Nicotinic acetylcholine receptors modulate the release of GABA, glutamate, acetylcholine and dopamine in the brain. Here we describe a novel choline-sensitive nicotinic acetylcholine receptor that mediates enhanced GABA release in the chick ventral lateral geniculate nucleus. Whole-cell recordings in slices demonstrated that choline (0.03-10 mM), generally considered an alpha7-selective agonist, and carbachol (3-300 microM), a non-selective cholinergic agonist, both increased the frequency of spontaneous GABAergic events in ventral lateral geniculate nucleus neurons. Tetrodotoxin (0.5 microM) partially reduced responses to carbachol, but eliminated responses to choline. During long-term (5 min) exposure to choline the GABA enhancement was maintained until choline was washed out. Choline (300 microM) enhanced the frequency of spontaneous GABAergic events by 4.28-fold in control artificial cerebrospinal fluid. This choline-mediated enhancement was significantly reduced by the following nicotinic acetylcholine receptor antagonists: 1 microM dihydro-beta-erythroidine (1.49-fold increase, P<0.001), 1 microM methyllycaconitine (1.53-fold, P<0.001) and 0.2 microM alpha-conotoxin ImI (1.84-fold, P<0.001). In contrast, no significant change was seen in the presence of 0.1 microM dihydro-beta-erythroidine, 0.1 microM methyllycaconitine, 0.1 microM alpha-bungarotoxin, 0.1 microM alpha-conotoxin MII, 0.1 microM kappa-bungarotoxin, or 1 microM alpha-conotoxin AuIB. These results indicate that choline, at concentrations as low as 100 microM, activates a nicotinic acetylcholine receptor that is distinct from the classical alpha7 nicotinic acetylcholine receptors previously known to be activated by choline.

Nicotine, the main addictive substance in tobacco, interacts with muscle and neuronal nicotinic acetylcholine receptors (nAChRs) that are also localized in astrocytes. We studied electrical effects elicited by nicotine in cultured astrocytes from the CA1 area of the rat hippocampus. Nicotine elicited different types of responses: sustained inward currents, decaying inward currents, and biphasic responses (an outward, followed by an inward current). Nicotine showed two opposite effects, an increase or a decrease of astrocyte membrane conductance, when voltage ramps were applied during sustained inward currents. The former was isolated by blocking K+ currents with Cs+ and was inhibited by mecamylamine. The latter was mimicked by tetraethylammonium ion, and was obtained in the presence of nAChR antagonists (mecamylamine, methyllycaconitine plus dihydro-beta-erythroidine). Thus, these results indicate that nicotine activates nAChRs and directly inhibits K+ currents in cultured astrocytes from the CA1 region of the rat hippocampus.

To demonstrate the antinociceptive and antihypersensitivity mechanisms of Cris-104 (1-{2-[5-(4-fluorophenyl)-1H-pyrazol-4-yl]ethyl}piperidine), a novel selective α4β2* nicotinic acetylcholine receptor (nAChR) agonist, in rodent acute/inflammatory and chronic pain models.

Cholinergic neurotransmission is a key signal pathway in the peripheral nervous system and in several branches of the central nervous system. Despite the fact that it has been studied extensively for a long period of time, some aspects of its regulation still have not yet been established. One is the relationship between the nicotine-induced autoregulation of acetylcholine (ACh) release with changes in the concentration of presynaptic calcium levels. The mouse neuromuscular junction of m. Levator Auris Longus was chosen as the model of the cholinergic synapse. ACh release was assessed by electrophysiological methods. Changes in calcium transients were recorded using a calcium-sensitive dye. Nicotine hydrogen tartrate salt application (10 μM) decreased the amount of evoked ACh release, while the calcium transient increased in the motor nerve terminal. Both of these effects of nicotine were abolished by the neuronal ACh receptor antagonist dihydro-beta-erythroidine and Cav1 blockers, verapamil, and nitrendipine. These data allow us to suggest that neuronal nicotinic ACh receptor activation decreases the number of ACh quanta released by boosting calcium influx through Cav1 channels.

