Effects of the DAG analogue 1,2-dioctanoyl-sn-glycerol (DiC8) on nicotine- and clothianidin-evoked currents through α-bungarotoxin-insensitive nicotinic acetylcholine receptors expressed on cockroach neurosecretory cells.

We previously demonstrated that the cockroach α-bungarotoxin-sensitive nicotinic acetylcholine receptors, nAChR1 and nAChR2 subtypes, are differently sensitive to intracellular calcium pathways. Here, using whole cell patch-clamp recordings, we studied the effects of the diacylglycerol (DAG) analogue 1,2-dioctanoyl-sn-glycerol (DiC8) on nicotine- and clothianidin-evoked currents under an α-bungarotoxin treatment. Our results demonstrated that DiC8 reduced nicotine and clothianidin evoked currents. 10 µM DiC8 suppressed the increase in nicotine-induced currents which was brought about by application of 5 mM caffeine or 9 mM Ca2+, whereas DiC8 did not affect the decrease in nicotine-induced currents induced by BAPTA. Similarly, bath application of caffeine or 9 mM Ca2+ did not change the clothianidin effects, and the amplitude of clothianidin-induced currents was not affected. However, co-application of both 10 µM DiC8 with 9 mM Ca2+, caffeine or BAPTA reduced clothianidin current amplitudes. We conclude that nicotine and clothianidin differently modulate nAChR1 and nAChR2 subtypes under DiC8 treatment, and that nicotine activates nAChR1, whereas clothianidin activates both nAChR1 and nAChR2 subtypes.

Ketamine inhibits synaptic transmission and nicotinic acetylcholine receptor-mediated responses in rat intracardiac ganglia in situ.

The intravenous anaesthetic ketamine, has been demonstrated to inhibit nicotinic acetylcholine receptor (nAChR)-mediated currents in dissociated rat intracardiac ganglion (ICG) neurons (Weber et al., 2005). This effect would be predicted to depress synaptic transmission in the ICG and would account for the inhibitory action of ketamine on vagal transmission to the heart (Inoue and König, 1988). This investigation was designed to examine the activity of ketamine on (i) postsynaptic responses to vagal nerve stimulation, (ii) the membrane potential, and (iii) membrane current responses evoked by exogenous application of ACh and nicotine in ICG neurons in situ. Intracellular recordings were made using sharp intracellular microelectrodes in a whole mount ICG preparation. Preganglionic nerve stimulation and recordings in current- and voltage-clamp modes were used to assess the action of ketamine on ganglionic transmission and nAChR-mediated responses. Ketamine attenuated the postsynaptic responses evoked by nerve stimulation. This reduction was significant at clinically relevant concentrations at high frequencies. The excitatory membrane potential and current responses to focal application of ACh and nicotine were inhibited in a concentration-dependent manner by ketamine. In contrast, ketamine had no effect on either the directly-evoked action potential or excitatory responses evoked by focal application of γ-aminobutyric acid (GABA). Taken together, ketamine inhibits synaptic transmission and nicotine- and ACh-evoked currents in adult rat ICG. Ketamine inhibition of synaptic transmission and nAChR-mediated responses in the ICG contributes significantly to its attenuation of the bradycardia observed in response to vagal stimulation in the mammalian heart.

α7 Nicotinic acetylcholine receptor-specific antibody induces inflammation and amyloid ß42 accumulation in the mouse brain to impair memory.

Nicotinic acetylcholine receptors (nAChRs) expressed in the brain are involved in regulating cognitive functions, as well as inflammatory reactions. Their density is decreased upon Alzheimer disease accompanied by accumulation of ß-amyloid (Aß42), memory deficit and neuroinflammation. Previously we found that α7 nAChR-specific antibody induced pro-inflammatory interleukin-6 production in U373 glioblastoma cells and that such antibodies were present in the blood of humans. We raised a hypothesis that α7 nAChR-specific antibody can cause neuroinflammation when penetrating the brain. To test this, C57Bl/6 mice were either immunized with extracellular domain of α7 nAChR subunit α7(1-208) or injected with bacterial lipopolysaccharide (LPS) for 5 months. We studied their behavior and the presence of α3, α4, α7, ß2 and ß4 nAChR subunits, Aß40 and Aß42 and activated astrocytes in the brain by sandwich ELISA and confocal microscopy. It was found that either LPS injections or immunizations with α7(1-208) resulted in region-specific decrease of α7 and α4ß2 and increase of α3ß4 nAChRs, accumulation of Aß42 and activated astrocytes in the brain of mice and worsening of their episodic memory. Intravenously transferred α7 nAChR-specific-antibodies penetrated the brain parenchyma of mice pre-injected with LPS. Our data demonstrate that (1) neuroinflammation is sufficient to provoke the decrease of α7 and α4ß2 nAChRs, Aß42 accumulation and memory impairment in mice and (2) α7(1-208) nAChR-specific antibodies can cause inflammation within the brain resulting in the symptoms typical for Alzheimer disease.

