoked release of NA and (iii). the choline acetyltransferase (ChAT) activity and the concentration of monoamines in homogenates. Saporin lesions reduced the accumulation of [3H]choline, the release of [3H]ACh and the ChAT activity, but increased the concentration of NA and facilitated the release of [3H]NA evoked by nicotine. 5,7-DHT lesions reduced the accumulation and the release of [3H]5-HT, the concentration of 5-HT, and also facilitated the release of [3H]NA evoked by nicotine. Accumulation and electrically evoked release of [3H]NA were not altered by either lesion. The combination of both toxins resulted in an addition of their particular effects. The 5-HT(1B) receptor agonist, CP 93129, and the muscarinic agonist, oxotremorine, reduced the release of [3H]ACh in control and 5,7-DHT-lesioned rats; in rats injected with saporin, their effects could not be measured reliably. CP 93129 and the alpha(2)-adrenoceptor agonist, UK 14304, reduced the release of [3H]5-HT in all groups by about 65%. In conclusion: (i). selective neurotoxins can be combined to enable controlled and selective damage of hippocampal transmitter systems; (ii). 5-HT exerts an inhibitory influence on the nicotine-evoked release of NA, but partial serotonergic lesions do not influence the release of ACh at a presynaptic level and (iii). presynaptic modulatory mechanisms involving auto- and heteroreceptors may be conserved on fibres spared by the lesions.     

The calcium-dependent [H]acetylcholine release from synaptosomes of brown trout (Salmo trutta) optic tectum is inhibited by adenosine A1 receptors: Effects of enucleation on A1 receptor density and cholinergic markers

Presynaptic inhibition is one of the major control mechanisms in the CNS. Previously we reported that A₁ adenosine receptors are highly concentrated in the brain, including optic tectum, of trout and that they inhibited the release of glutamate. The optic tectum is heavily innervated by cholinergic nerve terminals. We have investigated whether A₁ receptors inhibit the presynaptic release of acetylcholine and whether the inhibition is triggered by calcium. The release of [³H]ACh evoked by 30 mM KCl was Ca²⁺ dependent and it was dose-dependently inhibited by the A₁ adenosine receptor agonist 2-chloro-N⁶-cyclopentyladenosine (CCPA) ranging between 10 nM to 100 μM. The maximum of inhibition was reached at 10 μM. The A₁ receptor antagonist 8-cyclopentyltheopylline (CPT, 10 μM), reversed almost completely the inhibition induced by CCPA 10 μM. In Fura-2/AM loaded synaptosomes, K⁺ depolarization raised [Ca²⁺]_i by about 64%. CCPA (10 μM) reduced the K⁺-evoked Ca²⁺ influx increase by about 48% and this effect was completely antagonised by CPT 10 μM. Synaptosome pretreatment with different Ca²⁺ channel blockers differently affected K⁺-evoked Ca²⁺ influx. This was not significantly modified by nifedipine (1 μM, L-type blocker) nor by ω-agatoxin IVA (0.3 μM, P/Q-type blocker), whereas about 50% reduction was shown by 0.5 μM ω-conotoxin GVIA (N-type blocker). Neurochemical parameters associated with cholinergic transmission and the density of A₁ adenosine receptors were measured in the trout optic tectum 12 days after unilateral eye ablation. A significant drop of both acetylcholinesterase (AChE) activity (24%) and choline acetyltransferase (CAT) activity (32%) was observed in deafferentated optic tectum, whereas the high affinity choline uptake did not parallel the decrease in enzyme activity. Eye ablation caused a marked decrease (43%) of A₁ receptor density without changing the affinity. The K⁺-evoked release of [³H]ACh from synaptosomes of deafferentated was not modify as well as the efficacy of 10 μM CCPA in decreasing [³H]ACh release was not apparently modified.     

