Differential long-term effect of AF64A on [H]ACh synthesis and release in rat hippocampal synaptosomes

The activities of various presynaptic cholinergic parameters were determined in hippocampal synaptosomes of rats 29 weeks after intracerebroventricular injection of ethylcholine aziridinium (AF64A) (3 nmol/2 μl/side) or vehicle (saline). Synaptosomes were preloaded with [³H]choline ([³H]Ch), treated with diisopropyl fluorophosphate to inhibit cholinesterase activity and then were assayed for their content of [³H]Ch and [³H]acetylcholine ([³H]ACh) and for their ability to synthesize and release [³H]ACh. In synaptosomes from AF64A-treated rats compared with synaptosomes from vehicle-treated rats we observed that: (i) specific uptake of [³H]ACh was reduced to 60% of control; (ii) residing [³H]ACh levels were 43% of control while residing [³H]Ch levels were 72% of control; (iii) basal and K⁺-induced [³H]ACh release were 77% and 73% of control, respectively; (iv) high K⁺-induced synthesis of [³H]ACh was only 9% of control; (v) but, choline acetyltransferase activity remained relatively high, being 80% of control. These results suggest that AF64A-induced cholinergic hypofunction is expressed by both loss of some cholinergic neurons and impairment in the functioning of the spared neurons.     

Reduction in striatal D2 dopamine receptor mRNA and binding following AF64A lesions

Unilateral lesions by a cholinotoxin, receptor autoradiography, andin situ hybridization techniques were employed to determine if dopaminergic receptors are located on cholinergic interneurons in the caudate-putamen (CPu). Lesion of the CPu with small amounts of the cholinotoxin AF64A resulted in a significant decrease in D₂ receptor mRNA and D₂ receptor binding. The loss was more pronounced in lateral and central portions of the CPu. Results obtained using [³H] SCH23390 binding to D₁ receptors indicated that there was no change in this dopamine receptor subtype in the AF64A-lesioned CPu. A decrease in D₂ receptor mRNA and receptor binding in AF64A-lesioned animals indicates that a population of postsynaptic D₂ receptors is associated with the cholinergic interneurons. Lack of any change in [³H]SCH23390 binding in the AF64A-lesioned animals suggests that D₁ receptors are not located on cholinergic neurons. These results provide evidence to support the selectivity of the lesion when used as indicated.     

Differential long-term effect of AF64A on [3H]ACh synthesis and release in rat hippocampal synaptosomes.

The activities of various presynaptic cholinergic parameters were determined in hippocampal synaptosomes of rats 29 weeks after intracerebroventricular injection of ethylcholine aziridinium (AF64A) (3 nmol/2 microliters/side) or vehicle (saline). Synaptosomes were preloaded with [3H]choline ([3H]Ch), treated with diisopropyl fluorophosphate to inhibit cholinesterase activity and then were assayed for their content of [3H]Ch and [3H]acetylcholine ([3H]ACh) and for their ability to synthesize and release [3H]ACh. In synaptosomes from AF64A-treated rats compared with synaptosomes from vehicle-treated rats we observed that: (i) specific uptake of [3H]Ch was reduced to 60% of control; (ii) residing [3H]ACh levels were 43% of control while residing [3H]Ch levels were 72% of control; (iii) basal and K(+)-induced [3H]ACh release were 77% and 73% of control, respectively; (iv) high K(+)-induced synthesis of [3H]ACh was only 9% of control; (v) but, choline acetyltransferase activity remained relatively high, being 80% of control. These results suggest that AF64A-induced cholinergic hypofunction is expressed by both loss of some cholinergic neurons and impairment in the functioning of the spared neurons.     

