Timed pregnant Wistar female rats arrived at 5 d of gestation (Harlan, Horst, The Netherlands) and were housed individually in Macrolon cages under standard conditions and a reversed day-night cycle (lights on 7 PM–7 AM). On delivery, litters were culled to 8 pups/mother and preferably consisted of males only, but only occasionally were matched with females. At P21, animals were weaned and housed 2 rats/cage. Only males were used in these experiments. The experiments were approved by the Animal Users Care Committee of Vrije Universiteit (Amsterdam, The Netherlands).
Animals were injected subcutaneously with either nicotine (0.4 mg/kg, calculated as the base ([−]nicotine hydrogen tartrate salt; Sigma-Aldrich, St. Louis, MO, USA) or saline 3×/d (10 AM, 1 PM, and 3 PM) for 10 d (n=10 animals/group). A second control group consisted of animals that were not injected, but handled once a week. Nicotine was administered from P34 to P43 (adolescence) or P60 to P69 (adulthood). Saline and no-injection controls were littermates of nicotine-exposed animals in both age groups. Animals were decapitated without anesthesia by an experienced technician on P34 (before nicotine exposure), on the first day of withdrawal (P44/P70) and 5 wk following nicotine exposure (P78/P104). Following decapitation, the brains were removed and quickly frozen in ice-cold isopentane before storage (−80°C). For measurement of plasma nicotine and cotinine levels, 2 different groups of animals (n=8/group, adolescent and adult animals) were injected with nicotine and decapitated at 3 time points: 30 min following the first injection (P34/P60; 10:30 AM), 30 min following the third injection (P34/P60; 3:30 PM), and at the first withdrawal day (P44/P70). Following decapitation, trunk blood was collected and centrifuged at 600 g for 10 min to obtain plasma.
Plasma nicotine and cotinine levels
Extraction of nicotine and cotinine was done as described by O’Dell et al.
) with slight modifications. In short, 100 μl of heparinized plasma was spiked with 2-phenylimidazole to verify extraction efficiency. To this, 20 μl of 20% NaOH was added, before 400 μl of tert
-butyl methyl ether was added. After vortexing and centrifugation at 10,000 g
(3 min), the organic phase was transferred to a new tube. The extraction was repeated with 200 μl tert
-butyl methyl ether. Then MgSO4
was added to pellet any remaining proteins. Following vortexing and centrifugation, the aqueous phase was transferred to a new tube and evaporated to dryness under a gentle stream of nitrogen. The lyophilized samples were reconstituted in 25 μl mobile phase (acetonintrile/methanol/10 mM ammonium acetate, 53:32:15, v/v/v).
Liquid chromatography–electrospray ionization mass spectrometry (LC-ESI/MS)
A Shimadzu LC system of 2 pumps (LC-10ADvp), autosampler (SIL-10ADvp), system controller (SCL-10Avp), and degassing unit (DGU-14A; Shimadzu USA, Canby, OR, USA), was coupled to a Bruker micro-TOF-Q instrument (Bruker Daltonics, Bremen, Germany) equipped with an ESI source. Each LC separation lasted 15 min with a gradient:linear gradient (0% B to 100% B, 7 min), 3 min 100% B, and 5 min equilibration at 0% B, where A is 2 mM ammonium acetate (pH 6.8), and B is MeOH (flow rate 150 μl/min). For all separations, an XTerra MS C18 column was used (2.1×50 mm, 2.5 μm particle size). Positive-mode electrospray was performed at spray voltage 4.5 kV. Scanning was performed over an m/z range from 50 to 3000 Da. The instrument was calibrated by infusing 5 mM sodium formate in 50% MeOH with 0.1% FA at flow rate of 4 μl/min. The data were analyzed with Data Analysis 4.0 software (Bruker Daltonics).
