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Purpose: Status epilepticus (SE) has been considered an epileptogenic factor in humans. in the pilocarpine (PILO) model, after a brief period marked by SE, the rats exhibit recurrent spontaneous seizures, mimicking the clinical features of temporal lobe epilepsy. the aim of our study was to identify the molecular actions of PILO that could account for its ability to induce SE.Methods: Whole-cell mode of the patch-clamp technique was applied to cultured hippocampal neurons (2-3 weeks old) in the absence and in the presence of PILO (1-10 muM), to study the spontaneous activity, the evoked, and the miniature postsynaptic currents. the postsynaptic currents were isolated pharmacologically.Results: PILO (1 and 10 muM) caused an initial increase followed by a decrease in the frequency of spontaneous activity. the increase in the frequency of excitatory postsynaptic currents (EPSCs) and inhibitory PSCs (IPSCs) was blocked by atropine (1 muM), indicating that this effect is mediated through muscarinic receptors. PILO also promoted a brief increase of the amplitude of IPSCs indirectly evoked by stimulation of a neuron synaptically connected to the neuron under study. Conversely, PILO promoted a sustained increase on the amplitude of electrically evoked EPSCs. in presence of tetrodotoxin (TTX; 300 nM), PILO (1 muM) increased the frequency of miniature EPSCs and IPSCs without changing their amplitude during the first 3 min of application.Conclusions: These results indicate that PILO acting through muscarinic receptor causes an imbalance between excitatory and inhibitory transmission that can result in the generation of SE observed in animals acutely treated with PILO. Univ Maryland, Sch Med, Baltimore, MD 21201 USA Universidade Federal de São Paulo, EPM, São Paulo, Brazil Univ Maryland, Sch Med, Dept Pharmacol & Expt Therapeut, Baltimore, MD USA UFRJ, Ctr Ciencias Saude, Inst Ciencias Biomed, Dept Farmacol Basica & Clin, Rio de Janeiro, Brazil Universidade Federal de São Paulo, EPM, São Paulo, Brazil Web of Science

This study was designed to investigate the effects on single skeletal muscle fibers of a novel thienylhydrazone, referred to as LASSBio-294, which is a bioisoster of pyridazinone compounds that inhibit the cyclic AMP-specific phosphodiesterase (PDE) 4. Twitch and fatigue were analyzed in single skeletal muscle fibers isolated from either the semitendinous or the tibialis anterior muscles dissected from the frog Rana pipiens. LASSBio-294 (12.5-100 microM) increased twitch tension, accelerated the maximal rate of tension decay during relaxation, and had very little effect in the maximal rate of tension development of muscle fibers directly stimulated ator =30 Hz. The positive inotropic effect of LASSBio-294 developed slowly, reaching its maximum at 40 min and was inversely proportional to the frequency of stimulation, becoming negligible at 60 and 90 Hz. The concentration-response relationship for LASSBio-294-induced potentiation of twitch tension was bell-shaped, with maximal effect occurring at 25 microM. In addition, LASSBio-294 reduced development of fatigue induced by tetanic stimulation of the muscle fibers and reduced the time needed for 80% prefatigue tension recovery after fatigue had developed to 50% of the maximal pretetanic force. These effects of LASSBio-294 can be fully explained by stimulation of the sarcoplasmic reticulum Ca2+ pump and could be ascribed to an increase in cellular levels of cyclic AMP due to PDE inhibition. The novel thienylhydrazone LASSBio-294 may be useful for treatment of patients suffering from conditions in which muscle fatigue is a debilitating symptom (e.g., chronic heart failure).

Galantamine (Reminyl) is a novel drug treatment for mild to moderate Alzheimer's disease (AD). Originally established as a reversible inhibitor of the acetylcholine-degrading enzyme acetylcholinesterase (AChE), galantamine also acts as an allosterically potentiating ligand (APL) on nicotinic acetylcholine receptors (nAChR). Having previously established this second mode of action on nAChRs from murine brain, we demonstrate here the same action of galantamine on the most abundant nAChR in the human brain, the alpha4/beta2 subtype. This nAChR-sensitizing action is not a common property of all, or most, AChE inhibitors, as is shown by the absence of this effect for other therapeutically applied AChE inhibitors including tacrine, metrifonate, rivastigmine and donepezil. The possible benefits for therapy of AD of an APL action on nicotinic receptors is discussed.

In the present study, the whole-cell mode of the patch-clamp technique was applied to neurons of the CA1 pyramidal layer of rat hippocampal slices to investigate the effects of the organophosphate (OP) sarin on field stimulation-evoked and on tetrodotoxin (TTX)-insensitive postsynaptic currents (PSCs) mediated by activation of type A gamma-aminobutyric acid (GABA) receptors or AMPA-type glutamate receptors. At 0.3-1 nM, sarin reduced the amplitude of GABA-mediated PSCs and had no effect on the amplitude of glutamatergic PSCs evoked by field stimulation of neurons synaptically connected to the neuron under study. The effect of sarin on evoked GABAergic PSCs was unrelated to cholinesterase inhibition, was partially reversed upon washing of the neurons with sarin-free external solution, and was mediated by a direct interaction of the OP with muscarinic acetylcholine receptors present on presynaptic GABAergic neurons. Sarin had no effect on the amplitude or kinetics of GABA- or glutamate-mediated miniature postsynaptic currents (MPSCs) recorded in the presence of the Na+-channel blocker TTX (300 nM), indicating that the OP does not interact with GABA(A) or glutamate receptors. Further, sarin did not alter the frequency of GABAergic or glutamatergic MPSCs, a finding that led to the conclusion that this OP does not affect the TTX-insensitive release of neurotransmitters. A selective reduction by sarin of the action potential-dependent release of GABA in the hippocampus can account for the occurrence of seizures in intoxicated subjects.

We have studied, on the transcriptional, protein and functional level, the expression of alpha7 nicotinic acetylcholine receptors (nAChR) in the course of rat muscle development, denervation and renervation. At foetal day 13, alpha7 nAChR expression was observed in somites and developing muscles of the back, but not yet in migrating myoblasts. Two days later, concomitant with myoblast aggregation, the alpha7 isoform began to be expressed in isolated myoblasts, with the highest level of expression in the frontal zone of the migrating wave. On foetal day 18, a time when the myoblasts in the upper hindleg have fused, alpha7 nAChR expression was most prominent in the outer layer of muscle tissue. The highest level of expression was observed in the first postnatal week, when practically all muscle cells stained positively for alpha7 protein. During the following weeks, alpha7 nAChR expression slowly decreased and practically disappeared in adult hindleg muscle. Following chronic denervation of adult Soleus muscle fibres, expression of alpha7 nAChR returned within 2-4 weeks. Electrophysiological measurements showed that the alpha7 nAChR of chronically denervated soleus muscle fibres were functional and, in particular, that they could be activated by choline. The presence of the alpha7 nAChR in developing and denervated muscle suggests a role for this nicotinic receptor in neuronal pathfinding and/or endplate stabilization.

ACh receptors sensitive to nicotine (nAChR) are present in human skin keratinocytes and in bronchial epithelial cells. They are stimulated by ACh secreted by the same cells that express them, and they modulate cell motility and shape. A variety of non-neuronal tissues, including endothelial cells, synthesize ACh, which raises the possibility that they are sensitive to nicotine. We demonstrate here that endothelial cells that line blood vessels express functional nAChRs. Their structure and ion-gating properties are similar to those of the nAChRs expressed by ganglionic neurons and by skin keratinocytes and bronchial epithelial cells. In situ hybridization experiments using primary cultures of endothelial cells from human aorta demonstrated the presence in these cells of the subunits believed to contribute to ganglionic ACh receptors (AChRs) of the alpha3 subtype: alpha3, alpha5, beta2 and beta4. Binding of radiolabeled epibatidine-a high-affinity specific ligand of certain neuronal AChRs, including the alpha3 subtypes-revealed the presence of approximately 900 specific binding sites per cell. We assessed the presence of functional AChRs by patch-clamp experiments. Cultured human endothelial cells express ion channels that are opened by (+)-anatoxin-a and are blocked by dihydro-beta-erythroidine. These are specific agonist and antagonist, respectively, of neuronal AChRs of the alpha3 subtype. The ion-gating properties of the endothelial AChRs were similar to those of neuronal ganglionic AChRs. The presence of AChRs sensitive to nicotine in endothelial cells may be related to the toxic effects of nicotine on the vascular system.

