Horizontal brain slices were prepared from Sprague-Dawley rats (10–14 days of age). Following rapid decapitation, the brain was removed, the olfactory bulbs were cut away, and the midbrain was cut at the level of the 4th ventricle. Then the brain was placed in ice-cold artificial cerebrospinal fluid (aCSF) containing (in mM) NaCl 125, KCl 2.5, MgCl2 1, CaCl2 2.5, Glucose 20, NaH2PO4 1, NaHCO3 25, ascorbic acid 1; bubbled continuously with 95% O2/5% CO2, pH 7.3. From each brain, two or three slices (250–300 μm) were cut in cold aCSF and incubated for at least 1 hr (32–34°C). Slices were pretreated with bupropion at the test concentration during this incubation. For recording, the slice was transferred to a chamber superfused (~2 ml min−1) with aCSF lacking ascorbic acid at room temperature. Bupropion was included in all perfusion solutions at the test concentration.
Neurons were visualized under infrared illumination using an upright microscope (Axioskop, Zeiss). When recording GABAergic transmission, electrodes (2.5–4 MΩ) contained (in mM): K-Gluconate 78, KCl 77, EGTA 1, HEPES 10, Glucose 10, ATP 5, GTP 100 μM (pH 7.4 with KOH). When recording glutamatergic transmission, electrodes were filled with (in mM): K-Gluconate 154, KCl 1, EGTA 1, HEPES 10, Glucose 10, ATP 5 (pH 7.4 with KOH). Standard whole-cell voltage clamp recordings were made using an Axopatch 200B amplifier, a Digidata 1320A interface, and pCLAMP 8 software (Molecular Devices Corp. Sunnyvale, CA). In whole-cell recordings, DA neurons in the VTA can be distinguished from other cell types in the nucleus by action potential firing rates, action potential duration, soma diameter and the presence of a prominent hyperpolarization activated current, Ih
]. Recent findings suggest that non-DA neurons in the VTA may also express Ih
]. Cell morphology has some predictive value for identifying dopaminergic cells, but it is not entirely reliable [24
], but we contend that the combination of these parameters yields a population of neurons that is predominantly dopaminergic. Neurons were held at −60 mV to assess the presence of Ih
, but were held at −70 mV throughout the rest of the voltage clamp experiments. All experiments described here were performed on cells that expressed Ih
> 30 pA at −120 mV. Spontaneous transmission was filtered at 1 kHz and digitized at 5 kHz. In experiments measuring membrane potential in current clamp recording mode, we used perforated patch recording methods with amphotericin B (660 μg/ml final concentration, Sigma) dissolved in DMSO was added to the pipette solution. These experiments were started when the series resistance dropped below 40 MΩ, but was typically 15–30 MΩ. In whole-cell recordings, series resistance values were 4-6 MΩ. To isolate GABAergic transmission, experiments were done in the presence of 10 μM 6,7-Dinitroquinoxaline-2,3-dione (DNQX) to block glutamate transmission [26
]. Glutamatergic transmission was isolated by adding 20 μM bicuculline to the bath solution to block GABAergic synaptic inputs [27
]. Nicotine tartrate, bicuculline methiodide, (both from Sigma, St Louis, MO), DNQX (Tocris, Ellisville, MO) and bupropion HCl (Sigma) were applied through bath perfusion during recording. Bicuculline and DNQX were present in the bath at least 15 minutes before the effect of nicotine was assessed. A new slice was used for each experiment, so that neurons were exposed only once to nicotine.
Spontaneous IPSC and EPSC were recorded continuously at a holding current of −70 mV and data analysis was carried out using MiniAnalysis software (Synaptosoft, Inc., Decatur, GA). Amplitude, rise-time and area thresholds were used to acquire events and the amplitude threshold was set to five times the RMS noise of each recording, as determined by the software. Each acquired event was visually inspected to protect against software errors. Graphical representations of the results were constructed with SigmaPlot 7.0 (SPSS, Inc.).
After plotting synaptic activity frequency histograms determination of ‘responsive’ cells involved comparing, via Student’s t-test, the baseline frequency for a 1 min period immediately prior to nicotine application with a 1 min period centered on the peak nicotine effect. In the absence of a clear nicotine-induced change in frequency, the frequency data were sampled from a 1 min window centered 1 min after the start of the nicotine application. This time point most commonly corresponds to the maximal effect of nicotine on synaptic transmission [20
]. The magnitude of each change in synaptic transmission was defined as the difference between the average frequency 1 min immediately prior to nicotine application and an average of 30 sec of frequency data sampled around the peak nicotine response. Again, in the absence of a clear effect of nicotine, the baseline frequency was compared with the frequency during a 30 sec window centered 1 min after the start of the nicotine application. In bupropion pretreated samples, the same methods were used for determining response prevalence and magnitude.
Action potential frequencies were also determined using MiniAnalysis software. For examining the effects of each drug treatment on action potential activity, baseline firing rate was sampled and averaged over 1 min immediately prior to drug application, for nicotine, the response magnitude was determined comparing the baseline frequency determination with the average frequency during a 30 sec window centered on the peak effect. In the absence of an effect on firing rate, the baseline was compared with a 30 sec window centered 30 sec after the beginning of the nicotine application, which was generally the peak of the nicotine effect. Bupropion’s effects on firing rate were much slower to develop, as such, we perfused bupropion for at least 20 min prior to sampling the frequency during a 30 sec window. Determination of significant differences in the modulation of action potential frequency in , each treatment condition (nicotine, bupropion, and the combination of the two drugs) was compared to its own baseline control period (no drug for nicotine and bupropion, bupropion alone for the combination of nicotine and bupropion) using paired t-tests.
Figure 1 Bupropion inhibits nAChRs on VTA dopamine neurons. a) Bath application of 1 μM nicotine increases action potential frequency (n = 7). b) In slices pretreated with 1 μM bupropion nicotine does not alter action potential frequency in dopamine (more ...)
Data are presented as mean ± standard error. Statistical comparison of response magnitudes in the presence and absence of bupropion was evaluated using Student’s t-test. Multiple comparisons in were done using Bonferroni protection, where the three comparisons required p < 0.16 by Student’s t-test.