Summary We examined the contribution of endogenous cholinergic signaling to the acquisition and extinction of fear- related memory by optogenetic regulation of cholinergic input to the basal lateral amygdala (BLA). Stimulation of cholinergic terminal fields within the BLA in awake-behaving mice during training in a cued fear-conditioning paradigm slowed the extinction of learned fear as assayed by multi-day retention of extinction learning. Inhibition of cholinergic activity during training reduced the acquisition of learned fear behaviors. Circuit mechanisms underlying the behavioral effects of cholinergic signaling in the BLA were assessed by in vivo and ex vivo electrophysiological recording. Photo-stimulation of endogenous cholinergic input: (1) enhances firing of putative BLA principal neurons through activation of acetylcholine receptors (AChRs); (2) enhances glutamatergic synaptic transmission in the BLA and (3) induces LTP of cortical-amygdala circuits. These studies support an essential role of cholinergic modulation of BLA circuits in the inscription and retention of fear memories.

Nicotinic receptor activation is inextricably linked to desensitization. This duality affects our ability to develop useful therapeutics targeting nicotinic acetylcholine receptor (nAChR). Nicotine and some ␣7-selective experimental partial agonists produce a transient activation of ␣7 receptors followed by a period of prolonged residual inhibition or desensitization (RID). The object of the present study was to determine whether RID was primarily due to prolonged desensitization or due to channel block. To make this determination, we used agents that varied significantly in their production of RID and two ␣7-selective positive allosteric modulators (PAMs): 5-hydroxyindole (5HI), a type 1 PAM that does not prevent desensitization; and 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxanol-3-yl)-urea (PNU-120596), a type 2 PAM that reactivates desensitized receptors. The RID-producing compounds nicotine and 3-(2,4-dimethoxybenzylidene)anabaseine (diMeOBA) could obscure the potentiating effects of 5HI. However, through the use of nicotine, diMeOBA, and the RID-negative compound 3-(2,4-dihydroxybenzylidene)anabaseine (diOHBA) in combination with PNU-120596, we confirmed that diMeOBA produces short-lived channel block of ␣7 but that RID is because of the induction of a desensitized state that is stable in the absence of PNU-120596 and activated in the presence of PNU-120596. In contrast, diOHBA produced channel block but only readily reversible desensitization, whereas nicotine produced desensitization that could be converted into activation by PNU-120596 but no demonstrable channel block. Steady-state currents through receptors that would otherwise be desensitized could also be produced by the application of PNU-120596 in the presence of a physiologically relevant concentration of choline (60 M), which may be significant for the therapeutic development of type 2 PAMs.

Desensitization induced by chronic nicotine exposure has been hypothesized to trigger the up-regulation of the ␣4␤2 neuronal nicotinic acetylcholine receptor (nAChR) in the central nervous system. We studied the effect of acute and chronic nicotine exposure on the desensitization and up-regulation of different ␣4␤2 subunit ratios (1␣:4␤, 2␣:3␤, and 4␣:1␤) expressed in Xenopus oocytes. The presence of ␣4 subunit in the oocyte plasmatic membrane increased linearly with the amount of ␣4 mRNA injected. nAChR function and expression were assessed during acute and after chronic nicotine exposure using a two-electrode voltage clamp and whole-mount immunofluorescence assay along with confocal imaging for the detection of the ␣4 subunit. The 2␣4:3␤2 subunit ratio displayed the highest ACh sensitivity. Nicotine doseresponse curves for the 1␣4:4␤2 and 2␣4:3␤2 subunit ratios displayed a biphasic behavior at concentrations ranging from 0.1 to 300 M. A biphasic curve for 4␣4:1␤2 was obtained at nicotine concentrations higher than 300 M. The 1␣4:4␤2 subunit ratio exhibited the lowest ACh-and nicotine-induced macroscopic current, whereas 4␣4:1␤2 presented the largest currents at all agonist concentrations tested. Desensitization by acute nicotine exposure was more evident as the ratio of ␤2:␣4 subunits increased. All three ␣4␤2 subunit ratios displayed a reduced state of activation after chronic nicotine exposure. Chronic nicotine-induced up-regulation was obvious only for the 2␣4: 3␤2 subunit ratio. Our data suggest that the subunit ratio of ␣4␤2 determines the functional state of activation, desensitization, and up-regulation of this neuronal nAChR. We propose that independent structural sites regulate ␣4␤2 receptor activation and desensitization. Neuronal nicotinic acetylcholine receptors (nAChRs)1 belong to a superfamily of ligand-gated ion channels (e.g. ␥-aminobutyric acid, glutamate, 5-hydroxytryptamine, among others) and play an important role in modulating neurotransmitter release in distinct areas of the central and peripheral nervous system (1-5). Nicotine is the active ingredient of tobacco and specifically binds to nAChRs in the brain (3). One of the most remarkable effects of chronic nicotine exposure is the up-regulation of the ␣4␤2 subtype in the central nervous system (6 -10). Another important effect of chronic nicotine exposure is the long lasting functional deactivation of nAChR receptor (11-17). Chronic nicotine exposure produces a loss of nicotinic functional activity as a result of rapid and persistent desensitization (11, 18 -20). Desensitization induced by chronic exposure to nicotine has been hypothesized to trigger the up-regulation of the ␣4␤2 nAChR (3, 6, 21-23). The effect of chronic nicotine exposure on the activity of nAChR subtypes may be related to symptoms associated with nicotine addiction (3, 24, 25) such as tolerance, dependence, and withdrawal. In contrast to the aforementioned studies, a recent work suggests that the ␣4␤2 subtype expressed in the stable cell line K-177 functionally up-reg...

