KYNA is formed enzymatically by the irreversible transamination of L-kynurenine, a major peripheral tryptophan metabolite with ready access to the brain. Immunohistochemical and lesion studies demonstrated that cerebral KYNA synthesis takes place almost exclusively in astrocytes (129, 187, 199).
A link between alpha4 nAChRs and Cox2 was suggested by the observation that interneurons in the hippocampus coexpress both proteins (165). A mechanistic connection was inferred when long-term treatment of aged animals with NS398 promoted retention of alpha4 nAChR expression in the brain, an effect that was antagonized by the coadministration of nicotine.
Other endogenous ligands that impact on the activity of nAChRs noncompetitively and voltage independently include the amyloid beta peptide 1-42 (Abeta1-42; Refs. 123, 376) and the canabinoid anandamide (356, 442).
It is noteworthy that the alpha7 nAChR activity increases intracellular accumulation of Abeta in neurons (336), and Abeta peptides, in addition to modulating nAChR activity, downregulate the expression of nAChRs (197).
The Abeta1-42 peptide is one of the breakdown products of the proteolytic cleavage of the amyloid precursor protein by beta- and gamma-secretases. In biopsy samples of human brain tissue obtained from AD patients and in ectopic systems overexpressing either alpha7 nAChRs or APP, Abeta1-42 coimmunoprecipitates with alpha7 nAChRs (490). The Abeta1-42 peptide also displaces binding of [3H]MLA from alpha7 nAChRs in cerebral cortical and hippocampal synaptosomes (490).
More functional studies reported that while at picomolar concentrations Abeta1-42 activates alpha7 nAChRs ectopically expressed in Xenopus oocytes (123, 126), at nanomolar concentrations it inhibits alpha7 nAChRs present in different preparations (278, 376). The alpha7 nAChR inhibition by Abeta1-42 is noncompetitive with respect to the agonist, is voltage independent, and is therefore likely to be mediated by the interaction of the peptide with a site different from that for ACh on the nAChRs.
Other studies have reported that alpha4beta2 nAChRs are more sensitive than alpha7 nAChRs to inhibition by nanomolar concentrations of Abeta1-42 (506).
For example, during a low degree of activation of alpha7 and alpha3beta4 nAChRs, Ca2+ can enter the cells through nAChRs or NMDA receptors and favor activation (i.e., phosphorylation) of the transcription factor CREB, which in turn modifies gene expression (82).
There is evidence that anandamide is produced by postsynaptic neurons in response to elevated intracellular Ca2+ levels. For instance, concomitant activation of alpha7 nAChRs and NMDA receptors triggers the production of anandamine in postsynaptic neurons (448). Anandamine, then, functions as a retrograde messenger and regulates synaptic transmission by interacting with specific receptors in the presynaptic neurons/terminals (498).
It was then recognized that Ca2+ flux directly through nAChR channels or indirectly via voltage-gated Ca2+ channels is relevant for nicotinic modulation of transmitter release, synaptic plasticity, as well as neuronal viability, differentiation, and migration.
Third, nAChR-mediated GABA release can cause neuronal hyperpolarization, which in turn affects neuronal function via several mechanisms, including removal of inactivation of inward currents (89).
Other levels of regulation of dopaminergic transmission arise from alpha7 nAChRs located on cortical glutamatergic terminals; activation of these receptors increases glutamate release onto dopaminergic neurons in the VTA and, consequently, increases the their firing (344).
Of note is that in both of these catastrophic disorders, reduced nAChR activity/expression is accompanied by increased levels of kynurenic acid (KYNA), a tryptophan metabolite that in the brain is primarily produced and released by astrocytes (244, 419).
The neuroactive properties of KYNA have long been attributed to the inhibition of NMDA receptors (329). Electrophysiological studies, however, have demonstrated that physiologically relevant concentrations of KYNA block alpha7 nAChR activity noncompetitively and voltage independently (210).
This constituted the first evidence that in the hippocampus endogenous levels of KYNA are sufficient to directly modulate the activity of alpha7 nAChRs, but not that of NMDA receptors (31).
Acting as an endogenous regulator of the alpha7 nAChR activity, astrocyte-derived KYNA can modulate synaptic transmission, synaptic plasticity, neuronal viability, and neuronal connectivity in different areas of the brain (Fig. 8).
Not unlike snake toxins, conotoxins can disrupt multiple components of neurotransmission including voltage-gated Na+ and K+ channels in addition to nAChRs (132, 351).
