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The Journal of Neuroscience, August 1, 2007, 27(31):8202-8218; doi:10.1523/JNEUROSCI.2199-07.2007

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Behavioral/Systems/Cognitive
Chronic Nicotine Cell Specifically Upregulates Functional 4* Nicotinic Receptors: Basis for Both Tolerance in Midbrain and Enhanced Long-Term Potentiation in Perforant Path

Raad Nashmi,¹ Cheng Xiao,¹ Purnima Deshpande,¹ Sheri McKinney,¹ Sharon R. Grady,² Paul Whiteaker,² Qi Huang,¹ Tristan McClure-Begley,² Jon M. Lindstrom,³ Cesar Labarca,¹ Allan C. Collins,² Michael J. Marks,² and Henry A. Lester¹

¹Division of Biology, California Institute of Technology, Pasadena, California 91125, ²Institute of Behavioral Genetics, University of Colorado, Boulder, Colorado 80309, and ³Department of Neuroscience, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

Key words: nicotine addiction; GFP; knock-in mouse; nicotinic receptor; GABAergic; dopaminergic

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Drugs of abuse induce changes such as tolerance, dependence, sensitization, and pleasure-seeking behavior. Chronic exposure to the drug may lead to changed levels and/or responsiveness of drug receptors themselves. For example, desensitization, downregulation, and recycling of opioid receptors are thought to play key roles in opioid addiction (Waldhoer et al., 2004).

Chronic nicotine induces at least two phenomena that are challenging to explain in terms of changed receptor levels. The first is sensitization, both locomotor (Ksir et al., 1985; de Rover et al., 2004) and cognitive (Rezvani and Levin, 2001; Dani and Bertrand, 2007). In the human context, cognitive sensitization is epitomized by smokers' reports that they think better when they smoke; this anecdotal observation is confirmed by data that smokers who smoke nicotine cigarettes (but not nicotine-free cigarettes) display certain cognitive enhancements (Rusted and Warburton, 1992; Rusted et al., 1995). In the context of our studies on mice, rodents show more contextual fear conditioning if, 1 d after withdrawal from chronic nicotine, they receive an acute nicotine dose (Davis et al., 2005); also chronic nicotine produces better spatial working memory performance in the radial arm maze (Levin et al., 1990, 1996). The second phenomenon is tolerance to other effects such as reward; for instance, repeated nicotine treatment or nicotine self-administration results in chronic tolerance of mesolimbic dopamine release, which is closely associated with drug reward (Cadoni and Di Chiara, 2000; Di Chiara, 2000; Rahman et al., 2004).

Classical studies show that chronic nicotine exposure increases nicotinic acetylcholine receptor levels, but the increases differ among receptor subtypes and even vary among brain regions for the same subtype (Marks et al., 1983, 2004; Schwartz and Kellar, 1983; Pauly et al., 1996; Nguyen et al., 2004; McCallum et al., 2006). Data on isolated cells expressing nicotinic receptors also show that the upregulated receptors retain function and suggest that this upregulation arises primarily because more nicotinic receptors are assembled or stabilized in an easily activated state (Nashmi et al., 2003; Darsow et al., 2005; Kuryatov et al., 2005; Sallette et al., 2005; Vallejo et al., 2005). These changes may underlie mechanisms of sensitization. However, upregulation clearly disagrees with expectations that tolerance to nicotine is caused by fewer, or less functional, receptors. However, although the weight of evidence suggests that the upregulated receptors retain function (Nashmi et al., 2003; Nguyen et al., 2004; Darsow et al., 2005; Kuryatov et al., 2005; Sallette et al., 2005; Vallejo et al., 2005), it is still a viable hypothesis that most of the numerically upregulated receptors are actually desensitized, so that the upregulation would be masked or distorted by an overall decrease in sensitivity (Dani and Heinemann, 1996; Gentry and Lukas, 2002). The desensitization theory nicely explains tolerance, but it cannot directly explain sensitization.

This set of paradoxes, which we term the "upregulation dilemma," has led some researchers to suggest that nicotinic receptor upregulation is an epiphenomenon during chronic exposure to nicotine. Other signal transduction mechanisms, especially arising from Ca²⁺ influx through activated nicotinic acetylcholine receptors (nAChRs), apparently lead to adaptations in other proteins in addition to nAChRs (Chang and Berg, 2001; Ferrari et al., 2002; Brunzell et al., 2003; Marrero et al., 2004; Yeom et al., 2005; Marttila et al., 2006; Yamazaki et al., 2006b; Fornari et al., 2007). These adaptations, downstream from the receptors, could govern nicotine addiction at the cellular and circuit level (Koob et al., 2004; Laviolette and van der Kooy, 2004).

To assess the importance of receptor upregulation in the changes provoked by chronic nicotine, we asked whether upregulation can provide an explanation for chronic nicotine effects, both in the areas in forebrain thought to govern some simple aspects of cognition, as well as in the areas in midbrain thought to govern tolerance and reward. To monitor receptor changes, both numerically and functionally at a cellular level, we used an approach based on fluorescent receptors in knock-in mice. We studied receptors containing the 4 nicotinic receptor subunit (4* receptors, primarily 4ß2* receptors), because (1) these are among the most nicotine-sensitive subtypes (Luetje and Patrick, 1991), (2) these are also among those upregulated by chronic nicotine (Flores et al., 1992; McCallum et al., 2006), and (3) nicotine activation of these receptors is both necessary and sufficient for tolerance, sensitization, and reward behavior (Picciotto et al., 1998; Tapper et al., 2004; Maskos et al., 2005).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice. All experiments were conducted in accordance with the guidelines for care and use of animals provided by the National Institutes of Health, and protocols were approved by the Institutional Animal Care and Use Committee at the California Institute of Technology or the University of Colorado, Boulder. Mouse strains used in this study included 4YFP (see below), 4(Leu9'Ala) (Tapper et al., 2004), 4 knock-out (KO) (Ross et al., 2000), and GAT1-green fluorescent protein (GFP) (Chiu et al., 2002). Mice were kept on a standard 12 h light/dark cycle at 22°C and given food and water ad libitum. The 4YFP mice had a mixed Sv129/C57BL/6 background with one or more generations backcrossed to C57BL/6. Thus, all tissues studied were derived from mice with a mixed Sv129/C57BL/6 background, except that the slice experiments used inbred C57BL/6 mice.

4YFP mouse construction. The strategy used to generate the 4YFP line adapts the procedures used to generate the previous Leu9'Ser line (Labarca et al., 2001; Tapper et al., 2004; Fonck et al., 2005) (see Fig. 1A–D). From previous work, we have in hand a 129/SvJ 4 genomic clone containing exon 5 and exon 6 with the L9'S mutation in the vector pKO Scrambler V907 (15.2 kb in length; Lexicon Genetics, The Woodlands, TX). A neomycin resistance cassette with a phosphoglycerate kinase promoter and polyadenylation signal flanked by loxP sites is present 166 bp downstream from exon 5 for positive selection. The diphtheria toxin A chain gene with an RNA polymerase II promoter has also been added to provide negative selection for random insertion.

