Dietary restriction of choline reduces hippocampal acetylcholine release in rats: in vivo microdialysis study.

We fed rats with a diet deficient in choline for 12 weeks and studied how dietary choline deficiency affected their behavior and their ability to release acetylcholine in discrete regions of rat brain using step-through passive avoidance task and in vivo microdialysis. In comparison with the control, rats fed the choline-deficient diet showed poorer retention of nociceptive memory in the passive avoidance task. Average choline level in cerebrospinal fluid in the choline-deficient group was significantly less (33.1%) than that of control rats. In vivo microdialysis showed no difference in the pattern of acetylcholine release enhanced by intraperitoneal administration of scopolamine hydrochloride (2 mg/kg) in the striatum between the two groups, whereas in the hippocampus, the maximum and subsequent increase of acetylcholine from the baseline by scopolamine injection was significantly lower in the choline-deficient group than in the control. From the results of our study, we speculate that long-term dietary restriction of choline can affect extra- and intracellular sources of substrates required for acetylcholine synthesis, and eventually limit the ability to release acetylcholine in the hippocampus. Reduced capacity to release acetylcholine in the hippocampus implies that the mechanism, maintaining acetylcholine synthesis on increased neuronal demand, may vary in discrete regions of the brain in response to dietary manipulation. The vulnerability of the mechanism in the hippocampus to dietary choline restriction is indicated by impaired mnemonic performance we observed.     

CPP and amlodipine alter the decrease in basal acetylcholine and choline release by audiogenic stimulus in hippocampus of ethanol-withdrawn rats in vivo

Effects of N-methyl-D-aspartate (NMDA) receptor and Ca2+ channel antagonists on extracellular acetylcholine and choline release in the hippocampus of ethanol-withdrawn rats were investigated by in vivo microdialysis. Ethanol was administered to Wistar rats in a liquid diet for 28 days. Basal acetylcholine and choline levels significantly increased at the 24th hour of ethanol withdrawal syndrome (EWS). Either an NMDA receptor antagonist (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) or a calcium channel antagonist amlodipine was administered, and 15 min later, an audiogenic stimulus (100 dB, 1 min) was applied to rats. While audiogenic stimulus increased acetylcholine and had no effect on choline release in control rats, it decreased acetylcholine and increased choline release in ethanol-withdrawn rats. CPP (15 mg/kg) and amlodipine (20 mg/kg) reversed the decrement in acetylcholine and increment in choline release in EW rats. Their effects on acetylcholine and choline release were not different from saline in control rats. Therefore, our findings suggest that, (a) because of adaptive changes in EWS, decrease of the acetylcholine release following audiogenic stimulus may play a role in the triggering of seizures, (b) hippocampal glutamatergic pathway may play a role in the audiogenic stimulus induced decrement of acetylcholine release in EWS, (c) inhibition of this pathway by NMDA receptor and calcium channel antagonists may prevent triggering of the seizures.     

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

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

Veratridine-induced activation ofcholine-O-acetyltransferase activity in rat hippocampal tissue: relationship to the veratridine-induced release of acetylcholine

The effect of veratridine depolarization on the activity of 3 choline-O-acetyltransferase (ChAT) fractions in rat hippocampal tissue was investigated. Those concentrations of veratridine which augmented acetylcholine (ACh) release also increased the activity of water and detergent soluble ChAT fractions. These results may suggest that the depolarization induced release of ACh is linked to an activation of ChAT activity.     