As illustrated in Figure 8, KYNA-induced reduction of extracellular dopamine levels can be explained by the inhibition of tonically active alpha7 nAChRs in the dopaminergic neurons within the VTA and/or in cortical glutamatergic terminals that synapse onto striatal neurons. VTA dopaminergic neurons represent the major dopaminergic input to the nucleus accumbens.
Chronic alpha7 nAChR inhibition in the hippocampus by elevated levels of KYNA can contribute to auditory gating deficits, which appear to be associated with the development of schizophrenia (156). It is also feasible that KYNAinduced inhibition of alpha7 nAChRs contributes to the cognitive impairment observed in patients with AD and schizophrenia (273).
Mice with a null mutation in the gene that encodes KAT II became a unique tool to resolve this issue (31, 410, 516). Low levels of KYNA in these mutant mice lead to alpha7 nAChR disinhibition in hippocampal CA1 SR interneurons, thereby increasing the activity of GABAergic interneurons impinging onto CA1 pyramidal neurons (31)
Activation of alpha7 nAChRs is known to contribute to the regulation of extracellular dopamine levels in the rat striatum (81). Application via microdialysis of KYNA or alpha-BGT to the rat striatum significantly reduces the extracellular levels of dopamine, and the magnitude of the effect of either antagonist alone is comparable to that of both antagonists together (285).
Surprisingly, however, activation of nAChRs by galantamine or physostigmine was insensitive to blockade by competitive nAChR antagonists, was detected even when the receptors were desensitized by high agonist concentrations, and was inhibited by the monoclonal antibody FK1 (350, 370, 372, 413, 428, 429).
Thus inhibitors of proteasome function block endoplasmic reticulum-associated degradation of unassembled AChR subunits, which in turn increases the availability of subunits for assembly into mature receptors that are trafficked to the cell surface.
The metabotropic receptors are second messenger, G protein-coupled seven-transmembrane proteins. They are classically defined as being activated by muscarine, a toxin from the mushroom Amanita muscaria, and inhibited by atropine, a toxin from Atropa belladonna, a member of the nightshade family. Both toxins cross the blood-brain barrier poorly and were discovered primarily from their influences on postganglionic parasympathetic nervous system functions. Activation of muscarinic AChRs is relatively slow (milliseconds to seconds) and, depending on the subtypes present (M1- M5), they directly alter cellular homeostasis of phospholipase C, inositol trisphosphate, cAMP, and free calcium.
Acetylcholine receptors (AChRs), like many other ligand-activated neurotransmitter receptors, consist of two major subtypes: the metabotropic muscarinic receptors and the ionotropic nicotinic receptors. Both share the property of being activated by the endogenous neurotransmitter acetylcholine (ACh), and they are expressed by both neuronal and nonneuronal cells throughout the body (8, 113, 142, 184).
From the time of its discovery in 1914 by Henry H. Dale (109) and Otto Loewi (283) (the two shared the Nobel Prize in Physiology and Medicine in 1936) as an agent that decreases heart rate, ACh was recognized as an endogenous signaling compound, synthesized from choline and acetyl-CoA, through the action of choline acetyltransferase, that alters cell function.
Anandamide, a compound originally isolated from porcine brain extracts, is known to interact with canabinoid receptors 1 and 2 in the brain (120, 159). However, anandamide interacts with numerous other receptors, including voltage-gated Ca2+ channels (357), voltage-gated K+ channels (293), 5-HT3 receptors (358), kainate receptors (3), and nAChRs (356). At nanomolar concentrations, anandamine blocks noncompetitively and voltage independently the activation of alpha7 nAChRs ectopically expressed in Xenopus oocytes (356). It also inhibits the activity of alpha4beta2 nAChRs expressed in SH-EP1 cells (443).
Furthermore, pharmacological dissection of nicotine’s influence on cell cycle progression, apoptosis, and differentiation (43) indicate that alpha7 nAChRs expressed in keratynocytes are important. Other receptors are clearly involved in this process, since atropine, a muscarinic and sometimes nAChR inhibitor (531, 532), reduces cell adhesion through decreasing desmoligein expression.
Finally, bupropion (16, 294, 433) and UCI-30002 (514) are examples of synthetic compounds that act as noncompetitive inhibitors of different nAChRs, including those made up of the subunits alpha7, alpha4beta2, or alpha3beta4. Both compounds effectively decrease nicotine self-administration in rats (280, 514). Bupropion is presently approved as an adjunct therapy for smoking cessation.
For instance, studies carried out in PC12 cells demonstrated that codeine, a drug with no significant effect on ChE, can activate nicotinic single-channel currents and that this nicotinic agonist effect is sensitive to inhibition by FK1 while unaffected by classical nAChR antagonists (450).
