Inbred Substrain Differences Influence Neuroimmune Response and Drinking Behavior

Abstract

Background The inbred mouse strain C57BL/6 is widely used in both models of addiction and immunological disease. However, there are pronounced phenotypic differences in ethanol (EtOH) consumption and innate immune response between C57BL/6 substrains. The focus of this study was to examine the effects of substrain on innate immune response and neuroimmune‐induced escalation of voluntary EtOH consumption. The main goal was to identify whether substrain differences in immune response can account for differences in EtOH behavior. Methods We compared acute innate immune response with a viral dsRNA mimic, polyinosinic:polycytidylic acid (poly(I:C)), in brain using qRT‐PCR in both C57BL/6N and C57BL/6J mice. Next, we used a neuroimmune model of escalation using poly(I:C) to compare drinking behavior between substrains. Finally, we compared brain neuroimmune response with both EtOH and repeated poly(I:C) in both substrains as a way to account for differences in EtOH behavior. Results We found that C57BL/6 substrains have differing immune response and drinking behaviors. C57BL/6N mice have a shorter but more robust inflammatory response to acute poly(I:C). In contrast, C57BL/6J mice have a smaller but longer‐lasting acute immune response to poly(I:C). In our neuroimmune‐induced escalation model, C57BL/6J mice but not C57BL/6N mice escalate EtOH intake after poly(I:C). Finally, only C57BL/6J mice show enhanced proinflammatory transcript abundance after poly(I:C) and EtOH, suggesting that longer‐lasting immune responses are critical to neuroimmune drinking phenotypes. Conclusions Altogether, this work has elucidated additional influences that substrain has on both innate immune response and drinking phenotypes. Our observations highlight the importance of considering and reporting the source and background used for production of transgenic and knockout mice. These data provide further evidence that genetic background must be carefully considered when investigating the role of neuroimmune signaling in EtOH abuse.

Selective Inhibition of PDE4B Reduces Binge Drinking in Two C57BL/6 Substrains

“…Prior to the start of A33 pretreatment, B6NJ mice consumed on average 3.15 ± 0.51 g/kg of alcohol, whereas B6J mice consumed on average 5.17 ± 0.35 g/kg under our 3-bottle-choice procedures, a finding that aligns with prior comparisons of alcohol intake between these substrains [ 57 , 58 , 59 , 60 , 61 ]. There were no sex differences in the average baseline alcohol intake for either substrain (sex effects and sex X concentration interactions: for B6NJ, p > 0.016; for B6J, p > 0.20).…”

“…Consistent with the limited literature comparing alcohol intake between B6 mice from the Jackson lineage versus the NIH lineage [ 57 , 58 , 59 , 60 , 61 ], alcohol intake under our 3-bottle, 2 h, DID procedure was greater in B6J versus B6NJ mice, with a similar (albeit less robust) substrain difference observed under a more traditional single-bottle DID procedure [ 77 ]. Although blood ethanol concentrations (BECs) could not be determined in Experiment 1 (see Methods), the levels of alcohol intake exhibited by B6NJ and B6J mice are predicted, based on our published data using this model [ 76 , 78 , 79 , 80 ] and those of others employing single-bottle DID procedures e.g., [ 77 , 81 ] to result in BECs below and above, respectively, the 80 mg/dL criterion for binge drinking [ 82 ].…”

“…However, the temporal patterning of PDE4B2 and PDE4B3 mRNA expression following a challenge of the innate immune system (e.g., lipopolysaccharide/LPS administration) is sex dependent, with females exhibiting a shorter latency to peak mRNA expression, a longer duration of high mRNA expression and a compensatory downregulation of mRNA expression that is not observed in males [ 86 ]. Recently, a substrain difference in the neuroimmune response to a challenge injection of polyinosinic:polycytidylic acid (poly(I:C)) was reported between B6NJ vs. B6J mice, with B6J mice exhibiting a smaller, but longer-lasting, innate immune response and only B6J mice exhibiting a poly(I:C)-induced proinflammatory response following alcohol consumption [ 57 ]. While purely circumstantial at this time, the fact that sex and substrain differences exist with respect to neuroimmune responsiveness involving PDE4B isozyme activity, means that it is possible that sex- and substrain-related factors may interact to influence behavioral sensitivity to PDE4B inhibitors and their off-target effects.…”

“…To increase the scientific rigor of the study, we conducted parallel alcohol-drinking studies in mice of the related, but genetically distinct, B6J substrain. The B6NJ and B6J substrains diverged nearly 70 years ago and exhibit different behavioral response to drugs of abuse e.g., [ 51 , 52 , 53 , 54 , 55 ], divergent binge eating phenotypes [ 56 ] and neuroimmune responses [ 57 ]. Relative to other unrelated mouse strains, both B6J and B6NJ mice readily consume alcohol under both continuous- [ 58 ] and limited-access drinking procedures [ 59 ].…”

“…Relative to other unrelated mouse strains, both B6J and B6NJ mice readily consume alcohol under both continuous- [ 58 ] and limited-access drinking procedures [ 59 ]. Indeed, alcohol intake and preference is higher in B6J versus B6NJ mice [ 58 , 59 , 60 , 61 ] and only the B6J substrain escalates alcohol intake in response to a neuroimmune challenge [ 57 ]. Alcohol is well characterized to impact neuroimmune function [ 62 , 63 , 64 ].…”