Nicotinic acetylcholine receptors (nAChRs) are expressed widely in the CNS, and mediate both synaptic and perisynaptic activities of endogenous cholinergic inputs and pharmacological actions of exogenous compounds (e.g., nicotine and choline). Behavioral studies indicate that nicotine improves such cognitive functions as learning and memory. However, the mechanism of nicotine's action on cognitive function remains elusive. We performed patch-clamp recordings from hippocampal CA3 pyramidal neurons to determine the effect of nicotine on mossy fiber glutamatergic synaptic transmission. We found that nicotine in combination with NS1738, an α7 nAChR-positive allosteric modulator, strongly potentiated the amplitude of evoked EPSCs (eEPSCs), and reduced the EPSC paired-pulse ratio. The action of nicotine and NS1738 was mimicked by PNU-282987 (an α7 nAChR agonist), and was absent in α7 nAChR knock-out mice. These data indicate that activation of α7 nAChRs was both necessary and sufficient to enhance the amplitude of eEPSCs. BAPTA applied postsynaptically failed to block the action of nicotine and NS1738, suggesting again a presynaptic action of the α7 nAChRs. We also observed α7 nAChR-mediated calcium rises at mossy fiber giant terminals, indicating the presence of functional α7 nAChRs at presynaptic terminals. Furthermore, the addition of PNU-282987 enhanced action potential-dependent calcium transient at these terminals. Last, the potentiating effect of PNU-282987 on eEPSCs was abolished by inhibition of protein kinase A (PKA). Our findings indicate that activation of α7 nAChRs at presynaptic sites, via a mechanism involving PKA, plays a critical role in enhancing synaptic efficiency of hippocampal mossy fiber transmission.
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.
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.
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.
Nicotinic acetylcholine receptors (nAChRs) containing either the α4 and/or α6 subunit are robustly expressed in dopaminergic nerve terminals in dorsal striatum where they are hypothesized to modulate dopamine (DA) release via acetylcholine (ACh) stimulation from cholinergic interneurons. However, pharmacological blockade of nAChRs or genetic deletion of individual nAChR subunits, including α4 and α6, in mice, yields little effect on motor behavior. Based on the putative role of nAChRs containing the α4 subunit in modulation of DA in dorsal striatum, we hypothesized that mice expressing a single point mutation in the α4 nAChR subunit, Leu9′Ala, that renders nAChRs hypersensitive to agonist, would exhibit exaggerated differences in motor behavior compared to WT mice. To gain insight into these differences, we challenged WT and Leu9′Ala mice with the α4β2 nAChR antagonist dihydro-β-erythroidine (DHβE). Interestingly, in Leu9′Ala mice, DHβE elicited a robust, reversible motor impairment characterized by hypolocomotion, akinesia, catalepsy, clasping, and tremor; whereas the antagonist had little effect in WT mice at all doses tested. Pre-injection of nicotine (0.1 mg/kg) blocked DHβE-induced motor impairment in Leu9′Ala mice confirming that the phenotype was mediated by antagonism of nAChRs. In addition, SKF 82958 (1 mg/kg) and amphetamine (5 mg/kg) prevented the motor phenotype. DHβE significantly activated more neurons within striatum and substantia nigra pars reticulata in Leu9′Ala mice compared to WT animals, suggesting activation of the indirect motor pathway as the circuit underlying motor dysfunction. ACh evoked DA release from Leu9′Ala striatal synaptosomes revealed agonist hypersensitivity only at α4(non-α6)* nAChRs. Similarly, α6 nAChR subunit deletion in an α4 hypersensitive nAChR (Leu9′Ala/α6KO) background had little effect on the DHβE-induced phenotype, suggesting an α4(non-α6)* nAChR-dependent mechanism. Together, these data indicate that α4(non-α6)* nAChR have an impact on motor output and may be potential molecular targets for treatment of disorders associated with motor impairment.
nicotinic acetylcholine receptor; motor behavior; basal ganglia; dopamine
Smoking is a significant health concern and strongly correlated with clinical depression. Depression is associated with decreased extracellular NE concentrations in brain. Smokers may be self-medicating and alleviating their depression through nicotine stimulated norepinephrine (NE) release. Several antidepressants inhibit NE transporter (NET) function, thereby augmenting extracellular NE concentrations. Antidepressants, such as bupropion, also inhibit nicotinic receptor (nAChR) function. The current study determined if a recently discovered novel nAChR antagonist, N,N′-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), inhibits nicotine-evoked NE release from superfused rat hippocampal slices. Previous studies determined that bPiDDB potently (IC50 = 2 nM) inhibits nicotine-evoked striatal [3H]dopamine (DA) release in vitro, nicotine-evoked DA release in nucleus accumbens in vivo, and nicotine self-administration in rats. In the current study, nicotine stimulated [3H]NE release from rat hippocampal slices (EC50 = 50 μM). bPiDDB inhibited (IC50 = 430 nM; Imax = 90%) [3H]NE release evoked by 30 μM nicotine. For comparison, the nonselective nAChR antagonist, mecamylamine, and the α7 antagonist, methyllycaconitine, also inhibited nicotine-evoked [3H]NE release (IC50 = 31 and 275 nM, respectively; Imax = 91% and 72%, respectively). Inhibition by bPiDDB and mecamylamine was not overcome by increasing nicotine concentrations; Schild regression slope was different from unity, consistent with allosteric inhibition. Thus, bPiDDB was 200-fold more potent inhibiting nAChRs mediating nicotine-evoked [3H]DA release from striatum than those mediating nicotine-evoked [3H]NE release from hippocampus.
antidepressants; nicotine; nicotinic acetylcholine receptor; norepinephrine release; smoking cessation
Presynaptic effects of general anaesthetics are not well characterized. We tested the hypothesis that isoflurane exhibits transmitter-specific effects on neurotransmitter release from neurochemically and functionally distinct isolated mammalian nerve terminals.
