Timed-pregnant female Wistar rats arrived at 5 d of gestation. At postnatal day 21 (P21), litters were weaned and housed four per cage. Only males were used in these experiments. For a small number of control experiments, male Wistar rats were obtained directly from the breeder (Harlan or Charles River). All experiments were approved by the animal ethical committee of Vrije Universiteit (Amsterdam, The Netherlands). In total, 47 saline-treated rats, 40 nicotine-treated rats, and 7 control untreated rats were killed. The numbers of rats per experimental group are reported in the figure legends. To avoid litter effects, pups from the same mother were always divided between the experimental groups.
To test the long-term effects of nicotine, a rat model of nicotine exposure during adolescence was used (see , , ). Rats were injected subcutaneously with either nicotine [0.4 mg/kg; calculated as a base (–) nicotine hydrogen tartrate salt; Sigma-Aldrich] or saline three times a day (at 10:00 A.M., 1:00 P.M., and 4:00 P.M.) for 10 d. Nicotine/saline injections were administered during adolescence (P34–P43; see ) and for the control group of adult nicotine exposure during adulthood (P60–P69; see ). After the 10 d treatment, the injections stopped, and either 1–4 d or 5 weeks later the electrophysiological experiments were performed.
Figure 1 Nicotine bath application to PFC slices of adolescent rats strongly reduces tLTP. a, Biocytin-filled layer V pyramidal cell in rat PFC; the position of the recording and stimulating electrodes is schematically shown. Membrane potential in response to (more ...)
Figure 2 Nicotine exposure during adolescence leads to a short-term decrease in tLTP. a, Schematic representation of the experimental setup. Rats were treated with nicotine (red bar)/saline (blue bar) during adolescence, and the STDP recordings were performed (more ...)
Figure 3 Nicotine exposure during adolescence leads to a lasting increase in LTP in adult rats. a, Schematic representation of the experimental setup. Rats were treated with nicotine (red bar)/saline (blue bar) during adolescence, and the electrophysiological (more ...)
Figure 4 Nicotine exposure during adulthood does not lead to lasting changes in LTP. a, Schematic representation of the experimental setup. Rats were treated with nicotine (red bar)/saline (blue bar) during adulthood, and the electrophysiological recordings took (more ...)
Either 1–4 d (P44–P48) or 5 weeks after nicotine or saline exposure (starting at P78 for rats exposed during adolescence and at P104 for rats exposed during adulthood), rats were decapitated and their brains were rapidly removed. Coronal mPFC slices of 300 μm thickness were prepared in ice-cold artificial CSF (ACSF) consisting of 125 mm NaCl, 3 mm KCl, 1.2 mm NaH2PO4, 7 mm MgSO4, 0.5 mm CaCl2, 26 mm NaHCO3, and 10 mm d-glucose. Slices were transferred to holding chambers with ACSF containing 125 mm NaCl, 3 mm KCl, 1.2 mm Na2PO4, 1 mm MgSO4, 2 mm CaCl2, 26 mm NaHCO3, and 10 mm glucose, bubbled with carbogen gas (95% O2 and 5% CO2).
Whole-cell patch-clamp recordings were made from layer V pyramidal cells in prelimbic area of mPFC under visual guidance by differential interference contrast microscopy. Slices were kept in a submerged recording chamber at 32–35°C. Pipettes were made from standard borosilicate capillary glass tubing with resistance between 2.5 and 4 MΩ resistance and filled with intracellular solution containing the following: 110 mm K-gluconate, 10 mm KCl, 10 mm HEPES, 10 mm K2-phosphocreatine, 4 mm ATP-Mg, 0.4 mm GTP, 5 mg/ml biocytin, pH adjusted with KOH to 7.3, 290–300 mOsm. Series resistance was monitored but not compensated for. Pyramidal cells were identified based on their morphology and spiking profile. Cells were filled with biocytin (0.5%) and then processed for post hoc cell identification. All experiments were performed in the absence of blockers of GABAergic synaptic transmission. Recordings were made using Multiclamp 700B amplifier (Molecular Devices) sampling at 10 kHz, filtered at 3 kHz, and later analyzed off-line using custom-written MATLAB scripts (MathWorks). Whole-cell current injections and extracellular stimulation (both timing and levels) were controlled with a Master-8 stimulator (A.M.P.I.) triggered by the data acquisition software.
