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. Author manuscript; available in PMC: 2024 Sep 1.
Published in final edited form as: Physiol Behav. 2023 May 18;268:114249. doi: 10.1016/j.physbeh.2023.114249

Sex-dependent deficits in associative learning across multiple LPS doses

Reeva K Patel 1, Nicolas T Pirozzi 1, Tiffany G Hoefler 1, Meghan G Connolly 2, Lauren G Singleton 1, Rachel A Kohman 1,*
PMCID: PMC10330873  NIHMSID: NIHMS1904087  PMID: 37210020

Abstract

Activation of the immune system by administration of the bacterial endotoxin lipopolysaccharide (LPS) impairs cognitive and neural plasticity processes. For instance, acute LPS exposure has been reported to impair memory consolidation, spatial learning and memory, and associative learning. However, the inclusion of both males and females in basic research is limited. Whether LPS-induced cognitive deficits are comparable in males and females is currently unclear. Therefore, the present study evaluated sex differences in associative learning following administration of LPS at a dose (i.e., 0.25 mg/kg) that impairs learning in males and higher LPS doses (i.e., 0.325 – 1 mg/kg) across multiple experiments. Adult male and female C57BL/6J mice were trained in a two-way active avoidance conditioning task following their respective treatments. Results showed that LPS had sex-dependent effects on associative learning. The 0.25 mg/kg LPS dose impaired learning in males, consistent with prior work. However, LPS, at any of the doses employed across three experiments, did not disrupt associative learning in females. Female mice were resistant to learning deficits despite showing heightened levels of select proinflammatory cytokines in response to LPS. Collectively, these findings demonstrate that the learning impairments resulting from acute LPS exposure are sex-dependent.

Keywords: LPS, Lipopolysaccharide, avoidance learning, cytokine, female, sex differences, dose

1. Introduction

Males and females show distinct immune responses that are shaped by genetic, endocrine, and environmental factors (Klein and Flanagan, 2016). Though sex differences in immune function are complex, females often show a more potent immune response to infection compared to males. For instance, female rats mount a higher anti-viral response, resulting in faster viral clearance than male rats (Hannah et al., 2008). Moreover, the response to vaccination is higher in females, as indicated by antibody levels and adverse reactions, than in males (Fischinger et al., 2019). Importantly, the sex differences found in immune function contribute to variations in disease susceptibility and severity. Females have a higher incidence of autoimmune disorders, whereas males have higher mortality rates during sepsis (Angele et al., 2014; Klein and Flanagan, 2016). Though there are critical differences in immune function between males and females, how sex influences the functional consequences of immune activation is widely understudied.

Induction of an immune response, regardless of sex, commonly begins with the activation of pattern recognition receptors (PRR), which detect conserved molecular signatures associated with pathogens or cellular distress (Li and Wu, 2021). Toll-like receptors (TLRs) represent a class of PRRs with multiple subtypes that respond to different ligands associated with viral, fungal, or bacterial infections. Upon TLR activation, an inflammatory response tailored to combat that particular pathogen class is initiated (Li and Wu, 2021). For instance, TLR4 detects bacterial ligands, whereas TLR3 detects viral ligands. Administration of a TLR agonist is a common approach to elicit an inflammatory response. Lipopolysaccharide (LPS) is a TLR4 agonist that, when given systemically, transiently elevates proinflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α), among other inflammatory molecules. Cytokines released in the periphery induce inflammation within the central nervous system (CNS) through various signaling routes (Dantzer, 2004; Skelly et al., 2013).

LPS-induced central inflammation elicits sickness behaviors that include reduced food intake, locomotor behavior, and social interactions (Dantzer, 2004). Components of this behavioral and physiological response to LPS differ across males and females. For instance, LPS-induced sickness behavior persists longer in adult male CD-1 mice than in female mice, as females had lower sickness scores compared to males at 24 and 48 hours post-LPS (Cai et al., 2016). Similarly, males show greater LPS-induced alterations in body temperature compared to females, which may result from differences in cytokine levels (Ashdown et al., 2007; Cai et al., 2016). LPS-treated females had increased serum levels of the anti-inflammatory cytokine IL-1 receptor antagonist (IL-1ra) and lower hypothalamic expression of IL-1β and cyclooxygenase-2 (COX-2) compared to LPS-treated males three hours post-administration (Ashdown et al., 2007). Additionally, females, but not males, show selective reductions in sexual behavior following LPS or IL-1β administration (Avitsur et al., 1997). In contrast, males and females show similar decreases in locomotor behavior in an open field following a single injection of LPS (Avitsur et al., 1997; Engeland et al., 2003). For females, the LPS-induced reductions in locomotor behavior did not differ across the estrous cycle (Engeland et al., 2003). While males and females both express sickness-associated behaviors and physiological reactions following an immune challenge, depending on the timing and parameter measured, sex differences exist.

