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Vulnerability of hippocampal hemicholinium-3 (HC-3)-sensitive carriers to ethanol was evaluated in vitro during rat postnatal development. The high-affinity uptake of [3H]choline (HACU) and the specific binding of [3H]HC-3 were measured on synaptosomes from 7-, 14-, and 60-day-and 3-month-old male and female Wistar rats. Marked increases of basal (between 7 and 60 days of age) and of stimulated HACU levels via K(+)-depolarization (between 14 days and 3 months) but only a mild elevation in [3H]HC-3 binding (between 7 days and 3 months) associated with alterations in the binding site number were found. On the mature tissue, ethanol at high concentrations (5%) moderately inhibited the choline transport under basal conditions but totally eliminated depolarization effects. However, both age- and sex-dependent alterations in basal HACU mediated by high or low pharmacologically relevant alcohol concentrations (50-100 mM) were observed in the immature tissue. Namely, the dose- and incubation time-dependent inhibition of HACU associated with changes in the transport velocity was found in postnatal male but not female tissue. [3H]HC-3 binding site was not markedly sensitive to ethanol actions. Anisotropy measurements in the region of the hydrophilic heads of phospholipid bilayers and in the membrane hydrocarbon core indicated penetration of 100 mM ethanol to immature female but not male tissue. Our results suggest the noncompetitive binding of alcohol to choline carriers from immature male tissue and correspond with data reporting significant sexual dimorphism of postnatal hippocampal neurons. The direct effects of ethanol on male choline carriers can contribute to the inhibition of acetylcholine synthesis and to sex-dependent neurotoxic effects of alcohol applied in vivo during early and late postnatal period.

Evidence suggests that insulin-like growth factor-I (IGF-I) plays an important role during brain development and in the maintenance of normal as well as activity-dependent functioning of the adult brain. Apart from its trophic effects, IGF-I has also been implicated in the regulation of brain neurotransmitter release thus indicating a neuromodulatory role for this growth factor in the central nervous system. Using in vitro slice preparations, we have earlier reported that IGF-I potently inhibits K(+)-evoked endogenous acetylcholine (ACh) release from the adult rat hippocampus and cortex but not from the striatum. The effects of IGF-I on hippocampal ACh release was sensitive to the Na(+) channel blocker tetrodotoxin, suggesting that IGF-I might act indirectly via the release of other transmitters/modulators. In the present study, we have characterized the possible involvement of GABA in IGF-I-mediated inhibition of ACh release and measured the effects of this growth factor on choline acetyltransferase (ChAT) activity and high-affinity choline uptake in the hippocampus of the adult rat brain. Prototypical agonists of GABA(A) and GABA(B) receptors (i.e. 10 microM muscimol and 10 microM baclofen) inhibited, whereas the antagonists of the respective receptors (i.e. 10 microM bicuculline and 10 microM phaclofen) potentiated K(+)-evoked ACh release from rat hippocampal slices. IGF-I (10 nM) inhibited K(+)- as well as veratridine-evoked ACh release from rat hippocampal slices and the effect is possibly mediated via the activation of a typical IGF-I receptor and the subsequent phosphorylation of the insulin receptor substrate-1 (IRS-1). The inhibitory effects of IGF-I on hippocampal ACh release were not additive to those of either muscimol or baclofen, but were attenuated by GABA antagonists, bicuculline and phaclofen. Additionally, in contrast to ACh release, IGF-I did not alter either the activity of the enzyme ChAT or the uptake of choline in the hippocampus. These results, taken together, indicate that IGF-I, under acute conditions, can decrease hippocampal ACh release by acting on the typical IGF-I/IRS receptor complex while having no direct effect on ChAT activity or the uptake of choline. Furthermore, the evidence that effects of IGF-I could be modulated, at least in part, by GABA antagonists suggest that the release of GABA and the activation of its receptors may possibly be involved in mediating the inhibitory effects of IGF-I on hippocampal ACh release.