In our study, evidence is provided that strychnine, a competitive antagonist at glycine-gated Cl- channels, is also a potent competitive antagonist at native alpha-7-containing, alpha-bungarotoxin-sensitive nicotinic acetylcholine receptor (nAChRs). To address the effects of strychnine on two types of nicotinic responses, the whole-cell mode of the patch-clamp technique was applied to rat hippocampal neurons in culture. Type IA and type II nicotinic currents evoked by acetylcholine (ACh) were inhibited by strychnine in a concentration-dependent manner with IC50S of 1.2 and 38 microM, respectively. Strychnine (2 microM) decreased the peak amplitude of the alpha-bungarotoxin-sensitive type IA current in a voltage-independent manner and prolonged the decay phase of this current. The concentration-response curve for ACh in evoking type IA current showed a parallel shift to the right in the presence of strychnine (2 microM); the EC50 for ACh was increased from 0.4 to 0.8 mM. These findings suggest that strychnine acts as a competitive antagonist of ACh at the alpha 7 nAChRs that subserve type IA current. In contrast, the inhibition by strychnine of type II current was strongly voltage dependent, and the decay phase of this current was markedly accelerated by the toxin, suggesting an open-channel blockade by strychnine of the alpha 4 beta 2 nAChRs subserving type II currents. Preexposure of the neurons to strychnine enhanced its ability to decrease the peak amplitude of type II currents, indicating that the toxin may also act on alpha 4 beta 2 nAChR channels that are not open. It is concluded that strychnine is a potent competitive antagonist of ACh at neuronal alpha 7 nAChRs and a noncompetitive antagonist at the alpha 4 beta 2 nAChR.

In the present study we investigated electrophysiologically the nicotinic responses of pyramidal neurons and interneurons visualized by infrared-assisted videomicroscopy and fluorescence in the CA1 field of hippocampal slices obtained from 8- to 24-day-old rats. Application of nicotinic agonists to CA1 neurons evoked at least four types of nicotinic responses. Of major interest was the ability of these agonists to induce the release of gamma-aminobutyric acid (GABA) from interneurons. Slowly decaying ACh whole-cell currents and GABA-mediated postsynaptic currents could be recorded from pyramidal neurons and interneurons, whereas fast-decaying nicotinic currents and fast current transients were recorded only from interneurons. Nicotinic responses were sensitive to blockade by d-tubocurarine (10 microM), which indicated that they were mediated by nicotinic acetylcholine receptors (nAChRs). The slowly decaying currents, the postsynaptic currents and the fast current transients were insensitive to blockade by the alpha-7 nAChR-specific antagonist methyllycaconitine (up to 1 microM) or alpha-bungarotoxin (100 nM). On the other hand, the slowly decaying nicotinic currents recorded from the interneurons were blocked by the alpha4beta2 nAChR-specific antagonist dihydro-beta-erythroidine, and the fast-desensitizing nicotinic currents were evoked by the alpha-7 nAChR-specific agonist choline. In experimental conditions similar to those used to record nicotinic responses from neurons in slice (i. e., in the absence of tetrodotoxin), we observed that nicotinic agonists can also induce the release of GABA from hippocampal neurons in culture. In summary, these results provide direct evidence for more than one subtype of functional nAChR in CA1 neurons and suggest that activation of nAChRs present in GABAergic interneurons can evoke inhibitory activity in CA1 pyramidal neurons, thereby modulating processing of information in the hippocampus.

The effects of amantadine on nicotinic acetylcholine receptors (nAChRs) of hippocampal neurons were studied by recording three types of acetylcholine (ACh)-evoked currents, using the whole-cell patch-clamp technique. The rapidly desensitizing type IA nicotinic current, which is alpha-bungarotoxin-sensitive and is mediated by nAChRs bearing alpha 7 subunits, was inhibited by application of amantadine to neurons for 10 min (IC50 = 6.5 microM), but the potency of ACh (EC50 = 0.27 mM) was not affected by the drug. Amantadine (30-50 microM) attenuated the peak current amplitude in a voltage-dependent manner, with greater effect at negative than at positive membrane potentials. In contrast, the decay phase of the currents was shortened in a voltage-independent manner. When amantadine was coapplied briefly with ACh, the drug was markedly less potent (IC50 = 130 microM). Thus, the noncompetitive effects of amantadine on the type IA nicotinic current are complex, involving actions on the closed and desensitized states of the alpha 7 nAChR. The slowly desensitizing, alpha-bungarotoxin-insensitive nicotinic currents of type II, which is inhibited by dihydro-beta-erythroidine and is mediated by alpha 4 beta 2 nAChRs, and of type III, which is inhibited by mecamylamine and is mediated by alpha 3 beta 4 nAChRs, were also sensitive to inhibition by amantadine. The peak amplitude of type II current was reduced only slightly by 10 microM amantadine coapplied with ACh, but the decay-time constant and amplitude of the sustained current were markedly reduced. Type III current was also inhibited when amantadine was briefly coapplied with ACh. In contrast to its effects on nicotinic currents, amantadine at 10 microM did not affect currents evoked by N-methyl-D-aspartate plus glycine, gamma-aminobutyric acid, glycine or kainate. Thus, on cultured hippocampal neurons, amantadine preferentially inhibits nicotinic currents.

Similar to other neuroreceptors of the vertebrate central nervous system, the nicotinic acetylcholine receptor (nAChR) is subject to modulatory control by allosterically acting ligands. Of particular interest in this regard are allosteric ligands that enhance the sensitivity of the receptor to its natural agonist acetylcholine (ACh), as such ligands could be useful as drugs in diseases associated with impaired nicotinic neurotransmission. Here we discuss the action of a novel class of nAChR ligands which act as allosterically potentiating ligands (APL) on the nicotinic responses induced by ACh and competitive agonists. In addition, APLs also act as noncompetitive agonists of very low efficacy, and as direct blockers of ACh-activated channels. These actions are observed with nAChRs from brain, muscle and electric tissue, and they depend on the structure of the APL and the concentration range applied. We focus here on Torpedo nAChR because (i) the unusual pharmacology of these ligands was first discovered with this system, and (ii) large quantities of this receptor are readily available for biochemical studies.

To assess the density and distribution of functional nicotinic acetylcholine receptors (nAChRs) and gamma-aminobutyric acid (GABA) receptors on hippocampal neurons, we have combined infrared videomicroscopy with a nanorobotic micromanipulator system and studied the receptor-mediated currents by using the whole-cell patch-clamp technique. Acetylcholine or GABA was applied by pressure ejection onto a small segment of either cell soma or dendrite, and the resulting current was measured in cultured hippocampal neurons by using a whole-cell pipette positioned at the cell soma. Type IA nicotinic currents, sensitive to blockade by methyllycaconitine (1 nM) and alpha-bungarotoxin and associated with alpha 7-subunit-containing nAChRs, type II currents, sensitive to blockade by dihydro-beta-erythroidine (100 nM) subserved by alpha 4 beta 2 nAChRs, and GABA-mediated currents were evoked when the agonists were applied to either cell soma or the dendrites. Analysis of the current amplitude with respect to the membrane area covered by the applied agonist provided an estimation of the receptor density along the somato-dendritic axis of the hippocampal neurons. Such analysis revealed: 1) a nonuniform distribution for the receptor types studied; 2) a higher density of nAChR and GABA receptors at the dendrites than at the soma; and 3) an increasing density for both nAChR subtypes with distance from the center of the cell soma, but increasing and then decreasing density for the GABA receptor. Exposure of cultured hippocampal neurons to colchicine (100 nM for 3 days) produced a dramatic reduction in the dendritic branching, and this morphological feature was associated with a significant decrease in the receptor density, such an effect being more prominent for nAChRs than for GABA receptors. Given the high Ca+2 permeability of nAChRs, the dendritic localization of nAChRs suggest that they are involved in modulating the synaptic efficacy at the level of the dendrites.