Nicotine induction of theta frequency oscillations in rodent medial septal diagonal band in vitro.

AIM: This study aimed to examine the role of the nicotinic receptor (nAChR) in the generation of theta oscillations (4-12 Hz) in vitro. METHODS: Electrophysiological studies were performed on medial septal diagonal band area (MSDB) slices to measure theta oscillation. Immunofluorescence and confocal microscopy studies were carried out to detect α4 nAChR and ß2 nAChR subunits in perfused-fixed tissue from VGluT2-GFP and GAD67-GFP transgenic mice. RESULTS: Application of nicotine to MSDB slices produced persistent theta oscillations in which area power increased in a dose-responsive manner. This activity was inhibited by GABAA receptor antagonists and partially by ionotropic glutamate receptor antagonists, indicating the involvement of local GABAergic and glutamatergic neurons in the production of the rhythmic activity. The nicotine-induced theta activity was also inhibited selectively by non-α7*nAChR antagonists, suggesting the presence of these receptor types on GABAergic and glutamatergic neuron populations in the MSDB. This was confirmed by immunofluorescence and confocal microscopy studies in transgenic mice in which the GABAergic and glutamatergic neurons express green fluorescent protein (GFP), showing localisation of ß2 nAChR and α4 nAChR subunits, the most common constituents of non-α7*nAChRs, in both cell types in the MSDB. CONCLUSION: Theta activity in the MSDB may be generated by tonic stimulation of non-α7*nAChRs.

Experimental autoimmune myasthenia gravis in the mouse.

Myasthenia gravis (MG) is a T cell-dependent antibody-mediated autoimmune neuromuscular disease. Antibodies to the nicotinic acetylcholine receptor (AChR) destroy the AChR, thus leading to defective neuromuscular transmission of electrical impulse and to muscle weakness. This unit is a practical guide to the induction and evaluation of experimental autoimmune myasthenia gravis (EAMG) in the mouse, the animal model for MG. Protocols are provided for the extraction and purification of AChR from the electric organs of Torpedo californica, or the electric ray. The purified receptor is used as an immunogen to induce autoimmunity to AChR, thus causing EAMG. The defect in neuromuscular transmission can also be measured quantitatively by electromyography. In addition, EAMG is frequently characterized by the presence of serum antibodies to AChR, which are measured by radioimmunoassay and by a marked antibody-mediated reduction in the number of muscle AChRs. AChR extracted from mouse muscle is used in measuring serum antibody levels and for quantifying muscle AChR content. Another hallmark of the disease is complement and IgG deposits located at the neuromuscular junction, which can be visualized by immunofluorescence techniques.

Combined α7 nicotinic acetylcholine receptor agonism and partial serotonin transporter inhibition produce antidepressant-like effects in the mouse forced swim and tail suspension tests: a comparison of SSR180711 and PNU-282987.

