Exogenous Choline Enhances the Synthesis of Acetylcholine Only Under Conditions of Increased Cholinergic Neuronal Activity

Abstract: The effect of choline (60 mg/kg, i.p.) on fluphenazine- and pentylenetetrazol-induced alterations in the concentration of acetylcholine (ACh) and/or the rate of sodium-dependent high-affinity choline uptake (HACU) in rat striatum and hippocampus was studied. Systemic administration of the dopamine receptor blocking agent fluphenazine hydrochloride (0.5 mg/kg, i.p.) decreased the concentration of ACh in the striatum; this effect was prevented by the prior administration of choline. The central nervous system stimulant pentylenetetrazol (30 mg/kg, i.p.) reduced the concentration of ACh in both striatum and hippocampus and increased the velocity of HACU in the hippocampus. Pretreatment with choline totally prevented the depletion of ACh induced by pentylenetetrazol in the striatum. In the hippocampus, prior administration of choline prevented the pentylenetetrazol-induced increase in the rate of HACU and attenuated the effect of pentylenetetrazol on the levels of ACh. Results indicate that the acute administration of choline antagonizes pharmacologically induced alterations in cholinergic activity as assessed by the rate of HACU and the steady-state concentration of ACh. Furthermore, data support the hypothesis that the administration of choline increases the ability of central cholinergic neurons to synthesize ACh under conditions of increased neuronal activity.     

Determination of acetylcholine by on-line microdialysis coupled with pre- and post-microbore column enzyme reactors with electrochemical detection

A sensitive procedure consisting of a pre- and post-microbore column reactor sequence of a LC-electrochemical detection system coupled with on-line microdialysis system is described in the present study to measure endogenous acetylcholine concentration in freely moving rats. The pre-column packed, with immobilized choline oxidase and catalase, was used to remove choline, whereas the post-column, packed with immobilized acetylcholine oxidase and choline oxidase, was used to measure acetylcholine selectively. The detection limit of acetylcholine was found to be 5 fmol/μl (50 fmol/10 μl). The usefulness of the described methodology was evaluated by examining the change in the striatal acetylcholine concentration of freely moving rats after physostigmine (0.5 mg/kg, s.c.) administration.     

Relations Between the Extracellular Concentrations of Choline and Acetylcholine in Rat Striatum

Abstract: Changes in extracellular levels of acetylcholine (ACh) and choline (Ch) in the striatum of rats were examined by in vivo microdialysis after intraperitoneal injections of drugs. A dopamine D₂ antagonist, sulpiride (20 mg/kg), and a muscarinic antagonist, atropine (3.5 mg/kg), increased ACh levels and decreased Ch levels. On the contrary, the D₂ agonist (±)-2-( N -phenylethyl- N -propyl)amino-5-hydroxytetralin (N-434; 5 mg/kg) and an anesthetic, pentobarbital (50 mg/kg), decreased ACh levels and increased Ch levels. Perfusion of 10 µ M hemicholinium-3 (HC-3), a Ch uptake inhibitor, through the striatum induced a complete inhibition of ACh release and increased Ch levels in all drug-treated groups. The degree of relative increase in the level of Ch induced by HC-3 differed among the drug-pretreated groups; compared with the control group, the relative increase was larger in the sulpiride- and atropine-treated groups and smaller in the N-434 and pentobarbital-treated groups. Thus, we demonstrated reciprocal relations between extracellular concentrations of Ch and ACh after treatments by drugs. The data suggest that in the striatum, which is rich in cholinergic innervation, the extracellular Ch concentration is to a large extent determined by activity of the cholinergic transmission reflected in high-affinity choline uptake.     

Choline Administration Elevates Brain Phosphorylcholine Concentrations

Abstract: The phosphorylcholine concentration of rat brain rises and falls in response to parallel changes in the concentration of circulating choline. A single oral dose of choline chloride (20 mmol/kg) elevated whole-brain concentrations of both choline and phosphorylcholine 5 h after administration; a greater proportion of exogenously administered choline was retained by the brain in its phosphorylated form than as the free arnine. Striatal phosphorylcholine concentrations were elevated within 2 h of choline administration and continued to be significantly greater than control values for up to 34 h after treatment. The response of striatal choline levels to exogenous choline was of shorter duration than that of phosphorylcholine and was correlated with a significant increase in striatal acetylcholine concentrations. The consumption of a choline-free diet for 7 days lowered both serum choline and striatal phosphorylcholine concentrations, but had no effect on striatal choline or acetylcholine. These results suggest that choline kinase is unsaturated by its substrate in vivo and may thus serve to modulate the response of brain choline concentrations to alterations in the supply of circulating choline.     

