Perirhinal cortex abnormalities impair hippocampal plasticity and learning in Scn2a, Fmr1, and Cdkl5 autism mouse models
Data files
Jan 31, 2025 version files 502.06 KB
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I-O_Curve_-_Means___SEMs_Amp_-_Hippocampus.txt
4.63 KB
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I-O_Curve_-_Means___SEMs_Amp_-_PRC.txt
3.44 KB
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I-O_Curve_-_Means___SEMs_Slope_-_Hippocampus.txt
6.63 KB
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I-O_Curve_-_Means___SEMs_Slope_-_PRC.txt
2.81 KB
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I-O_Curve_-_Raw_EPSP_Amp_-_Hippocampus.txt
9.03 KB
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I-O_Curve_-_Raw_EPSP_Amp_-_PRC.txt
8.44 KB
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I-O_Curve_-_Raw_EPSP_Slope_-_Hipp.txt
10.06 KB
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I-O_Curve_-_Raw_EPSP_Slope_-_PRC.txt
7.97 KB
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I-O_Curve_-_Raw_FVs_-_Hippocampus.txt
10.99 KB
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I-O_Curve_-_Raw_FVs_-_PRC.txt
8.44 KB
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LTP_Amplitude_-_Raw_-_PRC.txt
43.03 KB
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LTP_Amplitude_-_Timepoints_-_PRC.txt
1.92 KB
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LTP_Slope_-_Raw_-_Hippocampus.txt
155.55 KB
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LTP_Slope_-_Raw_-_PRC.txt
167.10 KB
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LTP_Slope_-_Timepoints_-_Hippocampus.txt
7.31 KB
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LTP_Slope_-_Timepoints_-_PRC.txt
9.86 KB
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PPR_-_Amp_-_Hipp.txt
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PPR_-_Amp_-_PRC.txt
4.33 KB
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PPR_-_Slope_-_Hipp.txt
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PPR_-_Slope_-_PRC.txt
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README.md
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Abstract
Learning and memory deficits, including spatial navigation difficulties, are common in Autism Spectrum Disorder (ASD), yet the neurobiological underpinning of this phenotype remains unknown. Several ASD mouse models (Scn2a+/- , Fmr1-/-, Cdkl5-/-) exhibit impaired spatial learning, with these deficits often attributed to hippocampal dysfunction. However, we identify the perirhinal cortex (PRC) as a critical driver of these deficits. Cortical-wide Scn2a reduction in excitatory neurons replicated the spatial learning and long-term potentiation (LTP) impairments— a cellular correlate of learning—seen in Scn2a+/- mice, while hippocampal-wide reduction did not. PRC-specific viral-mediated Scn2a reduction in excitatory neurons decreased release probability, which consequently disrupted synaptic transmission and LTP in the hippocampus, as well as spatial learning. As PRC activity was reduced, chemogenetic activation of the PRC reversed these deficits in Scn2a+/- mice and rescued spatial learning and LTP impairments in Fmr1 and Cdkl5 knockout mice. Thus, in several genetic models of ASD, PRC abnormalities may disrupt hippocampal function to impair learning and memory.
https://doi.org/10.5061/dryad.6djh9w1c8
Description of the data and file structure
The data from the electrophysiology experiments were uploaded as .txt files, each with specific data included as delineated by the title. Specifically, the means and standard error (SEM) for the input-output curves for each genotype, the raw values for fiber volley amplitude, the raw values for fEPSP slope (and amplitude, in the case of a few datasets), raw paired-pulse ratios (PPR) from fEPSP amplitude (a few genotypes, supplemental) or fEPSP slope, and LTP as determined by normalizing either the fEPSP amplitude (Amp) or the fEPSP slope (Slope) to baseline. The LTP data also includes 'timepoints' data, which is data that is averaged from 20 - 30 min (second row) and data averaged from 50 - 60 min (third row) compared to baseline (first row).
