Genetic deletion of α7 nAChRs reduces hippocampal granule and pyramidal cell number in both sexes but impairs pattern separation in males only

Abstract

Introduction: Neurogenesis within the dentate gyrus is thought to play an important role in cognitive processes such as reversal learning and pattern separation. The α7 nicotinic acetylcholine receptor (α7 nAChR) is expressed early in newly formed granule cells of the dentate gyrus, though its role in neurogenesis and related cognitive function is not fully understood.

Methods: To better characterize relevant function of α7 nAChRs, we performed unbiased stereology to quantify hippocampal granule cells, pyramidal cells, and total volume and used a touchscreen operant spatial discrimination/reversal task to test pattern separation in a global α7 nAChR knockout mouse line.

Results: The knockout resulted in an ≈22% reduction in granule cells and a ≈ 20% reduction in pyramidal cells in both sexes, with no change in total hippocampal volume. However, the knockout impaired performance in the touchscreen task for males only. The sex-dependent difference in behavioral, but not stereological, results suggest a divergence in the structure-function relationship in males versus females. Detailed analyses revealed males were more biased by the initial reversal contingency relative to females indicating a potential source of the sex-specific interaction with the loss of α7 nAChRs.

Discussion: These findings argue that the α7 nAChR plays a critical role in hippocampal development, not just granule cell neurogenesis, and plays a sex-dependent role in cognitive function.

Keywords: CHRNA7; acetylcholine; cognitive flexibility; stereology; touchscreen.

Figure 1

Experimental timeline and touchscreen task paradigm. (A) The first cohort of mice were used for stereological analysis of total hippocampal granule cell count, pyramidal cell count, and volume at 3 months old. (B) The second cohort of mice were tested for pattern separation function at 4 months old. (C) The large separation task considered to not be dependent on dentate gyrus function. (D) The small separation task considered to be dependent on dentate gyrus function. WT, wildtype; KO, global α7 nAChR knockout.

Figure 2

Stereological granule cell count within the dentate gyrus. (A) A ROI (dashed green line) was manually generated around the dentate gyrus of the hippocampus. (B) Proportionator sampling was performed within the dentate gyrus at 40X magnification. (C) Granule cells (blue) were manually selected with the help of an analysis algorithm within the ROI. (D,E) Granule cells were counted and marked with a “C” when present in either the reference section (D) or the look-up section (E), but not present in both sections. (F) Granule cell counts. Boxplots represent median, interquartile range, absolute range, and individual points for each animal. ^*represents a significant effect at p < 0.05 for that effect.

Figure 3

Stereological pyramidal cell count within the CA1-3. (A) A ROI (dashed blue line) was manually generated around the CA1-3 of the hippocampus. (B) Proportionator sampling was performed within the CA1-3 at 40X magnification. (C) Pyramidal cells (yellow) were manually selected with the help of an analysis algorithm within the ROI. (D,E) Pyramidal cells were counted and marked with a “P” when present in either the reference section (D) or the look-up section (E), but not present in both sections. (F) Pyramidal cell counts. Boxplots represent median, interquartile range, absolute range, and individual points for each animal. ^*represents a significant effect at p < 0.05 for that effect.

Figure 4

Stereological hippocampal volume. (A) A ROI (green curvilinear shape) was manually generated around the CA1-3, dentate gyrus, and the subiculum of the hippocampus. (B) Points touching the hippocampus were tagged with an “H” and used to calculate the total volume. (C) Volume of the hippocampus. Boxplots represent median, interquartile range, absolute range, and individual points for each animal.

Figure 5

Pattern separation touchscreen task. (A) Large separation task for female mice. (B) Large separation task for male mice. (C) Small separation task for female mice. (D) Small separation task for male mice. Data in line graphs are represented by mean ± 95% CI. ^*represents a significant effect at p < 0.05 for that effect.

Figure 6

Behavior strategy for the small separation touchscreen task. (A) Percent change in correct touches before and after the first reversal. (B) Estimates of β₀. (C) Estimates of β₁. (D) Estimates of β₂. (E) Proportion of trials where mice shifted sides after an incorrect choice. (F) Proportion of trials where mice stayed on the same side after an incorrect choice. (G) Proportion of trials where mice shifted sides after a correct choice. (H) Proportion of trials where mice stayed on the same side after a correct choice. Boxplots represent median, interquartile range, absolute range, and individual points for each animal. ^*represents a significant effect at p < 0.05 for the related effect or a specific group versus zero for panels (B,D).