Drugs currently approved to treat mild-to-moderate AD, including galantamine, donepezil, and rivastigmine, all inhibit AChE, the enzyme that hydrolyzes ACh (462).
Studies from the early 1980s provided evidence that the cholinesterase (ChE) inhibitor physostigmine could interact directly with nAChRs at the frog neuromuscular junction and induce nicotinic single-channel currents (428, 429).
Parkinson’s disease (PD) is characterized by selective damage to dopaminergic nigrostriatal neurons and is clinically revealed by motor deficits, including rigidity, tremor, and bradykinesia. Dopamine replacement therapy (usually with L-dopa) is the most common treatment, although this drug loses efficacy over time.
An alternative means to increase nicotinic functions in the brain is to sensitize the nAChRs to activation by the endogenous agonist(s) using the so-called nicotinic allosteric potentiating ligands (APLs), which include drugs such as physostigmine and galantamine, a drug currently approved for the treatment of AD.
In the early 1990s, galantamine, an alkaloid originally extracted from the bulbs and flowers of the wild Caucasian snowdrop Galanthus nivalis and other related Amaryllidacea species, was found to act like physostigmine on muscle and neuronal nAChRs (370, 372).
The nicotinic APL action of galantamine appears to be an important determinant of its clinical effectiveness (reviewed in Refs. 98, 291, 371). Acting primarily as a nicotinic APL, galantamine improves synaptic transmission and decreases neurodegeneration, two effects essential for its cognitive-enhancing properties (40, 108, 241, 409, 521).
In the normal brain, 70% of KYNA formation is catalyzed by KAT II, one of the three cerebral KATs (199, 200). Systemic treatment of rats and mice with kynurenine leads to an elevation of brain levels of several neuroactive intermediates, including KYNA, the free radical generator 3-hydroxykynurenine, and the excitotoxic quinolinic acid (419).
The other subtype of AChR is the fast ionotropic cationic nicotinic receptor channel (nAChR). These receptors are sensitive to activation by nicotine and have ion channels whose activity is induced in the micro- to submicrosecond range.
Also, the continuous exposure of cells to nicotine increases nAChR surface expression by reducing degradation of the intracellular pool of receptors (367, 394).
In the smoker’s brain, upregulation can increase high-affinity nicotine binding by nearly fourfold relative to age- and gender-matched controls that have not been exposed to nicotine (373, 421). The mechanism by which nicotine increases the total number of high-affinity nAChRs, though poorly defined, is highly conserved among species.
Mechanistically, nicotine, acting through nAChRs, decreases keratinocyte migration (188, 189) and modifies the activity of PI3K/Akt, ERK, MEK, and JAK signaling pathways.
First, age-related nAChR subunit expression decline was observed in both strains, and this was dominated by diminished alpha4 nAChR expression. Second, long-term (12 mo) oral nicotine failed to reduce the age-related decline in the number of neurons expressing alpha4 nAChR subunits, although the neurons that remained exhibited larger processes with more varicosities than age-matched controls (165, 396). Acute nicotine treatment (alpha6 wk of oral nicotine) of aged mice had no measurable influence on nAChR expression, neuronal viability, or dendritic complexity (e.g., Ref. 396)
Nicotine is perhaps the most addictive drug that is widely used; 95% or more of its users with a strong desire to stop using it relapse within 1 yr (47, 203). Chronic nicotine use and the phenotypes of addiction are closely associated in humans and other animals with concurrent physiological changes in nAChR function and expression
One insect has escaped the ill effects of nicotine, Manduca sextans or the tobacco horn worm. While nicotine binds the nAChR to activate and subsequently desensitize it, this insect eats the tobacco plant without ill effects. Manduca exhibits two adaptations to tolerate the effects of nicotine. The first is altered nAChR amino acid sequences that limit the affinity of nicotine for the nAChR (136). The second is the development of the functional equivalent to a blood-brain barrier.
Metabolic degradation of nicotine and rapid clearance is a mechanism that protects neurons from greater nicotine concentrations, since nicotine readily crosses the mammalian blood-brain barrier and accumulates in the lipophilic brain environment to concentrations that may exceed plasma concentrations by one order of magnitude. Nevertheless, neurotoxicity to nicotine is not uncommon, as attested to by the recent increase in hospital emergency room visits by smokers who concurrently use the transdermal nicotine patch (503).
The receptor that exhibits the greatest upregulation when exposed to nicotine is the alpha4beta2 nAChR. Receptors assembled from this subunit combination form the highaffinity nicotine binding site (151, 215) and account for the vast majority of upregulated sites in the brain of smokers (55).
transfection of cells with the beta4 and alpha2 nAChR subunits or expression of these in Xenopus oocytes leads to high-affinity nicotine-binding receptors that upregulate in response to prolonged exposure to nicotine (113, 184, 215).
