Optogenetic examination identifies a context-specific role for orexins/hypocretins in anxiety-related behavior

Abstract

Maladaptation to stress is associated with psychopathology. However, our understanding of the underlying neural circuitry involved in adaptations to stress is limited. Previous work from our lab indicated the paraventricular hypothalamic neuropeptides orexins/hypocretins regulate behavioral and neuroendocrine responses to stress. To further elucidate the role of orexins in adaptation to stress, we employed optogenetic techniques to specifically examine the effects of orexin cell activation on behavior in the social interaction test and in the home cage as well as orexin receptor 1 internalization and ERK phosphorylation in brain regions receiving orexin inputs. In the social interaction test, optogenetic stimulation of orexin neurons decreased time spent in the interaction zone while increasing the frequency of entries into the interaction zone. In addition, optogenetic stimulation of orexin neurons increased the total distance traveled in the social interaction arena but had no effect on their home cage behavior. Together, these results suggest that orexin release increases anxiety in the social interaction test while increasing the salience of novel but not familiar environmental stimuli. Consistent with activation of orexin neurons, optogenetic stimulation increased orexin receptor1 internalization and ERK phosphorylation in the paraventricular thalamus (PVT) and locus coeruleus (LC), two regions heavily innervated by orexin neurons. Together these results show for the first time that elevation of orexin activity, possibly in the PVT and LC, is associated with increased anxiety, activity, and arousal in a context-specific manner.

Keywords: Anxiety; Hypocretins; Optogenetics; Orexins; Social interaction; Stress.

Fig. 1

A) Effect of hypothalamic photostimulation on average latency to movement following stimulation in Hcrt::YFP control (YFP; n = 4), and Hcrt::ChR2-YFP (ChR2; n = 6) transduced animals prior to 4 days of swim stress (Pre-swim, left bars) and after 4 days of swim stress (Post-swim, right bars). * = p < 0.05 pre-swim Hcrt::YFP control vs. pre-swim Hcrt::ChR2-YFP; ** = p < 0.05 Pre-swim vs. Post-swim. B) Effect of hypothalamic photostimulation on c-cFos expression in orexin positive cells in YFP and ChR2 animals on the virus injected side (left bars) and the non-virus injected side (right bars). * = p < 0.05 Hcrt::YFP control vs. Hcrt::ChR2-YFP.

Fig. 2

A) Effect of hypothalamic photostimulation on percent time spent in the social interaction zone in Hcrt::YFP control (YFP; n = 10), and Hcrt:: ChR2-YFP (ChR2; n = 15) transduced animals. * = p < 0.05 Hcrt::YFP control vs. Hcrt::ChR2-YFP. B) Effect of hypothalamic photostimulation on the number of entries into the social interaction zone of animals described above. * = p < 0.05 Hcrt::YFP control vs. Hcrt::ChR2-YFP. C) Effect of hypothalamic photostimulation on the total distance traveled during the social interaction test of animals described above. * = p < 0.05 Hcrt::YFP control vs. Hcrt::ChR2-YFP. D) Diagram of social interaction apparatus detailing the placement of the different zones, stimulus rat, and subject rat.

Fig. 3

A) Effect of hypothalamic photostimulation on total distance traveled in Hcrt::YFP control (YFP; n = 9), and Hcrt::ChR2-YFP (ChR2; n = 13) transduced animals following hypothalamic photostimulation of 20 Hz for 10 s at a time every minute for 30 min in their home cage. B) Effect of hypothalamic photostimulation on the number of rearings in the home cage of animals described above. C) Effect of hypothalamic photostimulation on the number of rearings in the home cage of animals described above.

Fig. 4

A) Western blot analysis of orexin1R expression in the cytosolic (left bars) and membrane (right bars) fractions of posteriorPVT homogenates of Hcrt::YFP control (YFP; n = 9), and Hcrt::ChR2-YFP (ChR2; n = 13) transduced animals following hypothalamic photostimulation of 20 Hz for 10 s at a time every minute for 30min in their home cage. * = p < 0.05 Hcrt::YFP control vs. Hcrt::ChR2-YFP. B) Western blot analysis of cFos expression in pPVT homogenates of animals described above. * = p < 0.05 Hcrt::YFP control vs. Hcrt::ChR2-YFP. C) Western blot analysis of ERK1 phosphorylation expressed as % total ERK in pPVT homogenates of animals described above. * = p < 0.05 Hcrt::YFP control vs. Hcrt::ChR2-YFP. D) Western blot analysis of ERK2 phosphorylation expressed as % total ERK in pPVT homogenates of animals described above. * = p < 0.05 Hcrt::YFP control vs. Hcrt::ChR2-YFP. In each graph, representative blots are shown for each group.

Fig. 5

A) Western blot analysis of orexin1R expression in the cytosolic (left bars) and membrane (right bars) fractions of LC homogenates of Hcrt::YFP control (YFP; n = 9), and Hcrt:: ChR2-YFP (ChR2; n=13) transduced animals following hypothalamic photostimulation of 20Hz for 10s at a time every minute for 30min in their home cage. *=p<0.05 Hcrt::YFP control vs. Hcrt::ChR2-YFP. B) Western blot analysis of cFos expression in LC homogenates of animals described above. *=p<0.05 Hcrt::YFP control vs. Hcrt::ChR2-YFP. C) Western blot analysis of ERK1 phosphorylation expressed as % total ERK in LC homogenates of animals described above. * = p < 0.05 Hcrt::YFP control vs. Hcrt::ChR2-YFP. D) Western blot analysis of ERK2 phosphorylation expressed as % total ERK in LC homogenates of animals described above. In each graph, representative blots are shown for each group.

Fig. 6

A) Western blot analysis of orexin1R expression in the cytosolic (left bars) and membrane (right bars) fractions of infralimbic (left graph) and prelimbic (right graph) PFC homogenates of Hcrt::YFP control (YFP; n = 9), and Hcrt::ChR2-YFP (ChR2; n = 13) transduced animals following hypothalamic photostimulation of 20Hz for 10s at a time every minute for 30min in their home cage. B) Western blot analysis of c-Fos expression in PFC homogenates of animals described above. C) Western blot analysis of ERK1 phosphorylation expressed as % total ERK in PFC homogenates of animals described above. D) Western blot analysis of ERK2 phosphorylation expressed as % total ERK in PFC homogenates of animals described above. * = p < 0.05 Hcrt::YFP control vs. Hcrt::ChR2-YFP. In each graph, representative blots are shown for each group.