Galantamine prevents long-lasting suppression of excitatory synaptic transmission in CA1 pyramidal neurons of soman-challenged guinea pigs.

Galantamine, a drug currently approved for the treatment of Alzheimer's disease, has recently emerged as an effective pretreatment against the acute toxicity and delayed cognitive deficits induced by organophosphorus (OP) nerve agents, including soman. Since cognitive deficits can result from impaired glutamatergic transmission in the hippocampus, the present study was designed to test the hypothesis that hippocampal glutamatergic transmission declines following an acute exposure to soman and that this effect can be prevented by galantamine. To test this hypothesis, spontaneous excitatory postsynaptic currents (EPSCs) were recorded from CA1 pyramidal neurons in hippocampal slices obtained at 1h, 24h, or 6-9 days after guinea pigs were injected with: (i) 1×LD50 soman (26.3µg/kg, s.c.); (ii) galantamine (8mg/kg, i.m.) followed 30min later by 1×LD50 soman, (iii) galantamine (8mg/kg, i.m.), or (iv) saline (0.5ml/kg, i.m.). In soman-injected guinea pigs that were not pretreated with galantamine, the frequency of EPSCs was significantly lower than that recorded from saline-injected animals. There was no correlation between the severity of soman-induced acute toxicity and the magnitude of soman-induced reduction of EPSC frequency. Pretreatment with galantamine prevented the reduction of EPSC frequency observed at 6-9 days after the soman challenge. Prevention of soman-induced long-lasting reduction of hippocampal glutamatergic synaptic transmission may be an important determinant of the ability of galantamine to counter cognitive deficits that develop long after an acute exposure to the nerve agent.

The herpes simplex virus type 2 R1 protein kinase (ICP10 PK) functions as a dominant regulator of apoptosis in hippocampal neurons involving activation of the ERK survival pathway and upregulation of the antiapoptotic protein Bag-1.

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) can trigger or block apoptosis in a cell type-dependent manner. We have recently shown that the protein kinase activity of the large subunit of the HSV-2 ribonucleotide reductase (R1) protein (ICP10 PK) blocks apoptosis in cultured hippocampal neurons by activating the extracellular signal-regulated kinase (ERK) survival pathway (Perkins et al., J. Virol. 76:1435-1449, 2002). The present studies were designed to better elucidate the mechanism of ICP10 PK-induced neuroprotection and determine whether HSV-1 has similar activity. The data indicate that apoptosis inhibition by ICP10 PK involves a c-Raf-1-dependent mechanism and induction of the antiapoptotic protein Bag-1 by the activated ERK survival pathway. Also associated with neuroprotection by ICP10 PK are increased activation/stability of the transcription factor CREB and stabilization of the antiapoptotic protein Bcl-2. HSV-1 and the ICP10 PK-deleted HSV-2 mutant ICP10DeltaPK activate JNK, c-Jun, and ATF-2, induce the proapoptotic protein BAD, and trigger apoptosis in hippocampal neurons. c-Jun activation and apoptosis are inhibited in hippocampal cultures infected with HSV-1 in the presence of the JNK inhibitor SP600125, suggesting that JNK/c-Jun activation is required for HSV-1-induced apoptosis. Ectopically delivered ICP10 PK (but not its PK-negative mutant p139) inhibits apoptosis triggered by HSV-1 or ICP10DeltaPK. Collectively, the data indicate that ICP10 PK-induced activation of the ERK survival pathway results in Bag-1 upregulation and overrides the proapoptotic JNK/c-Jun signal induced by other viral proteins.

The herpes simplex virus type 2 R1 protein kinase (ICP10 PK) blocks apoptosis in hippocampal neurons, involving activation of the MEK/MAPK survival pathway.

