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It has been demonstrated that the forebrain cholinergic system and the extracellular regulated kinase signal transduction pathway are involved in the mechanisms of learning, encoding, and storage of information. We investigated the involvement of the cholinergic and glutamatergic systems projecting to the medial prefrontal cortex and ventral hippocampus and of the extracellular regulated kinase signal transduction pathway in the acquisition and recall of the step-down inhibitory avoidance response in the rat, a relatively simple behavioral test acquired in a one-trial session. To this aim we studied by microdialysis the release of acetylcholine and glutamate, and by immunohistochemistry the activation of extracellular regulated kinase during acquisition, encoding and recall of the behavior. Cholinergic, but not glutamatergic, neurons projecting to the medial prefrontal cortex and ventral hippocampus were activated during acquisition of the task, as shown by increase in cortical and hippocampal acetylcholine release. Released acetylcholine in turn activated extracellular regulated kinase in neurons located in the target structures, since the muscarinic receptor antagonist scopolamine blocked extracellular regulated kinase activation. Both increased acetylcholine release and extracellular regulated kinase activation were necessary for memory formation, as administration of scopolamine and of extracellular regulated kinase inhibitors was followed by blockade of extracellular regulated kinase activation and amnesia. Our data indicate that a critical function of the learning-associated increase in acetylcholine release is to promote the activation of the extracellular regulated kinase signal transduction pathway and help understanding the role of these systems in the encoding of an inhibitory avoidance memory.

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The involvement of the forebrain cholinergic system in arousal, learning and memory has been well established. Other neurotransmitters such as GABA and glutamate may be involved in the mechanisms of memory by modulating the forebrain cholinergic pathways. We studied the activity of cortical and hippocampal cholinergic, GABAergic and glutamatergic systems during novelty and habituation in the rat using microdialysis. After establishing basal release of the neurotransmitters, the animals were transferred to a novel environment and allowed to explore it twice consecutively for 30 min (60 min apart; exploration I and II). The motor activity was monitored. Samples were collected throughout the experiment and the release of acetylcholine (ACh), GABA and glutamate was measured. During the two consecutive explorations of the arena, cortical and hippocampal, ACh release showed a significant tetrodotoxin-dependent increase which was higher during exploration I than II. The effect was more pronounced and longer-lasting in the hippocampus than in the cortex. Cortical GABA release increased significantly only during exploration II, while hippocampal GABA release did not increase during either exploration. Motor activity was higher during the first 10 min of exploration I and II and then gradually decreased during the further 20 min. Both cortical and hippocampal ACh release were positively correlated with motor activity during exploration II, but not during I. During exploration II, cortical GABA release was inversely correlated, while hippocampal GABA release was positively correlated to motor activity. No change in cortical and hippocampal glutamate release was observed. In summary, ACh released by the animal placed in a novel environment seems to have two components, one related to motor activity and one related to attention, anxiety and fear. This second component disappears in the familiar environment, where ACh release is directly related to motor activity. The negative relationship between cortical GABA levels and motor activity may indicate that cortical GABAergic activity is involved in habituation.

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The effects of neurotensin (NT) on the release of acetylcholine (ACh), aspartate (Asp), glutamate (Glu) and gamma-aminobutyric acid (GABA) from the hippocampus of freely moving rats were studied by transversal microdialysis. ACh was detected by High Performance Liquid Chromatography (HPLC) with electrochemical detection while GABA, glutamate and aspartate were measured using HPLC with fluorometric detection. Neurotensin (0.2 and 0.5 microM) administered locally through the microdialysis probe to the hippocampus produced a long-lasting and concentration-dependent increase in the basal extracellular levels of GABA and ACh but not of glutamate and aspartate. The increase in the extracellular levels of GABA and ACh produced by 0.5 microM neurotensin in the hippocampus reached a maximum of about 310% for GABA and 250% for ACh. This stimulant effect of NT was antagonized by the NT receptor antagonist SR 48692 (100 microg/kg, i.p.). Local infusion of tetrodotoxin (1 microM) decreased the basal release of ACh, GABA, Asp, Glu and prevented the 0.2 microM NT-induced increase in GABA and ACh release. The effect of NT on the release of ACh was blocked by the GABA(A) receptor antagonist bicuculline (2-10 microM). Our findings indicate for the first time that neurotensin plays a neuromodulatory role in the regulation of GABAergic and cholinergic neuronal activity in the hippocampus of awake and freely moving rats. The potentiating effects of neurotensin on GABA and ACh release in the hippocampus are probably mediated by (i) NT receptors located on GABAergic cell bodies and (ii) through GABA(A) receptors located on cholinergic nerve terminals.

