Adolescent stress and alcohol are associated with CX₃CR1-linked endocrine-cardiac signatures and anxiety-like behavior in mice

Adolescence is a vulnerable window in which stress and alcohol exposure can induce long-lasting neuroimmune, endocrine, and cardiac molecular alterations. The fractalkine axis (CX₃CL1/CX₃CR1) regulates neuron-microglia signaling and inflammatory responses, but its role in linking adolescent stress/alcohol exposure with adult anxiety-like behavior and cardiac biomarker/molecular signatures remains unclear.

To test whether genetic or pharmacological disruption of CX₃CR1 modifies adult anxiety-like behavior, circulating biomarkers, and cardiac transcriptional responses after adolescent stress and/or alcohol exposure.

Male and female C57BL/6J wild-type (WT) and CX₃CR1 knockout (KO) mice were exposed to restraint stress (90 min) and/or alcohol (2 g/kg, 14 days) during adolescence. In adulthood, elevated plus maze (EPM) behavior was assessed; plasma cTnI/cTnT, CX₃CL1, ACTH, and corticosterone were quantified; and cardiac mRNA expression of chemokines, inflammatory receptor/NF-κB-related genes, glucocorticoid/mineralocorticoid receptor-related genes, and RAAS-related transcripts was profiled. WT mice received the CX₃CR1 antagonist AZD8797 (20 mg/kg, i.p.) for behavioral validation.

CX₃CR1 loss or blockade was associated with increased anxiety index and reduced open-arm exploration under selected exposure conditions. CX₃CR1 KO mice showed higher plasma cTnI/cTnT, CX₃CL1, and ACTH, and lower corticosterone, stress and alcohol were also associated with lower corticosterone. In cardiac tissue, alcohol exposure, mainly under the no-stress condition, increased Cx3cl1, Ccl2, Ackr3, and selected RAAS-related transcripts, whereas CX₃CR1 deficiency was associated with broadly lower expression across chemokine, NF-κB-related, mineralocorticoid receptor-related, and RAAS-related targets. Correlation analyses indicated a chemokine/Cxcl12-anchored inflammatory-RAAS transcriptional architecture in WT mice that was attenuated and shifted toward glucocorticoid/mineralocorticoid receptor-NF-κB/IκBα-RAAS-related associations in CX₃CR1 KO mice.

These findings suggest that CX₃CR1 signaling contributes to the long-term coordination of behavioral stress-related responses, HPA-axis activity, circulating cardiac injury biomarkers, and cardiac inflammatory/RAAS-related transcriptional programs after adolescent stress and alcohol exposure. Because cardiac function and histopathology were not assessed, these results should be interpreted as evidence of cardiac biomarker and molecular vulnerability rather than definitive myocardial pathology.