Four lentiviral vectors were constructed. Control vector contains a H1 promoter followed by a U6 promoter and an ubiquitin promoter driving mcherry expression. To construct Syt1 KD vector, short hairpin sequence containing syt1 sequence 5'- gag caa atc cag aaa gtg caa -3' was cloned into Xho1-Xba1 locus downstream of the H1 promoter of control vector. In TetTox vector, tetanus toxin light chain (GenBank: L19522.1) was cloned into EcoR1 locus downstream of the ubiquitin promoter of FUW vector. Syt1 KD + rescue vector was constructed by changing mCherry in Syt1 KD vector to EGFP and shRNA-resistant syt1 gene linked in frame by 2A sequences derived from foot-and-mouth disease virus (Okita et al., 2008
). ShRNA-resistant syt1 was made by mutating syt1 sequence 5'- gag caa atc cag aaa gtg caa -3' into 5'- gaa cag att caa aag gtc caa -3'. Three AAV vectors were constructed. In control vector, the following components were arranged sequentially downstream of left-ITR of AAV2: CMV promoter and beta-globin intron, EGFP, hGH poly A sequence, H1 promoter, SV40 poly A sequence and right ITR. In Syt1 KD AAV vector, syt1 short hairpin sequence was cloned into the Xho1-Xba1 locus downstream of H1 promoter of control vector. In TetTox vector, the following components were arranged sequentially downstream of the left-ITR: CMV promoter and beta-globin intron, EGFP and TetTox linked in frame by 2A sequence as described above, hGH poly A and the right ITR.
Lentivirus packaging, infection and recording of cultured neurons
Lentivirus and cortical culture were prepared and recorded as described (Maximov et al., 2007
; Pang et al., 2010
). Neurons derived from P0 (postnatal day 0) or P1 mice were infected with lentivirus at 5–6 DIV (days in vitro) and recorded at 15–16 DIV. Cultured neurons were transferred to extracellular solution containing (in mM; at room temperature with pH 7.4) 140 NaCl, 5 KCl, 2 CaCl2
, 2 MgCl2
, 10 HEPES and 10 glucose. The neurons were patched with pipettes with resistance between 3 and 4 MΩ and clamped at −70 mV. The patch pipette was filled with intracellular solution containing the following components (in mM; at room temperature with pH 7.4): 135 CsCl, 10 HEPES, 1 EGTA, 1 Na-GTP, 4 Mg-ATP, and 10 QX-314 [N
-(2,6-dimethylphenylcarbamoylmethyl) triethylammonium bromide]. Synaptic current was evoked by 90 µA/1 ms current injections via concentric bipolar electrode (CBAEC75, FHC) placed ~150 µm to the patched neurons. IPSCs was isolated by adding AMPA and NMDA receptor blockers CNQX (20 µM) and AP-5 (50 µM) in the extracellular solution. The frequency, duration, and magnitude of the extracellular stimulus were controlled with a model 2100 Isolated Pulse Stimulator (A-M Systems) synchronized with Clampex 10.2 data acquisition software (Molecular Devices). Synaptic currents were monitored with a Multiclamp 700B amplifier (Molecular Devices). Synaptic currents were sampled at 10 kHz and analyzed offline using Clampfit 10.2 (Molecular Devices) software. For graphic representation, the stimulus artifacts of the current traces were removed.
AAVs were packaged with AAV-DJ capsids for high efficiency in vivo neuronal infection. Virus was prepared with a procedure as described (Zolotukhin et al., 1999). Briefly, AAV vectors were co-transfected with pHelper and pRC-DJ into AAV-293 cells. 72 hr later, cells were collected, lysed and loaded onto iodixanol gradient for centrifugation at 400,000g for 2 hrs. The fraction with 40% iodixanol of the gradient was collected, washed and concentrated with 100,000 MWCO tube filter. The infectious titer of virus was measured by infecting HEK293 cells. The concentrations of virus used for stereotaxic injection were adjusted to 1.0 × 107 infectious units/µl (except that for TetTox AAV, for which the highest titer obtained, 8.8 × 106, was used).
C57BL/6 mice were anesthetized with tribromoethanol (125–250 mg/kg). Viral solution was injected with a glass pipette at a flow rate of 0.15 µl/min. Coordinates used for the hippocampal injection were AP +1.95 mm, ML ±1.25 mm, DV −1.20 mm (for CA1) and −1.95 mm (for DG). 1 µl of viral solution was injected in CA1 and another 1 µl in DG. The coordinates used for the prefrontal injection were AP −1.0 mm, ML ±0.3 mm, DV−1.0 mm and −1.5 mm. The sites at DV−1.0 mm and −1.5 mm both received 1 µl of injection. The coordinates used for the entorhinal injection were AP +4.5 mm, ML ±3.5 mm, DV−4.0 mm. The injections were bilateral except otherwise noted.
2-month old C57BL/6 mice were injected with AAVs and were used for slice physiology 3–4 weeks after the infection. Transverse hippocampal slices or coronal prefrontal slices (250 µm) were cut in ice cold solution comprising (in mM): 75 Sucrose, 75 NaCl, 2.5 KCl, 1 NaH2PO4, 8 MgSO4, 0.5 CaCl2, 26.2 NaHCO3, 20 D-glucose saturated with 95% O2/ 5% CO2 and transferred to a holding chamber containing artificial cerebrospinal fluid (ACSF) composed of (in mM): 117.5 NaCl, 2.5 KCl, 1 NaH2PO4, 1.3 MgSO4, 2.5 CaCl2, 26.2 NaHCO3, 11 D-glucose to recover for at least one hour at room temperature before being transferred to a recording chamber continually perfused (1 ml/min) with oxygenated ACSF (maintained at 27–29° C) containing 50 µM of picrotoxin. Whole-cell voltage-clamp recordings were made with 3–5 MΩ pipettes filled with internal solution containing (in mM): 135 CsMeSO4, 10 HEPES, 8 NaCl, 0.25 EGTA, 2 MgCl2, 4 Mg ATP, 0.3 NaGTP and 5 Phosphocreatine (pH 7.3). Neurons were clamped at −65 mV for recording of EPSC in hippocampal slices. In the prefrontal slices neurons were clamped at +30 mV to record NMDAR-mediated EPSCs in the presence of 10 µM of NBQX.