The administration of LPS also disrupts learning and memory processes (Yirmiya and Goshen, 2011). For instance, LPS impairs associative learning in a two-way active avoidance conditioning task when administered before the first day of training (Kohman et al., 2010; Kohman et al., 2007; Sparkman et al., 2005a). LPS-induced deficits in memory consolidation, spatial learning and memory, and memory retrieval have also been reported (Czerniawski and Guzowski, 2014; Kranjac et al., 2012; Pugh et al., 1998; Sparkman et al., 2005a). While these data establish that systemic LPS administration disrupts cognitive function, one major limitation of the existing literature is the predominant use of males. Whether females develop similar LPS-induced learning and memory deficits has been relatively unexplored. A recent study by McNaughton and Williamson (2023) showed that context discrimination memory was disproportionately impaired in males under certain conditions following a single LPS injection. Additionally, two independent publications employing the same water maze protocol showed that LPS impaired spatial learning in males but not females (Sparkman et al., 2005b; Sparkman et al., 2005c). While these findings indicate potential sex differences in LPS-induced cognitive deficits, additional research comparing males and females is required to better characterize these sex differences and determine whether females’ resilience to LPS-induced cognitive disruption extends to other behavioral paradigms.

The current study sought to determine whether LPS had differential effects on associative learning across males and females. In response to LPS, we expected to replicate prior findings that LPS impairs acquisition in males (Kohman et al., 2007; Sparkman et al., 2005a). In contrast, based on prior reports, females were expected to show attenuated LPS-induced learning deficits. (McNaughton and Williamson, 2023; Sparkman et al., 2005b). Further, the current study evaluated whether males and females show variations in the magnitude of the peripheral and central cytokine response following LPS administration. After completing the first experiment that showed sex-dependent deficits in learning in response to LPS, we administered increasing doses of LPS to determine whether females required a higher dose of LPS to impair learning.

2. Methods

2.1. Subjects and housing

A total of 120 adult (5–6 month-old) C57BL/6J mice were evaluated across the three experiments. Mice were bred and housed in the University of North Carolina Wilmington (UNCW) animal facility from breeders obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were group housed (2–4 mice per cage) and given ad libitum access to food and water throughout the experiments. The colony room was maintained at 70° F ± 2° on a 12-hour reverse light/dark cycle. All procedures (i.e., injections, behavioral testing, and tissue collection) were completed during the animal’s dark cycle and did not start or end within an hour of the light cycle onset/offset. The individual animal was the unit of analysis. Treatments (i.e., saline or LPS) were distributed across mice within a cage. The cages were not changed during the five days of behavioral testing or within two days of sample collection. The experiments were approved by the UNCW Institutional Animal Care and Use Committee (IACUC) and followed the Guide for the Care and Use of Laboratory Animals.

2.2. Experimental design

2.2.1. Experiment 1 Sex differences

Fifty-one adult (5–6 month-old) C57BL/6J male (n=25) and female (n=26) mice were semi-randomly divided within sex and cages into the saline or LPS (0.25 mg/kg, from Escherichia coli, serotype 0111:B4; Sigma, St. Louis, MO) treatment groups, for a total of four groups (n=12–13 per group). To reduce animal numbers, mice were used for both behavioral testing and assessment of cytokine levels. Mice received two intraperitoneal (i.p.) injections of either LPS or saline spaced 32 days apart. Spacing the injections approximately one month apart was based on prior work and limits tolerance to multiple LPS injections (Setti et al., 2015). The first injection of LPS or saline was given four hours before the first day of testing in the two-way active avoidance conditioning task (described below). Injections were given in the morning, starting approximately one and a half hours after the onset of the dark cycle. Behavioral testing was conducted during the animal’s dark cycle in the afternoon at the same time each day. To blind the experimenters to the treatment conditions, different individuals administered the treatments and conducted behavioral testing. Mice were weighed throughout the five days of behavioral testing. A difference score between the current and the previous day’s weights were calculated to assess change in body weight following LPS or saline administration. The second injection was given four hours before tissue collection (described below), and mice received the same treatment (i.e., LPS or saline) as administered before behavioral testing. One male mouse was excluded from experiment 1 due to surface wounds on the mouse’s tail that likely resulted from cage-mate aggression.

2.2.2. Experiment 2 Assessment of LPS dose on associative learning in females

Experiment 2 was conducted to replicate the findings seen in the female mice in Experiment 1 and determine whether females would show learning deficits if administered higher LPS doses. Fifty female adult (5-month-old) C57BL/6J mice were divided into four treatment groups (n=12–13 per group): saline, LPS 0.25 mg/kg, LPS 0.325 mg/kg, and LPS 0.5 mg/kg. Female mice received a single i.p. injection of their respective treatment four hours before the first day of testing in the two-way active avoidance conditioning task (described below). Injections and behavioral testing occurred during the animal’s dark cycle, as described in Experiment 1. Body weight was measured daily and used to calculate a difference score.

