Mice

Two strains of mice were crossed for these experiments. The Nestin-CreERT2 driver line (congenic C57BL/6 background; Jackson Laboratories, stock #16261) expresses a Cre recombinase-ERT2 fusion protein under the control of the rat nestin promoter [42]. Nestin is an intermediate filament protein expressed in neural progenitor cells in the adult brain. This driver line was crossed with the Ai35D responder line (congenic C57BL/6 background; Jackson Laboratories, stock #12735), in which expression of a Archaerhodopsin-3-EGFP fusion protein (Arch) is regulated by a loxp-flanked STOP cassette [43]. Pulsing mice with tamoxifen activates CreERT2 recombinase activity, which initiates permanent Arch expression in the NPCs and their progeny. This line was selected based on evidence that recombination in this line is highly specific to adult neurogenic niches [44].

To generate mice for the experiments, hemizygous Nestin-CreERT2 mice were crossed with homozygous Ai35D mice (Fig. 1a, b). All mice used in experiments were heterozygous at the Ai35D locus (Arch+/−) and either CreERT2+ (Cre+) or CreERT2– (Cre–). One hundred and fifty-two female mice (67 Cre+ and 85 Cre–) were used in the experiments shown in Figs. 1, 3–5. The behavioral effect shown in Fig. 3 was replicated with an additional 32 male mice (13 Cre+ and 19 Cre–; Supplementary Figure 2). Three male Cre+ Arch mice were used for the electrophysiological recordings in Fig. 2.

Fig. 1 Arch-GFP is expressed in adult-born neurons in Nestin-CreERT2 mice. a The Cre driver line targeted Nestin-expressing neural stem and progenitor cells, which produce dentate granule cells. Immature adult-born granule cells are identified by doublecortin (DCX) expression. Mature granule cells express NeuN but not DCX. b Nestin-CreERT2 mice were crossed with the Ai35D Cre-dependent Archaerhodopsin-eGFP mouse line. TMX administration induces Cre expression in the Nestin+ neural stem/progenitor cells and their subsequent progeny. c Mice were implanted with optic fibers 4 weeks after the end of induction. Vaginal smears and handling started 5 weeks after induction, and CFC began 6 weeks after TMX induction. d Representative images of dorsal and ventral optic fiber implants. e, f Representative images of Arch-eGFP (green), DCX (red), and NeuN (blue) expression. In Cre+ mice, GFP+ neurons were observed in the GCL and subgranular zone of the DG (e, left) but were not present in CA3 (e, middle) or CA1 (e, right). GFP+ cells were not present in Cre– mice (f). Scale bars = 50 μm or 75 μm (inset). g Stereological estimates of the number of DCX+, GFP+, and co-labeled (DCX+/GFP+) cells in the DG 6 weeks after induction. h Percentage of DCX+ and GFP+ neurons co-expressing the other marker. Error bars represent ±1 SEM Full size image

Fig. 2 Whole-cell patch recordings demonstrate Arch-mediated silencing of adult-born neurons. a, b Representative images of a patched GFP+ granule cell. Arrowhead denotes patch pipet. Scale bars = 20 μm. c Photostimulation (2.9 mW, green bar) induced a sustained hyperpolarizing current. d Arch-mediated silencing was sufficient to block spiking for a range of depolarizing current injections (n = 3). e To determine the effectiveness during prolonged photostimulation similar to that used during behavior, membrane potential hyperpolarization was quantified using continuous illumination for 3 min. No rebound spiking was observed following any duration of photostimulation Full size image

Mice were maintained on a 12-h light/dark cycle in a temperature- and humidity-controlled room with food and water provided ad libitum. From birth until the implant surgery, mice were housed in plastic cages with wood chip bedding. After surgery, they were moved to a larger plastic cage with wood chip bedding to permit greater clearance between the cranial implants and cage lid. Experiments were conducted during the light cycle. All procedures were approved by the University of Texas at Austin Institutional Animal Care and Use Committee.

Tamoxifen administration

Mice (7–10 weeks old) were pulsed with tamoxifen (180 mg/kg/day; Sigma-Aldrich, T5648) for 5 days. Tamoxifen (in 10% ethanol/90% sunflower seed oil) was delivered per os by oral gavage.