A 4.1 kb cassette containing exon 5 and the L9'S mutation was removed from the pKO vector via digestion with SpeI and inserted into pBlueScript for modification (7.2 kb total length). The L9'S mutation was replaced with its wild-type (WT) codon by replacing an AvrII and SacI fragment (5.7 kb) by enzyme digestion and ligation from a WT 4 genomic clone in Bluescript. According to our previous work, we found that the optimal insertion site for yellow fluorescent protein (YFP) was a BstEII site in the M3–M4 cytoplasmic loop of 4 (Nashmi et al., 2003). There were two BstEII sites in the 4.1 kb SpeI fragment inserted in pBlueScript, however. Therefore, a partial digestion was performed with BstEII enzyme so that only one of the two sites was cut. YFP was cut from the previously published 4YFP cDNA construct with BstEII (Nashmi et al., 2003), and then ligated to the 4.1 kb cassette in pBlueScript. The YFP insert also contained an immediately upstream hemagglutinin (HA)-epitope tag. Restriction analysis with BsrGI and sequencing confirmed correct insertion orientation. The 4.1 kb exon 5 cassette was then removed by digestion with SpeI and ligated back into the 4(L9'S) knock-in construct, thus replacing the L9'S mutation with the WT L9'codon and inserting the YFP sequence. Clones were screened for correct orientation with restriction analysis using NsiI enzyme and sequencing. The 4YFP knock-in construct (15.9 kb) was linearized with NotI, chloroform/phenol extracted, precipitated in ethanol-ammonium acetate at –20°C, and resuspended in sterile water in preparation for introduction into mouse embryonic stem cells (ES cells).

CJ7 mouse ES cells were electroporated with 25 µg of linearized knock-in construct. Four rounds of electroporation were performed using 7 x 10⁷ ES cells per electroporation. After electroporation, each group of cells was plated on 10 cm dishes containing single layers of mitotically inactivated mouse primary fibroblasts. To select for recombinant clones, geneticin (G418) (180 µg/ml) was applied to ES cells 24 h after plating. Cell death was apparent 3 d after treatment. After 8 d of G418 treatment, 384 resistant colonies were picked and plated onto 96-well plates, grown for 72 h, trypsinized, and then split onto duplicate plates, one for freezing the other for screening. Correct recombinant colonies were screened for the presence of the neo cassette and YFP insert by a combination of PCR and automated sequence analysis. DNA for PCR and sequencing was isolated by applying lysis buffer (10 mM Tris, pH 7.5, 10 mM EDTA, 10 mM NaCl, 1% SDS, and 1 mg/ml proteinase K) to cells and incubating overnight at 60°C followed by ethanol precipitation. To confirm the correct position of the homologous recombination of the targeting construct within the genome and the insertion of YFP and neo cassette, ES cells were screened by PCR and sequencing. Two separate sets of PCRs were performed. One PCR included the forward primer (SEQS), 5'-GACCAACTGGACTTCTGGGAAAGTG-3', and the reverse primer (SEQYFPAS), 5'-CTGTTGTAGTTGTACTCCAGCTTGTG-3'. The second PCR included the forward primer (SEQYFPS), 5'-CACAAGCTGGAGTACAACTACAAC-3', and the reverse primer (S10L3'AS), 5'-CACTCCTACTGGTCTAGAGAGATG-3'. Karyotype analysis was used to assess chromosome number. Two positive clones with the highest percentage of proper chromosome number (90%) were selected for the subsequent round of electroporation.

Previous work with the Leu9'Ser mice has indicated that the presence of the neo cassette reduces expression of the target gene by approximately fourfold. To maximize expression of the mutated gene, we deleted the neo cassette. This was done by electroporating two clones of the neo-intact 4YFP ES cells with cytomegalovirus-Cre plasmid, leaving only a single 34 bp loxP. Electroporation was performed as described above. Cells were plated at a low density (600 cells/plate) onto 10 cm dishes containing single layers of mitotically inactivated mouse primary fibroblasts. A total of 178 colonies were picked and plated onto two 96-well plates, grown for 48 h, trypsinized, and split onto triplicate 96-well plates, one for freezing and two for screening. Clones were initially selected for neo cassette deletion by G418 (180 µg/ml) treatment. G418-sensitive clones were then further screened using a combination of PCR and automated sequencing. Primers were designed to anneal to a DNA sequence upstream of the 5' loxP site while inside the YFP sequence (SEQYFPS), 5'-CACAAGCTGGAGTACAACTACAAC-3', and downstream of the 3' loxP site (SwaI/loxPAS), 5'-CAGGTGTTAGAATCATAGGGCTAG-3'. All neo-deleted clones were sequenced to confirm the presence of the YFP mutation and deletion of the neo cassette. Karyotype analysis was used to verify normal chromosome number. Two neo-deleted 4YFP clones were injected into C57BL/6 blastocysts, and chimeras were established. Chimeras were mated with wild-type animals and germline transmission was established as assessed by PCR analysis of tail DNA and sequence analysis. Primers used for genotyping included the following: A4XFPF, 5'-CAACCGCATGGACACAGCAGTCGAGAC-3'; A4XFPM, 5'-GCACAAGCTGGAGTACAACTACAACAGC-3'; A4XFP2R, 5'-CTCAGTCAGGGAAGCAGCTCCATCTTG-3'.

RT-PCR. Total RNA was extracted from combined fresh frozen thalamus and ventral midbrain tissue from heterozygous (Het) and WT 4YFP mice (RNeasy Protect number 74124; Qiagen, Valencia, CA). The first-strand cDNA was synthesized using SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA) with the specific primer A4RTPCRAS, 5'-GATCATACCAGCCAACCATGGAG-3'. PCR amplification of 4YFP nAChR was done using the following primers: A4XFPF, 5'-CAACCGCATGGACACAGCAGTCGAGAC-3'; A4XFPM, 5'-GCACAAGCTGGAGTACAACTACAACAGC-3'; A4RTPCRAS, 5'-GATCATACCAGCCAACCATGGAG-3'.

Acetylcholine-stimulated ⁸⁶Rb⁺ efflux. Nicotine-stimulated ⁸⁶Rb⁺ efflux from thalamic and cortical synaptosomes of WT, Het, and homozygous (Hom) 4YFP mice was measured as described previously (Marks et al., 1999). Cortex and thalamus (excluding habenula) were dissected from adult mice killed by cervical dislocation. Tissue was homogenized by hand in 1 ml of ice-cold 0.32 M sucrose buffered to pH 7.5 with HEPES using a glass/Teflon tissue grinder. After homogenization, the grinder was rinsed three times with 0.5 ml of buffered sucrose solution. A crude synaptosomal pellet was obtained by centrifugation at 12,000 x g for 20 min. After removal of the sucrose, each pellet was resuspended in 0.35 ml (thalamus) or 0.8 ml (cortex) of load buffer (in mM: 140 NaCl, 1.5 KCl, 2 CaCl₂, 1 MgSO₄, 25 HEPES, and 22 glucose) and placed on ice until incubation with ⁸⁶RbCl. Thalamic and cortical synaptosomes were incubated with 4 µCi of ⁸⁶Rb⁺ for 30 min in a final volume of 35 µl of load buffer, after which samples were collected by gentle filtration onto a 6-mm-diameter Gelman-type A/E glass filter and washed once with 0.5 ml of load buffer. Filters containing the synaptosomes loaded with ⁸⁶Rb⁺ were transferred to a polypropylene platform and superfused for 5 min with effluent buffer (in mM: 135 NaCl, 1.5 KCl, 5 CsCl, 2 CaCl₂, 1 MgSO₄, 25 HEPES, 22 glucose, 50 nM tetrodotoxin, and 0.1% bovine serum albumin). A peristaltic pump applied buffer to the top of the synaptosome containing filter at a rate of 2 ml/min, and a second peristaltic pump set at a faster flow rate of 3 ml/min removed buffer from the bottom of the platform. The greater speed of the second pump prevented pooling of buffer on the filter. Effluent buffer was pumped through a 200 µl Cherenkov cell and into a ß-Ram detector (IN/US Systems, Tampa, FL). Radioactivity was measured for 3 min with a 3 s detection window providing 60 data points for each superfusion. Each aliquot was stimulated by 1 of 10 different acetylcholine (ACh) concentrations, with a 5 s exposure for each concentration.