The aim of this work was to assess whether nicotine prevents glutamate neurotoxicity in primary cultures of cerebellar neurons, to try to identify the receptor mediating the protective effect and to shed light on the step of the neurotoxic process which is prevented by nicotine. It is shown that nicotine prevents glutamate and NMDA neurotoxicity in primary cultures of cerebellar neurons. The protective effect of nicotine is not prevented by atropine, mecamylamine or dihydro-beta-erythroidine, but is slightly prevented by hexamethonium and completely prevented by tubocurarine and alpha-bungarotoxin, indicating that the protective effect is mediated by activation of alpha7 neuronal nicotinic receptors. Moreover, alpha-bungarotoxin potentiates glutamate neurotoxicity, suggesting a tonic prevention of glutamate neurotoxicity by basal activation of nicotinic receptors. Nicotine did not prevent glutamate-induced rise of free intracellular calcium nor depletion of ATP. Nicotine prevents glutamate-induced proteolysis of the microtubule-associated protein MAP-2 and disaggregation of the neuronal microtubular network. The possible mechanism responsible for this prevention is discussed.

The current study examined the effects of nicotine infusion into the dorsal hippocampus or anterior cingulate on fear conditioning and on ethanol-induced deficits in fear conditioning, and whether these effects involved receptor activation or inactivation. Conditioning consisted of two white noise (30 s, 85 dB)-foot-shock (2 s, 0.57 mA) pairings. Saline or ethanol was administered to C57BL/6 mice 15 min before training and saline or nicotine was administered 5 min before training or before training and testing. The ability of the high-affinity nicotinic acetylcholinergic receptor (nAChR) antagonist dihydro-beta-erythroidine (DHbetaE) to modulate the effects of ethanol and nicotine was also tested; saline or DHbetaE was administered 25 (injection) or 15 (infusion) minutes before training or before training and testing. Infusion of nicotine into the hippocampus enhanced contextual fear conditioning but had no effect on ethanol-induced learning deficits. Infusion of nicotine into the anterior cingulate ameliorated ethanol-induced deficits in contextual and cued fear conditioning but had no effect on learning in ethanol-naive mice. DHbetaE blocked the effects of nicotine on ethanol-induced deficits; interestingly, DHbetaE alone and co-administration of subthreshold doses of DHbetaE and nicotine also ameliorated ethanol-induced deficits but failed to enhance learning. Finally, DHbetaE failed to ameliorate ethanol-induced deficits in beta2 nAChR subunit knockout mice. These results suggest that nicotine acts in the hippocampus to enhance contextual learning, but acts in the cingulate to ameliorate ethanol-induced learning deficits through inactivation of high-affinity beta2 subunit-containing nAChRs.

Glucose improves memory for a variety of tasks when administered to rats and mice near the time of training. Prior work indicates glucose may enhance memory by increasing the synthesis and release of the neurotransmitter acetylcholine in the brain. To investigate if specific acetylcholine receptor subtypes may mediate some of the memory-enhancing actions of glucose, we examined the effects of subtype-specific nicotinic acetylcholine receptor antagonists on memory in Fischer-344 rats and also examined the ability of glucose to reverse drug-induced impairments. Pre-training peripheral injections of methyllycaconitine (MLA) or dihydro-beta-erythroidine (DHβE), which are specific α7 and α4β2 nicotinic receptor antagonists, respectively, dose-dependently impaired retention latencies in an inhibitory avoidance task when tested 7-days but not 1 h after training. Immediate post-training glucose injections attenuated the impairments, but were more effective in attenuating the DHβE-induced impairments. Likewise, peripheral or direct intrahippocampal injections of MLA or DHβE dose-dependently impaired spatial working memory scores on a spontaneous alternation task. Concurrent administration of glucose reversed DHβE- but not MLA-induced impairments. CREB phosphorylation downstream of cholinergic signaling was assessed 30 min after spontaneous alternation testing and intrahippocampal drug infusions. Both MLA and DHβE impaired hippocampal CREB phosphorylation; glucose reversed DHβE- but not MLA-induced deficits. The effectiveness of glucose in reversing DHβE- but not MLA-induced impairments in behavioral performance and CREB phosphorylation suggests that activation of α7 receptors may play an important role in memory enhancement by glucose.

