Presynaptic muscarinic acetylcholine receptors suppress GABAergic synaptic transmission in the intermediate grey layer of mouse superior colliculus

The intermediate grey layer (the stratum griseum intermediale; SGI) of the superior colliculus (SC) receives cholinergic inputs from the parabrachial region of the brainstem. It has been shown that cholinergic inputs activate nicotinic acetylcholine (nACh) receptors on projection neurons in the SGI. Therefore, it has been suggested that they facilitate the initiation of orienting behaviours. In this study, we investigated the effect of muscarinic acetylcholine (mACh) receptor activation on GABAergic synaptic transmission to SGI neurons using the whole-cell patch-clamp recording technique in slice preparations from mice. The GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) evoked in SGI neurons by focal electrical stimulation were suppressed by bath application of 10 microm muscarine chloride. During muscarine application, both the paired-pulse facilitation index and the coefficient of variation of IPSCs increased; however, the current responses induced by a transient pressure application of 1 mm GABA were not affected by muscarine. Muscarine reduced frequencies of miniature IPSCs (mIPSCs) while the amplitudes of mIPSCs remained unchanged. These results suggested that mAChR-mediated inhibition of IPSCs was of presynaptic origin. The suppressant effect of muscarine was antagonized by an M1 receptor antagonist, pirenzepine dihydrochloride (1 microM), and a relatively specific M3 receptor antagonist, 4-DAMP methiodide (50 nM). By contrast, an M2 receptor antagonist, methoctramine tetrahydrochloride (10 microM), was ineffective. These results suggest that the cholinergic inputs suppress GABAergic synaptic transmission to the SGI neurons at the presynaptic site via activation of M1 and, possibly, M3 receptors. This may be an additional mechanism by which cholinergic inputs can facilitate tectofugal command generation.     

Muscarinic receptor M(2) in cat visual cortex: laminar distribution, relationship to gamma-aminobutyric acidergic neurons, and effect of cingulate lesions.

Acetylcholine can have diverse effects on visual cortical neurons as a result of variations in postsynaptic receptor subtypes as well as the types of neurons and subcellular sites targeted. This study examines the cellular basis for cholinergic activation in visual cortex via M(2) type muscarinic receptors in gamma-aminobutyric acid (GABA)-ergic and non-GABAergic cells, using immunocytochemical techniques. At light microscopic resolution, M(2) immunoreactivity (-ir) was seen in all layers except area and sublayer specific bands in layer 4. Subcellularly, M(2)-ir occurred in both dendrites and terminals that form symmetric and asymmetric junctions. Layers 5 and 6 were characterized by axosomatic contacts that displayed labeling in the presynaptic component, and layer 6 displayed perikaryal postsynaptic staining, suggesting that corticofugal output neurons may be modulated particularly strongly via M(2). Infragranular layers differed from the supragranular layers in that more labeled profiles were axonal than dendritic, indicating a dominant presynaptic effect by acetylcholine via M(2) there. Unilateral cingulate cortex cuts caused reduction of cholinergic and noradrenergic fibers in the lesioned hemisphere at light microscopic resolution; at electron microscopic resolution, the synapse density and axonal M(2) labeling were reduced, suggesting that M(2) was localized presynaptically on extrathalamic modulatory inputs. Dual labeling with GABA in visual cortex layer 5 showed that half of M(2)-labeled dendrites originated from GABAergic neurons. Given that only one-fifth of all cortical dendritic profiles are GABAergic, this prevalence of dual labeling indicates an enrichment of M(2) within GABAergic dendrites and, thus, implicates abundant postsynaptic action on GABAergic neurons via M(2). In contrast, only one-tenth of M(2)-labeled terminals originated from GABAergic neurons, suggesting that the presynaptic action of acetylcholine via M(2) receptors would be more selective for non-GABAergic terminals.     