Effects of nootropic drugs on brain cholinergic and dopaminergic transmission

High affinity choline uptake (HACU) in the hippocampus and striatal concentration of dopamine (DA) and homovanillic acid (HVA) as measures of the in vivo acetylcholine and DA turnover, respectively, were estimated in male rats, Long-Evans, following 6-day administration of various nootropics in clinically relevant doses: piracetam and its derivatives pramiracetam and oxiracetam (100 mg/kg/day), pyritinol (50 mg/kg/day). Piracetam treatment was without effect on HACU, but induced significant increase of HVA in the striatum leaving striatal DA concentration unchanged. On the contrary, pyritinol, pramiracetam and oxiracetam increased HACU, but did not change striatal DA and HVA levels.     

Effects of streptozotocin-induced diabetes on acetylcholine metabolism in rat brain

The main objective of this study was to determine whether uncontrolled hyperglycemia, as a consequence of diabetes, altered the metabolism of acetylcholine (ACh) in rat brain. To accomplish this, rats received injections of streptozotocin (STZ, 60 mg/kg, i.v.) or vehicle, and were maintained for up to 7 weeks after the injections. Various indices of ACh metabolism were determined in striatum and hippocampus, two brain regions densely innervated by cholinergic neurons. STZ induced diabetes in 96% of the rats injected, as evidenced by glucose spillage into the urine within 48 hours. Serum glucose levels increased to 326% of control values by 1 week and remained at this level for the duration of the study. The steady-state concentrations of ACh and choline, determined in brain tissue from animals killed by head-focused microwave irradiation, did not differ between the control and STZ-injected groups. However, the synthesis and release of neurotransmitter by striatal slices, measured in vitro, decreased in a time-dependent manner. Although the basal release of ACh was unaltered at 1 week, neurotransmitter release decreased significantly by 21% at 5 weeks and by 26% at 7 weeks. The release of ACh evoked by incubation with 35 mM KCl was inhibited significantly by 20% at all time points studied. ACh synthesis by slices incubated under basal conditions decreased by 13% and 27% at 5- and 7-weeks, respectively, the latter significantly less than controls. Synthesis by striatal slices incubated with 35 mM KCl was inhibited by 17% at 7 weeks. Although the synthesis and release of ACh by hippocampal slices from diabetic animals tended to be less than controls, these alterations were not statistically significant. Investigations into the mechanism(s) mediating the deficit in ACh synthesis exhibited by striatal slices indicated that it did not involve alterations in precursor choline availability, nor could it be attributed to alterations in the activities of the synthetic or hydrolytic enzymes choline acetyltransferase or acetylcholinesterase; rather, the decreased turnover of ACh may be secondary to other STZ-induced, hyperglycemia-mediated neurochemical alterations.     

The Role of Chloride in Acetylcholine Metabolism

Abstract: The chloride dependence of acetylcholine (ACh) synthesis and release and of choline uptake was studied in synaptosomal preparations from rat brain. The substitution of propionate for chloride, in the presence of 35 m m -potassium, lowered the ACh content of the synaptosomes. However, in the presence of 5 m m -potassium, the ACh level in synaptosomes was reduced, but significantly less so. Propionate had no effect on choline acetyltransferase (EC 2.3.1.6) activity when measured in a standard chloride-containing medium. In the presence of propionate, the spontaneous release of ACh was unchanged, but potassium-stimulated release of ACh was markedly reduced as compared with a chloride-containing medium. The synthesis of ACh, as measured by the net increase in the amount of ACh in the synaptosomes and that released to the medium, was reduced with propionate at 5 m m -potassium and was totally inhibited when the potassium concentration was increased to 35 m m . Choline uptake studies revealed that with propionate only a low-affinity component of the choline transport system existed. Further, the V _max was markedly reduced when the potassium concentration was increased to 35 m m . The results suggest that under certain conditions choline transported by a low-affinity system might provide a substantial source of choline for ACh synthesis.     