1. Nicotinic effects on glycine release were investigated in slices of lumbar spinal cord using conventional whole-cell recordings. In most of the substantia gelatinosa (SG) neurons tested, nicotine increased the frequency of the glycinergic spontaneous miniature inhibitory postsynaptic currents (mIPSCs). In a smaller proportion, nicotine evoked not only this same presynaptic response but also a postsynaptic response. 2. Nicotinic facilitation of glycinergic mIPSCs was investigated in mechanically dissociated SG neurons using nystatin-perforated patch recordings. Nicotine (3 x 10(-6) to 10(-5) M) reversibly enhanced the frequency of glycinergic mIPSCs without altering their amplitudes, thus indicating that nicotine facilitates glycine release through a presynaptic mechanism. 3. Choline, a selective alpha7 subunit of nicotinic acetylcholine receptor (nAChR) agonist, had no effect on the mIPSC frequency while anatoxin A, a broad-spectrum agonist of nAChR, facilitated the mIPSC frequency. 4. alpha-Bungarotoxin, a selective alpha7 subunit antagonist, failed to block the nicotinic facilitatory action. Mecamylamine, a broad-spectrum nicotinic antagonist, reversibly inhibited nicotinic action. Dihydro-beta-erythroidine, a selective antagonist of nAChRs containing alpha4-beta2 subunits, completely blocked nicotinic action. 5. Ca(2+)-free but not Cd(2+)-containing bath solutions blocked nicotinic actions. 6. We therefore conclude that nicotine facilitates glycine release in the substantia gelatinosa of the spinal dorsal horn via specific nAChRs containing alpha4-beta2 subunits. This action on a subset of presynaptic nAChRs may underlie nicotine's modulation of noxious signal transmission and provide a cellular mechanism for the analgesic function of nicotine.

The presynaptic nicotinic modulation of glutamatergic transmission in the CNS has been associated with activation of the alpha7 subtype of nicotinic acetylcholine receptor (nAChR) in sub-cortical regions, whereas in the frontal cortex, non-alpha7 nAChRs have been implicated. The aim of this investigation was to directly characterise nAChR-evoked release of excitatory amino acids from rat frontal cortex, by monitoring the release of [3H]D-aspartate from superfused synaptosomes or minces. Co-administration of a nAChR agonist with a depolarising stimulus enhanced [3H]D-aspartate release above the effect of depolarising agent alone. This enhancement was blocked by the nicotinic antagonist mecamylamine. Other experiments revealed that in the absence of a depolarising stimulus, the nAChR agonists nicotine, epibatidine and anatoxin-a could evoke the release of [3H]D-aspartate in a Ca2+- and concentration-dependant manner. Differential sensitivity to the alpha7- and beta2*-selective nAChR antagonists alpha-bungarotoxin (alpha-Bgt) and dihydro-beta-erythroidine (DHbetaE) implicated two nAChR subtypes (alpha7 and beta2*), and this was supported by using the subtype-selective agonists choline (10 mM; alpha7 selective, blocked by alpha-Bgt but not by DHbetaE) and 5-Iodo-A-85380 (10 nM; beta2*-selective, blocked by DHbetaE but not by alpha-Bgt). Immunocytochemistry showed that alpha-Bgt labelling was associated with structures immunopositive for vesicular glutamate transporters, in both frontal cortex sections and synaptosome preparations, supporting the presence of alpha7 nAChR on glutamatergic terminals in rat frontal cortex.