The effects of lead (Pb++) on N-methyl-D-aspartate (NMDA) receptor function of rat hippocampal neurons in culture were studied by use of the whole-cell patch-clamp technique. Currents activated by NMDA (100 microM) in the presence of nonsaturating concentrations of glycine (0.01-0.05 microM) were potentiated in a voltage-independent manner by Pb++ (1-10 microM), and the potentiation was antagonized by 50 microM kynurenic acid. Increasing extracellular Ca++ from 1 to 10 mM similarly potentiated the NMDA-activated currents in the presence of a nonsaturating concentration of glycine (0.2 microM). The potentiation of NMDA-activated currents by low micromolar concentrations of Pb++ may be mediated by this cation's ability to increase the affinity of the NMDA receptor for glycine. In the presence of 10 microM glycine and 2 mM Ca++, Pb++ reduced the peak amplitudes of currents activated by NMDA (100 microM) in a voltage-independent manner (IC50 = 5.9 microM Pb++, Hill coefficient (nH) = 1.2). Also, steady-state currents activated by NMDA (50 microM) were inhibited by rapid application of Pb++ (IC50 = 3.2 microM, nH = 0.7). Increasing extracellular Ca++, in the presence of 10 microM glycine, reduced the NMDA-activated currents and shifted the Pb++ concentration-response curves to the right: at 0.2, 2 and 20 mM Ca++, the IC50 values of Pb++ were 3.0, 5.9 and 12.5 microM and the nH values were 0.9, 1.2 and 1.1, respectively. The finding that external Ca++ antagonized the inhibitory effect of Pb++ suggests that the noncompetitive inhibitory action of Pb++ with respect to glycine and NMDA may be mediated by Pb++ competition with Ca++ for a site on the NMDA receptor.

The presence of functional nicotinic acetylcholine receptors (nAChRs) on cultured neurons of the rat olfactory bulb was evaluated using the whole-cell patch-clamp technique. Application of acetylcholine (ACh) to 78% of the tested olfactory bulb neurons evoked whole-cell currents (referred to as direct response), which are very similar in characteristics to type IA currents. Their peak amplitude increased, while their rise-time and decay-time constants decreased with increasing agonist concentration. In 12% of the neurons, ACh evoked single or multiple miniature postsynaptic currents (referred to as indirect response) for which amplitude, rise time, and decay-time constants were not dependent upon the ACh concentration. Methyllycaconitine (1 nM), a selective competitive antagonist at the alpha-bungarotoxin-sensitive neuronal nAChR, reversibly blocked both responses, whereas 6-cyano-7-nitroquinoxaline-2,3-di-one (10 microM) reversibly blocked only the indirect responses. Whereas tetrodotoxin (0.2-2 microM) failed to affect the indirect response, Ca(+2)-free, Mg(+2)-containing external solution decreased reversibly and significantly the frequency of ACh-evoked miniature postsynaptic currents. The pharmacology and kinetics of the two types of responses are consistent with the existence in the olfactory bulb neurons of alpha-bungarotoxin-sensitive nAChRs at both postsynaptic and presynaptic sites, the presynaptic receptors being located on glutamatergic synapses where they modulate the release of the transmitter. The dimensions of the soma and dendrites of the neurons suggest that the direct response is obtained from periglomerular and/or granular neurons, and the indirect response from short-axon and/or external tufted cells. The present results suggest that 1) nicotinic synaptic transmission could play an important role in modulating the bulbar output at the glomerular level, and 2) a presynaptic modulatory effect is one of the functions for the alpha-bungarotoxin-sensitive nAChRs in the mammalian central nervous system.

In the present study, the patch-clamp technique was applied to rat hippocampal neurons or myoballs in culture to study the actions of alpha-conotoxin-ImI on the native alpha-bungarotoxin-sensitive, presumably alpha 7-bearing, neuronal nicotinic receptor and on other ligand-gated channels. Preexposure of the neurons for 5 min to alpha-conotoxin-ImI decreased the peak amplitude of alpha-BGT-sensitive currents (referred to as type IA currents) in a concentration-dependent fashion. Several lines of evidence revealed that the inhibitory effect of alpha-conotoxin-ImI was competitive with respect to the agonist (IC50 approximately 85 nM) and reversible by washing. At 300 nM, alpha-conotoxin-ImI decreased by only 15% the peak amplitude of ACh-evoked currents in rat myoballs, did not affect the activation of currents gated by gamma-aminobutyric acid, glycine, N-methyl-D-aspartate, kainate, or quisqualate in hippocampal neurons, but reduced to approximately 60% the peak amplitude and shortened the decay phase of curare-sensitive, serotonin-gated currents in these neurons. The competitive and reversible nature of the alpha-conotoxin-ImI-induced inhibition of native alpha 7-bearing neuronal nicotinic receptors makes this peptide a valuable new tool for the functional and structural characterization of these receptors in the central nervous system.

Applying the whole-cell mode of the patch-clamp technique to cultured hippocampal neurons, we demonstrated that extracellular Ca++ modulates the activation and inactivation of type IA nicotinic currents, ie., the currents subserved by alpha-bungarotoxin (alpha-BGT)-sensitive, alpha-7-containing nicotinic acetylcholine receptors (nAChRs). The rundown profile of acetylcholine (ACh)-induced type IA currents that were obtained using patch pipettes filled with F(-)-based internal solution had two components: the first component of rundown was counteracted by a more physiological internal solution containing an organic anion (malate or aspartate), suggesting energy dependence; the second component exhibited dependence on concentration of CaCl2 added to the external solution ([Ca++]o), with rundown minimized at 0.32 mM. The inward rectification of Ach-elicited type IA currents, induced by intracellular Mg++ was augmented by lowering [Ca++]o (from 2 to 0.32 mM). Moreover, extracellular Ca++ (0.01-10 mM) acted in a concentration-dependent manner (IC50 = 0.26 mM) to decrease the cooperativity induced by ACh (nH was reduced from 2.7 to 1). Extracellular Ca++ (EC50 = 0.1 mM) also increased the efficacy of ACh, but exposure to [Ca++]o from 1 to 32 mM decreased the efficacy of ACh and inactivated the alpha-BGT-sensitive nAChRs (IC50 = 11 mM). In conclusion, Ca++ regulates agonist efficacy and cooperativity at the alpha-BGT-sensitive neuronal nAChR, modulates rundown and counteracts Mg++ -dependent inward rectification of type IA currents. It is suggested that the regulation by Ca++ of the alpha-BGT-sensitive nAChR activity could modulate many neuronal functions.

Similar to the gamma-aminobutyric acidA receptor and the N-methyl-D-aspartate subtype of glutamate receptor, neuronal nicotinic acetylcholine receptors are subject to positive modulatory control by allosterically acting ligands. Exogenous ligands such as galanthamine and the neurotransmitter 5-hydroxytryptamine, when applied in submicromolar concentrations with nicotinic agonists, significantly increase the frequency of opening of nicotinic receptor channels and potentiate agonist-activated currents. Because these effects have been shown to be blocked by the monoclonal antibody FK1, they are mediated by binding sites that are located on alpha subunits of nicotinic receptors and distinct from those for acetylcholine and acetylcholine-competitive ligands. At higher concentrations, the potentiating effect of these ligands decreases and is eventually overcome by an inhibition of the agonist-induced response. The sensitizing actions of galanthamine, 5-hydroxytryptamine, and related compounds, at submicromolar concentrations, may reflect the existence of cross-talk between adjacent neuroreceptors and synapses in the central nervous system and thus suggests the formation of transiently active chemical networks in the vertebrate brain.