Files and variables
File: I-O_Curve_-Means___SEMs_Amp-_Hippocampus.txt
Description: Means and SEMs of input-output curve, as assessed by Fiber volley and fEPSP amplitude, collected from SC-CA1 for each genotype.
Variables
- Fiber Volley Amplitude as collected at 25, 50, 100, 200, 300 uA of stimulation
- fEPSP Amplitude as collected at 25, 50, 100, 200, 300 uA of stimulation
- Scn2a+/+Cre in PRh: Mean, SEM, N
- Scn2afx/+ Cre in PRh: Mean, SEM, N
- hM3Dqfx/+;Scn2a+/-Cre in PRh: Mean, SEM, N
- Drd3Cre;Scn2afx/+: Mean, SEM, N
- Scn2afx/+: Mean, SEM, N
- Drd3Cre;Scn2afx/+ w Cre-GFP in PRC: Mean, SEM, N
- Drd3Cre;Scn2afx/+ w GFP in PRC: Mean, SEM, N
- hM3Dqfx/+;Scn2a+/-Cre in PRC: Mean, SEM, N
- hM3Dqfx/+;Scn2a+/-Cre in PRC CNO: Mean, SEM, N
- hM3Dqfx/+;Scn2a+/+ Cre in PRC: Mean, SEM, N
- hM3Dqfx/+;Scn2a+/+ Cre in PRC CNO: Mean, SEM, N
File: I-O_Curve_-Means___SEMs_Amp-_PRC.txt
Description: Means and SEMs of input-output curve, as assessed by Fiber volley and fEPSP amplitude, collected from perirhinal cortex for each genotype.
Variables
- Fiber Volley Amplitude as collected at 25, 50, 100, 200, 300 uA of stimulation
- fEPSP Amplitude as collected at 25, 50, 100, 200, 300 uA of stimulation
- Scn2a+/+Cre in PRh: Mean, SEM, N
- Scn2afx/+ Cre in PRh: Mean, SEM, N
- hM3Dqfx/+;Scn2a+/-Cre in PRh: Mean, SEM, N
- Emx1Cre;Scn2a+/-: Mean, SEM, N
- Emx1Cre;Scn2a+/+: Mean, SEM, N
- hM3Dqfx/+;Scn2a+/-Cre in PRC: Mean, SEM, N
- hM3Dqfx/+;Scn2a+/-Cre in PRC CNO: Mean, SEM, N
- hM3Dqfx/+;Scn2a+/+ Cre in PRC: Mean, SEM, N
- hM3Dqfx/+;Scn2a+/+ Cre in PRC CNO: Mean, SEM, N
File: I-O_Curve_-Means___SEMs_Slope-_Hippocampus.txt
Description: Means and SEMs of input-output curve, as assessed by Fiber volley and fEPSP slope, collected from SC-CA1 for each genotype.
Variables
- Fiber Volley Amplitude as collected at 25, 50, 100, 200, 300 uA of stimulation
- fEPSP Slope as collected at 25, 50, 100, 200, 300 uA of stimulation
- Scn2a+/+Cre in PRh: Mean, SEM, N
- Scn2afx/+ Cre in PRh: Mean, SEM, N
- hM3Dqfx/+;Scn2a+/-Cre in PRh: Mean, SEM, N
- Drd3Cre;Scn2afx/+: Mean, SEM, N
- Scn2afx/+: Mean, SEM, N
- Drd3Cre;Scn2afx/+ w Cre-GFP in PRC: Mean, SEM, N
- Drd3Cre;Scn2afx/+ w GFP in PRC: Mean, SEM, N
- hM3Dqfx/+;Scn2a+/-Cre in PRC: Mean, SEM, N
- hM3Dqfx/+;Scn2a+/-Cre in PRC CNO: Mean, SEM, N
- hM3Dqfx/+;Scn2a+/+ Cre in PRC: Mean, SEM, N
- hM3Dqfx/+;Scn2a+/+ Cre in PRC CNO: Mean, SEM, N
- Scn2afx/+ Cre in VisAm: Mean, SEM, N
- Scn2afx/+ Cre in Retro: Mean, SEM, N
- Scn2afx/+ Cre in Ent: Mean, SEM, N
File: I-O_Curve_-Means___SEMs_Slope-_PRC.txt
Description: Means and SEMs of input-output curve, as assessed by Fiber volley and fEPSP slope, collected from perirhinal cortex for each genotype.
