All animal handling was performed in accordance with guidelines approved by the Harvard Institutional Animal Care and Use Committee and federal guidelines.
Dissociated cultures and western blotting
Primary hippocampal cultures were prepared from P0-1 Tsc1fl/fl mice of either sex using standard protocols. 2×105 cells were plated onto 24-well plates pre-coated with poly-d-lysine. On day 2 in culture, lentivirus expressing either Synapsin-driven GFP (6×108 IU/mL, Control) or GFP-IRES-Cre (2.1×108 IU/mL, Tsc1 KO) was added at 2-4 IU/cell. At 12–14 DIV, cells were harvested in lysis buffer containing 2mM EDTA, 2mM EGTA, 1% Triton-X, 0.5% SDS in PBS with Halt phosphatase inhibitor cocktail (Thermo) and Complete mini EDTA-free protease inhibitor cocktail (Roche). Total protein was determined by BCA assay (Pierce), and 12.5 μg of protein were loaded onto Tris-HCl gels (Bio-Rad). Proteins were transferred to PVDF membranes, blocked in 5% milk in TBS-Tween for one hour at room temperature (RT), and incubated with primary antibodies against Tsc1, Tsc2 (Bethyl Labs), β-Actin (Sigma), p-S6 (S240/244), total S6, p-p70S6K (T389), and total p-p70S6K (all from Cell Signaling) overnight at 4°C. Blots were incubated with HRP-conjugated secondary antibodies (Bio-Rad) for 1 hour at RT, washed, incubated with chemiluminesence substrate (Perkin-Elmer), and developed on Kodak Bio-Max film. Bands were quantified by densitometry using Image J software. Tsc1 and Tsc2 levels were normalized to a β-Actin loading control. p-p70S6K and p-S6 were normalized to total p70S6K and S6 levels respectively.
Mice were perfused transcardially with 1x PBS and 4% paraformaldehyde. Brains were post-fixed in 4% paraformaldehyde overnight and sectioned at 40 μm. Sections were blocked for 1 hour at RT in 2% normal serum and incubated overnight at 4°C with an antibody against phosphorylated (Ser240/244) S6 ribosomal protein (Cell Signaling). The following day, sections were washed and incubated for 1 hour at RT with an Alexa 594 conjugated secondary antibody (Invitrogen). Sections were mounted onto slides using ProLong Gold antifade reagent with DAPI (Invitrogen). Images were taken on an Olympus IX71 inverted microscope with an Andor camera using the same exposure and acquisition settings for each section.
Stereotaxic virus injection
mice of either sex were anesthetized with isofluorane and mounted on a stereotaxic frame (Stoetling) equipped with ear cups (Kopf). Unilateral injections into the CA1 region of the hippocampus were made at A/P −3.0 mm, M/L +/−3.0 mm, and D/V 2.2 mm relative to Bregma. 1.0 μl of an AAV-CreEGFP virus (1.2×1013
genome copy/ml, diluted 1:5 in PBS) was injected at a rate of 100nL/min. The viral construct was provided by Matthew During (Lu et al., 2009
), and the virus was prepared by the Harvard Gene Therapy Initiative using standard techniques. Unless otherwise specified, mice were used for experiments 10–14 days following the virus injection.
2-photon laser scanning microscopy and image analysis
Acute slices were prepared from virus-injected Tsc1fl/fl
mice as for electrophysiology (see below). CreEGFP-expressing (Tsc1 KO) and CreEGFP-negative (control) CA1 neurons were loaded with 40 μM Alexa 594 via a patch pipette and 2–5 images per neuron were taken from secondary apical dendrites (512 × 512 pixels; 36 × 36μm) using a custom-built 2-photon microscope and a Chameleon Ti-Sapphire laser tuned to 840 nm. Multiple slices at a separation of 1.0 μm were acquired to image the three-dimensional extent of the dendritic field. 6–12 neurons were imaged per genotype. Images were analyzed by an observer who was blind to genotype using custom software written in Matlab (Mathworks) (Tavazoie et al., 2005
). Spine lengths were measured from the junction with the dendritic shaft to the tip. Soma cross-sectional area was measured in the maximum intensity projection of a low-power image stack by quantifying the number of pixels within an outline drawn around the soma.
