This study investigated the effects of the orexin receptor antagonist suvorexant on cocaine-induced behaviors in rats. The results showed that: 1) Suvorexant dose-dependently decreased premature responses in the 5-choice serial reaction time task, attenuating cocaine-induced impulsivity. 2) Suvorexant caused hypolocomotor effects, but cocaine induced comparable hyperlocomotion irrespective of suvorexant pretreatment. 3) Suvorexant increased the proportion of orexin neurons in the lateral hypothalamus that were immunoreactive for the activity marker cFos.

1. Effects of Orexin and Kappa Opiate Receptor (KOR) Pharmacology on Cocaine-Induced Behaviors
Stacia I. Lewandowski; Taylor A. Gentile; Steven J. Simmons; John W. Muschamp, Ph.D.
Center for Substance Abuse Research (CSAR), Temple University School of Medicine
INTRODUCTION
• Orexins (hypocretins) are neuropeptides within the lateral hypothalamus (LH) that project to
the ventral tegmental area, nucleus accumbens, dorsal raphe and locus coeruleus.
• Orexin transmission is involved in arousal, wakefulness, and motivated behavior through
signaling via two G-protein coupled receptor subtypes (OX1R and OX2R).
• Orexin is co-localized with dynorphin, a neuropeptide involved in depressive-like states
through signaling via kappa opiate receptors (KORs).
• Preliminary evidence from our laboratory suggests that suvorexant, a dual orexin receptor
antagonist used to treat insomnia, attenuates affective reactivity to cocaine as assessed
through 50-kHz vocalizations, as well as effortful intravenous self-administration of cocaine.
AIMS
• The present study was designed to assess effects of suvorexant on attention and cocaine-
induced impulsivity using the 5-choice serial reaction time task (5-CSRTT), as well as to
examine effects of suvorexant on orexin cell activity within LH.
RESULTS
• Suvorexant dose-dependently decreases the number of premature responses in the 5-CSRTT
and attenuates cocaine-induced impulsivity.
• Suvorexant causes hypolomotor effects but cocaine induces comparable hyperlocomotor
effects irrespective of drug pre-treatment.
• Suvorexant increases cFos-immunoreactivity of orexin cells in LH..
ABSTRACT
5-Choice Serial Reaction Time Task (5-CSRTT):
• Food-restricted animals in a sound-attenuating chamber were provided with a light stimulus
to one of 5 apertures for a 1-s duration. Correct responses into the lit aperture after light
termination were rewarded with one sugar pellet. Trials were separated with a 5-s inter-trial
interval, and subjects were given 90 trials per training session (or 30 minutes).
Drug Administration and Tissue Preparation:
• Rats received a 30-minute pre-treatment of either suvorexant (30 mg/kg, i.p.), norBNI (10
mg/kg, i.p.) or vehicle (DMSO) followed by acute treatment of either cocaine (10 mg/kg, i.p.)
or vehicle (saline).
• All animals were immediately perfused with saline and 4% paraformaldehyde, and brains
were collected for sectioning and immunolabeling.
Immunofluoresence (IF):
• 40 μm coronal sections of LH were washed in 0.1M phosphate-buffered saline (PBS), blocked
in 5% donkey serum in PBS with 0.3% Triton X-100 (PBS+), incubated in primary antibody
solution for 72-hr at 4o C (polyclonal rabbit anti-cFos [1:1,000; SC-52, SantaCruz] in 1.5%
donkey serum in PBS+), incubated again overnight at 4o C (polyclonal goat anti-orexinA (OxA)
[1:2,000; SC-8070, SantaCruz] and washed in 0.1M PBS, incubated in secondary antibody
solution for 3-hr at room temperature (donkey anti-goat 488 [AlexaFluor] and donkey anti-
rabbit 555 [AlexFluor] in 1.5% donkey serum in PBS+), washed in 0.1M PBS, mounted, dried
for 24-hr and coverslipped on SuperFrost slides.
