Potassium-stimulated Acetylcholine Release Research Articles

Chronic treatment with a GPR30 antagonist impairs acquisition of a spatial learning task in young female rats

We hypothesize that the beneficial effects of estradiol on cognitive performance may be mediated through GPR30, a putative membrane target of estrogens. Recently we showed that administration of a selective GPR30 agonist (G-1) to ovariectomized rats enhanced acquisition of a delayed matching-to-position (DMP) T-maze task and increased potassium-stimulated acetylcholine release in the hippocampus, similar to estradiol (E2) (Hammond et al., 2009). The present study tested whether treating with a selective GPR30 antagonist (G-15) would impair spatial learning in gonadally intact rats and in ovariectomized (OVX) rats treated with E2. As predicted, G-15 dose-dependently impaired DMP acquisition both in gonadally intact rats and in OVX rats treated with E2. G-15 specifically reduced the rate of acquisition, and this effect was associated with an increased predisposition to adopt a persistent turn. In contrast, G-15 alone at the highest dose had no significant effect on DMP acquisition in OVX controls. The effects were task dependent, as similar effects of G-15 were not observed in gonadally intact rats tested on an operant discrimination/reversal learning task motivated by the same food reward. This suggests that the effects on DMP acquisition were not due to effects on motivation for food. Effects of G-15 on DMP acquisition were similar to previously published work showing significant impairment produced by selective cholinergic denervation of the hippocampus. These data suggest that GPR30 can play an important role in mediating the effects of estradiol on spatial learning, possibly by mediating estradiol effects on basal forebrain cholinergic function.

New Considerations on the Neuromodulatory Role of Thiamine

Background: A nonmetabolic role for thiamine in cholinergic neurotransmission has long been suggested. The mechanism remains unclear. We sought to extend our previous research to elucidate the effect of the thiamine metabolic antagonist, oxythiamine, on the release of acetylcholine from the brain. Methods: The potassium-stimulated release of acetylcholine from superfused rat brain slices was determined. Hand-cut slices of cerebral cortex were preincubated with tritiated choline to label acetylcholine stores. Two periods of stimulation (S1, S2) with 50 mmol/l solution for 3.5 min were performed as superfusate was collected. During S1, only 50 mmol/l potassium-containing Krebs-bicarbonate buffer with 2 mmol/l calcium was used. Using a two-by-two design, S2 consisted of exposure to 50 mmol/l potassium with or without 10^–4 mol/l oxythiamine, with or without calcium. The S2/S1 ratio was calculated. Results: Oxythiamine enhanced the potassium-evoked release of acetylcholine by 60% but only when calcium was present in the superfusing medium. Conclusion: These data confirm earlier findings with oxythiamine on the calcium-mediated synaptic transmission of acetylcholine and support a possible neuromodulatory role for thiamine distinct from its actions as a cofactor during metabolic processes.

GPR30 co-localizes with cholinergic neurons in the basal forebrain and enhances potassium-stimulated acetylcholine release in the hippocampus

GPR30 is a novel, membrane-bound, G-protein coupled estrogen receptor (Filardo et al., 2002; Prossnitz et al., 2008). We hypothesize that GPR30 may mediate effects of estradiol (E2) on basal forebrain cholinergic neurons and cognitive performance. Recently we showed that G-1, a selective GPR30 agonist, enhances the rate of acquisition on a delayed matching-to-position (DMP) T-maze task (Hammond et al., 2009). In the present study, we examined the distribution of GPR30 in the rat forebrain, and the effects of G-1 on potassium-stimulated acetylcholine release in the hippocampus. GPR30-like immunoreactivity was detected in many regions of the forebrain including the hippocampus, frontal cortex, medial septum/diagonal band of Broca, nucleus basalis magnocellularis and striatum. GPR30 mRNA also was detected, with higher levels in the hippocampus and cortex than in the septum and striatum. Co-localization studies revealed that the majority (63-99%) of cholinergic neurons in the forebrain expressed GPR30-like immunoreactivity. A far lower percentage (0.4-42%) of GABAergic (parvalbumin-containing) cells also contained GPR30. Sustained administration of either G-1 or E2 (5 μg/day) to ovariectomized rats produced a nearly 3-fold increase in potassium-stimulated acetylcholine release in the hippocampus relative to vehicle-treated controls. These data demonstrate that GPR30 is expressed by cholinergic neurons in the basal forebrain, and suggest that activation of GPR30 enhances cholinergic function in the hippocampus similar to E2. This may account for the effects of G-1 on DMP acquisition previously reported.