Central cholinergic neurotransmission was studied in learning-impaired transgenic mice expressing human acetylcholinesterase (hAChE-Tg). Total catalytic activity of AChE was approximately twofold higher in synaptosomes from hippocampus, striatum and cortex of hAChE-Tg mice as compared with controls (FVB/N mice). Extracellular acetylcholine (ACh) levels in the hippocampus, monitored by microdialysis in the absence or presence of 10(-8)-10(-3) M neostigmine in the perfusion fluid, were indistinguishable in freely moving control and hAChE-Tg mice. Muscarinic receptor functions were unchanged as indicated by similar effects of scopolamine on ACh release and of carbachol on inositol phosphate formation. However, when the mice were anaesthetized with halothane (0.8 vol. %), hippocampal ACh reached significantly lower levels in AChE-Tg mice as compared with controls. Also, the high-affinity choline uptake (HACU) in hippocampal synaptosomes from awake hAChE-Tg mice was accelerated but was reduced by halothane anaesthesia. Moreover, hAChE-Tg mice displayed increased motor activity in novel but not in familiar environment and presented reduced anxiety in the elevated plus-maze test. Systemic application of a low dose of physostigmine (100 microgram/kg i.p.) normalized all of the enhanced parameters in hAChE-Tg mice: spontaneous motor activity, hippocampal ACh efflux and hippocampal HACU, attributing these parameters to the hypocholinergic state due to excessive AChE activity. We conclude that, in hAChE-Tg mice, hippocampal ACh release is up-regulated in response to external stimuli thereby facilitating cholinergic neurotransmission. Such compensatory phenomena most likely play important roles in counteracting functional deficits in mammals with central cholinergic dysfunctions.

Three-month-old Long-Evans female rats sustained aspirative lesions of the dorsal septohippocampal pathways and, 2 weeks later, received intrahippocampal suspension grafts containing fetal cells from the mesencephalic raphe (rich in serotonergic neurons; RAPHE), the medial septum and the diagonal band of Broca (rich in cholinergic neurons; SEPT), or a mixture of both (COTR). Lesion-only (LES) and sham-operated rats (SHAM) were used as controls. Hippocampal slices of these rats (5-9 month after surgery) were preincubated with [3H]choline or [3H]5-HT, superfused continuously (in the presence of hemicholinium-3 or fluvoxamine) and stimulated electrically (360 pulses, 2 ms, 3 Hz, 26-28 mA) in order to study the presynaptic modulation of acetylcholine (ACh) and serotonin (5-HT) release. The accumulation of [3H]choline and the evoked overflow of [3H]ACh were significantly reduced in slices from LES and RAPHE rats, but reached a close-to-normal level in SEPT and COTR rats. As to accumulation and overflow of [3H]5-HT, the lesion-induced reduction was compensated for only in RAPHE and COTR rats. The relative amount of evoked [3H]5-HT release (in % of tissue-3H) was significantly increased in LES and SEPT rats. Only slight differences (group LES) were found in the sensitivity of muscarinic and serotonergic autoreceptors towards oxotremorine and CP 93,129, respectively. Moreover, CP 93,129 induced a significantly weaker inhibition of ACh release in slices of COTR rats than in all other groups. Using the 5-HT1A receptor agonist 8-OH-DPAT and antagonist Way 100,635, no evidence for a modulatory influence of 5-HT1A receptors was found in RAPHE and COTR rats. It is concluded that despite substantial lesion- and graft-induced changes in the amount of ACh and 5-HT released by hippocampal slices of lesion-only or grafted rats, the presynaptic modulation of these transmitters is only slightly affected by changes in the neuronal environment.