Nitric oxide increases stimulation-evoked acetylcholine release from rat hippocampal slices by a cyclic GMP-independent mechanism

Nitric oxide (NO) is an endothelium-derived relaxing factor and its main mechanism of action is activation of soluble guanylyl cyclase. NO and NO-related compounds have been reported to affect several neuronal functions in the central nervous system. In this study, we investigated the effects of NO donors (sodium nitroprusside (SNP) and (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (FK409)) on acetylcholine (ACh) release from rat hippocampal slices. SNP (10⁻⁵ M) and FK409 (10⁻⁴ M) increased electrical stimulation-evoked ACh release without affecting basal release. As dibutyryl cyclic GMP inhibited stimulation-evoked ACh release, the effects of these NO donors were not due to soluble guanylyl cyclase activation. Atropine increased stimulation-evoked ACh release by blocking presynaptic muscarinic autoreceptors, and SNP increased stimulation-evoked ACh release in the presence of atropine, suggesting that SNP and atropine increase stimulation-evoked ACh release by different mechanisms. The present results indicate that NO enhances some part of the excitation-secretion coupling pathway without inducing ACh release directly and these effects are mediated by cyclic GMP-independent mechanism.     

Ca-sensitive inhibition by Pb of α7-containing nicotinic acetylcholine receptors in hippocampal neurons

In the present study the patch-clamp technique was applied to cultured hippocampal neurons to determine the kinetics as well as the agonist concentration- and Ca²⁺-dependence of Pb²⁺-induced inhibition of α7 nicotinic receptors (nAChRs). Evidence is provided that more than two-thirds of the inhibition by Pb²⁺ (3–30 μM) of α7 nAChR-mediated whole-cell currents (referred to as type IA currents) develops rapidly and is fully reversible upon washing. The estimated values for τ_onset and τ_recovery were 165 and 240 ms, respectively. The magnitude of the effect of Pb²⁺ was the same regardless of whether acetylcholine or choline was the agonist. Pre-exposure of the neurons for 800 ms to Pb²⁺ (30 μM) decreased the amplitude and accelerated the decay phase of currents evoked by moderate to high agonist concentrations. In contrast, only the amplitude of currents evoked by low agonist concentrations was reduced when the neurons were exposed simultaneously to Pb²⁺ and the agonists. Taken together with the findings that Pb²⁺ reduces the frequency of opening and the mean open time of α7 nAChR channels, these data suggest that Pb²⁺ accelerates the rate of receptor desensitization. An additional reduction of type IA current amplitudes occurred after 2-min exposure of the neurons to Pb²⁺. This effect was not reversible upon washing of the neurons and was most likely due to an intracellular action of Pb²⁺. Pb²⁺-induced inhibition of α7 nAChRs, which was hindered by the enhancement of extracellular Ca²⁺ concentrations, may contribute to the neurotoxicity of the heavy metal.     

Raloxifene and estradiol benzoate both fully restore hippocampal choline acetyltransferase activity in ovariectomized rats

Selective estrogen receptor modulators (SERMs) demonstrate tissue-specific estrogen receptor (ER) agonist or antagonist properties. Raloxifene, a prototypical SERM, has ER agonist properties in bone and on cholesterol metabolism but full antagonist properties in the uterus and breast. To characterize the ER agonist/antagonist profile of raloxifene in the brain, we have examined its effect on the activity of a known estrogen-responsive gene product, choline acetyltransferase (ChAT), in the hippocampus and other brain regions of 6-month-old ovariectomized (OVX) Sprague–Dawley rats. Three weeks post-ovariectomy, animals received estradiol benzoate (EB, 0.03 mg or 0.3 mg kg^-1 day^-1 for 3 or 10 days); raloxifene HCl (3.0 mg kg^-1 day^-1 for 3 or 10 days), tamoxifen (3.0 mg kg^-1 day^-1 for 10 days) or vehicle (20% CDX). As previously reported, ChAT activity decreased by approximately 20%–50% in the hippocampus of OVX compared with SHAM-operated control rats with no change in ChAT activity observed in the hypothalamus. Raloxifene or EB reversed the OVX-induced decrease in ChAT activity in the hippocampus but did not change ChAT activity in the hypothalamus. Animals that received combined EB (0.03 mg/kg) plus raloxifene (1 mg/kg) or tamoxifen alone (3.0 or 10 mg/kg) also showed increased hippocampal ChAT activity. Raloxifene failed to increase uterine weight and blocked the estrogen-induced increase in uterine weight, while another SERM, tamoxifen, increased uterine weight. These data demonstrate that raloxifene has estrogen-like properties on hippocampal ChAT activity in vivo, and suggest that benzothiophene SERMs may exert estrogen-like beneficial effects on cholinergic neurotransmission in brain without producing peripheral stimulation of breast or uterine tissue.     