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The effect of the non-N-methyl-D-aspartate (NMDA) agonists (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and quisqualate (QUIS) on the release of acetylcholine (ACh), gamma-amino butyric acid (GABA), aspartate (Asp) and glutamate (Glu) from the hippocampus of freely moving rats was studied by transversal microdialysis. Intracerebroventricular (i.c.v.) administration of the non-NMDA receptor agonist AMPA (0.5 nmol) enhanced (by about 200%) ACh release from the hippocampus. The effect of AMPA was completely antagonized by 6-nitro-7-sulphamoyl-benz(f)quinoxaline-2,3-dione (NBQX; 2 nmol, i.c.v). No effect was seen when AMPA was perfused through the septum. However, AMPA (200 microM) locally applied to the hippocampus, increased (by about 200%) ACh release. QUIS (200 microM) applied locally to the hippocampus produced a long-lasting increase in the release of ACh (by about 215%) and GABA (by about 460%). Local infusion of tetrodotoxin (1 microM) decreased ACh and GABA basal extracellular levels, and abolished the QUIS-induced increase in ACh and GABA. Our results demonstrate that non-NMDA glutamatergic receptors in the hippocampus regulate hippocampal release of GABA and ACh.

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The release of [3H]gamma-aminobutyric acid ([3H]GABA) from rat striatal slices before and during electrical field stimulation (EFS) was measured. Electrical stimulation (10 Hz) induced an increase of Ca(++)- and tetrodotoxin-sensitive [3H]GABA release from the striatal slices. In the presence of sulphated octapeptide of cholecystokinin, CCK-8S (10(-9) M, 10(-8) M and 10(-7) M) both the basal and the electrically (10 Hz)-evoked release of [3H]GABA were dose-dependently increased. These effects of CCK-8S were abolished by tetrodotoxin (10(-6) M) and were not influenced by the CCK-A receptor antagonist loxiglumide (CR1505) (10(-7) M and 10(-6) M). The stimulant effect of CCK-8S was antagonized by the newly synthesized CCK-B selective receptor antagonist PD134308 (10(-7) M and 10(-6) M). These findings suggest that CCK-8 plays a neuromodulatory role in the regulation of GABAergic neuronal activity in the striatum. The activation of CCK-B receptors located on GABAergic neurons is involved in the GABA release-potentiating effect of CCK-8S in rat striatum.

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The effect of cholecystokinin octapeptide sulfated (CCK8S) on the basal and electrically evoked release of [3H]dopamine ([3H]DA) in striatal slices from the rat brain was studied. Cholecystokinin octapeptide did not influence the basal release of [3H]DA. Field electrical stimulation (FES) (2 Hz) induced an increase of dopamine release from striatal slices, which was Ca2+ dependent and was abolished by tetrodotoxin, 10(-6) M. Cholecystokinin octapeptide (10(-9) M, 10(-8) M and 10(-7) M) dose dependently reduced the electrically evoked release of [3H]DA. This effect was antagonized by the CCK-A receptor antagonists loxiglumide (10(-7) M, 10(-6) M and 10(-5) M) or proglumide (10(-5) M, 10(-4) M and 10(-3) M). The results suggest that CCK receptors type A are involved in this effect of CCK8S in the striatum.

