Sharon R. Grady

Guidelines on nicotine dose selection for in vivo research

RationaleThis review provides insight for the judicious selection of nicotine dose ranges and routes of administration for in vivo studies. The literature is replete with reports in which a dosaging regimen chosen for a specific nicotine-mediated response was suboptimal for the species used. In many cases, such discrepancies could be attributed to the complex variables comprising species-specific in vivo responses to acute or chronic nicotine exposure.ObjectivesThis review capitalizes on the authors’ collective decades of in vivo nicotine experimentation to clarify the issues and to identify the variables to be considered in choosing a dosaging regimen. Nicotine dose ranges tolerated by humans and their animal models provide guidelines for experiments intended to extrapolate to human tobacco exposure through cigarette smoking or nicotine replacement therapies. Just as important are the nicotine dosaging regimens used to provide a mechanistic framework for acquisition of drug-taking behavior, dependence, tolerance, or withdrawal in animal models.ResultsSeven species are addressed: humans, nonhuman primates, rats, mice, Drosophila, Caenorhabditis elegans, and zebrafish. After an overview on nicotine metabolism, each section focuses on an individual species, addressing issues related to genetic background, age, acute vs chronic exposure, route of administration, and behavioral responses.ConclusionsThe selected examples of successful dosaging ranges are provided, while emphasizing the necessity of empirically determined dose–response relationships based on the precise parameters and conditions inherent to a specific hypothesis. This review provides a new, experimentally based compilation of species-specific dose selection for studies on the in vivo effects of nicotine.

Characterization of Nicotinic Receptor-Mediated [3H]Dopamine Release from Synaptosomes Prepared from Mouse Striatum

Abstract: This study establishes that presynaptic nicotinic receptors modulate dopamine release in the mouse striatum. Nicotinic agonists elicit a dose‐dependent increase in the release of [3H]dopamine from synaptosomes prepared from mouse striatum. At low concentrations, this release is Ca2+ dependent, whereas at higher concentrations Ca2+‐independent, mecamylamine‐insensitive release was also observed. The Ca2+‐dependent nicotine‐evoked release was not blocked by α‐bungarotoxin but was effectively blocked by neuronal bungarotoxin as well as several other nicotinic receptor antagonists. The relationship between potency for stimulation of release for agonists and potency for inhibition of release for antagonists was compared to the affinity of these compounds for the [3H]nicotine binding site. The overall correlation between release and binding potency was not high, but the drugs may be classified into separate groups, each of which has a high correlation with binding. This finding suggests either that more than one nicotinic receptor regulates dopamine release or that not all agonists interact with the same receptor in an identical fashion.

Chronic Nicotine Cell Specifically Upregulates Functional α4* Nicotinic Receptors: Basis for Both Tolerance in Midbrain and Enhanced Long-Term Potentiation in Perforant Path

Understanding effects of chronic nicotine requires identifying the neurons and synapses whose responses to nicotine itself, and to endogenous acetylcholine, are altered by continued exposure to the drug. To address this problem, we developed mice whose α4 nicotinic receptor subunits are replaced by normally functioning fluorescently tagged subunits, providing quantitative studies of receptor regulation at micrometer resolution. Chronic nicotine increased α4 fluorescence in several regions; among these, midbrain and hippocampus were assessed functionally. Although the midbrain dopaminergic system dominates reward pathways, chronic nicotine does not change α4* receptor levels in dopaminergic neurons of ventral tegmental area (VTA) or substantia nigra pars compacta. Instead, upregulated, functional α4* receptors localize to the GABAergic neurons of the VTA and substantia nigra pars reticulata. In consequence, GABAergic neurons from chronically nicotine-treated mice have a higher basal firing rate and respond more strongly to nicotine; because of the resulting increased inhibition, dopaminergic neurons have lower basal firing and decreased response to nicotine. In hippocampus, chronic exposure to nicotine also increases α4* fluorescence on glutamatergic axons of the medial perforant path. In hippocampal slices from chronically treated animals, acute exposure to nicotine during tetanic stimuli enhances induction of long-term potentiation in the medial perforant path, showing that the upregulated α4* receptors in this pathway are also functional. The pattern of cell-specific upregulation of functional α4* receptors therefore provides a possible explanation for two effects of chronic nicotine: sensitization of synaptic transmission in forebrain and tolerance of dopaminergic neuron firing in midbrain.