Neurotransmitter release. After a mouse was killed by cervical dislocation, its brain was removed and placed immediately on ice. The striatal and hippocampal tissue dissected from each mouse was homogenized in 0.5 ml of ice-cold 0.32 M sucrose buffered with 5 mM HEPES, pH 7.5. A crude synaptosomal pellet was obtained by centrifugation at 12,000 x g for 20 min at 4°C. The pellets were resuspended in 0.8 ml of "uptake buffer" (in mM: 128 NaCl, 2.4 KCl, 3.2 CaCl_2, 1.2 KH₂PO₄, 1.2 MgSO₄, 25 HEPES, pH 7.5, 10 glucose). For [³H]dopamine uptake, the buffer was supplemented with 1 mM ascorbic acid and 0.01 mM pargyline. For [³H]GABA uptake, the buffer was supplemented with 1 mM amino-oxyacetic acid.

For [³H]GABA uptake, the crude hippocampal synaptosomes were incubated for 10 min at 37°C. [³H]GABA and unlabeled GABA were then added to final concentrations of 0.1 and 0.25 µM, respectively, and the suspension was incubated for another 10 min.

For [³H]dopamine uptake, striatal synaptosomes were incubated at 37°C in uptake buffer for 10 min before addition of 0.1 µM [³H]dopamine and the suspension was incubated for an additional 5 min (Grady et al., 1997). Because ACh was used as the agonist, the synaptosomes were treated with diisopropyl fluorophosphate (10 µM) during the last 5 min of the uptake procedure.

All release experiments were conducted at room temperature using methods described previously (Grady et al., 1997, 2001). In brief, after ³H-neurotransmitter uptake was complete, aliquots of synaptosomes (80 µl) were distributed onto filters and perfused with perfusion buffer [uptake buffer containing 0.1% bovine serum albumin, 1 µM atropine, and either 10 µM nomifensine for [³H]dopamine release or 0.1 µM NNC-711 [1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride] for [³H]GABA release] at 0.7 ml/min for 10 min before fractions were collected. Fractions were collected every 10 s into 96-well plates using a Gilson FC204 fraction collector (Gilson, Middleton, WI). Optiphase Supermix Scintillation Fluid (150 µl) was added to each well, and radioactivity was determined using a 1450 MicroBeta Trilux Scintillation Counter (PerkinElmer Life Sciences, Boston, MA). Instrument efficiency was 40%.

Basal ⁸⁶Rb⁺ efflux or neurotransmitter release was calculated by nonlinear least-squares curve fit of the samples before and after the ACh-stimulated peak to a first-order equation: C_t = C₀ x e^–kt, where C_t is baseline counts at time t, C₀ is baseline counts at t = 0, and k is the first-order constant for basal efflux or release. ACh-stimulated response was calculated as the difference between the actual sample counts after ACh stimulation and the estimated basal counts. ACh-stimulated response at each concentration was normalized by dividing by the calculated basal efflux; thus, ACh-stimulated ⁸⁶Rb⁺ efflux or transmitter release is expressed as the response relative to baseline. EC₅₀ values were calculated using the following Hill equation: E_ACh = E_max x [ACh]^nH/([ACh]^nH + EC₅₀), where E_ACh is the response measured after stimulation with ACh concentration [ACh], and n_H is the Hill coefficient. Concentration–response experiments were fitted with one Hill equation for transmitter release experiments and the sum of two Hill equations for ⁸⁶Rb⁺ efflux experiments.

¹²⁵I-Epibatidine binding. Epibatidine binding was used for quantitating nicotinic receptor levels in various brain regions of WT, Het, and Hom 4YFP mutant mice as described previously (Whiteaker et al., 2002). Tissue was collected from the thalamus, cerebral cortex, hippocampus, and striatum, and homogenized in ice-cold 0.1x binding buffer (in mM: 14.4 NaCl, 0.2 KCl, 0.2 CaCl₂, 1 MgSO₄, and 2 HEPES, pH 7.5). Homogenates were washed three times by centrifugation (12,000 x g; 15 min; 4°C) and resuspension into 0.1x binding buffer and stored at –70°C. For saturation binding experiments, ¹²⁵I-epibatidine concentrations were varied between 3 and 400 pM. The 200 pM ¹²⁵I-epibatidine (a saturating concentration) was used in inhibition binding experiments. Cytisine inhibition of ¹²⁵I-epibatidine was measured using drug concentrations of 0.1–3000 nM plus a no-drug control. The amount of membrane protein added was chosen to produce maximum ligand binding to the tissue of <1000 Bq/well (<5% of total ligand added, minimizing the effects of ligand depletion) and a minimum of 350 Bq of specific binding. Membranes were incubated for 2 h at 22°C in 30 µl of binding buffer. Nonspecific binding was determined in the presence of 1 mM (–)nicotine tartrate and fell in the range of 20–50 Bq. Incubations were terminated by filtration onto Gelman GF/F fiber filters using a cell harvester, followed by six washes with ice-cold binding buffer. Bound ligand was measured at 80–85% efficiency using a Packard Cobra gamma counter (PerkinElmer). Specific binding was calculated for each region from every animal by subtraction of the relevant nonspecific binding value. Saturation binding parameters were calculated by fitting data to the following Hill equation: B = B_maxLⁿ/(Lⁿ + K_Dⁿ), where B is the binding at the free ligand concentration L, B_max is the maximum number of binding sites, K_D is the equilibrium binding constant, and n is the Hill coefficient. Inhibition of ¹²⁵I-epibatidine binding was calculated using a two-site fit: B = B₁/(1 + (I/IC_50-1)) + B₂/(1 + (I/IC_50-2)), where B was ligand bound at inhibitor concentration I, and B₁ and B₂ represent ¹²⁵I-epibatidine binding to sites sensitive to cytisine inhibition with IC_50-1 and IC_50-2 (cytisine-sensitive and cytisine-resistant sites, respectively).

¹²⁵I-mAb 299 labeling. Three mice of each genotype were killed by cervical dislocation and decapitated, and whole brains were removed. Brains were rapidly frozen in isopentane (–35°C, 10 s) and stored, wrapped in aluminum foil, at –70°C until sliced. Coronal tissue sections (14 µm thick) were obtained using a Leica (Nussloch, Germany) CM 1850 cryostat/microtome, and thaw mounted onto SuperFrost glass slides (Fisher Scientific, Pittsburgh, PA). Sections were stored, desiccated, at –70°C until analyzed.