Variables
- Fiber Volley Amplitude as collected at 25, 50, 100, 200, 300 uA of stimulation
- fEPSP Slope as collected at 25, 50, 100, 200, 300 uA of stimulation
- Scn2a+/+Cre in PRh: Mean, SEM, N
- Scn2afx/+ Cre in PRh: Mean, SEM, N
- hM3Dqfx/+;Scn2a+/-Cre in PRh: Mean, SEM, N
- Emx1Cre;Scn2a+/-: Mean, SEM, N
- Emx1Cre;Scn2a+/+: Mean, SEM, N
- hM3Dqfx/+;Scn2a+/-Cre in PRC: Mean, SEM, N
- hM3Dqfx/+;Scn2a+/-Cre in PRC CNO: Mean, SEM, N
- hM3Dqfx/+;Scn2a+/+ Cre in PRC: Mean, SEM, N
- hM3Dqfx/+;Scn2a+/+ Cre in PRC CNO: Mean, SEM, N
File: I-O_Curve_-Raw_EPSP_Amp-_Hippocampus.txt
Description: Raw fEPSP values for all genotypes previously listed in I-O_Curve_-Means___SEMs_Amp-_Hippocampus.txt
Variables
- Genotypes previously listed in I-O_Curve_-Means___SEMs_Amp-_Hippocampus.txt
- Raw fEPSP amplitude values as collected at 25, 50, 100, 200, 300 uA of stimulation
File: I-O_Curve_-Raw_EPSP_Amp-_PRC.txt
Description: Raw fEPSP values for all genotypes previously listed in I-O_Curve_-Means___SEMs_Amp-_PRC.txt
Variables
- Genotypes previously listed in I-O_Curve_-Means___SEMs_Amp-_PRC.txt
- Raw fEPSP amplitude values as collected at 25, 50, 100, 200, 300 uA of stimulation
File: I-O_Curve_-Raw_EPSP_Slope-_Hipp.txt
Description: Raw fEPSP values for all genotypes previously listed in I-O_Curve_-Means___SEMs_Slope-_Hippocampus.txt
Variables
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- Genotypes previously listed in I-O_Curve_-Means___SEMs_Slope-_Hippocampus.txt
- Raw fEPSP slope values as collected at 25, 50, 100, 200, 300 uA of stimulation
File: I-O_Curve_-Raw_EPSP_Slope-_PRC.txt
Description: Raw fEPSP values for all genotypes previously listed in I-O_Curve_-Means___SEMs_Slope-_PRC.txt
Variables
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- Genotypes previously listed in I-O_Curve_-Means___SEMs_Slope-_PRC.txt
- Raw fEPSP slope values as collected at 25, 50, 100, 200, 300 uA of stimulation
File: I-O_Curve_-Raw_FVs-_Hippocampus.txt
Description: Raw fiber volley values for all genotypes previously listed in I-O_Curve_-Means___SEMs_Slope-_Hippocampus.txt
Variables
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- Genotypes previously listed in I-O_Curve_-Means___SEMs_Slope-_Hippocampus.txt
- Raw FV amplitude values as collected at 25, 50, 100, 200, 300 uA of stimulation
File: I-O_Curve_-Raw_FVs-_PRC.txt
Description: Raw fiber volley values for all genotypes previously listed in I-O_Curve_-Means___SEMs_Slope-_PRC.txt
Variables
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- Genotypes previously listed in I-O_Curve_-Means___SEMs_Slope-_PRC.txt
- Raw FV amplitude values as collected at 25, 50, 100, 200, 300 uA of stimulation
File: LTP_Amplitude_-Timepoints-_PRC.txt
Description: Normalized fEPSP amplitude values at baseline, 20 - 30 minutes, and 50 - 60 minutes for all genotypes included in the paper figures which have amplitude of fEPSP measured as opposed to slope (Supplementary figures).