Rodent Habenulo–Interpeduncular Pathway Expresses a Large Variety of Uncommon nAChR Subtypes, But Only the α3β4 and α3β3β4 Subtypes Mediate Acetylcholine Release

Recent studies suggest that the neuronal nicotinic receptors (nAChRs) present in the habenulo–interpeduncular (Hb–IPn) system can modulate the reinforcing effect of addictive drugs and the anxiolytic effect of nicotine. Hb and IPn neurons express mRNAs for most nAChR subunits, thus making it difficult to establish the subunit composition of functional receptors. We used immunoprecipitation and immunopurification studies performed in rat and wild-type (+/+) and β2 knock-out (−/−) mice to establish that the Hb and IPn contain significant β2* and β4* populations of nAChR receptors (each of which is heterogeneous). The β4* nAChR are more highly expressed in the IPn. We also identified novel native subtypes (α2β2*, α4β3β2*, α3β3β4*, α6β3β4*). Our studies on IPn synaptosomes obtained from +/+ and α2, α4, α5, α6, α7, β2, β3, and β4−/− mice show that only the α3β4 and α3β3β4 subtypes facilitate acetylcholine (ACh) release. Ligand binding, immunoprecipitation, and Western blotting studies in β3−/− mice showed that, in the IPn of these mice, there is a concomitant reduction of ACh release and α3β4* receptors, whereas the receptor number remains the same in the Hb. We suggest that, in habenular cholinergic neurons, the β3 subunit may be important for transporting the α3β4* subtype from the medial habenula to the IPn. Overall, these studies highlight the presence of a wealth of uncommon nAChR subtypes in the Hb–IPn system and identify α3β4 and α3β3β4, transported from the Hb and highly enriched in the IPn, as the subtypes modulating ACh release in the IPn.

Nicotinic agonists stimulate acetylcholine release from mouse interpeduncular nucleus: a function mediated by a different nAChR than dopamine release from striatum

Acetylcholine release stimulated by nicotinic agonists was measured as radioactivity released from perfused synaptosomes prepared from mouse interpeduncular nucleus (IPN) that had been loaded with [3H]choline. Agonist‐stimulated release was dependent upon external calcium and over 90% of released radioactivity was acetylcholine. The release process was characterized by dose response curves for 13 agonists and inhibition curves for six antagonists. α‐Conotoxin MII did not inhibit this release, while α‐conotoxin AuIB inhibited 50% of agonist‐stimulated release. Comparison of this process with [3H]dopamine release from mouse striatal synaptosomes indicated that different forms of nicotinic acetylcholine receptors (nAChRs) may mediate these processes. This was confirmed by assays using mice homozygous for the β2 subunit null mutation. The deletion of the β2 subunit had no effect on agonist‐stimulated acetylcholine release, but abolished agonist‐stimulated release of dopamine from striatal synaptosomes. Mice heterozygous for the β2 subunit null mutation showed decreased dopamine release evoked by l‐nicotine with no apparent change in EC50 value, as well as similar decreases in both transient and persistent phases of release with no changes in desensitization rates.

Pharmacology of α-conotoxin MII-sensitive subtypes of nicotinic acetylcholine receptors isolated by breeding of null mutant mice