Nerve terminals from adult male rat brain were prelabelled with [3H]glutamate and [14C]GABA (cerebral cortex), [3H]norepinephrine (hippocampus), [14C]dopamine (striatum), or [3H]choline (precursor of [3H]acetylcholine; striatum). Release evoked by depolarizing pulses of 4-aminopyridine (4AP) or elevated KCl was quantified using a closed superfusion system.
Isoflurane at clinical concentrations (<0.7 mM; ∼2 times median anaesthetic concentration) inhibited Na+ channel-dependent 4AP-evoked release of the five neurotransmitters tested in a concentration-dependent manner. Isoflurane was a more potent inhibitor [expressed as IC50 (sem)] of glutamate release [0.37 (0.03) mM; P<0.05] compared with the release of GABA [0.52 (0.03) mM], norepinephrine [0.48 (0.03) mM], dopamine [0.48 (0.03) mM], or acetylcholine [0.49 (0.02) mM]. Inhibition of Na+ channel-independent release evoked by elevated K+ was not significant at clinical concentrations of isoflurane, with the exception of dopamine release [IC50=0.59 (0.03) mM].
Isoflurane inhibited the release of the major central nervous system neurotransmitters with selectivity for glutamate release, consistent with both widespread inhibition and nerve terminal-specific presynaptic effects. Glutamate release was most sensitive to inhibition compared with GABA, acetylcholine, dopamine, and norepinephrine release due to presynaptic specializations in ion channel expression, regulation, and/or coupling to exocytosis. Reductions in neurotransmitter release by volatile anaesthetics could contribute to altered synaptic transmission, leading to therapeutic and toxic effects involving all major neurotransmitter systems.
acetylcholine; γ-aminobutyric acid; anaesthetics; dopamine; exocytosis; glutamate; Na+ channels; nerve terminal; neurotransmitter release; norepinephrine
Diverse nicotinic acetylcholine receptor (nAChR) subtypes containing different subunit combinations can be placed on nerve terminals or soma/dendrites in the ventral tegmental area (VTA). nAChR α6 subunit message is abundant in the VTA, but α6*-nAChR cellular localization, function, pharmacology, and roles in cholinergic modulation of dopaminergic (DA) neurons within the VTA are not well-understood. Here, we report evidence for α6β2*-nAChR expression on GABA neuronal boutons terminating on VTA DA neurons. α-Conotoxin (α-Ctx) MII labeling coupled with immunocytochemical staining localizes putative α6*-nAChRs to presynaptic GABAergic boutons on acutely dissociated, rat VTA DA neurons. Functionally, acetylcholine (ACh) induces increases in the frequency of bicuculline-, picrotoxin-, and 4-aminopyridine-sensitive miniature inhibitory postsynaptic currents (mIPSCs) mediated by GABAA receptors. These increases are abolished by α6*-nAChR-selective α-Ctx MII or α-Ctx PIA (1 nM), but not by α7-(10 nM methyllycaconitine) or α4*-(1 μM dihydro-β-erythroidine) nAChR-selective antagonists. ACh also fails to increase mIPSC frequency in VTA DA neurons prepared from nAChR β2 knockout mice. Moreover, ACh induces an α-Ctx PIA-sensitive elevation in intraterminal Ca2+ in synaptosomes prepared from the rat VTA. Subchronic exposure to 500 nM nicotine reduces ACh-induced GABA release onto the VTA DA neurons, as does 10 days of systemic nicotine exposure. Collectively, these results indicate that α6β2*-nAChRs are located on presynaptic GABAergic boutons within the VTA and modulate GABA release onto DA neurons. These presynaptic α6β2*-nAChRs likely play important roles in nicotinic modulation of DA neuronal activity.
α6-nicotinic acetylcholine receptor; miniature inhibitory postsynaptic current; ventral tegmental area; dopamine neuron; α-conotoxin; patch clamp
Despite the proven efficacy of current pharmacotherapies for tobacco dependence, relapse rates continue to be high, indicating that novel medications are needed. Currently, several smoking cessation agents are available, including varenicline (Chantix®), bupropion (Zyban®), and cytisine (Tabex®). Varenicline and cytisine are partial agonists at the α4β2* nicotinic acetylcholine receptor (nAChR). Bupropion is an antidepressant but is also an antagonist at α3β2* ganglionic nAChRs. The rewarding effects of nicotine are mediated, in part, by nicotine-evoked dopamine (DA) release leading to sensitization, which is associated with repeated nicotine administration and nicotine addiction. Receptor antagonists that selectivity target central nAChR subtypes mediating nicotine-evoked DA release should have efficacy as tobacco use cessation agents with the therapeutic advantage of a limited side-effect profile. While α-conotoxin MII (α-CtxMII)-insensitive nAChRs (e.g., α4β2*) contribute to nicotine-evoked DA release, these nAChRs are widely distributed in the brain, and inhibition of these receptors may lead to nonselective and untoward effects. In contrast, α-CtxMII-sensitive nAChRs mediating nicotine-evoked DA release offer an advantage as targets for smoking cessation, due to their more restricted localization primarily to dopaminergic neurons. Small drug-like molecules that are selective antagonists at α-CtxMII-sensitive nAChR subtypes that contain α6 and β2 subunits have now been identified. Early research identified a variety of quaternary ammonium analogs that were potent and selective antagonists at nAChRs mediating nicotine-evoked DA release. More recent data have shown that novel, non-quaternary bis-1,2,5,6-tetrahydropyridine analogs potently inhibit (IC50<1 nM) nicotine-evoked DA release in vitro by acting as antagonists at α-CtxMII-sensitive nAChR subtypes; these compounds also decrease NIC self-administration in rats.