Variables
- Emx1Cre: normalized fEPSP amplitude values at baseline, 20 - 30 minutes, and 50 - 60 minutes
- Scn2a+/+ Emx1Cre: normalized fEPSP amplitude values at baseline, 20 - 30 minutes, and 50 - 60 minutes
- Scn2a+/- Emx1Cre: normalized fEPSP amplitude values at baseline, 20 - 30 minutes, and 50 - 60 minutes
- Scn2a+/-: normalized fEPSP amplitude values at baseline, 20 - 30 minutes, and 50 - 60 minutes
File: LTP_Amplitude_-Raw-_PRC.txt
Description: Normalized fEPSP amplitude values for all sweeps, which includes the 15 minutes of baseline recording and the 60 minutes of post-induction recording. One sweep occurs every 30 seconds. This data is included for all genotypes included in the paper figures which have amplitude of fEPSP measured as opposed to slope (Supplementary figures).
Variables
- Emx1Cre: normalized fEPSP amplitude values from -15 to +60 minutes post-induction
- Scn2a+/+ Emx1Cre: normalized fEPSP amplitude values from -15 to +60 minutes post-induction
- Scn2a+/- Emx1Cre: normalized fEPSP amplitude values from -15 to +60 minutes post-induction
- Scn2a+/-: normalized fEPSP amplitude values from -15 to +60 minutes post-induction
File: LTP_Slope_-Timepoints-_Hippocampus.txt
Description: Normalized fEPSP slope values at baseline, 20 - 30 minutes, and 50 - 60 minutes for all genotypes included in the paper figures.
Variables
- Drd3Cre;Scn2afx/+: normalized fEPSP slope values at baseline, 20 - 30 minutes, and 50 - 60 minutes
- Scn2afx/+: normalized fEPSP slope values at baseline, 20 - 30 minutes, and 50 - 60 minutes
- Scn2afx/+ Cre in PRh: normalized fEPSP slope values at baseline, 20 - 30 minutes, and 50 - 60 minutes
- WTCre in PRh: normalized fEPSP slope values at baseline, 20 - 30 minutes, and 50 - 60 minutes
- hM3Dqfx/+;Scn2a+/-Cre in PRh +CNO: normalized fEPSP slope values at baseline, 20 - 30 minutes, and 50 - 60 minutes
- hM3Dqfx/+;Scn2a+/-Cre in PRh No CNO: normalized fEPSP slope values at baseline, 20 - 30 minutes, and 50 - 60 minutes
- Fmr1KO + hM3Dq-mCherry in PRC (No CNO): normalized fEPSP slope values at baseline, 20 - 30 minutes, and 50 - 60 minutes
- Fmr1KO + hM3Dq-mCherry in PRC (CNO): normalized fEPSP slope values at baseline, 20 - 30 minutes, and 50 - 60 minutes
- Cdkl5KO + hM3Dq-mCherry in PRC (No CNO): normalized fEPSP slope values at baseline, 20 - 30 minutes, and 50 - 60 minutes
- Cdkl5KO + hM3Dq-mCherry in PRC (CNO): normalized fEPSP slope values at baseline, 20 - 30 minutes, and 50 - 60 minutes
File: PPR_-Amp-_Hipp.txt
Description: The paired-pulse ratio collected at SC-CA1 from taking the ratio of the conditioning fEPSP amplitude to the test fEPSP amplitude for each genotype.