Effects of raloxifene and estradiol on hippocampal acetylcholine release and spatial learning in the rat

The effects of raloxifene on acquisition of a delayed matching to position (DMP) T-maze task and on hippocampal acetylcholine release were evaluated and compared with estradiol, to determine whether raloxifene has estrogenic effects on cognitive performance and hippocampal cholinergic activity. Ovariectomized rats received continuous treatment with raloxifene (one of two doses), estradiol, or vehicle for 30 days, followed by behavioral training, and then in vivo microdialysis assessment of basal and potassium-stimulated acetylcholine release. The data show that estradiol significantly enhanced DMP acquisition, whereas raloxifene did not. In contrast, both estradiol and the higher dose of raloxifene significantly increased potassium-stimulated acetylcholine release in the hippocampus. These data suggest that, despite increasing evidence for estrogenic effects of raloxifene in brain, raloxifene does not mimic the effects of estrogen on cognitive performance as assessed by acquisition of a simple spatial memory task in ovariectomized rats.

Estrogen enhances potassium-stimulated acetylcholine release in the rat hippocampus

Short-term estrogen replacement has been shown to enhance basal forebrain cholinergic function. Whether or not long-term estrogen replacement can enhance basal forebrain cholinergic function has been questioned in light of recent studies showing that several cholinergic measures which are increased following short-term treatment are not increased following longer-term (>30 days) treatment. In the present study, in vivo microdialysis was used to assess the effects of continuous estradiol replacement on basal forebrain cholinergic function. Our data show that 6–7 weeks of continuous estradiol replacement significantly enhanced potassium-stimulated acetylcholine release in the hippocampus of ovariectomized rats, and that this effect was reversed following discontinuation of the estrogen treatment. These data are consistent with the hypothesis that estrogen-mediated effects on cholinergic neurons can contribute to the effects of long-term estrogen replacement on cognitive performance recently described.

Oestrogen and the cholinergic hypothesis: implications for oestrogen replacement therapy in postmenopausal women.

Cholinergic deficits in the basal forebrain, hippocampus and cortex are thought to contribute to the risk and severity of cognitive decline associated with ageing and Alzheimer's disease. Work in our laboratory has demonstrated that in rats, basal forebrain cholinergic neurons are affected by physiological fluctuations in circulating oestrogen and progesterone, and that long-term loss of ovarian function produces decreases in cholinergic parameters and nerve growth factor receptor (trkA) mRNA beyond the effects of normal ageing. Conversely, short-term treatment with oestrogen or oestrogen plus progesterone produces increases in cholinergic parameters and trkA, as well as increases in potassium-stimulated acetylcholine release, that are consistent with an increase in basal forebrain cholinergic function. These findings are consistent with recent studies showing the ability of oestrogen and progesterone replacement to enhance spatial memory and reduce performance deficits associated with hippocampal cholinergic impairment. We hypothesize that similar effects of the ovarian hormones on basal forebrain cholinergic neurons in humans may contribute to the effects of hormone replacement on cognitive processes that have recently been described, and to the ability of oestrogen replacement to reduce the risk and severity of Alzheimer's-related dementia in postmenopausal women.

Effects of estrogen on potassium-stimulated acetylcholine release in the hippocampus and overlying cortex of adult rats

In vivo microdialysis techniques were used to examine the effects of estrogen on potassium-stimulated acetylcholine release in the hippocampus and overlying cortex of adult, ovariectomized rats. Estrogen treatment resulted in a significant increase in the percent change in acetylcholine release induced by potassium relative to controls, particularly after prolonged (90 min) exposure to high potassium. The data suggest that estrogen may help to maintain cholinergic function under conditions where cholinergic afferents to the hippocampal formation and cortex are challenged or impaired.

Inhibition of potassium-stimulated acetylcholine release from rat brain cortical slices by two high-affinity analogs of vesamicol

In this work, we investigated the effects of two structural analogs of the drug vesamicol, which inhibits the vesicular acetylcholine (ACh) transport, on the potassium-stimulated release of ACh from rat brain cortical slices. These vesamicol analogs, 4-aminobenzove-samicol (ABV) and ( trans)-cyclohexovesamicol (transDec), were almost as potent as vesamicol in inhibiting the evoked release of ACh from cortex slices. Similar to vesamicol, the presence of these analogues inhibited the ability of ACh newly-synthesized from [ 3H]choline to become releasable. However, vesamicol's action was reversible, while ABV and transDec caused a persistent block of this [ 3H]ACh release. In addition, vesamicol did not affect the release of pre-stored [ 3H]ACh, but ABV and transDec partially inhibited the release of [ 3H]ACh, in this condition, suggesting that the two latter drugs may alter some of the steps posterior to the entry of [ 3H]ACh, into synaptic vesicles. The rank order of potency for these drugs to reduce ACh release (vesamicol = transDec > ABV) is close to the rank order for inhibition of ACh vesicular transport (transDec > vesamicol > ABV), but is completely different from the order of affinities of these drugs for the vesamicol receptor (ABV > transDec > > vesamicol). These results suggest that although these two vesamicol analogs are able to block ACh release due to their effects on the vesicular transport system, they may have other unexpected actions not shared by vesamicol.

