Glutamatergic synapse formation is promoted by α7-containing nicotinic acetylcholine receptors

Glutamate is the primary excitatory transmitter in adult brain, acting through synapses on dendritic spines and shafts. Early in development, however, when glutamatergic synapses are only beginning to form, nicotinic cholinergic excitation is already widespread; it is mediated by acetylcholine activating nicotinic acetylcholine receptors (nAChRs) that generate waves of activity across brain regions. A major class of nAChRs contributing at this time is a species containing α7 subunits (α7-nAChRs). These receptors are highly permeable to calcium, influence a variety of calcium-dependent events, and are diversely distributed throughout the developing CNS. Here we show that α7-nAChRs unexpectedly promote formation of glutamatergic synapses during development. The dependence on α7-nAChRs becomes clear when comparing wild-type (WT) mice with mice constitutively lacking the α7-nAChR gene. Ultrastructural analysis, immunostaining, and patch-clamp recording all reveal synaptic deficits when α7-nAChR input is absent. Similarly, nicotinic activation of α7-nAChRs in WT organotypic culture, as well as cell culture, increases the number of glutamatergic synapses. RNA interference demonstrates that the α7-nAChRs must be expressed in the neuron being innervated for normal innervation to occur. Moreover, the deficits persist throughout the developmental period of major de novo synapse formation and are still fully apparent in the adult. GABAergic synapses, in contrast, are undiminished in number under such conditions. As a result, mice lacking α7-nAChRs have an altered balance in the excitatory/inhibitory input they receive. This ratio represents a fundamental feature of neural networks and shows for the first time that endogenous nicotinic cholinergic signaling plays a key role in network construction.     

A transgenic mouse model reveals fast nicotinic transmission in hippocampal pyramidal neurons

The relative contribution to brain cholinergic signaling by synaptic- and diffusion-based mechanisms remains to be elucidated. In this study, we examined the prevalence of fast nicotinic signaling in the hippocampus. We describe a mouse model where cholinergic axons are labeled with the tauGFP fusion protein driven by the choline acetyltransferase promoter. The model provides for the visualization of individual cholinergic axons at greater resolution than other available models and techniques, even in thick, live, slices. Combining calcium imaging and electrophysiology, we demonstrate that local stimulation of visualized cholinergic fibers results in rapid excitatory postsynaptic currents mediated by the activation of α7-subunit-containing nicotinic acetylcholine receptors (α7-nAChRs) on CA3 pyramidal neurons. These responses were blocked by the α7-nAChR antagonist methyllycaconitine and potentiated by the receptor-specific allosteric modulator 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxanol-3-yl)-urea (PNU-120596). Our results suggest, for the first time, that synaptic nAChRs can modulate pyramidal cell plasticity and development. Fast nicotinic transmission might play a greater role in cholinergic signaling than previously assumed. We provide a model for the examination of synaptic properties of basal forebrain cholinergic innervation in the brain.     

Lack of dystrophin functionally affects α3β2/β4-nicotinic acethylcholine receptors in sympathetic neurons of dystrophic mdx mice

In the sympathetic superior cervical ganglion (SCG), nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission. We previously demonstrated that in SCG neurons of mdx mice, an animal model for Duchenne muscular dystrophy, lack of dystrophin causes a decrease, compared to the wild-type, in post-synaptic nAChRs containing the α3 subunit associated with β2 and/or β4 (α3β2/β4-nAChRs), but not in those containing the α7 subunit. Here we show, by whole cell patch-clamp recordings from cultured SCG neurons, that both nicotine and acetylcholine-evoked currents through α3β2/β4-nAChRs are significantly reduced in mdx mice compared to the wild-type, while those through α7-nAChR are unaffected. This reduction associates with that of protein levels of α3, β2 and β4 subunits. Therefore, we suggest that, in mdx mouse SCG neurons, lack of dystrophin, by specifically affecting membrane stabilization of α3β2/β4-nAChRs, could determine an increase in receptor internalization and degradation, with consequent reduction in the fast intraganglionic cholinergic transmission.     