Variables
- Scn2afx/+: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
- Drd3Cre;Scn2afx/+: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
- Scn2afx/+ Cre in PRh: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
- Scn2a+/+Cre in PRh: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
- Drd3Cre;Scn2afx/+ w Cre-GFP in PRC: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
- Drd3Cre;Scn2afx/+ w GFP in PRC: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
- hM3Dqfx/+;Scn2a+/- Cre in PRC: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
- hM3Dqfx/+;Scn2a+/+ Cre in PRC: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
File: LTP_Slope_-Timepoints-_PRC.txt
Description: Normalized fEPSP slope values at baseline, 20 - 30 minutes, and 50 - 60 minutes for all genotypes included in the paper figures collected at the perirhinal cortex.
Variables
- Emx1Cre;Scn2a+/+: normalized fEPSP slope values at baseline, 20 - 30 minutes, and 50 - 60 minutes
- Emx1Cre;hM3Dqfx/+;Scn2a+/-: normalized fEPSP slope values at baseline, 20 - 30 minutes, and 50 - 60 minutes
- Emx1Cre;Scn2a+/-: normalized fEPSP slope values at baseline, 20 - 30 minutes, and 50 - 60 minutes
- Scn2afx/+ Cre in PRh: normalized fEPSP slope values at baseline, 20 - 30 minutes, and 50 - 60 minutes
- WTCre in PRh: normalized fEPSP slope values at baseline, 20 - 30 minutes, and 50 - 60 minutes
- hM3Dqfx/+;Scn2a+/-Cre in PRh +CNO: normalized fEPSP slope values at baseline, 20 - 30 minutes, and 50 - 60 minutes
- hM3Dqfx/+;Scn2a+/-Cre in PRh No CNO: normalized fEPSP slope values at baseline, 20 - 30 minutes, and 50 - 60 minutes
- Fmr1KO + hM3Dq-mCherry in PRC (No CNO): normalized fEPSP slope values at baseline, 20 - 30 minutes, and 50 - 60 minutes
- Fmr1KO + hM3Dq-mCherry in PRC (CNO): normalized fEPSP slope values at baseline, 20 - 30 minutes, and 50 - 60 minutes
- Cdkl5KO + hM3Dq-mCherry in PRC (No CNO): normalized fEPSP slope values at baseline, 20 - 30 minutes, and 50 - 60 minutes
- Cdkl5KO + hM3Dq-mCherry in PRC (CNO): normalized fEPSP slope values at baseline, 20 - 30 minutes, and 50 - 60 minutes
File: LTP_Slope_-Raw-_PRC.txt
Description: Normalized fEPSP slope values for all sweeps, which includes the 15 minutes of baseline recording and the 60 minutes of post-induction recording at the perirhinal cortex. One sweep occurs every 30 seconds. This data is included for all genotypes included in the paper figures.
Variables
- Emx1Cre;Scn2a+/+: normalized fEPSP slope values from -15 to +60 minutes post-induction
- Emx1Cre;hM3Dqfx/+;Scn2a+/-: normalized fEPSP slope values from -15 to +60 minutes post-induction
- Emx1Cre;Scn2a+/-: normalized fEPSP slope values from -15 to +60 minutes post-induction
- Scn2afx/+ Cre in PRh: normalized fEPSP slope values from -15 to +60 minutes post-induction
- WTCre in PRh: normalized fEPSP slope values from -15 to +60 minutes post-induction
- hM3Dqfx/+;Scn2a+/-Cre in PRh +CNO: normalized fEPSP slope values from -15 to +60 minutes post-induction
- hM3Dqfx/+;Scn2a+/-Cre in PRh No CNO: normalized fEPSP slope values from -15 to +60 minutes post-inductionn
- Fmr1KO + hM3Dq-mCherry in PRC (No CNO): normalized fEPSP slope values from -15 to +60 minutes post-induction
- Fmr1KO + hM3Dq-mCherry in PRC (CNO): normalized fEPSP slope values from -15 to +60 minutes post-induction
- Cdkl5KO + hM3Dq-mCherry in PRC (No CNO): normalized fEPSP slope values from -15 to +60 minutes post-induction
- Cdkl5KO + hM3Dq-mCherry in PRC (CNO): normalized fEPSP slope values from -15 to +60 minutes post-induction
File: LTP_Slope_-Raw-_Hippocampus.txt
Description: Normalized fEPSP slope values for all sweeps, which includes the 15 minutes of baseline recording and the 60 minutes of post-induction recording at SC-CA1. One sweep occurs every 30 seconds. This data is included for all genotypes included in the paper figures.