Analyses:
• Repeated-measures ANOVAs examining the number of premature responses in the 5-CSRTT
were used to determine main effects, and Newman-Keuls post hoc tests used for pairwise
comparisons when main effects were found.
• For cellular analyses, a single section containing the majority of orexin-immunoreactive cells
from a 1-in-3 collection was used for OxA+/cFos+ proportion estimations.
FIGURE 1
METHODS
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RESULTS
5-CSRTT:
• A repeated-measures ANOVAs examining premature responses revealed a significant main
effect of suvorexant dose [F(3, 40) = 2.900, p < 0.05] and of drug treatment [F(2, 18) = 8.962,
p < 0.01].
• Post-hoc tests demonstrated significantly greater PRs in the cocaine vs. vehicle group (p <
0.01) as well as significantly fewer cocaine-induced PRs in animals pretreated with
suvorexant relative to vehicle-pretreated control animals (p < 0.05). The number of cocaine-
induced PRs in suvorexant-pretreated animals, however, was significantly greater relative to
saline-induced PRs (p < 0.05).
• No significant differences in omissions, accuracy or latency to reward were demonstrated.
Locomotor Activity:
• A one-way ANOVA revealed a significant main effect of drug group on locomotor activity [F(2,
42) =7.714, p < 0.01]. While both cocaine and cocaine-suvorexant groups had significantly
greater locomotor activity relative to saline animals, no difference was found between
cocaine and cocaine-suvorexant groups.
OxA+/cFos+ Cell Counts:
• Acute suvorexant (30.0 mg/kg, i.p.) increased the proportion of OxA+/cFos+ cells in bilateral
LH compared to vehicle-treated control animals [p < 0.05].
Figure 1. (A) Diagram of 5-CSRTT apparatus and mean 5-CSRTT scores (B: premature responses, C: accuracy, D: latency, E: omissions) ± S.E.M.
* p < 0.05 compared to vehicle-treated control group.
Figure 2. Mean 5-CSRTT premature responses ± S.E.M.
* p < 0.05 compared to vehicle-treated control group.
FIGURE 3
CONCLUSION
Impulsivity in 5-CSRTT:
• Our laboratory has demonstrated that inhibition of orexin signaling via suvorexant administration
decreases motor impulsivity in a dose-dependent manner.
• Cocaine increases motor impulsivity while suvorexant can attenuate cocaine-induced impulsivity.
• We hypothesize that the orexin neuropeptide may affect baseline impulsivity regarding cocaine-
induced behaviors.
Locomotor:
• Locomotor activity is decreased when the orexin system is inhibited. However, suvorexant
administered 30-min prior to cocaine does not reduce cocaine-induced hyperactivity.
Orexin Cell Activity:
• Data is currently under analysis but preliminary evidence suggests that there is no difference in
orexin cell activity from animals who received different drug treatments.
• Our laboratory has demonstrated for the first time the potential therapeutic efficacy of suvorexant
in reducing impulsive behaviors associated with cocaine addiction.
• Suvorexant has sedative-like effect, but cocaine nonetheless elicits comparable hyperlocomotor
effects irrespective of drug pre-treatment.
• Future studies should investigate the mechanism behind orexin’s influences on motor impulsivity,
explore the role of co-localized dynorphin in impulsivity and explore suvorexant’s effects on other
brain regions.
The authors would like to acknowledge grant support from NIDA (R00DA031767,
JWM; T32DA007237 TG,SJS) for completion of this study.
Figure 3. Mean locomotor activity counts ± S.E.M. N’s = 6-8.
Figure 4. Representative photomicrograph of OxA+/cFos+ cells [black arrows] and
OxA+/cFos- cells [white arrows]. Image from an animal who received norBNI (10mg/kg,
i.p.) pretreatment followed by cocaine (10mg/kg, i.p.). N’s 2-3.
(A) (B)
*
*
(A) (B) (C) (D) (E)
FIGURE 2
FIGURE 4