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.

In nicotinic acetylcholine receptors (nAChR), as well as glycine, GABAA (gamma-aminobutyric acid), serotonin (5-HT3), and GluCl glutamate receptors, a leucine residue at the approximate midpoint of the M2 transmembrane domain (the 9' position) is conserved across most known subunits. Structural data for the nAChR suggest that the Leu 9' residues occupy a 'kink' in each of the five M2 helices and point into the closed channel; in the opening step, the M2 helices rotate so that Leu 9' side chains no longer occlude the conduction pathway. Mutation of Leu 9' to one of several other residues slows desensitization and increases sensitivity to agonist. We have exploited the alpha 2 beta gamma delta stoichiometry of muscle nAChR to express receptors with ms* = 0 to 5 Leu 9'Ser mutated subunits. Strikingly, each Leu 9'Ser mutation shifts the dose-response relation for ACh to the left by approximately 10-fold; a nAChR with ms* = 4 is 10(4)-fold more sensitive than the wild type. The results suggest that each of the five Leu 9' residues participates independently and symmetrically in a key step in the structural transition between the closed and open states.

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...

GABA transporter subtype 1 (GAT1) molecules were counted near GABAergic synapses, to a resolution of approximately 0.5 microm. Fusions between GAT1 and green fluorescent protein (GFP) were tested in heterologous expression systems, and a construct was selected that shows function, expression level, and trafficking similar to that of wild-type (WT) GAT1. A strain of knock-in mice was constructed that expresses this mGAT1-GFP fusion in place of the WT GAT1 gene. The pattern of fluorescence in brain slices agreed with previous immunocytochemical observations. [3H]GABA uptake, synaptic electrophysiology, and subcellular localization of the mGAT1-GFP construct were also compared with WT mice. Quantitative fluorescence microscopy was used to measure the density of mGAT1-GFP at presynaptic structures in CNS preparations from the knock-in mice. Fluorescence measurements were calibrated with transparent beads and gels that have known GFP densities. Surface biotinylation defined the fraction of transporters on the surface versus those in the nearby cytoplasm. The data show that the presynaptic boutons of GABAergic interneurons in cerebellum and hippocampus have a membrane density of 800-1300 GAT1 molecules per square micrometer, and the axons that connect boutons have a linear density of 640 GAT1 molecules per micrometer. A cerebellar basket cell bouton, a pinceau surrounding a Purkinje cell axon, and a cortical chandelier cell cartridge carry 9000, 7.8 million, and 430,000 GAT1 molecules, respectively; 61-63% of these molecules are on the surface membrane. In cultures from hippocampus, the set of fluorescent cells equals the set of GABAergic interneurons. Knock-in mice carrying GFP fusions of membrane proteins provide quantitative data required for understanding the details of synaptic transmission in living neurons.

Neuronal nAChRs in the medial habenula (MHb) to the interpeduncular nucleus (IPN) pathway are key mediators of nicotine's aversive properties. In this paper, we report new details regarding nAChR anatomical localization and function in MHb and IPN. A new group of knock-in mice were created that each expresses a single nAChR subunit fused to GFP, allowing high-resolution mapping. We find that ␣3 and ␤4 nAChR subunit levels are strong throughout the ventral MHb (MHbV). In contrast, ␣6, ␤2, ␤3, and ␣4 subunits are selectively found in some, but not all, areas of MHbV. All subunits were found in both ChAT-positive and ChAT-negative cells in MHbV. Next, we examined functional properties of neurons in the lateral and central part of MHbV (MHbVL and MHbVC) using brain slice patch-clamp recordings. MHbVL neurons were more excitable than MHbVC neurons, and they also responded more strongly to puffs of nicotine. In addition, we studied firing responses of MHbVL and MHbVC neurons in response to bath-applied nicotine. Cells in MHbVL, but not those in MHbVC, increased their firing substantially in response to 1 M nicotine. Additionally, MHbVL neurons from mice that underwent withdrawal from chronic nicotine were less responsive to nicotine application compared with mice withdrawn from chronic saline. Last, we characterized rostral and dorsomedial IPN neurons that receive input from MHbVL axons. Together, our data provide new details regarding neurophysiology and nAChR localization and function in cells within the MHbV.

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.

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