Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Rene Hen ( rh95@columbia.edu ).

For optogenetic manipulations, adeno-associated viruses (AAV5-CaMKIIa-hChR2(H134R)- eYFP; AAV5-CaMKIIa-eYFP; AAV5-CaMKII-ArchT-GFP; AAV5-CAG-Flex-ArchT-GFP) were packaged and supplied by the UNC Vector Core Facility at titers of ∼4-8 × 10 12 vg/ml. For calcium imaging, viruses (AAV1-Syn-GCaMP6f.WPRE.SV40; AAV1-Syn-Flex- GCaMP6f.WPRE.SV40) were packaged and supplied by UPenn Vector Core at titers ∼6 × 10 12 vg/ml and viral aliquots were diluted prior to use with artificial cortex buffer to ∼2 × 10 12 vg/ml.

All procedures were conducted in accordance with the U.S. NIH Guide for the Care and Use of Laboratory Animals and the New York State Psychiatric Institute Institutional Animal Care and Use Committees at Columbia University. Adult male C57BL/6J mice were supplied by Jackson Laboratory, and Vgat-IRES-Cre mice () were bred in-house on a C57BL/6J background, and used at 8 weeks of age. Mice were maintained with unrestricted access to food and water on a 12-hour light cycle, and experiments were conducted during the light portion.

Method Details

Stereotactic Surgeries For all surgical procedures, mice were anesthetized with 1.5% isoflurane at an oxygen flow rate of 1 L/min, and head-fixed in a stereotactic frame (David Kopf, Tujunga, CA). Eyes were lubricated with an ophthalmic ointment, and body temperature maintained at 37°C with a T/pump warm water recirculator (Stryker, Kalamazoo, MI). The fur was shaved and incision site sterilized prior to beginning surgical procedures, and subcutaneous saline and carpofen were provided peri-operatively and for 2 days post-operatively to prevent dehydration and for analgesia. 2+ imaging, mice underwent a single surgery in which 500nl of GCaMP6f virus was injected unilaterally with a Nanoject syringe (Drummond Scientific, Broomall, PA) prior to implanting a GRIN lens over the injection site. GRIN lenses were implanted with methods previously described ( Resendez et al., 2016 Resendez S.L.

Jennings J.H.

Ung R.L.

Namboodiri V.M.

Zhou Z.C.

Otis J.M.

Nomura H.

McHenry J.A.

Kosyk O.

Stuber G.D. Visualization of cortical, subcortical and deep brain neural circuit dynamics during naturalistic mammalian behavior with head-mounted microscopes and chronically implanted lenses. For in vivo Caimaging, mice underwent a single surgery in which 500nl of GCaMP6f virus was injected unilaterally with a Nanoject syringe (Drummond Scientific, Broomall, PA) prior to implanting a GRIN lens over the injection site. GRIN lenses were implanted with methods previously described (). Briefly, a craniotomy centered at the lens implantation site was made, and dura was removed from the brain surface and cleaned with a stream of sterile saline and absorptive spears (Fine Science Tools (FST), Foster City, CA) prior to lowering the GRIN lens (no tissue was aspirated out of site). 3 skull screws (FST, Foster City, CA) were inserted in evenly spaced locations around the implantation site, and the lens was slowly lowered in 0.1 mm DV steps and then fixed to the skull with dental cement (Dentsply Sinora, Philadelphia, PA). For vCA1 imaging, a ∼0.5 mm diameter, ∼6.1 mm long GRIN lens was used, and for dCA1 a ∼1.0 mm diameter, ∼4 mm long GRIN lens was used (Inscopix, Palo Alto, CA). Viral injection coordinates were (in mm, from brain tissue at site): (vCA1: −3.16 AP, 3.25 ML, −3.85, −3.50, −3.25 DV; dCA1: −2.15 AP, 1.85 ML, −1.55, −1.65 DV) and lens coordinates were (in mm, from skull at craniotomy): (vCA1: −3.16 AP, 3.50 ML, −3.50 DV; dCA1: −2.15 AP, 1.30 ML, −1.30 DV). At the completion of surgery, the lens was protected with liquid mold rubber (Smooth-On, Lower Macungie, PA), and imaging experiments commenced 3 weeks later. Kheirbek et al., 2013 Kheirbek M.A.