Hippocampal slices from P24–31 virus-injected Tsc1fl/fl or Tsc1wt/wt mice were cut (300 μm) in ice-cold external solution containing (in mM): 110 choline, 25 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 7 MgCl2, 0.5 CaCl2, 25 glucose, 11.6 sodium ascorbate, and 3.1 sodium pyruvate. Slices were transferred to ACSF containing (in mM): 127 NaCl, 25 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 1 MgCl2, 2 CaCl2, and 25 glucose.
For miniature excitatory postsynaptic current (mEPSC) recordings, the external solution was comprised of ASCF with (in μM): 1 TTX, 10 CPP, 50 picrotoxin, 10 mibefradil, 1 ω-conotoxin MVIIC, and 10 nimodipine to isolate AMPA receptor mediated EPSCs and to improve the quality of voltage- and space-clamp. ~3 MΩ recording pipettes were filled with cesium-based internal solution containing (in mM): 120CsMeSO3, 15 CsCl, 8 NaCl, 10 TEA-Cl, 10 HEPES, 2 QX 314 chloride, 4 Mg-ATP, 0.3 NaGTP, and 0.5 EGTA. Starting three minutes after break-in mEPSCs were recorded in voltage-clamp at −70mV for 10 minutes with the amplifier Bessel filter set at 3 kHz. Traces were analyzed in Igor Pro using custom software to identify miniature events and accumulate distributions of mEPSC amplitudes and inter-event intervals. Recordings with series resistance above 20MΩ or holding current above −200pA were rejected.
To measure evoked responses, paired voltage-clamp recordings were obtained from neighboring CreEGFP-positive and CreEGFP-negative CA1 neurons in ACSF containing 50 μM picrotoxin. Schaffer collaterals were stimulated at 0.33 Hz to evoke primarily AMPAR- (holding potential at −70mV) or NMDA-mediated (holding potential at +40mV) EPSCs. For this and all plasticity experiments, a cut was made between CA3 and CA1 to prevent recurrent excitation. Paired pulse ratio was determined in VC at −70mV by delivering two stimuli at various ISIs (10–500ms) and measuring the ratio of the peak amplitude of the second EPSC to the first EPSC, after subtracting the first EPSC from the second to remove any residual current.
For mGluR-LTD experiments, slices were allowed to recover 3–5 hours prior to LTD induction. For the DHPG-LTD experiment, VC recordings at −70mV in cesium-based internal solution were performed at RT in ACSF with 50 μM picrotoxin and 40 nM 2-chloroadenosine to reduce polysynaptic responses. LTD was induced by wash-in of 100 μM (RS)-3,5-DHPG (Tocris). Reconstituted DHPG solutions (50 mM) were kept at −80°C and used within 1 week of preparation. The PP-LFS-LTD experiments were performed at 30°C in ACSF with 50 μM picrotoxin and 50 μM D-APV (Tocris). Neurons were voltage clamped at −60mV in a potassium-based internal solution containing (in mM): 135 KMeSO4, 10 HEPES, 4 MgCl2, 4 NaATP, 0.4 NaGTP, 10 creatine phosphate, 125 EGTA, 0.2 EDTA. LTD was induced by 900 paired-pulse (50 ms ISI) stimuli delivered at low frequency (1 Hz) (Huber et al., 2001
For NMDAR-dependent LTD, neurons were voltage clamped at −70 mV in cesium-based internal solution and recordings were performed at 30°C in ACSF with 50 μM picrotoxin. NMDAR-dependent LTD was induced by 5 Hz stimulation for 3 minutes while cells were held at −40mV (Morishita et al., 2005
). For all plasticity experiments series resistance was 10–30 mΩ, remained stable during the course of the experiment, and was not compensated. The peak EPSC amplitude was calculated using Igor Pro and values were normalized to the 10 minute baseline period for each recording. The percent LTD was calculated from the average of the responses 35–45 (DHPG- and PP-LFS-LTD) or 25–35 (NMDAR-LTD) minutes after induction of LTD and expressed as a percentage of baseline.
Two-tailed, paired or unpaired t-tests were used for comparisons between two groups. In cases where the variance was found to be significantly different between two groups, a Welch’s t-test was used. To determine whether the late phase of plasticity was significantly different than baseline responses within an experiment, a one sample t-test was used.