Choline is a Selective Agonist of α7 Nicotinic Acetylcholine Receptors in the Rat Brain Neurons

In the present study, we demonstrate that choline, a precursor of acetylcholine (ACh) and a product of acetylcholine hydrolysis by acetylcholinesterase (AChE), acts as an efficient and relatively selective agonist of α7–containing nicotinic acetylcholine receptors (nAChR) in neurons cultured from the rat hippocampus, olfactory bulb and thalamus as well as in PC12 cells. Choline was able to activate postsynaptic and presynaptic α7 nAChRs, with the latter action resulting in the release of other neurotransmitters. Although choline was approximately one order of magnitude less potent than ACh (EC₅₀ of 1.6 mM for choline and 0.13 mM for ACh), it acted as a full agonist at α7 nAChRs. In contrast, choline did not activate α4β2 agonist-bearing nAChRs on hippocampal neurons, and acted as a partial agonist at α3β4-containing nAChRs on PC12 cells. The ethyl alcohol moiety of choline is required for the selective action on α7 nAChR. Exposure of cultured hippocampal neurons for 10 min to choline (10–100 μM) resulted in desensitization of the native α7 nAChRs. Moreover, chronic exposure (10 days) of the cultured hippocampal neurons to a desensitizing concentration of choline (∼30 μM) decreased their responsiveness to ACh. The selective action of choline on native α7 nAChRs suggests that this naturally occurring compound may act in vivo as an endogenous ligand for these receptors. Putative physiological actions of choline include retrograde messenger activity during the development of the mammalian central nervous system and during periods of elevated synaptic activity that leads to long-term potentiation.     

α7 and non-α7 nicotinic acetylcholine receptors modulate dopamine release in vitro and in vivo in the rat prefrontal cortex

Nicotine enhances attentional and working memory aspects of executive function in the prefrontal cortex (PFC) where dopamine plays a major role. Here, we have determined the nicotinic acetylcholine receptor (nAChR) subtypes that can modulate dopamine release in rat PFC using subtype-selective drugs. Nicotine and 5-Iodo-A-85380 (β2* selective) elicited [³H]dopamine release from both PFC and striatal prisms in vitro and dopamine overflow from medial PFC in vivo . Blockade by dihydro-β-erythroidine supports the participation of β2* nAChRs. However, insensitivity of nicotine-evoked [³H]dopamine release to α-conotoxin-MII in PFC prisms suggests no involvement of α6β2* nAChRs, in contrast to the striatum, and this distinction is supported by immunoprecipitation of nAChR subunits from these tissues. The α7 nAChR-selective agonists choline and Compound A also promoted dopamine release from PFC in vitro and in vivo , and their effects were enhanced by the α7 nAChR-selective allosteric potentiator PNU-120596 and blocked by specific antagonists. DNQX and MK801 inhibited [³H]dopamine release evoked by choline and PNU-120596, suggesting crosstalk between α7 nAChRs, glutamate and dopamine in the PFC. In vivo, systemic (but not local) administration of PNU-120596, in the absence of agonist, facilitated dopamine overflow in the medial PFC, consistent with the activation of extracortical α7 nAChRs by endogenous acetylcholine or choline. These data establish that both β2* and α7 nAChRs can modulate dopamine release in the PFC in vitro and in vivo . Through their distinct actions on dopamine release, these nAChR subtypes could contribute to executive function, making them specific therapeutic targets for conditions such as schizophrenia and attention deficit hyperactivity disorder.     

Nicotine-mediated plasticity in robust nucleus of the archistriatum of the adult zebra finch

Activation of neuronal nicotinic acetylcholine receptors (nAChRs) modulates the induction of long-term potentiation (LTP), a possible cellular mechanism for learning. This study was undertaken to determine the effects of activation of nAChRs by nicotine on long-term plasticity in the songbird zebra finch, which is a valuable model to study synaptic plasticity and its implications to behavioral learning. Electrophysiological recordings in the robust nucleus of the archistriatum (RA) in adult zebra finch brain slices reveal that tetanic stimulation alone does not produce LTP. However, LTP is induced by such stimulation in the presence of nicotine. The nicotine-mediated LTP is blocked by dihydro-beta-erythroidine (DHbetaE, 1 microM), an antagonist having a greater effect against nAChRs containing the alpha 4 subunit. In the presence of methyllcaconitine (MLA, 10 nM), an antagonist of nAChRs containing the alpha 7 subunit, a long-term depression (LTD) is unmasked, implicating a bi-directional type of plasticity in the zebra finch RA, which is modulated by differential activation of nAChR subtypes. Intracellular recordings from single neurons show a depression of the afterhyperpolarization (AHP) and an increase in frequency of evoked and spontaneous action potentials in the presence of nicotine. These results suggest that nicotinic cholinergic mechanisms may play a critical role in synaptic plasticity in the zebra finch song system and thereby influence song learning and plasticity.     