2.2.3. Experiment 3 Associative learning in females following 1 mg/kg of LPS

Based on the results of Experiments 1 and 2, we evaluated associative learning in female mice following a 1 mg/kg dose of LPS. Nineteen female adult (6-month-old) C57BL/6J mice were randomly divided within a cage into the saline or LPS treatment groups for a total of two groups (n=9–10 per group). Female mice received a single i.p. injection of LPS (1 mg/kg) or saline four hours prior to the first day of testing in the two-way active avoidance conditioning task (described below). Injections and behavioral testing occurred during the animal’s dark cycle, as described in Experiment 1. Body weight was measured daily and used to calculate a difference score.

2.3. Two-way active avoidance conditioning

For all three experiments, mice were trained for five consecutive days in four identical shuttle boxes (15 cm L × 30 cm W × 24 cm H; Coulbourn Instruments, Whitehall, PA, USA). Each shuttle box had two equal-sized compartments separated by a wall with an open doorway (5 cm W × 6.5 cm H) that allowed the mice to cross between the compartments freely. The transparent Plexiglas walls were covered with black film to darken the inside of the shuttle box and increase the salience of the discriminative stimulus (i.e., light). Shuttle boxes were housed in sound-attenuating cabinets. Each training session began with a five-minute habituation period, during which the mice could freely move about the dark shuttle box with crossing responses recorded to assess activity.

Following the habituation period, mice received 52 trials that consisted of turning on the side wall and overhead lights (i.e., discriminative stimulus) in the shuttle box compartment where the mouse was located. Photobeams on either side of the doorway monitored the mouse’s location and crossing behavior. The lights remained on for up to 10 seconds. If the mouse crossed to the opposite compartment within the initial 5 seconds of the lights turning on, the response was recorded as an avoidance response and prevented delivery of a scrambled footshock (0.4 mA). If the mouse remained in the lit compartment past 5 seconds, the aversive stimulus (i.e., footshock) was delivered through the grid floor. The aversive stimulus was presented for up to 5 additional seconds but could be shortened by the mouse crossing to the opposite compartment (i.e., escape response). If the mouse remained in the lit compartment, the footshock and lights turned off after 5 seconds (i.e., null response). Each trial was separated by a 20-second inter-trial-interval (ITI), during which the mouse’s crossing behavior was monitored. To evaluate response efficiency, a discrimination index score was calculated by dividing cued crossings (i.e., avoidance responses) by total crossing behavior (i.e., avoidance responses + escape response + ITI crossings) and multiplying by 100 to produce a percentage (Kohman et al., 2007; Sparkman et al., 2005a). The latency to perform avoidance and escape responses was recorded to monitor performance issues.

2.4. Experiment 1 Sex differences: Tissue collection, RT-PCR, and ELISAs

Four hours after the second i.p. injection of LPS (0.25 mg/kg) or saline, mice from Experiment 1 were euthanized via CO2 inhalation. Spleens were dissected and snap-frozen on dry ice. Brains were extracted, and the hippocampus was dissected on a chilled metal plate on ice. The hippocampus and the remaining brain tissue (subsequently referred to as the brain) were snap-frozen on dry ice and stored at ‒80 °C until assayed.

Brains were homogenized in a proteinase inhibitor solution containing 1 mM phenylmethylsulfonyl fluoride (PMSF) in 0.1 M phosphate buffer. Supernatants were collected after centrifuging homogenized samples at 3500 rpm at 4 °C for 30 min. Protein levels of IL-1β and IL-6 were measured by enzyme-linked immunosorbent assays (ELISAs) following the manufacturer’s instructions (eBioscience, Santa Clara, CA, USA). The IL-1β and IL-6 ELISAs had a detectable range of 7.8–1000 pg/ml and 7.8–500 pg/ml, respectively. Total protein levels were assessed by a Pierce protein assay (Thermo Scientific, Rockford, IL, USA) and used to normalize cytokine levels. ELISA data are expressed as picograms (pg) of the cytokine over micrograms (μg) of total protein. Samples that were below the detectable limit of the curve were replaced by the limit of detection (LOD) divided by two (LOD/2) (Hewett and Ganser, 2007).

RNA was isolated from hippocampal samples with the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). RNA purity and concentration were assessed with a Gen5 Epoch spectrophotometer (BioTek Instruments, Winooski, VT, USA). All samples had a 260/280 ratio above 2.0. Subsequently, cDNA was generated with the High-capacity cDNA reverse transcription kit (Life Technologies Carlsbad, CA, USA). Reverse-transcription polymerase chain reaction (RT-PCR) was employed to determine changes in hippocampal gene expression. TaqMan™ probes and primers (Applied Biosystems, Foster City, CA, USA) were used to assess changes in IL-6 (Mm00446190_m1), IL-1β (Mm00434228_m1), IL-10 (Mm00439614_m1), interleukin-1 receptor antagonist (IL-1ra, Mm00446186_m1), and IL-1 receptor 2 (IL-1R2, Mm00439629_m1) expression, with β-Actin (Mm00607939_s1) serving as the endogenous control gene. The cycling conditions were 50 °C for 2 min, 95 °C for 2 min, and then 40 cycles of 95 °C for 15 sec and 60 °C for 1 min. Data were analyzed using the 2−ΔΔCT method, with a sample from the saline-treated male group as the calibrator. No differences in β-Actin were detected between treatment groups.