Inhibition of high affinity choline transport attenuates both cholinergic and non-cholinergic effects of ethylcholine aziridinium (AF64A)

Ethylcholine aziridinium (AF64A) has been proposed as a specific cholinergic neurotoxin. In earlier studies, using AF64A, we reported that slow infusion of 1-2 nmol of this compound into each lateral ventricle of Sprague-Dawley rats resulted in small, and transient decreases in noradrenaline (NA) and serotonin (5-HT) levels in the hippocampus, while inducing a permanent and significant cholinergic hypofunction in the same brain region. The experiments described in this paper were designed to test the hypothesis that such noradrenergic and serotonergic changes after small doses of AF64A are secondary to the changes observed in cholinergic neurons. Levels of NA, and of 5-HT and its metabolite 5-hydroxyindole acetic acid (5-HIAA) were measured concurrently with levels of acetylcholine (ACh), in various brain regions of rats in which the effect of AF64A was attenuated, and in respective control animals. The effect of AF64A was diminished by inhibiting the interaction of AF64A with the high affinity transport site for choline (HAChT). This was achieved using hemicholinium-3 (HC-3), which does not cross the blood-brain barrier, and A-4 (a bis 4-methylpiperidine analog of HC-3), which is centrally active following its peripheral administration. A-4 (20 or 40 mg/kg i.p.) or HC-3 (10 micrograms/ventricle) had no effect on ACh, NA, 5-HT or 5-HIAA levels in saline-treated rats. However, all treatments significantly attenuated the decrease in ACh content produced by AF64A pretreatment. Transient decreases in NA, 5-HT and 5-HIAA contents after AF64A treatment were prevented or reduced by prior treatment with A-4 or HC-3. These results indicate that changes in noradrenergic and serotonergic neurons following AF64A administration are not due to non-specific toxicity of AF64A, but may be the result of adaptation of these neurons to withdrawal of cholinergic input, which would normally inhibit the release of NA and 5-HT. These results also indicate that AF64A can be used to produce specific lesions of hippocampal cholinergic nerve terminals.     

Presynaptic modulation of acetylcholine,noradrenaline, and serotonin release in the hippocampus of aged rats with various levels of memory impairments

Aged (25-27 months) Long-Evans female rats were distinguished according to whether they showed no significant impairment (AU), moderate impairment (AMI), or severe impairment (ASI) in a spatial reference-memory task. Young (3-5 months) rats served as controls. Electrically evoked overflow of tritium was assessed in hippocampal slices preloaded with [3H]choline or [3H]serotonin (5-HT). Nicotine-evoked overflow of tritium was measured after preloading with [3H]noradrenaline (NA). Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity, and concentration of monoamines were assessed in homogenates. Aged rats exhibited reduced accumulation of [3H]choline and [3H]5-HT, increased accumulation of [3H]NA, and weaker electrically evoked overflow of [3H]acetylcholine ([3H]ACh) and [3H]5-HT. The overflow of [3H]NA was not altered consistently by aging. Roughly, drugs acting presynaptically had comparable effects in aged rats: oxotremorine and CP 93,129 inhibited the overflow of [3H]ACh, CP 93,129 and UK 14,304 reduced that of [3H]5-HT. ChAT or AChE activity, and 5-HT concentration were not changed by age; NA concentration was reduced. When significant, changes were comparable in AU, AMI, and ASI rats. Data show that aging alters cholinergic and serotonergic hippocampal innervations, release of ACh and 5-HT, but not presynaptic release-modulating mechanisms. These alterations do not account for variability in water-maze performance of aged rats.     

