Central nervous system interaction and crosstalk between nAChRs and other ionotropic and metabotropic neurotransmitter receptors.

Neuronal nicotinic acetylcholine receptors (nAChRs) are widely distributed in both the peripheral and the central nervous systems. nAChRs exert a crucial modulatory influence on several brain biological processes; they are involved in a variety of neuronal diseases including Parkinson's disease, Alzheimer's disease, epilepsy, and nicotine addiction. The influence of nAChRs on brain function depends on the activity of other neurotransmitter receptors that co-exist with nAChRs on neurons. In fact, the crosstalk between receptors is an important mechanism of neurotransmission modulation and plasticity. This may be due to converging intracellular pathways but also occurs at the membrane level, because of direct physical interactions between receptors. In this line, this review is dedicated to summarizing how nAChRs and other ionotropic and metabotropic receptors interact and the relevance of nAChRs cross-talks in modulating various neuronal processes ranging from the classical modulation of neurotransmitter release to neuron plasticity and neuroprotection.

Long-term hippocampal glutamate synapse and astrocyte dysfunctions underlying the altered phenotype induced by adolescent THC treatment in male rats.

Cannabis use has been frequently associated with sex-dependent effects on brain and behavior. We previously demonstrated that adult female rats exposed to delta-9-tetrahydrocannabinol (THC) during adolescence develop long-term alterations in cognitive performances and emotional reactivity, whereas preliminary evidence suggests the presence of a different phenotype in male rats. To thoroughly depict the behavioral phenotype induced by adolescent THC exposure in male rats, we treated adolescent animals with increasing doses of THC twice a day (PND 35-45) and, at adulthood, we performed a battery of behavioral tests to measure affective- and psychotic-like symptoms as well as cognition. Poorer memory performance and psychotic-like behaviors were present after adolescent THC treatment in male rats, without alterations in the emotional component. At cellular level, the expression of the NMDA receptor subunit, GluN2B, as well as the levels of the AMPA subunits, GluA1 and GluA2, were significantly increased in hippocampal post-synaptic fractions from THC-exposed rats compared to controls. Furthermore, increases in the levels of the pre-synaptic marker, synaptophysin, and the post-synaptic marker, PSD95, were also present. Interestingly, KCl-induced [(3)H]D-ASP release from hippocampal synaptosomes, but not gliosomes, was significantly enhanced in THC-treated rats compared to controls. Moreover, in the same brain region, adolescent THC treatment also resulted in a persistent neuroinflammatory state, characterized by increased expression of the astrocyte marker, GFAP, increased levels of the pro-inflammatory markers, TNF-α, iNOS and COX-2, as well as a concomitant reduction of the anti-inflammatory cytokine, IL-10. Notably, none of these alterations was observed in the prefrontal cortex (PFC). Together with our previous findings in females, these data suggest that the sex-dependent detrimental effects induced by adolescent THC exposure on adult behavior may rely on its ability to trigger different region-dependent changes in glutamate synapse and glial cells. The phenotype observed in males is mainly associated with marked dysregulations in the hippocampus, whereas the prevalence of alterations in the emotional sphere in females is associated with profound changes in the PFC.

Metamodulation of presynaptic NMDA receptors: New perspectives for pharmacological interventions.

Metamodulation shifted the scenario of the central neuromodulation from a simplified unimodal model to a multimodal one. It involves different receptors/membrane proteins physically associated or merely colocalized that act in concert to control the neuronal functions influencing each other. Defects or maladaptation of metamodulation would subserve neuropsychiatric disorders or even synaptic adaptations relevant to drug dependence. Therefore, this "vulnerability" represents a main issue to be deeply analyzed to predict its aetiopathogenesis, but also to propose targeted pharmaceutical interventions. The review focusses on presynaptic release-regulating NMDA receptors and on some of the mechanisms of their metamodulation described in the literature. Attention is paid to the interactors, including both ionotropic and metabotropic receptors, transporters and intracellular proteins, which metamodulate their responsiveness in physiological conditions but also undergo adaptation that are relevant to neurological dysfunctions. All these structures are attracting more and more the interest as promising druggable targets for the treatment of NMDA receptor-related central diseases: these substances would not exert on-off control of the colocalized NMDA receptors (as usually observed with NMDA receptor full agonists/antagonists), but rather modulate their functions, with the promise of limiting side effects that would favor their translation from preclinic to clinic. This article is part of the Special Issue on "The receptor-receptor interaction as a new target for therapy".