2.5. Statistical analyses

Body weight and behavioral data were analyzed using repeated measures ANOVAs. Treatment and sex were between-subject factors, and day was the within-subject factor. Treatment in Experiment 1 consisted of LPS (0.25 mg/kg) or saline. In Experiment 2, treatment consisted of three LPS doses (i.e., 0.25 mg/kg. 0.325 mg/kg, or 0.5 mg/kg) or saline. For Experiment 3, treatment referred to saline or LPS (1 mg/kg). ELISA and RT-PCR data were analyzed with two-way ANOVAs with treatment and sex as the between-subject factors. Normality was tested by the Shapiro-Wilk test. Data that were not normally distributed were log-transformed (i.e., brain levels of IL-1β and IL-6 and spleen levels of IL-1β) to establish a normal distribution before analysis. Outliers were identified by Grubbs’ test, with a significance level of 0.05. One LPS-treated female was a significant outlier for hippocampal gene expression for IL-6, IL-1β, and IL-1R2 and was removed from those analyses. Additionally, one LPS-treated male was detected as an outlier for brain protein levels of IL-6 and was removed from the analysis. If a significant (i.e., p<0.05) difference was found, post hoc testing was conducted when appropriate using Fisher’s least significant difference test.

3. Results

3.1. Body weight

3.1.1. Experiment 1 Sex differences

For the change in body weight, there was a significant LPS treatment × day interaction (F(3,141)=159.03, p<0.001, see Figure 1A). Post hoc testing showed that LPS-treated mice, regardless of sex, lost more weight than saline-treated mice 24 hours after treatment (p<0.001). On the subsequent days (i.e., 48–96 hours post-treatment), the LPS-treated mice gained more weight than saline-treated mice (p<0.005). There was also a sex × day interaction (F(3,141)=4.37, p<0.05, see Figure 1A) for change in body weight. While females generally showed smaller gains/losses in weight than males (likely due to smaller body size), post hoc tests showed no significant differences in weight change between males and females on any day.

Figure 1.

Figure 1.

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Body weight change in experiments 1–3. Weight was measured at 24-hour intervals and subtracted from the previous day’s weight to calculate the change in weight. In experiment 1, LPS reduced body weight in males and females 24 hours after treatment (A; marked by *). On subsequent days, LPS-treated mice recovered the lost weight. The weight of saline-treated mice remained relatively constant (n=12–13/group). In experiment 2 (B), LPS-treated females, regardless of dose, lost weight 24 hours after treatment and then gained weight 48–96 hours after treatment relative to saline-treated females (marked by *). The LPS 0.5 mg/kg showed less weight gain than the LPS 0.325 mg/kg group 96 hours after treatment (marked by +; n=12–13/group). Graph C shows weight change in the female mice in experiment 3. A 1 mg/kg injection of LPS caused weight loss at 24 hours and then weight gain at 72–96 hours compared to saline-treated females (marked by *; n=9–10/group). Lines show means ± standard error of the means (SEM).

3.1.2. Experiment 2 Assessment of increasing LPS doses in females

Analysis of the change in body weight throughout the five days of behavior testing in experiment 2 showed a significant treatment × day interaction (F(9,138)=39.12, p<0.001, see Figure 1B). Post hoc testing showed that females given LPS, regardless of the dose (i.e., 0.25, 0.325, or 0.5 mg/kg), lost weight compared to saline-treated females 24 hours after the injection (p<0.001). Subsequently, 48–96 hours after the injection, the LPS-treated females gained more weight than saline-treated females (p<0.005). The only difference in weight change between the three doses of LPS was seen 96 hours after the injection, with the LPS 0.5 mg/kg females showing less weight gain than the LPS 0.325 mg/kg females.

3.1.3. Experiment 3 Females administered LPS at 1 mg/kg

Assessment of change in body weight in females treated with the highest LPS dose administered (i.e., 1 mg/kg) or saline showed a significant treatment × day interaction (F(3,51)=48.35, p<0.001, see Figure 1C). Twenty-four hours after administration, LPS-treated females lost more weight compared to saline-treated females (p<0.001) but gained more weight 72- and 96-hours post-treatment (p<0.001).