¹²⁵I-mAb autoradiography was performed as described (Whiteaker et al., 2006). Sections were allowed to equilibrate to room temperature, and then rehydrated for 15 min in 100 mM NaCl, 10 mM sodium phosphate buffer, pH 7.5 (PBS). The hydrated sections were then overlaid with ¹²⁵I-mAb 299 (0.3 nM) in PBS supplemented with 10 mM NaN₃, 10% (v/v) normal rat serum, and 5% (w/v) protease-free bovine serum albumin, and incubated for 48 h (humidified chambers, 4°C). A set of 4 KO sections was incubated in parallel, for each experiment, to define nonspecific ¹²⁵I-mAb 299 labeling. Incubated sections were washed in four changes of PBS (30 min each; 22°C), and then air-dried and desiccated overnight under vacuum before exposure to Super Resolution phosphorimaging screens (PerkinElmer Life Sciences; 2–4 d exposure). Images were collected using a Packard Cyclone system (PerkinElmer Life Sciences) and quantitated using the associated OptiQuant software. Regions were identified by reference to an atlas (Paxinos and Franklin, 2004). Generally, six separate samples were taken for each region in each mouse (exceptions were made for very small regions such as the interpeduncular nucleus, where fewer than six samples were available). For large regions such as striatum, thalamus, and cortex, samples were taken throughout the rostral–caudal span of the structure, to ensure that regional heterogeneity did not influence the results. Labeling intensity was measured in terms of digital light units per square millimeter. Specific ¹²⁵I-mAb 299 labeling in each region was defined by subtracting labeling in the corresponding region of 4 KO brain sections. Studies on 4 knock-out mice with this procedure show the specificity of our method (Whiteaker et al., 2006).

Chronic nicotine administration. Chronic nicotine or saline was administered to mice using miniosmotic pumps (model 2002; Alzet, Cupertino, CA). On the day of pump implantation, saline or (–)-nicotine hydrogen tartrate (Sigma, St. Louis) was prepared freshly and loaded into the pump to deliver nicotine at 2 mg · kg^–1 · h^–1 [which provides maximal nAChR upregulation (McCallum et al., 2006) and also provides a blood concentration of 590 nM (Marks et al., 2004), near the peak concentration in the blood of smokers] or 0.4 mg · kg^–1 · h^–1 with a flow rate of 0.5 µl/h. Surgery was identical with the description of telemetry probe implantation.

Hot plate. For antinociception experiments, a hot plate was set at 55°C. A baseline response was determined for each mouse and a cutoff time of 60 s was instituted. The nociceptive time point was indicated by jumping, paw licking, or rapid thumping of the paw, at which point the mouse was immediately removed from the hot plate. Mice were allowed to recover for one-half of an hour before the mouse was tested again but with subcutaneous injection with one dose of (–)-nicotine at either 0, 1, or 1.5 mg/kg (prepared in saline). Five minutes after the injection, the mouse was placed again on the hot plate, and the time was noted for reaction to the nociceptive stimuli of the hot plate. Antinociceptive response was measured as percentage of maximal possible effect (%MPE) (Marubio et al., 1999). %MPE = [(test – control)/(cutoff – control)] x 100.

Sample preparation for immunohistochemistry and spectral imaging. At day 10 of chronic infusion of saline or nicotine via miniosmotic pumps, mice were anesthetized with halothane (2-bromo-2-chloro-1,1,1-trifluorothane) and subjected to cardiac perfusion first with 20 ml of PBS, pH 7.6, with heparin, second with 30 ml of 4% paraformaldehyde in PBS, pH adjusted to 7.6 with Na₂HPO₄, and finally with 20 ml of 5% sucrose in PBS, pH 7.6. Thus, animals were perfused and fixed while the miniosmotic pumps were still active. All solutions were on ice. Brains were dissected and then incubated in 30% sucrose in PBS, pH 7.6, for 3 d at 4°C. The brains were embedded in OCT medium (Tissue-Tek, Torrance, CA) and rapidly frozen in 2-methylbutane on dry ice. Coronal and sagittal sections were sliced by cryostat at 30 µm and mounted on slides. Slides with mounted brain sections were stored at –20°C. Before imaging, slices were rinsed with PBS, pH 7.6, and mounted with Mowiol and coverslipped.

Immunohistochemistry. Brain sections on slides were rinsed twice for 10 min with PBS, pH 7.6, and then permeabilized with 0.25% Triton X-100 in PBS for 5 min. Cultures were rinsed twice for 10 min with PBS and blocked with 10% donkey serum (Jackson ImmunoResearch, West Grove, PA) in PBS for 30 min. The primary antibody was diluted in 3% donkey serum in PBS and incubated for 1 h at 37°C. Brain sections were then washed three times for 5 min with PBS. The secondary antibody was diluted in 3% donkey serum in PBS and incubated for 1 h at 37°C. The incubation and rinsing steps were repeated for second primary antibody labeling when triple-label immunohistochemistry was performed. Finally, brain sections were washed three times for 5 min with PBS and mounted with Mowiol and coverslipped.

The following primary and corresponding secondary antibodies and their dilutions were used in this study: rabbit anti-MAP2 (microtubule-associated protein 2) polyclonal antibody at 1:50 (AB5622; Chemicon, Temecula, CA), CY5-conjugated donkey anti-rabbit (Jackson ImmunoResearch) at 1:100, mouse anti-GAD67 monoclonal antibody at 1:50 (MAB5406; Chemicon), CY5-conjugated donkey anti-mouse (Jackson ImmunoResearch) at 1:100, rabbit anti-tyrosine hydroxylase (Pel-Freez, Rogers, AR) at 1:50, CY5-conjugated donkey anti-rabbit (Jackson ImmunoResearch) at 1:100, sheep anti-tyrosine hydroxylase polyclonal (AB1542; Chemicon), and Cy3-conjugated donkey anti-sheep IgG (Jackson ImmunoResearch).

Spectral confocal imaging. For quantification of 4YFP fluorescence, images were collected from either of two microscopes equipped with spectrally resolved multiple detector systems. Each pixel of the X–Y image has complete spectral emission data, comprising the stack. With an inverted LSM 510 Meta laser-scanning confocal microscope (Zeiss) using a Fluar 20x, 0.75 numerical aperture (NA) oil immersion objective (Dickinson et al., 2001; Lansford et al., 2001; Nashmi et al., 2005), images were collected at wavelengths between 495 and 602 nm with bandwidths of 10.7 nm during excitation of YFP with the 488 nm line of an argon laser (1–10%; 23–114 µW). Pinhole was 1.68 Airy units and a Z resolution of 2.9 µm. Images were collected at a 12-bit intensity resolution over 1024 x 1024 pixels at a pixel dwell time of 6.4 µs.

Other spectral images were taken, with excitation at 488 nm, with an inverted Nikon C1si laser-scanning confocal microscope (Nikon, Tokyo, Japan) over 512 x 512 pixels at 12-bit intensity resolution using a 60x plan apochromat water (1.2 NA) objective. The pinhole was set to a nominal diameter of 61.3 µm. The GAT1-GFP images were excited by a 440 nm modulated diode laser at 20% power. stacks of images were acquired at wavelengths between 479 and 634 nm, at 5 nm steps.

In all cases, detector gains and laser attenuators were adjusted to lie within the linear range of the detector. The output of the appropriate laser was calibrated at each session with a power meter.

The 4YFP knock-in mice present the advantage that the fluorescent moiety appears at WT levels rather than overexpressed levels, but this limits the fluorescence signal. Some brain regions had significant amount of autofluorescence. We used the capabilities of spectral imaging to deconvolve specific YFP fluorescence from background autofluorescence at each pixel using a linear unmixing algorithm based on the spectral signatures of YFP and autofluorescence created from reference stack images of HEK293T cells expressing soluble YFP and a fixed brain section from WT littermates of 4YFP mice, respectively (Dickinson et al., 2001; Lansford et al., 2001; Nashmi et al., 2005).