Muscarinic M1 and M3 receptors are present and increase intracellular calcium in adult rat anterior pituitary gland

Physiological and biochemical evidence indicates the existence of functional muscarinic cholinergic receptors in the anterior pituitary. The selectivity of these receptors has been characterised by studying the binding of [³H]quinuclidinyl benzilate ([³H]QNB) and [³H]diphenyl-acetoxy-N-methyl-piperidine ([³H]4-DAMP) in membrane preparation of male rat anterior pituitary at 25°C. Competition experiments with receptor selective muscarinic antagonists were used to characterise specific selective muscarinic receptor binding. Both [³H]QNB and [³H]4-DAMP bound to anterior pituitary membranes at low concentrations, binding was saturable and was potently displaced by 4-DAMP (M₁, M₃ subtypes selective antagonist) > atropine (general) > pirenzepine (M₁). Methoctramine (M₂) didn’t antagonise the [³H]QNB binding efficiently. Acetylcholine and carbachol increased the intracellular Ca²⁺ level in 62% and 65% of cultured rat anterior pituitary cells in a dose-dependent manner, and this effect was prevented by pirenzepine. Based on these results we suggest that both M₁ and M₃ muscarinic receptors are present and active in the majority of cells in the rat anterior pituitary gland, but their physiological role in the adult rat remains to be examined.     

Muscarinic and dopaminergic receptor subtypes on striatal cholinergic interneurons

Unilateral stereotaxic injection of small amounts of the cholinotoxin, AF64A, caused minimal nonselective tissue damage and resulted in a significant loss of the presynaptic cholinergic markers [3H]hemicholinium-3 (45% reduction) and choline acetyltransferase (27% reduction). No significant change from control was observed in tyrosine hydroxylase or tryptophan hydroxylase activity; presynaptic neuronal markers for dopamine- and serotonin-containing neurons, respectively. The AF64A lesion resulted in a significant reduction of dopamine D2 receptors as evidenced by a decrease in [3H]sulpiride binding (42% reduction) and decrease of muscarinic non-M1 receptors as shown by a reduction in [3H]QNB binding in the presence of 100 nM pirenzepine (36% reduction). Saturation studies revealed that the change in [3H]sulpiride and [3H]QNB binding was due to a change in Bmax not Kd. Intrastriatal injection of AF64A failed to alter dopamine D1 or muscarinic M1 receptors labeled with [3H]SCH23390 and [3H]pirenzepine, respectively. In addition, no change in [3H]forskolin-labeled adenylate cyclase was observed. These results demonstrate that a subpopulation of muscarinic receptors (non-M1) are presynaptic on cholinergic interneurons (hence, autoreceptors), and a subpopulation of dopamine D2 receptors are postsynaptic on cholinergic interneurons. Furthermore, dopamine D1, muscarinic M1 and [3H]forskolin-labeled adenylate cyclase are not localized to striatal cholinergic interneurons.     

Involvement of cholinergic neurons in the release of dopamine elicited by stimulation of μ-opioid receptors in striatum

The involvement of striatal cholinergic neurons in the release of dopamine (DA) elicited by the (μ-opioid receptor agonist DAGO [d-Ala², NMePhe⁴-Gly⁵(ol)]enkephalin) was explored. The striatal release of DA was measured by microdialysis in rats anesthetized with chloral hydrate. When infused in the striatum, through the microdialysis probe, DAGO increased the extracellular levels of DA. The previous injection in striatum of AF 64-A, a toxin for cholinergic neurons, or the concomitant infusion of the M₂-muscarinic antagonist methoctramine abolished the effect of DAGO on the DA release. It is concluded that stimulation of μ-opioid receptors, by inhibiting the acetylcholine release which stimulates tonically M₂-muscarinic receptors likely associated with dopaminergic nerve endings, indirectly increases the striatal DA release.     