Emerging evidence points to an involvement of nicotinic acetylcholine receptors (nAChRs) in major depression. Nicotine improves symptoms of depression in humans and shows antidepressant-like effects in rodents. Monoamine release is facilitated by nAChR stimulation, and nicotine-evoked serotonin (5-HT) release has been shown to depend on α7 nAChR activation. The α7 nAChR agonist PNU-282987 shows no antidepressant-like activity when tested alone in the mouse forced swim (mFST) or tail suspension tests (mTST). However, in combination with a sub-active dose of the selective 5-HT reuptake inhibitor citalopram, inducing ~50% 5-HT reuptake inhibition, PNU-282987 has shown marked antidepressant-like effects in the mFST. SSR180711 is a recently described α7 nAChR agonist that has shown antidepressant-like activity in the rat forced swim test. To address the possibility that 5-HT reuptake inhibition contributes to the antidepressant-like profile of SSR180711, we compared the behavioural and biochemical profiles of PNU-282987 and SSR180711. In the mFST and mTST, SSR180711 (3-30 mg/kg, s.c.) showed dose-dependent antidepressant-like activity, while PNU-282987 (3-30 mg/kg, s.c.) showed no significant effect. The ED(50) to displace [³H]α-bungarotoxin binding was 1.7 and 5.5 mg/kg for SSR180711 and PNU-282987, respectively, suggesting that both compounds produce near-maximal α7 nAChR occupancy at the highest dose. While PNU-282987 did not affect ex vivo [³H]5-HT uptake, SSR180711 inhibited [³H]5-HT uptake with an ED50 of 30 mg/kg. This degree of inhibition is similar to that observed with a citalopram dose of ~2.4 mg/kg, a dose that is normally not active in the mFST or mTST. This suggests that the antidepressant-like activity of SSR180711 may involve partial 5-HT reuptake inhibition. SSR180711 therefore represents a compound displaying the synergistic effect of α7 nAChR agonism combined with partial 5-HT reuptake inhibition previously described. The addition of α7 nAChR agonism to classical monoamine-based mechanisms may represent a novel option for the improved treatment of major depression.

Selective recognition and elimination of nicotinic acetylcholine receptor-reactive B cells by a recombinant fusion protein AChR-Fc in myasthenia gravis in vitro.

AChR-reactive B cells play a key role in the pathogenesis of myasthenia gravis (MG) by producing autoantibodies. Selective elimination of AChR-reactive B cells will be a promising way to treat MG. Thus, we generated a fusion protein (referred to as AChR-Fc) composed of the human extracellular domain of AChR α1 subunit and the Fc domain of the human IgG1 heavy chain, which could bind both to AChR-reactive BCR and FcγRIIB on the surface of AChR-reactive B cells. Our results showed that AChR-Fc inhibited the proliferation of AChR-specific hybridoma cells, promoted their apoptosis, and mediated cytotoxicity by cross-linking effector cells and complement. Likewise, AChR-Fc significantly reduced the number of AChR-reactive B cells from spleen of Lewis rats immunized with AChR ex vivo.

A human recombinant autoantibody-based immunotoxin specific for the fetal acetylcholine receptor inhibits rhabdomyosarcoma growth in vitro and in a murine transplantation model.

Rhabdomyosarcoma (RMS) is the most common malignant soft tissue tumor in children and is highly resistant to all forms of treatment currently available once metastasis or relapse has commenced. As it has recently been determined that the acetylcholine receptor (AChR) gamma-subunit, which defines the fetal AChR (fAChR) isoform, is almost exclusively expressed in RMS post partum, we recombinantly fused a single chain variable fragment (scFv) derived from a fully human anti-fAChR Fab-fragment to Pseudomonas exotoxin A to generate an anti-fAChR immunotoxin (scFv35-ETA). While scFv35-ETA had no damaging effect on fAChR-negative control cell lines, it killed human embryonic and alveolar RMS cell lines in vitro and delayed RMS development in a murine transplantation model. These results indicate that scFv35-ETA may be a valuable new therapeutic tool as well as a relevant step towards the development of a fully human immunotoxin directed against RMS. Moreover, as approximately 20% of metastatic malignant melanomas (MMs) display rhabdoid features including the expression of fAChR, the immunotoxin we developed may also prove to be of significant use in the treatment of these more common and most often fatal neoplasms.

Overexpression of IFN-induced protein 10 and its receptor CXCR3 in myasthenia gravis.

Myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG), are autoimmune disorders in which the acetylcholine receptor (AChR) is the major autoantigen. Microarray technology was used to identify new potential drug targets for treatment of myasthenia that would reduce the need for the currently used nonspecific immunosuppression. The chemokine IFN-gamma-inducible protein 10 (IP-10; CXCL10), a CXC chemokine, and its receptor, CXCR3, were found to be overexpressed in lymph node cells of EAMG rats. Quantitative real-time PCR confirmed these findings and revealed up-regulated mRNA levels of another chemoattractant that activates CXCR3, monokine induced by IFN-gamma (Mig; CXCL9). TNF-alpha and IL-1beta, which act synergistically with IFN-gamma to induce IP-10, were also up-regulated. These up-regulations were observed in immune response effector cells, namely, lymph node cells, and in the target organ of the autoimmune attack, the muscle of myasthenic rats, and were significantly reduced after suppression of EAMG by mucosal tolerance induction with an AChR fragment. The relevance of IP-10/CXCR3 signaling in myasthenia was validated by similar observations in MG patients. A significant increase in IP-10 and CXCR3 mRNA levels in both thymus and muscle was observed in myasthenic patients compared with age-matched controls. CXCR3 expression in PBMC of MG patients was markedly increased in CD4(+), but not in CD8(+), T cells or in CD19(+) B cells. Our results demonstrate a positive association of IP-10/CXCR3 signaling with the pathogenesis of EAMG in rats as well as in human MG patients.

Breakdown of tolerance to a self-peptide of acetylcholine receptor alpha-subunit induces experimental myasthenia gravis in rats.

Experimental autoimmune myasthenia gravis (EAMG), a model for human myasthenia (MG), is routinely induced in susceptible rat strains by a single immunization with Torpedo acetylcholine receptor (TAChR). TAChR immunization induces anti-AChR Abs that cross-react with self AChR, activate the complement cascade, and promote degradation of the postsynaptic membrane of the neuromuscular junction. In parallel, TAChR-specific T cells are induced, and their specific immunodominant epitope has been mapped to the sequence 97-116 of the AChR alpha subunit. A proliferative T cell response against the corresponding rat sequence (R97-116) was also found in TAChR-immunized rats. To test whether the rat (self) sequence can be pathogenic, we immunized Lewis rats with R97-116 or T97-116 peptides and evaluated clinical, neurophysiological, and immunological parameters. Clinical signs of the disease were noted only in R97-116-immunized animals and were confirmed by electrophysiological signs of impaired neuromuscular transmission. All animals produced Abs against the immunizing peptide, but anti-rat AChR Abs were observed only in animals immunized with the rat peptide. These findings suggested that EAMG in rats can be induced by a single peptide of the self AChR, that this sequence is recognized by T cells and Abs, and that breakdown of tolerance to a self epitope might be an initiating event in the pathogenesis of rat EAMG and MG.

B7-1 costimulatory molecule is critical for the development of experimental autoimmune myasthenia gravis.

Following immunization with acetylcholine receptor (AChR), MHC class II-restricted, AChR-specific CD4 cell activation is critical for the development of experimental autoimmune myasthenia gravis (EAMG) in C57BL/6 mice. To study the contributions of B7-1 and B7-2 costimulatory molecules in EAMG, B7-1, B7-2, and B7-1/B7-2 gene knockout (KO) mice were immunized with Torpedo AChR in CFA. Compared with wild-type C57BL6 mice, B7-1 and B7-1/2 KO mice were resistant to EAMG development. B7-1 KO mice had reduced anti-AChR Ab compared with C57BL/6 mice. However, neither B7-1 nor B7-2 gene disruption impaired AChR-induced or dominant alpha(146-162) peptide-induced in vitro lymphoproliferative responses. Blocking of the B7-1 or B7-2 molecule by specific mAbs in vivo led to a reduction in the AChR-specific lymphocyte response, and the reduction was more pronounced in mice treated with anti-B7-2 Ab. The findings implicate B7-1 molecules as having a critical role in the induction of EAMG, and the resistance of B7-1 KO mice is associated with suppressed humoral, rather than suppressed AChR-specific, T cell responses. The data also point to B7-2 molecules as being the dominant costimulatory molecules required for AChR-induced lymphocyte proliferation.

Nicotinic mechanisms of memory: effects of acute local DHbetaE and MLA infusions in the basolateral amygdala.

