Reduced Hippocampus-Dependent Fear Conditioning During Diestrus in Mice J.D. Cushman, M. Moore, S. Madsen, & M.S. Fanselow

Department of Psychology and Brain Research Institute, University of California Los Angeles, Los Angeles, CA, USA.

Support Contributed By: NINDS P01NS35985, NIGMS GM47818. This and all other Fanselow lab posters are available at http://www.psych.ucla.edu/fanselowlab

INTRODUCTION

RESULTS

CONCLUSIONS REFERENCES•The results indicated that there is a specific disruption of hippocampus dependent fear conditioning during diestrus relative to estrus in female C57Bl6 mice. This supports the hypothesis that the enhanced hippocampus dependent learning and memory in female δ-GABAAR KO mice is due to a loss of the δ-GABAAR mediated impairment that is normally present in wild-type females during diestrus. •In order to further test this hypothesis we will:

•Assess hippocampus dependent learning across estrus in female δ-GABAAR KO mice•Determine if male δ-GABAAR KO mice are less sensitive to the memory impairing effects of positive δ-GABAAR modulators, such as allopregnanalone•Examine estrus-cycle related changes in GABAAR expression levels throughout the brain

•Within the hippocampus, the δ-GABAAR is highly expressed in the dentate gyrus where it is thought to mediate a tonic inhibitory current•The δ-subunit is highly sensitive to enhancement by the neurosteroid allopregnanalone. •In female mice, the expression level of the δ-subunit within the dentate gyrus has been shown to fluctuate across the estrus cycle. Its expression is highest during diestrus resulting in enhanced tonic inhibition (Maguire et al, 2005).•Previous work in our lab (Wiltgen, et al, 2005) has shown that female δ-GABAAR KO mice show an enhancement in hippocampus dependent trace but not hippocampus independent delay fear conditioning whereas male δ-GABAAR KO’s are normal in both tasks. •In this study wild type females showed a range of freezing levels with some mice exhibiting very low levels of freezing suggesting that these particular mice failed to learn (Figure 1)•None of the female δ-GABAAR KO exhibited low freezing levels suggesting they all successfully learned the tone-shock association. •This previous study did not control for phase of the estrus cycle. •The current study, therefore, tested the hypothesis that the “failures to learn” in female wild-type mice were from mice that were trained during diestrus when δ-GABAAR subunit expression and neurosteroid mediated inhibition is high•Delay, trace and contextual fear conditioning were assessed in female C57Bl6 mice trained and tested in estrus versus mice trained and tested in diestrus

Chowdhury N, Quinn JJ, Fanselow MS. Dorsal hippocampus involvement in trace fear conditioning with long, but not short, trace intervals in mice. Behav Neurosci, 2005 Oct;119(5):1396-402.

Maguire JL, Stell GM, Rafizdeh M, Mody I. Ovarian cycle-linked changes in GABA(A) receptors mediating tonic inhibition alter seizure susceptibility and anxiety. Nat Neusroci, 2005 Jun;8(6):797-804. Epub 2005 May 15

Stell BM, Brikcley SG, Tang CY, Farrant M, Mody I. Neuroactive steroids reduce neuronal excitability by selectively enhancing tonic inhibition mediated by delta subunit-containing GABAA receptors. Proc Natl Acad Sci USA, 2003 Nov 25;100(24):14439-44. Epub 2003 Nov 17.

Wei W, Zhang N, Peng Z, Houser CR, Mody I. Perisynaptic localization of delta subunit-containing GABA(A) receptors and their activation by GABA spillover in the mouse dentate gyrus. J Neurosci, 2003 Nov 19;23(33):10650-61

Wiltgen BJ, Sanders MJ, Ferguson C, Homanics GE, Fanselow MS. Trace fear conditioning is enhanced in mice lacking the delta subunit of the GABAA receptor. Learn Mem, 2005 May-Jun;12(3):327-33. Epub 2005 May 16.

Estrus criteria:One day following peak vaginal impedance (which indicates proestrus)Large numbers of cornified cells

Diestrus II criteria:Second day of low vaginal impedanceVery few cells, mucous, streaks, some nucleated parabasal cells

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Figure 1: Freezing to a trace conditioned tone in female WT, Heterozygote and homozygote δ-GABAAR KO micePrevious data indicated that a significant proportion of female WT C57Bl6 mice failed to learn the trace conditioned tone-shock association. Delta GABA KO mice, however, did not show these failures to learn. Since the expression of the δ-GABAAR fluctuates across the estrus cycle we hypothesized that these failures to learn occurred in mice that were trained during diestrus when δ-GABAAR mediated inhibition is high. The present study examined trace fear conditioning across the estrus cycle in wild-type female C57Bl6 mice.

Trace Fear Conditioning in Female δ-GABAAR KO mice

Figure 2: Freezing during the tone test in mice that were trained and tested in estrus vs. diestrusFemale mice trained and tested in diestrus showed reduced levels of freezing during the post-tone interval after both trace and delay fear conditioning. Freezing during this interval has been shown to be disrupted by hippocampal lesions.

Figure 3: Post shock freezing during acquisition of contextual fear in estrus vs diestrusFemale mice in diestrus showed impaired acquisition of contextual fear as indicated by decreased freezing during post-shock periods 2 through 4

Figure 4: Freezing during the training and novel context tests in estrus vs. diestrus miceFemale mice in diestrus showed reduced freezing in both the original training context and in a novel context. This indicates impaired retention of hippocampus dependent contextual fear.

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Figure 5: Plasma hormone levels across estrus

There is a small but significant reduction in progesterone in diestrus relative to estrus. Estradiol did not differ between the two phases

Figure 6: Open field activity across estrus

The light in the experimental room was turned on halfway through the session in order to assess light induced locomotor suppression as well as responsiveness to stimulus change (i.e. the orienting response). There were no significant differences in locomotor activity in either lighting condition or in the orienting response.