Image analysis of cell counts, mean intensities, and area measurements were done with ImageJ, version 1.37, software (National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij/). Details of analyses of each region are shown in Tables 1 and 2. To calculate the mean spectrally unmixed YFP fluorescence from a particular brain region from a Hom or Het mouse, the corresponding spectrally unmixed residual background fluorescence from the same brain region of a WT mouse was subtracted.

View this table: [in this window] [in a new window]   Table 1. 4YFP intensities in ventral midbrain neurons

 

View this table: [in this window] [in a new window]   Table 2. 4YFP intensities in various brain regions

 
Calcium imaging in cultured ventral midbrain neurons. Changes in [Ca²⁺]_i were measured in ventral midbrain neuronal cultures using the ratiometric fura-2 method as described previously (Nashmi et al., 2003; Tapper et al., 2004; Fonck et al., 2005). Ventral midbrain tissue [including the substantia nigra (SN) and ventral tegmental area (VTA)] obtained from embryonic day 14 embryos was digested with 1 mg/ml papain, and cells were mechanically separated by gentle pipetting. Cells were plated onto 35 mm dishes with glass coverslip bottoms in Neurobasal culture medium containing 2% B27 supplement, 0.5 mM Glutamax, and 5% horse serum, all obtained from Invitrogen. Cell cultures were kept in a temperature-, humidity-, and CO₂/O₂-controlled incubator. Measurements were made at 28–35 d in culture. For experiments with chronic nicotine, 10 nM was applied for 3 d and removed 1 h before the imaging experiments. During this 1 h period, cells were loaded with 1.25 µM fura-2 AM (Invitrogen) in a 0.01% pluronic acid solution for 30 min at room temperature (22 ± 2°C) and in the dark. During experiments, cultures were continually perfused with an extracellular solution (in mM: 150 NaCl, 10 HEPES, 10 glucose, 4 KCl, 2 CaCl₂, and 2 MgCl₂). Various channel blockers to suppress nonspecific responses were included in the extracellular solution as follows (in µM): 0.5 TTX (voltage-gated Na⁺ channels), 0.5 atropine (muscarinic receptors), 10 CNQX (AMPA and kainate receptors), 20 bicuculline (GABA_A receptors), 50 AP-5 (NMDA receptors), and 0.01 methyllycaconitine (MLA) (7 receptors). Ascorbic acid at 1 mM was also added to the extracellular solution to protect neurons from photodamage. Imaging was performed with an inverted fluorescence microscope (IX71; Olympus, Melville, NY) using a 40x oil-immersed objective (UApo/340; 1.35 NA). To generate ratiometric estimates of [Ca²⁺]_i, pairs of images were obtained with alternate excitation wavelengths of 340/380 nm and emission wavelength of 510 nm. A U-tube connected to a pipette, placed 0.5 mm away from the targeted cell population, was used to deliver nicotine. Nicotinic receptors desensitize at agonist concentrations too low to cause appreciable activation; therefore, between applications, negative pressure was maintained on the U-tube to prevent nicotine from leaking out of the pipette. Nicotine was applied onto cultured cells when solution flow in the U-tube was momentarily (2 s) reversed by stopping outflow with a computer-controlled valve. Images were captured with a 16 bit CCD camera (Cascade 650; Photometrics, Tucson, AZ) and analyzed with Slidebook 4.1 imaging software (Intelligent Imaging Innovations, Denver, CO).

Whole-cell patch-clamp electrophysiology of cultured ventral midbrainneurons. Cultures were prepared and maintained as described in the fura-2 experiments. Whole-cell recordings were performed with glass electrodes (2–5 M) filled with an internal solution [in mM: 88 KH₂PO₄, 4.5 MgCl₂, 0.9 EGTA, 9 HEPES, 0.4 CaCl₂, 14 creatine phosphate, 4 Mg-ATP, and 0.3 GTP (Tris salt), pH 7.4 with KOH] (Nashmi et al., 2003; Fonck et al., 2005). Channel blockers to suppress nonspecific responses were included in the extracellular solution (in µM: 0.5 TTX, 0.5 atropine, 10 CNQX, 20 bicuculline, 50 AP-5, and 0.01 MLA). Recordings were made using an Axopatch 1D (Molecular Devices, Union City, CA) amplifier, low-pass filtered at 2–5 kHz, and digitized on-line at 20 kHz (pClamp 8; Molecular Devices). The membrane potential was held at –70 mV. The 3 µM ACh test pulses lasting 250 ms were delivered with a two-barrel glass theta tube pulled from borosilicate tubing and connected to a piezoelectric translator (LSS-3100; Burleigh Instruments, Fishers, NY).

Patch-clamp electrophysiology on midbrain slices. The patch-clamp experiments were conducted blind to the contents of the osmotic minipumps (nicotine vs saline) until the conclusion of recording from each group of implanted mice. P30–P38 mice were deeply anesthetized with sodium pentobarbital (40 mg/kg). Then, cardiac perfusion was performed using ice-cold glycerol-based artificial CSF (gACSF), containing 252 mM glycerol, 1.6 mM KCl, 1.2 mM NaH₂PO₄, 1.2 mM MgCl₂, 2.4 mM CaCl₂, 18 mM NaHCO₃, and 11 mM glucose, and oxygenated with 95%O₂/5%CO₂. After decapitation, the brain was removed and kept in gACSF (0–4°C), and was cut into 250–300 µm thick slices in the coronal plane (DTK-1000; Microslicer; Ted Pella, Redding, CA). The slices were allowed to recover for at least 1 h in regular artificial CSF (aCSF), containing 126 mM NaCl, 1.6 mM KCl, 1.2 mM NaH₂PO₄, 1.2 mM MgCl₂, 2.4 mM CaCl₂, 18 mM NaHCO₃, and 11 mM glucose, and oxygenated with 95% O₂/5% CO₂.

A single slice was transferred to the 0.8 ml recording chamber. Throughout the experiments, the bath was continually perfused with aCSF (1.5–2.0 ml/min). Cells were visualized with an upright microscope (BX 50WI; Olympus) and near-infrared illumination. The patch electrodes had a resistance of 5–8 M when filled with pipette solution: 135 mM potassium gluconate, 5 mM EGTA, 0.5 mM CaCl₂, 2 mM MgCl₂, 10 mM HEPES, 2 mM Mg-ATP, and 0.1 mM GTP, pH adjusted to 7.2 with Tris base, osmolarity adjusted to 280–300 mOsm with sucrose. Whole-cell current-clamp was performed at 32°C with a MultiClamp 700B amplifier (Molecular Devices), a Digidata 1200A A/D converter, and pCLAMP 9.2 software (Molecular Devices). Data were sampled at 10 kHz and filtered at 2 kHz. The junction potential between the patch pipette and the bath solution was nulled just before gigaseal formation.