Hippocampal sympathetic ingrowth and cholinergic denervation uniquely alter muscarinic receptor subtypes in the hippocampus

Following cholinergic denervation of the hippocampus by medial septal lesions, an unusual neoronal reorganization occurs, in which peripheral sympathetic fibers, originating from the superior cervical ganglia, grow into the hippocampus. Previously, we have found that both hippocampal sympathetic ingrowth (HSI) and cholinergic denervation (CD), alone, altered the total number and affinity of muscarinic cholinergic receptors (mAChR). In this study, we utilized the muscarinic antagonist [³H]Pirenzepine, in combination with membrane radioligand binding techniques, to determine the effects of HSI and CD on hippocampal M₁ and M₁ + M₃ mAChR subtypes, 4 weeks after MS lesions. In both the dorsal and ventral hippocampus, HSI was found to markedly diminish the number of M₁ AChRs, while CD was found to increase the number of M₁ AChRs. Neither treatment affected the affinity of the M₁ AChR. However, when M₁ + M₃ binding was assessed, CD was found to decrease the affinity in both hippocampal regions, without altering the number of receptors. Neither affinity nor number of M₁ + M₃ receptors was altered by HSI. The results of this study suggest that both cholinergic denervation and hippocampal sympathetic ingrowth uniquely affect hippocampal muscarinic receptors.     

Muscarinic M3 facilitation of acetylcholine release from rat myenteric neurons depends on adenosine outflow leading to activation of excitatory A2A receptors

Abstract Acetylcholine (ACh) is a major excitatory neurotransmitter in the myenteric plexus, and it regulates its own release acting via muscarinic autoreceptors. Adenosine released from stimulated myenteric neurons modulates ACh release preferentially via facilitatory A_2A receptors. In this study, we investigated how muscarinic and adenosine receptors interplay to regulate ACh from the longitudinal muscle–myenteric plexus of the rat ileum. Blockade of the muscarinic M₂ receptor with 11‐[[2‐1[(diethylamino) methyl‐1‐piperidinyl]‐ acetyl]]‐5,11‐dihydro‐6H‐pyrido [2,3‐b][1,4] benzodiazepine‐6‐one (AF‐DX 116), 4‐diphenylacetoxy‐N‐methylpiperidine methiodide (4‐DAMP) and atropine facilitated [³H]ACh release evoked by short stimulation trains (5 Hz, 200 pulses). Prolonging stimulus train length (>750 pulses) shifted muscarinic autoinhibition towards facilitatory M₃ receptors activation, as predicted by blockade with J104129 (a selective M₃ antagonist), 4‐DAMP and atropine, whereas the selective M₂ antagonist, AF‐DX 116, was without of effect. Blockade of A_2A receptors with ZM 241385, inhibition of adenosine transport with dipyridamole, and inhibition of ecto‐5′‐nucleotidase with concanavalin A, all attenuated release inhibition caused by 4‐DAMP. J104129 and 4‐DAMP, but not AF‐DX 116, decreased (～60%) evoked adenosine outflow (5 Hz, 3000 pulses). Oxotremorine (300 μmol L^?1) facilitated the release of [³H]ACh (34 ± 4%, n = 5) and adenosine (57 ± 3%, n = 6) from stimulated myenteric neurons. 4‐DAMP, dipyridamole and concanavalin A prevented oxotremorine‐induced facilitation. ZM 241385 blocked oxotremorine facilitation of [³H]ACh release, but kept adenosine outflow unchanged. Thus, ACh modulates its own release from myenteric neurons by activating inhibitory M₂ and facilitatory M₃ autoreceptors. While the M₂ inhibition is prevalent during brief stimulation periods, muscarinic M₃ facilitation is highlighted during sustained nerve activity as it depends on extracellular adenosine accumulation leading to activation of facilitatory A_2A receptors.     

Muscarinic cholinergic receptors in the human spinal cord: differential localization of [H]pirenzepine and [H]quinuclidinylbenzilate binding sites

The localization of muscarinic cholinergic receptor subtypes was studied in the human spinal cord using in vitro labelling of cryostat sections with [³H]quinuclidinylbenzilate (QNB) and [³H]pirenzepine (PZ) followed by autoradiography. The highest densities of [³H]QNB binding were localized in laminae II (substantia gelatinosa) and IX (motor neurons); in contrast, the highest density of [³H]PZ binding was localized to lamina II where the binding density was 22—32% higher than in lamina IX. These results suggest that the M₁ and M₂ muscarinic cholinergic receptor subtypes may be differentially localized in sensory and motor regions of the human spinal cord.     