Herpes simplex virus type 1 (HSV-1) and HSV-2 trigger or counteract apoptosis by a cell-specific mechanism. Our studies are based on previous findings that the protein kinase (PK) domain of the large subunit of HSV-2 ribonucleotide reductase (ICP10) activates the Ras/MEK/MAPK pathway (Smith et al., J. Virol. 74:10417, 2000). Because survival pathways can modulate apoptosis, we used cells that are stably or transiently transfected with ICP10 PK, an HSV-2 mutant deleted in ICP10 PK (ICP10DeltaPK) and the MEK-specific inhibitor U0126 to examine the role of ICP10 PK in apoptosis. Apoptosis was induced by staurosporine or D-mannitol in human (HEK293) cells or HEK293 cells stably transfected with the ICP10 PK-negative mutant p139 (JHL15), as determined by morphology, DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL), caspase-3 activation, and poly(ADP-ribose) polymerase (PARP) cleavage. HEK293 cells stably transfected with ICP10 (JHLa1) were protected from apoptosis. ICP10 but not p139 protected neuronally differentiated PC12 cells from death due to nerve growth factor withdrawal, and apoptosis (determined by TUNEL) and caspase-3 activation were seen in primary hippocampal cultures infected with ICP10DeltaPK but not with HSV-2 or a revertant virus [HSV-2(R)]. The data indicate that ICP10 has antiapoptotic activity under both paradigms and that it requires a functional PK activity. The apoptotic cells in primary hippocampal cultures were neurons, as determined by double immunofluorescence with fluorescein-labeled dUTP (TUNEL) and phycoerythrin-labeled antibodies specific for neuronal proteins (TuJ1 and NF-160). Protection from apoptosis was associated with MEK/MAPK activation, as evidenced by (i) increased levels of activated (phosphorylated) MAPK in HSV-2- but not ICP10DeltaPK-infected cultures and (ii) inhibition of MAPK activation by the MEK-specific inhibitor U0126. MEK and MAPK were activated by infection with UV-inactivated but not antibody-neutralized HSV-2, suggesting that activation requires cellular penetration but is independent of de novo viral protein synthesis.

Human bronchial epithelial and endothelial cells express alpha7 nicotinic acetylcholine receptors.

The epithelial or endothelial cells that line the human bronchi and the aorta express nicotinic acetylcholine receptors (nAChRs) of alpha3 subtypes. We report here that human bronchial epithelial cells (BEC) and aortic endothelial cells (AEC) express also the nAChR alpha7 subunit, which forms functional nAChRs. Polymerase chain reaction and in situ hybridization experiments detected alpha7 subunit mRNA in cultured human BEC and AEC and in sections of rat trachea. The binding of radiolabeled alpha-bungarotoxin revealed a few thousand binding sites per cell in cultured human BEC and human and bovine AEC. Western blot and immunohistochemistry experiments demonstrated that cultured BEC and AEC express a protein(s) recognized by anti-alpha7 antibodies. Whole-cell patch-clamp studies of cultured human BEC demonstrated the presence of fast-desensitizing currents activated by choline and nicotine that were blocked reversibly by methyllycaconitine (1 nM) and irreversibly by alpha-bungarotoxin (100 nM), consistent with the expression of functional alpha7 nAChRs. In some cells, choline activated also slowly decaying currents, confirming previous reports that BEC express functional alpha3beta4 nAChRs. Exposure of cultured BEC to nicotine (1 microM) for 3 days up-regulated functional alpha7 and alpha3 nAChRs, as indicated by the increased number of cells responding to acetylcholine and choline, with both fast-desensitizing currents, which were blocked irreversibly by alpha-bungarotoxin, and with slowly desensitizing currents, which are alpha-bungarotoxin-insensitive currents. The presence of alpha7 nAChRs in BEC and AEC suggests that some toxic effects of tobacco smoke could be mediated through these nicotine-sensitive receptors.

Modulation of nicotinic receptor activity in the central nervous system: a novel approach to the treatment of Alzheimer disease.