The nicotinic-agonist properties of epibatidine, an alkaloid originally isolated from the Ecuadorian frog Epipedobates tricolor, was investigated in rat hippocampal neurons grown in culture. The ability of epibatidine enantiomers to evoke nicotinic whole-cell currents of type IA, sensitive to blockade by alpha-bungaro-toxin and methyllycaconitine and presumably subserved by an alpha-7-based nAChR, and of type II, sensitive to blockade by dihydro-beta-erythroidine and presumably subserved by an alpha 4 beta 2-based nAChR, was studied and compared with that of other nicotinic agonists. Epibatidine elicited type IA currents with EC50 values of 2.9 and 4.3 microM for the (-)- and (+)- enantiomers, respectively. The potency of the agonists in evoking type IA currents was as follows: (-)-epibatidine(+)- anatoxin-a(+)-epibatidine(-)-nicotineDMPPcytisineacetylcholine. The EC50 values of (-)- and (+)- epibatidine in eliciting type II currents were 19 and 15 nM, respectively. The order of potency of the agonists in evoking type II currents was: (+)-epibatidine(-)-epibatidinecytisine(+)-anatoxin-a(-)-nicotineacetylcholineDMPP. Although these agonists produced nearly identical maximal type IA responses, the size of the maximal type II responses varied with the agonist. Cytisine and (+)-anatoxin-a were the least efficacious agonists in inducing type II currents. Enantiomers of epibatidine showed the least stereoselectivity, those of nicotine showed a moderate stereoselectivity, and those of anatoxin-a exhibited the highest stereoselectivity in inducing either type of currents. In summary, these results suggest that epibatidine can be used as a novel nicotinic agonist for the study of type II currents, and that the nicotinic acetylcholine receptor related to type II currents may be one of the key target sites through which agonists such as epibatidine and nicotine elicit their behavioral actions in vivo.

The sensitivity of N-methyl-D-aspartate- (NMDA) induced whole-cell and single-channel currents to Pb2+ was studied in neurons acutely dissociated from the hippocampus of 3- to 30-day-old rats. The amplitude of NMDA-induced whole-cell currents in the neurons increased with age of the animals until they were 10 to 12 days old, thereafter remaining nearly unchanged. As in cultured hippocampal neurons, Pb2+ at both 10 and 30 microM inhibited NMDA-induced currents, and the sensitivity of the currents to Pb2+ decreased with age of the rats. The sensitivity correlated with the age-related expression of two components of the NMDA-induced currents, one that was fast decaying and more prominent in younger neurons, and the other slowly decaying. Inhibition of the fast component by Pb2+ was always greater than that of the slow component, which may explain the greater sensitivity of younger neurones to Pb2+. may explain the greater sensitivity of younger neurons to Pb2+. The use of trypsin during acute dissociation of the neurons did not qualitatively alter the findings, although in the trypsin-treated neurons the currents were 65% smaller, and the EC50 for NMDA and glycine for the slow component were both about two times greater than in the untreated neurons; in the case of the fast component, the EC50s for NMDA and for glycine were not significantly altered. In outside-out patches excised from acutely dissociated hippocampal neurons, Pb2+ decreased the frequency of NMDA-activated channel openings without altering the mean channel open time. Our results are in agreement with those reported for embryonic neurons that developed in culture, and indicate that NMDA receptors expressed in hippocampal neurons of young animals are key target sites for the neurotoxic actions of Pb2+.

The patch-clamp technique was used to characterize the nicotinic currents in individual neurons acutely dissociated from the hippocampi of postnatal rats of different ages, and to investigate the effects of Pb2+ on these currents, both in acutely dissociated and in cultured hippocampal neurons. The effects of Pb2+ on nicotinic acetylcholine receptors (nAChR) expressed in frog muscle fibers were also investigated. Acetylcholine could activate fast- and slowly desensitizing whole-cell currents in neurons dissociated from the hippocampi of 4- to 20-day-old rats. Similar to the currents elicited in cultured hippocampal neurons, the rapidly desensitizing currents were blocked by methyllycaconitine, and were the nicotinic responses that could be elicited in most of the acutely dissociated neurons tested, whereas the slowly desensitizing currents could be evoked only in a few neurons. Although the peak amplitude of nicotinic currents recorded from acutely dissociated neurons increased with age of the neurons as it did in cultured neurons, the mean amplitude of the currents was at least an order of magnitude smaller in acutely dissociated neurons than in cultured neurons. Pb2+ could potently and specifically inhibit activation of fast-desensitizing nicotinic currents in hippocampal neurons. Pb2+ reduced the peak amplitude of fast-desensitizing currents with an IC50 of about 3 microM and an apparent Hill coefficient of 1.0, whereas only at 30 microM could Pb2+ decrease the peak amplitude of slowly desensitizing currents by about 50%. Inhibition of fast-desensitizing currents by Pb2+ was voltage independent and noncompetitive. However, Pb2+ had no effect on acetylcholine-activated single channels in frog muscle fibers. Our results indicate that the hippocampus of the developing rat expresses functional nAChRs of diverse types, and that methyllycaconitine-sensitive neuronal nAChRs, which are highly permeable to Ca2+, are much more sensitive to Pb2+ than other nAChR subtypes.

Our previous study demonstrated for the first time that nicotinic currents evoked in rat hippocampal neurons could be grouped into four categories (types IA, IB, II and III) according to their functional and pharmacological characteristics. In the second part of our continuing studies, the structural and functional diversity of nicotinic receptors expressed in hippocampal neurons was further explored. Type IA, the predominant and alpha-bungarotoxin-sensitive current, but not type II, the alpha-bungarotoxin-insensitive current, showed rundown in the peak amplitude during the whole-cell recording. The rundown of type IA currents could be prevented when the ATP-regenerating compound phosphocreatine, alone or in combination with ATP and creatine phosphokinase, was added to the internal recording solution. The addition to the internal solution of either the microfilament-stabilizing agent phalloidin (5 microM) or the microtubule-stabilizing agent taxol (50 microM) did not alter or prevent rundown in type IA currents. Type IA and type II currents showed inward rectification. The inward rectification of type IA currents was dependent on the presence of intracellular Mg++, whereas that of type II currents was independent of Mg++. When Mg++ was present in the internal pipette solution, the inward rectification of type IA currents was sustained throughout the recording time. However, when nominally Mg(++)-free internal solution was used, the inward rectification decreased with recording time in type IA currents, but not in type II currents, as a consequence of removal of intracellular Mg++. In situ hybridization demonstrated the presence of alpha 7-, alpha 4- and beta 2-nicotinic acetylcholine receptor subunit mRNAs in cultured hippocampal neurons. The distribution among the neurons of the mRNAs for alpha 7- and alpha 4-nicotinic acetylcholine receptor subunits, correlated with the frequency with which type IA and type II currents, respectively, could be evoked in these neurons. The present results provide evidence for 1) the presence of intracellular high-energy phosphate-dependent processes linked with the nicotinic acetylcholine receptor subserving type IA currents, 2) a requirement of intracellular Mg++ for the inward rectification of type IA currents and 3) a correlation between the distribution of nAChR subunits and the different probabilities of eliciting distinct types of nicotinic currents in hippocampal neurons.

In the present study, we demonstrated that the chicken alpha 4 beta 2 neuronal nicotinic receptor stably expressed in transfected mouse fibroblasts (M10 cells) can be activated via the acetylcholine-binding site or via a site that is distinct from that for acetylcholine and recognizes physostigmine and galanthamine as agonists. In outside-out patches excised from dexamethasone-induced M10 cells, (+)-anatoxin-a, physostigmine and galanthamine (each at 1 microM) activated single channels with conductances of 18 and 30 pS. Dihydro-beta-erythroidine (1-30 nM), but not the nicotinic receptor-specific monoclonal antibody FK1, reduced the frequency of channels activated by anatoxin (1 microM). On the other hand, the frequency of channel activity induced by physostigmine (1 microM) was unaffected by dihydro-beta-erythroidine and was markedly decreased by FK1. In uninduced M10 cells and in dexamethasone-treated untransfected fibroblasts, we observed that physostigmine, galanthamine and nicotinic agonists did not evoke whole-cell or single-channel currents. Also, neither [3H]L-nicotine nor FK1 was able to bind to uninduced M10 cells. In dexamethasone-induced M10 cells, the nicotinic agonists acetylcholine, anatoxin, 1,1-dimethyl-4-phenylpiperazinium, (-)-nicotine, and cytisine (each at 100 microM) activated whole-cell currents that showed a marked inward rectification and were sensitive to blockade by dihydro-beta-erythroidine (100 nM). However, neither galanthamine nor physostigmine could evoke whole-cell currents in cells that were responsive to nicotinic agonists. Other effects of physostigmine and galanthamine on the nicotinic receptor that outweight the agonist properties of these compounds could account for their inability to evoke whole-cell currents.

