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Synapse stabilization Early in brain development, neurons connect to each other enthusiastically. With development, an overabundance of synapses is winnowed down to refine efficiently connected circuits. Inactive synapses are prime targets for elimination, whereas active synapses tend to be retained. Gomez-Castro et al . took a closer look at how those choices are made (see the Perspective by Blum and Lopes). When postsynaptic adenosine receptors are muted or do not find enough extracellular adenosine, synapses get eliminated. Neurotransmitter-dependent signaling pathways drive protein kinase A to phosphorylate the postsynaptic scaffolding molecule gephyrin. Together with a partner synaptogenic membrane protein, gephyrin is required for the stabilization of γ-aminobutyric acid receptors. Adenosine receptors thus detect synaptic activity and in turn drive the stabilization of synapses that produce such activity. —PJH

Psychoactive drugs used during pregnancy can affect the development of the brain of offspring, directly triggering neurological disorders or increasing the risk for their occurrence. Caffeine is the most widely consumed psychoactive drug, including during pregnancy. In Wild type mice, early life exposure to caffeine renders offspring more susceptible to seizures. Here, we tested the long-term consequences of early life exposure to caffeine in THY-Tau22 transgenic mice, a model of Alzheimer’s disease-like Tau pathology. Caffeine exposed mutant offspring developed cognitive earlier than water treated mutants. Electrophysiological recordings of hippocampal CA1 pyramidal cells in vitro revealed that early life exposure to caffeine changed the way the glutamatergic and GABAergic drives were modified by the Tau pathology. We conclude that early-life exposure to caffeine affects the Tau phenotype and we suggest that caffeine exposure during pregnancy may constitute a risk-factor for early onset of Alzheimer’s disease-like pathology.

International audience; The consumption of psychoactive drugs during pregnancy can have deleterious effects on newborns. It remains unclear whether early-life exposure to caffeine, the most widely consumed psychoactive substance, alters brain development. We hypothesized that maternal caffeine ingestion during pregnancy and the early postnatal period in mice affects the construction and activity of cortical networks in offspring. To test this hypothesis, we first focused on primary visual cortex (V1) as a model six-layered neocortical region. In a study design mimicking the daily consumption of approximately three cups of coffee during pregnancy in humans, caffeine was added to the drinking water of female mice and their offspring were compared to control offspring. Caffeine altered the construction of GABAergic neuronal networks in V1, as reflected by a reduced number of somatostatincontaining GABA neurons at postnatal days 6-7, with the remaining ones showing poorly developed dendritic arbors. These findings were accompanied by increased synaptic activity in vitro and elevated network activity in vivo in V1. Similarly, in vivo hippocampal network activity was increased from the neonatal period until adulthood. Finally, caffeine-exposed offspring showed increased seizure susceptibility in a hyperthermia-induced seizure model. In summary, our results indicate detrimental effects of developmental caffeine exposure on mouse brain development.

In the adult brain, Adenosine, a degradation product of ATP, controls neurotransmitter release mainly through inhibitory A1 (A1R) and facilitatory A2A receptors (A2AR). Adenosine receptors also play an important role during the early phases of development. A1Rs and A2ARs are abundantly expressed in petrosal ganglion neurons, with a different pattern of distribution: A1 receptors show a cytoplasmic localization while A2 receptors are prevalently localized to the nucleus of the same neurons. Adenosine and its breakdown enzymes also play an important role in the regulation of growth and development of sympathetic neurons. Finally, A2ARs are present on some types of migrating γ-aminobutyric acid neurons, controlling their migration speed. At nontoxic doses adenosine receptors are antagonized by caffeine, the most widely used psychoactive drug, including during pregnancy. Both in vitro and in vivo recordings revealed network hyperactivity at postnatal day 6 in the offspring of female mice treated with caffeine in their drinking water (equivalent to 2–3 espressos/day in humans) during pregnancy and lactation. Perturbing adenosine at early stages of development may have deleterious consequences and predispose some individuals to the development of neurological disorders.