We verified the putative GABAergic neurons in the substantia nigra pars reticulata (SNR) and putative dopaminergic neurons in the substantia nigra pars compacta (SNC) (supplemental Fig. S6, available at www.jneurosci.org as supplemental material), according to generally accepted criteria (Lacey et al., 1989; Wooltorton et al., 2003): (1) narrow spikes in putative SNR GABAergic neurons, broad spikes in putative SNC dopaminergic neurons (supplemental Fig. S6A, available at www.jneurosci.org as supplemental material); (2) rapid firing in putative SNR GABAergic neurons, but slow firing in putative SNC dopaminergic neurons (supplemental Fig. S6B, available at www.jneurosci.org as supplemental material); (3) putative SNC dopaminergic neurons, but not putative SNR GABAergic neurons, express hyperpolarization induced cationic current (I_h) (supplemental Fig. S6C, available at www.jneurosci.org as supplemental material). In addition, in select experiments putative SNR GABAergic neurons and putative SNC dopaminergic neurons were respectively inhibited or disinhibited by DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-ol) (1 µM), a µ-opioid receptor agonist; and putative SNC dopaminergic neurons, but not putative SNR GABAergic neurons (supplemental Fig. S6D, available at www.jneurosci.org as supplemental material), are inhibited by quinpirole (0.2 µM), a D₂/D₃ receptor agonist (Lacey et al., 1989; Wooltorton et al., 2003) (supplemental Fig. S6E, available at www.jneurosci.org as supplemental material). Given the good agreement between these criteria and the localization of these neurons (SNC vs SNR), we drop the term "putative," and subsequently, neurons were routinely characterized by location and spike duration.

To examine the function of nAChRs in SNR GABAergic neurons, 1 µM nicotine was focally puffed (5 s), using a picospritzer driven pipette attached to a piezoelectric translator (Tapper et al., 2004). The pipette was moved within 20 µm of the recorded cell over a period of 250 ms starting 300 ms before drug application. Nicotine was then puffed at 10–20 psi for 5 s. Fifty milliseconds after the puff, the glass pipette was retracted over a period of 250 ms. For dopaminergic recordings of nicotinic responses, because we could not determine the connectivity between GABAergic to dopaminergic neurons, it was inappropriate to use a focal drug applicator such as a picospritzer. Therefore, on recording of dopaminergic neurons, nicotine (1 µM) was bath applied for 5 min. This enabled nicotinic receptors to be activated on all GABAergic and dopaminergic neurons within the slice.

Long-term potentiation electrophysiology. Adult Hom 4YFP mice (2–3 months), treated with saline or nicotine for 10 d, were killed by cervical dislocation, and the brain was rapidly removed (<50 s) and placed in aerated (95% O₂/5% CO₂) ice-cold aCSF of the following composition (in mM): 124 NaCl, 3 KCl, 2.4 CaCl₂, 1.3 MgSO₄, 1.25 NaH₂PO₄, 26 NaHCO₃, and 10 glucose. After 4 min, the brain was hemisected and glued with cyanoacrylate glue dorsal side down and rostral side closest to the blade. Horizontal sections (400 µm) of the hippocampus were made with a vibratome (DSK Microslicer; model DTK-1000; Ted Pella) and allowed to recover for >1 h at room temperature. The slice was transferred to a submersion chamber (catalog #65-0073 and 65-0074; Harvard Apparatus, South Natick, MA) maintained at 32°C. The GABA_A receptor blocker, bicuculline (10 µM), was added to the aerated aCSF during recording to relieve the strong GABAergic inhibition of the dentate gyrus (Colino and Malenka, 1993; Yun et al., 2005). Thus, brain tissue was removed from contact with the miniosmotic pump for periods of 2–8 h.

Stimulation of the medial perforant path was provided by bipolar stimulating electrodes fabricated from Formvar-coated Nichrome wires (50 µm bare diameter; catalog #762000; A-M Systems, Carlsborg, WA). Stimulating and recording electrodes were positioned in the suprapyramidal limb of the dentate gyrus in the middle one-third of the molecular layer to stimulate and record dendritic field EPSPs (fEPSPs). Recording electrodes (2–6 M) were pulled from borosilicate pipettes (catalog #1B150F-4; WPI, Sarasota, FL) and filled with aCSF. Stimulating electrodes were placed 200 µm from recording electrodes. Responses were recorded on an Axoclamp-2A amplifier (Molecular Devices), amplified 1000x, low-pass filtered at 3 kHz, and digitized at 10 kHz. An input–output relation for fEPSP slope was measured by varying the stimulus intensity (5–100 µA; 100 µs duration). The stimulus intensity that elicited 40% of the maximal slope was applied as test pulses and tetanic trains during long-term potentiation (LTP) experiments. Paired-pulse stimuli with a 50 ms interval were applied to check for fEPSP paired-pulse inhibition, indicating that stimulating and recording electrodes were located in the medial perforant path (Colino and Malenka, 1993; Yun et al., 2005). A steady baseline of fEPSP slopes was recorded for 20 min with a stimulation rate of 1 in 30 s. LTP was induced by three trains of 100 Hz, each 1 s in duration, at 4.5 min intervals. Responses were recorded thereafter for a period of 60 min. The magnitude of LTP was measured by averaging the percent increase of the fEPSP slope compared with baseline at 40–60 min after tetanic stimuli.

Statistical analysis. All values are reported as mean ± SEM. Significant differences (at p < 0.05) were determined between two groups using a t test for continuous data meeting parametric assumptions of normality and equal variance. Otherwise, the Mann–Whitney rank sum test was used for nonparametric data.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
We created a knock-in mouse strain whose 4 subunits are replaced by subunits containing yellow fluorescent protein (4YFP subunits), which was inserted in-frame within the M3–M4 cytoplasmic loop of 4 (Nashmi et al., 2003). Previous data in transfected systems show that receptor function remains unaffected by this insertion (Nashmi et al., 2003); the knock-in strategy now places the 4YFP subunit under the control of the same promoters, enhancers, and trafficking mechanisms as WT 4 subunits, and provides cellular and subcellular resolution in the normal physiological milieu of the intact animal.

Characterizing the 4YFP mice: normal nicotinic receptor function
We proceeded to quantify the relative amount of 4 nAChR subunits in various brain regions. Figure 1 displays a series of montages of confocal images of a coronal brain section showing the overall distribution of 4 at the level of the hippocampus/thalamus (Fig. 1E; bregma, –1.58). We imaged stacks over a spectral range (supplemental Fig. S1, available at www.jneurosci.org as supplemental material), enabling us to isolate specific YFP fluorescence from autofluorescent background using reference spectra of YFP (cells transfected with soluble YFP) and autofluorescence (a fixed brain slice of a wild-type mouse). Summarizing data from 16 different brain regions in WT, Het, and Hom mice, the highest level of 4 expression was in the medial habenula, followed by the thalamus, SNC, and the interpeduncular nucleus. Moderate levels of 4 expression were found in the superior colliculus, cerebral cortex, retrosplenial granular cortex, and subiculum. This pattern agrees well with previous data (Swanson et al., 1987; Marks et al., 1992; Whiteaker et al., 2006). When 4YFP fluorescence is totaled for these 16 brain regions, the Het mouse brain fluorescence is approximately one-half (45%) that of Hom, consistent with straightforward gene dosage.

