All procedures had prior approval from the Institutional Animal Care and Use Committee of Rosalind Franklin University, and followed the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). Care was taken to minimize animal distress and reduce the number of animals used. Male Sprague–Dawley rats (Harlan Laboratories, Indianapolis, IN) were weaned at postnatal day (P) 18, and upon arrival (P21–22) were housed at the Rosalind Franklin University animal facility (polycarbonate solid bottom cage, 43.2 × 21.5 × 20.3 height, in cm; Teklad Sani-Chips bedding). The rats were provided water and food (Rodent Diet 2020X pelleted feed, Harlan Teklad) ad libitum. The housing rooms were set to a 12 h/12 h reverse light–dark cycle with lights off at 0700 hours. Temperature was maintained between 64 and 79 °F and the humidity was maintained between 30 and 70%. Rats were randomly assigned to control group housing (2–3/cage) or social isolation single housing (1/cage). All other aspects of husbandry were the same. Rats were used for experiments after 4–5 weeks (P50–60 postadolescents), or at intermediate time points, as described below. Several studies indicate that social behavior in males may be more sensitive to postweaning social isolation (Ferdman et al, 2007; Wall et al, 2012; Ahern et al, 2016). Therefore, males were the focus of the current study.

Experimental Outline

Rats were group housed our single housed on the day of arrival, and then tested at noted timepoints. To test the effects of social isolation on MeA activity, in vivo electrophysiological measures were obtained from young adults (after more than 4 weeks; P50–60). Both single-unit (group housing n=9 rats; social isolation single housing n=10 rats) and evoked field potentials were measured from MeApd and MeApv (MeApd group housing n=9 rats, social isolation single housing n=8 rats; MeApv group housing, n=9 rats, social isolation single housing, n=7 rats). To test the proximal causes for a change in MeA activity, in vitro whole-cell recordings were also obtained from young adults to measure synaptic activity (8 rats per group) and membrane responsiveness (8 rats per group). To determine the time course of the changes over the postweaning period, in vitro whole-cell recordings were obtained after 1 day (P22–23, n=8 rats per group), 1 week (P28–29, group housing, n=8 rats, single housing, n=7 rats), 2 weeks (P35–36, group housing, n=7 rats, single housing, n=7 rats), or as young adults (P50–60, n=8 rats per group).

In vivo Electrophysiology

Rats were anesthetized with urethane (1.5 g/kg, intraperitoneally) and 2% lidocaine jelly was infiltrated into their ears. Rats were placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA or Stoelting Instruments, Wood Dale, IL), and core body temperature was maintained at ~37 °C (TC-1000 Temperature Controller; CWE, Ardmore, PA). Bore holes were drilled in the skull overlying the lateral nucleus of the amygdala (LAT; centered on −5.0 mm M–L, −3.0 mm A–P from bregma) and the MeA (centered on −3.2 mm M–L, −3.3 mm A–P from bregma, between 8.0 and 9.5 mm D–V). A concentric bipolar stimulation electrode (Rhodes Medical Instrument, Summerland, CA) was slowly lowered into the LAT to deliver stimulation and to record spontaneous local field potentials as a gauge of anesthesia state. After a minimum of 45 min, a glass microelectrode (2.0 mm outer diameter borosilicate; A–M Systems, Carlsborg, WA) was pulled (PE-2 microelectrode puller; Narishige Group, Tokyo, Japan) and filled with 2% Pontamine Sky Blue (Alfa Aesar, Ward Hill, MA) in 2 M NaCl (Thermo Fisher Scientific, Waltham, MA). The glass electrode was slowly lowered to the MeA to record neuronal activity. Signals were amplified (2400 Extracellular Preamplifier; Dagan, Minneapolis, MN) and digitized (5–10 kHz; InstruTECH ITC-18; HEKA Instruments, Bellmore, NY). Signals were monitored audially (AM10 amplifier; Grass Technologies, Warwick, RI) and visually (AxoGraph X software version 1.3.5; Axograph Scientific, Sydney, Australia) and saved for later analysis (Mac Pro; Apple, Cupertino, CA). Electrical stimulation was delivered (S88 Stimulator and PSIU6 Stimulation Isolation Unit; Grass Technologies, Warwick, RI) through the bipolar electrode with an intensity range of 0.1–0.9 mA, 0.2 ms duration, repeating at 0.2–40 Hz.

