Possible Synaptic NMDA-dependent Mechanism behind the Effects of Adolescent Chronic Stress on Potentiated Adult Fear Responses

HERMAN, JAMES; COTELLA, EVELYN; PAGLINI GABRIELA; FERNÁNDEZ, GUILLERMO; SCARPONI, ANTONELLA

Congreso; Society for Neurosciece world congress 2017; 2018

Adolescence is a period of active development of the brain. During this phase, the mechanisms that regulate stress response are not fully developed and stress response is more intense that in adults. As a consequence, it is believed that adolescent brains are more vulnerable to the effects of chronic stress. This can have particular relevance for the study of psychiatry disorder related symptoms. We hypothesize that fear responses throughout life are enhanced when facing chronic stress during adolescence. The goal of this study was to determine the long term-effects of adolescent chronic variable stress (CVS) on a contextual fear conditioning paradigm and to find a possible molecular mechanism by studying the cellular distribution and phosphorylation status of NR2B NMDA receptor subunit and other plasticity related molecules in the hippocampus after a fear conditioning training session. We also studied differences in sensitization and habituation of the fear response by exposing conditioned animals to a non-conditioned context. Methods: Adolescent male Wistar rats (PND40) were subjected to chronic variable stress (CVS) for 2 weeks: 5 stressors presented randomly twice daily and twice a week 2 overnight stressors. Controls underwent only normal animal handling. Animals were tested 5 weeks after the CVS (PND 90). Contextual fear conditioning: training (3-min exploration- 3 shocks 1mA, 1s, 1min apart - 3 min), 4 days extinction (5 min) and recall after a reminder 48 h after extinction (1-min, 1 shock: 1 mA, 1s, 3-min). Behavioral measurement: freezing, scored as general absence of movement except the necessary for respiration. Fear generalization and habituation: Animals previously stressed during adolescence along their corresponding controls were subjected to a conditioning trial as in the previous experiment, but the following day they were exposed to a different context during 5 minutes. This procedure was repeated during 4 days. 48 hs after the last habituation sessions the animals were re-exposed to the original conditioning context. Molecular mechanism: A different group of animals was subjected to a conditioning trial in the same context as the previous experiments and then returned to their home cage. 1 hour after, animals were decapitated and hippocampal tissue was dissected to be homogenized and subsequently processed to obtain protein samples from the nuclear, cytoplasmic and membrane fractions. Western blot was performed against total and phosphorylated NR2B, PSD95, p35 and Cdk5. Results: The analysis showed that rats with a previous history of adolescent CVS expressed increased freezing during the conditioning session (p<0.05), and one day after, during the retrieval session (p<0.05). No effects were observed during the extinction procedure or recall session. In the groups of animal exposed to a different context after extinction, animals subjected to adolescent CVS showed more freezing in the new context (p<0.05) and they habituated less than the control animals to this situation (p<0.05). They also exhibited increased freezing when re-exposed to the conditioning context a week after (p<0.05). Interestingly, animals submitted to the stress protocol during adolescence showed a significant increase in the expression level of NR2B receptor in the hippocampal membrane fraction compared with controls (p<0.05) however, there were no differences in the phosphorylation rate of the protein. No effect was observed in the other proteins tested. Conclusion: Chronic variable stress during adolescent enhances conditioned and unconditioned fear responses later in life. One of the mechanisms behind these behavioral effects could be by affecting the synaptic function, increasing availability of proteins related to the learning process, suggested by the enrichment of NR2B, an NMDA receptor subunit, in the membrane fraction of the dorsal hippocampal samples observed in our experiment. Further study in the area is needed to better understand the exact mechanism that might be contributing to our observations.