The effects of alkylene bis-9,9'-aminoacridines and 1,2,3,4-tetrahydro-9-aminoacridine (THA) were studied on single-channel currents activated by N-methyl-D-aspartate (NMDA) in outside-out patches from cultured rat hippocampal neurons. These compounds reduced the channel open times with concentration and voltage dependence, which was consistent with an open-channel blockade mechanism of action. In nominally Mg(2+)-free solutions, the forward blocking rate constants for 1,2-propane-bis-9,9'-aminoacridine, 1,4-butane-9,9'-aminoacridine, and THA were 1.1 x 10(8), 1.4 x 10(8), and 3.5 x 10(7) M-1 sec-1, respectively, at a holding potential of -80 mV. The unblocking rate constants for the bis-9-aminoacridines were similar and in the range of 7 sec-1, whereas THA had an unblocking rate constant of approximately 6.2 x 10(3) sec-1. In the presence of Mg2+ (approximately 5 microM), the predictions of the model for open-channel blockade by the 9-aminoacridines were invalid, because the relationships between the channel lifetimes and 9-aminoacridine concentrations were not linear. The effects of Mg2+ (approximately 0-50 microM) on the open-channel blockade of the NMDA receptor by the 9-aminoacridines were evaluated further by measuring the burst times in the presence of 1,2-propane-bis-9,9'-aminoacridine (5 microM). The results suggested that the interactions of 9-aminoacridines and Mg2+ with the ion channel of the NMDA receptor were not mutually exclusive. Simultaneous occupancy of the NMDA receptor ion channel by Mg2+ and a channel-blocking organic cation could be a common mechanism of channel blockade for this receptor under physiological conditions.

The study of the nicotinic acetylcholine receptor has since its very inception been associated with the use of toxins. The work by Langley in 1905 demonstrated (amongst many important and lasting findings) the presence of the “receptive substance” in striated muscle and sympathetic ganglia. Indeed, the receptor was named for its responsiveness to the tobacco alkaloid nicotine. Although the physiological transmitter in these and other tissues was later identified as acetylcholine (ACh), this receptor has been classically distinguished from other cholinergic receptors by their responses to nicotine and muscarine (Dale 1914). Langley also offered a precocious description of multiple sites or multiple ways in which a toxin might produce both agonist and antagonist effects

Publisher Summary This chapter highlights some of the electrophysiological techniques that have been used by many laboratories to study the structure and function of certain chemosensitive receptors in various tissues, including muscle and the central nervous system (CNS). The heterogeneity of structure and function of central neuronal nicotinic acetylcholine receptors (nAChRs) helps to determine the role that each neuron plays within the extremely complex fabric of the CNS. An end-plate potential is generated when the acetylcholine released from the nerve terminal interacts with the nAChR, located postjunctionally in the end-plate region of the muscle, causing the receptor ion channel to change conformation, open, and, thus conduct inward ionic currents that depolarize the region surrounding the end plate. This results in the propagation of an action potential and contraction of the muscle. Noise analysis is based on the baseline-corrected, random fluctuations in currents, induced by iontophoretic application of an agonist. Two methods are generally used in noise analysis: (1) autocovariance analysis, and (2) power spectrum analysis. The autocovariance analysis relies on a direct calculation of the average time dependence of relaxation. Power spectrum analysis, usually easier to implement, depends on a Fourier transform of the recording.

The actions of 1,2,3,4-tetrahydro-9-aminoacridine (THA) and 9-aminoacridine (9-AA) on glutamatergic receptors were studied using the whole-cell and outside-out variants of the patch-clamp technique. Typically, either N-methyl-D-aspartate (NMDA) or kainate alone or combined with various concentrations of THA or 9-AA was applied via a U tube to activate whole-cell currents. Other superfusion techniques were also used. THA (25-50 microM) and 9-AA (10-25 microM) reduced the peak and steady-state amplitudes of NMDA-activated whole-cell inward currents and had no significant effect on outward currents. At higher concentrations, these agents produced a delayed current peak in addition to a further depression of the current size. A delayed current peak was a high-amplitude current peak delayed in relation to the time course of control currents. THA and 9-AA were much less potent in blocking kainate-activated currents. Also, the blockade of kainate currents was voltage independent, and no delayed current peak was generated. With the same superfusion method, the antagonists APV (DL-2-amino-5-phosphonovaleric acid) and Mg++ were tested independently of THA or 9-AA and were found not to produce delayed current peaks or NMDA-activated whole-cell currents. Bath perfusion of THA (250 microM) abolished the delayed current peaks produced by pulse application of NMDA and THA, whereas bath perfusion of the competitive blocker APV did not have this effect. High concentrations of glycine (10 microM) did not alter THA's blocking effects or the production of delayed current peaks.(ABSTRACT TRUNCATED AT 250 WORDS)

Nicotinic acetylcholine receptors present on cultured hippocampal neurons from fetal rats were characterized by means of whole-cell patch-clamp technique, using a number of structurally divergent agonists and highly selective antagonists. Based upon the decay kinetics of the currents elicited by 3 mM acetylcholine (ACh) and their sensitivities to agonists and antagonists, the neurons were shown to exhibit four current types, IA, IB, II and III. Rapidly decaying currents (type IA) that were blocked by alpha-bungarotoxin (10 nM), kappa-bungarotoxin (10 nM) and methyllycaconitine (MLA, 1 nM) were the most frequent and were found in 83% of the neurons tested. Type II currents (found in 5% of the neurons) were blocked by dihydro-beta-erythroidine (10 nM), and by high concentrations of MLA and kappa-bungarotoxin (100 nM each) but not by alpha-bungarotoxin (100 nM). Type III currents (elicited in 2% of the neurons) decayed slowly and were blocked by (+/-)-mecamylamine (1 microM) but not by alpha-bungarotoxin, kappa-bungarotoxin or MLA (each at 100 nM). Some of the cells (10% of the neurons) had mixed responses (named type IB), which were only partially blocked by MLA (1 nM) or dihydro-beta-erythroidine (10 nM) alone and were completely blocked by combination of the two agents. The order of potency of agonists in activating the currents was the following: for type IA, (+)-anatoxin-a1,1-dimethyl-4-phenyl-piperazinium(-)-nicotinecystisineAChcarbachol(+)-nicotinearecolinesuberyldicholine; for type II, ACh(+)-anatoxin-a(-)-nicotine1,1-dimethyl-4-phenyl-piperaziniumcarbacholcytisine(+)-nicotinesuberyldicholinearecoline. Certain agonists were particularly useful in discriminating among the various types of currents: ACh, carbachol, (-)-nicotine and suberyldicholine for type II, and cytisine for type III currents. The EC50 of ACh was approximately 130 microM for type IA and approximately 2 microM for type II currents. A marked inward rectification was observed with type II, whereas type IA currents showed very little inward rectification. Differences observed in the pharmacological and functional properties of the nicotinic currents imply the expression of at least three structurally distinct nicotinic acetylcholine receptor subtypes in hippocampal neurons. The possible involvement of these currents in the transduction of signals is discussed.