Nicotine has been shown to improve working memory. The neural mechanisms underlying this effect are still being determined. The ventral hippocampus is critical for nicotinic effects on memory. Local ventral hippocampal infusions of either the nicotinic alpha7 nicotinic receptor antagonist methyllycaconitine (MLA) or the alpha4beta2 nicotinic receptor antagonist dihydro-beta-erythroidine (DHbetaE) caused working memory impairments, but no additive effects were seen. Other areas, such as the amygdala, also likely play important roles in nicotinic effects on memory. Amygdalar lesions cause memory impairment and there is a dense concentration of nicotinic receptors in the basolateral amygdala. The current study used local basolateral amygdalar infusions of the nicotinic antagonists MLA and DHbetaE to determine the involvement of alpha7 and alpha4beta2 nicotinic receptors in spatial working and reference memory. Rats (n=8) were trained in the 16-arm radial maze and were implanted with bilateral infusion cannulae into the basolateral amygdala. Acute infusions of MLA (6.75 micro g/side, P<0.0005) or DHbetaE (3.38 micro g/side, P<0.025) caused significant working memory impairments. When given together MLA and DHbetaE did not produce an additive effect. In fact, the 6.75 micro g/kg dose of DHbetaE produced a significant (P<0.0005) attenuation of the MLA-induced working memory impairment. Significant effects were not seen with reference memory or response latency. Nicotinic systems in the basolateral amygdala, as in the ventral hippocampus, are important for spatial working memory. In both the basolateral amygdala and the ventral hippocampus, MLA and DHbetaE individually caused working memory impairments. The lowest effective dose of DHbetaE was lower in the basolateral amygdala than in the ventral hippocampus. In both the basolateral amygdala and the ventral hippocampus, combined MLA and DHbetaE treatment did not produce additive working memory deficits. Unlike in the ventral hippocampus, the addition of DHbetaE to MLA in the basolateral amygdala significantly reduced the MLA-induced working memory deficit.

Inhibition of nicotinic acetylcholine receptors by bicuculline.

A study was made on the effects of bicuculline, the classical gamma-aminobutyric acid-A receptor antagonist, on heteromeric mouse muscle alphabetagammadelta, heteromeric neuronal rat alpha2beta4 and alpha4beta2 and homomeric human alpha7 nicotinic acetylcholine receptors (nAChRs), expressed in Xenopus oocytes. Bicuculline reduced the ACh-induced currents in a rapid and reversible way, with IC50 values of 34+/-1.5 microM for mouse muscle alphabetagammadelta and 12.4+/-0.7 and 18+/-1 microM for rat neuronal alpha2beta4 and alpha4beta2 nAChRs, respectively. Therefore, the three types of heteromeric receptors are inhibited by bicuculline but the neuronal alpha2beta4 and alpha4beta2 receptors were more sensitive than the muscle alphabetagammadelta receptor. The Hill coefficients for ACh-current inhibition were close to one for all types of receptors, suggesting a single site of action for bicuculline inhibition of nAChRs. Bicuculline shifted the ACh-dose-current response curve to the right and the maximal current was reduced, a reduction that for the heteromeric receptors was not overcome by high concentrations of ACh. The effect of bicuculline was examined at different membrane potentials, and the ACh-current-membrane potential relationships obtained indicate that the inhibition by bicuculline is voltage-dependent for muscle alphabetagammadelta and neuronal alpha2beta4 and alpha4beta2 nAChRs. All these results are consistent with the notion that bicuculline blocks the heteromeric muscle and neuronal nAChRs in a non-competitive way. Studies were also made on the wild type (wt alpha7) and mutant leu-to-threo (L248T) homomeric human neuronal alpha7-nAChRs. In sharp contrast to the heteromeric ACh receptors examined, bicuculline blocked in a competitive way the homomeric wt alpha7-nAChRs, as evidenced by a parallel shift of the bicuculline dose-ACh-current inhibition on raising the ACh concentration. Moreover, similar to the effects of serotonin on wt and mutant alpha7 ACh receptors, the mutation converted bicuculline from an antagonist into a competitive agonist. All this suggests that bicuculline may serve as a lead molecule to design new anticholinergic substances.

Functional incorporation of exogenous proteins into the Xenopus oocyte membrane does not depend on intracellular calcium increase.