Nicotine releases stereoselectively and Ca-dependently endogenous 3,4-dihydroxyphenylalanine from rat striatal slices

In superfused slices of rat striatum, nicotine-evoked release of endogenous 3,4-dihydroxyphenylalanine (DOPA) was studied in comparison with that of dopamine (DA). (if(±)-Nicotine (0.1–10 μM) constantly and repetitively released DOPA and DA over a similar time course in a concentration-dependent manner. The ratio of DOPA and DA evoked was approximately 1:2–3. The turnover rate of DOPA was about 300 times higher compared to DA. (±)-Nicotine (10 μM)-induced DOPA release was mecamylamine (20 μM)-sensitive, Ca²⁺-dependent and tetrodotoxin (0.3 μM)-insensitive. The (+)-isomer induced no DOPA release. These characteristics of DOPA release were almost the same as those of DA. Nicotine evokes endogenous DOPA via nicotinic cholinergic receptors in a manner similar to the transmitter DA. These findings further support a probable role of DOPA as a neuroactive substance in the rat central nervous system.     

Biochemical evidence for the presence of presynaptic receptors on dopaminergic nerve terminals

Two compartments of striatal synaptosome dopamine were identified by differential labelling with the isotopic presursors, L-tyrosine and Dopa, and from specific radioactivity measurement. Either, endogenous or exogenous L-tyrosine could provide a source for the dopamine pool synthesised and released in response to K+ depolarization, whereas external DOPA did not enter this pool. Acetylcholine (0.1 mM) in the presence of neostigmine (0.1 mg/ml) increased dopamine turnover as shown by increased formation of [14C] dopamine and [14C] DOPAC from [14C] DOPA. Haloperidol (0.65 mM) did not affect the size of dopamine pools but increased the conversion of [14C] DOPA to [14C] dopamine and the formation of [14C] DOPAC. Acetylcholine stimulated the release of dopamine from synaptosomes, which effect could be modified by both muscarinic and nicotinic antagonists. In the presence of the muscarinic antagonist, atropine, acetylcholine stimulated dopamine release, whereas in the presence of the nicotinic antagonists, hexamethonium (0.2 mM) or alpha-bungaro-toxin (0.188 muM), acetylcholine inhibited dopamine release. This showed that presynaptic cholinergic receptors were operational, excitatory nicotinic receptors in the former case and inhibitory muscarinic in the latter. The nicotinic receptor was shown to be saturable and to bind specifically 11.2 fmoles of [3H] alpha-bungarotoxin per mg. protein which could be prevented by hexamethonium or D-tubocurarine.     

Role of presynaptic nicotinic acetylcholine receptors in the regulation of gastrointestinal motility

Presynaptic nicotinic acetylcholine receptors (nAChRs) located on cholinergic terminals facilitate the release of acetylcholine (ACh), thereby constituting a fail-safe mechanism at strategic locations, such as the neuromuscular junction, where reliable transmission is vital. Accumulating data indicate that myenteric neurons in the enteric nervous system possess not only somatodendritic nAChRs, which mediate cholinergic transmission between neurons, but also presynaptic nAChRs. Functional evidence shows that these receptors mediate a positive feedback with respect to ACh release from myenteric motoneurons, and might therefore play an important role in the regulation of gastrointestinal motility. These presynaptic nAChRs were found to be more sensitive to nicotinic ligands than somatodendritic nAChRs and could therefore be primary targets of exogenous compounds, such as nicotine. This interaction might provide a neurochemical basis for the effect of smoking on gastrointestinal motility. Another important human pharmacological implication is based on our recent observation that monoamine uptake inhibitor-type antidepressant drugs are able to inhibit presynaptic nAChRs in the enteric nervous system. The disruption of the nAChR-mediated positive feedback modulation by antidepressants might explain the frequent occurrence of constipation, a common side effect, attributed to these drugs. Clarification of the role of presynaptic nAChRs in feedback mechanisms in the enteric nervous system might be instrumental in the development of new drugs affecting gastrointestinal motility.     

The cholinergic system and hippocampal plasticity

Acetylcholine is an essential excitatory neurotransmitter in the central nervous system and undertakes a vital role in cognitive function. Consequently, there is ample evidence to suggest the involvement of both nicotinic and muscarinic acetylcholine receptors in the modulation of synaptic plasticity, which is believed to be the molecular correlate of learning and memory. In the hippocampus in particular, multiple subtypes of both nicotinic and muscarinic receptors are present at presynaptic and postsynaptic loci of both principal neurons and inhibitory interneurons, where they exert profound bi-directional influences on synaptic transmission. Further evidence points to a role for cholinergic activation in the induction and maintenance of synaptic plasticity, and key influences on hippocampal network oscillations. The present review examines these multiple roles of acetylcholine in hippocampal plasticity.     