Reinforcement enhances hippocampal acetylcholine release in rats: an in vivo microdialysis study.

Rats were trained to press a lever under a 'Multiple FI-60s and Extinction' schedule with food reinforcements. After learning the task, an in vivo microdialysis probe was inserted into the dentate gyrus and CA3 regions of the hippocampus, and the sequential changes in the dialysate acetylcholine (ACh) concentration were analyzed. In the session, two fixed-interval of reinforcement (FI) components (for 20 min) and two extinction (EXT) components (for 30 min) were alternated to examine the correlation between behavioral and neurochemical outcomes. The dialysate ACh concentration increased during the FI component and returned to the baseline during the EXT component of the schedule. Next, in order to dissociate the effect of discrimination from the effect of rewarding on the neurochemical changes in the hippocampus, we used a final TEST period (for 20 min) during which the actual schedule was extinction but the discriminative stimulus was on, i.e. the manifest condition of the test period was reinforcement. In the TEST period, the animals pressed the lever with almost the same frequency as during the FI component; however, the dialysate ACh concentration did not increase above the baseline concentration. These results suggest that ACh release in the rat hippocampus is associated with reinforcement but not with discrimination in operant conditioning.     

In vivo acetylcholine release as measured by microdialysis is unaltered in the hippocampus of cognitively impaired aged rats with degenerative changes in the basal forebrain

Acetylcholine (ACh) release was studied in awake, freely moving animals using in vivo microdialysis in the hippocampus of young (3-month-old) and aged (24-month-old) female Sprague-Dawley rats. Two groups of aged rats were selected on basis of their spatial learning performance in the Morris water maze: non-impaired aged rats which performed as well as the young control animals, and impaired aged rats which learnt the task very poorly. Baseline ACh overflow (in the presence of 5 microM neostigmine) was 1.9 +/- 0.3 +/- pmol/15 min in the young animals and 1.6 +/- 0.4 pmol/15 min in both the impaired and the non-impaired aged rats; these levels did not differ from each other. Depolarization by KCl (100 mM) or muscarinic receptor blockade by atropine (3 microM) added to the perfusion fluid produced dramatic, 4-6-fold, increases in ACh overflow that was similar in magnitude in both the young and the aged impaired and non-impaired rats. Behavioral activation by either handling or electrical stimulation of the lateral habenula produced 2-3-fold increases in extracellular ACh-levels in the hippocampus similarly in all three groups. The results indicate that hippocampal ACh release is maintained in aged rats that exhibit severe spatial learning and memory impairments and that the septo-hippocampal cholinergic system retains its capacity to increase its ACh release in response to both K(+)-induced depolarization and behavioral activation in the aged rat.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular calcium alters frequency modulation of [H]acetylcholine release from rat hippocampal slices

The concentration of extracellular Ca²⁺ has been shown to enhance or attenuate [³H]acetylcholine (ACh) release subsequent to a conditioning stimulus in rat brain hippocampal slices. Slices were incubated in vitro in [³H]choline solution. Subsequently the slices were subjected to two consecutive electrical stimulations separated by 15 or 30 min at 0.25, 1, 4 and 16 Hz and [³H]ACh release was assessed. It was found that a conditioning stimulus may reduce [³H]ACh release during a second stimulation. This phenomenon is frequency related and disappears when the two stimulations are 30 min apart. High extracellular Ca²⁺ (4.0 mM) further attenuated [³H]ACh release during the second stimulation whereas low Ca²⁺ (0.32 mM) abolished the decrease in [³H]ACh release following the second stimulation in all frequencies tested.     