Turnover Rate of Brain Acetylcholine Using HPLC Separation of the Transmitter

A simple, reliable method was developed for measuring brain acetylcholine (ACh) turnover using HPLC methodology. Mice were injected intravenously with [3H]choline ([3H]Ch), and the turnover rate of ACh was calculated from the formation of [3H]ACh. Ch and ACh were separated from phosphorylcholine and from other radioactive compounds using tetraphenylboron extraction and counterion/reverse-phase chromatography. Endogenous Ch and ACh were quantified electrochemically through hydrogen peroxide production in a postcolumn reactor containing covalently bonded ACh esterase and Ch oxidase. Labeled Ch and ACh were quantified in the same sample by collecting the chromatographic fractions for radioactive content determinations. The method is rapid, well adapted to large series, and highly reproducible, with recoveries of 72.1% for Ch and 79.3% for ACh. The turnover value in mouse cerebral hemispheres was 16.02 nmol g-1 min-1 and decreased to 9.94 nmol g-1 min-1 in mice treated with oxotremorine.     

In Vivo Production and Release of Acetylcholine from Primary Fibroblasts Genetically Modified to Express Choline Acetyltransferase

Abstract: Primary rat fibroblasts genetically modified to express Drosophila choline acetyltransferase (dChAT) synthesize and release acetylcholine (ACh) in vitro. The ACh produced from the transduced fibroblasts was found to be enhanced by increasing amounts of choline chloride in the culture media. These dChAT-expressing cells were then implanted into the intact hippocampus of adult rats and in vivo microdialysis was performed 7–10 days after grafting to assess the ability of the cells to produce ACh and respond to exogenous choline in vivo. Samples collected from anesthetized rats revealed fourfold higher levels of ACh around dChAT grafts than from either non-grafted or control-grafted hippocampi. Localized choline infusion (200 μ) through the dialysis probes was found to induce a selective twofold increase in ACh release only from the dChAT-expressing fibroblasts. These results indicate not only that dChAT-expressing fibroblasts continue to synthesize and secrete ACh for at least 10 days after intracerebral grafting, but that the levels of ACh can be manipulated in vivo. The ability to regulate products within genetically modified cells in vivo may provide a powerful avenue for exploring the role of discrete substances within the CNS.     

Effect of oxotremorine,physostigmine, and scopolamine on brain acetylcholine synthesis: A study using HPLC

The synthesis rate of brain acetylcholine (ACh) was estimated in mice following i.v. administration of [³H]choline (Ch). The measurements were performed 1 min after the tracer injection, using the [³H]ACh/[³H]Ch specific radioactivity ratio as an index of ACh synthesis rate. Endogenous and labeled Ch and ACh were quantified using HPLC methodology. Oxotremorine and physostigmine (0.5 mg/kg, i.p.) increased the steady state concentration of brain ACh by +130% and 84%, respectively and of Ch by +60% (oxotremorine); they decreased ACh synthesis by 62 and 55%, respectively. By contrast, scopolamine (0.7 mg/kg, i.p.) decreased the cerebral content of Ch by –26% and of ACh by –23% without enhancing the synthesis of ACh. The results show the utility of HPLC methodology in the investigation of ACh turnover.     

Choline High-Affinity Uptake and Metabolism and Choline Acetyltransferase Activity in the Striatum of Rats Chronically Treated with Neuroleptics

High-affinity uptake of choline and choline acetyltransferase activity (ChAT) were measured in the striatum of rats treated for 45-60 days with haloperidol (1 mg/kg per os) and pimozide (1 mg/kg per os) daily and with fluspirilene (1 mg/kg i.m.) twice a week. Haloperidol and fluspirilene caused a 20%, and pimozide a 38%, increase in high-affinity uptake of choline. They also caused a significant decrease in ChAT activity: haloperidol, 20%; pimozide, 27%; and fluspirilene, 42%. In rats treated with fluspirilene for 65-80 days the metabolism of [3H] choline taken up by striatal synaptosomes was investigated. A 33% increase in total radioactivity, a significant increase in labelled acetylcholine (ACh), a relative decrease in labelled choline, and no change in labelled phosphorylcholine and betaine were found. It is concluded that the increase in high-affinity choline uptake caused by chronic administration of neuroleptic drugs is associated with a parallel increase in choline utilization for ACh formation. On the other hand, the decrease in ChAT activity does not appear to influence ACh formation.     

