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.     

Selective modulation of cholinergic properties in cultures of avian embryonic sympathetic ganglia

We have studied the expression of catecholaminergic and cholinergic phenotypes in sympathetic ganglia removed from 7- to 10-day-old quail embryos and grown in vitro under different conditions. Quantitative data were obtained by measuring the conversion of (³H) tyrosine and (³H) choline to catecholamines (CA) and acetylcholine (ACh), respectively. In explant cultures, large amounts of both neurotransmitters were synthesized from the onset, but CA generally predominated, the molar ratios of CA:ACh being, on average, of the order of 2:1. If the ganglia were dissociated before plating, there was a selective increase in ACh synthesis (three- to fivefold) such that the CA:ACh ratio fell strikingly. The early expression of the cholinergic phenotype appears to be species-specific in that, under identical conditions, dissociated cell cultures of newborn mouse superior cervical ganglia were overwhelmingly catecholaminergic (CA:ACh ratio of approximately 40:1) and ACh synthesis was only just detectable. Addition of veratridine (1.5 μM) either to explant or to dissociated cell cultures of embryonic quail sympathetic ganglia barely altered CA-synthesizing ability; in contrast, ACh synthesis and accumulation were stimulated about threefold. This effect, which we found to correspond to a quantitatively similar increase in the activity of choline acetyltransferase (ChAT), was completely blocked by tetrodotoxin, indicating that it was due to Na⁺-dependent depolarization. A preferential stimulation of ACh production was also observed when the concentration of K⁺ was raised to 20 mM. Veratridine treatment of cultures of presumptive sympathoblasts, in the form of sclerotome-associated neural crest cells, had identical effects. Our results reveal the quantitative importance of ACh-related properties in avian sympathetic ganglia from the earliest stages of their development and suggest that depolarization may be one of the factors selectively enhancing expression of the cholinergic phenotype during ontogeny. In these respects, the neurochemical differentiation of sympathetic neurons unfolds according to dissimilar scenarios in birds and mammals. © 1993 Wiley-Liss, Inc.     

Evidence to suggest that extracellular acetate is accumulated by rat hippocampal cholinergic nerve terminals for acetylcholine formation and release

It is well established that extracellular choline is transported into central cholinergic nerve terminals by `high' and `low' affinity processes to form the neurotransmitter acetylcholine (ACh). The intent of the present investigation was to ascertain whether extracellular acetate might also be transported into central cholinergic nerve terminals to form ACh. To test this possibility, rat hippocampal tissue was incubated with varying concentrations of extracellular [1-¹⁴C]acetate (0.1–100 μM) and the uptake of [1-¹⁴C]acetate and the amount of [¹⁴C]ACh formed by the tissue determined. The results indicated that the uptake of extracellular [1-¹⁴C]acetate was temperature-dependent and saturable having an apparent Michaelis constant (K_m) of 22 μM. The formation of [¹⁴C]ACh in the tissue as a function of extracellular [1-¹⁴C]acetate appeared to occur by both `high' and `low' affinity processes with apparent K_m values of 0.5 and 19.6 μM, respectively. In other experiments, three inhibitors (lithium, allicin and sodium) of acetyl CoA synthetase (EC 6.2.1.1 acetate: CoA ligase), the enzyme which converts acetate to acetyl CoA when ATP and CoA are present, inhibited [1-¹⁴C]acetate uptake and the amount of [¹⁴C]ACh formed from that [1-¹⁴C]acetate. Additionally, vesamicol, an inhibitor of ACh transport into synaptic vesicles, blocked the filling of a synaptic vesicle-enriched fraction of hippocampal tissue with newly synthesized [¹⁴C]ACh formed from extracellular [1-¹⁴C]acetate. High K⁺ depolarization of hippocampal tissue loaded with extracellular [1-¹⁴C]acetate not only increased the synthesis but also the release of [¹⁴C]ACh. These results suggest that extracellular acetate is recycled by rat hippocampal cholinergic nerve terminals for the formation and release of ACh. They also suggest that the enzyme acetyl CoA synthetase mediates extracellular acetate uptake into hippocampal cholinergic nerve terminals by metabolizing it to acetyl CoA and thereby creating a diffusion gradient for it to follow. © 1997 Elsevier Science B.V. All rights reserved.     

