Examining the impact of neuroimmune dysregulation on social behavior of male and female juvenile rats

Abstract

Many individuals diagnosed with neuropsychiatric disorders, such as autism, attention-deficit/hyperactivity disorder, schizophrenia, and social anxiety disorder, all share a common dimension of aberrant social behavior. Epidemiological data indicate that adverse environmental factors contribute to the risk for neurodevelopmental disorders, including those associated with aberrant social behavior. Early-life exposure to infectious pathogens is one of those adverse environmental factors, suggesting that activation of the immune system during early development may contribute to disease pathology associated with altered social behavior. In the current project, we examined the impact of neonatal infection, with or without juvenile immune activation, on the expression of juvenile social behavior and on the expression of inflammatory cytokines and microglial signaling molecules in the juvenile rat brain. The outcomes of these experiments revealed that neonatal infection significantly decreased juvenile social interaction, but significantly increased juvenile play behavior in male and female rats. Moreover, neonatal infection alone, juvenile immune activation alone, and neonatal infection plus juvenile immune activation all significantly impaired social recognition in juvenile male rats. Juvenile female rats (including controls) did not demonstrate social recognition as measured in our three-chamber social recognition test. Taken together, the behavioral and molecular data presented here support the sensitivity of the developing brain to immune activation, particularly in the expression of age-appropriate social behaviors. These data warrant the design of additional studies to examine the mechanistic relationship between early-life immune activation and aberrant social behavior to develop novel as well as modify existing therapeutic targets and preventative measures to help those who display aberrant social behavior.

Keywords: Juvenile; Microglia; Neurodevelopmental; Neuroimmune; Rat; Social behavior.

Figure 1.

Impact of neonatal infection and/or juvenile LPS administration on social interaction (seconds) in male and female juvenile rats during a three-chamber social interaction test. (A) Across the total ten-minute test, social interaction was unaffected by neonatal infection and/or LPS administration. (B) Within the first five minutes of the test, social interaction was significantly decreased in neonatally infected rats, independent of sex or juvenile LPS administration. (C) No significant main effects or interactions were found within the second five minutes of the test. N = 14-17/group. Error bars represent the ±SEM. *p < 0.05 indicates a main effect of neonatal infection.

Figure 2.

Impact of neonatal infection and/or juvenile LPS administration on social recognition (discrimination ratio) in male and female juvenile rats during a three-chamber social recognition test. (A) Across the total ten-minute test, only control male rats showed a significant preference for the novel stimulus rat, compared to chance. No other groups showed a significant preference for the novel stimulus rat, including control females. (B) Within the first five minutes of the test, no rats showed a significant preference for the novel stimulus rat compared to chance. (C) Within the second five minutes of the test, only control male rats showed a significant preference for the novel stimulus rat, compared to chance. No other groups showed a significant preference for the novel stimulus rat, including control females. Dotted red line = chance or 0.50; Discrimination Ratio = total time spent with novel stimulus rat (seconds) / total time spent with novel + familiar stimulus rat (seconds). N = 14-17/group. Error bars represent the ±SEM. *p < 0.05 indicates a group mean significantly different from chance: 0.05.

Figure 3.

Impact of neonatal infection and/or juvenile LPS administration on social play behavior (instances of behavior) of individual test animals within the home cage across a fifteen-minute interval. (A, B, C, & G) Neonatal infection significantly increased the total instances in which male and female test animals pounced, pinned, and chased their stimulus partners. (D, E, & F) Experimental manipulation had no significant impact on how often test animals were pounced, pinned, or chased, by their stimulus partners. N = 8/group. Error bars represent the ±SEM. *p < 0.05 indicates a main effect of neonatal infection.

Figure 4.

Summary of experimental conditions and significance markers used to indicate specific main effects and interactions within the relative gene expression (and behavior) figures.

Figure 5.