The review examines the multifaceted interactions between cholinergic transmission and beta-amyloid suggesting a continuum in the action of the peptide that at low concentrations (picomolar-low nanomolar) may directly stimulate nicotinic cholinergic receptor while desensitizing them at increasing concentrations (high nanomolar-low micromolar). In addition high beta-amyloid concentrations may reduce the synaptic release of several neurotransmitters, including glutamate, aspartate, GABA, glycine and dopamine, when the release is elicited through cholinergic stimulation but not following depolarization. The effect of beta-amyloid has been observed both in vitro and in vivo in at least three different brain areas (nucleus accumbens, striatum, hippocampus) suggesting that the peptide may exert some general effects even if not all the brain areas have been evaluated. In turn the activation of cholinergic receptors may affect the amyloid precursor protein processing diverting the metabolism toward non-amyloidogenic products. These actions, dissociated from those described in the case of high beta-amyloid concentrations leading to neurotoxic oligomers, may participate to cause dysfunctions in the neurotransmitter activity, in turn leading, at least from a theoretical point of view, to early neuropsychiatric disturbances in the disease. Complexively these observations underscore novel relationships between two main players in Alzheimer's disease pathogenesis that are beta-amyloid and cholinergic transmission. Also emerges the inherent difficulty of targeting beta-amyloid in a context in which the peptide exerts several actions beyond neurotoxicity.

We investigated on the neuronal nicotinic acetylcholine receptor subtypes involved in the cholinergic control of in vivo hippocampal glutamate (GLU), aspartate (ASP) and inhibitory γ-aminobutyric acid (GABA) overflow. We also investigated on the possible contribution of nicotinic acetylcholine receptors subtypes present on astrocytes in the regulation of the three neurotransmitter amino acids overflow using hippocampal gliosomes and on the effects of beta-amyloid (Aβ) 1–40 on the nicotinic control of amino acid neurotransmitter release. Nicotine was able to enhance the in vivo overflow of the three amino acids being more potent in stimulating GLU overflow. The α7 selective agonist PHA543613 induced an overflow very similar to that of nicotine. The α4β2 selective agonist 5IA85380 was significantly less potent in inducing GLU overflow while the overflow of ASP and GABA were almost inconsistent. Aβ1–40 inhibited the neurotransmitter overflow stimulated by PHA543613 but not the one evoked by 5IA85380. In hippocampal gliosomes nicotine elicited selectively GLU overflow which was also evoked by 5IA85380 and by the α7 selective agonist choline. Nicotine- and choline-induced glutamate overflow in gliosomes was inhibited by Aα1–40. In conclusion nicotine administration in vivo elicits hippocampal GLU release mostly through α7 nicotinic acetylcholine receptors likely present both on neurons and astrocytes. Aβ inhibitory effect on the nicotinic-control of GLU release seems to depend primarily to the inhibition of α7 nicotinic acetylcholine receptors functional responses.

BACKGROUND AND PURPOSE The present work aimed to investigate whether and through which mechanisms selective α7 and α4β2 nicotinic receptor (nAChR) agonists stimulate endogenous glutamate (GLU) and aspartate (ASP) release in rat hippocampus. EXPERIMENTAL APPROACH Rat hippocampal synaptosomes were purified on Percoll gradients and superfused in vitro to study endogenous GLU and ASP release. The synaptosomes were superfused with selective α7 and α4β2 nAChR agonists and antagonists. The excitatory amino acid (EAA) content of the samples of superfusate was determined by HPLC after pre-column derivatization and separation on a chromatographic column coupled with fluorimetric detection. KEY RESULTS Choline (Ch), a selective α7 receptor agonist, elicited a significant release of both GLU and ASP which was blocked by the α7 receptor antagonist methyllycaconitine (MLA), but was unaltered by the α4β2 receptor antagonist dihydro-β-erythroidine (DHβE). The stimulant effect of Ch was strongly reduced in a Ca2+-free medium, was not inhibited by Cd2+ and tetrodotoxin (TTX), but was antagonized by dantrolene, xestospongin C and thapsigargin. 5-Iodo-A-85380 dihydrochloride (5IA85380), a selective α4β2 receptor agonist, elicited EAA release in a DHβE-sensitive, MLA-insensitive fashion. The 5IA85380-evoked release was dependent on extracellular Ca2+, blocked by Cd2+ and TTX, but unaffected by dantrolene. CONCLUSIONS AND IMPLICATIONS Our study shows for the first time that rat hippocampal synaptosomes possess α7 and α4β2 nAChR subtypes, which can enhance the release of endogenous GLU and ASP via two distinct mechanisms of action. These results extend our knowledge of the nicotinic modulation of excitatory synaptic transmission in the hippocampus.