MKC-231, a choline uptake enhancer: (3) mode of action of MKC-231 in the enhancement of high-affinity choline uptake

MKC-231, a putative cholinergic activity, is reported to improve learning and memory impaired in AF64A-treated animals. MKC-231 enhances high-affinity choline uptake (HACU) known as the rate-limiting step of acetylcholine (ACh) synthesis. We investigated the mode of action (MOA) of HACU enhancement by MKC-231. Intracerebroventricular (i.c.v.) injections of AF64A (3 nmol/brain) resulted in significant HACU reduction in hippocampal synaptosomes. Treatment with MKC-231 increased Vmax of HACU and Bmax of [3H]-HC-3 binding 1.6 and 1.7-fold, respectively. In studies of [3H]-MKC-231 binding and Biacore analysis, MKC-231 showed noticeable affinity for cloned high-affinity choline transporters (CHT1). The present study suggests that MKC-231 directly affects trafficking of CHT1 and increases the numbers of transporter, working for HACU, at the synaptic membrane.     

In vivo and in vitro studies on the regulation of cholinergic neurotransmission in striatum, hippocampus and cortex of aged rats

Young (3 months) and senescent (23 months) rats were challenged with oxotremorine both in vivo, to determine its effects on acetylcholine content in hemispheric regions, and in vitro, to assess its action on K⁺-evoked release of ACh from brain synaptosomes. The drug failed to inhibit KCl-induced [³H]ACh release from the P₂ fraction of striatal and hippocampal homogenates of the senescent animals, whereas it was less efficient in increasing striatal ACh content. In contrast, oxotremorine was still able to stimulate an increase in ACh in the hippocampus and cerebral cortex of the aged rats to the same extent as it did in the young ones. The [³H]ACh output from striatal synaptosomes was lower in old rats with respect to young ones at low KCl depolarizing concentrations but was equal in the two groups at a high depolarizing concentration. In the hippocampus of the senescent rats, the release was significanly lower at each concentration of KCl used, resulting in a parallel downward-shift in the concentration-release plot. We also measured cholinergic muscarinic receptor binding in rat hemispheric regions using the radioligand [³H]dexetemide, a classical non-selective muscarinic receptor antagonist. It was found, in conformity with some of the literature, that receptor binding was decreased by about 32% in striatum of aged female rats as compared to younger rats. Changes were not observed in cortex and hippocampus. Analysis of the binding data indicated that the observed decrease in specific ligand binding was due to a decrease in the number of binding sites without a change in affinity. The results favor, once again, the cholinergic hypothesis for geriatric dysfunction. The possibility that the functional state of the cholinergic neuron depends also on a balance between the cholinergic system and other neurotransmitters known to regulate cholinergic activity is discussed.     

Effects of AF64A on [3H]acetylcholine synthesis in neuron-enriched primary brain cell cultures

[3H]Acetylcholine (ACh) synthesis was measured in primary neuronal cultures from neonatal rat brains. Neuronal [3H]ACh synthesis was blocked by hemicholinium-3 and depended on the age of the cultures, increasing for ca. 10 days, and eventually declining. The irreversible inhibitor AF64A (10 or 30 microM) inhibited [3H]ACh synthesis from [3H]choline at concentrations (10 or 30 microM) with affecting choline acetyltransferase activity. Nine-day-old cultures recovered 90% of their [3H]ACh synthesis within 7 days after AF64A, while 13-day-old cultures never recovered. These results suggest that the turnover of neuronal choline transporters is age-related.     

Compensatory elevation of acetylcholine synthesis in vivo by cholinergic neurons surviving partial lesions of the septohippocampal pathway

