The identity of nicotinic receptor subtypes sufficient to elicit both the acute and chronic effects of nicotine dependence is unknown. We engineered mutant mice with a4 nicotinic subunits containing a single point mutation, Leu9' --> Ala9' in the pore-forming M2 domain, rendering a4* receptors hypersensitive to nicotine. Selective activation of a4* nicotinic acetylcholine receptors with low doses of agonist recapitulates nicotine effects thought to be important in dependence, including reinforcement in response to acute nicotine administration, as well as tolerance and sensitization elicited by chronic nicotine administration. These data indicate that activation of a4* receptors is sufficient for nicotine-induced reward, tolerance, and sensitization.

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 ␣4 nicotinic receptor subunits are replaced by normally functioning fluorescently tagged subunits, providing quantitative studies of receptor regulation at micrometer resolution. Chronic nicotine increased ␣4 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 ␣4* receptor levels in dopaminergic neurons of ventral tegmental area (VTA) or substantia nigra pars compacta. Instead, upregulated, functional ␣4* 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 ␣4* 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 ␣4* receptors in this pathway are also functional. The pattern of cell-specific upregulation of functional ␣4* 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.

Fura-2 recording of Ca2+ influx was used to show that incubation in 1 microm nicotine (2-6 d) upregulates several pharmacological components of acetylcholine (ACh) responses in ventral midbrain cultures, including a MLA-resistant, DHbetaE-sensitive component that presumably corresponds to alpha4beta2 receptors. To study changes in alpha4beta2 receptor levels and assembly during this upregulation, we incorporated yellow and cyan fluorescent proteins (YFPs and CFPs) into the alpha4 or beta2 M3-M4 intracellular loops, and these subunits were coexpressed in human embryonic kidney (HEK) 293T cells and cultured ventral midbrain neurons. The fluorescent receptors resembled wild-type receptors in maximal responses to ACh, dose-response relations, ACh-induced Ca2+ influx, and somatic and dendritic distribution. Transfected midbrain neurons that were exposed to nicotine (1 d) displayed greater levels of fluorescent alpha4 and beta2 nicotinic ACh receptor (nAChR) subunits. As expected from the hetero-multimeric nature of alpha4beta2 receptors, coexpression of the alpha4-YFP and beta2-CFP subunits resulted in robust fluorescence resonance energy transfer (FRET), with a FRET efficiency of 22%. In midbrain neurons, dendritic alpha4beta2 nAChRs displayed greater FRET than receptors inside the soma, and in HEK293T cells, a similar increase was noted for receptors that were translocated to the surface during PKC stimulation. When cultured transfected midbrain neurons were incubated in 1 microm nicotine, there was increased FRET in the cell body, denoting increased assembly of alpha4beta2 receptors. Thus, changes in alpha4beta2 receptor assembly play a role in the regulation of alpha4beta2 levels and responses in both clonal cell lines and midbrain neurons, and the regulation may result from Ca2+-stimulated pathways.

The role of dopaminergic (DA) projections from the ventral tegmental area (VTA) in appetitive and rewarding behavior has been widely studied, but the VTA also has documented DA-independent functions. Several drugs of abuse, including nicotine, act on VTA GABAergic neurons, and most studies have focused on local inhibitory connections. Relatively little is known about VTA GABA projection neurons and their connections to brain sites outside the VTA. In this study, we employed viral-vector mediated cell-type specific anterograde tracing, classical retrograde tracing and immunohistochemistry to characterize VTA GABA efferents throughout the brain. We found that VTA GABA neurons project widely to forebrain and brainstem targets, including the ventral pallidum, lateral and magnocellular preoptic nuclei, lateral hypothalamus and lateral habenula. Minor projections also go to central amygdala, mediodorsal thalamus, dorsal raphe and deep mesencephalic nuclei, and sparse projections go to prefrontal cortical regions and to nucleus accumbens shell and core. Importantly, these projections differ from the major VTA DA target regions. Retrograde tracing studies confirmed results from the anterograde experiments and differences in projections from VTA subnuclei. Retrogradely-labeled GABA neurons were not numerous and most non-TH/retrogradely labeled cells lacked GABAergic markers. Many non-DA/retrogradely labeled cells projecting to several areas express VGluT2. VTA GABA and glutamate neurons project throughout the brain, most prominently to regions with reciprocal connections to the VTA. These data indicate that VTA GABA and glutamate neurons may have more dopamine-independent functions than previously recognized.

Several genetic strategies for inhibiting neuronal function in mice have been described, but no system that directly suppresses membrane excitability and is triggered by a systemically administered drug, has been validated in awake behaving animals. We expressed unilaterally in mouse striatum a modified heteromeric ivermectin (IVM)-gated chloride channel from C. elegans (GluClalphabeta), systemically administered IVM, and then assessed amphetamine-induced rotational behavior. Rotation was observed as early as 4 hr after a single intraperitoneal IVM injection (10 mg/kg), reached maximal levels by 12 hr, and was almost fully reversed by 4 days. Multiple cycles of silencing and recovery could be performed in a single animal. In striatal slice preparations from GluClalphabeta-expressing animals, IVM rapidly suppressed spiking. The two-subunit GluCl/IVM system permits "intersectional" strategies designed to increase the cellular specificity of silencing in transgenic animals.

