Adult male C57BL/6J and DBA/2J (6–9 weeks old, Jackson Laboratories) were group-housed in a colony room with 12:12 hr light-dark cycle with lights on at 7 a.m. Mice had ad libitum access to rodent chow and water. All procedures were approved by the Institutional Animal Care and Use Committee of the University of North Carolina at Chapel Hill and performed in accordance with the National Institutes of Health guide for the care and use of laboratory animals.
2.2 Chronic restraint stress
Mice were placed in ventilated 50 mL Falcon tubes for 2 hr/day (10:00 a.m. – 12:00 p.m.) for 10 consecutive days, while naïve mice remained in their home cages. Twenty-four hours after the final stress exposure, mice were sacrificed and their brains harvested for fluorescence immunohistochemistry or slice electrophysiology.
2.3 Brain slice preparation for electrophysiology
Mice were decapitated under isoflurane anesthesia. Brains were rapidly removed and placed in ice-cold sucrose-artificial cerebrospinal fluid (ACSF): (in mM) 194 sucrose, 20 NaCl, 4.4 KCl, 2 CaCl2, 1 MgCl2, 1.2 NaH2PO4, 10.0 glucose, and 26.0 NaHCO3 saturated with 95% O2/5% CO2. Coronal slices 300 μm in thickness containing the dorsal BNST (Bregma 0.26–0.02 mm) were identified using the internal capsule, anterior commissure, and stria terminalis as landmarks, according to the Allen mouse brain atlas, and prepared using a Leica VT1200 vibratome. Slices were stored in a heated (approximately 28 °C), oxygenated holding chamber containing ACSF [(in mM) 124 NaCl, 4.4 KCl, 2 CaCl2, 1.2 MgSO4, 1 NaH2PO4, 10.0 glucose, and 26.0 NaHCO3]. Slices were transferred to a submerged recording chamber (Warner Instruments, Hamden, CT), where they were perfused with heated, oxygenated ACSF at a rate of approximately 2 ml/min and allowed to equilibrate for 30 min before electrophysiological recordings.
2.4 Whole-cell voltage-clamp recordings
We performed our electrophysiological recordings on neurons throughout the dorsolateral BNST (dlBNST), a particularly important area for the actions of corticotrophin-releasing factor (CRF) related to stress and anxiety (Huang et al., 2010
). Because specific subnuceli of the dlBNST cannot be anatomically distinguished in this preparation (especially the oval nucleus), we performed our electrophysiological recordings on neurons throughout the dlBNST. Neurons were directly visualized with infrared video microscopy (Olympus). Recording electrodes (3–5 MΩ) were pulled with a Flaming-Brown Micropipette Puller (Sutter Instruments, Novato, CA) using thin-walled borosilicate glass capillaries. Electrodes were filled with (in mM) 70 KCl, 65 K+
-gluconate, 5 NaCl, 10 HEPES, 2 QX-314, 0.6 EGTA, 4 ATP, 0.4 GTP, pH 7.2, 290–295 mOsmol. Spontaneous and evoked GABA type A (GABAA
R)-mediated inhibitory postsynaptic currents (IPSCs) were pharmacologically isolated during all electrophysiological recordings by adding 3 mM kynurenic acid to block AMPA and NMDA receptor-dependent postsynaptic currents (Ascent Scientific, Princeton, NJ). Signals were acquired via a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA), digitized at 10 kHz, and analyzed using Clampfit 10.2 software (Molecular Devices, Sunnyvale, CA).
After entering a whole-cell configuration, cells were held at −70 mV, and initial membrane capacitance and resistance were noted. Immediately following, an IV plot with 12 10-mV steps beginning at −140 mV was recorded in order to determine the presence and magnitude of a hyperpolarization-activated current (Ih). After allowing the neuron to rest at −70 mV for several minutes, spontaneous IPSCs (sIPSCs) were recorded at a holding voltage of −70 mV for 120 s to determine the amplitude and frequency of these spontaneous inhibitory events. For evoked inhibitory postsynaptic currents (eIPSCs), twisted nichrome wire stimulating electrodes were placed dorsal to the recording electrode, 100–500 μm medial from the recorded neuron. IPSCs were evoked at 0.1 Hz by local fiber stimulation with bipolar electrodes (5–50 V with a 100–150 μs duration). After at least five minutes in which mean peak evoked IPSC (eIPSC) amplitude was stable (“baseline period”), drugs mixed in distilled water or dimethyl sulfoxide and diluted to appropriate concentrations in the bath solution (ACSF with 3 mM kynurenic acid) were applied to the bath for 10 minutes, followed by a five-minute washout period. eIPSC experiments were analyzed by measuring the peak amplitude of the synaptic response, which was normalized to the baseline period. Input resistance and access resistance were continuously monitored during all experiments, and those in which changes in access resistance were greater than 20% were not included in the data analysis. In addition, cells that had a membrane capacitance and Ih current magnitude greater than one standard deviation above the group mean were excluded from analysis (one cell per group), because these may represent a small, uncharacterized subpopulation of neurons that respond to NPY in the opposite fashion of other neurons in the dlBNST. Enough of these cells could not be found in order to characterize their response to include in the analyses.