Antibody production and characterization
The subunit-specific polyclonal antibodies were produced in rabbits against peptides derived from the C-terminal and/or intracytoplasmic loop regions of the rat, human, or mouse subunit sequences and affinity purified as described previously (29
). Two different peptides were chosen for all of the subunits: one located in the cytoplasmic loop between M3 and M4 (CYT), and the other located at the COOH-terminal (COOH). The antibodies raised against the peptides were purified on an affinity column made by coupling the corresponding peptide to cyanogen bromide-activated Sepharose 4B (Pharmacia, Uppsala, Sweden) according to the manufacturer’s instructions. Antibody specificity was checked by means of quantitative immunoprecipitation or immunopurification experiments using nAChRs from different areas of the CNS of wild-type α4, α5, α6, β2, β3, and β4 (+/+) and null mutant (−/−) mice, which allowed selection of antibodies specific for the subunit of interest, and assessing the immunoprecipitation capacity of each antibody. For full characterization of nAChR subunit antibodies, see Supplemental Table 1 in Grady et al.
Preparation of membranes and 2% Triton X-100 extracts
The mPFC (infralimbic and prelimbic cortex) and caudal (occipital) cortex were removed freehand at −20°C from 1-mm-thick slices. Dissected material was stored at −80°C until further use. In every experiment, tissue from 2 rats (0.04–0.05 g) from each experimental group was pooled and homogenized in 10 ml of 50 mM Na phosphate (pH 7.4), 1 M NaCl, 2 mM EDTA, 2 mM EGTA, and 2 mM PMSF using a potter homogenizer, and homogenates were diluted and centrifuged at 60,000 g (1 h). Total membrane homogenization, dilution, and centrifugation procedures were repeated, after which the cell membrane-enriched pellets were collected; rapidly rinsed with 50 mM TrisHCl (pH 7), 120 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, and 2 mM PMSF; and resuspended in the same buffer containing a mixture of 20 μg/ml of each of the following protease inhibitors: leupeptin, bestatin, pepstatin A, and aprotinin. Triton X-100, at a final concentration of 2%, was added to the washed membranes to extract membrane receptors and was incubated at 4°C (2 h). The extracts were centrifuged at 60,000 g (1.5 h) and recovered. An aliquot of the supernatants was collected for protein measurement (BCA protein assay; Pierce, Rockford, IL, USA) with bovine serum albumin as standard.
125I-αBgt (specific activity 200 Ci/mmol; PerkinElmer, Boston, MA, USA) binding experiments were performed by overnight incubation of mPFC and occipital cortex membranes dissolved in 50 mM Na phosphate (pH 7.4), 1 M NaCl, 2 mM EDTA, 2 mM EGTA, and 2 mM PMSF in a final volume of 100 μl containing a saturating concentration (5 nM) of 125I-αBgt at 20°C and 2 mg/ml bovine serum albumin. Nonspecific binding was determined in parallel by means of incubation in the presence of 1 μM unlabeled α-bungarotoxin. After incubation, the samples were filtered on GFC filters presoaked in polyethylenimine through a harvester apparatus, and the bound radioactivity was directly counted in a γ counter.
To ensure that the α7-containing receptor subtypes did not contribute to 3
H-Epi (specific activity 50–66 Ci/mmol; GE Healthcare, Little Chalfont, UK) binding to solubilized receptors (30
), the binding in the extract and immunoprecipitation experiments was performed in the presence of 2 μM 125
I-αBgt, which specifically binds to α7*-nAChRs (and thus prevents 3
H-Epi binding to these sites). Binding to the 2% Triton X-100 extracts from mPFC or occipital cortex was carried out overnight by incubating aliquots of extracts with 2 nM 3
H-Epi at 4°C. Nonspecific binding (on average 5–10% of total binding) was determined in parallel samples containing 100 nM unlabeled epibatidine. After incubation, the extracts were diluted to 200 μl with H2
O and applied to a 500 μl DE52 ion-exchange resin (Whatman, Maidstone, UK). After being washed with 10 ml of wash buffer (10 mM Na phosphate, pH 7.4; 50 mM NaCl; and 0.1% Triton X-100) to remove unbound 3
H-Epi, the bound receptors were eluted with 2 ml of 1 N NaOH and, after addition of 10 ml of the liquid scintillation Filter-Count solution (PerkinElmer), counted in a β counter.
Immunoprecipitation of 3H-Epi-labeled receptors by subunit-specific antibodies
Tissue extracts (100 μl) were preincubated with 2 μM 125
I-αBgt, labeled with 2 nM 3
H-Epi, and incubated overnight with a saturating concentration (20 μg) of anti-subunit-specific affinity-purified IgG produced and characterized by us (see Grady et al.
