Effects of transcranial photobiomodulation on anxiety-like behavior and spatial learning in adult and old female rats

Aging is associated with progressive cognitive decline and increased vulnerability to anxiety-related behaviors, partly due to mitochondrial dysfunction and reduced neuronal metabolic capacity. Transcranial photobiomodulation (tPBM) has emerged as a non-pharmacological intervention capable of stimulating mitochondrial cytochrome-c-oxidase and enhancing brain function. This study evaluated the effects of tPBM on anxiety-like behavior, spatial learning, and regional brain oxidative metabolism in adult and old female Wistar rats. Forty rats (10 or 18 months old) received 12 consecutive days of tPBM (810 nm; 20 J/cm²; 40 mW) or SHAM stimulation. Behavioral performance was assessed using the Elevated Zero Maze (EZM) and Morris Water Maze (MWM), followed by quantitative cytochrome-c-oxidase (CCO) histochemistry to evaluate regional brain metabolic activity. tPBM significantly reduced anxiety-like behavior in both age groups, evidenced by lower anxiety index scores and increased time in open arms in the EZM. In adult rats, tPBM improved spatial learning and memory performance in the MWM, whereas effects were limited in old animals. Analysis of CCO activity revealed increased metabolic activity in prefrontal, hippocampal, striatal, and somatosensory regions in adult rats versus the SHAM group. In old rats, higher levels of CCO activity as compared to an age-matched SHAM group were observed in the primary motor cortex, nucleus accumbens core, CA1, and somatosensory cortex. These findings suggest age-dependent responsiveness to tPBM, with greater behavioral and metabolic effects in adult compared with aged females. Overall, tPBM decreased anxiety-like behavior and selectively improved cognitive performance while enhancing metabolic activity in key brain regions such as the prefrontal cortex, dorsal hippocampus, and motor control regions. These results support tPBM as a promising neuromodulatory approach during aging and highlight the need for sex-specific and protocol-standardized research.