Supporting Information Bero et al. 10.1073/pnas.1408378111 SI Materials and Methods isolate was (13.5 ± 0.5) × 106 reads, of which (9.3 ± 0.3) × 106 Animals. Wild-type (B6SJL; Taconic Farms) male mice were used reads were aligned. Aligned reads were mapped to the RefSeq at 2.5 ± 0.5 mo of age for experimentation. Mice were given ad database and counted (HTSeq). Differential expression analysis libitum access to food and water. Mice were experimentally naïve was performed using DESeq (Bioconductor) followed by Student before the onset of the study and housed (4–5 mice per cage) in t test to model the experimental and gene-specific dispersion, 12 h light/12 h dark conditions (light phase beginning at 7:00 respectively. Genes were considered differentially expressed if P ≤ AM). All animal work was approved by the Committee for 0.05. Ontological and network analyses of differentially expressed Animal Care of the Division of Comparative Medicine at the genes were performed using Ingenuity Pathway Analysis software. Massachusetts Institute of Technology (MIT). Quantitative RT-PCR. Quantitative RT-PCR was performed es- Behavioral Analysis. All behavioral experiments were conducted sentially as described (2). Briefly, total mRNA was extracted during the light cycle and performed essentially as described (1). from mPFC isolates (anterior cingulate cortex; bregma +1.2 mm Briefly, mice were exposed to the conditioning chamber (TSE to +0.6 mm) (3) using the RNeasy kit (Qiagen), reverse tran- Systems) for 3 min (habituation phase), after which they received scribed (Invitrogen), and quantitatively amplified using a ther- two unsignaled foot shocks (2-s duration, 0.8 mA, 30-s intershock mal cycler (Bio-Rad), SsoFast EvaGreen (Bio-Rad) and gene- C interval). Following the foot shocks, mice remained in the specific primers (Table S3). The comparative t method was chamber for an additional 15 s. Control groups of mice were used to determine differences in gene expression. Values were treated identically, but either did not receive a foot shock normalized to expression levels of Gapdh. (context control group) or received foot shocks in the absence of context exploration (shock control group). Long-term memory Electron Microscopy. Mice were transcardially perfused with chilled tests were performed 1 d (recent memory) and 30 d later (re- PBS (pH 7.4) followed by 2.5% glutaraldehyde/2.5% para- mote memory) by returning the mice to the experimental cham- formaldehyde in 0.1 M sodium cacodylate buffer (pH 7.4; ber for 2 min. Freezing, defined as an absence of all movement Electron Microscopy Sciences). Brains were removed and post- except respiration, was scored every 10 s by an experimenter fixed in 2.5% glutaraldehyde/2.5% paraformaldehyde in 0.1 M blind to condition. Movement velocity was recorded using au- sodium cacodylate buffer at 4 °C overnight. Following fixation, μ tomated procedures (TSE Systems). brain sections (100 m thick) were cut on a vibratome (Leica), washed in 0.1 M sodium cacodylate buffer, postfixed in 1% os- Immunohistochemistry. Immunohistochemical staining was per- miumtetroxide/1.5% potassium ferrocyanide, incubated in 1% formed essentially as described (2). Briefly, mice were trans- aqueous uranyl acetate, and dehydrated. The samples were em- cardially perfused with chilled PBS (pH 7.4) followed by 10% bedded in TAAB Epon (Marivac), polymerized, and sectioned paraformaldehyde under deep anesthesia (ketamine, xylazine). (80 nm thick) using a microtome (Reichert). Resultant sections Brains were removed and postfixed in 10% paraformaldehyde at were placed onto copper grids, stained with lead citrate, and 4 °C overnight. Following fixation, serial coronal brain sections imaged using a transmission electron microscope (1200EX; JEOL). (40 μm thick) were cut on a vibratome (Leica) and collected Micrographs of mPFC synapses (anterior cingulate cortex; bregma from frontal cortex to caudal hippocampus. Sections were per- +1.2 mm to +0.6 mm) (3) were recorded using a CCD camera meabilized and blocked with 10% FBS/0.3% Triton-X 100 in (Advanced Microscopy Techniques). Synapses, active zones, post- PBS and incubated overnight with the following primary anti- synaptic densities, and docked synaptic vesicles were identified bodies: Zif268/Egr-1 (1:100; Santa Cruz, sc-101033), synapto- and defined as described (4, 5) and quantitative analysis of physin (1:1,000; Sigma, S5768) or CaMKIIα (1:100; Millipore, synapse ultrastructure was performed using ImageJ (NIH) by an MAB8699). Primary antibodies were visualized with a Cy5-con- experimenter blind to condition. jugated secondary antibody (1:400; Jackson ImmunoResearch, 115-175-146) and nuclei were visualized with Hoechst 33342 Golgi–Cox Impregnation. Golgi–Cox staining was performed es- (Invitrogen). A mouse brain atlas (3) was used to identify medial sentially as described (1). Briefly, mice were transcardially prefrontal cortex (mPFC) (anterior cingulate cortex; bregma + perfused with chilled PBS (pH 7.4) followed by 10% para- 1.2 mm to +0.6 