Lesion with the neurotoxin AF64A alters hippocampal cholinergic receptor function

The effect of selective lesion of cholinergic inputs to the hippocampus on the function of hippocampal cholinergic receptors was examined. Hippocampal cholinergic neurons were lesioned in the rat by administration of the selective cholinergic neurotoxin AF64A (ethylcholine mustard azirtdinium). Cholinergic receptor function was examined by assessing the ability of cholinergic agonists and antagonists to modulate the evoked release of radiolabelled acetylcholine (ACh) from hippocampal slices. Nicotine enhanced release, with a bell-shaped dose-response curve. The dose-response curve and EC₅₀ for nicotine was shifted 10-fold to the left in lesioned rats, suggesting an increased sensitivity to nicotine. However, there were no differences in either the number or affinity of nicotinic receptors as determined with binding studies. The muscarinic agonist oxotremorine inhibited the evoked release of ACh in control tissues, but had much less effect in AF64A-lesioned tissues. Binding to the M1 receptor subtype was not changed. However, the K_d for binding to the high affinity subtype of the M2 receptor was increased 10-fold, suggesting that the receptor has become less sensitive to stimulation. Loss of M2 function may allow an increase in the effect of stimulating nicotinic receptors that modulate ACh release.     

Septohippocampal adaptive GABAergic responses by AF64A treatment

A cholinergically disrupted laboratory animal has been produced by administration of the cholinotoxin ethylcholine aziridinium mustard (AF64A), which produced a dysfunction in the cholinergic forebrain system. After AF64A treatment, a reduction of choline acetyl transferase (ChAT) activity was measured in the hippocampal regions. ChAT activity was preferentially reduced in tissue samples of the dorsal with respect to the ventral hippocampus, and concomitantly with this reduction, a compensatory increase in ChAT activity in the medial septum was found. Tissue gamma‐aminobutyric acid (GABA) content in the hippocampal and septal brain areas was not affected by AF64A, indicating a specific effect on the cholinergic septohippocampal projection. The rate of GABA accumulation induced by aminooxyacetic acid administration was higher in the dorsal hippocampus and medial septum of AF64A‐treated animals, but not in their ventral hippocampus and lateral septum, where significant changes occurred in ChAT activity. Concomitantly with the changes in GABA metabolism, a significant B_max increase and K_d reduction of ³H‐flunitrazepam binding in the hippocampus of AF64A‐treated animals were associated with changes in the ChAT activity. This finding suggests an increase of GABA input on the cholinergic somas of the medial septum and an uncompensated GABAergic interneuron activity in the hippocampus. In this study, we present an adaptive mechanism of homotypic compensatory metabolism by cholinergic somas, and a heterotypic response of the GABAergic septohippocampal projection system, which was elicited by AF64A administration. J. Neurosci. Res. 55:178–186, 1999. © 1999 Wiley‐Liss, Inc.     

Crucial role of kainate receptors in mediating striatal kainate injection-induced decrease in acetylcholine M1 receptor binding in rat forebrain

