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

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 α4YFP (see below), α4(Leu9′Ala) (Tapper et al., 2004), α4 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 α4YFP 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.

α4YFP mouse construction.

The strategy used to generate the α4YFP 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 α4 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 α4 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 α4 (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 α4YFP 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 α4(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 α4YFP 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 × 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 α4YFP 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 α4YFP 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 α4YFP 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 α4YFP 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) α4YFP 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 × 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 β-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 × 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 × 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 × [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 α4YFP 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 0.1× 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 × g; 15 min; 4°C) and resuspension into 0.1× 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 α4 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 α4 KO brain sections. Studies on α4 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)] × 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 α4YFP 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 λ stack. With an inverted LSM 510 Meta laser-scanning confocal microscope (Zeiss) using a Fluar 20×, 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 × 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 × 512 pixels at 12-bit intensity resolution using a 60× 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. λ 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 α4YFP 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 λ stack images of HEK293T cells expressing soluble YFP and a fixed brain section from WT littermates of α4YFP 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.

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) (α7 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 40× 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 MΩ) 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 MΩ 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 α4YFP 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 MΩ) 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 1000×, 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.