Inhibition of hippocampal acetylcholine release after acute and repeated Δ-tetrahydrocannabinol in rats

The effects of acute and repeated administration of Δ⁹-tetrahydrocannabinol (Δ⁹-THC), the psychoactive principle of marijuana, on acetylcholine release in the hippocampus was studied in freely moving rats by microdialysis. The acute intraperitoneal (i.p.) administration of Δ⁹-THC at the doses of 2.5 and 5 mg/kg reduced acetylcholine release by about 25% and 45%, respectively. A dose of 7.5 mg/kg produced no further reduction. Δ⁹-THC effects were antagonized by the cannabinoid CB₁ antagonist SR141716A at the i.p. dose of 1 mg/kg, per se ineffective in modifying acetylcholine concentrations. After a repeated exposure (twice daily for up to seven days) to Δ⁹-THC (7.5 mg/kg, i.p.) or vehicle (0.3 ml/kg, i.p.), the inhibitory effect of Δ⁹-THC (2.5 and 5 mg/kg, i.p) on acetylcholine release was not reduced. The results confirm previous observations that cannabinoids inhibit acetylcholine release through cannabinoid CB₁ receptors, and indicate that no tolerance to this effects develops after a repeated Δ⁹-THC administration.     

Memory-related acetylcholine efflux from rat prefrontal cortex and hippocampus: a microdialysis study

To investigate the relationship between the prefrontal and hippocampal acetylcholine (ACh) systems and working memory, an in vivo microdialysis study was conducted. A group of rats was trained to perform a working memory task, delayed alternation, in an operant chamber for food reinforcement. The rats had to choose one of two response levers in an alternative manner in each trial, with a certain interval between trials. They had to remember which lever they chose in the previous trial without the assistance of external cues. Another group was trained to perform a reference memory task, cued alternation, in which the behavioral sequence was identical, but an external cue was provided. After stable behavior was established, a dialysis probe was implanted into the prefrontal cortex or the hippocampus of each rat. The extracellular concentration of ACh in the dialysates from the prefrontal cortex increased during performance of the delayed alternation task, while the hippocampal ACh showed a more distinct increase during performance of the cued alternation task. These results suggest that the prefrontal ACh is mainly related to working memory, whereas the hippocampal ACh is mainly related to reference memory.     

In vitro measurements of cholinergic activity in brain regions of hibernating ground squirrels

High affinity choline uptake (HACU) and choline acetyltransferase (ChAT) activity were measured in synaptosomal P2 fractions from four brain regions in a mammalian hibernator, the golden-mantled ground squirrel. The 14CO2 evolution from [6-14C]glucose was also measured. Comparisons were made across the euthermic (not hibernating) and hibernation state in synaptosomes from cortex (CTX), preoptic area and hypothalamus (POA/HYP), olfactory apparatus (OA), and hippocampus (HPC). HACU was significantly increased in the CTX, from hibernating ground squirrels compared to euthermic animals. ChAT activity was significantly increased in the CTX and OA from hibernating animals. No change in either cholinergic marker was evident for the POA/HYP and HPC. The evolution of 14CO2 from [6-14C]glucose was generally, though not significantly, higher for synaptosomes from euthermic animals compared to hibernating animals. The results are discussed with reference to the involvement of cholinergic mechanisms in the control of hibernation.     

Regulation of cortical acetylcholine release: Insights from in vivo microdialysis studies

Acetylcholine release links the activity of presynaptic neurons with their postsynaptic targets and thus represents the intercellular correlate of cholinergic neurotransmission. Here, we review the regulation and functional significance of acetylcholine release in the mammalian cerebral cortex, with a particular emphasis on information derived from in vivo microdialysis studies over the past three decades. This information is integrated with anatomical and behavioral data to derive conclusions regarding the role of cortical cholinergic transmission in normal behavioral and how its dysregulation may contribute to cognitive correlates of several neuropsychiatric conditions. Some unresolved issues regarding the regulation and significance of cortical acetylcholine release and the promise of new methodology for advancing our knowledge in this area are also briefly discussed.     