Tobacco smoking is the leading preventable cause of death in the United States. A major negative health consequence of chronic smoking is hypertension. Untoward addictive and cardiovascular sequelae associated with chronic smoking are mediated by nicotine-induced activation of nicotinic receptors (nAChRs) within striatal dopaminergic and hypothalamic noradrenergic systems. Hypertension involves both brain and peripheral angiotensin systems. Activation of angiotensin type-1 receptors (AT1) release dopamine and norepinephrine. The current study determined the role of AT1 and angiotensin type-2 (AT2) receptors in mediating nicotine-evoked dopamine and norepinephrine release from striatal and hypothalamic slices, respectively. The potential involvement of nAChRs in mediating effects of AT1 antagonist losartan and AT2 antagonist, 1-[[4-(dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid (PD123319) was evaluated by determining their affinities for α4β2* and α7* nAChRs using [3H]nicotine and [3H]methyllycaconitine binding assays, respectively. Results show that losartan concentration-dependently inhibited nicotine-evoked [3H]dopamine and [3H]norepinephrine release (IC50: 3.9±1.2 and 2.2±0.7 μM; Imax: 82±3 and 89±6%, respectively). In contrast, PD123319 did not alter nicotine-evoked norepinephrine release, and potentiated nicotine-evoked dopamine release. These results indicate that AT1 receptors modulate nicotine-evoked striatal dopamine and hypothalamic norepinephrine release. Furthermore, AT1 receptor activation appears to be counteracted by AT2 receptor activation in striatum. Losartan and PD123319 did not inhibit [3H]nicotine or [3H]methyllycaconitine binding, indicating that these AT1 and AT2 antagonists do not interact with the agonist recognition sites on α4β2* and α7* nAChRs to mediate these effects of nicotine. Thus, angiotensin receptors contribute to the effects of nicotine on dopamine and norepinephrine release in brain regions involved in nicotine reward and hypertension.
Angiotensin II receptors; dopamine; nicotinic acetylcholine receptors; norepinephrine; reward; smoking
Mouse superficial superior colliculus (SuSC) contains dense GABAergic innervation and diverse nicotinic acetylcholine receptor subtypes. Pharmacological and genetic approaches were used to investigate the subunit compositions of nicotinic acetylcholine receptors (nAChR) expressed on mouse SuSC GABAergic terminals. [125I]-Epibatidine competition binding studies revealed that the α3β2* and α6β2* nicotinic subtype-selective peptide α-conotoxinMII blocked binding to 40 +/- 5% of SuSC nAChRs. Acetylcholine-evoked [3H]-GABA release from SuSC crude synaptosomal preparations is calcium dependent, blocked by the voltage-sensitive calcium channel blocker, cadmium, and the nAChR antagonist mecamylamine, but is unaffected by muscarinic, glutamatergic, P2X and 5-HT3 receptor antagonists. Approximately 50% of nAChR-mediated SuSC [3H]-GABA release is inhibited by α-conotoxinMII. However, the highly-α6β2*-subtype-selective α-conotoxinPIA did not affect [3H]-GABA release. Nicotinic subunit-null mutant mouse experiments revealed that ACh-stimulated SuSC [3H]-GABA release is entirely β2 subunit-dependent. α4 subunit deletion decreased total function by >90%, and eliminated α-conotoxinMII-resistant release. ACh-stimulated SuSC [3H]-GABA release was unaffected by β3, α5 or α6 nicotinic subunit deletions. Together, these data suggest that a significant proportion of mouse SuSC nicotinic agonist-evoked GABA-release is mediated by a novel, α-conotoxinMII-sensitive α3α4β2 nAChR. The remaining α-conotoxinMII-resistant, nAChR agonist-evoked SuSC GABA release appears to be mediated via α4β2* subtype nAChRs.
Nicotinic acetylcholine receptors; GABA; synaptosome; subunit-null mutant; α-conotoxinMII; superior colliculus
In a previous study the simple, naturally derived coumarin scopoletin (SCT) was identified as an inhibitor of acetylcholinesterase (AChE), using a pharmacophore-based virtual screening approach. In this study the potential of SCT as procholinergic and cognition-enhancing therapeutic was investigated in a more detailed way, using different experimental approaches like measuring newly synthesized acetylcholine (ACh) in synaptosomes, long-term potentiation (LTP) experiments in hippocampal slices, and behavior studies. SCT enhanced the K+-stimulated release of ACh from rat frontal cortex synaptosomes, showing a bell-shaped dose effect curve (Emax: 4 μM). This effect was blocked by the nicotinic ACh receptor (nAChR) antagonists mecamylamine (MEC) and dihydro-β-erythroidine (DHE). The nAChR agonist (and AChE inhibitor) galantamine induced a similar increase in ACh release (Emax: 1 μM). SCT potentiated LTP in hippocampal slices of rat brain. The high-frequency stimulation (HFS)-induced, N-methyl-D-aspartate (NMDA) receptor dependent LTP of field excitatory postsynaptic potentials at CA3-CA1 synapses was greatly enhanced by pre-HFS application of SCT (4 μM for 4 min). This effect was mimicked by nicotine (2 μM) and abolished by MEC, suggesting an effect on nAChRs. SCT did not restore the total inhibition of LTP by NMDA receptor antagonist d, l-2-amino-5-phosphonopentanoic acid (AP-5). SCT (2 μg, i.c.v.) increased T-maze alternation and ameliorated novel object recognition of mice with scopolamine-induced cholinergic deficit. It also reduced age-associated deficits in object memory of 15–18-month-old mice (2 mg/kg sc). Our findings suggest that SCT possesses memory-improving properties, which are based on its direct nAChR agonistic activity. Therefore, SCT might be able to rescue impaired cholinergic functions by enhancing nAChR-mediated release of neurotransmitters and promoting neural plasticity in hippocampus.