Impaired cholinergic function in the central nervous system is an early feature of Alzheimer disease (AD). Currently, cholinergic deficit is usually corrected by increasing the amount of acetylcholine in the synapse by inhibiting acetylcholinesterase (AChE). One of the most consistent cholinergic deficits in AD is the reduced expression of nicotinic acetylcholine receptors (nAChR) in the brain. Since these receptors are essential for learning and memory, restoring nicotinic cholinergic function is a promising approach to treating AD. Allosteric modulation of nAChR is a novel approach, which circumvents development of tolerance through long-term use of conventional nicotinic agonists. Allosteric modulators interact with receptor-binding sites distinct from those capable of recognizing the natural agonist. Positive allosteric modulation of nAChR activity has no effect on conductance of single channels; instead, by facilitating channel opening, it potentiates responses evoked by the interaction of the natural agonist with presynaptic and postsynaptic nAChR. Allosteric modulation of nAChR activity could therefore potentially produce a significant benefit in AD. One such allosteric modulator is galantamine. In addition to increasing nAChR activity, galantamine also inhibits AChE. This novel, dual mechanism of action distinguishes galantamine from many other AChE inhibitors. Galantamine has been shown to improve cognitive and daily function for at least 6 months in placebo-controlled trials, and to maintain these functions at baseline levels for at least 12 months in a 6-month open-label extension study. Galantamine has positive effects on nAChR expression, which are likely to contribute to its sustained efficacy in the treatment of AD patients.

A novel thienylhydrazone, (2-thienylidene)3,4-methylenedioxybenzoylhydrazine, increases inotropism and decreases fatigue of skeletal muscle.

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 at < or =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).

The brain metabolite kynurenic acid inhibits alpha7 nicotinic receptor activity and increases non-alpha7 nicotinic receptor expression: physiopathological implications.

The tryptophan metabolite kynurenic acid (KYNA) has long been recognized as an NMDA receptor antagonist. Here, interactions between KYNA and the nicotinic system in the brain were investigated using the patch-clamp technique and HPLC. In the electrophysiological studies, agonists were delivered via a U-shaped tube, and KYNA was applied in admixture with agonists and via the background perfusion. Exposure (>/=4 min) of cultured hippocampal neurons to KYNA (>/=100 nm) inhibited activation of somatodendritic alpha7 nAChRs; the IC(50) for KYNA was approximately 7 microm. The inhibition of alpha7 nAChRs was noncompetitive with respect to the agonist and voltage independent. The slow onset of this effect could not be accounted for by an intracellular action because KYNA (1 mm) in the pipette solution had no effect on alpha7 nAChR activity. KYNA also blocked the activity of preterminal/presynaptic alpha7 nAChRs in hippocampal neurons in cultures and in slices. NMDA receptors were less sensitive than alpha7 nAChRs to KYNA. The IC(50) values for KYNA-induced blockade of NMDA receptors in the absence and presence of glycine (10 microm) were approximately 15 and 235 microm, respectively. Prolonged (3 d) exposure of cultured hippocampal neurons to KYNA increased their nicotinic sensitivity, apparently by enhancing alpha4beta2 nAChR expression. Furthermore, as determined by HPLC with fluorescence detection, repeated systemic treatment of rats with nicotine caused a transient reduction followed by an increase in brain KYNA levels. These results demonstrate that nAChRs are targets for KYNA and suggest a functionally significant cross talk between the nicotinic cholinergic system and the kynurenine pathway in the brain.

A galantamina no tratamento da doença de Alzheimer's: da pesquisa ao paciente / Galantamine in the treatment of Alzheimer disease: from research to the patient

