Perinatal asphyxia (PA) is a primary cause of neonatal morbidity, with persistent neurological sequelae, associated with mitochondrial dysfunction and redox imbalance. The lack of experimental approaches for region-specific evaluation of mitochondrial bioenergetics in structurally preserved neural tissues has limited the identification of selective vulnerabilities and redox-based therapeutic targets. Here, we introduce a real-time metabolic profiling strategy that integrates Seahorse XFe96 respirometry with Cytation 5 image-based quality control to quantify mitochondrial respiration and glycolytic flux in organotypic cultures derived from distinct brain regions of neonatal rats exposed to global PA. Using this strategy, we demonstrate that PA induces a profound and region-selective impairment of mitochondrial function, characterized by reduced basal, ATP-linked, maximal, and spare respiratory capacity in the substantia nigra and neostriatum, while neocortical bioenergetics remain largely preserved. These alterations are accompanied by a shift toward glycolysis, reflected by increased extracellular acidification rates and reduced OCR/ECAR ratios. Notably, in vivo administration of the NAD⁺ precursor nicotinamide riboside (0.8 mmol·kg⁻¹, i.p., 1 h after birth) effectively prevented PA-induced mitochondrial respiration deficits, attenuated oxidative stress-induced cell death, and preserved regional neurochemical integrity, particularly within the nigrostriatal neurocircuitry. Together, these findings reveal a heterogeneous mitochondrial redox and bioenergetic vulnerability of the developing brain to perinatal hypoxia. This integrated organotypic-metabolic approach may offer novel opportunities to dissect out region-specific mitochondrial dysfunction and to explore NAD⁺-based therapeutic strategies targeting neonatal brain injury.

Adversity is particularly pernicious in early life, increasing the likelihood of developing psychiatric disorders in adulthood. Juvenile and adult rats exposed to social isolation show differences in anxiety-like behaviors and significant changes in dopamine (DA) neurotransmission in the nucleus accumbens (NAc). Brain response to stress is partly mediated by the corticotropin-releasing factor (CRF) system, composed of CRF and its two main receptors, CRF-R1 and CRF-R2. In the NAc shell of adult rats, CRF induces anxiety-like behavior and changes local DA balance. However, the role of CRF receptors in the control of neurotransmission in the NAc is not fully understood, nor is it known whether there are differences between life stages. Our previous data showed that infusion of a CRF-R1 antagonist into the NAc of juvenile rats increased DA levels in response to a depolarizing stimulus and decreased basal glutamate levels. To extend this analysis, we now evaluated the effect of a CRF-R1 antagonist infusion in the NAc of adult rats. Here, we describe that the opposite occurred in the NAc of adult compared to juvenile rats. Infusion of a CRF-R1 antagonist decreased DA and increased glutamate levels in response to a depolarizing stimulus. Furthermore, basal levels of DA, glutamate, and γ-Aminobutyric acid (GABA) were similar in juvenile animals compared to adults. CRF-R1 protein levels and localization were not different in juvenile compared to adult rats. Interestingly, we observed differences in the signaling pathways of CRF-R1 in the NAc of juveniles compared to adult rats. We propose that the function of CRF-R1 receptors is differentially modulated in the NAc according to life stage.