▶The coumarin scopoletin has been described as AChE inhibitor. ▶Now we show it exerts promising procognitive properties via nAChRs. ▶It enhances release of ACh and potentiates hippocampal LTP. ▶It improves novel object recognition and T-maze alternation in scopolamine-amnestic mice and ameliorates object memory in age-impaired mice.
ACh release; long-term potentiation; hippocampus slice; nicotinic acetylcholine receptor; T-maze; object recognition; ACh, acetylcholine; AChE, acetylcholinesterase; AD, Alzheimer's dementia; AP-5, d,l-2-amino-5-phosphonopentanoic acid; CSF, cerebrospinal fluid; DHE, dihydro-β-erythroidine; DMSO, dimethyl sulfoxide; fEPSPs, field excitatory postsynaptic potentials; HFS, high-frequency stimulation; LTP, long-term potentiation; MAO, monoamino oxidase; MEC, mecamylamine; nAChR, nicotinic acetylcholine receptor; NMDA, N-methyl-D-aspartate; SCOP, scopolamine; SCT, scopoletin
The current study evaluated a new series of N,N′-alkane-diyl-bis-3-picolinium (bAPi) analogs with C6–C12 methylene linkers as nicotinic receptor (nAChR) antagonists, for nicotine-evoked [3H]dopamine (DA) overflow, for blood-brain barrier choline transporter affinity and for attenuation of discriminative stimulus and locomotor stimulant effects of nicotine. bAPi analogs exhibited little affinity forα4β2* andα7* high affinity ligand binding sites, nor for nAChRs modulating DA transporter function. With the exception of C6, all analogs inhibited nicotine-evoked [3H]DA overflow (IC50=2 nM–6μM; Imax=54–64%), with N,N′-dodecane-1,12-diyl-bis-3-picolinium dibromide (C12, bPiDDB) being most potent. bPiDDB did not inhibit electrically-evoked [3H]DA overflow, suggesting specific nAChR inhibitory effects and a lack of toxicity to DA neurons. Schild analysis suggested that bPiDDB interacts in an orthosteric manner at nAChRs mediating nicotine-evoked [3H]DA overflow. To determine if bPiDDB interacts with α-conotoxin MII-sensitive α6β2-containing nAChRs, slices were exposed concomitantly to maximally-effective concentrations of bPiDDB (10 nM) and α-conotoxin MII (1 nM). Inhibition of nicotine-evoked [3H]DA overflow was not different with the combination compared with either antagonist alone, suggesting that bPiDDB interacts with α6β2-containing nAChRs. C7, C8, C10 and C12 analogs exhibited high affinity for the blood-brain barrier choline transporter in vivo, suggesting brain bioavailability. Although, none of the analogs altered the discriminative stimulus effect of nicotine, C8, C9, C10 and C12 analogs decreased nicotine-induced hyperactivity in nicotine-sensitized rats, without reducing spontaneous activity. Further development of nAChR antagonists that inhibit nicotine-evoked DA release and penetrate brain to antagonize DA-mediated locomotor stimulant effects of nicotine as novel treatments for nicotine addiction is warranted.
Neuropeptides collaborate with conventional neurotransmitters to regulate synaptic output. Pituitary adenylate cyclase-activating polypeptide (PACAP) co-localizes with acetylcholine in presynaptic nerve terminals, is released by stimulation, and enhances nicotinic acetylcholine receptor- (nAChR-) mediated responses. Such findings implicate PACAP in modulating nicotinic neurotransmission, but relevant synaptic mechanisms have not been explored. We show here that PACAP acts via selective high-affinity G-protein coupled receptors (PAC1Rs) to enhance transmission at nicotinic synapses on parasympathetic ciliary ganglion (CG) neurons by rapidly and persistently increasing the frequency and amplitude of spontaneous, impulse-dependent nicotinic excitatory postsynaptic currents (sEPSCs). Of the canonical adenylate cyclase (AC) and phospholipase-C (PLC) transduction cascades stimulated by PACAP/PAC1R signaling, only AC-generated signals are critical for synaptic modulation since the increases in sEPSC frequency and amplitude were mimicked by 8-Bromo-cAMP, blocked by inhibiting AC or cAMP-dependent protein kinase (PKA), and unaffected by inhibiting PLC. Despite its ability to increase agonist-induced nAChR currents, PACAP failed to influence nAChR-mediated impulse-independent miniature EPSC amplitudes (quantal size). Instead, evoked transmission assays reveal that PACAP/PAC1R signaling increased quantal content, indicating it modulates synaptic function by increasing vesicular ACh release from presynaptic terminals. Lastly, signals generated by the retrograde messenger, nitric oxide- (NO-) are critical for the synaptic modulation since the PACAP-induced increases in spontaneous EPSC frequency, amplitude and quantal content were mimicked by NO donor and absent after inhibiting NO synthase (NOS). These results indicate that PACAP/PAC1R activation recruits AC-dependent signaling that stimulates NOS to increase NO production and control presynaptic transmitter output at neuronal nicotinic synapses.