Drew L.J.

Burghardt N.S.

Costantini D.O.

Tannenholz L.

Ahmari S.E.

Zeng H.

Fenton A.A.

Hen R. Differential control of learning and anxiety along the dorsoventral axis of the dentate gyrus. For optogenetic surgeries, mice underwent a single surgery in which 500nl of opsin virus was injected into the vCA1 subregion with a Nanoject syringe as described above, prior to implanting fiber optics at the target site. Fiber optics were made with procedures previously published (), and were cut at ∼5mm in length for implantation. A single skull screw was implanted to allow for better adherence of the dental cement to the skull surface. Virus was injected in vCA1 at the following coordinates for all optogenetic manipulations (in mm): (−3.16 AP, 3.30 ML, −3.85, −3.50, −3.00 DV from brain at craniotomy). vCA1 cell body silencing was done with bilateral virus and fiber optic implantation at the following coordinates (in mm): (−3.20 AP, 3.35 ML, −3.50 DV from brain at craniotomy). vCA1-BA terminal activation was done bilaterally with fiber optic implantation at (in mm, from brain at craniotomy): (−1.70 AP, 3.00 ML, −4.00 DV), and vCA1-LHA was done with unilateral virus and fiber optic implanted at: (−1.95 AP, 0.50 ML, −4.75 DV). vCA1-LHA-ArchT cell body silencing surgeries were done bilaterally with CAV2-Cre injection into LHA at the above coordinates. For vCA1 cell body silencing, mice were allowed to recover for 4 weeks prior to commencing behavior experiments. For terminal activation, experiments began 8 weeks after surgery to allow for sufficient viral expression and trafficking of opsin to axon terminals. For CTB retrograde studies, 290nl of conjugated CTB (Life Technologies, Carlsbad, CA) was injected unilaterally in the LHA and BA subregions or LHA and mPFC in a single surgery at the following coordinates (in mm from brain tissue at site): (LH: −2.0 AP, 0.75 ML, −5.25, −5.0, −4.75 DV; BA: −1.70 AP, 3.0 ML, −4.25, −4.0 DV; mPFC: +1.90 AP, 0.3 ML, −2.75, −2.50 DV), and mice were perfused 7 days after injection for histology.

