Prophylactic (R,S)-ketamine and (2S,6S)-hydroxynorketamine Decrease Fear Expression by Differentially Modulating Fear Neural Ensembles

Background: We previously reported that a single injection of (R,S)-ketamine or its metabolite (2S,6S)-hydroxynorketamine (HNK) prior to stress attenuated learned fear. However, whether these drugs attenuate learned fear through divergent or convergent effects on neural activity remains to be determined.

Methods: 129S6/SvEv male mice were injected with saline, (R,S)-ketamine, or (2S,6S)-HNK 1 week before a 3-shock contextual fear conditioning paradigm. Five days later, mice were re-exposed to the aversive context and euthanized 1 hour later to quantify active cells. Brains were processed for c-fos immunoreactivity, and neural networks were built with a novel, wide-scale imaging pipeline.

Results: We found that (R,S)-ketamine and (2S,6S)-HNK attenuated learned fear. Fear-related neural activity was altered in dorsal CA3 following (2S,6S)-HNK; ventral CA3 and CA1, infralimbic and prelimbic regions, insular cortex, retrosplenial cortex, piriform cortex, nucleus reuniens, and periaqueductal gray following both (R,S)-ketamine and (2S,6S)-HNK; and in the paraventricular nucleus of the thalamus (PVT) following (R,S)-ketamine. Dorsal CA3 and ventral hippocampus activation correlated with freezing in the (R,S)-ketamine group, and retrosplenial cortex activation correlated with freezing in both (R,S)-ketamine and (2S,6S)-HNK groups. (R,S)-ketamine increased connectivity between cortical and subcortical regions while (2S,6S)-HNK increased connectivity within these regions.

Conclusions: This work identifies novel nodes in fear networks that involve the nucleus reuniens, piriform cortex, insular cortex, periaqueductal gray, and retrosplenial cortex that can be targeted with neuromodulatory strategies or pharmaceutical compounds to treat fear-induced disorders. This approach could be used to optimize target engagement and dosing strategies of existing medications.