In this study we investigate on the effect of amyloid-beta1-40 (A beta 1-40) on the oxotremorine (OXO)-induced release of [(3)H] dopamine (DA), [(3)H]GABA and [(3)H]acetylcholine (ACh) from synaptosomes in the rat nucleus accumbens (NAc). OXO in presence of himbacine (HIMBA) was able to increase the basal release of [(3)H]GABA. The OXO-elicited [(3)H]GABA overflow was significantly antagonized by atropine (A; 94%), by the M3 antagonists DAU5884 (96%) and 4-DAMP (70%), and by A beta 1-40 (65%). Exposure of NAc synaptosomes to OXO produced a dose-dependent increase of [(3)H]DA overflow which was antagonized by A, partially inhibited by A beta 1-40 (100 nM) but unaffected by DAU5884 and 4-DAMP. The K(+)-evoked [(3)H]ACh overflow was inhibited by OXO. This effect was counteracted by the M2 antagonist AFDX-116 but not by the selective M4 antagonist mamba toxin 3 (MT3). The K(+)-evoked [(3)H]GABA overflow was also inhibited by OXO but conversely, this effect was counteracted by MT3 and not by AFDX-116. A beta 1-40 (100 nM) did not modify the inhibitory effect of OXO both on the K(+)-evoked [(3)H]ACh and [(3)H]GABA overflow. The results show that in the rat NAc, A beta 1-40 selectively inhibits the function of the muscarinic subtypes which stimulate neurotransmitter release and not those which modulate negatively the stimulated release.

We investigated the effects of salvinorin A on the basal and the 12 mM K(+)-evoked release of preloaded [(3)H]noradenaline ([(3)H]NA) and [(3)H]serotonin ([(3)H]5-HT) from mouse hippocampal nerve terminals (synaptosomes), as well as on the basal and 12mM K(+)-evoked release of preloaded [(3)H]dopamine ([(3)H]DA) from mouse striatal and prefrontal cortex (PFc) synaptosomes. Salvinorin A (0.1-1000 nM) failed to affect the basal release of amines, but inhibited the 12 mM K(+)-evoked, Ca(2+)-dependent, exocytotic-like release of [(3)H]5-HT and [(3)H]DA. At the same concentration, salvinorin A facilitated the 12 mM K(+)-evoked, Ca(2+)-dependent, exocytotic-like release of [(3)H]NA. These effects could not be observed in pertussis toxin (PTx) entrapped synaptosomes. The broad spectrum kappa-opioid receptor (KOR) antagonist norbinaltorphimine (norBNI, 1-100 nM) antagonized the inhibition of [(3)H]5-HT and [(3)H]DA exocytosis as well as the facilitation of [(3)H]NA overflow induced by 100 nM salvinorin A. The KOR agonist U69593 (1-100 nM) mimicked salvinorin A in inhibiting [(3)H]5-HT and of [(3)H]DA exocytosis, its effect being prevented by norBNI, but leaving unchanged the K(+)-evoked release of [(3)H]NA. The effects of Salvinorin A on neurotransmitter exocytosis were not prevented by the selective mu opioid (MOR) receptor antagonist CTAP (10-100 nM), whereas facilitation of [(3)H]NA exocytosis, but not inhibition of [(3)H]5-HT and [(3)H]DA K(+)-evoked release, was counteracted by the delta opioid receptor (DOR) antagonist naltrindole (1-100 nM). We conclude that salvinorin A presynaptically modulates central NA, 5-HT, and DA exocytosis evoked by a mild depolarizing stimulus by acting at presynaptic opioid receptors having different pharmacological profiles.

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.

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.