, ref. 29
The immunoprecipitation was recovered by incubating samples with beads containing bound goat anti-rabbit IgG (Technogenetics, Milan, Italy). The beads were washed with 10 ml of 10 mM Na phosphate (pH 7.4), 50 mM NaCl, and 0.1% Triton X-100 to remove unbound 3H-Epi, and the bound receptors were eluted with 2 N NaOH and counted in a β counter.
The level of antibody immunoprecipitation was expressed as percentage of 3H-Epi-labeled receptors immunoprecipitated by antibodies (taking the amount present in the Triton X-100 extracted solution before immunoprecipitation as 100%) or as femtomoles of immunoprecipitated receptors per milligram of protein.
Nicotine-induced changes in synaptic transmission
Adolescent (n=34) and adult (n=24) rats were exposed to nicotine or saline as described above and used for recording of nicotine-induced (10 μM, bath application) spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs), the latter in the presence of tetradotoxin (TTX). After 1 d (acute effect) or 5 to 6 wk (long-term effect) following nicotine exposure, rats were decapitated, and brains were rapidly removed and put in ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM) 3.5 KCl, 2.4 CaCl2, 1.3 MgSO4 · 7H2O, 1.2 KH2PO4, 215.5 sucrose, 26 NaHCO3, and 10 d-glucose, with osmolarity 300 mosmol.
Coronal mPFC slices of 300 μm thickness were prepared in the same sucrose-containing ACSF and then stored in holding chambers containing normal ACSF consisting of (in mM) 125 NaCl, 3 KCl, 1.25 NaH2PO4, 2 MgSO4, 1 CaCl2, 26 NaHCO3, and 10 glucose, bubbled with carbogen gas (95% O2/5% CO2). Pyramidal neurons in layer II/III in the mPFC were visualized using differential interference contrast microscopy, and whole-cell recordings from pyramidal neurons were made using Multiclamp 700B amplifier (Molecular Devices/Axon Instruments, Sunnyvale, CA, USA), digitized by the pClamp software (Molecular Devices/Axon Instruments), and later analyzed offline using Synaptosoft (Delaware, GA, USA) software. To record GABAergic activity at resting membrane potential (−70 mV), pipette medium contained elevated chloride concentration (in mM): 65 K gluconate, 70 KCl; 8 NaCl, 2 MgATP, 10 phosphocreatine, 0.2 EGTA, 10 HEPES, 0.3 Tris GTP, and 1 QX 314-Cl. Recordings were made (32°C) in the presence of 6,7-dinitroquinoxaline-2,3-dione to block glutamatergic currents measuring at baseline (7 min), nicotine wash-in (10 μM; 3 min), and wash-out (7 min). For estimation of the nicotine effect, average sIPSC frequency or amplitude was taken at the peak (last minute of nicotine wash-in and first minute of nicotine washout) and normalized to the last 5 min of baseline recording.
Data from plasma nicotine and cotinine measurements were subjected to univariate ANOVA with age of pretreatment (adolescent, adult) and time point as between-subject variables using SPSS 16 (SPSS Inc., Chicago, IL, USA). Developmental data on nAChR expression were subjected to a nonparametric ordered Jonckheere-Terpstra test due to unequal variance per brain region (Levene’s test). Due to their normal distribution, nicotine-dependent regulation of nAChR expression was analyzed by univariate factorial ANOVAs with age of treatment, time point after exposure, brain region, and treatment as between-subject variables. In case of statistically significant main effects and interactions, further breakdown in factors was performed, ultimately followed by post hoc comparisons (Student-Newman-Keuls tests). Due to independent data sets from electrophysiological experiments with different setups, data were analyzed separately for age and time using Student’s t test with nicotine and saline treatment as factors. For cumulative frequencies, a Kolmogorov-Smirnov (K-S) test was performed. The level of probability for statistically significant effects was set at 0.05. All data are displayed as mean values ± se. The effect size in terms of percentage change is calculated as the subtraction of the value from the normalized (vs. saline) nicotine sample minus the value of the saline sample.