Intact Female Mice Acquire Trace Eyeblink Conditioning Faster than Male and Ovariectomized Female Mice

Abstract

Female subjects have been widely excluded from past neuroscience work because of a number of biases, including the notion that cycling sex hormones increase variability. However, it is necessary to conduct behavioral research in mice that includes both sexes as mice are typically used for developing and evaluating future therapeutics. Understanding sex differences in learning is fundamental for the development of targeted therapies for numerous neurologic and neurodegenerative disorders, including Alzheimer's disease, which is more prevalent in females than males. This study set out to confirm the role of sex and necessity of circulating ovarian hormones in the acquisition of the temporal associative memory task trace eyeblink conditioning (tEBC) in C57BL/6J mice. We present evidence that sex and ovarian hormones are important factors in learning. Specifically, intact female mice learn significantly faster than both male and ovariectomized (ovx) female mice. Data from pseudoconditioned control mice indicate that sex differences are because of differences in learned associations, not sensitization or spontaneous blink rate. This study strengthens the idea that ovarian hormones such as estrogen and progesterone significantly influence learning and memory and that further research is needed to determine the underlying mechanisms behind their effects. Overall, our findings emphasize the necessity of including both sexes in future behavioral studies.

Figure 1.

Experimental paradigm and example EMG traces. A, Experimental timeline. B, Conditioning session protocol. CS in blue, US in gray. C, Pseudoconditioning session protocol. D, EMG trace of unconditioned response to airpuff during early training. E, EMG trace of CR to paired tone and airpuff during subsequent training sessions. F, EMG trace of response to tone alone pseudoconditioning trial.

Figure 2.

Schematic of EBC behavioral apparatus. Upper left. Video display of mouse with open eye during baseline. Lower left. Display depicting mouse with closed eyelids during a CR. Middle. Depiction of head-fixed mouse atop the freely rotating cylinder. Camera for visualizing mouse during conditioning task on left of cylinder frame. Speakers behind mouse are used to deliver tone CS. Blunted needle delivers aversive airpuff US to eye. Custom headbolt implanted on the mouse’s head connects to amplifier to receive EMG signal (depicted on the right).

Figure 3.

Upper. Percentage adaptive CRs for all conditioned animals (males, intact females, ovx) and pseudoconditioned animals across 2 d of habituation and 10 d of training. Mean ± SEM shown. * Šídák’s multiple comparisons test p < 0.05. Lower. Percentage adaptive CRs for intact females, male, and ovx female animals. Intact females learn significantly faster than males and ovx females. + Tukey’s multiple comparison test p < 0.05 intact female versus male; # Tukey’s multiple comparison test p < 0.05 intact female versus ovx.

Figure 4.

Intact females learn significantly faster than ovx females and males. Intact females reach eight consecutive CRs in 204 trials while males and ovx require 311 and 335 trials, respectively. Mean ± SEM shown; * Tukey’s multiple comparison test p < 0.05 for intact female versus male and ovx.

Figure 5.

Average CR onset latency. Mean ± SEM shown. Upper. Intact females respond earlier in the trial on average across all sessions. Lower. Average CR onset latency across all training sessions. Intact female n = 18, male n = 18, ovx n = 14, per training session.