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Animals: This study used male and female c57/BL6 mice, exposed to social isolation beginning PND 21 through adulthood. Mice were randomly assigned isolation or group housing at weaning (PND). All animals were housed in a temperature and humidity controlled animal care facility with a 12h light/dark cycle (lights on at 7:00 A.M.). The Temple University Animal Care and Use Committee approved all procedures.

Nucleus Accumbens Slices: Mice were decapitated following cervical dislocation. The brain was removed and coronal slices (250 μm) containing the nucleus accumbens were cut with a Vibratome (VT1000S, Leica Microsystems) in an ice-cold artificial cerebrospinal fluid solution (ACSF), in which NaCl was replaced by an equiosmolar concentration of sucrose. ACSF consisted of 130 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 10 mM glucose, 1 mM MgCl2, and 2 mM CaCl2 (pH 7.2–7.4 when saturated with 95% O2/5% CO2). Slices were incubated in ACSF at 32–34 °C for 25 min and kept at 22–25 °C thereafter, until transfer to the recording chamber. The osmolarity of all solutions was 305–315 mOsm. Slices were viewed using infrared differential interference contrast optics under an upright microscope (Slice Scope Pro, Scientifica) with a 10× objective.

Field potential recordings: The recording chamber was continuously perfused (1–2 ml/min) with oxygenated ACSF heated to 32±1 °C using an automatic temperature controller (Warner Instruments). Dendritic field excitatory postsynaptic potentials (fEPSPs) were recorded from the Nucleus Accumbens using micropipettes (resistance 1-4 MΩ) pulled from borosilicate glass capillaries (World Precision Instruments) filled with ACSF. All recordings were conducted with a MultiClamp700B amplifier (Molecular Devices). A bipolar tungsten stimulating electrode was placed within 100-300 μm from the recording electrode and used to stimulate excitatory afferents. Stimulations were applied as paired pulses (interval 20-240 ms) at 0.06 Hz. The initial slope of fEPSPs was used as a measure of synaptic response.

Whole cell recordings: Medium spiny neurons were patch in the voltage-clamp configuration and recording at holding potentials as indicated. Electrodes were filled with internal recording solution composed of (mM): 100 CH3O3SC5, 50 CsCl, 10 HEPES, 0.2 Bapta, 3 KCl, 1 MgCl2, 0.25 GTP tris, 2.5 Phosphocreatin 2 Na, 2 MgATP. Spontaneous excitatory synaptic currents (sEPSC): Currents were recorded at a holding potential of -70 mV for 10 min.

Optogenetic recordings: 0.4 μl of AAV9CamKIIa.hChR2(E123T/T159C).mCherry was injected in each half of the brain and various brain regions. Injected animals were sacrificed for recordings 14 days after the injections. asEPSC: Ca2+Cl2 was replaced with SrCl2 (4 mM). Amplitude and frequency was analyzed 50 ms after the light stimulation for 300 ms.

Data Analysis: Data were analyzed off-line using Clampfit and Graphpad prism.