Optic fiber construction and implantation

Optic fiber implants were constructed based on published protocols [45, 46], using 1.25 mm ceramic ferrules (Kientec Systems Inc.) and 200 μm core, 0.39 numerical aperture multimode fiber (ThorLabs). Four weeks after induction, mice underwent stereotaxic surgery for optic fiber implantation (Fig. 1c,d). Mice were anesthetized with isoflurane and placed into the stereotaxic frame. Fibers were implanted bilaterally with fiber tips targeted to the dorsal (from Bregma: A/P −2.0 mm; M/L ± 1.3 mm; D/V −1.5 mm; 20° angle) or the ventral DG (from Bregma: A/P −3.7 mm; M/L ± 2.6 mm; D/V −2.1 mm) and secured with dental cement (Fastray Regular Set, Interguide Dental and Medical Supply, 0921376). Coordinates were chosen based on previous studies [47, 48].

Electrophysiology

Six weeks after induction, mice were anesthetized with a lethal dose of ketamine/xylazine and transcardially perfused with a cold (~2 °C) modified artificial cerebral-spinal fluid (aCSF) consisting of the following (in mM): 210 sucrose, 1.25 NaH 2 PO 4 , 25 NaHCO 3 , 0.5 CaCl 2 , 7 MgCl 2 , 7 dextrose, 1.3 ascorbic acid, and 3 sodium pyruvate; bubbled with 95% O 2 –5% CO 2 . Brains were removed and hemisected along the longitudinal fissure. Slices were prepared from the intermediate hippocampus using a vibrating microtome. Slices were then placed in a warmed (~35 °C) holding chamber containing aCSF (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH 2 PO 4 , 25 NaHCO 3 , 2 CaCl 2 , 2 MgCl 2 , 12.5 dextrose, 1.3 ascorbic acid, and 3 sodium pyruvate; bubbled with 95% O 2 –5% CO 2 . After incubating in the holding chamber for 20 min, slices were removed and allowed to rest at room temperature for at least 40 min prior to recording.

Whole-cell current-clamp recordings were performed as previously described [49]. Briefly, hippocampal slices were submerged in a recording chamber continuously perfused with aCSF (32–34 °C). GFP+ neurons were identified using a Leica SP5 microscope using 930 nm 2-photon excitation. After establishing the whole-cell configuration, laser photostimulation (532 nm, 2.4–3.6 mW; Laserglow Technologies) was delivered to determine the effectiveness of the Arch construct. Action potentials were generated by somatic current injections (0–100 pA). The number of action potentials as a function of injected current was plotted before and during photostimulation. In addition, the efficacy during prolonged photostimulation was determined using continuous illumination for 3 min.

Optogenetic inhibition

For intracranial light stimulation, patch cables were connected via a rotary joint (Doric Lenses) to a 140 mW, 532 nm laser (Shanghai Dream Lasers Technology Co). Laser output was set to output 7–10 mW measured at the end of the implant.

For the Light Off condition, an opaque dummy ferrule was placed between the cranial implants and the patch cable, thereby blocking laser light entry into the brain. This configuration was used to equate the ambient light conditions between the Light On and Light Off conditions (due to any green light escaping from the patch cable or ferrule).

Fear conditioning

Apparatus

Fear conditioning was conducted in Med Associates chambers (30.5 cm × 24.0 cm × 21.0 cm) with three aluminum walls and a clear Plexiglas door and ceiling. Each chamber was housed within a larger, sound-attenuating cabinet equipped with a fan, which generated ambient noise (~65 dB). During all procedures, chambers were lighted from above with white and infrared lights. An infrared digital camera mounted on the inside of the door of the cabinet recorded mice during all sessions. The experiment room was illuminated with red light during all sessions.

The conditioning chambers could be configured into three contexts as previously described [50]. The training context (Context A) was comprised of a stainless steel rod floor (36 rods evenly spaced 8 mm from center to center) with a white paper towel covering the waste tray underneath the floor. The chamber was cleaned and scented with 70% ethanol. The similar alternate context (Context B) had a floor with staggered stainless steels rods (36 rods spaced 8 mm from center to center with alternating heights) and a brown paper towel in the waste tray underneath the floor and was cleaned and scented with Clorox Fresh Scent antiseptic wipes. The alternate context used for the tone test sessions (Context C) had a white Plexiglas floor covered with wood chip bedding and was cleaned and scented with Clorox Fresh Scent antiseptic wipes. Bedding was replaced between trials.

Common procedures

All experiments were run 6 weeks after TMX induction. Mice were handled for 5 days prior to the start of experiments. During each handling session, optic patch cords were connected, and the mice were allowed to explore a cage while tethered to the cables for ~5 min.

For behavioral experiments, mice were transported to the holding room adjacent to the testing room a minimum of 1 h prior to experimentation. For Contexts A and B, mice were individually transferred to and from the testing room in opaque white containers with clear lids. For Context C, mice were individually transferred in gray rectangular boxes with gray lids. Training and testing sessions were video recorded from the side. All handling and behavioral experiments were conducted by women.

