Nicotinic acetylcholine receptors in dorsal root ganglion neurons include the α6ß4* subtype.

The α6-containing nicotinic acetylcholine receptors (nAChRs) have recently been implicated in diseases of the central nervous system (CNS), including Parkinson's disease and substance abuse. In contrast, little is known about the role of α6* nAChRs in the peripheral nervous system (where the asterisk denotes the possible presence of additional subunits). Dorsal root ganglia (DRG) neurons are known to express nAChRs with a pharmacology consistent with an α7, α3ß4*, and α4ß2* composition. Here we present evidence that DRG neurons also express α6* nAChRs. We used RT-PCR to show the presence of α6 subunit transcripts and patch-clamp electrophysiology together with subtype-selective α-conotoxins to pharmacologically characterize the nAChRs in rat DRG neurons. α-Conotoxin BuIA (500 nM) blocked acetylcholine-gated currents (I(ACh)) by 90.3 ± 3.0%; the recovery from blockade was very slow, indicating a predominance of α(x)ß4* nAChRs. Perfusion with either 300 nM BuIA[T5A;P6O] or 200 nM MII[E11A], α-conotoxins that target the α6ß4* subtype, blocked I(ACh) by 49.3 ± 5 and 46.7 ± 8%, respectively. In these neurons, I(ACh) was relatively insensitive to 200 nM ArIB[V11L;V16D] (9.4±2.0% blockade) or 500 nM PnIA (23.0±4% blockade), α-conotoxins that target α7 and α3ß2*/α6ß2* nAChRs, respectively. We conclude that α6ß4* nAChRs are among the subtypes expressed by DRG, and to our knowledge, this is the first demonstration of α6ß4* in neurons outside the CNS.

A novel fluorescent alpha-conotoxin for the study of alpha7 nicotinic acetylcholine receptors.

Homomeric alpha7 nicotinic acetylcholine receptors are a well-established, pharmacologically distinct subtype. The more recently identified alpha9 subunit can also form functional homopentamers as well as alpha9alpha10 heteropentamers. Current fluorescent probes for alpha7 nicotinic ACh receptors are derived from alpha-bungarotoxin (alpha-BgTx). However, alpha-BgTx also binds to alpha9* and alpha1* receptors which are coexpressed with alpha7 in multiple tissues. We used an analog of alpha-conotoxin ArIB to develop a highly selective fluorescent probe for alpha7 receptors. This fluorescent alpha-conotoxin, Cy3-ArIB[V11L;V16A], blocked ACh-evoked alpha7 currents in Xenopus laevis oocytes with an IC(50) value of 2.0 nM. Observed rates of blockade were minute-scale with recovery from blockade even slower. Unlike FITC-conjugated alpha-BgTx, Cy3-ArIB[V11L;V16A] did not block alpha9alpha10 or alpha1beta1deltaepsilon receptors. In competition binding assays, Cy3-ArIB[V11L;V16A] potently displaced [(125)I]-alpha-BgTx binding to mouse hippocampal membranes with a K(i) value of 21 nM. Application of Cy3-ArIB[V11L;V16A] resulted in specific punctate labeling of KXalpha7R1 cells but not KXalpha3beta2R4, KXalpha3beta4R2, or KXalpha4beta2R2 cells. This labeling could be abolished by pre-treatment with alpha-cobratoxin. Thus, Cy3-ArIB[V11L;V16A] is a novel and selective fluorescent probe for alpha7 receptors.

The neuronal nicotinic acetylcholine receptors alpha 4* and alpha 6* differentially modulate dopamine release in mouse striatal slices.

Striatal dopamine (DA) plays a major role in the regulation of motor coordination and in the processing of salient information. We used voltammetry to monitor DA-release evoked by electrical stimulation in striatal slices, where interneurons continuously release acetylcholine. Use of the alpha6-selective antagonist alpha-conotoxin MII[E11A] and alpha4 knockout mice enabled identification of two populations of DA-ergic fibers. The first population had a low action potential threshold, and action potential-evoked DA-release from these fibers was modulated by alpha6. The second population had a higher action potential threshold, and only alpha4(non-alpha6) modulated action potential-evoked DA-release. Striatal DA-ergic neurons fire in both tonic and phasic patterns. When stimuli were applied in a train to mimic phasic firing, more DA-release was observed in alpha4 knockout versus wild-type mice. Furthermore, block of alpha4(non-alpha6), but not of alpha6, increased DA release evoked by a train. These results indicate that there are different classes of striatal DA-ergic fibers that express different subtypes of nicotinic receptors.

Mouse strain-specific nicotinic acetylcholine receptor expression by inhibitory interneurons and astrocytes in the dorsal hippocampus.