A doença de Alzheimer (DA) é uma doença neurodegenerativa progressiva, que representa um sério problema para saúde pública, particularmente nos países industrializados, onde a população de idosos vem crescendo de modo acentuado. O tratamento da doença, além de sintomático, visa retardar a progressão das deterioração mental dos pacientes. Recentemente, com bases no seu mecanismo de ação duplo, a galantamina foi adicionada ao arsenal terapêutico para tratamento da DA. Além de atuar como um anticolinesterásico fraco, a galantamina, associando-se diretamente a um sítio de ligação localizado nas subunidades alfa dos receptores nicotínicos, também age como "ligante potencializador alostérico" (LPA/APL) da atividade desses receptores. Esta revisão tem como objetivo central discutir as bases fisiopatológicas que levaram à introdução da galantamina na clínica para o tratamento de pacientes portadores da DA. Para tanto, apresenta uma abordagem geral dos clínicos da doença, incluído não somente uma descrição breve dos sintomas e diagnósticos da doença, como também um resumo dos processos celulares e moleculares alterados durante o curso da mesma. Ênfase é dada à hipótese colinérgica, que assume que a progressão da doença está associada à hipoatividade das funções colinérgicas, especialmente daquelas mediadas pelos receptores nicotínicos neuronais do cérebro. Estudos recentes indicam que pode haver uma relação causal entre a hipofunção colinérgica nicotínica no cérebro e a doença de Alzheimer. É com base nessa hipótese e na descoberta laboratorial da ação LPA/APL da galantamina sobre os receptores nicotínicos, que este alcalóide, originalmente isolado do bulbo da Galanthus nivalis, foi recentemente introduzido em vários países para o tratamento da DA

Multiplication and Movement of a Citrus Strain of Xylella fastidiosa Within Sweet Orange.

Populations of cultivable cells of a citrus variegated chlorosis (CVC) disease strain of Xylella fastidiosa in stems and leaf veins of sweet orange (Citrus sinensis (L.) Osbeck) seedlings were estimated by dilution plating at 1, 2, 4, 8, and 16 weeks after needle inoculation. Cell populations ranged from log 4 to log 5 CFU/g of tissue after 1 week and increased to log 5 to log 7 CFU/g (median log 6) after 8 to 16 weeks. Recovery of greater than log 5 CFU/g from stem nodes distal to the inoculation site indicated systemic movement of the bacteria. Foliar symptoms in inoculated seedlings first appeared after 8 weeks. Population estimates from leaf veins of CVC-affected trees in citrus groves were in the same range but slightly lower (average log 5.8 CFU/g). X. fastidiosa was isolated from citrus more efficiently in periwinkle wilt-GelRite (PWG) and periwinkle wilt (PW) media than in charcoal-yeast extract with ACES buffer (BCYE) medium The relatively lower populations of cultivable cells of X. fastidiosa in citrus with CVC symptoms, compared with those reported in grapevines with Pierce's disease, suggest that most cells of X. fastidiosa within symptomatic citrus may be dead, explaining in part the low rates of vector transmission from citrus to citrus.

Nicotine at concentrations found in cigarette smokers activates and desensitizes nicotinic acetylcholine receptors in CA1 interneurons of rat hippocampus.

Behavioral effects of cigarette smoking are attributed to the interactions of nicotine with brain nicotinic acetylcholine receptors (nAChRs). However, the mechanisms by which nAChR function in developing and mature brain is affected by a smoker's level of nicotine (50-500 nM) remain unclear. Thus, the objective of this study was to determine the concentration- and time-dependent effects of nicotine on alpha7 and alpha4beta2 nAChRs, the two major brain subtypes, natively expressed in CA1 interneurons of rat hippocampal slices. Only at concentrations > or =5 microM did nicotine (applied for 6-60 s) elicit action potentials or measurable whole-cell currents (EC(50)=158 microM) in stratum radiatum interneurons that express alpha7 nAChRs. Continuous exposure for 10-15 min of the neurons to nicotine (0.5-2.5 microM) inhibited alpha7 nAChR-mediated currents (IC(50)=640 nM) evoked by choline (10 mM). Nicotine (> or =0.125 microM) applied to the neurons for 1-5 min induced slowly desensitizing whole-cell currents (EC(50)=3.2 microM) in stratum lacunosum moleculare interneurons; this effect was mediated by alpha4beta2 nAChRs. Also via activation of alpha4beta2 nAChRs, nicotine (0.125-0.5 microM) increased the frequency and amplitude of GABAergic postsynaptic currents (PSCs) in stratum radiatum interneurons. However, exposure of the neurons for 10-15 min to nicotine (0.25-0.5 microM) resulted in desensitization of alpha4beta2 nAChRs. It is suggested that nanomolar concentrations of nicotine after acute intake suppress inhibitory inputs to pyramidal cells through a disinhibitory mechanism involving activation of alpha4beta2 nAChRs and desensitization of alpha7 nAChRs, and after chronic intake leads to up-regulation of both receptor subtypes via desensitization. These findings have direct implications to the actions of nicotine in cigarette smokers.