View larger version (43K): [in this window] [in a new window]   Figure 1. Construction of the 4YFP mouse and whole-brain fluorescence. A, Genomic map of the wild-type mouse 4 nAChR allele located on chromosome 2 (top). Targeting vector was constructed with the 4 gene including exons 5 and 6 and the YFP mutation inserted in the pKO V907 vector (second line; note an inserted HA-epitope tag immediately upstream of YFP is not shown). The neomycin-resistance gene (neo) flanked by loxP sites and the diphtheria toxin A (DT) gene were also inserted as positive and negative selection markers. Map of the recombined 4 gene (third line). The neo cassette was deleted by electroporating the neo-intact ES cells with a cytomegalovirus-Cre plasmid to generate neo-deleted ES cell lines (fourth line). Restriction sites: A, AvrII; B, BstEII; Sc, SacI; Sp, SpeI. B, Chromatogram showing the start of the YFP sequence and the upstream HA-epitope tag in the 4 nAChR gene. C, PCR genotyping of WT, Het, and Hom 4YFP mice. D, RT-PCR showing 4 mRNA expression from combined thalamic–ventral midbrain tissue from Het and WT mice. The higher molecular weight band in the Het is message from the 4YFP allele. Negative controls, lacking reverse transcriptase in the RT-PCR, were devoid of bands. E, Montage of tiled confocal images of a coronal brain section at the hippocampal level from a Hom 4YFP mouse showing overall brain distribution of 4YFP. F, Quantification of 4YFP fluorescence using spectral confocal imaging taken from various brain regions from brain sections of WT, Het, and Hom 4YFP mice. The horizontal bar plots show mean 4YFP intensity (±SEM) for Het and Hom mice for each brain region. Residual autofluorescence intensity from WT mice are shown in brackets and subtracted from the raw intensity values from Het and Hom. CauPut, Caudate–putamen; NuAcSh, nucleus accumbens shell; NuAcCo, nucleus accumbens core; VerDiaB, vertical diagonal band; Med Sep, medial septum; Cerebel, cerebellar cortex; S1 CerCort, primary somatosensory cortex of barrel and trunk; RGS Cer Cor, retrosplenial granular cerebral cortex; Sup Coll, superior colliculus; Red Nuc, red nucleus; Hip Neur, hippocampal interneurons; Hip PerfP, hippocampal medial perforant path; Thal, dorsolateral geniculate nucleus of thalamus; MHb, medial habenula; IPN, interpeduncular nucleus. The "Average" bars show the 4YFP fluorescence averaged for the various regions for both Het and Hom mice.

 
Detailed analyses of expression in ventral midbrain and in hippocampus are presented below (see Figs. 3–7); here, we present a survey of other areas. Most of the thalamus expressed strong 4YFP fluorescence (Fig. 1E). 4YFP was localized strongly in neuronal cell bodies, dendrites, and axons (supplemental Fig. S5, available at www.jneurosci.org as supplemental material). In addition to prominent fluorescence in the presumably glutamatergic neurons of most thalamic nuclei, we noted 4YFP in the cell bodies of GABAergic neurons of the reticular thalamus (supplemental Fig. S5, available at www.jneurosci.org as supplemental material). In most neocortical areas, neuronal cell bodies contained low 4YFP fluorescence levels (data not shown). However, significant 4YFP fluorescence was detected in diffuse axonal fibers innervating the cortex (data not shown). In the anterior cingulate cortex, two parallel medial bands of 4YFP fluorescence appeared outside the cell body layer (data not shown). 4YFP was also detectable in axonal fibers in the internal capsule (supplemental Fig. S5G, available at www.jneurosci.org as supplemental material), which comprise thalamic and cortical fibers.

We used a battery of functional, binding, immunolabeling, and behavioral assays to examine whether inserting YFP in the 4 nAChR subunit altered the function or expression in vivo. First, whole-cell patch-clamp recordings were performed on cultured ventral midbrain neurons from both Hom and WT mice. Nicotinic currents were elicited by application of a low (3 µM) and a nearly saturating dose (300 µM) of ACh, using a theta tube attached to a piezoelectric translator. Nicotinic currents were positively identified because the currents were reversibly blocked by the competitive antagonist DHßE (dihydro-ß-erythroidine) (1 µM) (supplemental Fig. S2A, available at www.jneurosci.org as supplemental material). Furthermore, other receptors and channels were blocked using atropine, TTX, AP-5, CNQX, bicuculline, and MLA. The average nicotinic peak current densities were about the same in WT as in Hom neuronal cultures (supplemental Fig. S2A, available at www.jneurosci.org as supplemental material).

Fura-2 imaging and dose–response relationships were performed on cultured ventral midbrain neurons. Various doses of ACh demonstrated nearly identical dose–response relationships of nicotinic responses between WT and Hom neuronal cultures in the presence of receptor and channel blockers that isolated nicotinic responses (supplemental Fig. S2B, available at www.jneurosci.org as supplemental material).

The effects of 4YFP expression on measures of nAChR levels and function in preparations from adult mouse brain are illustrated in Figure 2. Maximal response and EC₅₀ values for ACh-stimulated ⁸⁶Rb⁺ efflux measured in thalamus and cortex (data not shown), dopamine release in striatum, and GABA release in hippocampus were similar in 4YFP Hom and Het and WT mice (Fig. 2A). Total high-affinity ¹²⁵I-epibatidine binding as well as the cytisine-sensitive and cytisine-resistant components of these sites in striatum, cortex, thalamus, and hippocampus of Hom and Het 4YFP mice did not differ from that of WT mice (Fig. 2B), indicating that the expression of nicotinic binding sites was unaffected by YFP insertion. Consistent with these observations, binding of ¹²⁵I-mAb299, which specifically labels the 4 subunit (Whiteaker et al., 2006) was unaffected by YFP insertion (Fig. 2C).

View larger version (46K): [in this window] [in a new window]   Figure 2. Ion flux, transmitter release, agonist binding, and immunohistochemistry data. A, Left, Dose–response relationships for ACh evoked 86Rb+ efflux measurements from synaptosomes made from thalamic tissue from WT, Het, and Hom 4YFP mice. Data were fitted to two (H, L) components. For WT, EC50H = 2.6 ± 0.7 µM, EC50L = 79 ± 35 µM; Het, EC50H = 3.2 ± 0.4 µM, EC50L = 54 ± 9 µM; Hom, EC50H = 2.8 ± 2.7 µM, EC50L = 75 ± 110 µM. Middle, Right: Dose–response relationships of GABA (middle) and dopamine (right) release from the hippocampal and striatal synaptosomes, respectively, from WT, Het, and Hom 4YFP mice. Data were fitted to a single component including a Hill coefficient (n). GABA release: WT, EC50 = 3.7 ± 0.49 µM, n = 0.56 ± 0.25; Het, EC50 = 3.5 ± 1.34 µM, n = 0.94 ± 0.29; Hom, EC50 = 5.1 ± 1.8 µM, n = 1.0 ± 0.32. Dopamine release: WT, EC50 = 0.57 ± 0.09 µM, n = 0.93 ± 0.14; Het, EC50 = 0.56 ± 0.05 µM, n = 0.97 ± 0.09; Hom, EC50 = 0.61 ± 0.18 µM, n = 1.0 ± 0.29. B, Concentration dependence of 125I-epibatidine binding in the membrane fraction of thalamus, cortex, striatum, and hippocampus from WT, Het, and Hom 4YFP mice. 125I-Epibatidine binding was performed in the presence and absence of cytisine to determine the cytisine-sensitive and cytisine-resistant sites. The cytisine-sensitive binding sites are likely 4ß2 nAChRs. C, Quantitative autoradiography of 125I-mAb 299 labeling in WT, Het, and Hom 4YFP mice. Representative coronal sections are shown at the hippocampal level. Nonspecific labeling was defined in parallel sets of sections from 4–/– mice, and was subtracted from total signal to define regional specific labeling. The histogram shows mean regional specific labeling densities (±SD) for each genotype. Abbreviations are as in Figure 1H.