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.     

Acetylcholine synthesis and release in NIH3T3 cells coexpressing the high‐affinity choline transporter and choline acetyltransferase

Acetylcholine (ACh) is known to be a key neurotransmitter in the central and peripheral nervous systems, but it is also produced in a variety of non‐neuronal tissues and cells, including lymphocytes, placenta, amniotic membrane, vascular endothelial cells, keratinocytes, and epithelial cells in the digestive and respiratory tracts. To investigate contribution made by the high‐affinity choline transporter (CHT1) to ACh synthesis in both cholinergic neurons and nonneuronal cells, we transfected rat CHT1 cDNA into NIH3T3ChAT cells, a mouse fibroblast line expressing mouse choline acetyltransferase (ChAT), to establish the NIH3T3ChAT 112‐1 cell line, which stably expresses both CHT1 and ChAT. NIH3T3ChAT 112‐1 cells showed increased binding of the CHT1 inhibitor [³H]hemicholinium‐3 (HC‐3) and greater [³H]choline uptake and ACh synthesis than NIH3T3ChAT 103‐1 cells, a CHT1‐negative control cell line. HC‐3 significantly inhibited ACh synthesis in NIH3T3ChAT 112‐1 cells but did not affect synthesis in NIH3T3ChAT 103‐1 cells. ACh synthesis in NIH3T3ChAT 112‐1 cells was also reduced by amiloride, an inhibitor of organic cation transporters (OCTs) involved in low‐affinity choline uptake, and by procaine and lidocaine, two local anesthetics that inhibit plasma membrane phospholipid metabolism. These results suggest that CHT1 plays a key role in ACh synthesis in NIH3T3ChAT 112‐1 cells and that choline taken up by OCTs or derived from the plasma membrane is also utilized for ACh synthesis in both cholinergic neurons and nonneuronal cholinergic cells, such as lymphocytes. © 2009 Wiley‐Liss, Inc.     

Adenylate cyclase in striatal cholinergic interneurons regulates acetylcholine release

Fractional [³H]ACH efflux from dissociated rat striata tested whether tonic inhibition prevents stimulation of acetylcholine (ACH) release by adenylate cyclase. Forskolin stimulated release from the dissociated cells (threshold at 300 nM; EC₅₀ ≥ 1 μM). Release was also stimulated by 3-isobutyl-l-methylxanthine and was additive with forskolin. The 1,9-dideoxy forskolin analog that lacks cyclase-stimulating activity was ineffective. Thus, stimulation of adenylate cyclase within striatal cholinergic interneurons increases ACH secretion but is tonically inhibited by endogenous striatal transmitters. Disinhibition of the excitatory cyclase by denervation of striatal cholinergic interneurons in situ could contribute to supersensitivity without receptor upregulation.     

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.     

Differential long-term effect of AF64A on [H]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 μl/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]ACh 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.     

Small pial vessels, but not choroid plexus, exhibit specific biochemical correlates of functional cholinergic innervation