Inhibition by halothane of potassium-stimulated acetylcholine release from rat cortical slices.

1. Cholinergic neurones in the basal forebrain are linked to cortical activation and arousal. 2. The present study was designed to examine the hypothesis that clinically relevant doses of halothane (0.1 to 5%) would significantly reduce depolarization-evoked acetylcholine (ACh) release from rat cortical slices. 3. ACh release was measured from rat cortical slices by a chemiluminescent technique. 4. Depolarization-evoked ACh release was inhibited significantly by halothane with an IC50 of 0.38%. This value equates to 0.3 MAC (the minimum alveolar concentration at which no movement occurs to a standard surgical stimulus in 50% of subjects) for the rat. 5. The potent effect of halothane on ACh release suggests that this mechanism may be a target for the action of volatile anaesthetic agents. This in vitro effect on ACh release is consistent with effects of halothane reported in vivo.

Acetylcholine release from striatal slices of young adult and aged Fischer 344 rats

The release of endogenous and newly synthesized acetylcholine (ACh) was examined in neostriatal slices prepared from young adult (10-month) and aged (28-month) Fischer 344 rats. Both spontaneous and potassium-stimulated release were tested after various in vitro incubation times (1, 3 or 5 hr). The potassium-stimulated release of ACh from slices of 28-month rats was decreased by 53% when tested after incubating the slices 1 hr. The age-related differences in ACh release lessened if the slices were incubated for longer times (3 or 5 hr) before monitoring release. The spontaneous release of ACh was similar among the slices from both age-groups and at all times points monitored. When the neostriatal slices were incubated in medium supplemented with deuterated choline, the release of both the endogenous and newly synthesized ACh from slices of 28-month rats was decreased by 33% when tested after a 1-hr incubation, but was again similar to that released from slices of 10-month rats when tested after a 3-hr incubation. Choline release from slices of the 28-month rats was similar to that released from the slices of the 10-month rats when acetylcholinesterase (AChE) was inhibited during the release incubation. In slices with intact AChE activity, however, the age-related difference in choline release was similar to that observed for ACh release when AChE was inhibited. That is, when AChE activity was intact, the potassium-stimulated choline release from slices of 28-month rats was less than that released from slices of 10-month rats when release was tested after a 1-hr incubation. This difference in choline release was not apparent when release was tested after longer incubation times. The results show that in vitro age-related differences in neostriatal ACh release are apparent in early periods of incubation and reverse with longer incubation times.

Effects of lactic acidosis on the function of cerebral cortical synaptosomes.

Synaptosomes exposed to anoxic insult produce lactate at a slow rate (measured over 60 min). No measurable damaging effects were produced by prolonged depolarisation, anoxic insult, or exogenous lactate (2-32 mM) either on the synaptic plasma membrane (as judged by release of lactate dehydrogenase and soluble proteins), or on synaptosomal phospholipases (as judged by choline release from membrane phospholipids). Potassium-stimulated acetylcholine release was decreased by incubation in the presence of lactate (2-32 mM), as was potassium- and veratrine-stimulated calcium uptake and the calcium content of depolarised synaptosomes. The intrasynaptosomal pH was also reduced but there was no stimulation of oxygen radical production (as judged by H2O2 generation) by exogenous lactate. The role that lactic acidosis may play in giving rise to the altered calcium homeostasis and decreased acetylcholine release from synaptosomes exposed to anoxic insult is discussed.

Differential alteration of dopamine, acetylcholine, and glutamate release during anoxia and/or 3,4-diaminopyridine treatment.

The potassium-stimulated release of acetylcholine (ACh), glutamate (GLU) and dopamine (DA) from mouse striatal slices was studied during anoxia and/or 3,4-diaminopyridine (DAP) treatment. Anoxia, in the presence of calcium, increased DA and GLU release, but depressed ACh release. Omission of calcium from an anoxic incubation further stimulated GLU and DA release and impaired ACh release. Under normoxic conditions, DAP (100 microM) increased the release of all three neurotransmitters; the sensitivity of the slices to DAP changed with the presence or absence of an acetylcholinesterase inhibitor in the preincubation media. During an anoxic incubation, DAP did not ameliorate the anoxic-induced, K+-stimulated impairment of ACh release, but significantly reduced the K+-stimulated release of GLU and DA. These results are consistent with the hypothesis that hypoxia induces a presynaptic deficit that may underlie postsynaptic ischemic-induced changes. Amelioration of these presynaptic alterations in neurotransmitter release may be an effective approach to preventing hypoxic-induced damage.