Functional Properties of Neuronal Nicotinic Acetylcholine Receptor Channels Expressed in Transfected Human Cells

To study how subunit composition affects the functional properties of neuronal nicotinic acetylcholine receptors (nAChRs), we examined the behaviour of acetylcholine (ACh)-induced single-channel currents in human BOSC 23 cells transiently transfected with various subunit cDNA combinations. For all nAChRs examined (chick and rat α₃β₄, chick α_<3/β₂, α₄β₂, α₄β₄, α₇and α₈, expression levels were high enough to allow measurements of acetylcholine-evoked whole-cell currents and nicotine-elicited Ca²⁺ transients as well as the functional characterization of nAChR channels. Unitary acetylcholine-evoked events of α₈ nAChR had a slope conductance of 23 pS, whereas two conductance classes (19–23 and 32–45 pS) were identified for all other nAChR channels. The mean channel open times were significantly longer for homomeric α₇ and α₈ nAChRs (6–7 ms) than for heteromeric nAChRs (1–3 ms), with the exception of α₃α₄nAChRs (8.4 ms for rat, 7 ms for chick). At least two species of heterologously expressed nAChRs (α₃α₄and α₃α₂) exhibited single-channel characteristics similar to those reported for native receptors. The variety of nAChRs channel conductance and kinetic properties encountered in human cells transfected with nAChR subunits contributes to the functional diversity of nAChRs in nerve cells.     

α7-Containing and non-α7-containing nicotinic receptors respond differently to spillover of acetylcholine

We explored whether nicotinic acetylcholine receptors (nAChRs) might participate in paracrine transmission by asking if they respond to spillover of ACh at a model synapse in the chick ciliary ganglion, where ACh activates diffusely distributed α7- and α3-containing nAChRs (α7-nAChRs and α3*-nAChRs). Elevating quantal content lengthened EPSC decay time and prolonged both the fast (α7-nAChR-mediated) and slow (α3*-nAChR-mediated) components of decay, even in the presence of acetylcholinesterase. Increasing quantal content also prolonged decay times of pharmacologically isolated α7-nAChR- and α3*-nAChR-EPSCs. The effect upon EPSC decay time of changing quantal content was 5-10 times more pronounced for α3*-nAChR- than α7-nAChR-mediated currents and operated over a considerably longer time window: ≈ 20 vs ≈ 2 ms. Control experiments rule out a presynaptic source for the effect. We suggest that α3*-nAChR currents are prolonged at higher quantal content because of ACh spillover and postsynaptic potentiation (Hartzell et al., 1975), while α7-nAChR currents are prolonged probably for other reasons, e.g., increased occupancy of long channel open states. α3*-nAChRs report more spillover when α7-nAChRs are competitively blocked than under native conditions; this could be explained if α7-nAChRs buffer ACh and regulate its availability to activate α3*-nAChRs. Our results suggest that non-α7-nAChRs such as α3*-nAChRs may be suitable for paracrine nicotinic signaling but that α7-nAChRs may not be suitable. Our results further suggest that α7-nAChRs may buffer ACh and regulate its bioavailability.     

Expression of Neuronal Nicotinic Acetylcholine Receptor Subunit Genes in the Rat Autonomic Nervous System

In the autonomic nervous system efferent signals are relayed in sympathetic and parasympathetic ganglia. Fast synaptic transmission between pre- and postsynaptic neurons is achieved by neuronal nicotinic acetylcholine receptors (nAChRs). There is still little known about the subunit composition of these receptors. Establishing the subunit composition of native neuronal nAChRs is important for the understanding of their functional properties both in vivo and after expression in heterologous expression systems. We have combined in situ hybridization and autoradiography to detect the presence of mRNAs encoding subunits of neuronal nAChRs in sympathetic and parasympathetic ganglia. Inspection of the autoradiographs showed that the hybridization signal of five riboprobes (α3, 014–1, α7, β2 and β4) was significantly higher than the unspecific signal obtained with sense riboprobes. The distribution of α7 was tissue-dependent: a7 riboprobe binding was detected in the neurons of the superior cervical ganglion, adrenal medulla and ciliary ganglion. In contrast, the α7 hybridization signal was found only in a small fraction (1 -3%) of the neurons of the sphenopalatine and otic ganglia. Our results are consistent with the idea that α3 mRNA expression levels are somewhat higher than those of α7, α4 -1, β2 and β4.     

