Trace fear conditioning detects hypoxic-ischemic brain injury in neonatal mice

Trace fear conditioning is a well-established test for the assessment of learning deficits in rodents. The aim of this study was to determine whether hypoxia-ischemia (HI) on postnatal day 9 (P9) in mice prevents the acquisition and expression of cued and contextual fear learning in early adulthood. Brain injury was induced in mice on P9 by 30 min of HI. On P49 and P50, animals were tested for: (1) trace fear conditioning with a short delay (2 s) between a shock-paired tone plus light and shock, (2) trace fear conditioning with a longer delay (20 s) between a shock-paired tone and shock, and (3) trace fear conditioning with a 2-second delay between a shock-paired tone and shock with additional visual, olfactory and tactile contextual cues in the fear conditioning apparatus. Outcome was assessed as percent of time spent freezing during a 2-min test. Histological assessment of the hippocampus and amygdala was performed on P51 to determine the extent of HI injury. Both shock-paired tone plus light with a short delay and shock-paired tone with a short delay plus additional contextual cues enhanced tone-induced freezing behavior in a nonhandled control group, but not in the HI group. For trace fear conditioning with a 20-second delay between the tone and the shock, freezing behavior did not differ significantly between nonhandled control and HI animals. Dorsal hippocampal and amygdala volumes were smaller in the ischemic hemispheres of the HI mice that displayed impaired fear memory with shock-paired tone plus light. In summary, we have shown that trace fear conditioning is a sensitive method for detecting memory impairments in adolescent mice following mild HI injury during the neonatal period. Combining a discrete conditioned stimulus (shock-paired tone plus light) with a short trace delay was the most sensitive method for using the fear conditioning paradigm to detect mild HI damage to the hippocampus and amygdala.