GABAergic modulation of nociceptive threshold: effects of THIP and bicuculline microinjected in the ventral medulla of the rat

Neurons of the nucleus raphe magnus (NRM) and nucleus reticularis gigantocellularis pars alpha (NGCp) have been implicated in the regulation of nociceptive threshold and production of antinociception. Previous studies have shown that the activity of these neurons is modulated by noradrenergic, cholinergic and serotonergic afferents. The present study examined whether these neurons are additionally subject to regulation by a GABAergic input. Microinjection of the GABA_A receptor agonist 4 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP; 0.3 or 1.0 μg) in the NRM or NGCp significantly decreased tail flick latency (TFL) and increased responsiveness to noxious pinch. Hot plate latency (HPL) was not affected by microinjection of 0.3 μg THIP. Although HPL was increased after microinjection of 1.0 μg THIP, this effect may reflect motoric disturbances. In contrast to the hyperalgesia produced by THIP, microinjection of the GABA_A receptor antagonist bicuculline methiodide (0.04 or 0.1 μg) produced a small, but significant increase in TFL. Responsiveness to noxious pinch and HPL were not affected by either dose. These findings indicate that neurons of the NRM or NGCp involved in the regulation of nociceptive threshold are subject to an inhibitory GABAergic input mediated by a GABA_A receptor. However, in contrast to previously described inhibitory inputs, the GABAergic influence does not appear to be tonically active to a substantial extent in the unanesthetized rat.     

Effect of cholinergic compounds on spontaneous quantal mediator release at the neuromuscular synapse of the frog

The action of cholinergic drugs on spontaneous quantal transmitter release has been investigated in the frog sartorius muscle. Acetylcholine and carbacholine decreased the miniature end-plate potential frequency. These presynaptic effects had no dependence on the potassium concentration in the bath solution. Nicotinic agonists--nicotine, tetramethylammonium and suberyldicholine had a similar effect, while muscarinic agents--methylfurmetide, oxotremorine and F-2268 (L- and D-stereoisomers) did not affect the transmitter release. The presynaptic effects of carbacholine and acetylcholine were abolished neither by atropine nor by d-tubocurarine and bensohexonium. It is suggested that there are nicotinic receptors on the frog motor nerve terminals that modify spontaneous quantal transmitter release and differ pharmacologically from nicotinic end-plate, ganglionic and presynaptic receptors of higher vertebrates.     

γ-Aminobutyric acid-mediated inhibition at cholinergic synapses formed by cultured retinal neurons

The purpose of this study was to investigate the effect of γ-aminobutyric acid (GABA) on the function of synapses formed by cholinergic neurons derived from the chick retina. We used an experimental culture system in which striated muscle cells served as postsynaptic targets for cholinergic neurons of the retina. This cell culture system permitted the physiological monitoring of acetylcholine release at synapses formed by retinal neurons. By plating a low of density dissociated retinal cells with myotubes, it was possible to study relatively isolated, presynaptic cholinergic neurons. We found that GABA and agonists, muscimol and isoguvacine, inhibited spontaneous transmission at retina-muscle synapses. These inhibitory effects were reversibly blocked by bicuculline, a GABA receptor antagonist. The benzodiazepine, flurazepam, potentiated GABA-mediated inhibition. Overall, our findings suggest a direct inhibitory action of GABA on the cholinergic retinal neurons studied in our cell culture system.     