On the other hand, when alpha4beta2 nAChRs are activated, both SR and SLM interneurons are inhibited, resulting in disinhibition of dendritic areas innervated by both neuron types.
In particular, repeated self-administration produces the upregulation of high-affinity (alpha4beta2) nAChR expression, reduces receptor function due to desensitization and, in most cases, imparts developmental tolerance. Additional changes imposed by nicotine abuse range from reinforcement to physical discomfort associated with withdrawal including craving, anxiety, and a multitude of other less than desirable sensations of autonomic dysfunction when use is stopped.
For instance, prolonged exposure of HEK293 cells to saturating nicotine concentrations increased by 6- and 1.5-fold, respectively, the expression of alpha3beta2 and alpha3beta4 nAChRs.Similarly, while alpha4beta2 nAChRs upregulate strongly, alpha4beta4 nAChRs upregulate poorly in response to continuous exposure to nicotine.
Prolonged treatment of rodents and monkeys with nicotine downregulates the expression of alpha6beta3-containing nAChRs in the brain (257, 311, 332). However, in heterologous culture systems, nicotine appears to upregulate the expression of receptors assembled from alpha4/alpha6/beta2/beta3 input cDNA (363), and this may depend on numerous factors including ligand concentrations (483).
An ever-growing body of evidence indicates that in CNS and parasympathetic nervous system neurons and in heterologous systems expressing specific nAChR subtypes, nicotine stimulates several Ca2+-dependent kinases, including PI3K, protein kinase C (PKC), protein kinase A (PKA), calmodulin-dependent protein kinase II (CAM kinase II), and extracellular signal-regulated kinases (ERKs; Refs. 108, 112, 146, 318, 469).
Downstream from the nicotine-stimulated kinases, a number of transcription factors have been shown to be activated. Among these factors are the cAMP response element binding protein (CREB) and the activating transcription factor 2 (ATF-2) in PC12 cells (211, 337, 460), the Ets-like transcription factor Elk-1 in the rat hippocampus (349), and the signal transducer and activator of transcription (STAT3) in macrophages and skin cells (114, 354).
Recent studies have supported a role for ERK and CREB activity in neural plasticity associated with nicotine addiction (71, 381, 484). It has also been proposed that the ERK and JAK-2/STAT-3 signaling pathways contribute to the toxic effects of nicotine in skin cells (42), and other pathways contribute to the effects of nicotine and other nicotinic ligands on inflammatory responses as described below.
Modulation by nicotine of inflammatory responses in the intestines is much better reported. Early studies found that patients with ulcerative colitis who stopped smoking tobacco developed the disease or exhibited more severe disease progression, which was ameliorated by either returning to smoking (58, 401, 466), or, in some cases, administering nicotine through transdermal patches (313).In contrast, patients with Crohn’s disease experience much more severe disease when smoking (401).
Notably, mice with a null mutation in the gene that encodes the alpha5 nAChR subunit exhibit enhanced sensitivity to induction of inflammatory bowel disease relative to controls (353). Despite increased sensitivity to disease initiation, administration of transdermal nicotine remains effective in attenuating the disease process. Therefore, again nicotine appears to impact on inflammatory processes with considerable specificity and tissue dependency.
Notably, nicotine pretreatment of rat adipocytes (279) reduces the release of TNF-alpha as well as free fatty acids and the adipokine adiponectin (whose function is not known, although its levels change in metabolic syndrome).
Mice are particularly well-defined for their strain-specific complex genetic traits related to the effects of nicotine (105, 302) and morphological variations in the brain (e.g., Refs. 166, 167, 169).
In human trials, nicotine showed little efficacy in ameliorating AD symptoms (437). However, treatment was initiated after diagnosis of symptoms, and there is both epidemiological data and direct evidence from animal models that this is too late (106, 346, 396).
More direct evidence of the protective effects of nicotine in this disease process comes from studies in primates, where oral nicotine reduces the nigrostriatal neuronal loss observed in chemically induced PD (384, 385).
However, in rodent and nonprimate animal models, nicotine has been shown to enhance striatal dopamine release and to prevent toxin-induced degeneration of dopaminergic neurons (384, 385).
A principle component of genetic analysis of the contribution of alpha7 and alpha4beta2 nAChRs to the effects of nicotine was reported 15 years ago. The number of alpha-BGT binding sites (presumably alpha7 nAChRs) was shown to be highly correlated with sensitivity to nicotinic-induced seizures (105, 301, 303).