Context fear conditioning

Training occurred on day 1 in Context A. Mice were placed into the conditioning chamber immediately after the optic fiber was connected. A single footshock (2 s, 0.75 mA) was administered 180 s after the session started. The session ended 30 s after the footshock.

Mice that received Light On during training were tested in Context A on day 2 and were not tested in Context B. Mice that received Light On during context testing were tested in Contexts A and B in counterbalanced order on days 2 and 3. Context test sessions were 3 min without footshock. Fiber optic patch cables were connecting during all sessions.

Tone-shock fear conditioning

Training occurred on day 1 in Context A with Light Off. The tone (5000 Hz, 85 dB) was presented for 20 s starting at 121, 160, and 220 s into the 300-s session. Each tone co-terminated with a footshock (1 s, 0.75 mA). Each mouse received two tone test sessions in Context C: one session with Light On (day 2) and one with Light Off (day 3). Tone test sessions were 300 s in duration. The tone was presented three times during each session at the same time points as during conditioning (121, 160, and 220 s after session start). Patch cables were connected during all sessions.

Estrous cycle monitoring

Mice received daily vaginal smears beginning 1 week prior to fear conditioning and continuing through conditioning (~10 days total). The vaginal canal was flushed with 15 μl of sterile saline using a 20 μl pipette tip. Fluid was then smeared on a Superfrost plus slide (Fisher Scientific). The estrous stage at the time of training and testing was determined based on the pattern of changes in cytology over time [51, 52].

Tissue collection and immunohistochemistry

Mice were deeply anesthetized with a mixture of ketamine (150 mg/kg) and xylazine (15 mg/kg) and transcardially perfused with 25 ml of cold 0.1 M phosphate buffered saline (PBS) followed by 20 ml of 4% paraformaldehyde in 0.1 M PBS (PFA). Tissue was post-fixed for 24 h in 4% PFA at 4 °C then cryoprotected in 30% sucrose in 0.1 M PBS. Coronal sections (35 μm) were cut on a cryostat, and tissue sections were stored in cryoprotectant antifreeze [53].

DCX and NeuN immunohistochemistry were carried out as previously reported [54]. On day 1, sections were washed three times for 5 min each in 0.1 M PBS and blocked in 10% normal donkey serum in 0.1 M PBS with 0.25% Triton X-100 (PBST) for 1 h at room temperature. Sections were incubated overnight at room temperature with the primary antibodies in blocking solution (goat anti-DCX 1:1000, Santa Cruz Biotechnology, sc-8066; mouse anti-NeuN 1:1000, Millipore, MAB377). On day 2, tissue sections were washed three times for 5 min each in 0.1 M PBS followed by a 1-h incubation with secondary antibodies in blocking solution (Biotin-SP-conjugated Affinipure donkey anti-goat 1:500, Jackson Immuno Research, 705-065-003; donkey anti-mouse Cy3 1:500, Jackson Immuno Research, 715-165-150). Sections were next incubated with a tertiary antibody in blocking solution (Avidin DyLight 649 1:500, Jackson Immuno Research, 016-490-084) at room temperature for 60 min, followed by three 5-min washes in 0.1 M PBS. Sections were mounted onto Superfrost plus slides (Fisher Scientific) and coverslipped with Fluoromount-G (Southern Biotech).

Cell counting

GFP+ and DCX+ cells in the granule cell layer (GCL) and subgranular zone (SGZ) were counted by an experimenter blind to experimental condition. Cells were counted in every 12th section through the DG (7–8 sections total) under fluorescent illumination (Zeiss Axio Imager M2) using the optical fractionator (Stereo Investigator, MBF Bioscience). Counting was carried out using a 40× objective (Plan-Neofluar). The counting frame (70 μm x 70 μm) and sampling grids (~15 sites/section) were optimized to include at least 200 cells of each cell type of interest. Each GFP+ or DCX+ cell was examined for co-localization with the other antigen.

Data analysis

Freezing and distance traveled during the shock were quantified using AnyMaze software (Stoelting). Some videos were also scored by an experimenter blind to experimental condition using Stopwatch+ (Georgia State University, Center for Behavioral Neuroscience). Scoring by AnyMaze and the blind experimenter were highly correlated (r2 = 0.975, p < 0.001).

Statistical analyses were performed using Prism 6 (GraphPad software) and JMP PRO 12 (SAS Institute Inc.). Significant interactions were probed using pairwise Sidak’s multiple comparison test (post hoc comparisons).