We investigated the roles of kainate-, α-amino-3-hydroxy-5-methylisoxazol-4-propionate (AMPA)- and N-methyl- -aspartate (NMDA)-receptors in mediating striatal kainate injection-induced decrease in the binding of acetylcholine M₁ receptors in rat forebrain. After unilateral intrastriatal injection of kainate (4 nmol), the bindings of [³H]kainate (10 nM), [³H]MK-801 (4 nM) and [³H]pirenzepine (4 nM) to the rat ipsilateral forebrain membranes declined, reaching the lowest on day 2 to 4 and recovering on day 8. Saturation binding studies, performed on day 2 post-injection, showed that kainate (1, 2, 4 nmol) dose-dependently decreased B_max and K_d of the three ligands. (+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), a selective NMDA receptor channel blocker, antagonised (from a dose of 4 nmol) kainate-induced decreases in the bindings of [³H]kainate (up to 20%), [³H]MK-801 (up to 90%) and [³H]pirenzepine (up to 70%). In contrast, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a selective non-NMDA receptor antagonist, almost completely abolished (from a dose of 12 nmol) kainate-induced decreases in the bindings of all the three ligands (up to 95–98%). Cyclothiazide, a selective potentiator that enhances AMPA receptor-mediated responses, significantly enhanced (from a dose of 4 nmol) kainate-induced decrease in the binding of [³H]kainate but not that of [³H]pirenzepine or [³H]MK-801. In summary, these results indicate that striatal kainate injection-induced decrease in the binding of acetylcholine M₁ receptors in rat forebrain is dependent on activation of kainate receptors and, to a certain extent, a consequent involvement of NMDA receptors. These and previous studies provide some evidence showing that kainate receptors might play a crucial role in regulating excitatory amino acids (EAA)-modulated cholinergic neurotransmission in the central nervous system (CNS).     

Comparative ontogenic profile of cholinergic markers,including nicotinic and muscarinic receptors,in the rat brain

The ontogenic profiles of several cholinergic markers were assessed in the rat brain by using quantitative in vitro receptor autoradiography. Brain sections from animals at different stages of development were processed with [³H]AH5183 (vesamicol; vesicular acetylcholine transport sites), [³H]N-methylcarbamylcholine (α₄β₂ nicotinic receptor sites), [³H]hemicholinium-3 (high-affinity choline uptake sites), [³H]3-quinuclidinyl benzilate (total population of muscarinic receptor sites), [³H]4-DAMP (muscarinic M₁/M₃ receptor sites), [³H]pirenzepine (muscarinic M₁ receptor sites), and [³H]AF-DX 116 and [³H]AF-DX 384 (muscarinic M₂ receptor sites) as radiolabeled probes. The results revealed that, by the end of the prenatal period (embryonic day 20), the densities of nicotinic receptor and vesicular acetylcholine transport sites already represented a considerable proportion of those observed in adulthood (postnatal day 60) in different laminae of the frontal, parietal, and occipital cortices, in the layers of Ammon's horn fields and the dentate gyrus of the hippocampal formation, as well as in the amygdaloid body, the olfactory tubercle, and the striatum. In contrast, at that stage, the densities of total muscarinic, M₁/M₃, M₁, and possibly M₂ receptor and high-affinity choline uptake sites represent only a small proportion of levels seen in the adult. Differences were also observed in the postnatal ontogenic profiles of nicotinic, muscarinic, vesamicol, and high-affinity choline uptake sites. For example, between postnatal weeks 3 and 5, the levels of M₁/M₃ and M₁ sites were at least as high as in the adult, whereas M₂ and high-affinity choline uptake site densities appeared to be delayed and to reach adult values only after postnatal week 5. With regard to cholinergic innervation in the developing rat brain, the present findings suggest a temporal establishment of several components of the cholinergic systems. The first components are the vesicular acetylcholine transporter and nicotinic sites; these are followed by M₁/M₃ and M₁ sites and, finally, by M₂ and high-affinity choline uptake sites. © 1996 Wiley-Liss, Inc.     