The concept that beta2-containing nAChRs are involved in the reinforcing effects of nicotine was supported by the findings that these mice lacked the high-affinity nicotine binding site, exhibited poor nicotine self-administration, and failed to develop behaviors related to reinforcement (378). The demonstration that these mice developed symptoms of the nicotine withdrawal syndrome similar to those observed in wild-type mice led to the conclusion that beta2-containing nAChRs do not contribute to the physical dependence on nicotine (57).
In summary, while nicotine-induced upregulation requires at least the beta2 nAChR subunit, development of tolerance to nicotine requires neither the beta2 nor the alpha7 nAChR subunit; instead, it appears to be modulated by a beta4-containing nAChR and to require an alpha4-containing nAChR.
The second was the discovery of alpha-bungarotoxin (alpha-BGT), a component of krait snake venom that binds muscle-type nAChRs with near covalent affinity to inhibit their function and promote debilitating paralysis at the neuromuscular junction (6, 50, 149, 264).
The most valuable of these toxins to researchers proved to be alpha-BGT from the snake Bungarus multicinctus. Because this toxin binds to the muscle nAChR with great specificity and a near-covalent affinity, it was an invaluable tool in the purification of the first nAChRs (discussed above).
However, this appealing scenario is complicated by recent findings that beta-amyloid peptides directly modify alpha7 nAChR function (242, 278).
In contrast, the NMDA receptor antagonist 7-chloro-KYNA has no significant ef- fect on the extracellular levels of dopamine in the rat striatum (391).
NSAIDs [e.g., drugs such as ibuprofen and NS398 (celecoxib or Celebrex)] antagonize to varying degrees two related cyclooxygenase (Cox) enzymes, Cox1 and Cox2 (also termed, prostaglandin-endoperoxide synthase 2), that are rate-limiting in converting arachidonic acid to prostaglandin H2, a precursor to many additional prostaglandins (for review, see Ref. 436).
For example, Loring and colleagues (458) compared the relative expression of alpah4beta2 versus alpha7 nAChRs transfected into five different cell lines (GH4C1, SH-EP1, CV1, SN-56, and CHOCAR). Each cell line expressed appropriate mRNAs (indicating successful transfection); however, the relative levels of expression of each receptor subtype varied significantly among the various cell lines.
All cell lines appeared to produce alpha4beta2 nAChRs, although at considerably variable levels relative to each other. Therefore, cell and receptor identity combine to collectively determine the efficiency of nAChR expression on the cell surface.
In fact, as described above, AChE inhibitors do not affect alpha7 nAChR-mediated synaptic transmission evoked by low-frequency stimulation of cholinergic fibers in chick ciliary ganglia (522).
The first was the finding that the electric organ of a fish that produces an electric pulse to stun its prey, such as Torpedo, expresses nAChRs at densities that approach a crystalline array (245, 438). This provided an unprecedented source of starting material for receptor purification since nAChRs comprise 40% of the protein from this organ.
Another significant assembly checkpoint to ensure only correctly assembled nAChRs are transported to the cell surface is the endoplasmic reticulum. Most nAChRs are not constitutively sent to lysosomes. Instead, they are retained in intracellular pools that range from 65 to 85% of the total receptor number in a cell (147, 359, 397, 496).
In fact, 80% of the synthesized subunits appear to improperly assemble or never leave the endoplasmic reticulum where they are then degraded (485). The process of retaining subunits and possibly fully assembled receptors and then degrading them may be an important component of regulating receptor number.
Neuronal nAChRs are not expressed exclusively in neurons. Instead, they are expressed by multiple cell types of diverse origins and functions including glia (165, 167, 425), keratinocytes (44, 86, 95, 426), endothelial cells (290, 495), and multiple cell types of the digestive system, lungs, and immune system (e.g., Refs. 95, 309, 492, 495).
For instance, substantial strain-specific variability in nAChR expression has been observed in the striatum (34), retina (227), cerebellum (471), and dorsal hippocampus (164, 165, 167, 169) of mice.
These results suggest that mouse strains of different genetic backgrounds undergo dissimilar age-related changes in the expression of nAChR subunits.
Activation of alpha7 nAChRs in somatodendritic and preterminal/ terminal areas of interneurons in various strata of the CA1 region and in the dentate gyrus facilitates spontaneous quantal release of GABA (14, 25). Glutamate release from mossy fibers onto CA3 pyramidal neurons is also modulated by alpha7 nAChRs present in the mossy fiber terminals (190).
Strain-dependent variations in nAChR density in regions of the rat brain have also been reported.
For instance, the alpha3 nAChR transcript generally dominates in the prenatal brain or in injured neurons, whereas its expression tends to be downregulated in the adult or healthy neuron, and alpha4 transcription is increased.