Reversal of a selective decrease in hippocampal acetylcholine release, but not of the persistence of kindling, after discontinuation of long-term pentylenetetrazol administration in rats

The time course of the effect of pentylenetetrazol (PTZ)-induced kindling on acetylcholine release in the hippocampus of freely moving rats was investigated with the transversal microdialysis technique. The basal extracellular concentration of acetylcholine in the hippocampus was reduced significantly (−29%, P<0.05) after 3 weeks, and the effect was maximal (−52%, P<0.01) after 4 weeks and remained essentially unchanged during the remaining 4 weeks of PTZ treatment (30 mg/kg, i.p., 3 times/week), relative to vehicle-treated rats. The basal release of acetylcholine in the prefrontal cortex and in the striatum of kindled rats was unchanged compared with that of vehicle-treated rats. The specific binding of [³H]quinuclidinyl benzilate, a non-selective ligand of muscarinic receptors, was significantly increased (+29%, P<0.01) in hippocampal membranes, but not in membranes prepared from the prefrontal cortex or striatum, of PTZ-kindled rats. Thirty days after discontinuation of PTZ treatment, both hippocampal acetylcholine output and the density of muscarinic receptors had returned to values characteristic of vehicle-treated rats, whereas seizure susceptibility did not differ significantly from that apparent 4 days after PTZ administration. These results suggest that the selective and transient decrease in acetylcholine output and the parallel increase in the density of postsynaptic muscarinic receptors in the hippocampus may play a role in facilitating the development of kindling rather than in the maintenance of the kindled state. © 1997 Elsevier Science B.V. All rights reserved.     

Effects of supplemental choline on extracellular acetylcholine in the nucleus accumbens during normal behavior and pharmacological acetylcholine depletion

Brain microdialysis was used to determine whether systemic or local application of choline would modify the extracellular concentration of acetylcholine (ACh) in the nucleus accumbens (NAc) of freely moving rats. Supplemental choline given intraperitoneally or into the NAc of normal rats did not increase extracellular ACh. When local ACh interneurons in the NAc were treated pharmacologically to deplete the intracellular stores of ACh, then systemic choline (80 mg/kg) was an effective treatment. Specifically, )1) blockade of the high-affinity choline transporter with hemicholinium-3 (HC-3) to reduce ACh synthesis caused a decrease in extracellular ACh, but choline supplementation restored ACh toward its normal level in the NAc. (2) Local bicuculline treatment released ACh to the point of depletion, but systemic choline or locally infused choline helped maintain normal ACh levels. These results suggest that choline supplementation might be useful in preventing depletion of ACh in the nucleus accumbens during pathological conditions. © 1994 Wiley-Liss, Inc.     

Interleukin-1β decreases acetylcholine measured by microdialysis in the hippocampus of freely moving rats

Interleukin (IL-1) is a cytokine which plays an important role in the modulation of the acute in host defense. This cytokine is also increased in patients with Alzheimer's disease. In the present experiment systemic injection of IL-1β (7.5–50 μg/kg) decreased extracellular acetylccholine in the hippocampus. This effect could not be attributed entirely to general malaise since lithium chloride (130 mg/kg) had the opposite effect. Heat-inactivation of the cytokine eliminated the reduction of extracellular ACh. The results give further evidence of a relationship between the immune system and the central nervous system and suggest a possible relationship between IL-1 and cholinergic function or dysfunction in the hippocampus.     