EFFECT OF OXOTREMORINE ON THE APPARENT REGIONAL TURNOVER OF ACETYLCHOLINE IN MOUSE BRAIN

The effect of oxotremorine (1 mg kg^-1 i.p.) on the steady state concentration of acetylcholine (ACh) and choline (Ch) and the transformation of radioactive choline ([³H]Ch) was studied in different brain regions of the mouse following death by microwave irradiation of the head. Oxotremorine significantly increased the concentration of endogenous ACh in the cortex and hippocampus and of endogenous Ch in the cortex. Pretreatment with atropine (5 mg kg^-1 i.p.) prevented the increase in ACh. The biosynthesis of radioactive ACh ([³H]ACh) was decreased in all brain regions. Atropine (5 mg kg^-1) pretreatment counteracted this effect of oxotremorine (1 mg kg^-1), while methylatropine (5 mg kg^-1) had no effect except in the striatum. A calculation of the apparent turnover rate of ACh showed that oxotremorine (1 mg kg^-1) decreased the turnover in the cortex, hippocampus, midbrain. and striatum.     

Acetylcholine Synthesis and Release in the Extensor Digitorum Longus Muscle of Mature and Aged Rats

Uptake of labeled choline and its incorporation into acetylcholine (ACh) were assayed at the neuromuscular junction of the extensor digitorum longus (EDL) muscle of rats aged 11 (mature adult) and 27 (aged) months. Under resting conditions, there were no significant differences in muscle ACh or choline levels. Following a 1-h incubation in labeled choline, however, tissue from the younger rats contained significantly greater amounts of labeled choline and labeled ACh; the specific activities of ACh and choline were nearly 10-fold higher in the 11-month-old animals, indicating reduced uptake of labeled choline in the older animals. ACh and choline efflux rates under resting conditions did not change with age, indicating an uncoupling of exogenous choline uptake and ACh efflux in EDL during aging. During nerve stimulation (1 Hz), the amount of labeled choline incorporated into ACh was 150% greater in the aged animals. The specific activity of ACh released during stimulation was correspondingly greater in the 27-month-old animals, although total ACh released did not change appreciably with age. There were no age-related differences in choline acetyltransferase activity. Contrasting results were obtained from diaphragm in previous studies; the linkage between choline uptake and ACh efflux was maintained during rest and stimulation in the diaphragm. Hypothetically, these differences between EDL and diaphragm may be related to their diverse activation patterns: EDL is recruited much less frequently and less regularly than diaphragm, a continually active vital muscle.     

Dopaminergic Regulation of Septohippocampal Cholinergic Neurons

Abstract: The extent to which acetylcholine (ACh) release in the hippocampus is regulated by dopaminergic mechanisms was assessed using in vivo microdialysis in freely moving rats. Systemic administration of the dopamine (DA) receptor agonist apomorphine (1.0 mg/kg) or the specific D₁ agonist CY 208–243 (1.0 mg/kg) increased microdialysate concentrations of ACh in the hippocampus. The D₂ receptor agonist quinpirole (0.5 mg/kg) produced a small but statistically significant decrease in hippocampal ACh release. d -Amphetamine (2.0 mg/kg) increased ACh release, an effect that was blocked by the D₁ receptor antagonist SCH 23390 (0.3 mg/kg) but not by the D₂ antagonist raclopride (1.0 mg/kg). These findings suggest that endogenous DA stimulates septo-hippocampal cholinergic neurons primarily via actions at D₁ receptors. In addition, these results are similar to previous findings regarding the dopaminergic regulation of cortical ACh release, and suggest that the anatomical continuum formed by basal forebrain cholinergic neurons that project to the cortex and hippocampus acts as a functional unit, at least with respect to its regulation by DA.     

Nicotine Exposure Does not Alter Plasma to Brain Choline Transfer

Acute and chronic nicotine exposure in rats is associated with an increase in brain acetylcholine (ACh) transmission. The acquisition of choline for neuronal ACh synthesis occurs primarily via two pathways; first, free choline is transported from the blood across the blood-brain barrier (BBB) and/or second, from synaptic choline generated by either hydrolysis of non-bound ACh or membrane phosphatidylcholine catabolism. To determine if nicotine-induced cholinergic demand is associated with increased choline transport rates into brain, we measured BBB choline transport in naïve and S-(−) nicotine exposed rats (acute and chronic, 4.5 mg/kg/d for 1, 14, 21 and 28 d; osmotic minipumps) using the in situ rat brain perfusion technique. No significant changes in choline uptake after acute or chronic nicotine exposure were observed in whole brain or cortex. Of considerable interest was a significant decrease in regional brain choline uptake measured in the hippocampus after chronic nicotine exposure (28 d). Our data suggest that the increased ACh transmission observed after nicotine exposure does not correlate with increased blood-to-brain transfer of choline. Considering these data and previous literature reports, we propose that the additional free choline required under conditions of nicotine exposure (for ACh synthesis) is primarily recruited from membrane phospholipid metabolism.     