At the conclusion of experiments, Pontamine Sky Blue was iontophoresed through the recording electrode (−30 μA, >30 min; Constant Current Source, Fintronics, Orange, CT). The brain was removed and placed in 4% paraformaldehyde in 0.1 M phosphate buffer overnight, and then transferred to 25% sucrose (Thermo Fisher Scientific, Bannockburn, IL) in 0.1 M phosphate buffer. Brains were sectioned (60 μm thick) with a freezing microtome (Leica Microsystems, Buffalo Grove, IL) and stained with cresyl violet (Sigma-Aldrich, St Louis, MO). Recording and stimulation sites were verified by light microscopy.

In vitro Electrophysiology

Rats were deeply anesthetized with a mixture of ketamine (80–100 mg/kg; Webster Veterinary Supply, Devens, MA) and xylazine (10–20 mg/kg; Webster Veterinary Supply). Upon verification of deep anesthesia by the absence of response to foot pinch, rats were decapitated, and brains were rapidly immersed in ice-cold, aerated (95% O 2 /5% CO 2 ) high sucrose artificial cerebrospinal fluid (ACSF) containing (in mM) 2.5 KCl, 1.25 NaH 2 PO 4 , 25 NaHCO 3 , 7 dextrose, 7 MgCl 2 , 0.5 CaCl 2 , 210 sucrose, 1.3 ascorbic acid, 3 sodium pyruvate, and ~290 mOsm. The brain was sectioned at 250–300 μm (Vibratome 1000; Ted Pella, Redding, CA) in the same ACSF. Brain sections were then placed in physiological ACSF saturated with 95% O 2 /5% CO 2 , containing (in mM) 125 NaCl, 2.5 KCl, 1.25 NaH 2 PO 4 , 25 NaHCO3, 10 dextrose, 1 MgCl 2 , and 2 CaCl 2 , with the addition of 1.3 mM ascorbic acid and 3 mM sodium pyruvate for 50–60 min at 34 °C. Recordings were performed at 32–34 °C in submerged slices in physiological ACSF (as above, without ascorbic acid or sodium pyruvate). When specified, (+)-bicuculline (10 μM; Ascent Scientific, Princeton, NJ; dissolved in dimethyl sulfoxide), picrotoxin (10 μM; Sigma-Aldrich; dissolved in ethanol), 6-cyano-7-nitroquinoxaline-2,3-dion (CNQX) disodium salt (10 μM; Ascent Scientific; dissolved in ddH 2 O), and DL-2-amino-5-phosphonopentanoic acid sodium salt (50 μM; Abcam Biochemicals, Cambridge, MA; dissolved in 100 mM NaOH) were added to the ACSF to block GABAA-, AMPA- and NMDA receptor-mediated currents. Final solvent concentrations were <0.1% of the total ACSF volume.

Recording pipettes were pulled (borosilicate glass, 2.0 mm outer diameter, 2–8 MΩ open tip resistance; Sutter Instruments, Novato, CA). Electrodes were filled with (in mM) 120 K-gluconate, 20 KCl, 0.2 EGTA, 10 HEPES, 2 NaCl, 4 ATP-Mg, 0.3 GTP-Tris, 7 Tris-phosphocreatine, and 0.2% neurobiotin (Vector Laboratories, Burlingame, CA), with a pH of 7.3. Whole-cell recordings were performed from visualized neurons in the MeAp under IR-DIC conditions. Signals were amplified (AxoClamp 2B; Molecular Devices, Sunnyvale, CA) and low-pass-filtered at 3–5 kHz and digitalized at 10 kHz (InstruTECH ITC-18; HEKA Instruments). Mean series resistance for each group was below 25 MΩ. Data were monitored and saved for later analysis (AxoGraph X software v.1.3.5 (Axograph Scientific), on Mac Pro (Apple)). Upon completion of recordings, slices were fixed overnight and stored for up to 3 weeks at 4 °C (4% paraformaldehyde in 0.1 M phosphate-buffered saline, PBS). Sections were rinsed three times with PBS, treated with Triton X-100 (VWR International, Radnor, PA; 1% in PBS) for 6 to 8 h, and then incubated in the Vectastain ABC Reagent (Vector Laboratories) in PBS at room temperature overnight. After three rinses with PBS, sections were reacted with diaminobenzidine (DAB) and H 2 O 2 (Peroxidase Substrate Kit DAB; Vector Laboratories) in water to visualize the neurobiotin-filled neurons. Sections were washed in PBS repeatedly to stop the reaction. Sections were mounted, dried, and coverslipped. Stained sections were used to localize the recording sites.

Data Analysis and Statistics