The present study characterized the effects of partial destruction of the cholinergic septohippocampal pathway on transmitter functions of surviving cholinergic neurons in the hippocampus. Partial and full fimbrial transections were performed, and 3 weeks after lesioning, cholinergic functions were assessed in vivo and in vitro. Hippocampal ChAT activity and the capacity of hippocampal slices to synthesize [3H]ACh in vitro decreased by 35% and 45%, respectively, following partial fimbrial lesions and by 68% and 85%, respectively, following full fimbrial lesions. [3H]ACh release from hippocampal slices in vitro was decreased by 57% and 87%, respectively, following partial and full fimbrial lesions. Partial lesions decreased high-affinity choline uptake into hippocampal synaptosomes by 52%. In contrast to the significant reductions in cholinergic parameters measured in vitro after partial fimbrial lesions, such partial lesions did not significantly alter in vivo measures of hippocampal cholinergic function. Levels of endogenous ACh and choline measured in the hippocampus following partial lesions were similar to that of control values. Also, the hippocampal content of newly synthesized [2H4]ACh and the [2H4]ACh synthesis rate were not significantly different from control values. However, following full fimbrial lesions, in vivo measures of hippocampal cholinergic function were decreased to a degree similar to that observed in vitro. Hippocampal levels of endogenous ACh and [2H4]ACh and the synthesis rate for [2H4]ACh were decreased by 73%, 72%, and 83%, respectively. These results suggest that, following partial destruction of afferent cholinergic fibers that innervate the hippocampal formation, residual cholinergic neurons are able to upregulate their capacity to synthesize and store ACh in vivo, thus compensating for lesion-induced losses of cholinergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Muscimol increases acetylcholine release by directly stimulating adult striatal cholinergic interneurons

Because GabaA ligands increase acetylcholine (ACh) release from adult striatal slices, we hypothesized that activation of GabaA receptors on striatal cholinergic interneurons directly stimulates ACh secretion. Fractional [³H]ACh release was recorded during perifusion of acutely dissociated, [³H]choline-labeled, adult male rat striata. The GabaA agonist, muscimol, immediately stimulated release maximally 300% with EC₅₀=1 μM. This action was enhanced by the allosteric GabaA receptor modulators, diazepam and secobarbital, and inhibited by the GabaA antagonist, bicuculline, by ligands for D2 or muscarinic cholinergic receptors or by low calcium buffer, tetrodotoxin or vesamicol. Membrane depolarization inversely regulated muscimol-stimulated secretion. Release of endogenous and newly synthesized ACh was stimulated in parallel by muscimol without changing choline release. Muscimol pretreatment inhibited release evoked by K⁺ depolarization or by receptor-mediated stimulation with glutamate. Thus, GabaA receptors on adult striatal cholinergic interneurons directly stimulate voltage- and calcium-dependent exocytosis of ACh stored in vesamicol-sensitive synaptic vesicles. The action depends on the state of membrane polarization and apparently depolarizes the membrane in turn. This functional assay demonstrates that excitatory GabaA actions are not limited to neonatal tissues. GabaA-stimulated ACh release may be prevented in situ by normal tonic dopaminergic and muscarinic input to cholinergic neurons.     

Differential changes in rat cholinergic parameters subsequent to immunotoxic lesion of the basal forebrain nuclei

The degree of lesion produced by 192 IgG-saporin relative to controls was compared using three independent methods. Microdialyzed acetylcholine (ACh), choline acetyltransferase (ChAT) activity, and the rate of ACh synthesis were compared in the frontal cortex and hippocampus. Microdialysis of rats was performed 1 and 15 weeks post-lesion. In week 16, the rats were sacrificed after an injection of deuterated choline (Ch) for determination of the rate of ACh synthesis. ChAT activity was determined at the same timepoints in a separate set of rats. At 1 week, ChAT activity and microdialyzed ACh showed similar degrees of depletion. At 15 weeks, microdialyzed ACh was significantly lower than the synthesis rate in cortex, but not in hippocampus. A small increase in ChAT activity between 1 and 15 weeks was found in the cortex, but not hippocampus. In the hippocampus, however, the rate of ACh synthesis was significantly greater than ChAT activity. This was true for two doses of immunotoxin; the greater compensation occurring with the lesser lesion. Microdialyzed ACh levels were not different from the other measures in hippocampus. Residual cholinergic terminals in the hippocampus, but not frontal cortex, compensate for a selective cholinergic lesion by increasing the rate of synthesis and may thereby alleviate hippocampus-dependent behavioral deficits.     