α7 Nicotinic acetylcholine receptors and their role in cognition

The precise role of nicotinic acetylcholine receptors (nAChRs) in central cognitive processes still remains incompletely understood almost 150 years after its initial discovery. Central nAChRs are activated by acetylcholine, which functions in the extracellular space as a nonsynaptic messenger. Recently, a novel concept in the nAChR mode of operation has been described as a fast-type nonsynaptic transmission. In this review, we attempt to summarise the experimental findings that support the role of one of the most distributed receptor subtypes, the α7 nAChRs, and particularly focus on its procognitive effects following receptor activation. The basic characteristics of α7 nAChRs are discussed, from receptor homology to cellular-level functions. Synaptic plasticity is often implicated with α7 nAChRs on the basis of several diverse studies. Here, we provide a summary of the plastic features of the α7 receptor subtype and its role in higher level cognitive function. Finally, recent clinical evidence is reviewed, which demonstrates with increasing confidence the promise α7 nAChRs as a molecular target in future pharmacotherapy to prevent cognitive decline in various types of dementia, specifically, via the development of positive allosteric modulator compounds.     

Enhanced serum antigen-specific IgG1 and proinflammatory cytokine production in nicotinic acetylcholine receptor alpha7 subunit gene knockout mice

Human and murine immune cells such as mononuclear leukocytes consisting of mainly T and B cells, bone marrow derived dendritic cells (DCs) and macrophages all express various nicotinic acetylcholine (ACh) receptor (nAChR) subunits. Activated T cells and DCs have the ability to synthesize ACh by choline acetyltransferase, suggesting the role of non-neuronal cholinergic system expressed in immune cells in the regulation of immune cell function. Stimulation of human leukemic T and B cell lines with nicotine causes a transient Ca(2+)-signaling that is antagonized by alpha-bungarotoxin, suggesting the involvement of alpha7 subunit. Furthermore, alpha7 nAChRs have been shown to negatively regulate synthesis and release of tumor necrosis factor (TNF)-alpha in macrophages. These findings suggest that immune cell function is regulated by its own non-neuronal cholinergic system, at least in part, via alpha7 nAChR-mediated pathways. In the present study, we tested the role of alpha7 nAChRs in the regulation of immune function by measuring total serum and antigen-specific IgG(1) and IgM, and production of TNF-alpha, gamma interferon (IFN-gamma) and interleukin (IL)-6 in activated spleen cells of nAChR alpha7 subunit gene knockout (alpha7 KO) and wild-type C57BL/6J mice immunized with ovalbumin (OVA). We found that serum levels of total and anti-OVA-specific IgG(1) were significantly elevated in alpha7 KO mice, though there were no significant differences in serum levels of total and anti-OVA-specific IgM between the two genotypes. Production of TNF-alpha, IFN-gamma and IL-6 in spleen cells was significantly facilitated in alpha7 KO mice. Expression of AChE mRNA was not different between the two genotypes. These results suggest that alpha7 nAChRs are involved in the regulation of cytokine production, through which modulates TNF-alpha, IFN-gamma and IL-6 productions, leading to modification of antibody production, but are not involved in expression of cholinergic components in immune cells.     