Effects of nefiracetam on deficits in active avoidance response and hippocampal cholinergic and monoaminergic dysfunctions induced by AF64A in mice

Summary The effects of nefiracetam [DM-9384; N-(2,6-dimethyl-phenyl)-2-(2-oxo-pyrrolidinyl)acetamide] and of phosphatidylcholine on a step-up active avoidance response, locomotor activities and regional brain cholinergic and monoaminergic neurotransmitters in AF64A-treated mice were investigated. Intracerebroventricular (i.c.v.) injection of AF64A (ethylcholine mustard aziridinium ion; 8 nmol/ventricle) impaired acquisition and retention of the avoidance task, and increased vertical and horizontal locomotor activities. Regional levels of acetylcholine, noradrenaline, 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were significantly decreased and homovanillic acid (HVA) levels were increased in the hippocampus but not in the septum, cerebral cortex or striatum of AF64A-treated animals. Administration of nefiracetam (3 mg/kg, p.o.) twice daily for 9 days to AF64A-treated animals ameliorated the deficit in active avoidance response in addition to attenuating the increase in locomotor activities. In parallel with these behavioural effects, nefiracetam reversed AF64A-induced alterations in the hippocampal profiles of cholinergic and monoaminergic neurotransmitters and their metabolites. In contrast, administration of phosphatidylcholine (30 mg/kg, p.o.) twice daily for 9 days had no significant effect on the deficit in active avoidance response, despite significantly reversing the decrease in acetylcholine levels in the hippocampus. These results indicate that the effects of nefiracetam on AF64A-induced behavioural deficits are probably due to its ability to facilitate both cholinergic and monoaminergic neurotransmitter systems.     

Hippocampal muscarinic supersensitivity after AF64A medial septal lesion excludes M1 receptors

Stereotaxic injection of AF64A, into the medial septum of the rat, resulted in significant loss of presynaptic cholinergic markers in this structure. No significant change was observed for the presynaptic neuronal markers for dopamine- and serotonin-containing neurons in either the medial septum or hippocampus. The AF64A lesion also resulted in a significant reduction of muscarinic receptors as demonstrated by a loss of [3H]QNB binding in the medial septum. Subtype analysis showed the decrease of receptor binding in the medial septum to be due to a loss of M1 receptors as well as other muscarinic receptor subtypes. In the hippocampal formation, [3H]hemicholinium-3 binding was significantly reduced in the molecular layer of the dentate gyrus, and in the stratum oriens and stratum radiatum of the hippocampus. AF64A lesion resulted in a significant increase (Bmax) in non-M1 muscarinic receptors in hippocampal stratum oriens, in areas CA2, CA3, and CA4. AF64A lesion of the medial septum did not result in muscarinic receptor alterations in any other region of the hippocampal formation examined. These results indicate that postsynaptic muscarinic receptors in the stratum oriens of the CA2 to CA4 region of the hippocampus mediate primarily the function of the cholinergic cell bodies of the medial septum. These receptors are not of the M1 subtype.     

Subtype-specificity of the presynaptic α2-adrenoceptors modulating hippocampal norepinephrine release in rat

In vivo brain microdialysis and high-performance liquid chromatography with electrochemical detection were used to study the effect of different selective α₂-antagonists on hippocampal norepinephrine (NE) release in freely moving awake rat. Systemic administration (0.5 mg/kg i.p.) of either the α_2AD-antagonist BRL 44408 or the α-_2BC-antagonist ARC 239 did not significantly change the basal release of NE. At a higher dose (5 mg/kg i.p.) ARC 239 was still ineffective, whereas BRL 44408 caused a significant increase of the extracellular level of NE. Similar results were obtained from in vitro perfusion experiments. Rat hippocampal slices were loaded with [³H]NE and the electrical stimulation-evoked release of [³H]NE was determined. The α₂-antagonists were applied in a concentration range of 10⁻⁸ to 10⁻⁶ M. ARC 239 was ineffective, whereas BRL 44408 significantly increased the electrically induced release of [³H]NE. In agreement with the data of microdialysis and perfusion experiments. BRL 44408 displaced [³H]yohimbine from hippocampal and cortical membranes of rat brain with high affinity whereas ARC 239 was less effective. The pK_i values of eight different α₂-adrenergic compounds showed a very good correlation (r = 0.98, slope = 1.11 P < 0.0001) in hippocampus and frontal cortex where the α₂-adrenoceptors have been characterized as α_2D-subtype. Our data indicate that hippocampal NE release in rat is regulated by α_2D-adrenoceptors, a species variation of the human α_2A-subtype.     