 
Previous work showed that 4* nAChRs mediate nicotine-induced antinociception on the hot plate (Marubio et al., 1999; Damaj et al., 2007). WT, 4YFP, and 4 KO (Ross et al., 2000) mice showed similar baseline reaction times to sensing pain on the hot plate. Similar to the previous studies (Marubio et al., 1999; Damaj et al., 2007), we found that 4 KO mice (Ross et al., 2000) are relatively resistant to nicotine-induced antinociception, whereas 4YFP Hom, Het, and WT mice showed similar pharmacological sensitivity to nicotine-induced antinociception (supplemental Fig. S3, available at www.jneurosci.org as supplemental material). In data not shown, we also found that nicotine produces hypothermia equally in 4YFP Hom, Het, and WT mice.

Thus, using electrophysiology, fura-2 imaging, radiolabeled agonist binding, radiolabeled Rb⁺ efflux, neurotransmitter release, immunolabeling, and behavioral measurements, we found that 4YFP nAChRs in knock-in mice functioned and expressed like wild-type receptors. These data form the basis for examining changes produced by chronic exposure to nicotine.

Nicotine-induced 4YFP upregulation in midbrain GABAergic neurons
Dopaminergic reward systems include highly nicotine-sensitive neurons in the VTA and SNC (Stolerman and Shoaib, 1991; Mirenowicz and Schultz, 1996; Schultz et al., 1998; Hyman et al., 2006), and an accurate map of high-sensitivity nicotinic receptor expression in this region would further our understanding of the precise actions of nicotine. Therefore we labeled specific neuronal subpopulations within this region using cell-type specific markers, specifically dopaminergic neurons (tyrosine hydroxylase) versus GABAergic neurons (GAD67).

Figure 3 shows images of 4 expression in the ventral midbrain with tyrosine hydroxylase double label for dopaminergic neurons; triple labeling with GAD67 was also performed. The tabulated data (Tables 1, 2) show that nearly all neurons in the SNC are dopaminergic and >99% contain 4, as expected. In the VTA, 90% of neurons are dopaminergic, whereas 10% are GABAergic, as expected (Johnson and North, 1992). Of the GABAergic VTA neurons, 92% had detectable levels of 4YFP fluorescence (Fig. 4), extending previous data (Yin and French, 2000; Klink et al., 2001; Mansvelder et al., 2002). In the SNR, 99% of the GABAergic neurons contained 4, extending previous data (Klink et al., 2001) (Fig. 4) (described in more detail below). 4YFP fluorescence in dopaminergic neurons of the VTA and SNC had approximately twice the intensity of that found in GABAergic neurons in the SNR (Fig. 3B–D); but these data show, for the first time, that nearly all VTA and SNR GABAergic neurons express 4* receptors. 4YFP was also found in dopaminergic and nondopaminergic fibers of the dorsal striatum and was also detectable in dopaminergic fibers innervating the nucleus accumbens (supplemental Fig. S4, available at www.jneurosci.org as supplemental material).

View larger version (54K): [in this window] [in a new window]   Figure 3. Staining and quantification of dopaminergic and GABAergic 4YFP-containing neurons in the SN and VTA. A, Double labeling showing 4YFP (green) in most TH+ dopaminergic (red) neurons in the VTA and SNC. Intense 4YFP labeling is found in the interpeduncular nucleus (IPN) and light 4YFP fluorescence is found in the SNR. B, Triple labeling at higher magnification showing strong 4YFP (green) fluorescence in dopaminergic neurons (TH; blue) of the SNC and moderate 4YFP fluorescence in GABAergic neurons (GAD67; red) of the SNR. C, D, Quantification of 4YFP intensities per TH+ dopaminergic and GAD67+ GABAergic neuron in either the VTA, SNC, or SNR are shown with box plots (C) and histogram distributions (D).

 

View larger version (35K): [in this window] [in a new window]   Figure 4. Histograms and cumulative plots showing changes in 4YFP fluorescence in dopaminergic and GABAergic neurons of the SN and VTA with chronic nicotine. Histogram and cumulative plots of the mean 4YFP fluorescence per TH+ (dopaminergic) or GAD67+ (GABAergic) neuron from 4YFP Hom mice treated either with chronic saline or chronic nicotine (2 mg · kg–1 · h–1) for 10 d. 4YFP fluorescence was imaged from VTA, SNC, and SNR from brain sections triple labeled with TH, GAD67, and 4YFP. Dopaminergic neurons in SNC and VTA show very little change in 4YFP. However, GABAergic neurons in VTA and SNR show a shift (increase) in 4YFP intensity with chronic nicotine.

 
We performed experiments to quantitate the effect of chronic nicotine on 4* receptor levels in 4YFP mice. Nicotine at 2 mg · kg^–1 · h^–1 or saline was infused for 10 d via miniosmotic pumps to 4YFP Hom mice; and 4YFP fluorescence was measured in various brain regions to examine potential changes in 4* nAChR levels (Tables 1, 2). Surprisingly, 4YFP fluorescence did not change significantly from control to nicotine-treated mice in dopaminergic neurons of both SNC (106 ± 2%) and VTA (109 ± 3%). However, GABAergic neurons of both SNR (146 ± 4%) and VTA (136 ± 7%) showed significant (p < 0.001 and p < 0.002, respectively) increases in 4YFP fluorescence (Tables 1, 2; Fig. 4). With a lower dose of nicotine (0.4 mg · kg^–1 · h^–1), there was also a larger increase in 4YFP fluorescence in GABAergic neurons of SNR (117 ± 2%) compared with dopaminergic neurons of VTA (102 ± 4%) and SNC (110 ± 3%) (data not shown). These data in midbrain indicate differential receptor level changes in response to chronic nicotine, specific to neuronal subpopulations.

We acknowledge that the brain slice patch-clamp studies (described below) were performed on mice 30–38 d of age, and the experiments of Tables 1 and 2 on mice 67–69 d of age. To diminish the possibility that these two age groups respond differently to chronic nicotine, we performed pilot experiments on fluorescent imaging in 4YFP mice 42–52 d of age. We observed selective upregulation of 4YFP fluorescence in GABAergic but not dopaminergic neurons, as in Tables 1 and 2.

Chronic nicotine increases firing frequency in GABAergic, but not dopaminergic neurons, in response to acute nicotine
Because it is crucial to investigate the functional state of upregulated receptors, we sought to determine how neuronal signaling changed in these chronically treated circuits. We performed direct electrophysiological experiments on GABAergic compared with dopaminergic neurons in midbrain slices. The electrophysiological criteria for distinguishing dopaminergic versus GABAergic cells in rodent VTA are now uncertain (Ford et al., 2006; Margolis et al., 2006); we therefore studied neurons in the substantia nigra, where the dopaminergic and GABAergic cells occupy distinct subnuclei (SNC and SNR, respectively). We also found that SN neurons maintained their health much better than VTA neurons in slices from young animals that had been implanted for 10 d with osmotic minipumps. The recorded neurons in SNR and SNC did display the respective electrophysiological and pharmacological characteristics thought to distinguish GABAergic and dopaminergic neuron types (i.e., action potential duration, firing frequency, presence of I_h, ef