Behavioral recovery from an aversive stimulus corresponds with left habenular activity.

○Erik R. Duboué1, Akira Muto2, Koichi Kawakami2, Marnie Halpern1. 1) Carnegie Institution for Science, Baltimore, MD; 2) Molecular and Developmental Biology, National Institute of Genetics, Mishima, Shizuoka, Japan.

Throughout vertebrates the bilaterally paired habenular nuclei link the limbic forebrain with midbrain targets. Disruption of the dorsal habenular nuclei (analogous to the mammalian medial habenulae) has been correlated with increased fear/anxiety-related behaviors. In larval zebrafish, the dorsal habenulae exhibit prominent left-right differences in size, sub-nuclear and neuropil organization, and in connectivity with their shared target, the interpeduncular nucleus (IPN). We sought to determine how habenular asymmetry might influence behavioral responses by exposing 7-8 dpf larval zebrafish to an aversive stimulus, electrical shock (5V/cm2; 5 pulses; 1 pulse per sec). In freely moving wild-type (situs solitus) larvae, a mild electric shock results in a short period (~10 sec) of reduced activity followed by a return to baseline. In contrast, larvae with reversed habenular asymmetry (situs inversus) show a significantly longer period of immobility (~40 seconds) or enhanced freezing. To explore the respective contributions of each habenula in response to shock, we also assayed larvae with left or right isomerization of the habenular region. Larvae with left isomerization showed no significant difference from their WT siblings. However, larvae with right isomerization exhibited an extended period of freezing following shock similar to those with L-R reversed habenulae. To investigate whether neural activity in the habenular nuclei influences the response to shock, the heads of larvae expressing a genetically encoded calcium indicator were immobilized in agar. Electrical shock (5V/cm2; 1 pulse per sec; 1 pulse) was applied under a confocal microscope so that neural activity and locomotor responses could be simultaneously monitored in the head affixed larvae. We found that behavioral recovery from shock temporally correlates with an increase in neural activity in a subregion of the left habenula. The results demonstrate that fear/anxiety-related behavior can be assayed in larval zebrafish and reveal a preferential role for the left habenula in recovery from an aversive stimulus.