Patch-Clamp Electrophysiology For vCA1-ChR2 terminal slice recordings, mice with vCA1 viral expression of the excitatory ChR2-eYFP opsin (at 8 weeks post viral injection, to allow for trafficking of opsin to axon terminals) were anesthetized by halothane or isoflurane inhalation, decapitated, and brains rapidly removed. Coronal slices (350 μm) containing the BA and LHA were cut on a Leica VT1000S vibratome in ice cold partial sucrose artificial cerebrospinal fluid (ACSF) solution containing (in mM): 80 NaCl, 3.5 KCl, 4.5 MgSO 4 , 0.5 CaCl 2 , 1.25 H 2 PO 4 , 25 NaHCO 3 , 10 glucose, and 90 sucrose equilibrated with 95% O2 / 5% CO2 and stored in the same solution at 37°C for 30 minutes, then at room temperature until use. Recordings were made at 30-32°C (TC324-B; Warner Instrument Corp) in ACSF (in mM: 124 NaCl, 2.5 KCl, 1 NaH 2 PO 4 , 25 NaHCO 3 , 20 glucose, 1 MgCl 2 , 2 CaCl 2 ). Fluorescent vCA1-ChR2-eYFP axon terminals were first located within the BA and LHA on an upright microscope Axioskop-2 FS (Zeiss). Cells surrounded by these axons were then visualized via infrared-differential interference contrast (IR-DIC) optics and randomly selected for voltage-clamp recordings. A cesium-based internal solution was used (in mM): 125 Cs-methanesulfonate, 4 NaCl, 10 HEPES, 1 EGTA, 4 MgATP, 0.3 Na 2 GTP, 10 Na-phosphocreatine, 5 QX 314-Cl). Patch pipettes were made from borosciliate glass (A-M Systems) using a micropipette puller (Model P-1000; Sutter Instruments). In the bath, initial pipette resistance was 4.5-6.5 MΩ. Recordings were made without correction for junction potentials. Current and voltage signals were recorded with a MultiClamp 700B amplifier (Molecular Devices, USA), digitized at 5–10 kHz, and filtered at 2.5– 4 kHz. Data were acquired and analyzed using Axograph (Axograph Scientific, Sydney, Australia). For optical stimulation, 473 nm light pulses were generated using a 100 mW DPSS laser (Opto Engine LLC, Midvale, UT) and delivered through a 40X objective. Single light pulses (1 ms duration) delivered every 20 s were used to activate vCA1 fibers in the BA and LHA while recording light-evoked monosynaptic EPSCs in randomly chosen neurons. For vCA1-ArchT cell body slice recordings, mice with ArchT expression (4 weeks post viral injection) were anesthetized and perfused with modified sucrose ACSF containing (in mM) 75 NaCl, 2.5 KCl, 3.3 MgSO 4 , 0.5 CaCl 2 , 1NaH 2 PO 4 , 26.2 NaHCO 3 , 22 glucose, 52.6 sucrose, 10 HEPES, 10 choline chloride, 1 pyruvate, 1 L-ascorbic acid (∼300 mOsml, pH 7.4). The brain was dissected and 300 μm-thick slices were cut and placed in an interface chamber containing the same modified sucrose solution. Slices were incubated at 32°C for 30 min, then held at room temperature (23°C) in the interface chamber for at least 1 h. Recordings were made at room temperature and perfused with oxygenated ACSF containing (in mM) 119 NaCl, 2.5 KCl,1.3 MgCl 2 , 2.5 CaCl 2 , 1.3 NaH 2 PO 4 , 26.0 NaHCO 3 , 20 glucose (∼300 mOsml) at 23°C. Recordings were made at room temperature using pulled patch pipettes (5-7 MΩ) filled with internal solution containing (in mM) 150 K-Gluconate, 1.5 MgCl 2 , 5.0 HEPES, 1 EGTA, 10 phosphocreatine, 2.0 ATP, and 0.3 GTP. Green light was supplied via an arc lamp passed through a TRITC excitation filter, and delivered through a 40x objective centered on the soma of the patched cell. Patch-clamp recordings were obtained using Multiclamp 700B patch amplifiers, digitized using a Digidata 1322a, and data collected using pClamp 10 software (Molecular Devices).