Variables
- Drd3Cre;Scn2afx/+: normalized fEPSP slope values from -15 to +60 minutes post-induction
- Scn2afx/+: normalized fEPSP slope values from -15 to +60 minutes post-induction
- Scn2afx/+ Cre in PRh: normalized fEPSP slope values from -15 to +60 minutes post-induction
- WTCre in PRh: normalized fEPSP slope values from -15 to +60 minutes post-induction
- hM3Dqfx/+;Scn2a+/-Cre in PRh No CNO: normalized fEPSP slope values from -15 to +60 minutes post-induction
- hM3Dqfx/+;Scn2a+/-Cre in PRh +CNO: normalized fEPSP slope values from -15 to +60 minutes post-induction
- Fmr1KO + hM3Dq-mCherry in PRC (CNO): normalized fEPSP slope values from -15 to +60 minutes post-induction
- Fmr1KO + hM3Dq-mCherry in PRC (No CNO): normalized fEPSP slope values from -15 to +60 minutes post-induction
- Cdkl5KO + hM3Dq-mCherry in PRC (No CNO): normalized fEPSP slope values from -15 to +60 minutes post-induction
- Cdkl5KO + hM3Dq-mCherry in PRC (CNO): normalized fEPSP slope values at baseline, 20 - 30 minutes, and 50 - 60 minutes
File: PPR_-Slope-_Hipp.txt
Description: The paired-pulse ratio collected at SC-CA1 from taking the ratio of the conditioning fEPSP slope to the test fEPSP slope for each genotype.
Variables
- Scn2afx/+: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
- Drd3Cre;Scn2afx/+: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
- Scn2afx/+ Cre in PRh: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
- Scn2a+/+Cre in PRh: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
- Drd3Cre;Scn2afx/+ w Cre-GFP in PRC: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
- Drd3Cre;Scn2afx/+ w GFP in PRC: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
- hM3Dqfx/+;Scn2a+/- Cre in PRC: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
- hM3Dqfx/+;Scn2a+/- Cre in PRC CNO: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
- hM3Dqfx/+;Scn2a+/+ Cre in PRC: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
File: PPR_-Amp-_PRC.txt
Description: The paired-pulse ratio collected at perirhinal cortex from taking the ratio of the conditioning fEPSP amplitude to the test fEPSP amplitude for each genotype.
Variables
- Scn2afx/+ Cre in PRh: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
- Scn2a+/+Cre in PRh: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
- hM3Dqfx/+;Scn2a+/- Cre in PRC: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
- hM3Dqfx/+;Scn2a+/- Cre in PRC CNO: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
- hM3Dqfx/+;Scn2a+/+ Cre in PRC: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
- hM3Dqfx/+;Scn2a+/+ Cre in PRC CNO: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
File: PPR_-Slope-_PRC.txt
Description: The paired-pulse ratio collected at perirhinal cortex from taking the ratio of the conditioning fEPSP slope to the test fEPSP slope for each genotype.