In an attempt to provide the biochemical foundations for a putative cholinergic innervation of small pial vessels and choroid plexus, we have assessed their ability to specifically accumulate choline, synthesize and release acetylcholine (ACh) in response to depolarization. Our results show that both small pial vessels and choroid plexus avidly accumulate choline via a sodium-dependent mechanism which could be inhibited by hemicholinium-3 (IC50 in pial vessels = 47.8 microM). Light microscopic examination of radioautographs from vessels incubated with [3H]choline revealed two distinct sites of accumulation in the vessel wall. One site probably corresponded to nerve terminals and the other was closely associated with the endothelial cells. In small pial vessels, a major proportion (60%-70%) of the choline acetyltransferase (ChAT) activity could be inhibited by 4-naphthylvinylpyridine (4-NVP), a potent inhibitor of neuronal ChAT; and, following either K+ or veratridine depolarization, a Ca2(+)-dependent release of authentic [3H]ACh could be measured. In contrast, the choroid plexus exhibited a rather low ChAT activity which was not inhibited by 4-NVP and no release of ACh could be detected in this tissue following depolarization. Altogether, the results of the present study show that (1) small pial vessels exhibit all the most selective biochemical markers that are characteristic of cholinergic nerves; (2) [3H]choline in pial vessels can be accumulated in non-neuronal elements which probably correspond to the endothelial cells; and (3) the choroid plexus failed to exhibit convincing biochemical markers that would attest in favor of a functional cholinergic innervation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Acetylcholine Metabolism in the Inflamed Rat Intestine

Acetylcholine (ACh) is a major neurotransmitter in the enteric nervous system. Since increasing evidence suggests that inflammation alters neural regulation of intestinal function, we examined the synthesis and breakdown of ACh in smooth muscle/myenteric plexus (SM/MP) preparations from the jejunum of the rat during inflammation caused by infection with the nematode parasiteTrichinella spiralis. Both total and neuron-specific uptake of the ACh precursor [³H]choline into SM/MP preparations was increased by over twofold on Day 6 postinfection. Further, a radiochemical assay of choline acetyltransferase activity showed significant increase by Day 1, with peak values reached by Day 3 and maintained without reversal thereafter. Despite the enhancement of these steps, measurement of the conversion of [³H]choline into [³H]ACh in SM/MP preparationsin vitroshowed a nearly fourfold decrease by Day 6, implying a large decrease in ACh production in the inflamed jejunum. Examination of acetylcholinesterase in the rat jejunum showed decreased histochemical staining intensity in the muscle wall, and quantitative evaluation showed significantly decreased (>50%) acetylcholinesterase activity in SM/MP preparations. These results show that cholinergic innervation of the intestine can undergo rapid and long-lasting alterations during inflammation. Upregulation of major steps in the synthetic pathway for ACh was not matched by increased ACh production, suggesting that defects in ACh packaging, storage, and granule exocytosis may also be present.     

Differential acetylcholine and GABA release from cultured chick retina cells

In the present work we investigated the mechanisms controlling the release of acetylcholine (ACh) and of γ-aminobutyric acid (GABA) from cultures of amacrine-like neurons, containing a subpopulation of cells which are simultaneously GABAergic and cholinergic. We found that 81.2 ± 2.8% of the cells present in the culture were stained immunocytochemically with an antibody against choline acetyltransferase, and 38.5 ± 4.8% of the cells were stained with an antibody against GABA. Most of the cells containing GABA (87.0 ± 2.9%) were cholinergic. The release of acetylcholine and GABA was mostly Ca²⁺-dependent, although a significant release of [³H]GABA occurred by reversal of its transporter. Potassium evoked the Ca²⁺-dependent release of [³H]GABA and [³H]acetylcholine, with EC₅₀ of 31.0 ± 1.0 mm and 21.6 ± 1.1 mm , respectively. The Ca²⁺-dependent release of [³H]acetylcholine was significantly inhibited by 1 μm tetrodotoxin and by low (30 nm ) ω-conotoxin GVIA (ω-CgTx GVIA) concentrations, or by high (300 nm ) nitrendipine (Nit) concentrations. On the contrary, the release of [¹⁴C]GABA was reduced by 30 nm nitrendipine, or by 500 nm ω-CgTx GVIA, but not by this toxin at 30 nm . The release of either transmitters was unaffected by 200 nm ω-Agatoxin IVA (ω-Aga IVA), a toxin that blocks P/Q-type voltage-sensitive Ca²⁺ channels (VSCC). The results show that Ca²⁺-influx through ω-CgTx GVIA-sensitive N-type VSCC and through Nit-sensitive L-type VSCC induce the release of ACh and GABA. However, the significant differences observed regarding the Ca²⁺ channels involved in the release of each neurotransmitter suggest that in amacrine-like neurons containing simultaneously GABA and acetylcholine the two neurotransmitters may be released in distinct regions of the cells, endowed with different populations of VSCC.     