Ultrastructural and neurochemical effects of the presumed cholinergic toxin AF64A in the rat interpeduncular nucleus

We have studied the effect of the presumptive cholinergic neurotoxin, ethylcholine mustard aziridinium ion (compound AF64A), on ultrastructure and neurochemical markers in the rat interpeduncular nucleus (IPN). Stereotaxic injections of 1 nmol of AF64A resulted in extensive degeneration of synaptic terminals within 40 h. Ultrastructural damage to neuronal cell bodies, dendrites and axons was also sometimes observed at this stage. Five days after the injection, more severe degenerative changes were observed in a larger number of neuronal cell bodies, axons and dendrites. High affinity uptake of [³H]choline, but not [³H]GABA, was significantly decreased 24 h after toxin injection. Five days after the injection, not only choline acetyltransferase but also glutamate decar☐ylase levels were significantly decreased. Our results suggest that, in addition to presynaptic cholinergic neurotoxicity, AF64A also leads to degenerative alterations of non-cholinergic neurons. Our electron microscopic observations constitute the first ultrastructural report on neuropathological damage caused by AF64A.     

Veratridine-induced breakdown of cytosolic acetylcholine in rat hippocampal minces: An intraterminal form of acetylcholinesterase or choline O-acetyltransferase?

Rat hippocampal minces were loaded with N-methyl-[3H]acetylcholine ([3H]ACh) in the presence of the 'poorly penetrating' acetylcholinesterase (EC 3.1.1.7, AChE) inhibitor echothiophate and the effect of the depolarizing agent veratridine determined on the subcellular storage and release of [3H]ACh and [3H]choline. Results indicated that veratridine stimulated the release of [3H]ACh from a crude vesicular fraction (P3) by a Ca2+-dependent process, while simultaneously accelerating the breakdown of cytosolic (S3) [3H]ACh. A portion of the [3H]choline derived from the hydrolyzed S3 [3H]ACh was donated to the P3 fraction for [3H]ACh formation and release. When the identical experiment was done using hippocampal minces from septal lesioned rats, veratridine did not stimulate either the Ca2+-dependent release of [3H]ACh or the hydrolysis of cytosolic [3H]ACh. Incubation of control hippocampal minces with paraoxon, an AChE inhibitor which can penetrate cholinergic nerve terminals more rapidly than echothiophate, prevented veratridine from stimulating the Ca2+-dependent release of [3H]ACh from the P3 fraction. Instead, it then stimulated the Ca2+-independent release of [3H]ACh from the S3 fraction. When minces were incubated with the choline O-acetyltransferase (EC 2.3.1.6, ChAT) inhibitor 4-(1-naphthyl)vinyl pyridine (NVP), veratridine was no longer able to stimulate the Ca2+-dependent release of labelled ACh either. Instead, veratridine stimulated the Ca2+-independent release of labelled ACh from the S3 fraction. NVP also abolished the veratridine-induced, Ca2+-dependent release of total ACh. Both paraoxon and NVP inhibited the reversible reaction of ionically bound ChAT prepared from rat brain when tested in vitro, yet paraoxon was much less potent than NVP, and was unable to inhibit this reaction at the low concentration which prevented the veratridine induced breakdown of S3 [3H]ACh during mince incubation. Veratridine depolarization of hippocampal minces stimulated the activity of a membrane-bound fraction of ChAT associated with the P3 fraction, but this fraction of ChAT did not become more sensitive to inhibition by paraoxon during tissue incubation. Veratridine depolarization of minces also increased the activity of membrane-bound AChE, but this enzyme was not inhibited by the low NVP concentration which prevented the veratridine-induced breakdown of S3 [3H]ACh. The veratridine-induced increase in membrane-bound ChAT activity was dependent on the presence of extracellular Ca2+ in the incubation medium.(ABSTRACT TRUNCATED AT 400 WORDS)