The presence of liquid near the larynx of immature mammals triggers prolonged apneas with significant O2 desaturations and bradycardias. When excessive, this reflex (the laryngeal chemoreflex; LCR) can be fatal. Our understanding of the origins of abnormal LCR are limited; however, perinatal stress and male sex are risk factors for cardio-respiratory failure in infants. Because exposure to stress during early life has deleterious and sex-specific consequences on brain development it is plausible that respiratory reflexes are vulnerable to neuroendocrine dysfunction. To address this issue, we tested the hypothesis that neonatal maternal separation (NMS) is sufficient to exacerbate LCR-induced cardio-respiratory inhibition in anesthetized rat pups. Stressed pups were separated from their mother 3 h/d from postnatal days 3 to 12. At P14-P15, pups were instrumented to monitor breathing, O2 saturation (Spo2), and heart rate. The LCR was activated by water injections near the larynx (10 µl). LCR-induced apneas were longer in stressed pups than controls; O2 desaturations and bradycardias were more profound, especially in males. NMS increased the frequency and amplitude of spontaneous EPSCs (sEPSCs) in the dorsal motor nucleus of the vagus (DMNV) of males but not females. The positive relationship between corticosterone and testosterone observed in stressed pups (males only) suggests that disruption of neuroendocrine function by stress is key to sex-based differences in abnormal LCR. Because testosterone application onto medullary slices augments EPSC amplitude only in males, we propose that testosterone-mediated enhancement of synaptic connectivity within the DMNV contributes to the male bias in cardio-respiratory inhibition following LCR activation in stressed pups.

Brain networks underlying states of social and sensory alertness are normally adaptive, influenced by serotonin and dopamine (DA), and abnormal in neuropsychiatric disorders, often with sex-specific manifestations. Underlying circuits, cells, and molecules are just beginning to be delineated. Implicated is a subtype of serotonergic neuron denoted Drd2-Pet1, distinguished by expression of the type-2 DA receptor (Drd2) gene, inhibited cell-autonomously by DRD2 agonism in slice, and, when constitutively silenced in male mice, affects levels of defensive and exploratory behaviors (Niederkofler et al., 2016). Unknown has been whether DRD2 signaling in these Pet1 neurons contributes to their capacity for shaping defensive behaviors. To address this, we generated mice in which Drd2 gene sequences were deleted selectively in Pet1 neurons. We found that Drd2Pet1-CKO males, but not females, demonstrated increased winning against sex-matched controls in a social dominance assay. Drd2Pet1-CKO females, but not males, exhibited blunting of the acoustic startle response, a protective, defensive reflex. Indistinguishable from controls were auditory brainstem responses (ABRs), locomotion, cognition, and anxiety-like and depression-like behaviors. Analyzing wild-type Drd2-Pet1 neurons, we found sex-specific differences in the proportional distribution of axonal collaterals, in action potential (AP) duration, and in transcript levels of Gad2, important for GABA synthesis. Drd2Pet1-CKO cells displayed sex-specific differences in the percentage of cells harboring Gad2 transcripts. Our results suggest that DRD2 function in Drd2-Pet1 neurons is required for normal defensive/protective behaviors in a sex-specific manner, which may be influenced by the identified sex-specific molecular and cellular features. Related behaviors in humans too show sex differences, suggesting translational relevance.