Acute nicotine enhances contextual fear conditioning, whereas withdrawal from chronic nicotine produces impairments. However, the nicotinic acetylcholine receptors (nAChR) that are involved in nicotine withdrawal deficits in contextual fear conditioning are unknown. The present study used genetic and pharmacological techniques to investigate the nAChR subtype(s) involved in the effects of nicotine withdrawal on contextual fear conditioning. beta2 or alpha 7 nAChR subunit knockout (KO) and corresponding wild-type (WT) mice were withdrawn from 12 days of chronic nicotine treatment (6.3mg/kg/day), and trained with 2 conditioned stimulus (CS; 85 dB white noise)--unconditioned stimulus (US; 0.57 mA footshock) pairings on day 13. On day 14, mice were tested for contextual and cued freezing. beta2 KO mice did not show nicotine withdrawal-related deficits in contextual fear conditioning, in contrast to WT mice and alpha 7 KO mice. A follow-up study investigated if nicotine withdrawal disrupts acquisition or recall of contextual fear conditioning. The high affinity nAChR antagonist dihydro-beta-erythroidine (DH beta E; 3mg/kg) was administered prior to training or testing to precipitate withdrawal in chronic nicotine-treated C57BL/6 mice. Deficits in contextual fear conditioning were observed in chronic nicotine-treated mice when DH beta E was administered prior to training, but not when administered at testing. These results indicate that beta2-containing nAChRs, such as the alpha 4 beta 2 receptor, mediate nicotine withdrawal deficits in contextual fear conditioning. In addition, nicotine withdrawal selectively affects acquisition but not recall or expression of the learned response.

1. Site-directed mutagenesis was used to create an altered form of the chicken alpha7 nicotinic acetylcholine (ACh) receptor subunit (alpha7x61) in which a leucine residue was inserted between residues Leu9' and Ser10' in transmembrane domain 2. The properties of alpha7x61 receptors are distinct from those of the wild-type receptor. 2. Oocytes expressing wild-type alpha7 receptors responded to 10 microM nicotine with rapid inward currents that desensitized with a time-constant of 710+/-409 ms (mean+/-s.e.mean, n=5). However in alpha7x61 receptors 10 microM nicotine resulted in slower onset inward currents that desensitized with a time-constant of 5684+/-3403 ms (mean+/-s.e.mean, n = 4). No significant difference in the apparent affinity of nicotine or acetylcholine between mutant and wild-type receptors was observed. Dihydro-beta-erythroidine (DHbetaE) acted as an antagonist on both receptors. 3. Molecular modelling of the alpha7x61 receptor channel pore formed by a bundle of M2 alpha-helices suggested that three of the channel lining residues would be altered by the leucine insertion i.e.; Ser10 would be replaced by the leucine insertion, Val13' and Phe14' would be replaced, by Thr and Val, respectively. 4 When present in the LEV-1 nicotinic ACh receptor subunit from Caenorhabditis elegans the same alteration conferred resistance to levamisole anthelmintic drug. Levamisole blocked responses to nicotine of wild-type and alpha7x61 receptors. However, block was more dependent on membrane potential for the alpha7x61 receptors. 5. We conclude that the leucine insertion in transmembrane domain 2 has the unusual effect of slowing desensitization without altering apparent agonist affinity.