2.5 Fluorescence immunohistochemistry
Mice were anesthetized using Avertin and perfused transcardially with chilled PBS, followed by a 4% paraformaldehyde solution. Brains were post-fixed with 4% paraformaldehyde for 24 hours and immersed in a sucrose solution for a minimum of 24 hours until slices were collected. Brains were hemisected and coronal slices 45 μm in thickness containing the BNST were prepared using a Leica VT1200 vibratome (Leica Microsystems, Nussloch, Germany). One hemisphere of each brain was used for NPY-IR and the other for Y2R-IR, and this was randomly selected and counterbalanced within each group of mice. Because 6–8 slices were used for immunohistochemistry per subject for each type of stain, all brains could not be processed at the same time. Thus, examination of basal differences between C57BL/6J and DBA/2J mice were assessed first and separately from the effects of stress within each strain. For examination of the effects of chronic stress on NPY and Y2R expression within each strain, C57BL/6J and DBA/2J brains were processed and imaged separately to ensure a complete and meaningful analysis void of inter-run variability.
All slices containing the BNST were first rinsed in PBS three times for 10 min, incubated in 50% methanol for 30 min and then 3% hydrogen peroxide for 5 min to quench endogenous peroxidases, and then rinsed in PBS three more times. Slices used for NPY-IR were incubated in PBS containing 0.5% Triton X-100 for 30 min, rinsed with PBS for 10 min, and incubated in a blocking solution of PBS containing 0.1% Triton X-100 and 10% normal donkey serum for 60 min. Slices were then directly transferred to the blocking solution also containing a 1:1,250 concentration of anti-NPY (Abcam) for 48 hrs at 4°C. Slices were rinsed with PBS four times, incubated in a secondary solution containing Cy2 (1:200) for 2 hrs at RT, and rinsed in PBS four times.
Because Y2R-IR is less easily detected, slices used for Y2R immunohistochemistry underwent an amplification procedure using a Tyramide Signal Amplfication (TSA) detection kit (Jackson Laboratories) to increase signal sensitivity. While it should be noted that there is a possibility that TSA amplification may not be linear and thus may indicate larger differences in protein signal than are actually present, this amplification procedure is commonly used for low signal antibodies, including the Y2R antibody used here (e.g., Stanic et al., 2006
). This antibody has been shown to be selective, as there is an absence of all staining in the Y2R KO mouse (Stanic et al., 2006
). Slices were incubated in primary solution containing 0.3% Triton X-100, 0.5% bovine serum albumin, and a 1:3,000 concentration of anti-Y2R (Neuromics, Edina, MN) for 24 hrs at 4°C. Slices were washed in TNT buffer solution containing Tris/HCl, NaCl, and Tween20 for 10 minutes followed by TNB blocking solution containing TNT buffer with 0.5% blocking reagent provided in the TSA kit for 30 minutes (as described in TSA kit manual). Slices were then washed in TNB solution containing horseradish peroxidase (1:200) for 30 minutes and rinsed in TNT buffer four times (five minutes each). Sections were incubated in Cy3 (1:50) in amplification diluents provided in the TSA kit for 10 minutes and then rinsed in TNT buffer two times (10 minutes each). All slices were mounted on glass slides, air-dried, and coverslipped. Images of NPY-IR and Y2R-IR in the BNST were obtained with a Leica SP2 upright confocal microscope with 10X objective and Leica Confocal Software.
All serial sections throughout the BNST (6–8 per hemisphere) were used for quantification of NPY-IR and Y2R-IR by a researcher blind to experimental conditions using Image J Software (Image J, National Institute of Health, Bethesda, MD). The experimenter drew a contour around each subregion of BNST to be analyzed (oval, dorsal non-oval, and ventral), as illustrated for NPY-IR in . We elected to analyze these specific subregions because they have distinct inputs (Dong et al., 2001
). From our images and other published images (Stanic et al., 2006
), it was clear that the oval nucleus had a qualitatively smaller signal. The intensity value was obtained by calculating the percent of the total area studied that shows staining relative to sub-threshold background. The size of the area was held constant between animals and groups for each subregion. Intensity values for all serial sections for each mouse were averaged to obtain one value per mouse for each subregion of the BNST analyzed.
Representative confocal image of the BNST delineating subregions in which IR was quantified.
2.6 Statistical analysis
Appropriate statistical analyses were performed using Graphpad Prism. Basal strain differences in NPY-IR and Y2R-IR were evaluated using unpaired t-tests between strains, and basal strain differences in NPY signaling were examined using 2×2 analysis of variance (ANOVA). Effects of chronic stress on NPY-IR, Y2R-IR, and NPY’s ability to modulate evoked IPSC amplitude were determined using unpaired t-tests between conditions within each strain, as initial experiments showed that there were no differences between C57BL/6J and DBA/2J control subject values for any of these measures, and because C57BL/6J and DBA/2J brains had to be processed for immunohistochemistry separately due to the large number of slices in each group. When variances were significantly different between groups for t-tests, Welch’s correction was employed in order to maintain a conservative interpretation of the results. Effects of drugs and chronic stress during electrophysiological recordings were evaluated via t-tests between groups during the washout period or between baseline and washout periods. All values given for IR and drug effects are presented as mean ± S.E.M.