Electrophysiological and biochemical techniques were used to demonstrate that alpha-bungarotoxin-, methyllycaconitine-sensitive neuronal nicotinic acetylcholine receptors (nAChRs) can be activated via a novel agonist site(s). The residue proposed to be essential to this site is the amino acid Lys-125 of the receptor alpha subunit. In outside-out patches excised from cultured hippocampal neurons, physostigmine (PHY) and 1-methyl-PHY activated single channels whose main conductances were 46 and 23 pS. This action was insensitive to DL-2-amino-5-phosphonovaleric acid, atropine, tetrodotoxin and competitive nicotinic antagonists, but blocked by benzoquinonium or FK1, a nAChR-specific antibody raised against rat muscle nAChR alpha subunits that binds to the novel site. Indirect immunofluorescence staining demonstrated that FK1 binds to the hippocampal neurons, as would be expected based on the high degree of homology among nAChR alpha subunits from diverse sources in the region surrounding Lys-125. PHY prevented the binding of FK1, thus supporting that FK1 is a specific probe for the PHY site. High-affinity sites (KD approximately 35 nM) for 1-methyl-PHY were identified in hippocampal neurons. Similar to PHY, benzoquinonium (0.1-10 microM) and galanthamine (1-10 microM) activated nicotinic single channels. The agonists benzoquinonium and PHY were also open-channel blockers at the neuronal nAChRs, whereas galanthamine was predominantly a desensitizing agent. In mouse fibroblasts transfected with cDNAs of alpha 4 and beta 2 neuronal nAChR subunits, PHY also activated single channels that were blocked by FK1. In these cells, dihydro-beta-erythroidine blocked single channels activated by (+)-anatoxin-a and did not affect those opened by PHY. Thus, the present results suggest that the novel agonist site located on the receptor alpha subunit is a common feature of neuronal nAChRs.

We describe a new method, RNA amplification with oocyte expression, for efficient expression of proteins in the Xenopus oocyte after PCR amplification of cDNA coding regions, using as examples mouse GABAA receptor alpha 1 and beta 2 subunits. RNA is reverse-transcribed and the cDNAs are amplified using 5' primers containing a T7 RNA polymerase promoter and a consensus ribosome binding site and 3' primers giving a short poly(A) tail. This is followed by direct in vitro transcription of the PCR products and injection of the resulting mRNAs into Xenopus oocytes. The method gave abundant GABA-gated chloride channels in the oocyte membrane, as measured by recording agonist-induced currents. It promises to be a rapid route to expression of cloned proteins in oocytes, without requiring actual clones, and is free of possible variations in efficiency from untranslated regions.

The ontogeny of the N-methyl-D-aspartate (NMDA) subtype of glutamatergic receptor/ion channel was studied by examining whole cell currents evoked by NMDA in cultured hippocampal neurons 1 to 30 days after plating of cells from 18- to 20-day-gestation rat fetuses. We observed a maturation-dependent increase in conductance, compatible with an increased density of NMDA receptors, which is in agreement with previous binding data. The whole cell currents evoked by NMDA (10-100 microM) in the presence of glycine (1-100 microM) had two components that contributed to the peak amplitude. The first was a rapidly decaying current (fast component) and the second a slowly decaying current (slow component), their ratio depending upon glycine concentration. The EC50 values for glycine were 1.8 and 0.3 microM for the fast and slow components of the current, respectively. The quantitative analysis of these components indicated the existence of two distinct glycine sites, which differ in their affinity for glycine. The fast component originates from the action of glycine at the site with lower affinity. Moreover, the ratio of the fast to the slow component was also dependent on the time lapsed after plating of the fetal hippocampal neurons. The slow component became more predominant and the fast component less predominant along with cell maturation in culture, a phenomenon which reflects a change in the ratio of high- to low-affinity glycine binding sites. In addition, studies on Zn2+ gave further evidence of a change in the NMDA receptor/channel properties related to maturation of the cultured neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

The inhibition of N-methyl-D-aspartate (NMDA)-activated current in cultured fetal rat hippocampal neurons by Pb2+ was investigated at various stages of cell development. Pb2+ selectively inhibited NMDA currents recorded from young cultured neurons. In the first week of culture, Pb2+ showed the most prominent inhibition, which was gradually attenuated in the following weeks. Pb2+'s action was selective for NMDA- as opposed to either kainate- or quisqualate-induced currents. The current-voltage relationship for NMDA-induced currents in the presence of Pb2+ revealed that the effect of this cation was voltage-independent, which suggested that the site of interaction of Pb2+ with the NMDA receptor/channel is located outside the membrane electric field. Single channel studies showed that Pb2+ reduced the frequency but not the lifetime of the NMDA-activated single channel currents. Further evaluation of the mechanism of action of Pb2+ on the NMDA receptor demonstrated that this cation is a noncompetitive antagonist of both NMDA and glycine. We have demonstrated that the NMDA-induced whole cell currents change along with cell development, and the effects of Pb2+ are also dependent upon age of culture. The NMDA-induced currents in cultured rat hippocampal neurons had two components, one that decayed rapidly and another that decayed slowly. The fast component was clearly observed at concentrations of glycine higher than 1 microM, whereas the slow component reached its maximum amplitude at the glycine concentration of 1 microM. Moreover, the rapidly decaying component of NMDA-evoked whole cell currents was predominant in young cultured neurons, and its contribution to the total current was reduced in old cultured neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

The need to treat diseases affecting the nicotinic AChR is great, but therapeutic options are few. Through careful correlation of structure-activity relationships of AnTX analogs, we may ultimately be led to the development of diagnostic and therapeutic drugs with specific nicotinic agonist or antagonist activities in the central nervous system that would be of major importance in the treatment of Alzheimer's disease.

Methyllycaconitine, a toxin isolated from the seeds of Delphinium brownii, inhibited acetylcholine- and anatoxin-induced whole-cell currents in cultured fetal rat hippocampal neurons, at picomolar concentrations. This antagonism was specific, concentration dependent, reversible, and voltage independent. Furthermore, methyllycaconitine inhibited 125I-alpha-bungarotoxin binding to adult rat hippocampal membranes, protected against the alpha-bungarotoxin-induced pseudoirreversible blockade of nicotinic currents, and shifted the concentration-response curve of acetylcholine to the right in fetal rat hippocampal neurons, suggesting a possible competitive mode of action for this toxin. Remarkably low concentrations of methyllycaconitine (1-1000 fM) decreased the frequency of anatoxin-induced single-channel openings, with no detectable decrease in the mean channel open time. These actions of methyllycaconitine commend this neurotoxin for the characterization of the alpha-bungarotoxin-sensitive subclass of neuronal nicotinic receptors, which has hitherto eluded functional demonstration.

Electrophysiological and biochemical studies demonstrated that pyrazole, an inhibitor of alcohol dehydrogenase and a proposed therapeutic agent for treatment of alcoholic intoxication, activated and blocked the N-methyl-D-aspartate (NMDA) receptor and did not interact significantly with the end-plate nicotinic acetylcholine receptor (AChR). Pyrazole, at concentrations as low as 0.5 microM, applied to outside-out patches excised from the membrane of cultured rat hippocampal neurons, elicited single-channel currents of 48 pS which were blocked by DL-2-amino-5-phosphorovaleric acid, a competitive antagonist of NMDA. In addition, binding studies showed that pyrazole displaced 1-(cis-2-carboxypiperidine-4-yl)methyl-1-phosphoric acid from the agonist recognition site of the NMDA receptor in a concentration-dependent manner and enhanced the binding of (+)-5-methyl-10,11-dihydro-5H- dibenzo[a,d]cyclohepten-5,10-imine to this complex. These data indicate that pyrazole is an agonist at NMDA receptors. However, at higher concentrations, open and burst times as well as the frequency of single-channel currents activated by pyrazole were reduced significantly, a finding which suggests that this compound is also an open channel blocker. In agreement with these results, it was shown biochemically that pyrazole was able to stimulate influx of Ca++ into rat brain microsomes via NMDA receptors and on the other hand to block the influx of Ca++ induced by NMDA. Pyrazole was unable to affect the neuromuscular transmission of frog sartorius muscle-sciatic nerve preparations. Additionally, pyrazole did not interact either with the agonist recognition site or with noncompetitive sites of the AChR. However, this drug had a very weak agonist-like action on the AChR of the Torpedo electric organ, most likely via binding sites different from those described previously for acetylcholine. Therefore, the therapeutic efficacy of pyrazole may be related at least in part to its effects on the NMDA receptor. Furthermore, this compound, because of the small size and rigidity of its molecular structure, becomes a promising drug for the study of the NMDA receptor. Indeed its use may allow a better understanding of the physiological and pathological processes involving this receptor.