The influence of acetylcholine,dopamine, and GABA on the functioning of the corticostriatal neuronal network in Alzheimer’s and Parkinson’s diseases: A hypothetical mechanism

A proposed model of the functioning of the basal ganglia complements the existing opinions about the complex interaction between cholinergic and dopaminergic systems. A hypothesis is proposed that one of the means of interaction between these systems is the operation of a negative feedback loop. In this loop, a conditioned stimulus evokes the excitation of dopaminergic neurons and GABAergic cells with long axons in the dopaminergic nuclei, which leads to an increase in the influence on dopamine D2 receptors on striatal cholinergic interneurons; an increase in their inhibition can lead to a pause in their responses. In turn, during this pause reduced action on presynaptic nicotinic receptors at axon terminals of dopaminergic neurons results in a decrease in dopamine release. In addition, dopaminergic neurons are under the inhibitory action of GABAergic striatonigral cells in the striosomes of the dorsal striatum and clusters in the ventral striatum. During the pause, stimulation of M2/M4 receptors located on these striatonigral cells weakens, which should promote potentiation of their excitation, subsequent enhancement of the inhibition of dopaminergic cells, and a decrease in the dopamine concentration in the striatum. In addition, a decrease in the stimulation of M1 receptors on striatopallidal cells and M2 receptors on striatonigral cells of the matrix during the pause should promote synergistic disinhibition through the direct and indirect pathways via the basal ganglia of certain groups of thalamic neurons and enhancement of the excitation of neocortical neurons connected with them. This interaction between cholinergic and dopaminergic systems contributes to the normal functioning of the various parallel cortico–basal ganglia–thalamocortical loops, which play a determining role in movement choice, sensory perception, learning, and intentional behavior. The proposed model implies that cholinergic and dopaminergic denervation of different structures, as well as changes in the density and affinity of receptors that are sensitive to acetylcholine, dopamine, and NMDA, which are typical for Alzheimer’s and Parkinson’s diseases, should lead to abnormal functioning of these loops. This may underlie various cognitive and motor disorders.     

幼年非洲爪蟾视顶盖区突触前、后膜上受体间的相互作用

目的　 记录幼年非洲爪蟾视顶盖区第六层神经元的自发性微突触后电流 (mPSCs)。 方法 　应用盲法电压膜片钳全细胞记录技术。 结果　 观察到用谷氨酸受体激动剂NMDA灌流脑片后先引起mIPSCs的频率明显增加并出现内向膜电流及高频的mEPSCs,经一段时间洗脱后mIPSCs和mEPSCs又均完全消失 ,而膜电流恢复到原来未加NMDA前的水平。GABA受体的激动剂GABA可诱发明显的外向膜电流。GABAa受体拮抗剂荷包牡丹碱 (bicucullin ,BM)不仅能将mIPSCs全部抑制掉 ,并且还可以诱发mEPSCs。谷氨酸受体的拮抗剂APV对mPSCs亦有类似的作用 ,不仅可以抑制顶盖神经元的mEPSCs,而且可以使原有的mIPSCs的频率和振幅均增加。 结论　幼年期的突触前、后膜上既有兴奋性谷氨酸能受体也有抑制性γ 氨基丁酸能受体 ,而且在突触前膜上受体可以调制突触末梢神经递质的释放。因此突触前、后膜上的受体间存在相互作用 ,以确保突触前后活动和功能上的稳定 ,从而达到神经网络的平衡。     

Stimulation by interleukin-1 (IL-1) of the release of rat corticotropin-releasing factor (CRF), which is independent of the cholinergic mechanism, from superfused rat hypothalamo-neurohypophysial complexes

The effects of interleukin-1 (IL-1) and interferon-γ (Ifn-γ) on the release of corticotropin-releasing factor (CRF) from superfused hypothalamo-neurohypophysial complexes (HNC) of rats were examined in the present study. In this in vitro system, the release of CRF from HNC was not affected by any dose of human recombinant Ifn-γ tested (0.1, 1 and 10 nM). In contrast, a rapid increase of CRF from HNC was elicited in a dose-dependent manner by human recombinant IL-1 and -1β in concentrations of 0.1–10 nM. The involvement of the cholinergic system in the mediation of the stimulatory effect of IL-1 on CRF release was evaluated. Acetylcholine in concentrations of 1–100 nM also elicited a rapid increase of CRF. The increase in CRF release induced by 10 nM of acetylcholine was completely suppressed in the presence of both hexamethonium (10 μM) and atropine (50 μM), a nicotinic and a muscarinic receptor antagonist, respectively. On the other hand, the increase in CRF release induced by 10 nM IL-1 or -1β was not affected by these two antagonists. These results indicate that IL-1 stimulates of CRF release through an action on the hypothalamo-neurohypophysial system, most likely on the hypothalamus, and that the stimulatory effect of IL-1 is probably independent of the cholinergic system.     