Ciliary ganglion; quantal analysis; patch-clamp; neuropeptide; nitric oxide
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 [3H]GLY and [3H]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.
β amyloid; glycine release; nicotinic receptors; muscarinic receptors; microdialysis
We previously showed that beta-amyloid (Aβ), a peptide considered as relevant to Alzheimer's Disease, is able to act as a neuromodulator affecting neurotransmitter release in absence of evident sign of neurotoxicity in two different rat brain areas. In this paper we focused on the hippocampus, a brain area which is sensitive to Alzheimer's Disease pathology, evaluating the effect of Aβ (at different concentrations) on the neurotransmitter release stimulated by the activation of pre-synaptic cholinergic nicotinic receptors (nAChRs, α4β2 and α7 subtypes). Particularly, we focused on some neurotransmitters that are usually involved in learning and memory: glutamate, aspartate and GABA.
We used a dual approach: in vivo experiments (microdialysis technique on freely moving rats) in parallel to in vitro experiments (isolated nerve endings derived from rat hippocampus). Both in vivo and in vitro the administration of nicotine stimulated an overflow of aspartate, glutamate and GABA. This effect was greatly inhibited by the highest concentrations of Aβ considered (10 µM in vivo and 100 nM in vitro). In vivo administration of 100 nM Aβ (the lowest concentration considered) potentiated the GABA overflow evoked by nicotine. All these effects were specific for Aβ and for nicotinic secretory stimuli. The in vitro administration of either choline or 5-Iodo-A-85380 dihydrochloride (α7 and α4β2 nAChRs selective agonists, respectively) elicited the hippocampal release of aspartate, glutamate, and GABA. High Aβ concentrations (100 nM) inhibited the overflow of all three neurotransmitters evoked by both choline and 5-Iodo-A-85380 dihydrochloride. On the contrary, low Aβ concentrations (1 nM and 100 pM) selectively acted on α7 subtypes potentiating the choline-induced release of both aspartate and glutamate, but not the one of GABA.
The results reinforce the concept that Aβ has relevant neuromodulatory effects, which may span from facilitation to inhibition of stimulated release depending upon the concentration used.
A recently developed α-conotoxin, α-CtxArIB[V11L,V16D] is a potent and selective competitive antagonist at rat recombinant α7 nicotinic acetylcholine receptors (nAChRs), making it an attractive probe for this receptor subtype. α7 nAChRs are potential therapeutic targets that are widely expressed in both neuronal and non-neuronal tissues where they are implicated in a variety of functions. Here we evaluate this toxin at rat and human native nAChRs. Functional α7 nAChR responses were evoked by choline plus the allosteric potentiator PNU-120596 in rat PC12 cells and human SHSY5Y cells loaded with calcium indicators. α-CtxArIB[V11L,V16D] specifically inhibited α7 nAChR-mediated increases in Ca2+ in PC12 cells. Responses to other stimuli (5-iodo-A-85380, nicotine or KCl) that did not activate α7 nAChRs were unaffected. Human α7 nAChRs were also sensitive to α-CtxArIB[V11L,V16D]: ACh-evoked currents in X. laevis oocytes expressing human α7 nAChRs were inhibited by α-CtxArIB[V11L,V16D] (IC50 3.4 nM) in a slowly reversible manner, with full recovery taking 15 min. This is consistent with the timecourse of recovery from blockade of rat α7 nAChRs in PC12 cells. α-CtxArIB[V11L,V16D] inhibited human native α7 nAChRs in SHSY5Y cells, activated by either choline or AR-R17779 plus PNU-120596. Rat brain α7 nAChRs contribute to dopamine release from striatal minces: α-CtxArIB[V11L,V16D] (300 nM) selectively inhibited choline-evoked dopamine release without affecting responses evoked by nicotine that activates heteromeric nAChRs. This study establishes that α-CtxArIB[V11L,V16D] selectively inhibits human and rat native α7 nAChRs with comparable potency, making this a potentially useful antagonist for investigating α7 nAChR functions.
Presynaptic nicotinic acetylcholine receptors (nAChRs) have long been implicated in the modulation of CNS circuits. We previously reported that brief exposure to low concentrations of nicotine induced sustained potentiation of glutamatergic transmission at ventral hippocampal (vHipp)-striatal synapses. Here, we exploited nAChR subtype-selective antagonists and agonists and α7*nAChR knockout mutant mice (α7-/-) to elucidate the signaling mechanisms underlying nAChR-mediated modulation of synaptic transmission. Using a combination of micro-slices culture from WT and α7-/-mice, calcium imaging, and immuno-histochemical techniques, we found that nicotine elicits localized and oscillatory increases in intracellular Ca2+ along vHipp axons that persists for up to 30 minutes. The sustained phase of the nicotine-induced Ca2+ response was blocked by α-BgTx but not by DHβE and was mimicked by α7*nAChR agonists but not by non-α7*nAChR agonists. In vHipp slices from α7-/- mice, nicotine elicited only transient increases of axonal Ca2+ signals and did not activate CaMKII. The sustained phase of the nicotine-induced Ca2+ response required localized activation of CaMKII, phospholipase C, and IP3 receptor mediated Ca2+-induced Ca2+ release (CICR). In conclusion, activation of presynaptic nAChRs by nicotine elicits Ca2+ influx into the presynaptic axons, the sustained phase of the nicotine-induced Ca2+ response requires that axonal α7*nAChR activate a downstream signaling network in the vHipp axons.