Behavioral Assays Elevated Plus Maze. Mice were placed in a standard EPM sized maze (13.5” height of maze from floor, 25” full length of each arm-type, 2” arm width, 7” tall closed arms, with 0.5” tall/wide ledges on the open arms), with ∼650 light lux centered over the open arms to promote avoidance. Mice were placed in the center region of the maze, and were allowed to explore for 10 minutes while recording behavior with a webcam EthoVision XT 10 (Noldus, Leesburg, VA) or a digital camera (Carl Zeiss), and analyzed with EthoVision software or TopScan tracking software (Clever Sys, Reston, VA). Headdip behaviors in the EPM were hand-scored with Observer XT software (Noldus, Leesburg, VA). For ArchT-GFP silencing experiments, mice were run for 20 minutes in the EPM to allow for a sufficient number of open arm entries/ laser triggering events. Novel Object Task. Mice were placed in a familiar arena (22 × 16 × 6” length-width-height) which they were allowed to explore for 20 minutes on the previous day, in low light lux condition (∼50 lux). Behavior during the initial exposure to the arena was recorded and tracked with EthoVision XT 10 software, and 4 corner zones of equal size were drawn to determine the relative baseline preference for each location (∼6 × 5.5” length-width). During the novel object session, a novel object that elicited approach (a funnel taped down with colored tape) was placed into the least preferred corner zone of the arena (from day 1 tracking). Mice were allowed to explore the familiar arena for 10 minutes and behavior was recorded with EthoVision XT 10 software and webcam. Open Field Test. Mice were placed in an arena (18 × 18 × 12” length-width-height; Kinder Scientific, Poway, CA) with bright light (∼650 lux) centered over the center zone, and allowed to explore for 10 minutes while behavior was recorded and analyzed with MotorMonitor software. Context Exploration Task for Place Field Analysis. For place field analysis, mice were allowed to explore a novel arena (Context A- Context B- and Context A) (9.5 × 18” length-width) for 10 minutes each in low light lux conditions, with a 10 minute rest in a transfer cage in between sessions. Context A was a plain arena with short walls (6” height) while Context B was generated by placing standard mouse bedding, and tall, rounded yellow walls (10” height) within the same arena as Context A. The arena was kept in the same location for all 3 imaging sessions, and behavior was recorded and tracked with EthoVision XT 10 software. Real-Time Place Preference. Mice were placed in an identical 2-chamber arena (18.5 × 10 × 8” length-width-height) with standard mouse bedding and low light lux, and allowed to freely explore both chambers for 20 minutes while behavior was recorded with EthoVision XT 10 software. Contextual Fear Conditioning. Mice were run through a 2-day contextual fear conditioning paradigm. On day 1, mice were placed in a standard fear conditioning shock box (Coulbourn Instruments, Holliston, MA) with the following contextual cues: (anise scent, white noise, and a light on within the chamber), and were allowed to explore the context for 3 minutes prior to receiving a 2 s 0.7mA strength foot shock. On day 2, mice were placed back into the same context for 3 minutes to assess for freezing during CFC retrieval. For vCA1-LHA terminal modulation, at the end of day 2 retrieval, mice were given an additional 2 s foot shock to re-train them for day 3 retrieval testing. Behavior was recording with FreezeFrame video software (Coulbourn Instruments, Holliston, MA), and freezing was hand-scored by a blinded experimenter using Stopwatch scoring software (Center for Behavioral Neuroscience).

Freely Moving Ca2+ imaging Resendez et al., 2016 Resendez S.L.

Jennings J.H.

Ung R.L.

Namboodiri V.M.

Zhou Z.C.

Otis J.M.

Nomura H.

McHenry J.A.

Kosyk O.

Stuber G.D. Visualization of cortical, subcortical and deep brain neural circuit dynamics during naturalistic mammalian behavior with head-mounted microscopes and chronically implanted lenses. 3 weeks after surgery, mice were checked for GCaMP expression with a miniaturized microscope (Inscopix, Palo Alto, CA) and procedures previously described (). Mice were briefly anesthetized with 1.5% isoflurane at 1 L/min oxygen flow, and head- fixed into a stereotactic frame. The protective rubber mold was removed from the lens, and a magnetic baseplate was attached to a microscope and lowered over the implanted GRIN lens to assess the FOV for GCaMP+ neurons. If GCaMP+ neurons were visible, the baseplate was dental cemented in place onto the mouse headcap to allow for re-imaging of the same FOV for several weeks. Once baseplated, the same microscope was used for every imaging session with that mouse, and the focal plane on the hardware of the miniscope was not altered throughout the imaging experiments to ensure a constant FOV across sessions. Awake- behaving imaging sessions were commenced the day after baseplating, and mice were briefly anesthetized (< 5mins) in order to attach the miniscope to the baseplate each imaging session day. Mice were allowed to recover from anesthesia for 30 minutes before beginning imaging. Ca2+ videos were recorded with nVista acquisition software (Inscopix, Palo Alto, CA), and triggered with a TTL pulse from EthoVision XT 10 and Noldus IO box system to allow for simultaneous acquisition of Ca2+ and behavioral videos. Ca2+ videos were acquired at 15 frames per second with 66.56 ms exposure. An optimal LED power was selected for each mouse based on GCaMP expression in the FOV (pixel values), and the same LED settings were used for each mouse throughout the series of imaging sessions.

Optogenetic Manipulations Kheirbek et al., 2013 Kheirbek M.A.

Drew L.J.

