In vivo Calcium Imaging Reveals That Cortisol Treatment Reduces the Number of Place Cells in Thy1-GCaMP6f Transgenic Mice

Abstract

The hippocampus, a structure essential for spatial navigation and memory undergoes anatomical and functional changes during chronic stress. Here, we investigate the effects of chronic stress on the ability of place cells to encode the neural representation of a linear track. To model physiological conditions of chronic stress on hippocampal function, transgenic mice expressing the genetically encoded calcium indicator GCaMP6f in CA1 pyramidal neurons were chronically administered with 40 μg/ml of cortisol for 8 weeks. Cortisol-treated mice exhibited symptoms typically observed during chronic stress, including diminished reward seeking behavior and reduced adrenal gland and spleen weights. In vivo imaging of hippocampal cellular activity during linear track running behavior revealed a reduced number of cells that could be recruited to encode spatial position, despite an unchanged overall number of active cells, in cortisol-treated mice. The properties of the remaining place cells that could be recruited to encode spatial information, however, was unperturbed. Bayesian decoders trained to estimate the mouse's position on the track using single neuron activity data demonstrated reduced performance in a cue richness-dependent fashion in cortisol-treated animals. The performance of decoders utilizing data from the entire neuronal ensemble was unaffected by cortisol treatment. Finally, to test the hypothesis that an antidepressant drug could prevent the effects of cortisol, we orally administered a group of mice with 10 mg/kg citalopram during cortisol administration. Citalopram prevented the cortisol-induced decrease in single-neuron decoder performance but failed to significantly prevent anhedonia and the cortisol-induced reduction in the proportion place cells. The dysfunction observed at the single-neuron level indicates that chronic stress may impair the ability of the hippocampus to encode individual neural representations of the mouse's spatial position, a function pivotal to forming an accurate navigational map of the mouse's external environment; however, the hippocampal ensemble as a whole is resilient to any cortisol-induced insults to single neuronal place cell function on the linear track.

Keywords: GCaMP6f; calcium imaging; citalopram; cortisol; miniature microscopes; place cells; transgenic mice.

FIGURE 1

Experimental timeline. Timeline of the experimental design. Treatment groups consisted of mice that were administered either cortisol only, cortisol with citalopram, citalopram only, or control vehicles. All four treatment groups had an n = 6 mice. After cannulation surgery, mice were allowed to recover for 2 weeks before drug treatment was initiated (weeks 0–2). To ensure effects of cortisol treatment were sufficient to model conditions of chronic stress, a 3-week period of drug treatment was maintained prior to exposing the mice to the linear track (weeks 2–5). Following 2 weeks of training on the linear track (weeks 5–7), mice were imaged during a 2-week period of daily linear track running (weeks 7–9). Mice recovered from water restriction for 1 week (weeks 9–10) before female urine sniffing test (FUST) administration and organ harvest. Cortisol (40 μg/ml) was added into the drinking water during ad lib water consumption (weeks 2–5 and 10) or administered subcutaneously (10 mg/kg) (weeks 5–9). Likewise, citalopram (10 mg/kg) was administered daily by oral gavage (weeks 2–5 and 10) and injected subcutaneously (10 mg/kg) during weeks 5–9. Untreated mice received drinking water or subcutaneous saline injections during water restriction sessions.

FIGURE 2

Cortisol treatment induced stress-like changes in physiology and behavior. (A,B) Organs were harvested and weighed after 8 weeks of drug treatment. Cortisol-treated mice and cortisol + citalopram-treated mice had significantly reduced adrenal (sum of left and right adrenals) and spleen weights compared to untreated and citalopram-treated mice. Numbers in bars on the graphs indicate the number of animals used. Note that adrenals and spleens were not harvested from all mice. (C) Female urine sniffing test administered to mice following chronic drug treatment. Untreated and citalopram-treated mice exhibited urine sniffing preferences. Chronic cortisol treatment eliminated this preference, which was not rescued by citalopram co-administration. (D) Linear track runs averaged over all recording sessions (left). Cortisol-treated mice and cortisol + citalopram-treated mice had significantly fewer linear track runs compared to untreated and citalopram-treated mice. Daily linear track runs plotted over time (right). A total of six mice per treatment group, with nine recording sessions per mouse, for a maximal N of 54 were used. (E) Average velocity during linear track running (left) and running velocity plotted over time for individual recording sessions (right). Cortisol-treated mice showed significantly reduced running velocity compared to untreated mice, an effect that was not reversed with citalopram co-administration. Cort, cortisol; Cit, citalopram. Asterisks show group comparisons with ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.005.

FIGURE 3

Imaged place cell properties from a representative mouse. (A) Linear track with feature-rich contextual cues (textures walls, scored floor, shower curtain design) for the left running direction and feature-space cues (plain walls and shower curtains, smooth floor) for the right running direction. (B) Miniature microscope and placement schematic. (C) A raw fluorescence field of view (left) and projection map of all cells identified using PCA-ICA (right). Non-place cells (white) and place cells for right (blue) and left running direction (red) derived by spatial information analysis. (D) Daily changes in the proportion of place cells within the population of imaged cells superimposed onto the number of linear track runs per imaging session to illustrate correlated behavior and place cell function (Pearson’s r = 0.77). The proportion of place cells (linear regression: β = 0.01, SE = 0.003, p = 0.018) and the number of linear track runs improved significantly with time (linear regression: β = 6.07, SE = 1.66, p = 0.008). (E) Place cell tuning curves from a mouse for left and right running directions, sorted for place field locations during imaging. (F) Raw ΔF/F trace of a single place cell (top panel) and representative firing location from a single place cell during linear track running across multiple recording sessions (bottom panels). Events from a single place cell (filled colored circles) overlaid with mouse position on the linear track (gray dashed lines). Data were acquired at a rate of 10 frames per second. Recording sessions 1–2 were excluded for display purposes. Cort, cortisol; Cit, citalopram.

FIGURE 4

Cortisol treatment induces place cell dysfunction. (A) Place cell tuning curves from 24 mice, separated by treatment group (six mice per treatment group), concatenated for left and right running directions. Place cells, identified by shuffle analysis, were sorted by linear track place field location on recording session 6. (B) Place field centroid shift distributions between recording session (colored by inter-recording session interval) were indistinguishable within treatment groups (K-S tests, untreated: p = 0.58, cortisol: p = 0.41, cortisol + citalopram: p = 0.27, citalopram: p = 0.41) and between groups (p = 0.99). Centroid shift distributions peaked at zero centimeters and were distinct from the null hypothesis that place fields would randomly relocate (p = 4e^-05). (C) Proportion of identified place cells for individual recording sessions (left) and averaged over all recording sessions (right). Cortisol treatment significantly reduced the proportion of identified place cells as compared to untreated control animals. Cort, cortisol; Cit, citalopram. Asterisks show group comparisons with ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.005.

FIGURE 5

Cortisol treatment increases the single neuron prediction error. (A) Bar graphs of single neuron mouse location prediction errors for all neurons (457 ± 25 cells per group) averaged over all recording sessions (8.58 ± 0.16 sessions) [mean ± SEM]. Calculations were averaged by mice (n = 6) per group for left and right running directions separately. Cortisol-treated mice had a significantly higher prediction error (p < 0.05) compared to untreated mice for the rightward running direction only, but a higher prediction error for both running directions when compared to citalopram-treated mice. Asterisks show group comparisons with ^∗p < 0.05 and ^∗∗∗p < 0.005. (B) Mouse location prediction errors using Bayesian decoders trained on ensemble neural data. No significant group effects were found for the ensemble decoders. Data shown as mean ± SEM, (n = 6 mice).