We examined the role of α7- and β2-containing nicotinic acetylcholine receptors (nAChRs) in the induction of long-term potentiation (LTP). Theta-burst stimulation (TBS), mimicking the brain's naturally occurring theta rhythm, induced robust LTP in hippocampal slices from α7 and β2 knockout mice. This suggests TBS is capable of inducing LTP without activation of α7- or β2-containing nAChRs. However, when weak TBS was applied, the modulatory effects of nicotinic receptors on LTP induction became visible. We showed that during weak TBS, activation of α7 nAChRs occurs by the release of ACh, contributing to LTP induction. Additionally, bath-application of nicotine activated β2-containing nAChRs to promote LTP induction. Despite predicted nicotine-induced desensitization, synaptically mediated activation of α7 nAChRs still occurs in the presence of nicotine and contributed to LTP induction. Optical recording of single-stimulation-evoked excitatory activity with a voltage-sensitive dye revealed enhanced excitatory activity in the presence of nicotine. This effect of nicotine was robust during high frequency stimulation, and was accompanied by enhanced burst excitatory postsynaptic potentials. Nicotine-induced enhancement of excitatory activity was observed in slices from α7 knockout mice, but was absent in β2 knockout mice. These results suggest that the nicotine-induced enhancement of excitatory activity is mediated by β2-containing nAChRs, and is related to the nicotine-induced facilitation of LTP induction. Thus, our study demonstrates that the activation of α7-and β2-containing nAChRs differentially facilitates LTP induction via endogenously released ACh and exogenous nicotine, respectively, in the hippocampal CA1 region of mice.
nicotinic acetylcholine receptors; α7 knockout mice; β2 knockout mice; optical recording; voltage-sensitive dye; synaptic plasticity
Rapid synaptic transmission at the calyciform synapse in the embryonic chicken ciliary ganglion is mediated by two classes of nicotinic receptors; those containing α3 subunits (α3-nAChRs) and those containing α7 subunits (α7-nAChRs). α3-nAChRs and α7-nAChRs are differentially distributed on the cell surface; α3-nAChRs are concentrated at postsynaptic densities, while both α7-nAChRs and α3-nAChRs are found extrasynaptically on somatic spines. I explored the contribution of α3-nAChRs and α7-nAChRs to uniquantal responses, measured as mEPSCs or as evoked responses under low release probability conditions. The contribution that each nAChR makes to uniquantal response shape was determined by blocking one nAChR type; pharmacologically isolated α7-nAChR responses were kinetically fast (rise time: 0.32 ± 0.02 ms; decay time: 1.66 ± 0.18 ms; mean ± SD, n=6 cells), while pharmacologically isolated α3-nAChR responses were slow (rise time: 1.28 ± 0.35 ms; decay time: 6.71 ± 1.46 ms; n=8 cells). In the absence of antagonists, most cells (11/14) showed heterogeneity in the kinetics of uniquantal responses, with ∼25% of events exhibiting fast, α7-nAChR-like kinetics and ∼75% of events exhibiting the kinetics expected of co-activation of α7-nAChRs and α3-nAChRs. Cells rarely showed significant numbers of uniquantal responses with slow, α3-nAChR-like kinetics, which was unexpected given that α3-nAChRs alone are concentrated at postsynaptic densities. The only site where ACh quanta can activate both α3-nAChRs and α7-nAChRs readily is on the somatic spines, where α7-nAChRs and α3-nAChRs are present extrasynaptically. At the calyciform synapse, rapid synaptic transmission is mediated apparently without participation of ionotropic receptors concentrated at postsynaptic densities.
synaptic transmission; nicotinic receptor; release; ciliary; ganglion cell; acetylcholine receptor
A variety of peptide ligands are known to inhibit the function of neuronal nicotinic acetylcholine receptors (nAChRs), including small toxins and brain-derived peptides such as β-amyloid1–42 and synthetic apolipoproteinE peptides. The myristoylated alanine-rich C kinase substrate (MARCKS) protein is a major substrate of protein kinase C and is highly expressed in the developing and adult brain. The ability of a 25-amino acid synthetic MARCKS peptide, derived from the effector domain (ED), to modulate nAChR activity was tested. To determine the effects of the MARCKS ED peptide on nAChR function, receptors were expressed in Xenopus laevis oocytes, and two-electrode voltage-clamp experiments were performed. The MARCKS ED peptide completely inhibited acetylcholine (ACh)-evoked responses from α7 nAChRs in a dose-dependent manner, yielding an IC50 value of 16 nM. Inhibition of ACh-induced responses was both activity- and voltage-independent. The MARCKS ED peptide was unable to block α-bungarotoxin binding. A MARCKS ED peptide in which four serine residues were replaced with aspartate residues was unable to inhibit α7 nAChR-mediated currents. The MARCKS ED peptide inhibited ACh-induced α4β2 and α2β2 responses, although with decreased potency. The effects of the MARCKS ED peptide on native nAChRs were tested using acutely isolated rat hippocampal slices. In hippocampal interneurons, the MARCKS ED peptide was able to block native α7 nAChRs in a dose-dependent manner. The MARCKS ED peptide represents a novel antagonist of neuronal nAChRs that has considerable utility as a research tool.