The mechanisms regulating the compartmentation of acetylcholine (ACh) and the relationship between transmitter release and ACh stores are not fully understood. In the present experiments, we investigated whether the inhibitors of serine/threonine phosphatases 1 and 2A, calyculin A and okadaic acid, alter subcellular distribution and the release of ACh in rat hippocampal slices. Calyculin A and okadaic acid significantly (p < 0.05) depleted the occluded ACh of the vesicular P3 fraction, but cytoplasmic ACh contained in the S3 fraction was not significantly affected. The P3 fraction is known to be heterogeneous; calyculin A and okadaic acid reduced significantly (p < 0.05) the amount of ACh recovered with a monodispersed fraction (D) of synaptic vesicles, but the other nerve terminal bound pools (E-F and G-H) were not so affected. K+-evoked ACh release decreased significantly (p < 0.01) in the presence of calyculin A and okadaic acid, suggesting that fraction D's vesicular store of ACh contributes to transmitter release. The loss of ACh from synaptic vesicle fractions prepared from tissue exposed to phosphatase inhibitors appeared not to result from a reduced ability to take up ACh. Thus, when tissue was allowed to synthesize [3H]ACh from [3H]choline, the ratio of [3H]ACh in the S3 to P3 fractions was not much changed by exposure of tissue to calyculin A or okadaic acid; furthermore, the specific activity of ACh recovered from the D fraction was not reduced disproportionately to that of cytosolic ACh. The changes are considered to reflect reduced synthesis of ACh by tissue treated with the phosphatase inhibitors, rather than an effect on vesicle uptake mechanisms. Thus, exposure of tissue to calyculin A or okadaic acid appears to produce selective depletion of tissue ACh content in a subpopulation of synaptic vesicles, suggesting that phosphatases play a role in ACh compartmentation.

We studied the postnatal development of the release of acetylcholine (ACh) and of presynaptic, release-inhibiting muscarinic autoreceptors in the rat hippocampus. To this end, hippocampal slices (350 microns thick) from rats of various postnatal ages (postnatal day 3 [P3] to P16) were preincubated with [3H]choline and stimulated twice (S1, S2: 360 pulses, 2 ms, 3 Hz, 60 mA) during superfusion with physiological buffer containing hemicholinium-3 (10 microM). In parallel, the activities of hemicholinium-sensitive high-affinity choline uptake (HACU, in synaptosomes) and of choline acetyltransferase (ChAT, in crude homogenates) were determined as markers for the cholinergic ingrowth. In hippocampal slices preincubated with [3H]choline, the electrically evoked overflow of 3H at S1 increased from 0.11 (P3) to 0.81% of tissue 3H (P16), the latter value being still much lower than that of hippocampal slices from adult rats (2.89% of tissue 3H). Already at P3 the evoked overflow of 3H was Ca(2+)-dependent and sensitive to tetrodotoxin, indicating an action potential-evoked exocytotic mechanism of ACh release. The muscarinic agonist oxotremorine (1 microM) significantly inhibited the evoked ACh release in hippocampal slices with increasing effectivity from P4 to P16; no significant effect was detectable at P3. The ACh esterase inhibitor physostigmine and the muscarinic antagonist atropine (1 microM, each) exhibited significant inhibitory and facilitatory effects, respectively, only at P15-16. The specific activities of both hippocampal HACU (pmoles/mg protein/min) and ChAT (nmoles/mg protein/min) continuously increased from P3 to P16. It is concluded (1) that cholinergic nerve terminals arriving at the hippocampal formation during postnatal ingrowth are already endowed with the apparatus for action potential-induced, Ca(2+)-sensitive (exocytotic) ACh release; (2) that, in contrast, the expression of presynaptic muscarinic autoreceptors on these cholinergic axon terminals is delayed; and (3) that autoinhibition due to endogenous ACh develops even later, probably when the density of presynaptic terminals in the hippocampus and hence, the concentration of released ACh has reached a suprathreshold value.