This is also true of alpha3, alpha4, beta2, and beta4 nAChR subunits, which can freely interact to form receptors but appear to exhibit considerable preference in the brain as well as ganglia to form mostly receptors of alpha3beta4 and alpha4beta2 subunit composition (150, 471).
However, in hippocampal neurons expressing the alpha7, alpha4, and beta2 nAChR subunits, the vast majority of functional nAChRs are pharmacologically identified as being distinctly alpha4beta2 and alpha7 nAChRs (12).
In the muscle, for example, despite the coexpression of as many as five distinct subunits, only receptors of well-defined stoichiometries are expressed: (alpha1)2beta1deltagamma in noninnervated muscle and (alpha1)2beta1deltaepsilon at mature neuromuscular synapses.
In the immature muscle alpha1, beta1, delta and gamma nAChR subunit transcripts are made and receptors from these subunits are synthesized and transported to the cell surface.
For example, while the alpha7 nAChR is primarily a homomeric receptor in neurons (127), combinations of alpha7 nAChR subunits with alpha5, beta2, or beta3 nAChR subunits have been reported to form functional heteromeric receptors in some systems (240, 360, 515).
Interestingly, astrocytic KYNA production is regulated by neuronal activity (187) and cellular energy metabolism (213). This dependence of extracellular KYNA concentrations on the functional interplay between neurons and astrocytes is in line with the postulated neuromodulatory role of KYNA (418) and adds to the complexity of the neurochemical networks in the brain.
A similar level of fidelity in nAChR assembly is achieved by cells of the brain. For example, the alpha4, alpha7, and beta2 nAChR interact with each other to form functional receptors in heterologous systems such as oocytes.
Because of the absence of reuptake or degradation mechanisms, subsequent KYNA removal is accomplished exclusively by probenecid-sensitive brain efflux (330, 473).
In addition to nicotine, an nAChR agonist of considerable commercial importance is anatoxin-a (Fig. 3). This toxin is a product of the blue-green algae, Anabaena, and can reach high concentrations during algal blooms common to ponds that serve as the summer water source of livestock. While this toxin exerts much of its effect through targeting muscle nAChRs, it was recognized over two decades ago to also interact with nAChRs expressed by ganglionic receptors (38). Its ability to activate in central nervous system (CNS) neurons nicotinic currents sensitive to alpha-BGT was among the first indicators that functional alpha7 nAChRs could be distinguished from other nAChRs in neurons of the mammalian brain (38).
More recently, epibatidine, an alkaloid from the skin of the Ecuadorain tree frog Epipedobates tricolor, revealed another example of how a nicotinic agonist can produce toxic effects (111, 130). In addition to being a potent analgesic, when injected into mice at a relatively low dose (0.4 microg/mouse), this compound produced straub tail reaction. The major target of epibatidine is the alpha4beta2 high-affinity nAChR, although other nAChRs are targeted with various affinities (e.g., Ref. 507).
Furthermore, TNF-alpha strongly promotes ligand-mediated upregulation of alpha4beta2 nAChRs through a mechanism that requires p38 mitogen-activated protein kinase (MAPK) signaling (163).
In cell lines, this interaction of trans-activating components is also under the regulation of the Ras-dependent MAPK and pathways related to phosphoinositide-3-kinase (PI3K) and MEK activation whose response to trophic factors such as nerve growth factor (NGF) contributes to regulating transcript initiation.
It is noteworthy that, via such mechanisms, alpha7 nAChR activation could trigger rebound burst firing in SLM interneurons even in the absence of excitation (256). Burst firing in SLM interneurons suppresses spikes in pyramidal neurons evoked by stimulation of Schaffer collaterals (134), and, thereby allows selective activation of the pyramidal cells via the perforant pathway.
In receptors harboring the gamma subunit, agonist-induced receptor activation results in a long-lasting open channel time. The large agonist-induced current in turn leads to local intermittent depolarization and adjustments to protein-protein interactions that favor receptor clustering. As the depolarization increases, transcription of the epsilon subunit is increased dramatically (183).
If there is intense stimulation of all three nAChRs, the resulting depolarization can trigger activation of voltage- gated Ca2+ channels (VGCC), which in turn would activate the calcineurin pathway and prevent CREB activation.
Activation of somatodendritic alpha7 nAChRs increases the action potential-dependent release of dopamine, while activation of presynaptic alpha6 and/or alpha4 nAChRs increases action potential-independent dopamine release.