Variables
- Scn2afx/+ Cre in PRh: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
- Scn2a+/+Cre in PRh: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
- hM3Dqfx/+;Scn2a+/- Cre in PRC: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
- hM3Dqfx/+;Scn2a+/- Cre in PRC CNO: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
- hM3Dqfx/+;Scn2a+/+ Cre in PRC: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
- hM3Dqfx/+;Scn2a+/+ Cre in PRC CNO: Paired-pulse ratio at 15, 25, 50, 100, 200 ms interstimulus interval
File: README.md
Description: This is the README file for the R code. It contains the following: Light Sheet Image Analysis. This repository contains code used to munge, statistically analyze, and visualize data obtained from light-sheet imaging of whole brains.
File: RegionsTblFunction.R
Description: This function, when given as input a data frame in R containing LifeCanvas light-sheet information, will output a data table containing Means, SDs, SEMs, and N values for each brain region from a set of whole-brain data.
File: BrainsDataFunction.R
Description: This function, when given as input a file path to Excel spreadsheets containing LifeCanvas light-sheet information for single brains, will create as output a list of 3 tibbles, one "LeftHemisphere", one "RightHemisphere", and one "WholeBrain". Each tibble contains five columns: "Name" [Brain Region], "Count" [Cell counts], "VolumeMm3" [Volume of Brain Region], "DensityCellsMm3" [Cell density of expression target of interest, such as FosGFP or NeuN], and "BrainId" [Each individual Excel sheet uploaded]. The WholeBrain tibble, which combines information from the left and right hemisphere tibbles, is then used for further analysis.
File: FeverConditionAnalysis.R
Description: This code is a basic analysis pipeline for the Life Canvas data and includes use of the BrainsDataFunction and RegionsTbl function above.
File: InterestList.R
Description: This code allows for the subsetting of data derived from the BrainsDataFunction based on brain regions of interest, and also permits analysis and data visualization.
File: LICENSE.txt
Description: This includes the GNU Affero General Public License v3.0.
Code/software
For the code, R and R Studio were used to view and run. The code itself is in .R files and was run on Windows 10 and 11 successfully. All code is commented with explanations for ease of use.
For the electrophysiology data listed above, any point and click or script-based statistics program can be used, but our group used Prism, up to version 10.4.0. Minor changes in p-values and test statistics can occur if the data is run in different softwares, depending on the forms of the posthocs/statistics used, as well as how the data is formatted.
For the R code also uploaded onto GitHub:
Light-Sheet Imaging
Mice aged postnatal day 21 to postnatal day 24 received injections of either saline or CNO (0.25 mg/kg) 30 minutes before exposure to a novel open field. After one hour, mice were anesthetized with 50 mg/kg pentobarbital, then transcardially perfused with phosphate buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde (PFA). After overnight shaking in 4% PFA at 4°C, brains were washed with 1X PBS, then transferred to 0.02% sodium azide/1X PBS solution. LifeCanvas Technologies provided light-sheet images of the entire brain and cell density counts. Antibodies used were Goat GFP antibody (#GPCA-GFP, EnCor Biotechnology) and Rabbit NeuN antibody (#24307, Cell Signaling Technology). Data contained the identified region (verified via Mouse Brain Atlas overlay), cell count, volume (in mm3) of the region, cell density (cells per mm3), and the genotype of the animal. Cells were analyzed in three channels: coexpression of cFos and NeuN, cFos alone, and NeuN alone. Cell density averages were evaluated per brain region across genotypes using an in-house developed R pipeline (Github link). Layer 1 of the cortex was excluded from analysis due to high levels of autofluorescence resulting from light-scattering. Heatmaps were generated with average cell density values in brain regions that showed significant differences between genotypes. Significance was set at p-values < 0.05. Regions with cell counts of zero were omitted from the analysis. Each experiment was conducted in triplicate, with n representing each assessed brain.