Involvement of GABA systems in acetylcholine release induced by 5-HT3 receptor blockade in slices from rat entorhinal cortex

The aim of the present study was to examine the role of 5-HT₃ receptors in spontaneous and K⁺-evoked acetylcholine (ACh) release from rat entorhinal cortex and striatal slices. The 5-HT3 receptor antagonists ondansetron and granisetron (0.01–10 μM) produced a concentration-dependent increase in both spontaneous and K⁺-evoked [³H]ACh release in the two brain regions studied. The release of ACh was Ca²⁺-dependent and tetrodotoxin-sensitive. 5-HT₃ receptor agonists, such as 2-methyl-5-HT and 1-phenylbiguanide, at concentrations up to 1 μM, did not show any intrinsic effect on [³H]ACh release in both rat brain regions. However, 2-methyl-5-HT, 1 μM, fully blocked the ondansetron-induced enhancement in both basal and K⁺-evoked ACh release, suggesting that 5-HT₃ through 5-HT₃ receptor activation, tonically inhibits ACh release. The possible implication of interposed inhibitory systems in ACh release after 5-HT₃ receptor blockade was subsequently analyzed. While the effect of ondansetron was not modified by haloperidol or naloxone, the GABA_A receptor antagonist bicuculline produced a marked potentiation of ACh release in the entorhinal cortex but not in the striatum. The results suggest that in this cortical area 5-HT activates 5-HT₃ receptors located on GABAergic neurons which in turn inhibit cholinergic function.     

Pre- and postsynaptic effects of norepinephrine on γ-aminobutyric acid-mediated synaptic transmission in layer 2/3 of the rat auditory cortex

Noradrenergic terminals from the locus coeruleus release norepinephrine (NE) throughout most brain areas, including the auditory cortex, where they affect neural processing by modulating numerous cellular properties including the inhibitory γ-aminobutyric acid (GABA)ergic transmission. We recently demonstrated that NE affects GABAergic signaling onto cortical pyramidal cells in a complex manner. In this study, we used a combination of patch-clamp recording and immunohistochemical techniques to identify the synaptic site and the location of the adrenergic receptors involved in the modulation of GABAergic signaling in cortical layer 2/3 of the rat. Our results showed that NE increases the frequency of spike-independent miniature inhibitory postsynaptic currents (mIPSCs), as well as the probability of release of unitary inhibitory postsynaptic currents (IPSCs) obtained with patch-clamp pair-recordings. The pharmacology of mIPSCs and the identification of adrenergic receptors in neurons containing the GABAergic marker parvalbumin (PV) suggest that NE increases the presynaptic probability of GABA release by activating α(2) - and β-receptors on PV-positive neurons. On the contrary, bath-applied NE or phenylephrine, decreased the current mediated by pressure application of the GABA(A) -receptor agonist muscimol, as well as the amplitude-but not the frequency-of mIPSCs, indicating that activation of postsynaptic α(1) adrenoceptors reversibly depressed GABAergic currents. We speculate that while a generalized postsynaptic decrease of GABAergic inhibition might decrease the synaptic activation threshold for pyramidal neurons corresponding to an alert state, NE might promote perception and sensory binding by facilitating lateral inhibition as well as the production of γ-oscillations by a selective enhancement of perisomatic inhibition.     