Correction: Prophylactic versus Therapeutic Fingolimod: Restoration of Presynaptic Defects in Mice Suffering from Experimental Autoimmune Encephalomyelitis.

[This corrects the article DOI: 10.1371/journal.pone.0170825.].

Urinary secretion and extracellular aggregation of mutant uromodulin isoforms.

Uromodulin is exclusively expressed in the thick ascending limb and is the most abundant protein secreted in urine where it is found in high-molecular-weight polymers. Its biological functions are still elusive, but it is thought to play a protective role against urinary tract infection, calcium oxalate crystal formation, and regulation of water and salt balance in the thick ascending limb. Mutations in uromodulin are responsible for autosomal-dominant kidney diseases characterized by defective urine concentrating ability, hyperuricemia, gout, tubulointerstitial fibrosis, renal cysts, and chronic kidney disease. Previous in vitro studies found retention in the endoplasmic reticulum as a common feature of all uromodulin mutant isoforms. Both in vitro and in vivo we found that mutant isoforms partially escaped retention in the endoplasmic reticulum and reached the plasma membrane where they formed large extracellular aggregates that have a dominant-negative effect on coexpressed wild-type protein. Notably, mutant uromodulin excretion was detected in patients carrying uromodulin mutations. Thus, our results suggest that mutant uromodulin exerts a gain-of-function effect that can be exerted by both intra- and extracellular forms of the protein.

Primary hyperoxaluria Type 1: indications for screening and guidance for diagnosis and treatment.

Primary hyperoxaluria Type 1 is a rare autosomal recessive inborn error of glyoxylate metabolism, caused by a deficiency of the liver-specific enzyme alanine:glyoxylate aminotransferase. The disorder results in overproduction and excessive urinary excretion of oxalate, causing recurrent urolithiasis and nephrocalcinosis. As glomerular filtration rate declines due to progressive renal involvement, oxalate accumulates leading to systemic oxalosis. The diagnosis is based on clinical and sonographic findings, urine oxalate assessment, enzymology and/or DNA analysis. Early initiation of conservative treatment (high fluid intake, pyridoxine, inhibitors of calcium oxalate crystallization) aims at maintaining renal function. In chronic kidney disease Stages 4 and 5, the best outcomes to date were achieved with combined liver-kidney transplantation.

Synaptosomes and Metamodulation of Receptors.

Synaptosomes are re-sealed pinched off nerve terminals that maintain all the main structural and functional features of the original structures and that are appropriate to study presynaptic events. Because of the discovery of new structural and molecular events that dictate the efficiency of transmitter release and of its receptor-mediated control in the central nervous system, the interest in this tissue preparation is continuously renewing. Most of these events have been already discussed in previous reviews, but few of them were not and deserve some comments since they could suggest new functional and possibly therapeutic considerations. Among them, the "metamodulation" of receptors represents an emerging aspect that dramatically increased the complexity of the presynaptic compartment, adding new insights to the role of presynaptic receptors as modulators of chemical synapses. Deciphering the mechanism of presynaptic metamodulation would permit indirect approaches to control the activity of presynaptic release-regulating receptors that are currently orphans of direct ligands/modulators, paving the road for the proposal of new therapeutic approaches for central neurological diseases.

The Anti-Aggregative Peptide KLVFF Mimics Aß1-40 in the Modulation of Nicotinic Receptors: Implications for Peptide-Based Therapy.

In recent years, the inhibition of beta-amyloid (Aß) aggregation has emerged as a potential strategy for Alzheimer's disease. KLVFF, a small peptide corresponding to the aminoacidic sequence 16-20 of Aß, reduces Aß fibrillation dose dependently. Therefore, the toxic and functional characterization of its brain activity is fundamental for clarifying its potential therapeutic role. Accordingly, we studied the modulatory role of KLVFF on the cholinergic receptors regulating dopamine and noradrenaline release in rat synaptosomes. Nicotinic receptors on dopaminergic nerve terminals in the nucleus acccumbens are inhibited by KLVFF, which closely resembles full-length Aß1-40. Moreover, KLVFF entrapped in synaptosomes does not modify the nicotinic receptor's function, suggesting that external binding to the receptor is required for its activity. The cholinergic agent desformylflustrabromine counteracts the KLVFF effect. Remarkably, muscarinic receptors on dopaminergic terminals and nicotinic receptors regulating noradrenaline release in the hippocampus are completely insensitive to KLVFF. Based on our findings, KLVFF mimics Aß1-40 as a negative modulator of specific nicotinic receptor subtypes affecting dopamine transmission in the rat brain. Therefore, new pharmacological strategies using the anti-aggregative properties of KLVFF need to be evaluated for potential interference with nicotinic receptor-mediated transmission.