Intracerebroventricular administration of ethylcholine mustard aziridinium ion (AF64A) reduces release of acetylcholine from rat hippocampal slices

Ethylcholine mustard aziridinium ion (AF64A), or vehicle, was infused bilaterally (3 nmol/3 μl per side) into the lateral ventricles of rats. The effect of such treatment on various cholinergic responses was measured in the hippocampus, cortex and striatum. Potassium-stimulated release of acetylcholine from superfused slices of hippocampus was reduced, 7 and 21 days after treatment with AF64A, to 24 and 35% of control, respectively. The activity of choline acetyltransferase in the hippocampus also decreased, to 42% of control, both 7 and 21 days after treatment with AF64A. Similarly, the activity of acetylcholinesterase and the high affinity transport of choline in the hippocampus were reduced, to 40 and 30% of control; and to 33 and 48% of control, respectively, 7 and 21 days after treatment with AF64A. Synthesis of acetylcholine in slices of hippocampus was also decreased after treatment with AF64A (71 and 51% of control, 7 and 21 days post-AF64A respectively). Only the binding of [ 3h]qnb in the hippocampus was unchanged at 7 days after treatment with AF64A, although a small reduction (11%) was noted 21 days after treatment with AF64A. The activity of choline acetyltransferase and acetylcholinesterase, the high-affinity transport of choline and the binding of fhiqnb in cortex and striatum were unaffected by treatment with AF64A under the same experimental conditions. Using a substantially smaler dose than that earlier reported in mice, the earlier finding was thus confirmed, and extended, in rats, of a highly selective effect of AF64A on several components of the cholinergic system. Under the conditions of this study, these effects appeared to be confined to the hippocampus.

Selective alteration of neurotransmitter release by low oxygen in vitro.

The potassium-stimulated release of acetylcholine, norepinephrine, serotonin, glutamate, and 4-aminobutyrate from superfused rat cortical slices was studied during hypoxia. A reduction in oxygen tensions from 603 +/- 6 to 22 +/- 2 mm Hg selectively altered the calcium-dependent efflux of these neurotransmitters, but did not change their calcium-independent release. The calcium-dependent release of [14C]acetylcholine decreased (39%), while that of glutamate increased (66%) and 4-aminobutyrate, [3H]norepinephrine, and [3H]serotonin were unaffected. Thus, low oxygen reveals variations in the calcium-dependent release mechanisms of several neurotransmitters. These differences may have important implications for pharmacological intervention of neurotransmitter release.

Temporal characteristics of potassium-stimulated acetylcholine release and inactivation of calcium influx in rat brain synaptosomes.

The time course of Ca2+-dependent [3H]acetylcholine [( 3H]ACh) release and inactivation of 45Ca2+ entry were examined in rat brain synaptosomes depolarized by 45 mM [K+]0. Under conditions where the intrasynaptosomal stores of releasable [3H]ACh were neither exhausted nor replenished in the course of stimulation, the K+-evoked release consisted of a major (40% of the releasable [3H]ACh pool), rapidly terminating phase (t1/2 = 17.8 s), and a subsequent, slow efflux that could be detected only during a prolonged, maintained depolarization. The time course of inactivation of K+-stimulated Ca2+ entry suggests the presence of fast-inactivating, slow-inactivating, and noninactivating, or very slowly inactivating, components. The fast-inactivating component of the K+-stimulated Ca2+ entry into synaptosomes appears to be responsible for the rapidly terminating phase of transmitter release during the first 60 s of K+ stimulus. The noninactivating Ca2+ entry may account for the slow phase of transmitter release. These results indicate that under conditions of maintained depolarization of synaptosomes by high [K+]0 the time course and the amount of transmitter released may be a function of the kinetics of inactivation of the voltage-dependent Ca channels.

The role of chloride in acetylcholine metabolism.

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 mM-potassium, lowered the ACh content of the synaptosomes. However, in the presence of 5 mM-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 mM-potassium and was totally inhibited when the potassium concentration was increased to 35 mM. Choline uptake studies revealed that with propionate only a low-affinity component of the choline transport system existed. Further, the Vmax was markedly reduced when the potassium concentration was increased to 35 mM. The results suggest that under certain conditions choline transported by a low-affinity system might provide a substantial source of choline for ACh synthesis.