Effects of sex and pro-inflammatory cytokines on context discrimination memory

Abstract

In learning and memory tasks, immune overactivation is associated with impaired performance, while normal immune activation is associated with optimal performance. In one specific domain of memory, context discrimination memory, peripheral immune stimulation has been shown to impair performance on the context-object discrimination memory task in male rats. In order to evaluate potential sex differences in this task, as well as potential mechanisms for the memory impairment, we evaluated the ability of peripheral immune stimulation to impair task performance in both males and females. Next, we examined whether treatment with interleukin-1 receptor antagonist (IL-1ra), a receptor antagonist for the pro-inflammatory cytokine interleukin (IL)-1β, was able to rescue the memory deficit. We examined microglial morphology in the hippocampus and cytokine mRNA and protein expression in the hippocampus and the periphery. Male rats displayed memory impairment in response to LPS, and this impairment was not rescued by IL-1ra. Female rats did not have significant memory impairments and IL-1ra administration improved memory following inflammation. A subset of cytokines and chemokines were increased only in LPS-treated males. Inflammation alone did not alter microglia morphology, but IL-1ra did in certain sub-regions of the hippocampus. Together, these results indicate that sex differences exist in the ability of a peripheral immune stimulus to influence context discrimination memory and specific cytokine signals may be altered in impaired males. This study highlights the importance of sex differences in response to inflammatory challenges, especially related to memory impairments in context discrimination memory.

Keywords: Cytokines; Hippocampus; Interleukin-1; Learning; Memory; Sex differences.

Figure 1.

A) Context A and its associated objects. B) Context B and its associated objects. C) For the COD task, rats were exposed to two days of training during which they encountered each context and its associated objects. On the third day of testing, they encountered a familiar context with one in-context object and one out-of-context object. Rats with intact context discrimination memory would be expected to show a preference for the out-of-context object.

Figure 2.

Timeline of behavioral testing. For Cohort 1.1, males and females were trained on a COD task for two days, exposed to an immune challenge 6H prior to testing on Day 3, and then tested on the COD task. Cohort 1.1 examined sex differences in the influence of neuroinflammation on context discrimination memory. For Cohort 1.2, males and females were trained on a COD task for two days. On Day 3, they were injected with LPS 6H prior to tissue collection. Cohort 1.2 assessed the neuroinflammatory state of the brain at the time of memory testing. For Cohort 2.1, rats were trained on a COD task for two days similar to Cohort 1. They were then exposed to an immune challenge 6H prior to testing on Day 3, followed by intra cisterna magna (i.c.m.) injections with IL-1ra 3H prior to testing and then tested on the COD task. Cohort 2.1 examined the ability of IL-1ra to rescue a memory impairment following a peripheral immune challenge. For Cohort 2.2, rats were exposed to handling but no behavioral task training or testing. Following an immune challenge and intra cisterna magna injection with IL-1ra, rats were killed, and tissue was collected to examine inflammatory status in the brain following the injections.

Figure 3.

Immune-challenged males demonstrate impaired context discrimination memory but explore the objects similarly to controls. A) Males injected with LPS prior to memory testing on Day 3 demonstrated impaired context-object memory. B) All groups explored both objects in the testing context equally during the trials. Asterisks (*) indicate groups that demonstrated intact context discrimination memory (p < 0.05), as measured by context discrimination scores that differed significantly from zero under the Benjamini-Hochberg procedure. Columns indicate group mean, n = 13–14 per group.

Figure 4.

Sex and treatment altered gene expression within the hippocampus. A) Ccl11 expression was significantly higher in saline-treated females than LPS-treated females (p=0.042). B) LPS-treated males had higher csf2 mRNA expression than LPS-treated females (p<0.026). C) LPS-treated males had higher il1α expression than LPS-treated females (p<0.022). Columns indicate group mean, n = 4 per group.

Figure 5.

A subset of inflammatory genes were significantly higher in LPS-treated males than all other groups (indicated by *). LPS-treated males had higher A) ccl12 expression (p<0.006), B) ccl4 expression (p<0.004), C) ccl7 expression (p<0.004), and D) il6 expression (p<0.004). Columns indicate group mean, n = 4 per group.

Figure 6.

IL-1ra rescues inflammation-induced context discrimination memory deficits in females, but not males, while exploration of the objects remains the same. A) Females given LPS and IL-1ra prior to testing had intact context discrimination memory, while females given only LPS did not show intact memory. Males injected with LPS followed by either SAL or IL-1ra prior to memory testing on Day 3 had impaired context-object memory. B) All groups explored both objects in the testing context equally during the trials. Asterisks (*) mark groups that demonstrated intact context discrimination memory (p<0.05). Columns indicate group mean, n = 6–8 per group.

Figure 7.

Immune challenge increased serum IL-1β concentrations, but not IL-1β protein levels within the hippocampus. A) Serum concentrations of IL-1β were measured using ELISA. Rats injected with LPS had significantly higher concentrations of serum IL-1β than controls six hours post-injection (* indicates p < 0.001). B) LPS administration, IL-1ra administration, and sex do not significantly impact hippocampal concentrations of IL-1β. Columns indicate group mean, n = 5–6 per group.

Figure 8.

A) Regions of interest in hippocampus with Iba1 stain at 4X magnification. Integrated area density was measured as an estimate of microglia morphology, with higher values indicating denser microglia staining. B) Region of interest for entorhinal cortex of Iba1 stain at 4X magnification. C) In the CA1 sub-region of the hippocampus, there is no significant effect of LPS, IL-1ra, or sex on microglial density. D) In the CA3 sub-region of the hippocampus, intra cisterna magna injection of IL-1ra significantly lowers microglia density. Animals treated with IL-1ra had significantly reduced staining compared to saline-treated (SAL) animals (* indicates p < 0.05). E) In the DG sub-region of the hippocampus, there is no significant effect of LPS, IL-1ra, or sex on microglial density. F) In the lateral entorhinal cortex, there is no significant effect of LPS, IL-1ra, or sex on microglial density. Columns indicate group mean, n = 4–6 per group.