Effect of neostigmine on the hippocampal noradrenaline release: role of cholinergic receptors

The effect of the cholinesterase inhibitor neostigmine on hippocampal noradrenaline (NA) release was studied using in vivo microdialysis. Local application of neostigmine significantly increased the release of NA. The effect was potentiated by coperfusion of the nicotinic antagonist mecamylamine but was completely blocked by the muscarinic antagonist atropine. The neostigmine-evoked NA release was not affected by the M2-selective muscarinic antagonist gallamine but was completely blocked by the M1-selective muscarinic antagonist pirenzepine. While muscarinic antagonists had no effect on the resting release of NA, mecamylamine increased it. Our data indicate that acetylcholine can stimulate the hippocampal NA release via M1 muscarinic receptors and that a population of nicotinic receptors mediate inhibitory tone on hippocampal NA release. The fact that neostigmine is able to enhance both cholinergic and noradrenergic neurotransmission may help to understand the beneficial effect of cholinesterase inhibitors in Alzheimer's disease.     

Prostaglandin E2 Inhibits Spontaneous Inhibitory Postsynaptic Currents in Rat Supraoptic Neurones via Presynaptic EP Receptors

Prostaglandin E2 (PGE2) has been implicated in the excitatory regulation of magnocellular neurones in the supraoptic nucleus (SON). We have recently reported that PGE2 excited SON neurones by directly activating postsynaptic PGE2 receptors (EP receptors) of a subclass other than EP1-3, but did not affect excitatory postsynaptic currents (EPSCs). In the present study, we examined presynaptic effects of PGE2 on rat SON neurones by measuring spontaneous inhibitory postsynaptic currents (IPSCs) by a slice patch-clamp technique. PGE2 inhibited spontaneous IPSCs in a dose-dependent and reversible manner. PGE2 selectively suppressed the frequency of IPSCs without affecting the amplitude of IPSCs in the presence of tetrodotoxin, a blocker of Na+ channels, indicating that the effects were presynaptic. The inhibitory effects of PGE2 on the frequency of IPSCs were mimicked by the EP1/EP3 agonists, 17PT-PGE2 and sulprostone, and the EP2/EP3 agonist, misoprostol, whereas the EP2 agonist, butaprost, or the FP agonist, fluprostenol, had little effect. The effects of PGE2 on IPSCs were unaffected by the selective EP1 antagonist, SC-51322. They were unaffected also by antagonists of GABAB and alpha2 adrenergic receptors, which are present at presynaptic terminals of GABA neurones in the SON and cause suppression of spontaneous IPSCs. The inhibitor of PG synthesis, indomethacin, had little effect on spontaneous IPSCs and on the inhibitory effects of PGE2 as well as of the GABAB agonist, baclofen, and noradrenaline. These results suggest that PGE2 inhibits release of GABA from the GABAergic terminals innervating SON neurones by activating presynaptic EP receptors, presumably of the EP3 subclass, and that such a presynaptic mechanism may play a role in the excitatory regulation of SON neurones by PGE2.     

Allosteric modulation of alpha 7 nicotinic receptors selectively depolarizes hippocampal interneurons, enhancing spontaneous GABAergic transmission

The role of postsynaptic nicotinic receptors for acetylcholine (nAChRs) in mediating fast neurotransmission processes in the CNS is controversial. Here we have studied the modulation of synaptic transmission by an agonist (choline) and an allosteric modulator (5-OH-indole) of alpha7 nAChRs in rat hippocampal neuronal cultures. Choline evoked a fast inactivating inward current, causing neuron depolarization and action potential discharge, thereby enhancing the spontaneous postsynaptic current activity (sPSCs). This effect was markedly enhanced when both choline and 5-OH-indole were applied together and was blocked by the selective alpha7 nAChR antagonist methyllycaconitine. This choline action was suppressed by the GABA(A) receptor antagonist bicuculline, while the glutamatergic receptor antagonist kynurenic acid had no effect. Frequency, but not amplitude or area, of both excitatory and inhibitory miniature postsynaptic currents (mEPSCs and mIPSCs) were drastically reduced when Ca(2+) influx was blocked by Cd(2+). Additionally, nAChR activation did not modify the mIPSCs. These data suggest that Ca(2+) influx through the highly Ca(2+)-permeablealpha7 nAChRs was insufficient to directly activate neurotransmitter release, suggesting that a tight colocalization of this receptor with secretory hot spots is unlikely. In a few cases, the activation of alpha7 AChRs led to a suppression of spontaneous synaptic transmission. This effect may be related to the potentiation of GABAergic interneurons that inhibit the spontaneous activity of neurons making synapses with the cell under study. We suggest that GABA release is modulated by alpha7 nAChRs. Thus, selective allosteric modulators of alpha7 nAChRs could have potential therapeutic applications in brain disorders such as epilepsy and schizophrenia and in alterations of cognition and sensory processing.     