The opioid antagonist naltrexone inhibits activity and alters expression of alpha7 and alpha4beta2 nicotinic receptors in hippocampal neurons: implications for smoking cessation programs.

This study was designed to investigate whether naltrexone, an opioid antagonist that has been evaluated clinically as a co-adjuvant in smoking cessation programs, affects function and expression of neuronal nicotinic receptors (nAChRs). Whole-cell current recordings from rat hippocampal neurons in culture and in slices demonstrated that alpha7 nAChRs can be inhibited non-competitively by naltrexone (IC(50) approximately 25 microM). The voltage dependence of the effect suggested that naltrexone acts as an open-channel blocker of alpha7 nAChRs. Naltrexone also inhibited activation of alpha4beta2 nAChRs in hippocampal neurons; however its IC(50) was higher ( approximately 141 microM). At a concentration as high as 300 microM (which is sufficient to block by 100% and 70% the activity of alpha7 and alpha4beta2 nAChRs, respectively), naltrexone had no effect on kainate and AMPA receptors, blocked by no more than 20% the activity of NMDA and glycine receptors, and reduced by 35% the activity of GABA(A) receptors. A 3-day exposure of cultured hippocampal neurons to naltrexone (30 microM) or nicotine (10 microM, a concentration that fully desensitized alpha7 nAChRs) resulted in a 2-fold increase in the average amplitude of alpha7 nAChR-subserved currents. Naltrexone did not augment the maximal up-regulation of alpha7 nAChRs induced by nicotine, indicating that both drugs act via a common mechanism. In addition to increasing alpha7 nAChRs-mediated responses per neuron, nicotine increased the number of neurons expressing functional non-alpha7 nAChRs (probably alpha4beta2 nAChRs); this effect was blocked by naltrexone (0.3 and 30 microM). Therefore, naltrexone may affect dependence on cigarette smoking by differentially altering function and expression of alpha7 and alpha4beta2 nAChRs in the central nervous system.

Ca(2+)-sensitive inhibition by Pb(2+) of alpha7-containing nicotinic acetylcholine receptors in hippocampal neurons.

In the present study the patch-clamp technique was applied to cultured hippocampal neurons to determine the kinetics as well as the agonist concentration- and Ca(2+)-dependence of Pb(2+)-induced inhibition of alpha7 nicotinic receptors (nAChRs). Evidence is provided that more than two-thirds of the inhibition by Pb(2+) (3-30 microM) of alpha7 nAChR-mediated whole-cell currents (referred to as type IA currents) develops rapidly and is fully reversible upon washing. The estimated values for tau(onset) and tau(recovery) were 165 and 240 ms, respectively. The magnitude of the effect of Pb(2+) was the same regardless of whether acetylcholine or choline was the agonist. Pre-exposure of the neurons for 800 ms to Pb(2+) (30 microM) decreased the amplitude and accelerated the decay phase of currents evoked by moderate to high agonist concentrations. In contrast, only the amplitude of currents evoked by low agonist concentrations was reduced when the neurons were exposed simultaneously to Pb(2+) and the agonists. Taken together with the findings that Pb(2+) reduces the frequency of opening and the mean open time of alpha7 nAChR channels, these data suggest that Pb(2+) accelerates the rate of receptor desensitization. An additional reduction of type IA current amplitudes occurred after 2-min exposure of the neurons to Pb(2+). This effect was not reversible upon washing of the neurons and was most likely due to an intracellular action of Pb(2+). Pb(2+)-induced inhibition of alpha7 nAChRs, which was hindered by the enhancement of extracellular Ca(2+) concentrations, may contribute to the neurotoxicity of the heavy metal.

Neuronal nicotinic receptors in synaptic functions in humans and rats: physiological and clinical relevance.