Effects of cholinergic enhancing drugs on cholinergic transporters in the brain and peripheral blood lymphocytes of spontaneously hypertensive rats

Cholinergic hypofunction is a trait of Alzheimer's disease and vascular dementia and countering it is one of the main therapeutic strategies available for these disorders. Cholinergic transporters control cellular mechanisms of acetylcholine (ACh) synthesis and release at presynaptic terminals. This study has assessed the influence of 4 week treatment with two different cholinergic enhancing drugs, the cholinergic precursor choline alphoscerate (alpha-glyceryl-phosphorylcholine) or the acetylcholinesterase (AChE) inhibitor galantamine on high affinity choline uptake transporter (CHT) and vesicular ACh transporter (VAChT) expression in the brain of spontaneously hypertensive rats (SHR). SHR represent an animal model of cerebrovascular injury characterized by cholinergic hypofunction. Analysis was performed by immunochemistry, ELISA and immunohistochemistry on frontal cortex, striatum and hippocampus. Immunochemical and ELISA analysis was extended to peripheral blood lymphocytes (PBL), used as a peripheral reference of changes of brain cholinergic markers. An increased expression of VAChT and CHT was observed in brain areas investigated and in PBL of SHR. The similar trend for cholinergic transporters observed in brain and PBL suggests these cells may represent a marker of brain cholinergic transporters. Treatment with choline alphoscerate increased CHT and to a greater extent VAChT expression. Treatment with galantamine countered the increase of CHT and VAChT. The different activity of the cholinergic precursor and of the AChE inhibitor on parameters investigated is likely related to their mechanism of action. Choline alphoscerate increases ACh synthesis and release. This requires an augmentation of systems regulating neurotransmitter uptake and storage. The effect of choline alphoscerate on CHT and VAChT observed in this study suggests an improved synaptic efficiency elicited by the compound. The AChE inhibitor slows-down ACh degradation in the synaptic cleft. A greater availability of neurotransmitter elicited by galantamine counters the enhanced activity of cholinergic transporters compensating cholinergic deficits. Differences in the activity of the cholinergic precursor and AChE inhibitor investigated on CHT and VAChT suggests that association between choline alphoscerate and AChE/cholinesterase inhibitors may represent a strategy for potentiating deficient cholinergic neurotransmission worthwhile of being investigated in clinical trials.     

Neurochemical aspects of the ontogenesis of cholinergic neurons in the rat brain

Ontogenic development of central cholinergic neurons in rat brain was examined by measuring the activity of choline acetyltransferase (CAT), concentration of acetylcholine (ACh) after focused microwave irradiation, the activity of the high affinity uptake process for choline and the apparent muscarinic receptor as quantified by specific binding of [³H]3-quinuclidinyl benzilate (QNB). For whole brain, the specific activity of CAT increases from 1 to 8% of adult between 15 days gestation and 7 days postpartum and then increases linearly to 83% by 4 weeks postpartum. The concentration of ACh is 22% of adult at 15 days gestation, rises to 29% by birth and attains adult levels by 4 weeks postpartum. The developmental rise in specific binding of [³H]QNB is intermediate between CAT and ACh with 10% of adult concentration of receptor at birth and a linear increase to 90% by 4 weeks postpartum. The development of the uptake of [³H]choline parallels that of CAT. In all regions of the neonatal rat brain, the relative level (% adult) of ACh is higher than [³H]QNB binding, which is higher than CAT. The neonatal medulla-pons has higher levels of [³H]QNB binding and activity of CAT (% adult) and develops more rapidly than the parietal cortex and corpus striatum; the hypothalamus and midbrain-thalamus exhibit intermediate rates of development.     