Nicotine addiction and other forms of drug addiction continue to be significant public health problems in the United States and the rest of the world. Accumulated evidence indicates that brain nicotinic acetylcholine receptors (nAChRs) are a heterogenous family of ion channels expressed in the various parts of the brain. A growing body of preclinical studies suggests that brain nAChRs are critical targets for the development of pharmacotherapies for nicotine and other drug addictions. In this review, we will discuss the nAChR subtypes, their function in response to endogenous brain transmitters, and how their functions are regulated in the presence of nicotine. Furthermore, we will discuss the role of nAChRs in mediating nicotine-induced addictive behavior in animal models. Additionally, we will provide an overview of the effects of nicotine and nicotinic compounds on the mesolimbic dopamine system, part of the reinforcement/reward circuitry of the brain, as an example of the neurochemical basis of nicotine addiction and other drug addictions. An appreciation of the complexity of nicotinic receptors and their regulation will be necessary for the development of nicotinic receptor modulators as potential pharmacotherapy for drug addiction.

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SummaryOne approach for the identification of therapeutic agents for Alzheimer's disease has focused on the research of α7 nAChR-selective agonists such as the partial agonists 3-(4-hydroxy,2-methoxybenzylidene)anabaseine (4OH-GTS-21) and, more recently, 2-[2-(4-bromophenyl)-2-oxoethyl]-1-methyl pyridinium (S 24795). An alternative approach for targeting α7 nAChR has been the development of positive modulators for this receptor. In this study we examined the interactions between full or partial agonists and positive modulators of α7 nAChRs in situ in brain tissue. Three positive modulators were used, 5-hydroxyindole (5-HI), 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxanol-3-yl)-urea (PNU-120596), and genistein. Whole-cell recordings were performed in stratum radiatum interneurons from rat brain slices. Hippocampal interneurons were stimulated by ACh, choline, S 24795, or 4OH-GTS-21, before and after bath perfusion with the positive modulators. 5-HI was not effective at potentiating 200 μM 4OH-GTS-21-evoked responses, however 5-HI induced a sustained potentiation of responses evoked by 30 μM 4OH-GTS-21. When 1 mM ACh and 200 μM 4OH-GTS-21 were applied alternately α7-mediated responses to both agonists were reduced, suggesting that high concentration of 4OH-GTS-21 produces residual inhibition or desensitization and that 5-HI is not effective at overcoming receptor desensitization. Similar results were obtained with α7 receptors expressed in Xenopus oocytes. Interestingly, responses evoked by S 24795 were potentiated by 5-HI but not by genistein. Additionally, PNU-120596 was able to potentiate α7-mediated responses, regardless of the nature of the agonist. We demonstrated that the potentiation of α7 nAChR response would depend on the nature and the effective concentration of the agonist involved and its particular interaction with the positive modulator.