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Recent years have seen an increase in the information concerning the mechanisms of action of brain-gut neuropeptides (cholecystokinin (CCK), vasoactive intestinal peptide (VIP), somatostatin (SS)) on the biliary tract motility. This article is intended to extend our knowledge of the problem and based on our recent studies. Researchers and students interested in an historical overview of the subject, as well as in the information on the physiology and pharmacology of biliary smooth muscle are referred to earlier reviews (Ryan, 1981, 1987). The article focuses on the involvement of cholinergic mechanisms in the action of CCK, SOM and VIP on the gallbladder motility under in vivo and in vitro conditions. Some species differences in the responses of the gallbladder to CCK, VIP and ACh have also been described. Furthermore, new data about the interactions between CCK, SOM and VIP in the regulation of the gallbladder motility are presented.

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Release of [3H]-acetylcholine (3H-ACh) and muscle contractions in response to cholecystokinin (CCK) were measured and recorded simultaneously from isolated guinea-pig gallbladder. Cholecystokinin octapeptide (CCK8) (10(-10)-10(-7) M) enhanced the release of [3H]ACh and the contractions of the muscle. TTX (10(-6) M) inhibited the CCK-induced release of 3H-ACh by only 30%. In Ca(2+)-free medium CCK8 had no effect. Loxiglumide, (CR 1505), a newly synthesized nonpeptide CCK-A-receptor antagonist, D.L-(3,4-dichlorbenzoilamino)-5-/N-(3-methoxypropyl)-pentylamin o-5-oxo-pentanoi c acid, antagonized both the ACh-releasing effect of CCK and the contractions in a dose-dependent manner. The affinity (pA2) of CR 1505 to CCK-receptors, determined by the shift of the concentration-response curves for CCK8 was 8.36. It was 5 logarithmic orders higher than the pA2 of proglumide. The IC50 value of CR 1505 calculated by the CCK-induced release of 3H-ACh was 10 nM. The results suggest the existence not only of muscular CCK receptors but also neuronal receptors for CCK probably located on cholinergic nerves.

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The effect of neurotensin (NT) on [3H]acetylcholine release and contraction from isolated longitudinal muscle strip of guinea-pig ileum was examined. Neurotensin dose-dependently enhanced the release of [3H]-acetylcholine. This effect of neurotensin was inhibited by stimulation of alpha 2-adrenoceptors: noradrenaline, clonidine, xylazine or dexmedetomidine (alpha 2-adrenoceptor agonists) inhibited neurotensin-induced release of acetycholine (ACh) as well as the contractions, while CH-38083 or yohimbine (alpha 2-adrenoceptor antagonist) prevented this inhibitory effect. Our findings suggest that neurotensin may play a neuromodulatory role in the regulation of cholinergic neuronal activity in the gut and this modulatory effect is continuously controlled by the tonic activity of the sympathetic nervous system: endogenous noradrenaline release is capable of reducing the release of ACh and the consequent contraction of the gut enhanced by neurotensin.

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The effect of neurotensin (NT) on the release of acetylcholine (ACh) and dopamine (DA) from striatal slices of the rat brain was studied. Neurotensin, 1-150 nM, was able to release ACh from cholinergic interneurons of the striatum. Like the response to electrical stimulation, the ACh-releasing effect of NT was completely inhibited by tetrodotoxin indicating that neuronal firing is involved in its effect. Immunneutralization reduced the stimulation-evoked release of ACh, an effect that was much marked when the inhibitory dopaminergic input was suspended by sulpiride-selective antagonists of D2 receptors. Sulpiride, 0.1 mM, induced a 2-fold increase in the NT- and electrically-induced release of ACh. A quantitatively similar increase was also observed after degeneration of the nigrostriatal DA pathway with 6-hydroxydopamine (6-OHDA) (2 x 250 micrograms/animal, i.c.v.). However, the D2 receptor agonist quinpirole, 0.01 mM, significantly reduced the NT-induced release of ACh by 77%. Neurotensin enhanced the stimulation-evoked release of [3H]DA. These findings indicate that, using field stimulation when dopaminergic, cholinergic and NT-containing neurons are stimulated in concert, NT is capable of releasing both ACh and DA in the striatum, but its effect on ACh release is masked unless the D2 receptor-mediated tonic inhibitory effect of DA released from the nigro-striatal pathway is attenuated. Thus, in Parkinson's disease where the dopaminergic input is impaired, NT may be involved in producing cholinergic dominance.