4-(1-Naphthylvinyl)pyridine Decreases Brain Acetylcholine In Vivo, but Does Not Alter the Level of Acetyl-CoA

The effects of intraperitoneally administered 4-(1-naphthylvinyl)pyridine (NVP; 200 mg/kg) on the concentrations of acetylcholine (ACh), choline (Ch), and acetyl-CoA (AcCoA) in rat striatum, cortex, hippocampus, and cerebellum were investigated. Twenty minutes after treatment, the content of ACh was significantly diminished, whereas that of Ch was increased. In response to stress (swimming for 20 min), these changes were enhanced. However, the AcCoA content did not change in any of the brain regions. It is thus very likely that the decrease of brain ACh concentration induced by NVP is due to the drug's effect on choline acetyltransferase (ChAT) and/or the reduction of the high-affinity Ch uptake, and not on the availability of AcCoA. Presumably, the pharmacologically diminished activity of ChAT may become the rate-limiting factor in the maintenance of ACh levels in cholinergic neurons.     

High-Affinity Choline Transport Regulation by Drug Administration During Postnatal Development

High-affinity choline transport sites specifically bind [3H]hemicholinium-3. Hemicholinium-3 binding sites are regulated by in vivo drug treatments in the same manner as these drugs alter acetylcholine release and high-affinity choline transport. The current study examines regulation of binding sites by in vivo drug administration for adult, day 15, and day 5 rats. Drugs or saline were administered intraperitoneally, and striatal and cortical membrane preparations were assayed. Control [3H]hemicholinium-3 binding increases twofold between postnatal days 5 and 15 only in striatum. After day 15, binding increases 2.7-fold in cortex and striatum. Nicotine treatment increases striatal and cortical hemicholinium-3 binding at all three ages, with greater percent increases at day 5. Haloperidol increases binding only in striatum, again with larger effects at day 5. Both striatal and cortical binding are reduced by oxotremorine; however, the magnitude of this effect is unchanged during development. Pentobarbital reduces binding only in striatum, with no developmental change. Atropine and apomorphine do not change binding from control values. In summary, all drug treatments effective in adults were already effective by day 5. Cholinergic terminals present early in development are regulated by similar nicotinic and muscarinic cholinergic, dopaminergic, and sedative-hypnotic mechanisms as the adult. Changes in magnitude may be due to changes in drug metabolism or to developmental differences in regulation.     

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)     

Examination of the Role of Central Cholinergic Mechanisms in the Therapeutic Effects of HI-6 in Organophosphate Poisoning

Abstract: In atropine-pretreated rats, HI-6 (125 mg/kg i.p.) raised the LD₅₀ of Soman (subcutaneous) 5.7 times. Addition of HI-6 (25 μg i.c. v.) failed to enhance this protection further. HI-6 (intraperitoneal) also protected animals from intracerebroventricular Soman. HI-6, administered intracerebroventricularly either alone or in combination with intraperitoneal HI-6, failed to increase protection, nor did it reactivate Soman-inhibited acetylcholinesterase (AChE) in several brain areas. HI-6 (125 or 62.5 mg/kg i.p.) protected rats from Sarin lethality, but only the higher dose significantly altered the brain AChE activity. Furthermore, HI-6 (intraperitoneal) failed to block the Soman-induced increase in acetylcholine (ACh) or choline (Ch) levels in any of the brain areas examined. These data indicate that HI-6 is a very beneficial therapy against Soman, but that no definitive central anticholinergic activity of the compound could be found to explain its protective effects. It is possible that HI-6 acts by noncholinergic central mechanisms, or that it produces its beneficial effects outside the CNS. Furthermore, brain AChE activity does not appear to be indicative of protective effects of this oxime. ACh or Ch levels in this study were not good parameters to predict the outcome of Soman poisoning.     

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.