Cholinergic direct inhibition of N‐methyl‐D aspartate receptor‐mediated currents in the rat neocortex

Acetylcholine (ACh) and N‐methyl‐D aspartate receptors (NMDARs) interact in the regulation of multiple important brain functions. NMDAR activation is indirectly modulated by ACh through the activation of muscarinic or nicotinic receptors. Scant information is available on whether ACh directly interacts with the NMDAR. By using a cortical brain slice preparation we found that the application of ACh and of other drugs acting on muscarinic or nicotinic receptors induces an acute and reversible reduction of NMDAR‐mediated currents (I_NMDA), ranging from 20 to 90% of the control amplitude. The reduction displayed similar features in synaptic I_NMDA in brain slices, as well as in currents evoked by NMDA application in brain slices or from acutely dissociated cortical cells, demonstrating its postsynaptic nature. The cholinergic inhibition of I_NMDA displayed an onset–offset rate in the order of a second, and was resistant to the presence of the muscarinic antagonist atropine (10 μM) in the extracellular solution, and of G‐protein blocker GDP_βS (500 μM) and activator GTP_γS (400 μM) in the intracellular solution, indicating that it was not G‐protein dependent. Recording at depolarized or hyperpolarized holding voltages reduced NMDAR‐mediated currents to similar extents, suggesting that the inhibition was voltage‐independent, whereas the reduction was markedly more pronounced in the presence of glycine (20 μM). A detailed analysis of the effects of tubocurarine suggested that at least this drug interfered with glycine‐dependent NMDAR‐activity. We conclude that NMDAR‐mediated current scan be inhibited directly by cholinergic drugs, possibly by direct interaction within one or more subunits of the NMDAR. Our results could supply a new interpretation to previous studies on the role of ACh at the glutamatergic synapse. Synapse 63:308–318, 2009. © 2009 Wiley‐Liss, Inc.     

Cholinergic differentiation triggered by blocking cell proliferation and treatment with all-trans-retinoic acid

This study determined whether the effect of all-trans-retinoic acid (t-RA) on markers of cholinergic differentiation in a murine septal cell line, SN56.B5.G4, differed depending upon the cell’s proliferative status. To develop a model of non-proliferating cells, aphidicolin, a DNA α-polymerase inhibitor, was used. Cessation of proliferation by aphidicolin increased intracellular choline and acetylcholine (ACh) levels in the absence of change to choline acetyltransferase (ChAT) activity and mRNA and vesicular ACh transporter (VAChT) mRNA. Importantly, the response to t-RA differed depending upon proliferative status. Consistent with previous reports, t-RA increased ChAT and VAChT mRNA, ChAT activity and intracellular ACh levels in proliferating SN56 cells with no effect on intracellular choline levels. When cells were treated with t-RA while undergoing proliferative arrest, an additive effect of combined treatment was observed on ACh levels; nevertheless, this was only accompanied by an increase in choline levels, VAChT and ChAT mRNAs, but not ChAT activity. Indeed, aphidicolin treatment completely suppressed the t-RA-induced increase in ChAT activity observed in proliferating cells. To explore the response to t-RA in post-mitotic cells, a sequential treatment of aphidicolin and t-RA was employed. t-RA treatment was ineffective in increasing ACh and choline levels, over and above that observed with the aphidicolin treatment alone. Comparable to the combined treatment, sequential treatment lead to an increase in ChAT mRNA without any increase in ChAT activity. In conclusion, both the magnitude and the mechanism(s) of action whereby t-RA enhances the cholinergic phenotype of SN56 cells is dependent upon the cell’s proliferative status.     