Immobilization stress-induced increase of hippocampal acetylcholine and of plasma epinephrine, norepinephrine and glucose in rats

We investigated the role of the hippocampal cholinergic neurons during immobilization stress in rats using a microdialysis technique. Blood levels of glucose, epinephrine and norepinephrine during immobilization stress were also determined. Acetylcholine release was initially increased by immobilization stress, then gradually decreased. Plasma level of epinephrine increased gradually and reached significance at 30 min after the start of immobilization and remained at the elevated level during immobilization. Plasma level of norepinephrine initially increased and reached significance at 30 min after the start of immobilization and remained at the elevated level during immobilization. Plasma level of glucose increased gradually and reached maximum and significance 45 min after the start of immobilization, then decreased. Fifteen min after immobilization, acetylcholine release increased again, while concentrations of epinephrine and norepinephrine were still elevated. Thus the response of acetylcholine and the other responses to immobilization stress were not parallel.     

Relations between acetylcholine release and electrophysiological characteristics of theta rhythm: A microdialysis study in the urethane-anesthetized rat hippocampus

In urethane-anesthetized rats, recording electrodes were implanted in the left dorsal hippocampus and a dialysis probe was placed in the contralateral dorsal or ventral hippocampus. Samples of extracellular acetylcholine (ACh) levels were assessed at 10-min intervals over a period of 30 min using microdialysis with high-performance liquid chromatography with electrochemical detection. EEG was recorded during the same period and amplitude, frequency, and duration of theta rhythm were calculated for each of the three 10-min intervals. Data were analyzed using the two-tailed Spearman rank-order correlation test. A positive and high rank correlation coefficient (rho = 0.90, p < 0.01, N = 8) was seen between the average ACh cutflow in the dorsal hippocampus and the average theta amplitude, both being calculated for the entire collection period. A lower but statistically significant positive correlation (rho = 0.59, p < 0.01) between dorsal hippocampus ACh output and theta amplitude was also found when the couples of values collected for the 30-min period were pooled (n = 20). In contrast, frequency and duration of theta were not significantly correlated with dorsal hippocampus ACh release. Also, no statistically significant correlation (p > 0.05) was found between ACh output in the ventral hippocampus and theta parameters. Because changes in hippocampal ACh outflow are believed to be the reflection of changes in number and/or level of activity of cholinergic afferents to the dorsal hippocampus, our present findings support the view that, at least in the dorsal hippocampus of the urethane-anesthetized rat, the septohippocampal cholinergic projection regulates the theta amplitude but not frequency. Finally, the possibility that ACh outflow increase and tonic release in the hippocampus is not a sufficient condition to induce and maintain theta in the urethane-anesthetized rat is discussed.     

Involvement of medial septal glutamate and GABAA receptors in behaviour-induced acetylcholine release in the hippocampus: A dual probe microdialysis study

In the present study, the role of medial septal receptors in behaviour-induced increase in acetylcholine (ACh) release in hippocampus was investigated using dual-probe microdialysis in combination with a simple behavioural procedure. γ-Aminobutyric acid (GABA) and glutamate receptor agonists and antagonists were administered by retrograde dialysis into the medial septal area, while ACh was measured in the ventral hippocampus. Rats were behaviourally activated by immobilization or handling, but only handling was performed during drug-treatment. The GABA_A receptor agonist muscimol did not affect ACh release, but blocked the handling-induced increase in ACh release completely. In addition, muscimol administration induced behavioural activity. Administration of the GABA_A receptor antagonist bicuculline increased ACh release more than 2-fold. However, handling-induced increase in ACh release, expressed as percentage of drug-induced release, was similar to that of controls. Administration of the glutamate receptor agonists N-methyl-

Full-size image

-aspartate (NMDA) and (S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) in the septal area increased hippocampal ACh levels, but reduced the handling-induced increase in ACh release. The NMDA antagonist, 3-((R)-2-carboxypiperazin-4-yl) (CPP) increased ACh levels moderately, and reduced handling-induced increase in ACh release. However, similarly to muscimol, CPP administration induced behavioural activity. The AMPA/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) did not affect behaviour or basal ACh levels, but attenuated the handling-evoked ACh release. We conclude that the handling-induced increase in hippocampal ACh levels is mediated via stimulation of septal non-NMDA, and possibly NMDA receptors, whereas GABA_A receptors are probably not involved. The feasibility of the experimental design is further discussed.