mm), hippocampal area cornu ammonis 1 (CA1; formaldehyde under deep anesthesia (ketamine, xylazine). bregma −1.8 mm to −2.3 mm), and entorhinal cortex (bregma −2.2 Brains were removed and postfixed in 10% paraformaldehyde at mm to −2.5 mm), and images were acquired using a confocal mi- 4 °C overnight. Following fixation, whole-brain Golgi–Cox im- croscope (LSM 510; Zeiss) at identical settings for each condition. pregnation was performed using the Rapid GolgiStain kit (FD Images were quantified using ImageJ (National Institutes of Health, Neurotechnologies) as per the manufacturer’s instructions and NIH) by an experimenter blind to condition groups, whenever serial brain sections (60 μm thick) were cut on a vibratome possible. (Leica). Using a confocal microscope (LSM 510; Zeiss), den- dritic spines present on visually unobstructed apical and basal Genome-Wide RNA Sequencing. RNA sequencing was performed dendrites of layer II/III mPFC pyramidal neurons (anterior essentially as described (1). Briefly, total mRNA was extracted cingulate cortex; bregma +1.2 mm to +0.6 mm) (3) were imaged from mPFC isolates (anterior cingulate cortex; bregma +1.2 mm by an experimenter blind to condition. To capture the morpho- to +0.6 mm) (3) using the RNeasy kit (Qiagen). Purified mRNA logical extent of all spines on each dendritic segment of interest, was quality controlled (Agilent Bioanalyzer), poly-A purified, a z-stack image series was acquired from the anterior to poste- and converted to cDNA using the Illumina TruSeq protocol as rior limits of each spine set (incremental z-step distance, 0.25–0.5 per the manufacturer’s instructions. High-throughput sequencing μm). Quantitative morphological analysis of individual dendritic was performed using the Illumina HiSeq 2000 platform at the spines was performed using ImageJ (NIH) and spines were classi- MIT BioMicro Center. Sequence reads were aligned to the fied as thin, mushroom, or stubby using previously described cri- mouse mm9 genome using TopHat. The mean yield per mPFC teria (6). Briefly, thin spines lack a definable head or possess Bero et al. www.pnas.org/cgi/content/short/1408378111 1of6 a head whose diameter is less than 120% of the stem diameter. Comparative Medicine at MIT. Mice were anesthetized under Mushroom spines exhibit a bulbous, mushroom-shaped head volatile isoflurane (0.5–2%) with the head secured in a stereo- whose diameter is equal to or greater than 120% of the stem taxic frame (Leica). The skin and periosteum were removed to diameter. Stubby spines lack a definable neck and their length expose the skull, and either the AAV5-CaMKIIα-eNpHR3.0- approximately equals their width. Spine analysis was performed EYFP or the control AAV5-CaMKIIα-EYFP vector was ste- by an experimenter blind to condition. reotaxically injected into the bilateral mPFC (1.0 μL hemi- − − sphere 1; flow rate, 0.075 μL min 1; bregma +1.0 mm, 0.35 mm Electrophysiology. For whole-cell patch-clamp recordings, coronal lateral to midline, 1.3 mm below the dura) (3) using a motorized brain slices (250 μm thick) containing the mPFC (anterior cin- injector (Stoelting). Following viral injection, injection needles gulate cortex; bregma +1.2 mm to +0.6 mm) (3) were prepared in ice-cold dissection buffer containing (in mM) 211 sucrose, 3.3 remained in place for 5 min and were then slowly withdrawn. Optic fibers (300-μm diameter, NA = 0.39; Thorlabs) held in KCl, 1.3 NaH2PO4, 0.5 CaCl2, 10 MgCl2, 26 NaHCO3, and 11 μ glucose using a vibratome (VT1000S; Leica). Slices were re- a stainless steel ferrule (330- m diameter; Precision Fiber Products) were implanted into the unilateral mPFC (counter- covered in a submerged chamber with 95% O2–5% CO2-satu- rated artificial cerebrospinal fluid (aCSF) consisting of (in mM) balanced for hemisphere) as close as possible to the midline + 124 NaCl, 3.3 KCl, 1.3 NaH2PO4, 2.5 CaCl2, 1.5 MgCl2,26 (bregma 1 mm, 0.2 mm lateral to midline, 0.8 mm below the NaHCO3, and 11 glucose for 1 h at 28–30 °C. Intracellular re- dura) and secured to the skull using dental cement (Stoelting). cordings were made from layer II/III mPFC pyramidal neurons After drying, the incision was closed and sealed with tissue ad- using recording pipettes (3–5MΩ) filled with an internal solu- hesive (Vetbond). Animal body temperature was maintained at tion containing (in mM) 145 CsCl, 5 NaCl, 10 Hepes-CsOH, 10 37 °C until fully recovered from anesthesia. All experiments were EGTA, 4 MgATP, and 0.3 Na2GTP. For miniature excitatory conducted 4 wk following surgery. postsynaptic current (mEPSC) measurements, tetrodotoxin (1 μM) and picrotoxin (50 μM) were added to the perfusion In Vivo Laser Delivery. To permit optogenetic inhibition of mPFC solution (aCSF) and cells were held at −70 mV. For ex vivo neurons in vivo, a 200-mW 593-nm DPSS laser (Opto Engine) was optical inhibition experiments, 20 mW of constant optical (593 connected to a patch cord with a fiber channel/physical contact nm) stimulation was generated by a 200-mW 593-nm diode- connector at each end (Doric Lenses).