Orexin A modulates neuronal activity of the rodent suprachiasmatic nucleus in vitro

We have previously shown that activation of nicotinic acetylcholine receptors (nAChRs) enhanced long-term potentiation (LTP) in the rat dentate gyrus in vitro via activation of α7 nAChR. In the present studies, mechanisms underlying the acute and chronic nicotinic enhancement of LTP were examined. In particular, the involvement of activation of intracellular kinases was examined using selective kinase antagonists, and the effects of enhancing cholinergic function with positive allosteric modulators of the α7 nAChR and with acetylcholinesterase (AChE) inhibitors were also investigated. Activation of extracellular signal-regulated kinase (ERK) and cAMP-dependent protein kinase (PKA) was found to be involved in the induction of the acute nicotinic enhancement of LTP, although not control LTP. In contrast, activation of the tyrosine kinase Src, Ca²⁺-calmodulin-dependent protein kinase II, Janus kinase 2 and p38 mitogen-activated protein kinase was not involved in the acute nicotinic enhancement of LTP, although Src activation was necessary for control LTP. Moreover, activation of phosphoinositide 3-kinase was involved in the acute nicotinic enhancement of LTP to a much lesser extent than in control LTP. Chronic nicotine enhancement of LTP was found to be dependent on PKA, ERK and Src kinases. Acute nicotinic enhancement of LTP was occluded by chronic nicotine treatment. The positive allosteric modulator PNU-120596 was found to strongly reduce the threshold for nicotinic enhancement of LTP, an affect mediated via the α7 nAChR as it was blocked by the selective antagonist methyllycaconitine. The AChE inhibitors tacrine and physostigmine enhanced control LTP.     

Regional differential effects of chronic nicotine on brain alpha 4-containing and alpha 6-containing receptors

Chronic nicotine upregulates central nicotinic acetylcholine receptors (nAChRs), a plasticity process thought to contribute to its addictive properties. To analyze this process in vivo, we chronically exposed mice to nicotine using minipump delivering nicotine at concentrations close to those found in tobacco smokers. Binding studies show upregulation of high-affinity nAChRs after 21 days of treatment in cortical areas, caudate putamen, nucleus accumbens, hippocampus, ventral tegmental area, and superior colliculi. No upregulation was observed in thalamus and discrete cortical areas. Using wild type and alpha 6-/- mice, we observed a downregulation of alpha 6*-nAChRs in superior colliculi and no effects in other structures. The complex pattern of upregulation/downregulation observed in this study depends on both nAChR composition and regional distribution.     

Nicotinic receptor agonists as neuroprotective/neurotrophic drugs. Progress in molecular mechanisms

Summary. In the present work we reviewed recent advances concerning neuroprotective/neurotrophic effects of acute or chronic nicotine exposure, and the signalling pathways mediating these effects, including mechanisms implicated in nicotine addiction and nAChR desensitization. Experimental and clinical data largely indicate long-lasting effects of nicotine and nicotinic agonists that imply a neuroprotective/neurotrophic role of nAChR activation, involving mainly α7 and α4β2 nAChR subtypes, as evidenced using selective nAChR agonists. Compounds interacting with neuronal nAChRs have the potential to be neuroprotective and treatment with nAChR agonists elicits long-lasting neurotrophic effects, e.g. improvement of cognitive performance in a variety of behavioural tests in rats, monkeys and humans. Nicotine addiction, which is mediated by interaction with nACh receptors, is believed to involve the modification of signalling cascades that modulate synaptic plasticity and gene expression. Desensitization, in addition to protecting cells from uncontrolled excitation, is recently considered as a form of signal plasticity. nAChR can generate these longe-lasting effects by elaboration of complex intracellular signals that mediate medium to long-term events crucial for neuronal maintenance, survival and regeneration. Although a comprehensive survey of the gene-based molecular mechanisms that underlie nicotine effects has yet not been performed a growing amount of data is beginning to improve our understanding of signalling mechanisms that lead to neurotrophic/neuroprotective responses. Evidence for an involvement of the fibroblast growth factor-2 gene in nAChR mechanisms mediating neuronal survival, trophism and plasticity has been obtained. However, more work is needed to establish the mechanisms involved in the effects of nicotinic receptor subtype activation from cognition-enhancing and neurotrophic effects to smoking behaviour and to determine more precisely the therapeutic objectives in potential nicotinic drug treatments of neurodegenerative diseases.     

