LFPI decreases theta and gamma power across CA1 lamina

(A) Silicon probe recording from dorsal CA1. Schematic of electrode shank widened for visualization but accurately scaled along CA1 lamina. The peak of ripple frequency (100-250 Hz) power across channels was used to define the st. pyr channel (marked with *). Channels within ±80 µm of the defined st. pyr channel were assigned to the pyramidal cell layer (denoted by bracket).

(B) Mean waveforms from 6 single units identified as putative pyramidal cells recorded on the shank shown in A across all electrode contacts.

(C) Mean SWR waveform from the shank shown in A and its associated current source density (CSD)

(D) Schematic of an electrode array with shanks positioned along the proximal-distal axis of CA1. Heatmap contours show the mean SWR CSD for each shank. Normalized ripple frequency power and edges of t e pyramidal cell layer are shown as solid and dashed lines respectively for each shank. Individual recorded units are shown at the depth of the channel containing their max amplitude (putative cell types: interneuron=magenta circle, pyramidal cell=green triangle, unclassified=yellow square). Shank 2 shown in A-C.

(E) Example recording from a shank spanning both st. pyr and st rad. The peak of ripple frequency power occurs at channel 3 (identified as pyr.). The st. rad channel was defined as the channel closest to the peak sink in the CSD of the mean SWR waveform (channel 13 denoted by rad.). Note theta (5-10 Hz) and gamma (30-59 Hz) oscillations especially apparent in st. rad.

(F) Power spectra (mean±SEM) from st. pyr (top) and st. rad (bottom) for sham and injured rats moving (>10 cm/sec) in the familiar environment. Theta (5-10 Hz) and gamma (30-59 Hz) bands denoted by dashed lines. Statistically significant differences between sham and injured are outlined in gray (p<0.01 for multiple comparisons; t-test).

(G) Integral of power in the theta and gamma frequency bands (mean±SEM; individual animals labeled by points) from sham and injured rats in st. pyr (top; theta: sham=-7.65±0.09 log10V2, injured=-8.38±0.10 log10V2, p=0.001; gamma: sham=-7.64±0.05 log10V2, injured=-7.90±0.04 log10V2, p=0.009; t-test) and st. rad (bottom; theta: sham=-7.15±0.04 log10V2, injured=-7.51±0.07 log10V2, p=0.001; gamma: sham=-7.20±0.05 log10V2, injured=-7.42±0.07 log10V2, p=0.022; t-test).

LFPI decreases theta-gamma phase-amplitude coupling (PAC) in st. pyr but not st. rad

(A) Visualization of theta-gamma PAC. Raw 3 sec trace recorded from st. pyr (top), the same trace filtered for theta (5-10 Hz; middle) and for gamma (30-59 Hz; bottom). Envelope amplitude of gamma is plotted in red and peaks in gamma amplitude >1SD above mean are marked with a dashed line. Note that most peaks align to a similar phase of theta (∼90°).

(B) Averaged PAC heatmap contours from sham (left) and injured (right) animals in st. pyr (top) and st. rad (bottom).

(C) Difference between sham and injured PAC heatmap contours in both st. pyr (top) and st. rad (bottom).

(D) PAC modulation index values from broadband theta and gamma filtered signals (mean±SEM; individual animals labeled by points) in st. pyr (top; sham=0.00130±0.00016, injured=0.00051±0.00012, p=0.006, t-test) and st. rad (bottom; sham=0.00121±0.00032, injured=0.00145±0.00032, p=0.623, t-test).

(E) Normalized gamma amplitudes across theta phase bins (2 cycles of theta shown in black for reference). Peak gamma in st. pyr (sham=89.2°, injured=115.2°, Δ=26°) and st. rad (sham=200.0°, injured=255.4°, Δ=55.4°).

LFPI minimally affects single unit firing rates but increases recruitment of pyramidal cells

(A) Waveforms (mean±SD) from an example putative pyramidal cell (top) and interneuron (bottom) and their associated autocorrelograms. W=width, FR=firing rate

(B) Scatterplot of spike width, first moment of the autocorrelogram, and firing rate of all cells located within the pyramidal cell layer from both sham and injured animals. Note separation of putative pyramidal cells and interneurons with unclassified cells typically lying between these 2 populations.

(C) Location of all cells relative to the defined st. pyr channel (based on peak ripple frequency power). Note the peak of the pyramidal cell distribution is located on the defined st. pyr channel, and the bimodal distribution of interneurons reflects populations localized to st. oriens and st. pyr.

