Imaging Neuronal Calcium Dynamics in Brain Slices

We made recordings from CA1 cells in hippocampal slice culture, and cortical layer 2/3 pyramidal cells (S1) in acute brain slices at room temperature (22–24 °C). Patch pipettes were pulled from borosilicate glass (standard wall with filament) and had 4–6 MΩ resistance when filled with internal solution (128 K-methylsulfate or K-gluconate, 10 HEPES, 10 Na-phosphocreatine, 4 MgCl₂, 4 Na₂ATP, 0.4 Na₂GTP, 3 ascorbic acid (pH 7.25, 290 mOsm), in mM). Slice recording and simultaneous line scan imaging were performed as before 24 (link). During recording, slices were bathed in ACSF (127 NaCl, 25 NaHCO₃, 1.25 NaH₂PO₄, 25 glucose, 2.5 KCl, 2 CaCl₂, 1 MgCl₂, in mM) bubbled with carbogen. Cells were selected for data analysis if they had nuclear exclusion of GECI fluorescence, input resistances of at least 100 MΩ, and resting potentials ≤ −50 mV in cultured slice, or ≤ −65 mV in acute slice. For experiments with evoked-action potential stimuli, 10 μM (R)-CPP (Tocris) and 10 μM NBQX (Sigma) were added to the bath to block glutamate receptors. Action potentials were triggered at 83 Hz by current injection (1–4 nA, 2 ms) through the patch pipette.
Imaging was performed in line-scan mode (500 Hz) across the apical dendrite, 20–50 μm from the base (Fig. 2a). The Ti:Sapphire laser (Mai Tai, Spectro-Physics, CA) was tuned to 910 nm for GCaMPs imaging and 860 nm to excite FRET indicators. For GCaMPs co-expressed with mCherry, we separated fluorescence into green and red channels with a 565 nm dichroic, and BG22 (green channel) and HQ620/90 (red channel) emission filters. For the FRET-based GECIs, we separated fluorescence with a 505 nm dichroic, and HQ480/80 (cyan channel) and HQ535/50 (yellow channel) emission filters. The PMT dark current was subtracted from all traces. In slice culture recordings, mean baseline fluorescence (F₀) was calculated from the filter raw trace (20 Hz) prior to the action potential stimuli, as in 24 (link). Peak fluorescence was determined by averaging 30 ms of the raw fluorescence time series about the peak of the trace linearly filtered at 20 Hz. For acute slices, response baseline was defined as the mean of the 250 ms window immediately prior to stimulation. Peak response was calculated as the maximum value of the filtered trace (100 ms moving window) within 500 ms of stimulation cessation. This method gave ~3% ΔF/F for 0 AP traces. Noise was calculated on a per cell level, as the mean standard deviation of stimulation-free, one second, bleach-corrected trace segments. For display, example traces were filtered with a Savitzky-Golay filter (2^nd order, 50 ms span). Action potential detection was quantified both by a double blind psychometric test and by algorithmic template matching. In the psychometric test, eight volunteers were shown a response template and asked if it was present in randomly ordered, sequentially presented traces. False positive rate was determined by the response to 0 AP traces. The algorithmic method computed the maximum cross-correlation between a template and the fluorescence trace lagged 200 ms about the stimulus onset. Detection success was defined as a cross-correlation value greater than 95% of baseline traces. The baseline trace set consisted of all recorded 0 AP traces plus those traces reversed and/or inverted. The template was the first 1.5 seconds of the mean 3 AP response (GCaMP3) or the mean 5AP response (D3cpV, TN-XXL). Rise T_1/2 of hippocampal neurons was measured as the time between the onset of current injection and the half peak response. Decay T_1/2 was measured as the time of half decay of a single exponential fit of the recovery from peak response to baseline. All analysis was performed with MATLAB (Mathworks, Natick, MA).