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. 2022 Jul 23;13(1):42.
doi: 10.1186/s13293-022-00450-2.

Sex differences in contextual pattern separation, neurogenesis, and functional connectivity within the limbic system

Shunya Yagi  1 , Amanda Lee  2 , Nadine Truter  2 , Liisa A M Galea  3   4   5
Affiliations

Sex differences in contextual pattern separation, neurogenesis, and functional connectivity within the limbic system

Shunya Yagi et al. Biol Sex Differ. .

Abstract

Background: Females are more likely to present with anxiety disorders such as post-traumatic stress disorder (PTSD) compared to males, which are associated with disrupted hippocampal integrity. Sex differences in the structure and function of hippocampus exist. Here, we examined sex differences in contextual pattern separation, functional connectivity, and activation of new neurons during fear memory.

Methods: Two-month-old male and female Sprague-Dawley rats were injected with the DNA synthesis markers, iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU) 3 weeks and 4 weeks before perfusion, respectively. One week after CldU injection, the rats underwent a context discrimination task in which rats were placed in context A (shock) and context A' (no shock) every day for 12 days. On the test day, rats were placed in the shock context (context A) to measure fear memory and expression of zif268, an immediate early gene across 16 different limbic and reward regions. Repeated-measures or factorial analysis of variance was conducted on our variables of interest. Pearson product-moment calculations and principal component analyses on zif268 expression across regions were also performed.

Results: We found that females, but not males, showed contextual discrimination during the last days of training. On the test day, both sexes displayed similar levels of freezing, indicating equivalent fear memory for context A. Despite similar fear memory, males showed more positive correlations of zif268 activation between the limbic regions and the striatum, whereas females showed more negative correlations among these regions. Females showed greater activation of the frontal cortex, dorsal CA1, and 3-week-old adult-born dentate granular cells compared to males.