Also central to restricting (or at least limiting) the expression of these transcripts to predominantly neuronal-like cell lines (Neuro2A and NGF-treated PC12) are interactions among other factors including SCIP/Tst- 1/Oct-6 and transactivation by Sox10 (66, 268, 317, 513).
AD is the most common form of dementia in the elderly population. The histopathology of this disease is well known to have at least four components: 1) loss of cholinergic neurotransmission, 2) deposition of extracellular Abeta peptides into plaques, 3) hyperphosphorylation of the tau protein that leads to excessive formation of neurofibrillar tangles, and 4) increased local inflammation.
In particular, the association of the alpha7 nAChR gene with a sensory gating deficit that is similar to attention deficits in patients with schizophrenia (157), and the degree of alpha4beta2 nAChR loss and altered alpha7 expresson correlate well with the magnitude of progressive cognitive decline in mild-to-moderate AD patients (46).
When cRNAs encoding specific nAChR subunits are introduced into Xenopus oocytes, simple (alpha3beta4) as well as more complex (muscle alpha1beta1deltagamma) heteromeric receptors are assembled and expressed on the cell surface (341). In Xenopus oocytes, these heteromeric nAChRs are assembled and expressed with almost equivalent efficiencies as the homomeric 5HT3A receptor (341).
In the basal ganglia, for instance, dopaminergic transmission is ultimately regulated by the activity of specific nAChR subtypes in different neurons and neuronal compartments (Fig. 5). Thus evidence exists that in the VTA,alpha6- andalpha4-containing nAChRs are mainly located on dopaminergic nerve terminals, whereas alpha7 nAChRs are primarily expressed on the soma of dopaminergic neurons (Fig. 5).
Reduced nAChR function/expression in the brain has been associated with the pathophysiology of catastrophic disorders, including AD and schizophrenia (discussed in later sections, and see Refs. 277, 432).
However, loss of brain nAChRs precedes that of muscarinic receptors during normal aging, and it is often much more extensive in human brains afflicted with AD relative to age-matched controls (236, 308, 373, 374, 416, 519). In fact, alpha4 nAChR expression can decrease by >80% in the AD brain (306, 374).
It is noteworthy that nAChR expression by astrocytes in brains afflicted with AD is increased (463, 518), and astrocytes in general have been reported to be more plentiful in the hippocampus of some rat strains with age (35, 284).
It has long been recognized that nAChR activation in mammalian sympathetic neurons induces the opening of a nonselective cation channel that leads to Na+ influx, membrane depolarization, and consequently activation of voltage-gated Ca2+ channels (92, 119).
Long before the identification of the high Ca2+ permeability of alpha7 nAChR channels, different studies reported significant Ca2+ influx through nAChRs in muscle, parasympathetic neurons, pheochromocytoma cells, and human neuroblastoma cells (115, 321, 347, 407, 411, 459, 468).
There is current evidence that nAChRs present in skin cells modulate the responses triggered by inflammatory stimuli applied to the skin (354). Smoking is a welldefined risk factor in delayed wound healing and possibly the development of premature facial wrinkling (226).
In CA1 and CA3 pyramidal neurons of the developed hippocampus, alpha7 nAChRs are expressed primarily on axon terminals whereby their activation modulates the efficacy of glutamate synaptic transmission.
Several possibilities exist for the identity of the nAChR important to tolerance development. The strong positive correlation between alpha-BGT site number and sensitivity to nicotine-induced seizures among multiple mouse strains led to the suggestion that alpha7 nAChRs are critical to limit oral nicotine self-administration in mice (105, 301).
While development of tolerance does not seem to be regulated by alpha7 nAChRs, a recent study of alpha7 nAChR-null mice indicates that these receptors control the severity of the nicotine withdrawal syndrome (402).
As will be returned to below, it is also the first nAChR subtype to exhibit measurable decline in expression in the aged mammalian brain and especially in neurodegenerative disorders such as AD (236, 374).
Genetic deletion of the alpha4 or the alpha2 nAChR subunit abolishes essentially all high-affinity nicotine binding to brain tissue and upregulation in response to chronic exposure to nicotine (151, 311).
A concurrent activation of preterminal alpha4beta2 nAChRs would hyperpolarize the neuron via GABAergic inhibition and prevent activation of the VGCC.
Activation of alpha4beta2 nAChRs on GABAergic interneurons in the VTA relieves the inhibitory control they exert on dopaminergic neurons (295, 380).
Several E26 transformation-specific sequence (ETS) factor binding sites were identified that upon deletion led to substantially diminished expression of both alpha3 and beta4, and to direct transgene expression of the reporter gene, LacZ, to major sites of gene cluster expression in multiple brain regions, ganglia, and peripheral systems.