Access information
Other publicly accessible locations of the data:
- Github link to the code: https://github.com/NeuroResearcher/LightSheetImageAnalysis
Slice Preparation
Using standard methods, horizontal brain sections (350 µM) containing hippocampus and perirhinal cortex were collected from mice aged postnatal day 21 (surgically naïve mice) to postnatal day 70 (mice which underwent viral injection and behavior testing). For slice preparation from surgically naïve mice, the cutting solution contained (in mM): 85 NaCl, 2.5 KCl, 1.25 NaH2PO4-H2O, 3 MgSO4·7H2O, 0.5 L-ascorbic acid, 0.5 CaCl2, 75 sucrose, 25 D-(+)-glucose, and 25 NaHCO3. For slice preparation from mice which underwent viral injection, the cutting solution contained (in mM): 2.5 KCl, 1.25 NaH2PO4-H2O, 0.4 L-ascorbic acid, 3 MgSO4·7H2O, 200 sucrose, 20 D-(+)-glucose, and 26 NaHCO3. Both solutions were neutral pH (i.e., 7.3), 310 mOsm, and saturated with 95% O2 and 5% CO2. Once cut, slices were transferred to an incubation chamber (Campden Instruments, Model 7470) filled with oxygenated artificial cerebrospinal fluid (ACSF) (Standard Ringer’s Solution, 300 mOsm, in mM: 119 NaCl, 1.3 MgSO4-7H2O, 2.5 KCl, 1 NaH2PO4-H2O, 2.5 CaCl2, 11 D-(+)-glucose, and 26.2 NaHCO3, pH 7.4) and then incubated at 32°C for 30 minutes. Slices were kept at room temperature for 30 minutes before transfer to the recording chamber (30°C oxygenated ACSF, 2 – 3 mL/min). Heating was maintained with LinLab 2 (Scientifica, Version 1.0.19.50).
Field Potential Recording Electrophysiology
Recordings utilized bipolar electrodes for stimulation and 2-6 megaohm micropipettes containing ACSF for recording. For perirhinal cortex, the stimulating electrode was positioned into layer II/III on the temporal side of the rhinal sulcus, while the recording electrode was placed in the same layer 400 µm away in the rostral direction (54). For hippocampal recordings, stimulation was applied to Schaffer collaterals from area CA3, and stratum radiatum in area CA1 was recorded. A stimulus generator (Model DS3, Digitimer Ltd.) delivered constant-current square pulses (0.1 ms, 25 - 300 µA), and signals were lowpass filtered at 1 kHz. Parameters were recorded and analyzed utilizing the Clampex suite (Molecular Devices, Version 11.1) Field excitatory postsynaptic potentials (fEPSPs) were recorded every 10 seconds. Input-output curves were determined by measuring the fiber volley (FV) and fEPSP slope at 25 µA, 50 µA, 100 µA, 200 µA and 300 µA for 10 sweeps. Stimulus intensity was adjusted to induce a half-maximal fEPSP amplitude. Paired pulse ratios were determined by calculating the ratio of the slope of the test EPSP (second response) to the conditioning EPSP (first response) at interstimulus intervals of 15, 25, 50, 100, and 200 milliseconds (ms). For long-term potentiation, a baseline was established by recording responses every 30 seconds for fifteen minutes before applying theta-burst stimulation (TBS: four trains every 15 seconds, each train comprising 10 bursts of 5 pulses at 100 Hz, inter-burst interval 150 ms). After TBS, responses were recorded every 30 seconds for 60 min. For studies involving hM3Dq, 1 µM CNO was bath applied to activate this designer receptor. FV amplitude and fEPSP slope were compared using a two-way repeated measures ANOVA followed by a Tukey post-hoc test across genotypes and stimulus intensities to derive input-output curves. A two-way repeated measures ANOVA with a Fisher’s LSD post-hoc test was used to assess the effect of genotype and interstimulus interval on the paired-pulse ratio. Baseline and post-LTP induction responses across genotypes and time were also compared by repeated measures ANOVA. As reported in figure captions, n represents the number of slices recorded, followed by the number of animals utilized. Significance was defined as p-values < 0.05, with α = 0.05.