(D) Left: firing rates (mean±SEM) of putative interneurons in the familiar and novel environments across sham and injured animals (familiar: sham=20.3±3.8 Hz, n=20, injured=13.0±1.3 Hz, n=88, p=0.116; novel: sham=17.4±3.3 Hz, n=19, injured=12.6±1.4 Hz, n=89, p=0.185; ks-test). Right: cumulative distributions of interneuron firing rates.

(E) Left: firing rates (mean±SEM) of putative pyramidal cells in the familiar and novel environments across sham and injured animals (familiar: sham=1.63±0.23 Hz, n=51, injured=2.11±0.20 Hz, n=141, p=0.446; novel sham=1.84±0.18 Hz, n=55, injured=2.23±0.21 Hz, n=134, p=0.170, ks-test). Right: cumulative distributions of pyramidal cell firing rates.

(F) Recruitment of putative pyramidal cells across environments (sham=74%, injured=87%, p=0.025, Fisher’s exact test).

LFPI disrupts spike field coherence to hippocampal oscillations

(A) Example 2 sec recording trace containing a large amplitude interneuron entrained to theta (all spikes are from the same unit). Right: probability of spike times across theta phase bins (line shows 2 cycles of theta for reference).

(B) Entrainment strength of all interneurons across frequencies in both environments while the rat was still <10 cm/sec; top) or moving (>10 cm/sec; bottom).

(C) Percent of interneurons significantly entrained to theta (5-10 Hz) and gamma (30-59 Hz) while animals were still or moving (theta still: sham=97.44%, injured=91.53%, p=0.315; theta moving: sham=97.44%, injured=91.53%, p=0.315; gamma still: sham=89.74%, injured=67.80%, p=0.006; gamma moving: sham=89.74%, injured=65.54%, p=0.002; Fisher’s exact test).

(D) Entrainment strength of all pyramidal cells across frequencies in both environments while the rat was still (top) or moving (bottom).

(E) Percent of pyramidal cells significantly entrained to theta (5-10 Hz) and gamma (30-59 Hz) while animals were still or moving (theta still: sham=71.57%, injured=67.17%, p=0.454; theta moving: sham=58.82%, injured=65.28%, p=0.277; gamma still: sham=25.49%, injured=37.74%, p=0.028; gamma moving: sham=29.41%, injured=30.19%, p>0.999; Fisher’s exact test).

LFPI impairs single unit entrainment to theta oscillations

(A) Cumulative distributions of all interneurons significantly entrained to theta across all conditions.

(B) Left: average entrainment (mean±SEM) of interneurons significantly entrained to theta while rats were moving in the familiar (sham=0.31±0.03, n=19, injured=0.21±0.01, n=83, p=0.004, ks-test) and novel (sham=0.27±0.03, n=19, injured=0.20±0.01, n=79, p=0.026, ks-test) environment. Right: average entrainment of interneurons while rats were still or moving in each environment (sham familiar: still=0.27±0.02, n=19, moving=0.31±0.03, n=19, p=0.462; sham novel: still=0.23±0.02, n=19, moving=0.27±0.03, n=19, p=0.462; injured familiar: still=0.14±0.01, n=79, moving=0.21±0.01, n=83, p<0.001; injured novel: still=0.13±0.01, n=83, moving=0.20±0.01, n=79, p<0.001; ks-test).

(C) Polar histograms showing the mean angle of entrainment for all significantly entrained interneurons while animals were moving in the familiar or novel environment (familiar: sham=173.7°, injured=196.6°, p=0.446; novel: sham=167.6°, injured=190.2°, p=0.443; circular Kruskal-Wallis test).

(D) Cumulative distributions of pyramidal cells significantly entrained to theta across all conditions.

(E) Left: average entrainment (mean±SEM) of pyramidal cells significantly entrained to theta while rats were moving in the familiar (sham=0.19±0.02, n=25, injured=0.20±0.01, n=89, p=0.518, ks-test) and novel (sham=0.16±0.01, n=35, injured=0.19±0.01, n=84, p=0.191, ks-test) environment. Right: average entrainment of pyramidal cells while rats were still or moving in each environment (sham familiar: still=0.18± 0.02, n=35, moving=0.19±0.02, n=25, p=0.780; sham novel: still=0.15±0.01, n=38, moving=0.16±0.01, n=35, p=0.908; injured familiar: still=0.12±0.01, n=85, moving=0.2±0.01, n=89, p<0.001; injured novel: still=0.14±0.01, n=93, moving=0.19±0.01, n=84, p<0.001; ks-test).