3.2. Assessment of associative learning in the two-way active avoidance conditioning task

3.2.1. Experiment 1 Sex differences

A significant treatment × sex × day (F(4,188)=3.88; p<0.005, see Figure 2A) interaction for the number of avoidance responses showed that LPS-treated males made fewer avoidance responses on days 3–5 of testing relative to saline-treated males. LPS-treated males also made fewer avoids than LPS-treated females on days 4 and 5 (p<0.05). There were no significant differences in the number of avoidance responses between LPS- and saline-treated females on any of the five testing days. For saline-treated mice, females made fewer avoidance responses than males only on day 1 (p<0.05). For escape responses, there was a main effect of treatment (F(1,47)=21.25; p<0.0001) that showed LPS-treated mice made more escape responses overall than saline-treated mice. The sex × LPS treatment interaction for escape responses did not reach statistical significance with p=0.08. For null responses, there was a treatment × day interaction (F(4,188)=15.26; p<0.0001) that showed LPS-treated mice made more null responses than saline-treated mice, regardless of sex, on day 1 of testing (p<0.05). For the discrimination index scores, there was a significant treatment × sex × day interaction (F(4,188)=3.76; p<0.05, see Figure 2B). Post hoc analyses showed that males treated with LPS had lower discrimination scores compared to saline-treated males on days 2–5 of testing (p<0.05). LPS-treated males also had lower discrimination scores than LPS-treated females on days 4 and 5 (p<0.05). LPS administration did not affect the discrimination scores of female mice, as there was no difference between LPS- and saline-treated females (p<0.05). For saline-treated mice, females had lower discrimination scores than males on day 1 (p<0.05) but did not differ throughout the remaining four days of testing.

Figure 2.

Figure 2.

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LPS selectively impairs acquisition in males. In males, LPS decreased avoidance responses (A) and discrimination index scores (B) during later days of testing relative to saline-treated males (marked by *) and LPS-treated females (marked by +). Conversely, LPS did not affect avoidance responses or discrimination index scores in females. For saline-treated mice, females made fewer avoidance responses and had lower discrimination scores than males only on day 1 (marked by ^). There was no difference in latency to perform an avoidance response (C) between groups, but avoidance latency generally decreased across days (main effect of day marked by >). LPS decreased habituation crossings relative to saline in males and females selectively on day 1 of testing (D). Lines show means ± SEMs (n=12–13/group).

There was a significant main effect of testing day for latency to perform an avoidance response (F(1,47)=6.72,p<0.0001, see Figure 2C), with latency to avoid generally decreasing across test days. However, there were no significant differences in the latency to perform an avoidance response between LPS- and saline-treated mice nor between males and females. For latency to perform an escape response, there was a main effect of treatment (F(1,47)=8.46,p<0.05), as LPS-treated mice (LPS mean 0.74 seconds) overall had longer latencies to perform an escape response compared to saline-treated mice (saline mean 0.60 seconds). In addition, there was a main effect of test day (F(4,188)=146.87, p<0.001) that showed latency to perform an escape response decreased across testing. There was no significant main effect or interaction with sex for escape latency.

There was a significant main effect of test day (F(4,188)=7.53, p<0.001) for ITI crossings that showed ITI crossings tended to increase across the five days of testing. For crossing during the five-minute habituation period, there was a significant treatment × day interaction (F(4,188)=24.69, p<0.0001, see Figure 2D). LPS-treated mice, regardless of sex, made fewer habituation crossings compared to saline-treated mice on day 1 (p<0.0001); there were no differences on days 2–5 of testing.

3.2.2. Experiment 2 Assessment of increasing LPS doses in females

There were significant main effects of test day for the number of avoidance responses, number of escape responses, number of null responses, and discrimination index scores (F(4,184)=239.77, p<0.0001; F(4,184)=205.56, p<0.0001; F(4,184)=32.04, p<0.001, F(4,184)=206.70, p<0.0001, respectively). The number of avoidance responses (see Figure 3A) and discrimination index scores (see Figure 3B) increased across testing, whereas the number of escapes and null responses decreased. There were no differences between the treatment groups (i.e., saline, LPS 0.25, LPS 0.325, or LPS 0.5 mg/kg) for these four measures. Latency to perform an avoidance response (see Figure 3C) and escape response decreased across the testing days, as shown by main effects of day (F(4,184)=26.10, p<0.001, F(4,184)=175.65, p<0.0001, respectively). There were no differences in response latencies between the saline and three LPS treatment groups. A significant treatment × day interaction for ITI crossings (F(12,184)=2.10, p<0.05) showed that LPS-treated females made more ITI crossings than saline-treated mice on day 2 (p<0.05). On day 4, the females in the LPS 0.5 mg/kg group made more ITI crossings than the LPS 0.325 and saline females (p<0.05). On day 5, the LPS 0.5 mg/kg females had higher ITI crossing compared to the saline-treated females (p<0.05). For habituation crossings, there was a significant treatment × day interaction (F(12,184)=3.29, p<0.0005, see Figure 3D). Post hocs showed that all three LPS doses decreased habituation crossings relative to saline-treated females on day 1 (p<0.0005). There were no differences in habituation crossings between treatment groups on days 2–5.

Figure 3.

Figure 3.

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Females show no dose-dependent changes in associative learning. LPS- and saline-treated females showed equivalent increases in avoidance responses (A) and discrimination index scores (B), as well as decreases in latency to perform an avoidance response (C) across the five testing days (significant main effects of day marked by >). LPS at all three doses decreased habituation crossings (D) relative to saline-treated females selectively on day 1 of testing (marked by *). Lines show means ± SEMs (n=12–13/group).