The presynaptic control of dopamine release in the nucleus accumbens (NAc) by glutamate and acetylcholine has a profound impact on reward signaling. Here we provide immunocytochemical and neurochemical evidence supporting the co-localization and functional interaction between nicotinic acetylcholine receptors (nAChRs) and N-methyl-D-aspartic acid (NMDA) receptors in dopaminergic terminals of the NAc. Most NAc dopaminergic terminals possessed the nAChR α4 subunit and the pre-exposure of synaptosomes to nicotine (30 μM) or to the α4β2-containing nAChR agonist 5IA85380 (10 nM) selectively inhibited the NMDA (100 μM)-evoked, but not the 4-aminopyridine (10 μM)-evoked, [ 3 H] dopamine outflow; this inhibition was blunted by mecamylamine (10 μM). Nicotine and 5IA85380 pretreatment also inhibited the NMDA (100 μM)-evoked increase of calcium levels in single nerve terminals, an effect prevented by dihydro-β-erythroidine (1 μM). This supports a functional interaction between α4β2-containing nAChR and NMDA receptors within the same terminal, as supported by the immunocytochemical co-localization of α4 and GluN1 subunits in individual NAc dopaminergic terminals. The NMDA-evoked [ 3 H]dopamine outflow was blocked by MK801 (1 μM) and inhibited by the selective GluN2B-selective antagonists ifenprodil (1 μM) and RO 25-6981 (1 μM), but not by the GluN2A-preferring antagonists CPP-19755 (1 μM) and ZnCl 2 (1 nM). Notably, nicotine pretreatment significantly decreased the density of biotin-tagged GluN2B proteins in NAc synaptosomes. These results show that nAChRs dynamically and negatively regulate NMDA receptors in NAc dopaminergic terminals through the internalization of GluN2B receptors.

We here provide functional and immunocytochemical evidence supporting the co-localization and functional interaction between nicotinic acetylcholine receptors (nAChRs) and N-methyl-D-aspartic acid receptors (NMDARs) in glutamatergic terminals of the nucleus accumbens (NAc). Immunocytochemical studies showed that a significant percentage of NAc terminals were glutamatergic and possessed GluN1 and α7-containing nAChR. A short-term pre-exposure of synaptosomes to nicotine (30 µM) or choline (1 mM) caused a significant potentiation of the 100 µM NMDA-evoked [(3)H]D-aspartate ([(3)H]D-Asp) outflow, which was prevented by α-bungarotoxin (100 nM). The pre-exposure to nicotine (100 µM) or choline (1 mM) also enhanced the NMDA-induced cytosolic free calcium levels, as measured by FURA-2 fluorescence imaging in individual NAc terminals, an effect also prevented by α-bungarotoxin. Pre-exposure to the α4-nAChR agonists 5IA85380 (10 nM) or RJR2429 (1 µM) did not modify NMDA-evoked ([(3)H]D-Asp) outflow and calcium transients. The NMDA-evoked ([(3)H]D-Asp) overflow was partially antagonized by the NMDAR antagonists MK801, D-AP5, 5,7-DCKA and R(-)CPP and unaffected by the GluN2B-NMDAR antagonists Ro256981 and ifenprodil. Notably, pre-treatment with choline increased GluN2A biotin-tagged proteins. In conclusion, our results show that the GluN2A-NMDA receptor function can be positively regulated in NAc terminals in response to a brief incubation with α7 but not α4 nAChRs agonists. This might be a general feature in different brain areas since a similar nAChR-mediated bolstering of NMDA-induced ([(3)H]D-Asp) overflow was also observed in hippocampal synaptosomes.

The effect of chronic nicotine treatment on the release of endogenous glutamate (GLU), aspartate (ASP) and GABA evoked in vitro by KCl, 4-aminopyridine (4AP) and nicotinic agonists in synaptosomes of rat hippocampus was investigated. Rats were chronically administered with nicotine bitartrate or saline vehicle each for 14 days using osmotic mini-pumps. Hippocampal synaptosomes were stimulated with KCl, 4AP, nicotine or with choline (Ch) and 5-iodo-A-85380 dihydrochloride (5IA85380). The GLU and ASP overflow evoked by Ch, nicotine, KCl and 4AP were increased in treated animals while the nicotine-evoked GABA overflow was reduced and that evoked by Ch, KCl and 4AP was unaffected. The 5IA85380-evoked overflow of the three aminoacids (AAs) was always reduced. The increase of ASP and GLU overflow evoked by KCl, 4AP or Ch was blocked by dl-threo-β-benzyloxyaspartic acid (dl-TBOA), a carrier transporter inhibitor, and by inhibitors of the Na(+)/Ca(2+) exchangers 2-[[4-[(4-nitrophenyl)methoxy]phenyl]methyl]-4-thiazolidinecarboxylic acid ethyl ester (SN-6) and 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea mesylate (KB-R7943). In conclusion long-term nicotine treatment may selectively increase GLU and ASP overflow elicited by KCl, 4AP and Ch through the activation of a carrier-mediated release mechanism and completely abolished the stimulatory effects of α4β2 nAChRs which modulate the release of all the three AA.