Knock-in mice were generated that harbored a leucine-to-serine mutation in the ␣4 nicotinic receptor near the gate in the channel pore. Mice with intact expression of this hypersensitive receptor display dominant neonatal lethality. These mice have a severe deficit of dopaminergic neurons in the substantia nigra, possibly because the hypersensitive receptors are continuously activated by normal extracellular choline concentrations. A strain that retains the neo selection cassette in an intron has reduced expression of the hypersensitive receptor and is viable and fertile. The viable mice display increased anxiety, poor motor learning, excessive ambulation that is eliminated by very low levels of nicotine, and a reduction of nigrostriatal dopaminergic function upon aging. These knock-in mice provide useful insights into the pathophysiology of sustained nicotinic receptor activation and may provide a model for Parkinson's disease.T he mechanism leading from nicotine intake to addiction is not known in detail, but it begins with the activation of neuronal nicotinic acetylcholine receptors (nAChRs). Nicotine elicits dopamine release in several regions of the brain, presumably leading to the reward, motor, and addictive effects (1, 2). The highest-affinity and most abundant nicotine binding in the brain corresponds to the ␣4␤2 nAChR (3). The ␣4 subunit is the principal partner for the ␤2 subunit in brain; ␤2-containing receptors play an important role in nicotine self-administration, in nicotine-stimulated electrophysiological responses in midbrain neurons, and in nicotine-stimulated dopamine release in the ventral striatum (4, 5). The ␣4 subunit and tyrosine hydroxylase are colocalized in dopaminergic neurons (6). Epidemiological studies show that smokers have a lower incidence of Parkinson's disease (7,8), suggesting a protective effect of nicotine via modulation of the dopaminergic system. Both ␣4 and ␤2 knockout mice show only subtle alterations in their physiology or behavior until they reach old age (4, 9, 10). We have used a complementary strategy to understand the roles of ␣4-containing nAChRs. We reasoned that gain of function mutations might generate more noticeable phenotypes, and that these phenotypes would gain relevance from the fact that nicotine and some candidate analgesics (11) are agonists. We have generated lines of knock-in mice by introducing a point mutation into the M2 transmembrane region of the ␣4 subunit to produce a hypersensitive receptor. Materials and MethodsXenopus Oocyte Injections and Electrophysiology. The ␣4 and ␤2 subunits were subcloned into pAMV-PA (12). Capped mRNA transcripts were prepared, and ␣4͞␤2 (2 ng, 1:1) or ␣4L9ЈS͞␤2 (0.2-0.5 ng, 1:1) were microinjected into Xenopus oocytes (12). Twenty-four to seventy-two hours later, two-electrode voltage clamp recordings (12) were made in solutions containing zero Ca 2ϩ .Knock-in Mouse Construction. A 129͞SvJ ␣4 genomic clone containing exon 5 and the L9ЈS mutation was inserted into pKO Scrambler V907 (Lexicon-Genetics, The Woodland...

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A leucine to alanine substitution (L9ЈA) was introduced in the M2 region of the mouse ␣4 neuronal nicotinic acetylcholine receptor (nAChR) subunit. Expressed in Xenopus oocytes, ␣4(L9ЈA)␤2 nAChRs were Ն30-fold more sensitive than wild type (WT) to both ACh and nicotine. We generated knock-in mice with the L9ЈA mutation and studied their cellular responses, seizure phenotype, and sleepwake cycle. Seizure studies on ␣4-mutated animals are relevant to epilepsy research because all known mutations linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) occur in the M2 region of ␣4 or ␤2 subunits. Thalamic cultures and synaptosomes from L9ЈA mice were hypersensitive to nicotine-induced ion flux. L9ЈA mice were ϳ15-fold more sensitive to seizures elicited by nicotine injection than their WT littermates. Seizures in L9ЈA mice differed qualitatively from those in WT: L9ЈA seizures started earlier, were prevented by nicotine pretreatment, lacked EEG spike-wave discharges, and consisted of fast repetitive movements. Nicotine-induced seizures in L9ЈA mice were partial, whereas WT seizures were generalized. When L9ЈA homozygous mice received a 10 mg/kg nicotine injection, there was temporal and phenomenological separation of mutant and WT-like seizures: an initial seizure ϳ20 s after injection was clonic and showed no EEG changes. A second seizure began 3-4 min after injection, was tonic-clonic, and had EEG spike-wave activity. No spontaneous seizures were detected in L9ЈA mice during chronic video/EEG recordings, but their sleep-wake cycle was altered. Our findings show that hypersensitive ␣4* nicotinic receptors in mice mediate changes in the sleep-wake cycle and nicotineinduced seizures resembling ADNFLE.