Spike timing-dependent plasticity
EPSPs were evoked every 10 s using an extracellular stimulation electrode positioned at ~200 μ
m along the somatodendritic axis of the cell (see ). During the pairing period, presynaptic extracellular stimulations were paired to a single postsynaptic action potential (AP) (50 times at 0.1 Hz) or a burst of five action potentials at 100 Hz (50 times at 0.1 Hz). During the induction protocol pairing intervals of 5 ms were used (pre-before-post), timed from the onset of the evoked EPSP to the onset of the action potential. The slope of the initial 2 ms of the EPSP was analyzed to ensure that the data reflected only the monosynaptic component of each EPSP (Couey et al., 2007
). Synaptic gain was measured as the percentage change in EPSP slope: the average EPSP slope during 10 min in the period 35–45 min (where a different time window was used, it is specified in the figure legends) after pairing compared with the average baseline EPSP slope. Mean baseline EPSP slopes were averaged from at least 30 sweeps. Cell input resistance was monitored by applying a –100 pA, 200–500 ms hyperpolarizing pulse at the end of each sweep. Cells in which input resistance change during the experiment was >30% were discarded from the analysis. No blockers of GABAergic transmission were used during the STDP recordings.
For experiments in adolescent mPFC (see ), nicotine (Sigma-Aldrich; 10 μm) was bath applied for 5 min (2 min prior and 3 min during pairing). To measure mGluR2-dependent inhibition (see ), EPSCs were recorded in ACSF containing gabazine (1 μm). Stimulation intensity–response amplitude curves were taken for each cell before the recording. Baseline EPSCs were recorded for 10 min; mGluR2-specific agonist (1S,2R,5R,6R)-2-amino-4-oxabicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY379268) (Tocris; 5 μm) was bath applied for 7 min (the last 10 EPSCs were averaged for each cell and used as estimation of mGluR2 effect on EPSC amplitude) and then washed out for 25 min. For estimation of mGluR2 involvement in STDP after nicotine/saline exposure during adolescence, mGluR2 agonist LY379268 (Tocris; 1 μm) or antagonist (RS)-α-methyl-4-phosphonophenylglycine (MPPG) (Tocris; 100 μm) were bath applied 5 min prior and for the whole duration of the recordings.
Figure 5 mGluR2 signaling is reduced in adult rats after nicotine exposure during adolescence. a, EPSC traces recorded in PFC layer V pyramidal neurons 5 weeks after saline (black) and nicotine (gray) treatment during adolescence. mGluR2 agonist LY379268 leads (more ...)
GraphPad Instat software was used to assess statistical significance (GraphPad Software). Data were first tested for deviations from Gaussian distribution using the Kolmogorov–Smirnov normality test. For normally distributed data, Student’s t tests were applied.
To determine significance in EPSP slope change for individual cells, the EPSP slopes within a 10 min window 35–45 min after pairing were compared with baseline EPSP slopes using Student’s t test. On the basis of this test, the experiments were divided into three categories: timing-dependent long-term potentiation (tLTP) (significant increase in EPSP slope), timing-dependent long-term depression (tLTD) (significant decrease in EPSP slope), and neurons with no change in plasticity (nonsignificant EPSP slope change). To assess the effects of nicotine treatment on STDP, all cells recorded within the treatment group were compared with all cell recorded from saline-treated controls (individual data points are shown in summary plots in –, ). In the experiment in which MPPG was applied in nicotine-treated rats (see ), statistical significance was assessed using the χ2 test of independence (SPSS software) to compare the proportion of cells with tLTP, tLTD, and no change in presence and absence of MPPG.
Figure 6 Impaired mGluR2 signaling is responsible for short- and long-term changes in LTP after nicotine exposure during adolescence. a, Schematic representation of the experimental setup for data shown in b–d. Rats were treated with nicotine (red bar)/saline (more ...)