The present report describes the participation of nicotinic receptors (nAChRs) in controlling the excitability of local neuronal circuitries in the rat hippocampus and in the human cerebral cortex. The patch-clamp technique was used to record responses triggered by the non-selective agonist ACh and the alpha7-nAChR-selective agonist choline in interneurons of human cerebral cortical and rat hippocampal slices. Evidence is provided that functional alpha7- and alpha4beta2-like nAChRs are present on somatodendritic and/or preterminal/terminal regions of interneurons in the CA1 field of the rat hippocampus and in the human cerebral cortex and that activation of the different nAChR subtypes present in the preterminal/terminal areas of the interneurons triggers the tetrodotoxin-sensitive release of GABA. Modulation by nAChRs of GABAergic transmission, which can result either in inhibition or disinhibition of pyramidal neurons, depends both on the receptor subtype present in the interneurons and on the agonist acting upon these receptors. Not only do alpha7 nAChRs desensitize faster than alpha4beta2 nAChRs, but also alpha7 nAChR desensitization induced by ACh lasts longer than that induced by choline. These mechanisms, which appear to be retained across species, might explain the involvement of nAChRs in cognitive functions and in such neurological disorders as Alzheimer's disease and schizophrenia.

alpha7 nicotinic acetylcholine receptors and modulation of gabaergic synaptic transmission in the hippocampus.

The present report provides new findings regarding modulation of gamma-aminobutyric acid (GABA) transmission by alpha7 nicotinic receptor activity in CA1 interneurons of rat hippocampal slices. Recordings were obtained from tight-seal cell-attached patches of the CA1 interneurons, and agonists were delivered to the neurons via a modified U-tube. Application for 6 s of the alpha7 nicotinic receptor-selective agonist choline (> or =1 mM) to all CA1 interneurons tested triggered action potentials that were detected as fast current transients. The activity triggered by choline terminated well before the end of the agonist pulse, was blocked by the alpha7 nicotinic receptor antagonist methyllycaconitine (50 nM) and was concentration dependent; the higher the concentration of choline the higher the frequency of events and the shorter the delay for detection of the first event. In 40% of the neurons tested, choline-triggered action potentials decreased in amplitude progressively until no more events could be detected despite the presence of the agonist. Primarily, this finding could be explained by Na(+)-channel inactivation associated with membrane depolarization induced by alpha7 nicotinic receptor activation. In 60% of the neurons, the amplitude of choline-induced action potentials was sustained at the intial level, but again the activity did not last as long as the agonist pulse, in this case apparently because of agonist-induced receptor desensitization. These results altogether demonstrate that agonists interacting with alpha7 nicotinic receptors, including the natural transmitter acetylcholine and its metabolite choline, influence GABAergic transmission, not only by activating these receptors, but also by controlling the rate of Na(+)-channel inactivation and/or by inducing receptor desensitization.

Nicotinic receptor activation in human cerebral cortical interneurons: a mechanism for inhibition and disinhibition of neuronal networks.

Cholinergic control of the activity of human cerebral cortical circuits has long been thought to be accounted for by the interaction of acetylcholine (ACh) with muscarinic receptors. Here we report the discovery of functional nicotinic receptors (nAChRs) in interneurons of the human cerebral cortex and discuss the physiological and clinical implications of these findings. The whole-cell mode of the patch-clamp technique was used to record responses triggered by U-tube application of the nonselective agonist ACh and of the alpha7-nAChR-selective agonist choline to interneurons visualized by means of infrared-assisted videomicroscopy in slices of the human cerebral cortex. Choline induced rapidly desensitizing whole-cell currents that, being sensitive to blockade by methyllycaconitine (MLA; 50 nM), were most likely subserved by an alpha7-like nAChR. In contrast, ACh evoked slowly decaying whole-cell currents that, being sensitive to blockade by dihydro-beta-erythroidine (DHbetaE; 10 microM), were most likely subserved by an alpha4beta2-like nAChR. Application of ACh (but not choline) to the slices also triggered GABAergic postsynaptic currents (PSCs). Evidence is provided that ACh-evoked PSCs are the result of activation of alpha4beta2-like nAChRs present in preterminal axon segments and/or in presynaptic terminals of interneurons. Thus, nAChRs can relay inhibitory and/or disinhibitory signals to pyramidal neurons and thereby modulate the activity of neuronal circuits in the human cerebral cortex. These mechanisms, which appear to be retained across species, can account for the involvement of nAChRs in cognitive functions and in certain neuropathological conditions.