Role of presynaptic nicotinic acetylcholine receptors in the regulation of gastrointestinal motility

Presynaptic nicotinic acetylcholine receptors (nAChRs) located on cholinergic terminals facilitate the release of acetylcholine (ACh), thereby constituting a fail-safe mechanism at strategic locations, such as the neuromuscular junction, where reliable transmission is vital. Accumulating data indicate that myenteric neurons in the enteric nervous system possess not only somatodendritic nAChRs, which mediate cholinergic transmission between neurons, but also presynaptic nAChRs. Functional evidence shows that these receptors mediate a positive feedback with respect to ACh release from myenteric motoneurons, and might therefore play an important role in the regulation of gastrointestinal motility. These presynaptic nAChRs were found to be more sensitive to nicotinic ligands than somatodendritic nAChRs and could therefore be primary targets of exogenous compounds, such as nicotine. This interaction might provide a neurochemical basis for the effect of smoking on gastrointestinal motility. Another important human pharmacological implication is based on our recent observation that monoamine uptake inhibitor-type antidepressant drugs are able to inhibit presynaptic nAChRs in the enteric nervous system. The disruption of the nAChR-mediated positive feedback modulation by antidepressants might explain the frequent occurrence of constipation, a common side effect, attributed to these drugs. Clarification of the role of presynaptic nAChRs in feedback mechanisms in the enteric nervous system might be instrumental in the development of new drugs affecting gastrointestinal motility.     

Alpha7 nicotinic acetylcholine receptors targeted by cholinergic developmental neurotoxicants: nicotine and chlorpyrifos

Alpha7 nicotinic acetylcholine receptors (nAChRs) play a role in axonogenesis, synaptogenesis and synaptic plasticity, and are therefore potential targets for developmental neurotoxicants. We administered nicotine to neonatal rats during discrete periods spanning the onset and peak of axonogenesis/synaptogenesis, focusing on three brain regions with disparate distributions of cell bodies and neural projections: brainstem, forebrain and cerebellum. Nicotine treatment on postnatal days (PN) 1-4 had little or no effect on alpha7 nAChRs but treatment during the second (PN11-14) or third (PN21-24) weeks elicited significant decrements in receptor expression in brainstem and cerebellum, regions containing cell bodies that project to the forebrain. Exposure to chlorpyrifos, a neurotoxicant pesticide that acts partially through cholinergic mechanisms, also elicited deficits in alpha7 nAChRs during the second postnatal week but not the first week. For both nicotine and chlorpyrifos, the effects on alpha7 nAChRs were distinct from those on the alpha4beta2 subtype. Continuous prenatal nicotine exposure, which elicits subsequent, postnatal deficits in axonogenesis and synaptogenesis, also produced delayed-onset changes in alpha7 nAChRs, characterized by reductions in the forebrain and upregulation in the brainstem and cerebellum, a pattern consistent with impaired axonogenesis/synaptogenesis and reactive sprouting. Males were more sensitive to the persistent effects of prenatal nicotine exposure on alpha7 nAChRs, a pattern that mimics neurobehavioral deficits resulting from this treatment. The present findings reinforce the mechanistic involvement of alpha7 nAChRs in the actions of developmental neurotoxicants, and its biomarker potential for neuroteratogens that target neuritic outgrowth.     

Preclinical characterization of A-582941: a novel alpha7 neuronal nicotinic receptor agonist with broad spectrum cognition-enhancing properties

Among the diverse sets of nicotinic acetylcholine receptors (nAChRs), the α7 subtype is highly expressed in the hippocampus and cortex and is thought to play important roles in a variety of cognitive processes. In this review, we describe the properties of a novel biaryl diamine α7 nAChR agonist, A-582941. A-582941 was found to exhibit high-affinity binding and partial agonism at α7 nAChRs, with acceptable pharmacokinetic properties and excellent distribution to the central nervous system (CNS). In vitro and in vivo studies indicated that A-582941 activates signaling pathways known to be involved in cognitive function such as ERK1/2 and CREB phosphorylation. A-582941 enhanced cognitive performance in behavioral models that capture domains of working memory, short-term recognition memory, memory consolidation, and sensory gating deficit. A-582941 exhibited a benign secondary pharmacodynamic and tolerability profile as assessed in a battery of assays of cardiovascular, gastrointestinal, and CNS function. The studies summarized in this review collectively provide preclinical validation that α7 nAChR agonism offers a mechanism with potential to improve cognitive deficits associated with various neurodegenerative and psychiatric disorders.