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Release of [3H]-acetylcholine ([3H]ACh) and muscle contractions in response to caerulein was measured and recorded simultaneously from isolated guinea-pig gallbladder. Caerulein (5 x 10(-9) M) enhanced the release of [3H]ACh and the contractions. Tetrodotoxin (10(-6) M) inhibited the caerulein-induced release of [3H]ACh by only 30%. Proglumide and loxiglumide (CR 1505) antagonized the effect of caerulein on both ACh-release and contractions. Neither proglumide nor loxiglumide affected the DMPP-induced contractions and the ACh release. The results suggest not only muscular but also neuronal receptors for caerulein in guinea-pig gallbladder.

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The release of [3H]ACh and the contractions of guinea-pig ileal longitudinal muscle preparations, with myenteric plexus attached were measured and recorded simultaneously. Caerulein in concentrations of 10(-11) M to 10(-8) M caused dose-dependent increase of the contractions and the [3H]ACh release. This excitatory effect of caerulein was sensitive to TTX (10(-6) M). Proglumide selectively antagonized both the contractions and the [3H]ACh-releasing effect of caerulein. Electrical field stimulation (O.1 Hz)-evoked contractions were sensitive to atropine. Caerulein (10(-9) M) did not influence the electrically-evoked release of [3H]ACh and the electrically-evoked contractions. Nifedipine (10(-6)M) decreased them about 50%. In the presence of nifedipine caerulein produced an inhibitory effect on the electrically-induced contractions. This nifedipine-unmasking inhibitory effect of caerulein was accompanied by a decrease of the [3H]ACh release and was prevented by proglumide.

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Suc-Tyr-(SE)-Met-Gly-Trp-Met-Asp-beta-phenethylamide (GE 410) competitively antagonized the contractions of smooth muscle strips from guinea pig ileum (pA2 = 7.6, n = 0.95) induced by cholecystokinin-octapeptide (CCK8). GE 410 inhibited the electrically-induced cholinergically mediated contractile responses and the [3H]ACh release in the ileum, as well as the CCK-stimulated electrical contractile responses and the [3H]ACh release in the cholinergic nerve terminals. The results suggest the existence of CCK-receptors not only in the smooth muscles but also on the neurons.

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Somatostatin (SOM) at doses up to 1 microgram was not effective on the motility of canine and guinea pig gallbladder smooth muscle preparations in vitro. When the preparations were contracted by field electrical stimulation (0.7 ms, 40 Hz) the cholecystokinin octapeptide (CCK OP) enhanced these contractions while SOM inhibited them. These effects were accompanied, respectively, by an increase or a decrease in [3H] acetylcholine (ACh) release in the intrinsic cholinergic nerve terminals. SOM (0.5 to 2 micrograms/kg i.v.) inhibited the spontaneous and the CCK OP-activated gallbladder pressure in conscious dogs. The effect of atropine (10-50 micrograms/kg) was similar to that of SOM when injected intravenously in conscious dogs. It is suggested that the inhibitory effect of SOM on gallbladder pressure in conscious dogs is probably mediated by a decrease in ACh release by cholinergic neurons.

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1. Electrically-induced contractions and acetylcholine (Ach) release in two parts of the ileum: 5 and 25 cm proximal to the ileo-colic sphincter (ICS) were compared. 2. The amplitude of electrically- and Ach-induced contractions of the ileum 5 cm proximal to the ICS (terminal ileum) was much higher than the amplitude of the contractions of the ileum 25 cm proximal to the ICS. 3. The amount of Ach released in the ileum 25 cm proximal to the ICS accounted for 58% of the amount of Ach released in the terminal ileum, which was taken to be 100%.

