¹Ribeirao Preto Medical School, University of Sao Paulo, Pharmacology, Ribeirao Preto, Brazil

Background: Adolescence is a critical period of neurodevelopment during which exposure to stress is a major risk factor for psychiatric disorders. Using rodent models, we demonstrate that adolescent stress leads to long-lasting behavioral, molecular, and electrophysiological abnormalities associated with schizophrenia. These include increased anxiety-like behavior, impaired sociability and cognition, heightened responsiveness to psychostimulants, and elevated activity of dopamine neurons in the ventral tegmental area (VTA). These changes were associated with deficits in parvalbumin GABAergic interneurons (PVIs) and their associated perineuronal nets (PNNs) in the ventral hippocampus (vHipp). Multiple mechanisms have been proposed to contribute to PVI/PNN deficits, including oxidative stress and increased activity of matrix metalloproteinases (MMPs), particularly MMP-9.

Aims & Objectives: We investigated the impact of adolescent stress on oxidative stress markers, especially within PVIs, and on MMP-9 activity across development, and changes in MMP-9 activity induced by adolescent stress. Furthermore, we tested whether pharmacological interventions targeting oxidative stress and MMP-9, and viral-mediated knockdown of MMP-9 in the vHipp prevent stress-induced PVI/PNN deficits and behavioral abnormalities.

Method: MMP-9 activity in the vHipp of male Sprague Dawley rats was assessed by gel zymography at postnatal days (PND) 31, 41, 51, 75, 85, and 110. To examine the effects of stress on MMP-9 activity, animals were subjected to a stress protocol starting on PND 31, consisting of daily sessions for 10 days, combined with restraint sessions on PND 31, 32, and 40. Stress-induced changes in MMP-9 activity were assessed on PND 41, 51, and 75. To evaluate the effects of the pan-MMP inhibitor doxycycline on behavioral and molecular outcomes, treatment was initiated concurrently with the stress protocol (PND 31-40). Doxycycline (2 mg/mL) were administered via drinking water. We also investigated whether viral-mediated knockdown of MMP-9 in the vHipp could prevent the deleterious effects of adolescent stress. In adulthood, animals underwent behavioral testing, including the light-dark box, elevated plus maze, social interaction, and novel object recognition tests.

Results: MMP-9 activity increased from PND 31 to later time points. Adolescent stress increased MMP-9 activity at PND 45 and 51. Behaviorally, stressed animals showed reduced social interaction and a lower discrimination index in the novel object recognition test, indicating deficits in sociability and cognition. There was a significant reduction in the number of PV+ cells and PV+/PNN+ cells in the vHipp of adult rats subjected to adolescent stress. These behavioral and molecular alterations were prevented by treatment with doxycycline during the stress protocol, as well as by viral-mediated MMP-9 knockdown in the vHipp prior to stress exposure. Additionally, doxycycline attenuated the stress-induced increase in MMP-9 activity.

Discussion & Conclusions: Our results indicate dynamic changes in MMP-9 activity in the vHipp during development. Adolescent stress increases MMP-9 activity and reduces the number of PV+ and PV+/PNN+ cells in the vHipp, while also producing lasting behavioral impairments. Importantly, interventions with doxycycline or local MMP-9 knockdown conferred protection against these effects. Although further studies are needed to elucidate the precise mechanisms linking MMP-9 dysregulation to the effects of adolescent stress, our findings suggest that it represent a promising target for the prevention of stress-related neurodevelopmental abnormalities.