Burghardt N.S.

Costantini D.O.

Tannenholz L.

Ahmari S.E.

Zeng H.

Fenton A.A.

Hen R. Differential control of learning and anxiety along the dorsoventral axis of the dentate gyrus. Mice were handled and habituated to fiber optic adaptor cables for 3 days prior to commencing behavioral experiments. For ArchT-GFP silencing experiments, ∼10mW of constant light were delivered via a 523nm 100mW laser (Opto Engine, Midvale, UT; a 594 nm laser was used in vCA1-BA Arch silencing experiments) to fiber optics implanted in mouse brain using a fiber optic patch cable as previously described (). For ChR2-eYFP experiments, ∼5-8mW of 5ms 10hz or 20hz light pulses were delivered via a 473nm 100mW laser (Opto Engine, Midvale, UT), and light delivery protocol was controlled via a Master-8 stimulator (AMPI, Jerusalem, Israel). For closed-loop ArchT-GFP silencing experiments, EthoVision XT 10 software and Noldus IO box system were used to record live- tracking of mice while they explored the EPM, OFT, and RTPP tasks. The laser was triggered- ON when mice were live-tracked in EthoVision in a pre-drawn stimulation zone (open arms for EPM, center for OFT, and a randomly selected chamber for RTPP). For ChR2-eYFP experiments, OFT optogenetic manipulations were ran in 3 minute laser epochs (light on-off-on). RTPP was ran as described above. In CFC, on light-ON days, light was delivered for the entire session (including through the end of the foot shock on day 1 light-ON cohorts).

Histology and Confocal and Epifluorescent Microscopy For all histology, mice were perfused transcardially with 4% (weight/volume) paraformaldehyde in 1X phosphate buffer solution (PBS) and brains were then removed and post-fixed in 4% PFA for 24 hours, after-which they were transferred to a 30% sucrose solution in PBS for 2 days. Sucrose-saturated brains were then flash-frozen and sliced in 50um thick coronal sections on a cryostat (Leica CM 3050S). Sections were incubated with 1:1000 Hoechst in 1x PBS (Invitrogen, Carlsbad, CA) for 10 minutes to label cell nuclei, and mounted and coverslipped with ProLong Gold antifade reagent (Invitrogen, Carlsbad, CA). Endogenous viral expression of fluorophores was used in all histology preparations (no immunolabeling was required to visualize fluorophores). Histology slides were imaged on a (Leica TCS SP8) confocal microscope using a 10x or 20x objective, or a (Zeiss Axiovert 200) epifluorescent microscope using a 2.5x or 10x objective. Appropriate GRIN lens and fiber optic placements were determined by post-fixing brains with head-caps and skulls intact for 1 week in 4% PFA to improve the clarity of the GRIN lens and fiber optic tracts. Brains were then placed into 30% sucrose solution as described above, and slices were collected in individual culture wells to maintain the accurate AP order of sections for fiber optic/ lens placement reconstructions. Sections were then mounted in AP order, and the bottom location of fiber optic tips and GRIN lenses were visually determined for each mouse by inspecting sections on an epifluorescent microscope. For CTB retrograde studies, tiled images were captured on a confocal microscope with a 10x objective, and red, green, and yellow cells were counted with an ImageJ Cell Counter toolbox. Lamination of CTB labeled neurons was determined by measuring the distance of counted cells to the Pyr/Rad border in ImageJ. For anterograde terminal fluorescence measurements, the vCA1 terminal fields in the BA and LHA were visualized in the same AP location (−1.70 mm) in vCA1-ChR2-eYFP expressing mice (8 weeks post-viral injection to allow for sufficient trafficking of opsin to axon terminals). The BA and LHA subfields were then imaged in the same section with identical exposure times using a 2.5x objective on an upright epifluorescent microscope. This allowed us to control for differences in baseline background fluorescence between sections, as BA and LHA terminal images were taken from the same sections, and fluorescence levels compared in a pairwise fashion. ROIs of the BA and LHA were hand-drawn in ImageJ, and the mean fluorescence value was used for comparisons.