Effects of verapamil on the release of different neurotransmitters

The effect of verapamil on resting and depolarization-induced monoamine release was investigated in rat hippocampal synaptosomes prelabeled with [³H]-5-hydroxytryptamine (HT) or [³H]-norepinephrine (NE) and rat striatal synaptosomes prelabeled with [³H]-dopamine (DA). Verapamil (50 μM) completely abolishes high K⁺-induced [³H]-NE release, but paradoxically facilitates high K⁺-induced [³H]-5-HT and [³H]-DA release. All these high K⁺-evoked responses were Ca²⁺ dependent. Verapamil does not modify [³H]-NE baseline release, but increases dose dependently [³H]-5-HT and [³H]-DA baseline release. Verapamil (10 μM, for 5 min) increases endogenous DA release (70%) and endogenous 5-HT release (40%) independently on the presence of external Ca²⁺. The total amount of these monoamines (released plus retained by the preparation) and their metabolites (DOPAC and 5-HIAA) was similar in control and verapamil-treated synaptosomes. Verapamil displaces [³H]-spiroperidol specific binding (K_i of 2.4 × 10^?6M) and [³H]-SCH-23390 specific binding (K_i of 9 × 10^?6M) from striatal synaptosomal membranes, and [³H]-5-HT specific binding (K_i of 3 × 10^?5M) from hippocampal synaptosomal membranes. It is concluded that in addition to the Ca²⁺ antagonistic properties of verapamil on the Ca²⁺-dependent, depolarization-induced release of some neurotransmitters [gamma aminobutyric acid (GABA and NE)], another mechanism probably mediated by presynaptic receptors underlies the effects of verapamil on DA and 5-HT release from discrete brain regions. © 1995 Wiley-Liss, Inc.     

Bidirectional modulation of long-term potentiation by carbachol via M1 and M2 muscarinic receptors in guinea pig hippocampal mossy fiber-CA3 synapses

Participation of muscarinic M₁ and M₂ receptors in the modulation of long-term potentiation (LTP) was studied in the mossy fiber-CA3 synapse of guinea pig hippocampal slices. The magnitude of tetanus-induced LTP was attenuated in the presence of 0.01–0.1 μM carbachol, at which concentration the pre-tetanus amplitude of field excitatory postsynaptic potential (fEPSP) was not affected. The attenuation of LTP by the low concentration of carbachol was reversed by an M₂ muscarinic antagonist, AF-DX 116, but not by an M₁ antagonist, pirenzepine. On the contrary, a high concentration (10 μM) of carbachol decreased the pre-tetanic amplitude of fEPSP, however, the magnitude of LTP was significantly larger than that in control slices in which pre-tetanic amplitude of fEPSP was reduced to the level of carbachol-treated slices by reducing the intensity of stimulation or extracellular Ca²⁺ concentration. The augmentation of LTP by 10 μM carbachol was blocked by pirenzepine but not by AF-DX 116. These results suggest that the synaptic plasticity in the guinea pig hippocampal mossy fiber-CA3 synapse is inhibited and facilitated by muscarinic agonist through muscarinic M₂ and M₁ receptors to inhibit and facilitate the LTP, respectively.     