(F) Polar histograms showing the mean angle of entrainment for all significantly entrained pyramidal cells while animals were moving in the familiar or novel environment (familiar: sham=197.4, injured=250.1, p=0.003; novel: sham=214.5, injured=267.9, p<0.001: circular Kruskal-Wallis test).

LFPI decreases SWR amplitude

(A) Example SWR event recorded across all electrode contacts from a single shank (st. pyr channel labeled and colored blue), the associated time-frequency map from the st. pyr channel (red contour outlines where ripple event exceeds amplitude threshold; white dashed lines mark edges of ripple event in time), and the frequency-amplitude spectrum of the event averaged over time between white dashed lines.

(B) Percentage of SWRs that were detected while the animal was still (sham: familiar=96.7±0.7%, novel=97.5±0.3%, injured: familiar=97.9±0.7%, novel=96.9±0.7%; mean±SEM; points represent individual animals).

(C) SWR event rate normalized to the amount of time animals were still (sham: familiar=0.26±0.03 Hz, novel=0.56±0.05 Hz, p=0.011; injured: familiar=0.36±0.03 Hz, novel=0.52±0.03 Hz, p=0.052; t-test; mean±SEM; points represent individual animals).

(D) Average (mean±SEM) frequency-amplitude spectrum of all ripples across conditions.

(E) Left: cumulative distributions of ripple amplitudes across conditions (familiar: sham=91.2±1.7 mV, n=490, injured=73.3±0.9 mV, n=1082, p<0.001; novel: sham=126.2±1.6 mV, n=1285, injured=98.4±1.3 mV, n=1464, p<0.001; sham still vs moving: p<0.001; injured still vs moving: p<0.001; mean±SEM; ks-test). Right: cumulative distributions of ripple durations across conditions (familiar: sham=34.3±0.9 ms, injured=34.7±0.6 ms, p=0.019; novel: sham=36.9±0.5 ms, injured=38.5±0.5 ms, p=0.062; sham still vs moving: p<0.001; injured still vs moving: p<0.001; mean±SEM; ks-test).

Comparison of manual and automated clustering of cell types

Scatter plots of spike width, first moment of the autocorrelogram, and firing rate for all single units located within the defined pyramidal cell layer. Left side shows manual clustering (matching Fig 3B) used for all analyses. Right side shows automated clustering (k-means consensus clustering with 2 groups). There was a 95.47% agreement between the two methods when unclassified cells were not included in the comparison. We chose to use manual clustering because inclusion of the unclassified group allowed us to be more conservative, and manual clustering is more robust to outliers in automated clustering such as the cell classified as a pyramidal cell with a firing rate of ∼30 Hz.

Cells above the defined pyramidal cell layer have features similar to interneurons

Scatter plots of spike width, first moment of the autocorrelogram, and firing rate for all single units. Cells in purple were >80 µm above the defined st. pyr channel and were automatically identified as interneurons. These cells have firing properties matching interneurons and cluster around the interneuron group (see Fig 3).

LFPI disrupts spatial memory and induces stereotypic pathologies

(A) Morris water maze memory scores (mean±SEM; individual animals labeled by points) from sham and injured rats tested at 48hr post-injury (sham=114.8±21.8, n=9, injured=51.5±6.8, n=14, p=0.020; Welch’s t-test). (B) H&E-stained sections showing hemorrhagic contusion in the ipsilateral white matter, including the corpus callosum at 48 hr post-LFPI. Note, the underlying hippocampus appears grossly intact (scale bar: 500 µm). (C-D) APP immunoreactive axonal pathology in the angular bundle (C) and fimbria-fornix (D) at 48 hrs post-LFPI (scale bars: 50 µm).

(E) An absence of axonal pathology in the fimbria-fornix 48 hrs following sham procedures (scale bar: 100 µm).

(F) Fluoro-Jade C positive neurons in the peri-lesional cortex at 48 hrs post-LFPI (scale bar: 50 µm).

(G) An absence of Fluoro-Jade C positive cells in the CA1 region of hippocampus at 48 hrs post-LFPI (scale bar: 50 µm).

(H) Ipsilateral cortex displaying an absence of Fluoro-Jade C positive cells following sham procedures (scale bar: 50 µm).