3.2.3. Experiment 3 Females administered LPS at 1 mg/kg

There were significant main effects of test day for the number of avoidance responses, number of escape responses, number of null responses, and discrimination index scores (F(4,68)=58.38, p<0.001; F(4,68)=47.66, p<0.001; F(4,68)=40.68, p<0.001; F(4,68)=52.87, p<0.001, respectively). Data show that females performed more avoidance responses (see Figure 4A), increased their discrimination scores (see Figure 4B), and performed fewer escape and null responses across the five days of testing. There were no significant differences in avoidance, escape, null responses, or discrimination index scores between LPS- and saline-treated female mice. Analysis of latency to perform an avoidance response showed that females, regardless of their treatment, were faster at avoiding during later days of testing, as shown by a main effect of test day (F(4,68)=4.83, p<0.01, see Figure 4C). For latency to perform an escape response, there was a significant treatment × day interaction (F(4,68)=3.64, p<0.01). Post hoc testing showed that females injected with 1 mg/kg of LPS had longer escape latencies on the first day of testing compared to saline-treated females (p<0.05) but did not differ during the remaining four days of testing. Crossing during the ITI showed a significant treatment × day interaction (F(4,68)=3.32, p<0.05), with LPS-treated females performing more ITI crossings than saline-treated females on days 2–5 (p<0.05), there was no difference between treatments on day 1. There was also a significant treatment × day interaction (F(4,68)=17.37, p<0.0001, see Figure 4D) for crossings during the habituation period. LPS-treated females had fewer habituation crossings than saline-treated females on day 1 (p<0.0001) and more habituation crossings on day 4 (p<0.05).

Figure 4.

Figure 4.

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Females showed no evidence of impaired learning following a 1 mg/kg dose of LPS, as there were no differences in avoidance responses (A), discrimination index scores (B), or avoidance latencies (C) between the treatment groups. All measures showed changes across testing days (marked by >). LPS decreased habituation crossings on day 1 of testing and increased habituation crossings (D) on day 4 relative to saline-treated females (marked by *). Lines show means ± SEMs (n=9–10/group).

3.3. Experiment 1 Sex differences: Hippocampal gene expression.

Treatment × sex interactions for hippocampal expression IL-1β and IL-6 were significant (F(1,46)=7.61, p<0.005; F(1,46)=12.90, p<0.001, respectively, see Figures 5A and 5B). Both LPS-treated males and females had higher expression of IL-1β and IL-6 relative to their respective saline-treated controls (p<0.001). LPS-treated females had higher expression of IL-1β and IL-6 relative to LPS-treated males (p<0.01). There were no differences in IL-1β and IL-6 between saline-treated males and females. There were significant main effects of treatment for hippocampal expression of IL-10, IL-1ra, and IL-1R2 (F(1,47)=11.03, p<0.005; F(1,47)=57.99, p<0.0001, F(1,46)=47.84, p<0.0001, respectively). LPS-treated mice, regardless of sex, had higher hippocampal expression of IL-10, IL-1ra (see Figure 5C), and IL-1R2 (see Figure 5D) than saline-treated mice.

Figure 5.

Figure 5.

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Hippocampal cytokine expression. LPS increased hippocampal expression of IL-1β (A), IL-6 (B), IL-1ra (C), and IL-1R2 (D) in both males and females relative to saline-treated controls of the same sex. Females showed higher LPS-induced IL-1β and IL-6 expression than LPS-treated males. Data are shown as means overlaid with individual values (n=12–13/group). Brackets mark significant differences.

3.4. Experiment 1 Sex differences: Protein levels of IL-1β and IL-6 in the brain.

There was a significant main effect of treatment for brain levels of IL-1β (F(1,47)=12.19, p<0.005, see Figure 6A). LPS-treated mice had higher levels of IL-1β compared to saline-treated mice 4 hours after administration. There was a significant sex × treatment interaction for brain IL-6 (F(1,46)=10.08, p<0.005, see Figure 6B). LPS-treated mice had higher levels of IL-6 compared to saline-treated mice (p<0.05). Additionally, the LPS-treated females had higher brain levels of IL-6 compared to LPS-treated males (p<0.05).

Figure 6.

Figure 6.

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Brain and splenic protein levels of IL-1β and IL-6. LPS increased protein levels of IL-1β (A) and IL-6 (B) in brain samples from males and females relative to saline controls. Females showed higher brain levels of IL-6 than males in response to LPS (B). Splenic IL-1β (C) and IL-6 (D) were elevated in both LPS-treatment groups relative to saline-treated controls. Data are shown as means overlaid with individual values (n=12–13/group). Brackets mark significant differences.

3.5. Experiment 1 Sex differences: Protein levels of IL-1β and IL-6 in the spleen.

There were significant main effects of treatment for splenic levels of IL-1β (F(1,47)=22.85, p<0.0001, see Figure 6C) and IL-6 (F(1,47)=20.53, p<0.001, see Figure 6D). As expected, LPS-treated mice had higher splenic levels of IL-1β and IL-6 compared to saline-treated mice four hours after administration. The sex × treatment interactions for splenic IL-1β and IL-6 were not significant.