Presynaptic 5-HT2A-mGlu2/3 Receptor-Receptor Crosstalk in the Prefrontal Cortex: Metamodulation of Glutamate Exocytosis.

The glutamatergic nerve endings of a rat prefrontal cortex (PFc) possess presynaptic 5-HT2A heteroreceptors and mGlu2/3 autoreceptors, whose activation inhibits glutamate exocytosis, and is measured as 15 mM KCl-evoked [3H]D-aspartate ([3H]D-asp) release (which mimics glutamate exocytosis). The concomitant activation of the two receptors nulls their inhibitory activities, whereas blockade of the 5-HT2A heteroreceptors with MDL11,939 (1 µM) strengthens the inhibitory effect elicited by the mGlu2/3 receptor agonist LY329268 (1 µM). 5-HT2A receptor antagonists (MDL11,939; ketanserin; trazodone) amplify the impact of low (3 nM) LY379268. Clozapine (0.1-10 µM) mimics the 5-HT2A agonist (±) DOI and inhibits the KCl-evoked [3H]D-asp overflow in a MDL11,939-dependent fashion, but does not modify the (±) DOI-induced effect. mGlu2 and 5-HT2A proteins do not co-immunoprecipitate from synaptosomal lysates, nor does the incubation of PFc synaptosomes with MDL11,939 (1 µM) or clozapine (10 µM) modify the insertion of mGlu2 subunits in synaptosomal plasma membranes. In conclusion, 5-HT2A and mGlu2/3 receptors colocalize, but do not physically associate, in PFc glutamatergic terminals, where they functionally interact in an antagonist-like fashion to control glutamate exocytosis. The mGlu2/3-5-HT2A metamodulation could be relevant to therapy for central neuropsychiatric disorders, including schizophrenia, but also unveil cellular events accounting for their development, which also influence the responsiveness to drugs regimens.

p53 Arg72Pro and MDM2 309 SNPs in hereditary retinoblastoma.

The tumor suppressor p53 and its negative regulator MDM2 have crucial roles in a variety of cellular functions such as the control of the cell cycle, senescence, genome stability and apoptosis, and are frequently deregulated in carcinogenesis. Previous studies have highlighted the contribution of the common functional polymorphisms p53 p.Arg72Pro and MDM2 309SNP to the risk of both common cancers and Li-Fraumeni syndrome. Their possible role in retinoblastoma has recently been addressed by Castéra et al, who however only studied the MDM2 309SNP. Here, for the first time, we analyzed both single nucleotide polymorphisms (SNPs) in a case-control study of 111 Italian hereditary retinoblastoma patients. We found a significant association of the p53 Pro/Pro genotype with the disease (odds ratio=3.58, P=0.002). The MDM2 309SNP showed a weak negative association of allele G that deserves further investigation. These findings further support the hypothesis that genetic variability of the p53 pathway contributes to the individual susceptibility to retinoblastoma, as shown for Li-Fraumeni syndrome and a variety of non-hereditary cancers.

Modeling the effect of 3 missense AGXT mutations on dimerization of the AGT enzyme in primary hyperoxaluria type 1.

INTRODUCTION: Mutations of the AGXT gene encoding the alanine:glyoxylate aminotransferase liver enzyme (AGT) cause primary hyperoxaluria type 1 (PH1). Here we report a molecular modeling study of selected missense AGXT mutations: the common Gly170Arg and the recently described Gly47Arg and Ser81Leu variants, predicted to be pathogenic using standard criteria. METHODS: Taking advantage of the refined 3D structure of AGT, we computed the dimerization energy of the wild-type and mutated proteins. RESULTS: Molecular modeling predicted that Gly47Arg affects dimerization with a similar effect to that shown previously for Gly170Arg through classical biochemical approaches. In contrast, no effect on dimerization was predicted for Ser81Leu. Therefore, this probably demonstrates pathogenic properties via a different mechanism, similar to that described for the adjacent Gly82Glu mutation that affects pyridoxine binding. CONCLUSION: This study shows that the molecular modeling approach can contribute to evaluating the pathogenicity of some missense variants that affect dimerization. However, in silico studies--aimed to assess the relationship between structural change and biological effects--require the integrated use of more than 1 tool.