The response by individuals to nicotine is likely to reflect the interaction of this compound with target nAChRs. However, resolving how different genetic backgrounds contribute to unique mouse strain-specific responses to this compound remains an important and unresolved issue. To examine this question in detail, expression of the nicotine acetylcholine receptor (nAChR) subunits alpha3, alpha4, alpha5, alpha7, beta2, and beta4 was measured in the dorsal hippocampus using immunohistochemistry in mouse strains or lines BALB/c, C3H/J, C57BL/6, CBA/J, DBA/2, Long Sleep (LS), Short Sleep (SS), and CF1. The nAChRs in all mice colocalized with glutamic acid decarboxylase (GAD)-positive interneurons that were subclassified into at least four groups based on nAChR subunit heterogeneity. A notable difference between mouse strains was the expression of nAChRs by astrocyte subpopulations in CA1 subregions whose numbers vary inversely with nAChR-immunostained neurons. This novel relationship also correlated with published parameters of strain sensitivity to nicotine. Attempts to identify the origin of this significant difference in nAChR expression among strains included comparison of the entire nAChRalpha4 gene sequence. Although multiple polymorphisms were identified, including two that changed nAChRalpha4 amino acid coding, none of these clearly correlate with strain-related differences in cell type-specific nAChR expression. These findings suggest that mouse strain-specific behavioral and physiological responses to nicotine are likely to be a reflection of a complex interplay between genetic factors that shape differences in expression and cellular architecture of this modulatory neurotransmitter system in the mammalian nervous system.

Nicotine-induced neuroprotection against N-methyl-D-aspartic acid or beta-amyloid peptide occur through independent mechanisms distinguished by pro-inflammatory cytokines.

Nicotine, the causative agent of addiction to tobacco, can also be a neuroprotectant. Nicotine-induced neuroprotection against different toxins is imparted through pharmacologically distinct neuronal nicotinic acetylcholine receptors (nAChR) where protection against chronic N-methyl-d-aspartic acid (NMDA) exposure is through nAChRalpha7 but protection against the toxic peptide of amyloid precursor protein, Abeta25-35, is through nAChRalpha4beta2. The inflammatory cytokine tumor necrosis factor alpha (TNFalpha) is also neuroprotective, however, in the presence of nicotine, neuroprotection against NMDA is abolished. The specificity of nicotine-TNFalpha antagonism was further refined using a mouse transgenic dominant negative of nAChRalpha7 in which nicotine failed to induce neuroprotection against NMDA and antagonism of TNFalpha was absent. However, nicotine-mediated neuroprotection against Abeta25-35 was unaffected and, therefore, did not require the expression of functional nAChRalpha7s. The mechanism of TNFalpha-mediated neuroprotection and antagonism by nicotine was independent of caspase 8 activation or nuclear factor kappa B translocation in neurons but C6-ceramide addition to neuronal cultures subsequently exposed to NMDA mimicked the neuroprotective effect of TNFalpha and, like TNFalpha, it was antagonized by cotreatment with nicotine. Therefore, the neuroprotective effects of nicotine against differing toxic assaults requires distinct nAChR subtypes and proceeds through intracellular pathways that overlap with similarly different mechanisms initiated by pro-inflammatory cytokines. These results provide insight into how nicotine imparts neuroprotection and modulates inflammatory responses.

Nicotine preconditioning antagonizes activity-dependent caspase proteolysis of a glutamate receptor.

Neuronal excitation is required for normal brain function including processes of learning and memory, yet if this process becomes dysregulated there is reduced neurotransmission and possibly death through excitotoxicity. Nicotine, through interaction with neuronal nicotinic acetylcholine receptors, possesses the ability to modulate neurotransmitter systems through numerous mechanisms that define this critical balance. We examined the modulatory role of nicotine in primary mixed cortical neuronal-glial cultures on activity-dependent caspase cleavage of a glutamate receptor, GluR1. We find that GluR1, but not GluR2 or GluR3, is a substrate for agonist (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid)-initiated rapid proteolytic cleavage at aspartic acid 865 through the activation of caspase 8-like activity that is independent of membrane fusion and is not coincident with apoptosis. Dose-dependent nicotine preconditioning for 24 h antagonizes agonist-initiated caspase cleavage of GluR1 through a mechanism that is coincident with desensitization of both nAChRalpha4beta2 and nAChRalpha7 receptors and the delayed activation of a caspase 8-like activity. The modulation of GluR1 agonist-initiated caspase-mediated cleavage by nicotine preconditioning offers a novel insight into how this agent can impart its numerous effects on the nervous system.

Glutamate receptor subunit 3 is modified by site-specific limited proteolysis including cleavage by gamma-secretase.

Ionotropic glutamate receptor (GluR) expression and function is regulated through multiple pre- and post-translational mechanisms. We find that limited proteolytic cleavage of GluR3 at two distinct sites generates stable GluR3 short forms that are glycosylated and found in association with other full-length GluRs in the mouse brain and cultured primary neurons. A combination of mutagenesis and transfection into HEK293 cells revealed cleavage by a gamma-secretase-like activity within the membrane-localized re-entry loop at or near the leucine-glycine pair (amino acids 585-586, GluR3sbeta) and a second site within a proline-rich PEST-like sequence in the first cytoplasmic loop (Asp570-Pro571, GluR3salpha). Generation of the prominent GluR3salpha form was effectively abolished in the mutant, GluR3D570A, but inhibitors of lysosomes, the proteasome, caspases, or calpains had no effect. The possible impact of cleavage on receptor function was suggested when the co-expression of the GluR3P571Stop mutant (creating GluR3salpha) co-assembled with other GluR subunits and decreased receptor function in Xenopus oocytes. In transiently transfected HEK293 cells, co-expression of GluR3salpha alters the relative association between GluR1 and GluR3 during assembly, and the presence of the novel C-terminal proline-rich domain of GluR3salpha imparts lateral membrane mobility to GluR complexes. These results suggest that limited proteolysis is another post-translational mechanism through which functional diversity and specialization between closely related GluR subunits is accomplished.