4. Discussion

Eliciting an inflammatory response through LPS has well-described disruptive effects on learning and memory (Yirmiya and Goshen, 2011), but the predominant use of males limit the generalizability of these findings. Therefore, the present study sought to determine whether males and females show differences in LPS-induced cognitive deficits. Results demonstrate a sexually dimorphic response, as males showed associative learning deficits in response to LPS. In contrast, females showed no evidence of disrupted learning following a range of LPS doses (0.25 – 1 mg/kg). Despite the sex-related differences in learning impairments, assessment of LPS-induced cytokines showed that males and females generally had comparable increases in splenic and brain cytokines, with select elevation of IL-6 in the brains of females following LPS. These findings provide novel evidence that females are resilient to LPS-induced learning deficits across multiple doses.

LPS-induced learning and memory deficits have been replicated in various behavioral paradigms (Czerniawski and Guzowski, 2014; Kohman et al., 2010; Kranjac et al., 2012; Pugh et al., 1998; Sparkman et al., 2005b). However, the majority of these experiments were conducted with males. The present results demonstrate that the disruptive effects of acute LPS exposure on cognitive performance may be specific to males. Female mice administered LPS before the first day of testing showed no changes in associative learning, as they performed comparable numbers of avoidance responses as saline-treated females. The data from the LPS-treated males replicates prior reports that show LPS impairs acquisition of the two-way active avoidance conditioning task (Kohman et al., 2010; Kohman et al., 2007; Sparkman et al., 2005a). Males treated with LPS show no differences in latency to perform an avoidance response, demonstrating that the impaired acquisition does not relate to motor issues. Notably, while LPS-treated females showed no evidence of impaired learning, they showed a comparable sickness-like response as the males. Both sexes showed transient weight loss and reduced activity on the first day of testing as measured by crossing behavior during the habituation period. Evidence of sickness-behavior in both sexes demonstrates that the lack of a learning deficit in females does not result from insensitivity to LPS.

The absence of LPS-induced learning deficits in females agrees with prior reports, though few studies have directly compared males and females. Studies by Sparkman et al. reported that LPS (0.25 mg/kg) impaired spatial learning and memory in males (Sparkman et al., 2005b) but had no effect in females (Sparkman et al., 2005c). Similarly, acute LPS (0.167 mg/kg) was reported to impair context discrimination memory selectively in male rats, with female rats showing no deficits (McNaughton and Williamson, 2023). In addition, following a lower dose of LPS (0.1 mg/kg), female mice showed no changes in working memory relative to controls in a water T-maze (O’Neill et al., 2021). Cunningham et al. (2009) reported that acute LPS administration produced spatial memory deficits in females infected with a prion, but no cognitive deficits were observed in uninfected control females exposed to LPS (0.5 mg/kg), in agreement with the present results. These findings indicate that acute LPS can exacerbate cognitive impairments in the presence of an ongoing disease state in females but has no observable effect on spatial memory in healthy females. In contrast, a study conducted with female rats showed that administration of LPS (0.25 mg/kg) prior to training in a passive avoidance task impaired memory (Pourganji et al., 2014). However, the female rats that showed memory deficits in passive avoidance underwent anesthesia and surgery (i.e., ovariectomy or sham) that may have altered the response to LPS (Tanaka et al., 2013). In response to repeated LPS administration, both males and females show impaired memory in a novel object recognition task eight weeks after LPS exposure though sex differences in the onset and the extent of deficits were found (Tchessalova and Tronson, 2019). Similarly, Cloutier et al. (2017) reported that repeated injections of LPS (0.2 mg/kg) impaired acquisition of context-conditioned disgust in both male and female rats. However, chronic LPS infusion into the fourth ventricle of female rats did not affect water maze performance (Marriott et al., 2002). These findings indicate that impairments in cognitive performance are generally observed in both sexes following repeated exposure to LPS. However, in response to acute LPS exposure, males are more vulnerable to LPS-induced cognitive deficits, whereas females are resilient.