The characteristic pathological features of the postmortem brain of Alzheimer's disease (AD) patients include, among other features, the presence of neuritic plaques composed of amyloid beta-peptide (A beta) and the loss of basal forebrain cholinergic neurons, which innervate the hippocampus and the cortex. Studies of the pathological changes that characterize AD and several other lines of evidence indicate that A beta accumulation in vivo may initiate and/or contribute to the process of neurodegeneration and thereby the development of AD. However, the mechanisms by which A beta peptide influences/causes degeneration of the basal forebrain cholinergic neurons and/or the cognitive impairment characteristic of AD remain obscure. Using in vitro slice preparations, we have recently reported that A beta-related peptides, under acute conditions, potently inhibit K+-evoked endogenous acetylcholine (ACh) release from hippocampus and cortex but not from striatum. In the present study, we have further characterized A beta-mediated inhibition of ACh release and also measured the effects of these peptides on choline acetyltransferase (ChAT) activity and high-affinity choline uptake (HACU) in hippocampal, cortical, and striatal regions of the rat brain. A beta(1-40) (10(-8) M) potently inhibited veratridine-evoked endogenous ACh release from rat hippocampal slices and also decreased the K+-evoked release potentiated by the nitric oxide-generating agent, sodium nitroprusside (SNP). It is interesting that the endogenous cyclic GMP level induced by SNP was found to be unaltered in the presence of A beta(1-40). The activity of the enzyme ChAT was not altered by A beta peptides in hippocampus, cortex, or striatum. HACU was reduced significantly by various A beta peptides (10(-14) to 10(-6) M) in hippocampal and cortical synaptosomes. However, the uptake of choline by striatal synaptosomes was altered only at high concentration of A beta (10(-6) M). Taken together, these results indicate that A beta peptides, under acute conditions, can decrease endogenous ACh release and the uptake of choline but exhibit no effect on ChAT activity. In addition, the evidence that A beta peptides target primarily the hippocampus and cortex provides a potential mechanistic framework suggesting that the preferential vulnerability of basal forebrain cholinergic neurons and their projections in AD could relate, at least in part, to their sensitivity to A beta peptides.

Choline supplementation during fetal development [embryonic days (E) 11-17] permanently enhances memory performance in rats. To characterize the neurochemical mechanisms that may mediate this effect, we investigated the development of indices of the cholinergic system in the hippocampus: choline acetyltransferase (ChAT), acetylcholinesterase (AChE), synthesis of acetylcholine (ACh) from choline transported by high-affinity choline uptake (HACU), and potassium-evoked ACh release. During E11-E17, Sprague-Dawley pregnant rats consumed 0 [choline-deficient (ChD)], 1.3 [control (ChC)], and 4.6 [choline-supplemented (ChS)] mmol/(kg x day) of choline, respectively. On postnatal days 17 and 27, hippocampi of the ChD animals had the highest AChE and ChAT activities, and increased synthesis of ACh from choline transported by HACU, concomitant with reductions of tissue ACh content relative to the ChC and ChS rats and an inability to sustain depolarization-evoked ACh release relative to the ChS animals. In contrast, AChE and ChAT activities, and ACh synthesized from choline transported by HACU, were lowest in ChS rats whereas depolarization-evoked ACh release was the highest. This pattern of changes suggests that the hippocampus of the ChD animals is characterized by fast ACh recycling and efficient choline reutilization for ACh synthesis, presumably to maintain adequate ACh release despite the decrease of the ACh pool, whereas in the ChS animals ACh turnover and choline recycling is slower while the evoked release of ACh is high. Together, the data show a complex adaptive response of the hippocampal cholinergic system to prenatal choline availability and provide a novel example of developmental plasticity in the nervous system governed by the supply of a single nutrient.

A second messenger role for arachidonic acid (AA) in the regulation of the high-affinity choline uptake (HACU) was suggested. It was reported that micromolar concentrations of AA applied in vitro decreased the HACU values and increased the specific binding of [3H]hemicholinium-3 ([3H]HCh-3). It was published that L-glutamic acid (GA) applied in vivo produced a fall in the HACU values. In addition, GA liberates free AA. In this study, an ability of GA to influence in vitro the activity of presynaptic cholinergic nerve terminals via its effect on the release of AA is investigated in hippocampal synaptosomes of young Wistar rats. Millimolar concentrations of GA decrease both the high- and low-affinity choline uptake, the specific as well as nonspecific binding of [3H]HCh-3 and the activity of Na+, K(+)-ATPase. Kinetic analysis (Lineweaver-Burk and Scatchard plots) reveals a change in Vmax and Bmax, but not in KM and KD. It appears very likely that under normal conditions GA applied in vitro is not able to change markedly the choline transport via its effect on the release of AA. Results confirm the hypothesis about an indirect inhibitory role for glutamatergic receptors on cholinergic cells.