Here we provide functional and immunocytochemical evidence supporting the presence on Nucleus Accumbens (NAc) dopaminergic terminals of cyclothiazide-sensitive, alfa-amino-3-hydroxy-5-methyl-4-isoxazolone propionate (AMPA) receptors, which activation causes Ca2+-dependent [3H]dopamine ([3H]DA) exocytosis. These AMPA receptors cross-talk with co-localized nicotinic receptors (nAChRs), as suggested by the finding that in vitro short-term pre-exposure of synaptosomes to 30 ?M nicotine caused a significant reduction of both the 30 ?M nicotine and the 100 ?M AMPA-evoked [3H]DA overflow. Entrapping pep2-SVKI, a peptide known to compete for the binding of GluA2 subunit to scaffolding proteins involved in AMPA receptor endocytosis, in NAC synaptosomes prevented the nicotine-induced reduction of AMPA-mediated [3H]DA exocytosis, while pep2-SVKE, used as negative control, was inefficacious. Immunocytochemical studies showed that a significant percentage of NAc terminals were dopaminergic and that most of these terminals also posses GluA2 receptor subunits. Western blot analysis of GluA2 immunoreactivity showed that presynaptic GluA2 proteins in NAc terminals were reduced in nicotine-pretreated synaptosomes when compared to the control. The nACh-AMPA receptor-receptor interaction was not limited to dopaminergic terminals since nicotine pre-exposure also affected the presynaptic AMPA receptors controlling hippocampal noradrenaline release, but not the presynaptic AMPA receptors controlling GABA and acetylcholine release. These observations could be relevant to the comprehension of the molecular mechanisms at the basis of nicotine rewarding.

Using both in vitro (hippocampal synaptosomes in superfusion) and in vivo (microdialysis) approaches we investigated whether and to what extent β amyloid peptide 1-40 (Aβ 1-40) interferes with the cholinergic modulation of the release of glycine (GLY) in the rat hippocampus. The nicotine-evoked overflow of endogenous GLY in hippocampal synaptosomes in superfusion was significantly inhibited by Aβ 1-40 (10 nM) while increasing the concentration to 100 nM the inhibitory effect did not further increase. Both the Choline (Ch; α7 agonist; 1 mM) and the 5-Iodo-A-85380 dihydrochloride (5IA85380, α4β2 agonist; 10 nM)-evoked GLY overflow were inhibited by Aβ 1-40 at 100 nM but not at 10 nM concentrations. The KCl evoked [(3)H]GLY and [(3)H]Acetylcholine (ACh) overflow were strongly inhibited in presence of oxotremorine; however this inhibitory muscarinic effect was not affected by Aβ 1-40. The effects of Aβ 1-40 on the administration of nicotine, veratridine, 5IA85380, and PHA543613 hydrochloride (PHA543613; a selective agonist of α7 subtypes) on hippocampal endogenous GLY release in vivo were also studied. Aβ 1-40 significantly reduced (at 10 μM but not at 1 μM) the nicotine-evoked in vivo release of GLY. Aβ 1-40 (at 10 μM but not at 1 μM) significantly inhibited the PHA543613 (1 mM)-elicited GLY overflow while was ineffective on the GLY overflow evoked by 5IA85380 (1 mM). Aβ 40-1 (10 μM) did not produce any inhibitory effect on nicotine-evoked GLY overflow both in the in vitro and in vivo experiments. Our results indicate that (a) the cholinergic modulation of the release of GLY occurs by the activation of both α7 and α4β2 nicotinic ACh receptors (nAChRs) as well as by the activation of inhibitory muscarinic ACh receptors (mAChRs) and (b) Aβ 1-40 can modulate cholinergic evoked GLY release exclusively through the interaction with α7 and the α4β2 nAChR nicotinic receptors but not through mAChR subtypes.