Activation of spinal nicotinic receptors evokes a prominent algogenic response. Recently, epibatidine, a potent nicotinic agonist, was found to display an antinociceptive response after systemic administration. To examine the spinal component of this action, effects of three nicotinic agonists epibatidine, cytisine and nicotine--were given intrathecally (IT) and their antinociceptive activity was subsequently assessed. All agents elicited dose-dependent algogenic activity, characterized at lower doses by touch-evoked hyperactivity and at higher doses by intermittent vocalization and marked behavioral activity, with the rank order of potency being epibatidine > cytisine > nicotine. In addition, intrathecal epibatidine elicited a short-lasting, dose-dependent thermal antinociception. In contrast, the other nicotinic agonists at the highest usable dose failed to produce a significant antinociception. Mecamylamine, a nicotinic channel blocker, completely abolished the antinociceptive and algogenic responses of epibatidine. The competitive antagonist, alpha-lobeline, blocked both the analgesic and algogenic responses, but methyllycaconitine inhibited only the algogenic effects of epibatidine. Dihydro-beta-erythroidine, also a competitive antagonist, had no effect on the initial intense algogenic responses. The analgesic response to epibatidine was neither inhibited by naloxone nor by atropine. 2-Amino-5-phosphopentanoic acid, a competitive N-methyl-D-aspartate receptor antagonist, did not affect the analgesic response to intrathecal epibatidine or the intense initial algogenic response. Upon repeated administration (30-min interval), epibatidine (1 microg, IT) exhibited marked and rapid desensitization to both the analgesic and algogenic responses which recovered within 8 h. Pretreatment with two consecutive doses of cytisine (5 microg, IT, 30-min apart) inhibited the agitation and analgesic actions of intrathecal epibatidine. Thus, we contend that in addition to the typical nociceptive response elicited by spinal nicotinic agonists, intrathecal epibatidine also exhibits a pronounced but short-lasting antinociception. The analgesic and algogenic responses to intrathecal epibatidine may be mediated by distinct subtypes of spinal nicotinic receptors as suggested by the antagonist studies.

Anatomical studies indicate that synaptic inputs from many cortical and subcortical structures converge on neurons of the ventral tegmental area (VTA). Although in vitro electrophysiological studies have examined synaptic inputs to dopamine (DA) and non-DA neurons in the VTA, they have largely relied upon local electrical stimulation to activate these synapses. This provides little information regarding the distinct properties of synapses originating from different brain areas. Using whole-cell recordings in parasagittal rat brain slices that preserved subcortical axons from the pedunculopontine nucleus (PPN) to the VTA, we compared these synapses with those activated by intra-VTA stimulation. PPN-evoked currents demonstrated longer latencies than intra-VTA-evoked currents, and both VTA and PPN responses were mediated by GABA(A) and AMPA receptors. However, unlike VTA-evoked currents, PPN currents were exclusively mediated by glutamate in 25-40% of the VTA neurons. Consistent with a cholinergic projection from the PPN to the VTA, nicotinic acetylcholine receptors (nAChR) were activated by endogenous acetylcholine released during PPN, but not VTA, stimulation. This was seen as a reduction of PPN-evoked, and not VTA-evoked, synaptic currents by the alpha7-nAChR antagonist methyllycaconitine (MLA) and the agonist nicotine. The beta2-nAChR subunit antagonist dihydro-beta-erythroidine had no effect on VTA- or PPN-evoked synaptic currents. The effects of MLA on PPN-evoked currents were unchanged by the GABA(A) receptor blocker picrotoxin, indicating that alpha7-nAChRs presynaptically modulated glutamate and not GABA release. These differences in physiological and pharmacological properties demonstrate that ascending PPN and presumed descending inputs to VTA utilize distinct mechanisms to differentially modulate neuronal activity and encode cortical and subcortical information.

Nicotine enhances Pavlovian conditioned responses to reward-associated cues. We investigated through which nicotinic acetylcholine receptor (nAChR) subtypes nicotine acts to produce this behavioral effect to an alcohol-associated cue. Male Long-Evans rats with freely available food and water were first accustomed to drinking 15% ethanol in their home cages using an intermittent access, two-bottle choice procedure. Then the rats were given 15 Pavlovian conditioning sessions in which a 15-s audiovisual conditioned stimulus (CS) predicted the delivery of 0.2 ml of ethanol, the unconditioned stimulus (US). Each session contained 12 CS-US trials. A control group received explicitly unpaired presentations of the CS and US. We measured Pavlovian conditioned approach to the site of US delivery during presentations of the CS, accounting for pre-CS baseline activity. Before each conditioning session, rats were injected subcutaneously with nicotine (0.4 mg/kg) or saline (1 ml/kg). During nAChR antagonist test sessions, rats were first injected systemically with the β2*-selective nAChR antagonist dihydro-beta-erythroidine (DHβE; 3 mg/kg) or the α7-selective nAChR antagonist methyllycaconitine (MLA; 6 mg/kg), followed by their assigned nicotine or saline injection before assessing their conditioned response to the alcohol-associated cue. Consistent with previous reports, nicotine enhanced the Pavlovian conditioned response to the alcohol-paired cue. DHβE attenuated this enhancement, whereas MLA did not. These results suggest that nicotine acts via β2*, but not α7, nAChRs to amplify Pavlovian conditioned responding to an alcohol cue. These findings contribute to a growing literature that identifies nAChRs as potential targets for pharmacological treatment of co-morbid alcohol and tobacco use disorders.