Pre-synaptic nicotinic and D receptors functionally interact on dopaminergic nerve endings of rat and mouse nucleus accumbens.

The existence of pre-synaptic auto- and hetero receptors which modulate neurotransmitter release is well documented. Emerging evidence show that in some cases these pre-synaptic receptors may also cross-talk with each other. The aim of the present work was to investigate whether acetylcholine receptors (nAChRs) and dopamine (DA) autoreceptors, which are both able to modulate DA release, functionally interact on the same nerve endings. We used rat and mouse nucleus accumbens synaptosomes pre-labeled with [(3)H]DA and exposed to nicotinic and dopaminergic receptor ligands. Both nicotinic agonists and 4-aminopyridine (4-AP) provoked [(3)H]DA release which was inhibited by quinpirole and blocked by sulpiride and raclopride. Both the inhibitory effect of quinpirole and the stimulatory effect of (-)nicotine did not change when the nAChRs or the DA receptors were desensitized. (-)Nicotine and 4-AP were able to stimulate [(3)H]DA overflow also in mouse synaptosomes and this overflow was partially inhibited by quinpirole. In the beta(2) knockout mice quinpirole was still able to inhibit the [(3)H]DA overflow elicited by 4-AP. To conclude: in rat and mouse the (-)nicotine evoked-release can be modulated by D(2)/D(3) autoreceptors present on the DA terminals and nAChRs function is independent from D(2)/D(3) autoreceptors which themselves may function independently from the activation of nAChRs.

NMDA-mediated modulation of dopamine release is modified in rat prefrontal cortex and nucleus accumbens after chronic nicotine treatment.

In this study, we investigate the effects of chronic administration of (-)nicotine on the function of the NMDA-mediated modulation of [(3)H]dopamine (DA) release in rat prefrontal cortex (PFC) and nucleus accumbens (NAc). In the PFC synaptosomes NMDA in a concentration-dependent manner evoked [(3)H]DA release in rats chronically treated with vehicle (14 days) with an EC(50) of 13.1 +/- 2.0 microM. The NMDA-evoked overflow of the [(3)H]DA in PFC nerve endings of rats treated with (-)nicotine was significantly lower (-43%) than in vehicle treated rats. The EC(50) was 9.0 +/- 1.4 microM. Exposure of NAc synaptosomes of rats treated with vehicle to NMDA produced an increase in [(3)H]DA overflow with an EC(50) of 14.5 +/- 5.5 microM. This effect was significantly enhanced in chronically treated animals. The EC(50) was 10.5 +/- 0.5 microM. The K(+)-evoked release of [(3)H]DA was not modified by the (-)nicotine administration. Both the changes of the NMDA-evoked [(3)H]DA overflow in the NAc and PFC disappeared after 14 days withdrawal. The results show that chronic (-)nicotine differentially affects the NMDA-mediated [(3)H]DA release in the PFC and NAc of the rat.

Exposure to an enriched environment selectively increases the functional response of the pre-synaptic NMDA receptors which modulate noradrenaline release in mouse hippocampus.

We evaluated the impact of environmental training on the functions of pre-synaptic glutamatergic NMDA and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and nicotinic receptors expressed by hippocampal noradrenergic nerve terminals. Synaptosomes isolated from the hippocampi of mice housed in enriched (EE) or standard (SE) environment were labeled with [(3)H]noradrenaline ([(3)H]NA) and tritium release was monitored during exposure in superfusion to NMDA, AMPA, epibatidine or high K(+). NMDA -evoked [(3)H]NA release from EE hippocampal synaptosomes was significantly higher than that from SE synaptosomes, while the [(3)H]NA overflow elicited by 100 muM AMPA, 1 muM epibatidine or (9, 15, 25 mM) KCl was unchanged. In EE mice, the apparent affinity of NMDA or glycine was unmodified, while the efficacy was significantly augmented. Sensitivity to non-selective or subtype-selective NMDA receptor antagonists (MK-801, ifenprodil and Zn(2+) ions) was not modified in EE. Finally, the analysis of NMDA receptor subunit mRNA expression in noradrenergic cell bodies of the locus coeruleus showed that NR1, NR2A, NR2B and NR2D subunits were unchanged, while NR2C decreased significantly in EE mice as compared to SE mice. Functional up-regulation of the pre-synaptic NMDA receptors modulating NA release might contribute to the improved learning and memory found in animals exposed to an EE.