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The actions of substance P (SP) and met-enkephalin (ME) on the motility of stomach and small intestine were compared in conscious dogs. In stomach the two peptides induced different effects on the motility whereas in small intestine SP and ME caused similar actions. The effects of SP were not influenced significantly by ME or naloxone applied before. Furthermore, SP desensitization did not influence the typical response to ME. Our results indicate no interactions between SP and ME in modulation of gastrointestinal motility. The presence of two peptidergic systems existing independently is discussed.

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Species differences have been observed in the effect of cholecystokinin octapeptide (CCK OP) on the canine and guinea pig gallbladder smooth muscle motility. 1. CCK OP was more potent stimulant in canine than in guinea pig gallbladder smooth muscles. Its pD2 values were 10 and 9.2, respectively. 2. The acetylcholine (10(-4) M)-induced maximum contractions in canine gallbladder muscle strips were by 50% lower as compared to the CCK OP (10(-8) M) maximum responses while in guinea pig gallbladder muscle strips the acetylcholine (ACh) maximum responses were by 20% lower than the CCK OP maximum responses. 3. CCK OP increased [3H]ACh release by 27% in canine gallbladder and by 40% in guinea pig gallbladder. 4. Somatostatin (SOM) had not any direct myogenic effect in guinea pig and canine gallbladder but it decreased [3H]ACh release from gallbladder intrinsic cholinergic neurons.

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The participation of cholinergic mechanisms in cholecystokinin octapeptide (CCKOP) action on canine gall bladder was studied in vivo and in vitro, using three different experimental conditions. In vitro the responses of canine gall bladder smooth muscle to CCKOP (0.01 to 10 nm) were insensitive to atropine (1 to 10 microM) and tetrodotoxin (3 microM). When gall bladder muscle preparations were contracted by field electrical stimulation (0.7 ms, 40 Hz) CCKOP (0.001 to 0.1 nM) enhanced these contractions while atropine (1 microM) abolished them. This suggests that CCKOP is able to influence acetylcholine (ACH)-release from intrinsic cholinergic nerve terminals. In vivo the responses of canine gall bladder smooth muscle to CCKOP (1 to 10 ng/kg i.v.) were reduced and even abolished by atropine (10 to 50 micrograms/kg i.v.) and hexamethonium (0.5 to 3 mg/kg i.v.). The results suggest the participation of at least two mechanisms in CCKOP action on canine gall bladder motility: a direct action on smooth muscle cells, insensitive to atropine or tetrodotoxin, and an indirect action, which is dependent on pre- and post ganglionic cholinergic pathways.

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Cholecystokinin 7 (CCK 7), a synthetic analogue of cholecystokinin/pancreozymin (CCK 33), increased in a dose-dependent manner the tone of the guinea-pig ileal, gastric and gall bladder smooth muscle preparations. In all these preparations CCK 7 was more potent than CCK 8 and CCK 33 and all three cholecystokinins were more potent than acetylcholine (ACH). Atropine and tetrodotoxin (TTX) did not influence the CCK 7 action in fundus and gall bladder muscle strips but reduced non-competitively its effect in ileal muscle strips. Neither GE 410 nor dbcGMP affected the ACH and histamine (His) response of the muscle strips but both antagonists shifted the dose-response curve of CCK 7 to the right, GE 410 (cholecystokinin antagonist) being a much more potent antagonist of CCK 7 as compared to dbcGMP. In all muscle strips a competitive action on the CCK 7 responses was found for GE 410. In gastric muscle strips a competitive influence on the CCK 7 responses was found for dbcGMP at low concentration (1 x 10(-5)M) and a non-competitive influence at high concentration (5 x 10(-4)M). The results suggest that the contractile effects of CCK 7 in the isolated ileal smooth muscle are realized by cholinergic and direct myogenic mechanisms, whereas in the isolated gall bladder and gastric smooth muscles, by a direct myogenic mechanism only.

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