recording glass patch pipettes  (World Precision Instruments)

Journal: bioRxiv

Article Title: Heterogeneity and variability in short term synaptic plasticity and implications for signal transformation

doi: 10.1101/2025.06.28.662098

Figure Lengend Snippet: A. Schematic illustrating cellular heterogeneity, referring to between-cell variability; pattern heterogeneity, referring to within-cell variability reflected in responses to distinct optical patterns; and trial-to-trial variability, referring to fluctuations in response when repeating the same pattern (12 repeats). B. Schematic showing acute transverse slices of mouse hippocampus expressing channelrhodopsin specifically in CA3. Optogenetic activation of CA3 was delivered using a DMD device (Polygon) integrated into the light path of the microscope. CA3 activity was recorded with a field electrode placed near the cell body layer, while CA1 responses were recorded using whole-cell patch clamp. C. Fluorescence image showing channelrhodopsin expression (Td-Tomato) in CA3. CA3 provides direct excitatory input and feedforward inhibition to CA1. D. Optical patterns were either 1, 5 or 15 non-overlapping squares, randomly chosen in a 24x24 optical grid. STP was tested by pairing the same optical pattern at 50 ms interval. CA1 was voltage clamped at -70 mV for recording Excitatory PostSynaptic Currents (EPSC traces in blue) and at 0 mV for recording Inhibitory PostSynaptic Currents (IPSC traces in red). E. There were fifteen 1-square patterns which were then clubbed into three 5-square and one 15-square pattern. F. Representative raw traces from a single CA1 pyramidal cell recording in response to a 15 square (15 SQ) stimulation pattern. From top to bottom: CA1 inhibitory postsynaptic current (IPSC, orange) recorded at 0 mV; CA1 excitatory postsynaptic current (EPSC, blue) recorded at -70 mV; frame trigger to switch optical patterns; photodiode signal for stimulus timing; and the corresponding local field potential recorded in the CA3 pyramidal layer. G. Ridgeline density plots showing the distribution of response amplitudes across the entire population for the first (P1) and second (P2) peaks. Distributions are shown for inhibitory currents (P1 and P2 Inh), excitatory currents (P1 and P2 Exc), and CA3 field potentials (Field P1 and P2). Responses to 1, 5, and 15 SQ stimuli are color-coded as indicated in the legend. The probability density was estimated using a Gaussian Kernel Density Estimation (KDE) with a bandwidth adjustment factor of 0.1. H. Summary of peak synaptic currents across all recorded cells. Split violin plots show the distribution of the first peak amplitudes as a function of the number of stimulated squares (1, 5, 15 Squares). Orange and blue represent IPSCs and EPSCs, respectively. Statistical comparisons between 1, 5 and 15 patterns are shown (Mann-Whitney U-test: *p<0.05, **p<0.01, ***p<0.001). I. Same as H for second peak amplitudes J. Summary of CA3 field potential amplitude (1, 5, 15 SQ). The violin plot shows the distribution of the first peak-to-trough amplitude (Field P1) for selected cells. K. Correlation between EPSCs and local field potentials. The scatter plot shows the relationship between Cell P1 and Field P1. The red line indicates the linear regression, with the coefficient of determination (R 2 ) and p-value shown. L. Desensitization of CA3 field potential: distribution of the first peak (Field P1) and second peak (Field P2) amplitudes across all conditions M. Stability of the field potential response over time: Field potential peak (Field P1) between the first and last trials (Trial 1 vs. Trial 12) of the experiment.

Article Snippet: Whole cell recording pipettes of 4-6 MO were pulled from thick-walled borosilicate glass capillaries from Sutter Instruments (1.50mm O.D., 0.86mm I.D., 1Ocm long) on a P-97 Flaming/Brown micropipette puller (Sutter Instrument, Novato, CA).

Techniques: Expressing, Activation Assay, Microscopy, Activity Assay, Patch Clamp, Fluorescence, Inhibition, MANN-WHITNEY