Anatoxin analogs were designed to evaluate the importance of H-bonding, planarity, size and steric configuration of the anatoxin side chain moiety with regard to nicotinic potency and efficacy. This report examines the actions of these analogs on the somatic nicotinic acetylcholine receptor at two different loci: the agonist recognition site and the ion channel site. Agonist effects were evaluated using stimulation of contracture and radioligand binding competition for [125I]alpha bungarotoxin sites in Rana pipiens muscle, and stimulation of [3H]perhydrohistrionicotoxin binding and competition for [125I]alpha bungarotoxin sites in Torpedo californica electric organ. Antagonist effects were evident in the inhibition of neurally evoked twitch of the frog sciatic nerve-sartorius muscle preparation and in inhibition of [3H]perhydrohistrionicotoxin binding to Torpedo receptors. The affinity of these analogs for the agonist locus was consistently associated with activation of the AChR. Our results show that side chain steric configuration has an important role in affinity of the (+)-anatoxin-a analogs for the nicotinic acetylcholine receptor ion channel sites. Several analogs also revealed stereospecific noncompetitive actions. The (+)-anatoxin-a-related structures are important probes for characterizing both agonist and ion channel target sites on the peripheral nicotinic receptor.

Eighteen analogs of (+)-anatoxin-a were evaluated for nicotinic potency at two putative nicotinic acetylcholine receptor sites in the central nervous system. The affinities of the analogs for [3H]nicotine and [125I]alpha-bungarotoxin binding sites were compared. This series of analogs, with modifications to the side chain moieties of the parent structure, enables the importance (for nicotinic binding) of hydrogen bonding strength, planarity, size and steric configuration of this region of the molecule to be assessed. These studies confirm the importance of the side chain stereochemistry and the subordinate role of H-bonding strength of anatoxin analogs. Of all the analogs tested, the parent compound (+)-anatoxin-a is the most potent competitor of ligand binding. Although all analogs have higher affinity at the [3H](-)-nicotine site compared to the alpha-[125I]bungarotoxin site, the rank order of potency is generally the same at both central nervous system sites, and agrees with the order at the muscle nicotinic receptor. However, the simple methoxyamide and the isoxazolidide analogs appear more selective for the neuronal nicotinic receptor subtype identified by [3H](-)-nicotine, indicative of structural differences among the agonist recognition sites.

The effects of MK-801 [( +]-5-methyl-10,11-dihydro-5H-di-benzo[a, d]cyclohepten-5,10-imine) on peripheral and central nicotinic receptors were studied using electrophysiological and biochemical techniques. MK-801 depressed the peak amplitude and accelerated the decay of end-plate currents. The drug (1-10 microM) decreased the frequency of activation of acetylcholine (ACh)-induced single-channel currents in addition to shortening the mean open and burst times of channels activated by either ACh or (+)anatoxin-a (AnTX). MK-801 (10-40 microM) depressed the single potentials and trains of ACh and AnTX-induced potentials in chronically denervated rat soleus muscles. MK-801 blocked the twitch responses (20-100 microM) of both frog sartorius and rat diaphragm muscles evoked by stimulation of their respective nerves. Also this drug (less than 1 microM) decreased the frequency of channels activated by AnTX or ACh in outside-out patch membranes of rat retinal ganglion cells with minimal changes in the channel open time. MK-801 (10-25 microM) depressed (-)nicotine-evoked gamma-amino[2,3-3H]butyric acid release from rat hippocampal synaptosomes; however, it failed to affect the binding of [3H](-)nicotine to brain membranes and also failed to interfere with the binding of [125I]alpha-bungarotoxin to either frog muscle or Torpedo membranes. On the other hand, MK-801 inhibited the binding of [3H]perhydrohistrionicotoxin to Torpedo membranes and such an effect was more pronounced in the presence of carbamylcholine. Neither AnTX nor any other nicotinic agonist increased the binding of [3H]MK-801 to the N-methyl-D-aspartate receptor ion channel complex. The actions of MK-801 were evident at concentrations comparable with those needed to block N-methyl-D-aspartate receptors. These results demonstrate the existence of at least three different types of nicotinic AChR, all of which were blocked noncompetitively by MK-801.

The effects of (+)- and (-)-anatoxin-a (AnTX) and N-methylanatoxin (M-AnTX) on peripheral nicotinic ion channel activity were studied using high micromolar concentrations. Whereas (+)-AnTX is an effective agonist at nanomolar concentrations, (-)-AnTX and M-AnTX were effective at low micromolar concentrations. The binding of [3H]perhydrohistrionicotoxin to the nicotinic acetylcholine receptor-ion channel was stimulated by the above agonist concentrations, but [3H]perhydrohistrionicotoxin binding was inhibited at high micromolar concentrations of each of the toxins. In single channel recordings, these toxins exhibited ion channel blocking properties; the concentration- and voltage-dependent kinetics of each were essentially the same. In the case of (+)-AnTX, desensitization was also present at micromolar concentrations. These data show that ion channel blockade may be a property of many anatoxin-a analogs, and that in the particular case of analogs with low agonist potency, ion channel blockade may be a concomitant primary effect of the toxins. Stereospecificity and number of amine moieties did not influence the ion channel blocking characteristics in this series of molecules, although these factors strongly modified agonist potency.

The present study demonstrates that the reversible and irreversible anti-ChE agents have direct actions on the nicotinic acetylcholine receptor-ionic channel (AChR) and on the locust glutamatergic neuromuscular junction. In addition, the prophylaxis of lethality of organophosphorus anti-ChE compounds was studied. The lethality of VX and sarin was diminished when the rats were pretreated with physostigmine and atropine. The effectiveness of this protection, however, was markedly increased when a ganglionic blocker, either mecamylamine or chlorisondamine, was added, such that all the animals survived after receiving four times a lethal dose of VX. Pretreated animals receiving sarin showed significant recovery of morphological and functional properties of the neuromuscular junction as compared to the damage of structures from animals without pretreatment. Blood ChE inhibition was slightly decreased while brain and muscle AChE levels were significantly recovered (from 98 and 70% to 56 and 32%, respectively) by the pretreatment. This effect may partially explain the protection given by physostigmine but not that afforded by addition of a non-anti-ChE agent. Physostigmine, at concentrations greater than 20 microM, showed both a marked depression of the peak amplitudes of the endplate current (EPC) and a shortening of the decay time constants tau EPC. These effects were mostly due to a direct drug interaction with the nicotinic AChR blocking the ionic channel in its open conformation. Single-channel recordings showed that physostigmine decreases conductance and open times of the channels activated in the presence of ACh and in addition has an agonistic property on the nicotinic AChR. VX, on the other hand, only shortened the open times of ACh-activated channels without affecting the conductance. No agonist property was detected with VX. On glutamatergic synapses, the ChE inhibitors generated spontaneous firing of end-plate potentials (EPPs) and action potentials (APs). This effect was blocked in the presence of low external Ca2+ concentration or tetrodotoxin. It seems that the spontaneous EPP and AP firing resulted from an increased transmitter release induced by an increase in Na+ influx at the presynpatic nerve terminal. Physostigmine and some irreversible ChE inhibitors (VX and DFP) also blocked the postjunctional glutamate receptors. Similar to the nicotinic AChR, this effect was mostly related to a blockade of the open channels. In conclusion, the present studies showed significant protection of rats by physostigmine in combination with some ganglionic antagonists against lethality by organophosphate agents.(ABSTRACT TRUNCATED AT 400 WORDS)

Perhydrohistrionicotoxin, at concentrations of 10(-12)-10(-7) M, depressed the current generated by iontophoretic application of acetylcholine to endplate regions of soleus and extensor digitorum longus muscles of rats. However, no changes in the amplitude or time course of spontaneous miniature endplate potentials or currents were seen with these concentrations of toxin. Evoked endplate currents were also unaffected by the toxin. Similarly, the responses to iontophoretic acetylcholine were depressed by these concentrations of perhydrohistrionicotoxin in chronically denervated muscles. Depression of responses in both normal and chronically denervated muscles developed gradually, was greater at higher concentrations, and was reversible. The different effects of the toxin on neurally evoked currents and currents produced by iontophoretic application of acetylcholine raise the possibility of the existence of two different population of receptor complexes.