Local Field Recording
2-month old mice were injected with AAVs and were implanted with recording electrodes 2–3 weeks later. Field potential recordings were obtained from the CA1 field of the right, dorsal hippocampus. To implant electrodes, mice were sedated with diazepam (10 mg/kg, i.p.), anesthetized with isoflurane (1–3%), placed in a stereotaxic frame, maintained on a heating pad, and prepared for aseptic surgery. A hole was drilled 2.2 mm posterior and 1.6 mm right of bregma. An insulated, 50 µm diameter stainless steel wire (California Fine Wire) was implanted 1.7 mm below the surface of the brain. The reference electrode was placed in the cerebellum. Two screws were placed in the skull. Electrode leads were connected to pins that were inserted into a strip connector, which was attached to the screws and skull with cranioplastic cement. Following surgery, mice were kept warm and received lactated ringer’s with dextrose, antibiotic (enrofloxacin, 10 mg/kg, s.c.), and analgesic (buprenorphine, 0.05 mg/kg, s.c.). After recovering for 7 d, mice were monitored for at least 6 h by EEG recording and simultaneous videotaping. Recordings were obtained with epoch transmitter and receiver tray for wireless EEG (Ripple LLC) and a Cyberamp 380 (Molecular Devices). Signals were amplified, filtered (1–100 Hz), and sampled at 200 Hz (PClamp, Molecular Devices). The whole 6-hr recording was divided into 5-min sessions. Based on the behavioral states of the mice, each session was classified as “exploration”, “motionlessness” or a situation which cannot be categorized into these two situations with the following criteria: if a mouse was exploring the recording chamber for more than 3 minutes in a 5-min session, this session will be classified as "exploration"; if a mouse was immobile (no apparent movement except breathing and slight shaking of head or body) for more than 4 minutes in a 5-min session, this session will be classified as "motionlessness”. The power spectrums of each 5-min session were generated and the power spectrums from the same behavioral category were averaged together with Clampfit10.2 (Axon Laboratory). The wavelet spectrums of representative traces were produced by AutoSignal1.7.
2 month-old male C57BL/6 mice (Charles River) were housed individually with normal 12/12 hr daylight cycle. They were handled daily for 5 days prior to training. On training day, mice were placed in fear conditioning chamber (H10–11M-TC, Coulbourn Instruments, PA) located in the center of a sound attenuating cubicle (Coulbourn Instruments). The conditioning chamber was cleaned with 10% ethanol to provide a background odor. A ventilation fan provided a background noise at ~55 dB. After a 2 min exploration period, 3 tone-footshock pairings separated by 1 min intervals were delivered. The 85 dB 2 kHz tone lasted for 30 s and the footshocks were 0.75 mA and lasted for 2 s. The foot shocks co-terminated with the tone. The mice remained in training chamber for another 30 seconds before being returned to home cages. In context test, mice were placed back into the original conditioning chamber for 5 min. The altered context and tone tests were conducted in a new room. The same conditioning chamber were moved to this room and modified by changing its metal grid floor to a plastic sheet, white metal side walls to plastic walls decorated with red stripes, background odor of ethanol to vanilla. The ventilation fan was turned off to reduce background noise. Mice were placed in the altered chamber for 5 min to measure the freeze level in the altered context and after this 5-min period a tone (85 dB, 2 kHz) was delivered for 1 min to measure the freeze to tone. The behavior of the mice was recorded with the Freezeframe software and analyzed with Freezeview software (Coulbourn Instruments). Motionless bouts lasting more than 1 second were considered as freeze. Animal experiments were conducted following protocols approved by Administrative Panel on Laboratory Animal Care at Stanford University.
Mice were anesthetized with tribromoethanol and perfused with 10 ml of PBS followed by 50 ml of fixative (4% paraformaldehyde diluted in PBS). The brains were removed and post-fixed for 3 hours at room temperature and then immersed in 30% sucrose solution overnight before being sectioned at 30 µm-thickness on a cryostat. The free-floating brain sections were collected in PBS and counterstained with DAPI. The brain sections were mounted onto glass slides with Vectashield mounting medium (Vector Laboratories, CA). Micoscopic photos were taken with a Leica DM IRE2 microscope. Photos taken with 10X objective were tiled to generate the image of the whole brain sections.
Cultured neurons were homogenized in lysis buffer (1% SDS, 10 mM Tris), mixed with 6× loading buffer (0.5 M tris, 60% glycerol, 10% SDS, 10% Beta-Mercaptoethanol and 0.01% bromphenol blue) and denatured at 100°C for 20 min. After centrifugation at 14,000 rpm for 30 min the supernatants were loaded for SDS-PAGE and immunoblotted with standard chemiluminescence protocols. The primary antibodies used in the study include: anti-syt1 (CL41.1), anti-syb2 (CL69.1) and Synx1 (U6251). Blots were digitized and quantified with NIH image software. All band intensities were normalized to that of control samples.
- Burst-evoked synaptic transmission suffices to entrain hippocampus-dependent memory
- Precisely timed spikes are indispensible for prefrontal cortex-dependent memory
- Prefrontal cortex and hippocampus determine the precision of contextual memories
- AAV DJ allows efficient brain region-specific manipulation of gene expression