Such toxins are not limited to the muscle receptor as seen in the Taiwanese krate snake. This snake produces “neuronal bungarotoxin” (also referred to as 3.1 toxin or kappa-bungarotoxin; Ref. 286), which preferentially binds to and inactivates neuronal nAChRs that contain the alpha3 and beta4 subunits. In this case, the specificity of the toxin appears to in part be controlled by the subtype of beta nAChR subunit; beta2-containing nAChRs are less sensitive than beta4-containing nAChRs to inhibition by neuronal BGT.
Additional examples of snake toxins include alpha-cobratoxin (Fig. 3), which binds to the agonist binding site of the receptor and blocks receptor activation.
Finally, the alkaloid methyllycaconitine (MLA) emerged as a potent and specific competitive antagonist that inhibits muscle, alpha7-, alpha6-, and alpha3-containing nAChRs (30, 326, 445). The alkaloid is derived from the larkspur (genus Delphinium), which is of great economic interest since estimates of its cost to ranchers in poisoned livestock exceeds many millions of dollars annually. Similar to most nAChR poisons, MLA binds to the receptor agonistbinding site (Fig. 3) in a manner similar to that of alpha-BGT to block agonist binding and receptor activation.
The epsilon subunit protein outcompetes the gamma subunit for assembly into the receptor. The receptors assembled with the epsilon subunit are more stable to degradation, aggregate at the neuromuscular junction to greater density and exhibit a more rapid response to agonist (96, 275, 324).
Green and colleagues (191, 365, 485) report that nAChR assembly proceeds in the endoplasmic reticulum where specific subunits are added sequentially to the receptor complex according to the conformations the complex assumes. In this model, nAChR subunits are synthesized, and initial polypeptide folding favors the rapid recognition and interaction between alpha-beta-gamma subunits to produce trimers that in turn form a structure favorable to the addition of the delta subunit and finally the second alpha subunit.
Subsequent studies have revealed that the DNA binding Sp-1 transcriptional factor interacts in response to NGF with the c-Jun coactivator (317) to increase beta4 transcription.
Upon transfection of the cDNA encoding alpha7 nAChR subunits, HEK293 cells reportedly express the corresponding transcripts and even make considerable protein. Yet, the number of functional receptors expressed on the cell surface was low and could vary by three orders of magnitude.
Only two of these cell lines expressed alpha7 nAChRs: GH4C1 cells expressed substantially greater numbers of surface receptors than did SH-EP1 cells, which exhibited poor assembly efficiency.
In another model, a somewhat different route to assembly is proposed (59, 435, 493). In this scenario, dimers between alpha-gamma and alpha-delta subunits are formed before these paired subunits subsequently interact with the beta subunit to assemble the mature pentamer.
For example, in the original PC12 line (194), NGF is a potent inducer of beta4 transcription (217), but in PC12 lines that are defective in the expression of functional alpha7 nAChRs, NGF decreases beta4 nAChR subunit transcription (60, 397).
As was noted above, in different laboratories, these cells were reported to regulate nAChR mRNA expression differently in response to nerve growth factor, and to exhibit dramatically different expression of alpha7 nAChRs.
In the basal ganglia, including the ventral tegmental area (VTA) and substantia nigra, the alpha6 and possibly the beta3 nAChR subunits are included in alpha4beta2 nAChR complexes to generate highaffinity receptors. At present, this is the only brain area identified where alpha6 and beta3 are coexpressed with alpha4 and beta2 nAChR subunits. This finding is highly relevant for Parkinson’s disease (385, 386).
Receptors composed of alpha7 subunits are known to desensitize rapidly and to have a high Ca2+:Na+ permeability ratio that exceeds that of the glutamate NMDA receptor, and the 3-4:1 ratio of most other nAChRs (8, 68, 78, 387). As a result, quite distinctly from other nAChRs and even other ligand-activated ion channels, the opening of alpha7 nAChR channels can impact on several Ca2+-dependent mechanisms, including activation of second messenger pathways (328, 456).
At the neuromuscular junction, nicotinic function is enhanced by inhibition of acetylcholinesterase (AChE), the enzyme that metabolizes the endogenous neurotransmitter ACh.
Studies of recombinant chimeric subunits containing sequences of the NH2-terminal domains of the alpha7 and the alpha3 (M1-S232) nAChR subunits indicated that a 23- amino acid region (glycine-23 to asparagine-46) contained residues required for correct association of the alpha7 subunit into a homopentameric receptor.
In the brain, however, Cox2 is constitutively expressed by neurons (212, 512), participates in modulating synaptic plasticity (53, 464), and conditionally can either inhibit or promote cell death (74, 85, 237, 322, 451).