The biochemical mechanisms involved in the regulation of acetylcholine (ACh) turnover are poorly understood. In the experiments reported here, we examined whether inhibition of the serine/threonine phosphatases 1 and 2A by calyculin A or okadaic acid alters ACh synthesis by rat hippocampal preparations. With hippocampal slices, calyculin A (50 nM) and okadaic acid (50 nM) reduced significantly (p < 0.01) the synthesis of [3H]ACh from [3H]choline. Both calyculin A and okadaic acid produced significant depletion of endogenous tissue ACh in a concentration-dependent manner (p < 0.01). This depletion was not the result of a drug-induced increase of spontaneous ACh release, which was not changed significantly (p > 0.7) by either drug. Choline acetyltransferase (ChAT) activity from tissue exposed to calyculin A or okadaic acid was reduced in a concentration-dependent manner (p < 0.05), but these phosphatase inhibitors did not act directly on ChAT in vitro; i.e., enzymatic activity was not altered significantly (p > 0.4) in the presence of calyculin A or okadaic acid. Both high-affinity and low-affinity [3H]choline uptake by hippocampal synaptosomes were reduced significantly in a concentration-dependent manner in the presence of calyculin A or okadaic acid; these agents reduced Vmax values for high- and low-affinity choline uptake (p < 0.01) with no significant change in Km values (p > 0.1), indicating a noncompetitive inhibition. Taken together, these data suggest that phosphatase activity plays a role in presynaptic central cholinergic nerve terminal function, in particular in the modulation of ACh synthesis.

Hippocampal cholinergic hypofunction may contribute to memory deficits following experimental traumatic brain injury. These studies examined two important factors in acetylcholine synthesis: choline availability and neuronal uptake. No reductions in basal extracellular choline levels, using microdialysis, were observed 2 weeks after cortical impact injury. However, studies of high affinity [3H]choline uptake in the hippocampus, measured in a synaptosomal preparation, found a reduction in the maximum velocity of choline uptake (Vmax), while no differences in affinity constants (Km) were found. The results suggest that post-traumatic cholinergic deficits are not attributable to decreased availability of choline, but may be associated with either a decreased ability of cholinergic neurons to take up choline and/or a loss of cholinergic neurons.

We performed experiments to investigate subtypes of opioid receptors in the brain involved in the effect of acute (45 min) pulsed microwave exposure (2,450-MHz, 2-microseconds pulses, 500 pps, average power density 1 mW/cm2, peak-power density, 1 W/cm2, average whole body SAR 0.6 W/kg) on cholinergic activity in the rat brain. Rats were pretreated by microinjection of specific antagonists of mu, delta, and kappa opioid-receptors into the lateral cerebroventricle before exposure to microwaves. The data showed that all three subtypes of opioid receptors are involved in the microwave-induced decrease in cholinergic activity in the hippocampus. However, the microwave-induced decrease in cholinergic activity in the frontal cortex was not significantly affected by any of the drug treatments, confirming our previous conclusion that the effect of microwaves on the frontal cortex is not mediated by endogenous opioids.

Normal aging has been associated with a progressive decline in hippocampal cholinergic function. In the present study, specific markers of hippocampal cholinergic function, high-affinity choline uptake (HACU) and muscarinic quinuclidinylbenzilate (QNB) binding, were shown to be altered by endurance training (6 months of treadmill running, 5 days/week, 30 min/day). HACU and QNB binding were determined in synaptosomes of endurance trained F344 rats and their age-matched sedentary controls. Comparison of synaptosomes of sedentary rats ages 3 months, 12 months and 25 months (distinguished in this paper as young (Y), middle age (MA) and old (O), respectively) showed maximum HACU at 12 months and subsequent reduction in HACU and QNB binding at 25 months (P less than 0.05). This decline at 25 months is consistent with previous reports of an age-related decline in cholinergic function. Endurance trained rats (trained from 6 months to 12 months of age) showed a reduction (P less than 0.02) in HACU and an increase (P less than 0.05) in QNB binding compared to their age-matched sedentary controls whereas endurance trained rats (trained from 19 months to 25 months of age) showed no significant difference in either parameter from their age-matched sedentary controls. From these results, it appears that while both training and normal aging reduce HACU, the reductions may be different in presynaptic mechanism and postsynaptic consequence.