The influx of Ca and the release of noradrenaline evoked by the stimulation of presynaptic nicotinic receptors of chick sympathetic neurons in culture are not mediated via L-, N-, or P-type calcium channels

We have shown earlier that nicotinic agonists induce the release of noradrenaline from chick sympathetic neurons in culture in two ways: (a) by activating the postsynaptic nicotinic receptors on nerve cell bodies, giving rise to spreading electrical activity and opening of voltage operated calcium channels in neuronal processes; (b) by activating the presynaptic nicotinic receptors on neuronal processes. In the present work, we investigated the contribution of various pathways to the observed Ca²⁺ influx and subsequent noradrenaline release. Sympathetic neurons in culture were stimulated either by the nicotinic agonist dimethylphenylpiperazinium or electrically, in the presence or absence of tetrodotoxin and of specific blockers of calcium or nicotinic channels, and the effects on [Ca²⁺]_i in the area of neuronal processes and on noradrenaline release were measured. Under control conditions, the N-type channel blocker ω-conotoxin (0.1 μmol/1) diminished the release of noradrenaline and the increase of intraterminal Ca²⁺ by 48% and 55%, respectively, whereas the L-type channel blocker (+)Bay k 8644 (1 μmol/1) diminished the release of noradrenaline by 25% and the increase of [Ca²⁺]_i by 39%. The P-type channel blocker ω-agatoxin (0.3 μmol/1) had no effect. The effects of the L-type channel ligands were complex and could only be explained on the assumption that, at high concentrations, these drugs also act as nicotinic antagonists. Tetrodotoxin blocked the Ca²⁺ response evoked by electrical stimulation whereas DMPP applied in the presence of tetrodotoxin still evoked an increase of [Ca²⁺]_i and the release of noradrenaline (27% and 30% of control without tetrodotoxin, respectively). These residual responses were not blocked by any of the calcium channel blockers used or by their combination. Apparently, a substantial part of the influx of Ca²⁺ induced by the activation of presynaptic nicotinic receptors is not carried by the N-, L- or P-type channels and probably occurs directly via the open channels of nicotinic receptors.     

Postsynaptic M1 and M3 receptors are responsible for the muscarinic enhancement of retrograde endocannabinoid signalling in the hippocampus

The cholinergic system is crucial for higher brain functions including learning and memory. These functions are mediated primarily by muscarinic acetylcholine receptors (mAChRs) that consist of five subtypes (M(1)-M(5)). A recent study suggested a novel role of acetylcholine as a potent enhancer of endocannabinoid signalling that acts retrogradely from postsynaptic to presynaptic neurons. In the present study, we further investigated the mechanisms of this cholinergic effect on endocannabinoid signalling. We made paired whole-cell recordings from cultured hippocampal neurons, and monitored inhibitory postsynaptic currents (IPSCs). The postsynaptic depolarization induced a transient suppression of IPSCs (DSI), a phenomenon known to involve retrograde signalling by endocannabinoids. The cholinergic agonist carbachol (CCh) markedly enhanced DSI at 0.01-0.3 microM without changing the presynaptic cannabinoid sensitivity. The facilitating effect of CCh on DSI was mimicked by the muscarinic agonist oxotremorine-M, whereas it was eliminated by the muscarinic antagonist atropine. It was also blocked by a non-hydrolizable analogue of GDP (GDP-beta-S) that was applied intracellularly to postsynaptic neurons. The muscarinic enhancement of DSI persisted to a substantial degree in the neurons prepared from M1-knockout and M3-knockout mice, but was virtually eliminated in the neurons from M1/M3-compound-knockout mice. CCh still enhanced DSI significantly under the blockade of postsynatpic K(+) conductance, and did not significantly influence the depolarization-induced Ca(2+) transients. These results indicate that the activation of postsynaptic M1 and M3 receptors facilitates the depolarization-induced release of endocannabinoids.