Subtypes of nicotinic acetylcholine receptors (nAChR) containing α6 subunits comprise 25 to 30% of the presynaptic nAChRs expressed in striatal dopaminergic terminals in rodents and 70% in monkeys. This class of receptors, potentially important in nicotine addiction, binds α-conotoxin MII (α-CtxMII) with high affinity and is heterogeneous, consisting of several subtypes in mice, possibly an important consideration for the design of compounds that selectively activate or antagonize the α6 subclass of nAChRs. Selected-null mutant mice were bred to generate isolated subtypes of α6β2* nAChRs expressed in vivo for assessing pharmacology of α6β2* nAChRs. Binding to striatal membranes and function in synaptosomes from (α4–/–)(β3+/+) and (α4–/–)(β3–/–) mice were measured and compared with wild-type (α4+/+)(β3+/+) mice. Gene deletions (α4 and β3) decreased binding of 125I-α-CtxMII without affecting affinity for α-CtxMII or inhibition of α-CtxMII binding by epibatidine or nicotine. Deletion of the α4 subunit substantially increased EC50 values for both nicotine- and cytisine-stimulated α-CtxMII-sensitive dopamine release from striatal synaptosomes. A further increase in EC50 values was seen upon the additional deletion of the β3 subunit. The data indicate that one α-CtxMII-sensitive nAChR subtype, prevalent on wild-type dopaminergic terminals, has the lowest EC50 for a nicotine-mediated function so far measured in mice. In conclusion, the gene deletion strategy enabled isolation of α6* subtypes, and these nAChR subtypes exhibited differential activation by nicotine and cytisine.

Desensitization of Nicotine‐Stimulated [3H]Dopamine Release from Mouse Striatal Synaptosomes

Potential desensitization of brain nicotinic receptors was studied using a [3H]dopamine release assay. Nicotine‐stimulated [3H]dopamine release from mouse striatal synaptosomes was concentration‐dependent with an EC50 of 0.33 ± 0.13 μM and a Hill coefficient of 1.44 ± 0.18. Desensitization by activating concentrations of nicotine had a similar EC50 and a half‐time of 35 s. Concentrations of nicotine that evoked little release also induced a concentration‐dependent desensitization (EC50=6.9 plusmn; 3.6 nM, t1/2= 1.6‐2.0 min, nH=1.02 ± 0.01). Both types of desensitization produced a maximum 75% decrease in [3H]dopamine release. Recovery from desensitization after exposure to low or activating concentrations of nicotine was time‐dependent with half‐times of 6.1 min and 12.4 min, respectively. Constants determined for binding of [3H]nicotine to striatal membrane at 22°C included a KDof 3.7 ± 0.5 nM, Bmax of 67.5 ± 2.2 fmol/mg, and Hill coefficient of 1.07 ± 0.06. Association of nicotine with membrane binding sites was biphasic with half‐times of 9 s and 1.8 min. The fast rate process contributed 37% of the total reaction. Dissociation was a uniphasic process with a half‐time of 1.6 min. Comparison of constants determined by the release and binding assays indicated that the [3H]‐nicotine binding site could be the presynaptic receptor involved in [3H]dopamine release in mouse striatal synaptosomes.

Cholinergic Modulation of Locomotion and Striatal Dopamine Release is Mediated by α6α4* Nicotinic Acetylcholine Receptors

Dopamine (DA) release in striatum is governed by firing rates of midbrain DA neurons, striatal cholinergic tone, and nicotinic ACh receptors (nAChRs) on DA presynaptic terminals. DA neurons selectively express α6* nAChRs, which show high ACh and nicotine sensitivity. To help identify nAChR subtypes that control DA transmission, we studied transgenic mice expressing hypersensitive α6L9′S* receptors. α6L9′S mice are hyperactive, travel greater distance, exhibit increased ambulatory behaviors such as walking, turning, and rearing, and show decreased pausing, hanging, drinking, and grooming. These effects were mediated by α6α4* pentamers, as α6L9′S mice lacking α4 subunits displayed essentially normal behavior. In α6L9′S mice, receptor numbers are normal, but loss of α4 subunits leads to fewer and less sensitive α6* receptors. Gain-of-function nicotine-stimulated DA release from striatal synaptosomes requires α4 subunits, implicating α6α4β2* nAChRs in α6L9′S mouse behaviors. In brain slices, we applied electrochemical measurements to study control of DA release by α6L9′S nAChRs. Burst stimulation of DA fibers elicited increased DA release relative to single action potentials selectively in α6L9′S, but not WT or α4KO/α6L9′S, mice. Thus, increased nAChR activity, like decreased activity, leads to enhanced extracellular DA release during phasic firing. Bursts may directly enhance DA release from α6L9′S presynaptic terminals, as there was no difference in striatal DA receptor numbers or DA transporter levels or function in vitro. These results implicate α6α4β2* nAChRs in cholinergic control of DA transmission, and strongly suggest that these receptors are candidate drug targets for disorders involving the DA system.