Manganese (Mn) and iron (Fe) are trace elements that are essential for proper growth and physiological functions as both play critical role in a variety of enzymatic reactions. At high concentrations, however, they can be toxic and cause neurodegenerative disorders, particularly Parkinson-like syndromes. Nicotine, on the other hand, has been shown to have neuroprotective effects against various endogenous or exogenous toxins that selectively damage the dopaminergic cells. These cells include neuroblastoma-derived SH-SY5Y cells which express significant dopaminergic activity. However, practically no information on possible neuroprotective effects of nicotine against toxicity induced by trace elements is available. Therefore, in this study we investigated the effects of nicotine on toxicity induced by manganese or iron in these cells. Exposure of SH-SY5Y cells for 24 h to manganese (20 μM) or iron (20 μM) resulted in approximately 30% and 35% toxicity, respectively. Pretreatment with nicotine (1 μM) completely blocked the toxicities of Mn and Fe. The effects of nicotine, in turn, were blocked by selective nicotinic receptor antagonists. Thus, dihydro-beta erythroidine (DHBE), a selective alpha 4-beta 2 subtype antagonist and methyllycaconitine (MLA), a selective alpha7 antagonist, as well as mecamylamine, a non-selective nicotinic antagonist all dose-dependently blocked the protective effects of nicotine against both Mn and Fe. These findings provide further support for the potential utility of nicotine or nicotinic agonists in Parkinson's disease-like neurodegenerative disorders, including those that might be precipitated by trace elements, such as Fe and Mn. Moreover, both alpha4-beta2 and alpha7 nicotinic receptor subtypes appear to mediate the neuroprotective effects of nicotine against toxicity induced by these two trace metals.

DYT1 dystonia is an early-onset, hyperkinetic movement disorder caused by a deletion in the gene TOR1A, which encodes the protein torsinA. Several lines of evidence show that in animal models of DTY1 dystonia, there is impaired basal dopamine (DA) release and enhanced acetylcholine tone. Clinically, anticholinergic drugs are the most effective pharmacological treatment for DYT1 dystonia, but the currently used agents are non-selective muscarinic antagonists and associated with side effects. We used a DYT1 ∆GAG knock-in mouse model (DYT1 KI) to investigate whether nicotine and/or a non-desensitizing nicotinic agonist, AZD1446, would increase DA output in DYT1 dystonia. Using in vivo microdialysis, we found that DYT1 KI mice showed significantly increased DA output and greater sensitivity to nicotine compared to wild type (WT) littermate controls. In contrast, neither systemic injection (0.25-0.75 mg/kg) or intrastriatal infusion (30 μM-1 mM) of AZD1446 had a significant effect on DA efflux in WT or DYT1 KI mice. In vitro, we found that AZD1446 had no effect on the membrane properties of striatal spiny projection neurons (SPNs) and did not alter the spontaneous firing of ChI interneurons in either WT or DYT1 KI mice. We did observe that the firing frequency of dopaminergic neurons was significantly increased by AZD1446 (10 μM), an effect blocked by dihydro-beta-erythroidine (DHβE 3 μM), but the effect was similar in WT and DYT1 KI mice. Our results support the view that DYT1 models are associated with abnormal striatal cholinergic transmission, and that the DYT1 KI animals have enhanced sensitivity to nicotine. We found little effect of AZD1446 in this model, suggesting that other approaches to nicotinic modulation should be explored.