1. A study has been made of the effects of tetrodotoxin on the impulse activity, resting membrane potential, input resistance, and the generator potential and its after-hyperpolarization of the slowly adapting stretch receptor neurone of the lobster.2. Tetrodotoxin was able to abolish completely within about 2 min the impulse activity in most cells, when given in a dose of 2 x 10(-8) g/ml., but in all cells, when administered in a dose of 4 x 10(-8) g/ml. After blockage by the toxin in concentrations as high as 4 x 10(-6) g/ml. for periods of up to 30 min the action potential was restored by washing the preparation in physiological solution for 1 hr.3. In a concentration of 4 x 10(-8) g/ml. tetrodotoxin produced within 1-2 min an average increase of 4.8 mV of the resting membrane potential and a simultaneous 47% reduction of the resting input resistance. These effects were reversed by washing the preparation in physiological solution for 1 hr.4. Tetrodotoxin administered in doses as high as 4 x 10(-6) g/ml. for periods of up to 30 min had no effect on the amplitude of the steady phase of the generator potential.5. In a concentration of 4 x 10(-8) g/ml. tetrodotoxin produced within 5 min a 65% reduction of the amplitude of the hyperpolarization following the generator potential. This effect was reversed by washing the preparation in physiological solution for 1 hr.6. The simultaneous increase in resting membrane potential and decrease in membrane resistance is suggested to be due to an elevated potassium permeability besides a reduced sodium conductance. The constancy in height of the generator potential in the presence of a decreased membrane resistance makes necessary the assumption of an augmented generator current. The decrease in amplitude of the hyperpolarization following the generator potential may be the result of an increase in potassium conductance and/or a reduction in acceleration of an electrogenic pump in consequence of a diminished sodium influx during the generator potential.

Tetrodotoxin in concentrations of 0.8–1.0 × 10-7 g/ml completely blocked conducted activity of the afferent nerve of isolated frog muscle spindle without affecting the receptor potential. Development of block was characterized by an increase in threshold of the nerve, a gradual delay of the onset of the impulse response and a reduction and decomposition of the individual action potentials into smaller spikes. The spike decomposition appeared to be due to a de-synchronization of the conducted activity in the branched afferent terminals. After block by tetrodotoxin for up to 20 min the normal response could be restored by washing the preparation with Ringer's solution. The blocking action of tetrodotoxin was delayed and reduced by increasing calcium; removal of calcium had the opposite effect. Tetrodotoxin (1000 ng/ml) had no significant effect on the receptor potential. With low sodium plus tetrodotoxin the receptor potential was reduced by about 50 %; this reduction was reversed by addition of normal sodium. It is suggested that the sodium carrying system of the generator potential differs from that underlying the regenerative activity.

The effects of phospholipase C on the muscle membrane were studied by the recording of the resting membrane potential, the input resistance and the action potential generation in single fibres of the isolated extensor digitorum longus and soleus muscles of the rat. Incubation with phospholipase C abolished spike generation, depolarized the fibres by 15–20 mV and increased the ionic permeability of the membrane as shown by a fall of about 40 per cent of the “input” resistance. Removal of the enzyme by washing the preparation with Krebs-Ringer solution did not restore membrane exitability, the resting membrane potential nor the input resistance of the fibres. When the external calcium concentration was raised from the normal 2 mM to 15 mM the muscle fibres repolarized and action potentials with a reduced rate of rise and amplitude were recorded. No recovery of the input resistance occurred. When the calcium concentration was brought back to 2 mM the fibres again depolarized and became inexcitable. Potassium (10 or 15 mM), caesium (10 mM) or rubidium (6 mM) antagonized the aforementioned effects of 15 mM calcium. It was concluded that phospholipase C irreversibly affected the muscle membrane and that an increase in the external calcium ion concentration only functionally restored some of the membrane properties, i.e., spike generation and the resting membrane potential. The results support the view that the hydrophilic head of certain membrane phospholipids is connected with passive ion transport in the electrogenic membrane.

Albuquerque, E. X. and S. Thesleff. Effects of phospholipase A and lysolecithin on some electrical properties of the muscle membrane. Acta physiol. scand. 1968. 72. 248–252. The effects of incubation with phospholipase A and lysolecithin on the resting membrane potential, action potential, input resistance and acetyleholine sensitivity of isolated extensor digitorum longus and soleus muscles of the rat were compared with those previously obtained with phospholipase C. A common action of the agents studied were that they in sufficient doses depolarized the muscle fibres and reduced the input resistance. In contrast to phospholipase C which irreversibly blocked action potential generation, phospholipase A and lysolecithin had little effect on membrance excitability. The sensitivity of chronically dencrvated muscle fibres to locally applied acetylcholine was unaffected by the two phospholipascs and by Iysolccithin. The difference in action between phospholipasr C and phospholipase A on electrogenic membrane excitability is suggested to be explained by the respective mode of lipolytic action of the two enzymes. Phospholipasc C hydrolyses the diglyceride — phosphate, ester linkage in phosphatides and thereby removes the phosphate head from the molecult while phospliolipase A cleaves one fatty acid ester linking leaving the polar heads of phospholipids intact. Lysolecithin possibly rearranges membrane structures by its high surface activity.

The first alteration noted after denervation of the extensor digitorum longus muscles of rats was a decrease in resting membrane potential (RMP) which occurred at about 2h. The exact time course of this membrane depolarization was dependent upon the intramuscular length of the degenerating nerve stump. The decrease in RMP occurred prior to any detectable alteration in adenosine triphosphate and phosphocreatine content of the muscle. Prior to failure of spontaneous transmitter release, which occurred 10 h after denervation, some muscle fibres showed an increase in frequency of the miniature end-plate potentials (m.e.p.p.) with no alterations in amplitude and shape of the single potentials. Appearance of areas sensitive to acetylcholine (ACh) on the extra-junctional membrane occurred at 24 h after crushing the motor nerve. At 48 h after denervation a high ACh-sensitivity appeared at the muscle-tendon region, but was not detected in the majority of the muscle fibres studied midway between end-plate region and muscle-tendon area. The transverse resistance of a unit area of the muscle membrane was increased 3 days after denervation. These results provide evidence that the processes of post-denervation changes occur in the following temporal sequence: a) partial depolarization of the postsynaptic muscle membrane; b) a decrease and subsequent cessation of the spontaneous transmitter release preceded in some fibres by a transient increase in m.e.p.p. frequency; c) appearance of extrajunctional ACh sensitivity; d) increase in the transverse resistance of a unit area of the muscle membrane. It is suggested that the motor nerve releases more than one neurotrophic substance.

Triphenylmethylphosphonium (TPMP) blocked the nerve-elicited twitch tension of frog sartorius muscles by 50% at a concentration of about 20 microM. This neuromuscular blockade by TPMP, which originated at the level of the nicotinic receptor, was due in part to the ability of the drug to block the quiescent receptor by degrees that depended on the TPMP concentration and on membrane potential. In addition, the blockade was markedly enhanced when the receptor was activated by acetylcholine. Finally, TPMP shortened the lifetimes of ACh-activated ion channels. These findings were interpreted as follows. Under resting conditions, TPMP shifted the equilibrium of the receptor channel complex toward the desensitized state. TPMP united with the activated ion channel to shorten channel lifetime and to deepen the blockade. High concentrations (greater than or equal to 50 microM) of TPMP altered muscle action potentials and often increased, by about 30-fold, the frequency of miniature endplate potentials.