Fusion of membranes occurs in diverse biological events and, in most cases, Ca2+ greatly augments its rate. The aim of this work was to study the role played by Ca2+ when transplanting exogenous proteins into Xenopus oocyte membranes. Lipid vesicles carrying nicotinic acetylcholine (ACh) receptors (nAChRs) from Torpedo electroplaques were injected into oocytes. The time course of nAChR incorporation was assessed by recording ACh-evoked currents at different times from injection. An incorporation peak was found at 16 h, but responses were maintained for over 48 h. To assess the role played by Ca2+, two groups were considered: control and chelator-loaded oocytes. In the latter group, cells were incubated with 50 microM 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetrakis(acetoxymethyl)ester (BAPTA-AM) or loaded with ca. 5 nmol ethyleneglycol-bis (beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) 2 h before nAChR injection. Both groups responded to ACh, although the current amplitude was smaller in chelator-loaded than in control cells. These results indicate that the slow fusion of lipoproteosome vesicles with the oocyte plasma membrane does not depend on intracellular Ca2+ increase and therefore belongs to the type called "constitutive". This membrane fusion process is thus different from those involved in resealing of disrupted oocyte membranes or in the fusion of cortical granules with the egg membrane.

Role of tolerogen conformation in induction of oral tolerance in experimental autoimmune myasthenia gravis.

We recently demonstrated that oral or nasal administration of recombinant fragments of the acetylcholine receptor (AChR) prevents the induction of experimental autoimmune myasthenia gravis (EAMG) and suppresses ongoing EAMG in rats. We have now studied the role of spatial conformation of these recombinant fragments in determining their tolerogenicity. Two fragments corresponding to the extracellular domain of the human AChR alpha-subunit and differing in conformation were tested: Halpha1-205 expressed with no fusion partner and Halpha1-210 fused to thioredoxin (Trx), and designated Trx-Halpha1-210. The conformational similarity of the fragments to intact AChR was assessed by their reactivity with alpha-bungarotoxin and with anti-AChR mAbs, specific for conformation-dependent epitopes. Oral administration of the more native fragment, Trx-Halpha1-210, at the acute phase of disease led to exacerbation of EAMG, accompanied by an elevation of AChR-specific humoral and cellular reactivity, increased levels of Th1-type cytokines (IL-2, IL-12), decreased levels of Th2 (IL-10)- or Th3 (TGF-beta)-type cytokines, and higher expression of costimulatory factors (CD28, CTLA4, B7-1, B7-2, CD40L, and CD40). On the other hand, oral administration of the less native fragments Halpha1-205 or denatured Trx-Halpha1-210 suppressed ongoing EAMG and led to opposite changes in the immunological parameters. It thus seems that native conformation of AChR-derived fragments renders them immunogenic and immunopathogenic and therefore not suitable for treatment of myasthenia gravis. Conformation of tolerogens should therefore be given careful attention when considering oral tolerance for treatment of autoimmune diseases.

Oral administration of a dual analog of two myasthenogenic T cell epitopes down-regulates experimental autoimmune myasthenia gravis in mice.

Myasthenia gravis (MG) and experimental autoimmune MG (EAMG) are T cell-regulated, antibody-mediated autoimmune diseases. The major autoantigen in MG is the nicotinic acetylcholine receptor (AChR). Two peptides, representing sequences of the human AChR alpha-subunit, p195-212 and p259-271, were previously shown to be immunodominant T cell epitopes in MG patients as well as, respectively, in SJL and BALB/c mice. A dual analog (termed Lys-262-Ala-207) composed of the tandemly arranged two single amino acid analogs of p195-212 and p259-271 was shown to inhibit, in vitro and in vivo, MG-associated autoimmune responses. Furthermore, the dual analog could down-regulate myasthenogenic manifestations in mice with EAMG that was induced by inoculation of a pathogenic T cell line. In the present study, the ability of the dual analog to treat EAMG induced in susceptible C57BL/6 mice by native Torpedo AChR was evaluated. Mice that were diagnosed to have clinical symptoms of EAMG were treated with the dual analog by oral administration, 500 microg per mouse three times a week for 5-8 weeks. Treatment with the dual analog down-regulated the clinical manifestations of the ongoing disease as assessed by the clinical score, grip strength (measured by a grip strength meter), and electromyography. The effects on the clinical EAMG correlated with a reduced production of anti-AChR antibody as well as a decrease in the secretion of interleukin-2 and, more dramatically, interferon-gamma, in response to AChR triggering. Thus, the dual analog is an efficient immunomodulator of EAMG in mice and might be of specific therapeutic potential for MG.