The nucleus of the solitary tract (NTS) is the principal integrating relay in the processing of visceral sensory and gustatory information. In the present study, patch-clamp electrophysiological experiments were conducted using rat horizontal brainstem sections. Pre-synaptic and somatic/dendritic nicotinic acetylcholine receptors (nAChRs) expressed in neurons of the caudal NTS (cNTS) were found to be randomly distributed between pre-synaptic and somatic/dendritic sites (chi(2)=0.72, df=3, p>0.87, n=200). Pre-synaptic nAChRs were detected by their facilitating effects on glutamatergic neurotransmission of a sub-population of cNTS neurons (categorized as "effect-positive") upon brief picospritzer applications of 0.1-0.5mM nicotine. These effects were resistant to inhibition by 20nM methyllycaconitine (MLA) and 4muM dihydro-beta-erythroidine (DHbetaE), and were replicated by brief picospritzer applications of 0.2-1mM cytisine. Picospritzer applications of 0.2mM RJR-2403, a potent agonist of alpha4beta2 nAChRs, did not facilitate synaptic release of glutamate in effect-positive cNTS neurons. The population of somatic/dendritic nAChRs has been found to be heterogeneous and included nAChRs that were activated by RJR-2403 and/or cytisine, or insensitive to cytisine, or inhibited by MLA. The presented results are consistent with the expression of beta4-containing (i.e., beta4*) nAChRs, likely alpha3beta4*, in pre-synaptic terminals of effect-positive cNTS neurons. Somatic/dendritic nAChRs appear to involve both alpha7 and non-alpha7 subunits. Heterogeneity in the subunit composition of pre-synaptic and somatic/dendritic nAChRs may underlie diverse roles that these receptors play in regulation of behavioral and visceral reflexes, and may reflect specific targeting by endogenous nicotinic agents and nicotine.

The alpha 7 nicotinic acetylcholine receptor (nAChR) has been implicated widely in behavioural functions and dysfunctions related to the hippocampus, but the detailed mechanisms by which this receptor contributes to these behavioural processes have yet to be elucidated. In the present study, sustained application (5 min) of nicotine significantly lowered the threshold for synaptic plasticity, and thus a long-lasting potentiation was induced by a stimulus that would normally evoke only a short-term potentiation. This effect appeared to be mediated by alpha 7 nAChRs, as it was inhibited by the alpha 7 nAChR-specific antagonist alpha-bungarotoxin (100 nM), but not by mecamylamine (50 microM) or dihydro-beta-erythroidine (DH beta E; 1 microM) at concentrations known to be selective for non-alpha 7 nAChRs. Further pharmacological dissection revealed that the effect was also abolished by the NMDA receptor antagonist, D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5; 50 microM). This blockade, however, unmasked a slowly developing nicotine-induced potentiation of field excitatory postsynaptic potential that appeared to be dependent on both alpha 7 nAChR activation and non-alpha 7 nAChR desensitisation. This secondary effect of nicotine was blocked by a combination of picrotoxin (50 microM) and saclofen (100 microM), and thus appeared to be mediated via GABAergic interneurons. The important implication of this study was that the sustained application of alpha 7 nAChR agonists could modulate the conditions for synaptic plasticity through multiple transduction pathways, and not simply the inactivation of alpha 7 nAChRs. These alpha 7-nAChR-dependent mechanisms could reconcile the discrepancies between the previously reported behavioural versus electrophysiological effects of nicotine in the hippocampus. Effects of sustained alpha 7 nAChR stimulation Effects of sustained alpha 7 nAChR stimulation Effects of sustained alpha 7 nAChR stimulation Effects of sustained alpha 7 nAChR stimulation Effects of sustained alpha 7 nAChR stimulation