The behavioral and physiological response to LPS can vary by the LPS dose (Biesmans et al., 2013; Teeling et al., 2007). For instance, the onset and duration of sickness-like behavior can vary by dose, as locomotor behavior in an open field is reduced twenty-four hours after administration of LPS at doses of 0.63 mg/kg or 1.25 mg/kg, but not 0.31 mg/kg. However, all three doses reduced locomotor behavior at earlier time points (i.e., 2–6 hours) (Biesmans et al., 2013), indicating that higher LPS doses may extend the sickness-like response. Similar dose-dependent effects of LPS on learning have been reported. Pugh et al. (1998) report that in juvenile rats, administration of 0.125 mg/kg of LPS, but not 0.5 mg/kg, consistently impaired memory in the contextual fear task. In adult rats, context memory was impaired by a 1 mg/kg LPS dose but not 0.5 or 2 mg/kg (Pugh et al., 1998). Thomson and Sutherland (2005) replicated these dose-dependent effects on contextual memory. The prior literature evaluating cognitive changes in females after LPS has predominately used LPS doses at or below 0.25 mg/kg. To determine whether females require a higher LPS dose before learning deficits are observed, we acutely administered a range of LPS doses (i.e., 0.25, 0.325, 0.5, and 1 mg/kg) to females. In male mice, comparable deficits in associative learning in the two-way active avoidance conditioning paradigm are detected after a range of LPS doses (0.1–0.325 mg/kg) (Sparkman et al., 2005a), indicating that after a minimum threshold dose is delivered, learning deficits are apparent in males in this task. Our data demonstrate that female mice showed no evidence of LPS-induced learning deficits even after administration of an LPS dose of the same serotype ten times higher (1 mg/kg) than the dose needed to impair learning in males (0.1 mg/kg). Notably, females showed evidence of sickness behavior at all LPS doses, with potential evidence of extended anorexia following the 1 mg/kg LPS dose as weight was slower to recover. Additionally, the administration of higher LPS doses was associated with increased ITI crossings during later testing days. While dose-dependent effects of LPS occurred, our data indicate that the lack of LPS-induced associated learning deficits in females is unlikely to be dose-dependent.

LPS-induced increases in inflammatory molecules, such as proinflammatory cytokines in the brain, have been implicated in driving the cognitive deficits following LPS. This is based on data showing that individual cytokine administration produces similar cognitive deficits as those seen after LPS exposure (Barrientos et al., 2002; Gibertini, 1996). Further inhibiting IL-1 signaling by administering an interleukin-1 receptor antagonist (IL-1Ra) or lentivirus knock-down in the hippocampus prevents LPS-induced memory deficits (Li et al., 2017; Pugh et al., 1998). The present data demonstrate that females show comparable or higher LPS-induced cytokine levels than males when assessed in the spleen, brain, and hippocampus. The heightened brain and hippocampal levels of IL-6 in response to LPS seen in the females are consistent with prior work showing that females often mount a heightened inflammatory response relative to males (vom Steeg and Klein, 2016). Both males and females showed similar increases in the immunoregulatory molecules IL-10, IL-1ra, and IL-1R2 relative to saline-treated mice. Despite the central inflammatory response to LPS, females showed no evidence of impaired learning in the active avoidance conditioning paradigm. Potentially, brain regions engaged in cognitive function in the male and female brain show differential sensitivity during acute inflammation. Sex-related differences in the duration of inflammation may also contribute, as females have been reported to clear infections faster than males (Barna et al., 1996; Briggs et al., 2020; Hannah et al., 2008). Moreover, LPS-induced sickness behaviors have been reported to last longer in males than females (Cai et al., 2016; Tesfaigzi et al., 2001). The present data indicate that although LPS administration produced heightened and/or comparable levels of inflammatory cytokines, females showed no associative learning disruption.

The sex-specific sensitivity to the learning alterations resulting from LPS may stem from differences in the response strategy or neuromechanisms engaged when acquiring the active avoidance task. Saline-treated males and females showed minimal differences in learning. In experiment 1, saline-treated females made fewer avoidance responses and had lower discrimination index scores than saline-treated males on the first day of testing, with no differences detected during the remaining four days. While this indicates subtle differences in acquisition between control males and females, the overall performance was comparable. Despite the limited differences in performance between the control groups, this does not eliminate the potential for sex differences in the response strategy, as female rats have previously been found to respond with active behaviors when exposed to a threat (Shansky, 2018). Whether females and males engaged in different response strategies to acquire the active avoidance task cannot be determined from the present data but should be considered in future studies evaluating sex differences. Sex-specific molecular mechanisms of learning and memory also exist. Males and females show anatomical differences in the hippocampus that may contribute to variations in hippocampal-dependent learning (Mizuno and Giese, 2010). Moreover, males and females are reported to show unique neural activation patterns, brain region engagement, and transcriptional profiles during learning and memory (Mizuno and Giese, 2010). The utilization of unique molecular processes may produce differential sensitivity of males and females to LPS.

Our findings demonstrate that vulnerability to LPS-induced learning deficits is sex-dependent, as females showed no evidence of disrupted associative learning. Adding to the novelty is that females are resistant to LPS-induced learning deficits at a range of LPS doses. Moreover, these LPS-induced learning impairments were specific to males, despite comparable or heightened central cytokine levels in females. Collectively, the findings demonstrate that sex is a critical determinant of the cognitive effects of acute LPS, as females are resilient and males are susceptible.

Highlights.

  • LPS impairs associative learning in males but has no effect in females

  • Females are resistant to LPS-induced learning deficits across multiple doses

  • LPS elevates cytokines in both sexes, with greater IL-6 in female brains

Funding

This work was supported by a grant from the National Institute on Aging [R15AG052935] awarded to RAK. The funding source had no involvement in the experimental design, interpretation of the results, or manuscript preparation.

Footnotes

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Declaration of Competing Interest

None

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