Effects of choline and glucose on atropine-induced alterations of acetylcholine synthesis and content in the caudate nuclei of rats

Atropine-induced decrease in the concentration of acetylcholine (ACh) in the brain can be diminished by pretreating the animals with a large dose of choline^72,73. The injected choline might act by improving the supply of substrate for the synthesis of ACh, or by competing with atropine for presynaptic muscarinic receptors and thus decreasing the release of ACh. Since, under in vitro conditions, changes in the release of ACh are reflected by changes in its synthesis, experiments have been performed on slices of rat caudate nuclei to check whether an increase in the extracellular concentration of choline causes a decrease in the atropine-induced stimulation of ACh synthesis. The rate of [¹⁴C]ACh synthesis from [¹⁴C]glucose was faster in the presence of 530 μM than 30 μM choline, and 5 μM atropine stimulated the synthesis of [¹⁴C]ACh more at 530 μM than at 30 μM choline in the medium. These observations substantiate the view that the administration of choline in vivo acts by supporting the synthesis of ACh, rather than by inhibiting its release.

In subsequent experiments on rats in vivo, an attempt has been made to influence the ACh-depleting action of atropine by pretreatment with a large dose of glucose as a distant precursor of acetyl-CoA, the second substrate for the synthesis of ACh. The administration of atropine (25 mg/kg) diminished the content of ACh in the caudate nuclei by 48%; if the injection of atropine followed after an injection of glucose (20.2 mmol/kg), the content of ACh was diminished by only 25%. It appears likely that glucose acted by improving the availability of acetyl-CoA for the synthesis of ACh and that the supply of either precursor of ACh (i.e. choline and acetyl-CoA) may become rate-limiting when the demands on the synthesis of ACh in the brain are increased.     

Modulation of striatal acetylcholine concentrations by NMDA and the competitive NMDA receptor-antagonist AP-5: an in vivo microdialysis study

Summary. The effects of local perfusion with the competitive NMDA receptor antagonist 2-amino-5-phosphonovalerate (AP-5) and the glutamate receptor agonist N-methyl-D-aspartate (NMDA) on release of extracellular acetylcholine (ACh) and choline (Ch) in the dorsolateral striatum were studied using in vivo microdialysis in freely moving rats. AP-5 caused a dose-dependent decrease in ACh release that was counteracted by the addition of NMDA. Perfusion with AP-5 also decreased Ch levels. Local perfusion with NMDA induced an elevation of ACh release in low (10⁻⁵ M), but not high (10⁻² M and 10⁻³ M) concentrations, that were associated with massive cellular death. These inhibitory effects of AP-5 and the stimulatory effect of NMDA in non-neurotoxic dosages on ACh release provide further evidence for a tonic stimulation of striatal cholinergic interneurons by glutamatergic neurons via NMDA receptors. Received May 21, 1998; accepted July 8, 1998     

Veratridine-induced release of acetylcholine from mouse forebrain minces: Dependence on the hydrolysis of cytoplasmic acetylcholine for a source of choline

The importance of depolarization induced hydrolysis of cytoplasmic acetylcholine (ACh) in providing choline for the veratridine- and high K⁺-induced release of acetylcholine was studied in mouse forebrain minces. Results indicated that a loss of hydrolyzable cytoplasmic ACh prior to depolarization reduced the amount of ACh released by veratridine but not the amount released by high K⁺. The reduction in the veratridine-induced release of ACh did not occur during the first 5 min of incubation. Loss of vesicular ACh prior to depolarization reduced both the veratridine- and K⁺-induced release of ACh during the first 5 min of incubation. Blockade of extra-cellular choline transport by hemicholinium (HC-3) did not affect the veratridine-induced release of ACh during a 10 min incubation period unless the cytoplasmic pool of ACh had first been depleted and was unavailable as a source of choline. In contrast, HC-3 reduced the K⁺-induced release of ACh from brain tissue with normal stores of cytoplasmic ACh. These results indicate that both depolarizing agents primarily stimulate the release of preformed ACh from a vesicular fraction during the first 5 min of mince incubation. Thereafter, they both stimulate the release of newly synthesized ACh, however, they differ in one important respect. The principal source of choline for the veratridine-induced release of newly synthesized ACh appears to be the cytoplasmic pool of ACh, whereas the major source of choline for the K⁺-induced release of newly synthesized ACh appears to be extracellular choline.     

Presynaptic muscarinic modulation of nicotinic excitation in the rat neostriatum

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