We have earlier reported that Abeta were significantly reduced in brains of smoking Alzheimer patients and control subjects compared with non-smokers, as well as in nicotine treated APPsw transgenic mice. To examine the mechanisms by which nicotine modulates APP processing we here measured levels of secreted amyloid precursor protein (sAPPalpha), total sAPP, Abeta40 and Abeta42 in different cell lines expressing different nicotinic receptor (nAChR) subtypes or no nAChRs. Treatment with nicotine increased release of sAPPalpha and at the same time lowered Abeta levels in both SH-SY5Y and SH-SY5Y/APPsw cells expressing alpha3 and alpha7 nAChR subtypes. These effects could also be evoked by co-treatment with the competitive alpha7 nAChR antagonists alpha-bungarotoxin and methyllycaconitine (MLA), and by these antagonists alone, suggesting that binding to the agonist binding site, rather than activation of the receptor, may be sufficient to trigger changes in APP processing. The nicotine-induced increase in sAPPalpha could only be blocked by co-treatment with the open channel blocker mecamylamine. In addition to nicotine, the agonists epibatidine and cytisine both significantly increased the release of sAPP in M10 cells expressing the alpha4/beta2 nAChR subtype, and this effect was blocked by co-treatment with mecamylamine but not by the alpha4/beta2 competitive antagonist dihydro-beta-erythroidine. The lack of effect of nicotine on sAPPalpha and Abeta levels in HEK 293/APPsw cells, which do not express any nAChRs, demonstrates that the nicotine-induced attenuation of beta-amyloidosis is mediated by nAChRs and not by a direct effect of nicotine. Our data show that nicotinic compounds stimulate the non-amyloidogenic pathway and that alpha4 and alpha7 nAChRs play a major role in modulating this process. Nicotinic drugs directed towards specific nAChR subtypes might therefore be beneficial for the treatment of AD not only by lowering Abeta production but also by enhance release of neuroprotective sAPPalpha.

Previous work by our group demonstrated that homomeric alpha7 nicotinic acetylcholine receptors (nAChR) play a role in the neurotoxicity induced by 3,4-methylenedioxymethamphetamine (MDMA), as well as the binding affinity of this drug to these receptors. Here we studied the effect of MDMA on the activation of nAChR subtypes, the consequent calcium mobilization, and calpain/caspase 3 activation because prolonged Ca(2+) increase could contribute to cytotoxicity. As techniques, we used fluorimetry in Fluo-4-loaded PC12 cells and electrophysiology in Xenopus oocytes. MDMA produced a rapid and sustained increase in calcium without reaching the maximum effect induced by ACh. It also concentration-dependently inhibited the response induced by ACh, nicotine, and the specific alpha7 agonist PNU 282987 with IC(50) values in the low micromolar range. Similarly, MDMA induced inward currents in Xenopus oocytes transfected with human alpha7 but not with alpha4beta2 nAChR and inhibited ACh-induced currents in both receptors in a concentration-dependent manner. The calcium response was inhibited by methyllycaconitine (MLA) and alpha-bungarotoxin but not by dihydro-beta-erythroidine. These results therefore indicate that MDMA acts as a partial agonist on alpha7 nAChRs and as an antagonist on the heteromeric subtypes. Subsequently, calcium-induced Ca(2+) release from the endoplasmic reticulum and entry through voltage-operated calcium channels are also implicated as proved using specific antagonists. In addition, treatment with MDMA for 24 h significantly increased basal Ca(2+) levels and induced an increase in alpha-spectrin breakdown products, which indicates that calpain and caspase 3 were activated. These effects were inhibited by pretreatment with MLA. Moreover, pretreatment with MDMA induced functional upregulation of calcium responses to specific agonists of both heteromeric and alpha7 nAChR. Sustained calcium entry and calpain activation could favor the activation of Ca(2+)-dependent enzymes such as protein kinase C and nitric oxide synthase, which are involved in the generation of ROS and the blockade of the dopamine transporter. This, together with caspase 3 activation, must play a role in MDMA-induced cytotoxicity.