Prejunctional nicotinic acetylcholine receptors (nAChRs) amplify postganglionic sympathetic neurotransmission, and there are indications that intraterminal Ca2+ stores might be involved. However, the mechanisms by which nAChR activation stimulates neurotransmitter release at such junctions is unknown. Rapid local delivery (picospritzing) of the nAChR agonist epibatidine was combined with intracellular sharp microelectrode recording to monitor spontaneous and field-stimulation-evoked neurotransmitter release from sympathetic nerve terminals in the mouse isolated vas deferens. Locally applied epibatidine (1 µM) produced ‘epibatidine-induced depolarisations’ (EIDs) that were similar in shape to spontaneous excitatory junction potentials (SEJPs) and were abolished by nonselective nAChR antagonists and the purinergic desensitizing agonist α,β-methylene ATP. The amplitude distribution of EIDs was only slightly shifted towards lower amplitudes by the selective α7 nAChR antagonists α-bungarotoxin and methyllcaconitine, the voltage-gated Na+ channel blocker tetrodotoxin or by blocking voltage-gated Ca2+ channels with Cd2+. Lowering the extracellular Ca2+ concentration reduced the frequency of EIDs by 69%, but more surprisingly, the Ca2+-induced Ca2+ release blocker ryanodine greatly decreased the amplitude (by 41%) and the frequency of EIDs by 36%. Ryanodine had no effect on electrically-evoked neurotransmitter release, paired-pulse facilitation, SEJP frequency, SEJP amplitude or SEJP amplitude distribution. These results show that activation of non-α7 nAChRs on sympathetic postganglionic nerve terminals induces high-amplitude junctional potentials that are argued to represent multipacketed neurotransmitter release synchronized by intraterminal Ca2+-induced Ca2+ release, triggered by Ca2+ influx directly through the nAChR. This nAChR-induced neurotransmitter release can be targeted pharmacologically without affecting spontaneous or electrically-evoked neurotransmitter release.
Limitations in efficacy and high relapse rates of currently available smoking cessation agents reveal the need for more efficacious pharmacotherapies. One strategy is to develop subtype-selective nicotinic receptor (nAChR) antagonists that inhibit nicotine-evoked dopamine (DA) release, the primary neurotransmitter involved in nicotine reward. Simple alkylation of the pyridino N-atom converts nicotine from a potent agonist into a potent antagonist. The classical antagonists, hexamethonium and decamethonium, differentiate between peripheral nAChR subtypes. Using a similar approach, we interconnected varying quaternary ammonium moieties with a lipophilic linker to provide N,N′- bis-nicotinium analogs, affording a lead compound, N,N′-dodecyl-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), which inhibited nicotine-evoked DA release and decreased nicotine self-administration. The current work describes a novel compound, 1-(3-picolinium)-12-triethylammonium-dodecane dibromide (TMPD), a hybrid of bPiDDB and decamethonium. TMPD completely inhibited (IC50 = 500 nM) nicotine-evoked DA release from superfused rat striatal slices, suggesting that TMPD acts as a nAChR antagonist at more than one subtype. TMPD (1 μM) inhibited the response to acetylcholine at α3β4, α4β4, α4β2, and α1β1εδ receptors expressed in Xenopus oocytes. TMPD had a 2-fold higher affinity for the blood-brain barrier choline transporter, suggesting that is brain bioavailable. TMPD did not inhibit the hyperactivity in nicotine sensitized rats, but significantly and specifically decreased nicotine self-administration. Together, the results suggest that TMPD may have the ability to reduce the rewarding effect of nicotine with minimal side effects, a pharmacological profile indicative of potential clinical utility for the treatment of tobacco dependence.
dopamine; nicotine; nicotinic receptors; blood-brain barrier choline transporter; smoking cessation; TMPD
The novel nicotinic receptor (nAChR) antagonist, N,N’-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), and its chemically-reduced analog, r-bPiDDB, potently inhibit nicotine-evoked dopamine (DA) release from rat striatal slices. Since tobacco smokers self-administer nicotine repeatedly, animal models incorporating repeated nicotine treatment allow for mechanistic evaluation of therapeutic candidates following neuroadaptive changes. The current study determined the ability of bPiDDB, r-bPiDDB and α-conotoxin MII (α-CtxMII), a peptide antagonist selective for α6β2-containing nAChRs, to inhibit nicotine-evoked [3H]DA release from striatal slices from rats repeatedly administered nicotine (0.4 mg/kg for 10 days) or saline (control). Concomitant exposure to maximally effective concentrations of r-bPiDDB (1 nM) and α-CtxMII (1 nM) resulted in inhibition of nicotine-evoked [3H]DA release no greater than that produced by either antagonist alone, suggesting that r-bPiDDB inhibits α6β2-containing nAChRs. Repeated nicotine treatment increased locomotor activity, demonstrating behavioral sensitization. Concentration-response curves for nicotine-evoked [3H]DA release were not different between nicotine-treated and control groups. Maximal inhibition for α-CtxMII was greater following repeated nicotine compared to control (Imax=90% vs. 62%), with no change in potency. bPiDDB was 3-orders of magnitude more potent in inhibiting nicotine-evoked [3H]DA release in nicotine-treated rats compared to control rats (IC50=5 pM vs 6 nM), with no change in maximal inhibition. Neither a shift to left in the concentration response nor a change in maximal inhibition was observed for r-bPiDDB following repeated nicotine. Thus, repeated nicotine treatment may differentially regulate the stoichiometry, conformation and/or composition of α6β2-containing nAChRs mediating nicotine-evoked striatal DA release. Therefore, bPiDDB and r-bPiDDB appear to target different α6β2-containing nAChR subtypes.