Conclusions: These results highlight the importance of studying sex differences in fear memory and the contribution of adult neurogenesis to the neuronal network and may contribute to differences in susceptibility to fear-related disorders such as post-traumatic stress disorder. Highlights Female rats, but not male rats, show faster discrimination during a contextual pattern separation task. Three-week-old adult-born neurons are more active in response to fear memory in females compared to males. Females had greater neural activation compared to males in the frontal cortex and dorsal CA1 region of the hippocampus in response to fear memory. Males and females show distinct patterns in functional connectivity for fear memory across limbic regions. Males have many positive correlations between activated new neurons of different ages between the dorsal and ventral hippocampus, while females show more correlations between activated new neurons and other limbic regions.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Experimental design. A Schematic illustration of experimental timeline: subjects received one intraperitoneal injection of 5-chloro-2'-deoxyuridine on Experimental Day 1 and one intraperitoneal injection of 5-iodo-2'-deoxyuridine on Experimental Day 8. Then, subjects were tested in the contextual pattern separation task for a total of 12 days (Experimental Day 16–28), followed by an activation trial in which the rats were placed in the context previously paired with shock but received no shock (Experimental Day 29). During the contextual pattern separation task, subjects were exposed to two different contexts each day; context A a shock-paired context (context paired with foot shocks) and context A’ a neutral context (context with no foot shock). B Brain regions that were examined for functional connectivity using zif268. ACC cingulate cortex (Cg1), PrL prelimbic cortex, IL infralimbic cortex, lDS lateral dorsal striatum, mDS medial dorsal striatum, NAc nucleus accumbens core, NAs nucleus accumbens shell, LA lateral amygdala, BLA basolateral amygdala, CeA central amygdala, dDG dorsal dentate gyrus, vDG ventral dentate gyrus, dCA1 dorsal cornu ammonis 1, vCA1 ventral cornu ammonis 1, dCA3 dorsal cornu ammonis 3, vCA3 ventral cornu ammonis 3
Fig. 2
Fig. 2
Sex differences in contextual fear discrimination. Mean (± SEM) percentage of freezing in Context A (shock) and Context A’ (non-shock) in males (A) and females (B). Mean (± SEM) discrimination index of the trial block 6 (Trial Day 11 + 12) (C). Mean (± SEM) percentage of freezing in males and females during the activation trial on Day 29 (D). Females exhibited significantly greater percentage of freezing in the shock-paired context (Context A) than in the neutral context (Context A’) on Trial Day 9 and Trial Day 12, whereas there was no significant difference in percentage of freezing between the two contexts in any days in males. Furthermore, females showed greater discrimination based on the index on the last trial block (Trial Day 11 + 12) compared to males. There was no significant sex difference in the percentage of freezing during the Activation Trial. *Indicates p < 0.05
Fig. 3
Fig. 3
Sex differences in zif268 activation of dentate adult-born cells. Photomicrographs of doublecortin-immunoreactive (ir) cells (DCX-ir: green) and zif268-ir cells (red) were taken under Zeiss Axio Scan.Z1 with a 40 × objective lens (E). Photomicrographs of IdU-ir cells (green) and zif268-ir cells (white) were taken under Olympus FV1000 confocal microscope with a 40 × objective lens (F). Photomicrographs of CldU-ir cells (red) and zif268-ir (white) cells were taken under Olympus FV1000 confocal microscope with a 40 × objective lens (G). Scale bars indicate 50 μm. Mean (± SEM) percentage of double-labeled cells in the dorsal (H) and ventral (I) dentate gyrus. Females, compared to males, had greater percentage of IdU/zif268-ir cells in the dorsal and ventral dentate gyrus, whereas there was a significant interaction effect of sex by region for the percentage of CldU/zif268-ir cells, with females having a greater percentage of CldU/zif268 in the dorsal dentate gyrus but males having a greater percentage of CldU/zif268-ir in the ventral dentate gyrus. Mean (± SEM) density of double-labeled cells in the dorsal (J) and ventral (K) dentate gyrus. There was a significant interaction effect of sex by region for the density of CldU/zif268-ir cells, with females having a greater density of CldU/zif268-ir in the dorsal dentate gyrus but males having a greater density in of CldU/zif268-ir in the ventral dentate gyrus. *Indicates p < 0.05
Fig. 4
Fig. 4
Sex differences in zif268 activation in the brain, and correlations with fear response. Photomicrographs of zif268 immunoreactivity in the frontal cortex (A) and in the dorsal hippocampus (B) in male (left) and female rats (right). Mean (± SEM) density of zif268-ir cells in the frontal cortex (C) and in the dorsal hippocampus (D). Females, compared to males, had greater density of zif268-ir cells in the anterior cingulate cortex (ACC), in the prelimbic cortex (PrL) and in the dorsal CA1. *Indicates p < 0.05. Heat maps generated based on correlations coefficients between the amount of freezing and activated cells (zif268-ir) in different regions of the limbic system (E). Males and females had significant correlations of zif268-ir cell density in different brain regions with the percentage of freezing (*indicates correlations with p < 0.05). There were significant sex differences in the correlations between the percentage of freezing and the density of zif268-ir cells in four different brain regions (Green boxes indicates significant sex differences with p < 0.05). Scatter plots for correlations between the percentage of freezing and the density of zif268-ir cells in the dorsal CA1 (dCA1) (F) and in the lateral dorsal striatum (lDS) (G), and correlations between the discrimination index on the last trial day and the percentage of IdU/zif268-ir cells in the dorsal dentate gyrus (H) in males (blue) and females (red)
Fig. 5
Fig. 5
Sex differences in inter-regional correlations of zif268-ir cell density. A A heatmap showing correlation coefficients (r) of the density of zif268-ir cells between each brain region in males and females. Males and females showed distinct patterns of significant inter-regional correlations of zif268-ir cell density. * Indicates significant correlations (p < 0.05) and green boxes indicate sex differences between the correlations (p < 0.05). B Brain network maps were generated with correlations with coefficients larger than 0.67 or smaller than − 0.67 in males (left) and females (right) with p < 0.1. Red lines indicate positive correlations with wider lines indicating larger coefficients and blue lines indicate negative correlations with wider lines indicating smaller coefficients
Fig. 6
Fig. 6
A–C Sex difference in the principal component analysis (PCA). Factor coordinates of the variables in the first three factors identified using principal component analysis (PCA) for zif268 activation (A). Scree plot for eigenvalues with the total percentage of variance for the first three factors (B). Eigenvalues for the first three factors were significant based on Horn’s parallel analysis. The first three factors explained 70.45% of the variances. A graph showing sex difference in the factor scores of individual samples (C). ANOVA and post hoc revealed males and females showed significant sex difference in the first factor. *Indicates p < 0.05 and ** indicates p < 0.01
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