Nicotinic and muscarinic cholinergic receptors coexist on GABAergic nerve endings in the mouse striatum and interact in modulating GABA release.

Muscarinic cholinergic receptors (mAChRs) and nicotinic cholinergic receptors (nAChRs) regulating GABA release from striatal nerve endings were studied by monitoring release of previously accumulated [(3)H]GABA or endogenous GABA from superfused mouse striatal synaptosomes. Oxotremorine inhibited the release of [(3)H]GABA elicited by depolarization with 4-aminopyridine (4-AP), an effect antagonized by atropine. Agonists at nAChRs, including the alpha(4)beta(2)( *) subunit-selective RJR2403, provoked the release of [(3)H]GABA as well as of the endogenous transmitter; these effects also were prevented by oxotremorine and pilocarpine suggesting coexpression of functional mAChRs and alpha(4)beta(2)( *) nAChRs on GABAergic nerve endings. The inhibitory effects of oxotremorine on the release of [(3)H]GABA evoked by 4-AP or by RJR2403 were: (i) prevented by the M(2)/M(4) mAChR antagonist himbacine; (ii) insensitive to the M2 antagonist AFDX116; (iii) blocked by the selective M(4) mAChR antagonists MT3, thus indicating the involvement of receptors of the M(4) subtype. In conclusion, in the corpus striatum, acetylcholine released from cholinergic interneurons can activate alpha(4)beta(2)( *) nAChRs mediating release of GABA; this evoked release can be negatively modulated by M(4) mAChRs coexpressed on the same GABAergic terminals.

The cyclin-dependent kinase inhibitor 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole induces nongenotoxic, DNA replication-independent apoptosis of normal and leukemic cells, regardless of their p53 status.

BACKGROUND: Current chemotherapy of human cancers focuses on the DNA damage pathway to induce a p53-mediated cellular response leading to either G1 arrest or apoptosis. However, genotoxic treatments may induce mutations and translocations that result in secondary malignancies or recurrent disease. In addition, about 50% of human cancers are associated with mutations in the p53 gene. Nongenotoxic activation of apoptosis by targeting specific molecular pathways thus provides an attractive therapeutic approach. METHODS: Normal and leukemic cells were evaluated for their sensitivity to 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) through cell viability and caspase activation tests. The apoptotic pathway induced by DRB was analysed by immunfluorescence and immunoblot analysis. H2AX phosphorylation and cell cycle analysis were performed to study the dependance of apoptosis on DNA damage and DNA replication, respectively. To investigate the role of p53 in DRB-induced apoptosis, specific p53 inhibitors were used. Statistical analysis on cell survival was performed with the test of independence. RESULTS: Here we report that DRB, an inhibitor of the transcriptional cyclin-dependent kinases (CDKs) 7 and 9, triggers DNA replication-independent apoptosis in normal and leukemic human cells regardless of their p53 status and without inducing DNA damage. Our data indicate that (i) in p53-competent cells, apoptosis induced by DRB relies on a cytosolic accumulation of p53 and subsequent Bax activation, (ii) in the absence of p53, it may rely on p73, and (iii) it is independent of ATM and NBS1 proteins. Notably, even apoptosis-resistant leukemic cells such as Raji were sensitive to DRB. CONCLUSION: Our results indicate that DRB represents a potentially useful cancer chemotherapeutic strategy that employs both the p53-dependent and -independent apoptotic pathways without inducing genotoxic stress, thereby decreasing the risk of secondary malignancies.

An in vitro model of T cell receptor revision in mature human CD8+ T cells.