smoking cessation; nicotine; nicotinic acetylcholine receptor; dopamine release; nicotinic receptor antagonist
The nicotine metabolite cotinine is an abundant long-lived bio-active compound that may contribute to the overall physiological effects of tobacco use. Although its mechanism of action in the central nervous system has not been extensively investigated, cotinine is known to evoke dopamine release in the nigrostriatal pathway through an interaction at nicotinic receptors (nAChRs). Because considerable evidence now demonstrates the presence of multiple nAChRs in the striatum, the present experiments were done to determine the subtypes through which cotinine exerts its effects in monkeys, a species that expresses similar densities of striatal α4β2* (nAChR containing the α4 and β2 subunits, but not α3 or α6) and α3/α6β2* (nAChR composed of the α3 or α6 subunits and β2) nAChRs. Competition binding studies showed that cotinine interacts with both α4β2* and α3/α6β2* nAChR subtypes in the caudate, with cotinine IC50 values for inhibition of 5-[125I]iodo-3-[2(S)-azetinylmethoxy]pyridine-2HCl ([125I]A-85380) and 125I-α-conotoxinMII binding in the micromolar range. This interaction at the receptor level is of functional significance because cotinine stimulated both α4β2* and α3/α6β2* nAChR [3H]dopamine release from caudate synaptosomes. Our results unexpectedly showed that nicotine evokes [3H]dopamine release from two α3/α6β2* nAChR populations, one of which was sensitive to cotinine and the other was not. This cotinine-insensitive subtype was only present in the medial caudate and was preferentially lost with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced nigrostriatal damage. In contrast, cotinine and nicotine elicited equivalent levels of α4β2* nAChR-mediated dopamine release. These data demonstrate that cotinine functionally discriminates between two α3/α6β2* nAChRs in monkey striatum, with the cotinine-insensitive α3/α6β2* nAChR preferentially vulnerable to nigrostriatal damage.
Stimulus evoked neurotransmitter release requires that Na+ channel-dependent nerve terminal depolarization be transduced into synaptic vesicle exocytosis. Inhaled anesthetics block presynaptic Na+ channels and selectively inhibit glutamate over GABA release from isolated nerve terminals, indicating mechanistic differences between excitatory and inhibitory transmitter release. We compared the effects of isoflurane on depolarization-evoked [3H]glutamate and [14C]GABA release from isolated nerve terminals prepared from four regions of rat CNS evoked by 4-aminopyridine (4AP), veratridine (VTD), or elevated K+. These mechanistically distinct secretegogues distinguished between Na+ channel- and/or Ca2+ channel-mediated presynaptic effects. Isoflurane completely inhibited total 4AP-evoked glutamate release (IC50=0.42 ± 0.03 mM) more potently than GABA release (IC50=0.56 ± 0.02 mM) from cerebral cortex (1.3-fold greater potency), hippocampus and striatum, but inhibited glutamate and GABA release from spinal cord terminals equipotently. Na+ channel-specific VTD-evoked glutamate release from cortex was also significantly more sensitive to inhibition by isoflurane than was GABA release. Na+ channel-independent K+-evoked release was insensitive to isoflurane at clinical concentrations in all four regions, consistent with a target upstream of Ca2+ entry. Isoflurane inhibited Na+ channel-mediated (tetrodotoxin-sensitive) 4AP-evoked glutamate release (IC50=0.30 ± 0.03 mM) more potently than GABA release (IC50=0.67 ± 0.04 mM) from cortex (2.2-fold greater potency). The magnitude of inhibition of Na+ channel-mediated 4AP-evoked release by a single clinical concentration of isoflurane (0.35 mM) varied by region and transmitter: Inhibition of glutamate release from spinal cord was greater than from the three brain regions and greater than GABA release for each CNS region. These findings indicate that isoflurane selectively inhibits glutamate release compared to GABA release via Na+ channel-mediated transduction in the four CNS regions tested, and that differences in presynaptic Na+ channel involvement determine differences in anesthetic pharmacology.
Na+ channels; glutamate; GABA; nerve terminal; tetrodotoxin; rat
Identifying the neurological mechanisms underlying nicotine reinforcement is a healthcare imperative if society is to effectively combat tobacco addiction. The majority of studies of the neurobiology of addiction have focused on dopamine (DA)-containing neurons of the ventral tegmental area (VTA). However, recent data suggests that neurons of the laterodorsal tegmental (LDT) nucleus which sends cholinergic, GABAergic- and glutamatergic-containing projections to DA-containing neurons of the VTA are critical to gating normal functioning of this nucleus. The actions of nicotine on LDT neurons are unknown. We addressed this issue by examination of nicotinic effects on identified cholinergic and non-cholinergic LDT neurons using whole-cell patch clamp and Ca2+ imaging methods in brain slices from mice (P12-P45). Nicotine applied via puffer pipette or bath superfusion elicited membrane depolarization that often induced firing and TTX-resistant inward currents. Nicotine also enhanced sensitivity to injected current; and, baseline changes in intracellular calcium were elicited in the dendrites of some cholinergic LDT cells. Additionally, activity-dependent calcium transients were increased, suggesting that nicotine exposure sufficient to induce firing may lead to enhancement of levels of intracellular calcium. Nicotine also had strong actions on glutamate and GABA-releasing presynaptic terminals since it greatly increased the frequency of miniature EPSCs and IPSCs to both cholinergic and non-cholinergic neurons. Utilization of nAChR subunit antagonists revealed that presynaptic, inhibitory terminals on cholinergic neurons were activated by receptors containing α7, β2 and non-α7 subunits; whereas, presynaptic glutamatergic terminals were activated by nAChRs comprised of non-α7 subunits. We also found that direct nicotinic actions on cholinergic LDT neurons were mediated by receptors containing α7, β2 and non-α7 subunits. These findings lead us to suggest that nicotine exposure from smoking will enhance both the excitability and synaptic modulation of cholinergic and non-cholinergic LDT neurons and increase their signature neurotransmitter outflow to target regions including the VTA. This may reinforce the direct actions of this drug within reward circuitry and contribute to encoding stimulus saliency.
REM sleep; pontine; nNOS+; cholinergic; patch clamp