In previous research, we found that sodium-dependent high-affinity choline uptake in the hippocampus and frontal cortex of the rat was lowered after acute (45 min) exposure to low-level 2450-MHz pulsed microwaves (power density 1 mW/cm2; average whole body specific absorption rate, 0.6 W/kg; 2 mu sec pulses, 500 pps). In the present experiment, we investigated developments of tolerance and classical conditioning to these effects of microwaves. Rats were exposed to microwaves in cylindrical waveguides in 10 daily sessions (45 min per session). In an 11th session, we subjected the rats to either microwave (study of tolerance) or sham exposure (study of conditioned effect) for 45 min, and immediately measured choline uptake in the hippocampus and frontal cortex. We found that tolerance, a decrease in response to microwaves, developed to the effect of microwaves on choline uptake in the hippocampus, but not in the frontal cortex. Conditioned effects were also observed: an increase in choline uptake in the hippocampus and a decrease in uptake in the frontal cortex. These data suggest that the effects of microwaves on choline uptake in the hippocampus and frontal cortex are classically conditionable, probably to cues in the exposure environment.

In vivo studies have shown that high blood concentrations of pituitary-adrenocortical hormones can activate the hippocampal cholinergic terminals. Incubation of hippocampal synaptosomal preparations with methylprednisolone, or with ACTH at concentrations comparable to stress-induced high concentrations in plasma, did not have any significant effects on the cholinergic parameters measured under unactivated conditions. In the presence of either high K+ or of ACh, choline uptake was decreased. This decrease was not affected by methylprednisolone. However, methylprednisolone did enhance ACh release both after a previous increase (induced by K+) or a decrease (induced by ACh) of ACh release. In contrast, ACTH had no direct effects on either unactivated or K+-stimulated synaptosomes. Thus, a differential effect was exerted by methylprednisolone on the two presynaptic regulatory mechanisms: choline uptake (no change) and ACh release (increase). We suggest that the activation, observed in vivo, resulted mainly from indirect action of the hormones on the hippocampal cholinergic terminals, in view of the fact that the direct effect in vitro was partial.

We have compared the effects of tetrahydroaminoacridine (THA), 4-aminopyridine (4-AP) and tetraethylammonium (TEA) on high affinity choline uptake and the release of newly synthesized acetylcholine (ACh) from striatal and hippocampal synaptosomes, and on choline acetyltransferase (ChAT) activity in rat striatal synaptosomes. Incubation of the striatal synaptosomes with various THA concentrations (5-100 microM) caused a concentration-dependent inhibition in their accumulation of soluble 14C-labeled compounds ([14C]choline and [14C]ACh); at concentrations of 50 microM or greater the THA also completely suppressed the release of newly synthesized [14C]ACh. 4-AP slightly but significantly decreased the accumulation of the [14C]ACh in the striatal synaptosomes, without affecting that of [14C]choline, but markedly increased the release of [14C]ACh into the medium; hence the drug stimulated net choline uptake (by 19, 20 and 31%, respectively, in the presence of 5, 50 and 100 microM concentrations). Like THA, but not 4-AP, TEA decreased both the accumulation of soluble 14C-compounds in the striatal synaptosomes and the release of newly synthesized [14C]ACh. Similar effects of THA and 4-AP on HACU and the release of [14C]ACh were also observed when hippocampal synaptosomes were used. THA and 4-AP are structurally similar, and share with TEA the ability to block certain potassium channels. Present data show that, in spite of these similarities, these compounds produce opposite effects on net choline uptake by striatal and hippocampal synaptosomes.