The effect of time following exposure to trimethyltin (TMT) on cholinergic muscarinic receptor binding in rat hippocampus

Adult male Long-Evans rats were given 6 mg/kg trimethyltin (TMT). Rats were killed 1, 3, 7, 14, 21, 35 or 60 d later. An untreated control group was included. Brain sections were processed using film autoradiography to visualize in the hippocampus either total muscarinic receptor binding ([³H]quinuclidiny] benzilate: [³H]QNB), or M₁ receptors ([³H]pirenzepine; [³H]PZ), or M₂ receptors ([³H]oxotremorine-M; [³H]OXO-M). A reduction in [³H]QNB binding was found in CA1 and CA3c 7 d after TMT, but not in CA3a,b, or the dentate gyrus. [³H]PZ binding was decreased throughout Ammon’s horn by 14 d after treatment. [³H]OXO-M binding decreased 1 d after exposure in CA1 and in all subfields of Ammon’s horn by d 3. Neither [³H]PZ or [³H]OXO-M binding decreased in the dentate gyrus of TMT-treated rat at any time point. The temporal patterns of receptor loss may be explicable by reference to timing of fiber and cell body degeneration reported in previous studies and the regional differences may account for discrepancies between reports of either substantial decreases or no loss in hippocampal muscarinic receptors after TMT exposure.     

Downregulation of muscarinic- and 5-HT1B-mediated modulation of [H]acetylcholine release in hippocampal slices of rats with fimbria-fornix lesions and intrahippocampal grafts of septal origin

Adult Long-Evans female rats sustained electrolytic fimbria-fornix lesions and, two weeks later, received intrahippocampal suspension grafts of fetal septal tissue. Sham-operated and lesion-only rats served as controls. Between 6.5 and 8 months after grafting, both the [³H]choline accumulation and the electrically evoked [³H]acetylcholine ([³H]ACh) release were assessed in hippocampal slices. The release of [³H]ACh was measured in presence of atropine (muscarinic antagonist, 1 μM), physostigmine (acetylcholinesterase inhibitor, 0.1 μM), oxotremorine (muscarinic agonist, 0.01 μM–10 μM), mecamylamine (nicotinic antagonist, 10 μM), methiothepin (mixed 5-HT₁/5-HT₂ antagonist, 10 μM), 8-OH-DPAT (5-HT_1A agonist, 1 μM), 2-methyl-serotonin (5-HT₃ agonist, 1 μM) and CP 93129 (5-HT_1B agonist, 0.1 μM–100 μM), or without any drug application as a control. In lesion-only rats, the specific accumulation of [³H]choline was reduced to 46% of normal and the release of [³H]ACh to 32% (nCi) and 43% (% of tissue tritium content). In the grafted rats, these parameters were significantly increased to 63%, 98% and 116% of control, respectively. Physostigmine reduced the evoked [³H]ACh release and was significantly more effective in grafted (−70%) than in sham-operated (−56%) or lesion-only (−54%) rats. When physostigmine was superfused throughout, mecamylamine had no effect. Conversely, atropine induced a significant increase of [³H]ACh release in all groups, but this increase was significantly larger in sham-operated rats (+209%) than in the other groups (lesioned: +80%; grafted: +117%). Oxotremorine dose-dependently decreased the ([³H]ACh) release, but in lesion-only rats, this effect was significantly lower than in sham-operated rats. Whatever group was considered, 8-OH-DPAT, methiothepin and 2-methyl-serotonin failed to induce any significant effect on [³H]ACh release. In contrast, CP 93129 dose-dependently decreased [³H]ACh release. This effect was significantly weaker in grafted rats than in the rats of the two other groups. Our data confirm that cholinergic terminals in the intact hippocampus possess inhibitory muscarinic autoreceptors and serotonin heteroreceptors of the 5-HT_1B subtype. They also show that both types of receptors are still operative in the cholinergic terminals which survived the lesions and in the grafted cholinergic neurons. However, the muscarinic receptors in both lesioned and grafted rats, as well as the 5-HT_1B receptors in grafted rats show a sensitivity which seems to be downregulated in comparison to that found in sham-operated rats. In the grafted rats, both types of downregulations might contribute to (or reflect) an increased cholinergic function that results from a reduction of the inhibitory tonus which ACh and serotonin exert at the level of the cholinergic terminal.