Expression of nicotinic acetylcholine receptor subunits in the cerebral cortex in Alzheimer's disease: histotopographical correlation with amyloid plaques and hyperphosphorylated-tau protein.

Impairment of cholinergic transmission and decreased numbers of nicotinic binding sites are well-known features accompanying the cognitive dysfunction seen in Alzheimer's disease (AD). In order to elucidate the underlying cause of this cholinoceptive dysfunction, the expression of two pharmacologically different nicotinic acetylcholine receptor (nAChR) subunits (alpha4, alpha7) was studied in the cerebral cortex of Alzheimer patients as compared to controls. Patch-clamp recordings of 14 dissociated neurons of control cortices showed responses suggesting the existence of alpha4- and alpha7-containing functional nAChRs in the human cortex. In cortices of Alzheimer patients and controls, the pattern of distribution and the number of alpha4 and alpha7 mRNA-expressing neurons were similar, whereas at the protein level a decrease in the density of alpha4- and alpha7-expressing neurons of approximately 30% was observed in Alzheimer patients. The histotopographical correlation of nAChR expression with accompanying pathological changes, e.g. accumulation of hyperphosphorylated-tau (HP-tau) protein and beta-amyloid showed that neurons in the vicinity of beta-amyloid plaques bore both nAChR transcripts. Neurons heavily labelled for HP-tau, however, expressed little or no alpha4 and alpha7 mRNA. These results point to an impaired synthesis of nAChRs on the protein level as a possible cause of the cholinoceptive deficit in AD. Further investigations need to elucidate whether interactions of HP-tau with nAChR mRNA, or alterations in the quality of alpha4 and alpha7 transcripts give rise to decreased protein expression at the level of individual neurons.

Lead increases tetrodotoxin-insensitive spontaneous release of glutamate and GABA from hippocampal neurons.

This study was aimed at investigating the effects of the environmental pollutant lead (Pb2+) on the tetrodotoxin (TTX)-insensitive release of neurotransmitters from hippocampal neurons. Evidence is provided that Pb2+ (>/=100 nM) increases the frequency of gamma-aminobutyric acid (GABA)- and glutamate-mediated miniature postsynaptic currents (MPSCs) recorded by means of the patch-clamp technique from cultured hippocampal neurons. Because Pb2+ changed neither the amplitude nor the decay-time constant of the MPSCs, Pb2+-induced changes in MPSC frequency are exclusively due to a presynaptic action of this heavy metal. Increase by Pb2+ of the action potential-independent release of GABA and glutamate was concentration dependent and was only partially reversible upon washing of the neurons with nominally Pb2+-free external solution. This effect was also Ca2+ independent and began approximately after 1-2-min exposure of the neurons to Pb2+. The latency for the onset of the Pb2+'s effect on the MPSC frequency and the inability of the chelator ethylenediaminetetraacetic acid (100 microM) to reverse the effect that remained after washing of the neurons with external solution suggested that Pb2+ acted via an intracellular mechanism. Of interest also was the finding that Pb2+ simultaneously increased the release of GABA and glutamate, overriding the ability of these neurotransmitters to decrease the release of one another. Given that synaptic activity is a key mechanism for the establishment of stable synaptic connections early in the development, it is possible that, by interfering with spontaneous transmitter release, Pb2+ has lasting effects on neuronal maturation and plasticity.

Nanomolar concentrations of lead inhibit glutamatergic and GABAergic transmission in hippocampal neurons.