Nicotine-Mediated Activation of Dopaminergic Neurons in Distinct Regions of the Ventral Tegmental Area

Nicotine activation of nicotinic acetylcholine receptors (nAChRs) within the dopaminergic (DAergic) neuron-rich ventral tegmental area (VTA) is necessary and sufficient for nicotine reinforcement. In this study, we show that rewarding doses of nicotine activated VTA DAergic neurons in a region-selective manner, preferentially activating neurons in the posterior VTA (pVTA) but not in the anterior VTA (aVTA) or in the tail VTA (tVTA). Nicotine (1 μM) directly activated pVTA DAergic neurons in adult mouse midbrain slices, but had little effect on DAergic neurons within the aVTA. Quantification of nAChR subunit gene expression revealed that pVTA DAergic neurons expressed higher levels of α4, α6, and β3 transcripts than did aVTA DAergic neurons. Activation of nAChRs containing the α4 subunit (α4* nAChRs) was necessary and sufficient for activation of pVTA DAergic neurons: nicotine failed to activate pVTA DAergic neurons in α4 knockout animals; in contrast, pVTA α4* nAChRs were selectively activated by nicotine in mutant mice expressing agonist-hypersensitive α4* nAChRs (Leu9′Ala mice). In addition, whole-cell currents induced by nicotine in DAergic neurons were mediated by α4* nAChRs and were significantly larger in pVTA neurons than in aVTA neurons. Infusion of an α6* nAChR antagonist into the VTA blocked activation of pVTA DAergic neurons in WT mice and in Leu9′Ala mice at nicotine doses, which only activate the mutant receptor indicating that α4 and α6 subunits coassemble to form functional receptors in these neurons. Thus, nicotine selectively activates DAergic neurons within the pVTA through α4α6* nAChRs. These receptors represent novel targets for smoking-cessation therapies.

α4β2 Nicotinic Acetylcholine Receptors on Dopaminergic Neurons Mediate Nicotine Reward and Anxiety Relief

Nicotine is the primary psychoactive substance in tobacco, and it exerts its effects by interaction with various subtypes of nicotinic acetylcholine receptors (nAChRs) in the brain. One of the major subtypes expressed in brain, the α4β2-nAChR, endogenously modulates neuronal excitability and thereby, modifies certain normal as well as nicotine-induced behaviors. Although α4-containing nAChRs are widely expressed across the brain, a major focus has been on their roles within midbrain dopaminergic regions involved in drug addiction, mental illness, and movement control in humans. We developed a unique model system to examine the role of α4-nAChRs within dopaminergic neurons by a targeted genetic deletion of the α4 subunit from dopaminergic neurons in mice. The loss α4 mRNA and α4β2-nAChRs from dopaminergic neurons was confirmed, as well as selective loss of α4β2-nAChR function from dopaminergic but not GABAergic neurons. Two behaviors central to nicotine dependence, reward and anxiety relief, were examined. α4-nAChRs specifically on dopaminergic neurons were demonstrated to be necessary for nicotine reward as measured by nicotine place preference, but not for another drug of addiction, cocaine. α4-nAChRs are necessary for the anxiolytic effects of nicotine in the elevated plus maze, and elimination of α4β2-nAChRs specifically from dopaminergic neurons decreased sensitivity to the anxiolytic effects of nicotine. Deletion of α4-nAChRs specifically from dopaminergic neurons also increased sensitivity to nicotine-induced locomotor depression; however, nicotine-induced hypothermia was unaffected. This is the first work to develop a dopaminergic specific deletion of a nAChR subunit and examine resulting changes in nicotine-related behaviors.