A family of 20 tris-azaaromatic quaternary ammonium (AQA) compounds were tested for their inhibition of ␣7 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus laevis oocytes. The potency of inhibitory activity was related to the hydrophobic character of the tris head groups. Two tris-AQA compounds were studied in detail: the highly effective inhibitor 1,3,5-tri-[5-(1-quinolinum)-pent-1-yn-1-yl]-benzene tribromide (tPyQB) and the less potent antagonist 1,3,5,-tri-{5-[1-(2-picolinium)]-pent-1-yn-1-yl}benzene tribromide (tPy2PiB). In addition, we evaluated 1,2,4,5-tetra-{5-[1-(3-benzyl)pyridinium]pent-1-yl}benzene tetrabromide (tkP3BzPB), a tetrakis-AQA with very hydrophobic headgroups. We compared the activity of the AQA compounds to the frequently used ␣7-antagonist methyllycaconitine (MLA). Both tPyQB and tkP3BzPB were selective antagonists of ␣7. However, although inhibition by tPyQB was reversible within 5 min, the recovery time constant for tkP3BzPB inhibition was 26.6 Ϯ 0.8 min, so that the equilibrium inhibition in the prolonged presence of nanomolar concentrations of tkP3BzPB was nearly 100%. The potency, selectivity, and slow reversibility of tkP3BzPB were comparable with or greater than that of MLA. The inhibitory actions of tPyQB, tPy2PiB, and tkP3BzPB were evaluated on the acetylcholine (ACh)-evoked responses of native nAChRs in rat brain slices. The ␣7-mediated responses of hippocampal interneurons were effectively reduced by 1 M tPyQB and tkP3BzPB but not tPy2PiB. In rat medial septum, tkP3BzPB produced a greater inhibition of ACh-evoked responses of cells with fast inward currents (type I) than of cells with predominantly slow kinetics (type II), suggesting that tkP3BzPB can block ␣7 yet preserve the responsiveness of non-␣7 receptors. These agents might be helpful in elucidating complex receptor responses in brain regions with mixed populations of nAChRs.Nicotinic acetylcholine receptors (nAChRs) are distributed throughout the central and peripheral nervous systems (Role and Berg, 1996;Wonnacott, 1997). Nine neuronal ␣ subunits (␣2-␣10) and three neuronal ␤ subunits (␤2-␤4) have thus far been identified and cloned in vertebrate systems. One type of neuronal nAChR is formed by the assembly of ␣ and ␤ subunits, with functional properties depending on both ␣ and ␤ subunits within the receptor complex (Buisson and Bertrand, 2002). In Xenopus laevis oocytes, pairwise combinations of some neuronal ␣ and ␤ subunits form functional receptors. However, the existence of complex subtypes consisting of more than two different subunits has been documented in native brain regions. In addition to the heteromeric recep-

Recent developments in the generation of neuronal population-specific, genetically modified mouse lines have allowed precise identification and selective stimulation of cholinergic neurons in vivo. Although considerably less laborious than studies conducted with post hoc identification of cholinergic neurons by immunostaining, it is not known whether the genetically based labeling procedures that permit in vivo identification are electrophysiologically benign. In this study, we use mice carrying a bacterial artificial chromosome transgene that drives expression of a tau-green fluorescent fusion protein specifically in cholinergic neurons. This allowed us to visualize basal forebrain cholinergic neurons in acute slice preparations. Using whole cell, patch clamp electrophysiological recording in acute brain slices, here we present original data about the basic electrical properties of these genetically tagged cholinergic neurons including firing rate, resting membrane potential, rheobase, and various characteristics of their action potentials and after-hyperpolarization potentials. The basic electrical properties are compared (i) with non-cholinergic neurons in the same brain regions; (ii) in cholinergic neurons between immature animals and young adults; and (iii) with cholinergic neurons that are expressing light-sensitive channels. Our conclusions based on these data are (i) cholinergic neurons are less excitable then their noncholinergic neighbors, (ii) the basic properties of cholinergic neurons do not significantly change between adolescence and young adulthood and (iii) these properties are not significantly affected by chronic expression of the excitatory opsin, oChIEF.