To investigate whether lead (Pb2+) affects the tetrodotoxin (TTX)-sensitive release of neurotransmitters, the whole-cell mode of the patch-clamp technique was applied to cultured hippocampal neurons. Pb2+ (>/=10 nM) reversibly blocked the TTX-sensitive release of glutamate and gamma-aminobutyric acid (GABA), as evidenced by the reduction of the amplitude and frequency of glutamate- and GABA-mediated postsynaptic currents (PSCs) evoked by spontaneous neuronal firing. This effect of Pb2+, which occurred 2-3 s after exposure of the neurons to Pb2+-containing external solution, was not related to changes in Na+-channel activity, and was quantified by measurements of changes in the amplitude of PSCs evoked when a 50-micros, 5-V stimulus was applied via a bipolar electrode to a neuron synaptically connected to the neuron under study. With an IC50 of approximately 68 nM, Pb2+ blocked the evoked release of glutamate and GABA. This effect was most likely mediated by Pb2+'s actions on extracellular targets, because there was a very short delay (<3 s) for its onset, and it could be completely reversed by the chelator ethylene diaminetetraacetic acid (EDTA). Given that Pb2+-induced blockade of evoked transmitter release could be reversed by 4-aminopyridine, it is suggested that the effect on release was mediated via the binding of Pb2+ to voltage-gated Ca2+ channels. Thus, it is most likely that the neurotoxic effects of Pb2+ in the mammalian brain involve a decrease of the TTX-sensitive, Ca2+-dependent release of neurotransmitters.

Choline and selective antagonists identify two subtypes of nicotinic acetylcholine receptors that modulate GABA release from CA1 interneurons in rat hippocampal slices.

Neuronal nicotinic receptors (nAChR) are known to control transmitter release in the CNS. Thus, this study was aimed at exploring the diversity and localization of nAChRs present in CA1 interneurons in rat hippocampal slices. The use of a U-tube as the agonist delivery system was critical for the reliable detection of nicotinic responses induced by brief exposure of the neurons to ACh or to the alpha7 nAChR-selective agonist choline. The present study demonstrated that CA1 interneurons, in addition to expressing functional alpha7 nAChRs, also express functional alpha4beta2-like nAChRs and that activation of both receptors facilitates an action potential-dependent release of GABA. Depending on the experimental condition, one of the following nicotinic responses was recorded from the interneurons by means of the patch-clamp technique: a nicotinic whole-cell current, depolarization accompanied by action potentials, or GABA-mediated postsynaptic currents (PSCs). Responses mediated by alpha7 nAChRs were short-lasting, whereas those mediated by alpha4beta2 nAChRs were long-lasting. Thus, phasic or tonic inhibition of CA1 interneurons may be achieved by selective activation of alpha7 or alpha4beta2 nAChRs, respectively. It can also be suggested that synaptic levels of choline generated by hydrolysis of ACh in vivo may be sufficient to control the activity of the alpha7 nAChRs. The finding that methyllycaconitine and dihydro-beta-erythroidine (antagonists of alpha7 and alpha4beta2 nAChRs, respectively) increased the frequency and amplitude of GABAergic PSCs suggests that there is an intrinsic cholinergic activity that sustains a basal level of nAChR activity in these interneurons.

alpha-bungarotoxin- and methyllycaconitine-sensitive nicotinic receptors mediate fast synaptic transmission in interneurons of rat hippocampal slices.

This study demonstrates for the first time that alpha7 nicotinic receptors (nAChRs) mediate fast synaptic transmission in conventional hippocampal slices. In the presence of antagonists of muscarinic, AMPA, NMDA, GABAA, ATP, and 5-HT3 receptors, spontaneous and evoked postsynaptic currents (PSCs) recorded from CA1 interneurons were blocked by the alpha7 nAChR antagonists methyllycaconitine and alpha-bungarotoxin and by a desensitizing concentration of the alpha7 nAChR agonist choline. Spontaneous nicotinic PSCs were also accompanied by Na+ transients, indicating that alpha7 nAChR-mediated transmission serves as an excitatory signal to the CA1 interneurons in the hippocampus.