Stimulation of Hypothalamic Orexin Neurons using DREADD Technology Increases Intraocular Pressure

Abstract

Purpose : Orexins, a novel class of neuropeptides, play a key role in regulating circadian behaviors and autonomic functions. Previously, we showed that chemical stimulation of neurons in the dorsomedial/perifornical hypothalamus (DMH/PeF), the location of orexin neurons, evoked increases in intraocular pressure (IOP), intracranial pressure (ICP), and the translaminar pressure gradient. Pre-treatment with an orexin receptor antagonist attenuates the IOP and ICP response. Given that orexin neurons have extensive efferent projections to autonomic relays, we hypothesize that fluctuations in IOP are regulated, at least in part, by orexin neurons. To further test this hypothesis, we used chemogenetic DREADD technology (Designer Receptors Exclusively Activated by Designer Drugs) to stimulate DMH/PeF orexin neurons and examined the resulting IOP and cardiovascular effects.

Methods : Orexin neurons of Sprague-Dawley rats (n=9) were selectively transfected by microinjecting a DREADD adeno-associated virus (AAV) under the orexin promoter into the DMH/PeF region. One month was allowed for excitatory M3GqR DREADD receptor expression. Isoflurane anesthtized rats then received either clozapine-n-oxide (CNO; selective DREADD M3GqR agonist that is otherwise inert; 3mg/kg i.p.) or vehicle control in a crossover design. An iCareLab tonometer and a tail pressure cuff attached to a PowerLab system was used to record IOP and heart rate (HR), respectively. Group differences in IOP and HR response were evaluated by paired t-test (significance set at P≤0.05). Immunohistochemistry was then performed to confirm selective transfection and activation of orexin neurons and downstream nuclei by c-fos analysis.

Results : Microinjection resulted in selective transduction and expression of M3GqR DREADD receptors onto ~80-90% of local orexin neurons. CNO caused a greater increase in IOP (4.1±2.4 vs 2.0±1.2 mmHg; p=0.02) and HR (96±29 vs 29±19 beats/min; p=0.001) compared to vehicle control. CNO injection caused a significant increase in c-fos⁺ orexin neurons in the DMH/PeF and other downstream targets of the orexin neurons compared to vehicle control.

Conclusions : These data further support the hypothesis that orexin neurons located in the DMH/PeF region play a role in mediating circadian fluctuations in IOP. Further, the orexin system may provide a novel target for future glaucoma therapies aimed at reducing circadian fluctuation of IOP.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.