V(D)J recombination is a mechanism peculiar to the somatic rearrangement of antigen receptor genes. It requires both expression of the RAG-1 and RAG-2 recombinases and accessibility of the substrate to its recombinase and post-cleavage/DNA repair stage. TCR revision is a genetic correction mechanism that changes T cell specificity by re-activating V(D)J recombination in peripheral T cells. This process is now well described in both normal or pathological murine and human settings. Many of its features, such as the question of whether it occurs in truly mature T cells, remain to be elucidated. Its occurrence in human CD8+ T cells is also an open question. We have therefore established an in vitro model of TCR revision in mature human CD8+ T cells to determine whether down-regulation of the TCR/CD3 complex from the cell surface in the presence of IL7 as a factor favouring chromatin remodelling initiates a TCR revision pathway. Only mature CD8+ T cells carrying already-formed antigen receptors were used. CD8+ T cells treated with anti-CD3 and IL7 showed rearrangement intermediates and expressed new Vbeta-chains on their surface. Investigation of the molecular pathway thus induced disclosed up-regulation of the RAG-2 transcript, but absence of the 'canonical' RAG-1 mRNA. A surprising finding was the demonstration of alternative splice forms of this mRNA, already expressed in untreated CD8+ T cells, encoding for the full-length RAG-1 protein, which was increased three-fold in the treated cells. All the V(D)J requirements were thus fulfilled when mature human CD8+ T cells were stimulated with anti-CD3 and IL7. Induction of TCR revision in vitro in mature T cells is an easily controllable system that could be employed in further studies to elucidate the molecular pathways involved in secondary V(D)J rearrangements in peripheral cells.

Release-enhancing pre-synaptic muscarinic and nicotinic receptors co-exist and interact on dopaminergic nerve endings of rat nucleus accumbens.

Dopaminergic nerve endings in the corpus striatum possess nicotinic (nAChRs) and muscarinic cholinergic receptors (mAChRs) mediating release of dopamine (DA). Whether nAChRs and mAChRs co-exist and interact on the same nerve endings is unknown. We here investigate on these possibilities using rat nucleus accumbens synaptosomes pre-labeled with [(3)H]DA and exposed in superfusion to cholinergic receptor ligands. The mixed nAChR-mAChR agonists acetylcholine (ACh) and carbachol provoked [(3)H]DA release partially sensitive to the mAChR antagonist atropine but totally blocked by the nAChR antagonist mecamylamine. Addition of the mAChR agonist oxotremorine at the minimally effective concentration of 30 micromol/L, together with 3, 10, or 100 micromol/L (-)nicotine provoked synergistic effect on [(3)H]DA overflow. The [(3)H]DA overflow elicited by 100 micromol/L (-)nicotine plus 30 micromol/L oxotremorine was reduced by atropine down to the release produced by (-)nicotine alone and it was abolished by mecamylamine. The ryanodine receptor blockers dantrolene or 8-bromo-cADP-ribose, but not the inositol 1,4,5-trisphosphate receptor blocker xestospongin C inhibited the (-)nicotine/oxotremorine evoked [(3)H]DA overflow similarly to atropine. This overflow was partly sensitive to 100 nmol/L methyllycaconitine which did not prevent the synergistic effect of (-)nicotine/oxotremorine. Similarly to (-)nicotine, the selective alpha4beta2 nAChR agonist RJR2403 exhibited synergism when added together with oxotremorine. To conclude, in rat nucleus accumbens, alpha4beta2 nAChRs exert a permissive role on the releasing function of reportedly M(5) mAChRs co-existing on the same dopaminergic nerve endings.

Acute beta-amyloid administration disrupts the cholinergic control of dopamine release in the nucleus accumbens.

The clinical presentation of Alzheimer's disease is characterized by memory deficits but it also involves the impairment of several cognitive functions. Some of these cognitive and executive functions are mediated by limbic areas and are regulated by dopaminergic neurotransmission. Furthermore, literature data suggest that beta-amyloid (Abeta) can influence synaptic activity in absence of neurotoxicity and in particular can impair cholinergic modulation of other neurotransmitter actions. In the present study, we evaluated whether small concentrations of Abeta could disrupt cholinergic control of dopamine (DA) release in nucleus accumbens using in vivo (brain dialysis) and in vitro (isolated synaptosomes) models. The cholinergic agonist carbachol (CCh) greatly enhanced DA release from dopaminergic nerve endings in nucleus accumbens both in vivo and in vitro. This effect was mainly exerted on muscarinic receptors because it was inhibited by the muscarinic antagonist atropine and it was unaffected by the nicotinic antagonist mecamylamine. Also the nicotinic agonists epibatidine and nicotine evoked a dopaminergic outflow in nucleus accumbens, which, however, was lower. Abeta 1-40 in absence of neurotoxicity fully inhibited the DA release evoked by CCh and only marginally affected the DA release evoked by epibatidine. The PKC inhibitor GF109203X mimicked the effect of Abeta on DA release and, in turn, Abeta impaired PKC activation by CCh. We can suggest that, in nucleus accumbens, Abeta disrupted in vivo and in vitro cholinergic control of DA release by acting on muscarinic transduction machinery.