Background Although converging evidence has suggested that nicotinic acetylcholine receptors (nAChR) play a role in the modulation of GABA release in rat hippocampus, the specific involvement of different nAChR subtypes at presynaptic level is still a matter of debate. In the present work we investigated, using selective α7 and α4β2 nAChR agonists, the presence of different nAChR subtypes on hippocampal GABA nerve endings to assess to what extent and through which mechanisms they stimulate endogenous GABA release. Methodology/Findings All agonists elicited GABA overflow. Choline (Ch)-evoked GABA overflow was dependent to external Ca2+, but unaltered in the presence of Cd2+, tetrodotoxin (TTX), dihydro-β-erythroidine (DHβE) and 1-(4,4-Diphenyl-3-butenyl)-3-piperidinecarboxylic acid hydrochloride SKF 89976A. The effect of Ch was blocked by methyllycaconitine (MLA), α-bungarotoxin (α-BTX), dantrolene, thapsigargin and xestospongin C, suggesting that GABA release might be triggered by Ca2+ entry into synaptosomes through the α7 nAChR channel with the involvement of calcium from intracellular stores. Additionally, 5-Iodo-A-85380 dihydrochloride (5IA85380) elicited GABA overflow, which was Ca2+ dependent, blocked by Cd2+, and significantly inhibited by TTX and DHβE, but unaffected by MLA, SKF 89976A, thapsigargin and xestospongin C and dantrolene. These findings confirm the involvement of α4β2 nAChR in 5IA85380-induced GABA release that seems to occur following membrane depolarization and opening calcium channels. Conclusions/Significance Rat hippocampal synaptosomes possess both α7 and α4β2 nAChR subtypes, which can modulate GABA release via two distinct mechanisms of action. The finding that GABA release evoked by the mixture of sub-maximal concentration of 5IA85380 plus sub-threshold concentrations of Ch was significantly larger than that elicited by the sum of the effects of the two agonists is compatible with the possibility that they coexist on the same nerve terminals. These findings would provide the basis for possible selective pharmacological strategies to treat neuronal disorders that involve the dysfunction of hippocampal cholinergic system.

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.

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.

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.

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.

In the present study, using an in vivo approach (a microdialysis technique associated to HPLC with fluorimetric detection) and in vitro purified hippocampal synaptosomes in superfusion, we investigated the glycinergic transmission in the hippocampus, focusing on the nicotinic control of glycine (GLY) release. The acute administration of nicotine in vivo was able to evoke endogenous GLY release in the rat hippocampus. The specific nicotinic agonists PHA-543613 hydrochloride (PHA543613) selective for the α7 nicotinic receptor subtype administered in vivo also elicited GLY release in a similar extent, while the α4β2 agonist 5-IA85380 dihydrochloride (5IA85380) was less effective. Nicotine elicited GLY overflow also from hippocampal synaptosomes in vitro. This overflow was Ca2+-dependent and inhibited by methyllycaconitine (MLA), but was not modified by dihydro-beta-erythroidine (DHβE, 1 μM). Choline(Ch)-evoked GLY overflow was Ca2+ dependent, unaltered in presence of DHβE and blocked by methyllycaconitine (MLA). Additionally, 5IA85380 elicited a GLY overflow, which in turn was Ca2+ dependent, was significantly inhibited by DHβE but was unaffected by MLA. The GLY overflow produced by these nicotinic agonists quantitatively resembles that evoked by 9 mM KCl. The effects of a high concentration of 5IA85380 (1 mM), in the presence of 2 μM DHβE, on the release of GLY was also studied comparatively to that on glutamate and aspartate release. The nicotinic agonist 5IA85380 tested at high concentration (1 mM) was able to produce a stimulatory effect of endogenous release of the three amino acids, even in the presence of 2 μM DHβE, indicating the existence of a DHβE resistant, α4β2 nAChR subtype with a functional role in the modulation of GLY, ASP, and GLU release. Our results show that in the rat hippocampus the release of GLY is, at least in part, of neuronal origin and is modulated by the activation of both α7 and α4β2 (low and high affinity) nAChR subtypes.