Impact of neonatal infection and/or juvenile LPS administration on IL-1β relative gene expression in the male and female rat brain at 4, 24, and 72 hours post-LPS administration on P25. Unless otherwise stated, changes are relative to control rats. (A) At 4 hours, IL-1β was significantly increased in LPS-injected rats. (B) At 24 hours, IL-1β was significantly increased in LPS-injected rats that were not neonatally infected, but not in neonatally infected plus LPS-injected rats. (C) At 72 hours, IL-1β was significantly increased in LPS-injected male rats, but not in LPS-injected female rats. (D and E) At 4 & 24 hours, IL-1β was significantly increased in LPS-injected rats. (F) At 72 hours, IL-1β was significantly increased in neonatally infected plus LPS-injected female rats, but only relative to female rats receiving either an LPS injection or neonatal infection, and relative to control male rats. (G) At 4 hours, IL-1β was significantly increased in LPS-injected male rats that were not neonatally infected, but not in neonatally and LPS-injected male rats. However, IL-1β relative gene expression was significantly increased in LPS-injected plus neonatally infected female rats, but not in female rats administered LPS alone. (H) At 24 hours, IL-1β was significantly increased in LPS-injected rats. (I) At 72 hours, IL-1β was significantly increased in LPS-injected plus neonatally infected rats, but not in rats administered LPS alone. (J and K) At 4 & 24 hours post-LPS administration, IL-1β was significantly increased in LPS-injected rats. (L) At 72 hours, IL-1β was significantly increased in neonatally infected plus LPS-injected rats, but not in rats administered LPS alone. N = 8-10/group. Error bars represent the ±SEM. *p < 0.05 indicates a significant main effect (juvenile LPS administration, neonatal infection, or sex – depending on * placement); +p < 0.05 indicates a significant interaction between neonatal infection and juvenile LPS administration; ^p < 0.05 indicates a significant interaction between sex and juvenile LPS administration; ¤p < 0.05 indicates a significant interaction between sex and neonatal infection; individual letters indicate a significant three-way interaction between sex, neonatal infection, and juvenile LPS administration: bars with the same letter do not significantly differ from one another; and “ns” indicates no significant differences.

Figure 6.

Impact of neonatal infection and/or juvenile LPS administration on IL-6 relative gene expression in the male and female rat brain at 4, 24, and 72 hours post-LPS administration on P25. Unless otherwise stated, changes are relative to control rats. (A) At 4 hours, IL-6 was significantly increased in neonatally infected plus LPS-injected rats, but not in rats administered LPS alone. (B) At 24 hours, IL-6 was significantly greater in juvenile male rats than in juvenile female rats. (C) At 72 hours, IL-6 was significantly increased in neonatally infected female rats, relative to female rats that were not neonatally infected. (D) At 4 hours, IL-6 was significantly increased in LPS-injected male rats, but not in LPS-injected female rats. (E) At 24 hours, IL-6 was significantly increased in LPS-injected rats. (F) At 72 hours, IL-6 was significantly increased in neonatally infected female rats, relative to female rats that were not neonatally infected. (G) At 4 hours, IL-6 was significantly increased in male rats administered LPS alone, but not in neonatally infected plus LPS-injected male rats. (H) At 24 hours, post-hoc comparisons indicated a trending increase in IL-6 in LPS-injected male rats, but no significant or trending differences in LPS-injected female rats. (I) At 72 hours, there were no significant differences in IL-6 expression. (J) At 4 hours, IL-6 was significantly increased in LPS-injected rats. (K) At 24 hours, IL-6 was significantly decreased in neonatally infected male rats, relative to male rats that were not neonatally infected. (L) At 72 hours, IL-6 was significantly increased in neonatally infected female rats, relative to female rats that were not neonatally infected. IL-6 was also significantly decreased in LPS-injected rats, relative to control rats. N = 8-10/group. Error bars represent the ±SEM. *p < 0.05 indicates a significant main effect (juvenile LPS administration, neonatal infection, or sex – depending on * placement); +p < 0.05 indicates a significant interaction between neonatal infection and juvenile LPS administration; ^p < 0.05 indicates a significant interaction between sex and juvenile LPS administration; ¤p < 0.05 indicates a significant interaction between sex and neonatal infection; individual letters indicate a significant three-way interaction between sex, neonatal infection, and juvenile LPS administration: bars with the same letter do not significantly differ from one another; and “ns” indicates no significant differences.

Figure 7.

Impact of neonatal infection and/or juvenile LPS administration on C3 relative gene expression in the male and female rat brain at 4, 24, and 72 hours post-LPS administration on P25. Unless otherwise stated, changes are relative to control rats. (A) At 4 hours, there were no significant differences in C3 expression. (B) At 24 hours, C3 was significantly increased in male rats administered LPS alone, but not in male rats neonatally infected plus LPS-injected. (C) At 72 hours, C3 was significantly increased in neonatally infected plus LPS-injected rats, but not in rats administered LPS alone. C3 was also significantly increased in all LPS-injected male rats, but not in all LPS-injected female rats. (D) At 4 hours, there were no significant differences in C3 expression. (E) At 24 hours, C3 was significantly increased in LPS-injected male rats, but not in LPS-injected female rats. (F) At 72 hours, C3 was significantly increased in neonatally infected plus LPS-injected rats, but not in rats administered LPS alone. (G and H) At 4 & 24 hours, C3 was significantly increased in LPS-injected rats. (I) At 72 hours, increases in C3 were more robust in neonatally infected plus LPS-injected rats than in rats administered LPS alone. (J) At 4 hours, C3 was significantly increased in LPS-injected rats. C3 was also significantly increased in neonatally infected rats, relative to rats that were not neonatally infected. (K) At 24 hours, C3 was significantly increased in male rats administered LPS alone, but not in neonatally infected plus LPS-injected male rats. (L) At 72 hours, increases in C3 were more robust in neonatally infected plus LPS-injected rats than in rats administered LPS alone. N = 8-10/group. Error bars represent the ±SEM. *p < 0.05 indicates a significant main effect (juvenile LPS administration, neonatal infection, or sex – depending on * placement); +p < 0.05 indicates a significant interaction between neonatal infection and juvenile LPS administration; ^p < 0.05 indicates a significant interaction between sex and juvenile LPS administration; ¤p < 0.05 indicates a significant interaction between sex and neonatal infection; individual letters indicate a significant three-way interaction between sex, neonatal infection, and juvenile LPS administration: bars with the same letter do not significantly differ from one another; and “ns” indicates no significant differences.

Figure 8.

Impact of neonatal infection and/or juvenile LPS administration on CX3CR1 relative gene expression in the male and female rat brain at 4, 24, and 72 hours post-LPS administration on P25. Unless otherwise stated, changes are relative to control rats. (A, B, & C) At 4 hours, 24, and 72 hours, there were no significant differences in CX3CR1 expression. (D) At 4 hours, CX3CR1 was significantly decreased in LPS-injected rats. (E) At 24 hours, CX3CR1 was significantly decreased in neonatally infected rats. (F) At 72 hours, there were no significant differences in CX3CR1 expression. (G and H) At 4 & 24 hours, there were no significant differences in CX3CR1 expression. (I) At 72 hours, post-hoc analyses did not reveal any significant group differences but did reveal a trending increase in CX3CR1 expression in control females relative to control males. (J) At 4 hours, there were no significant differences in CX3CR1 expression. (K) At 24 hours, CX3CR1 was significantly increased in neonatally infected and LPS-injected female rats relative to neonatally infected and LPS-injected male rats. However, neither group significantly differed from its sex-specific control. (L) At 72 hours, CX3CR1 expression was significant greater in female rats than in male rats. N = 8-10/group. Error bars represent the ±SEM. *p < 0.05 indicates a significant main effect (juvenile LPS administration, neonatal infection, or sex – depending on * placement); individual letters indicate a significant three-way interaction between sex, neonatal infection, and juvenile LPS administration: bars with the same letter do not significantly differ from one another; and “ns” indicates no significant differences.

Figure 9.

Impact of neonatal infection and/or juvenile LPS administration on CX3CL1 relative gene expression in the male and female rat brain at 4, 24, and 72 hours post-LPS administration on P25. Unless otherwise stated, changes are relative to control rats. (A, B, & C) At 4 hours, 24, and 72 hours, there were no significant differences in CX3CL1 expression. (D) At 4 hours, there were no significant differences in CX3CL1 expression. (E) At 24 hours, post-hoc comparisons did not reveal any significant or trending group differences. (F) At 72 hours, neonatally infected females had significantly greater CXCL1 expression than neonatally infected males. However, neither group significantly differed from their sex-specific controls. (G) At 4 hours, post-hoc comparisons did not reveal any significant or trending group differences. (H) At 24 hours, there were no significant differences in CX3CL1 expression. (I) At 72 hours, CX3CL1 expression was significantly increased in female rats compared to male rats. Neonatally infected females had significantly greater CXCL1 expression than neonatally infected males. However, neither group significantly differed from their sex-specific controls. (J, K, & L) At 4 hours, 24, and 72 hours, there were no significant differences in CX3CL1 expression. N = 8-10/group. Error bars represent the ±SEM. *p < 0.05 indicates a significant main effect (juvenile LPS administration, neonatal infection, or sex – depending on * placement); individual letters indicate a significant three-way interaction between sex, neonatal infection, and juvenile LPS administration: bars with the same letter do not significantly differ from one another; and “ns” indicates no significant differences.