Mu opioid receptor knockdown in the substantia nigra/ventral tegmental area by synthetic small interfering RNA blocks the rewarding and locomotor effects of heroin

doi:10.1016/j.neuroscience.2008.09.039

Neuroscience

Volume 158, Issue 2, 23 January 2009, Pages 474-483

https://doi.org/10.1016/j.neuroscience.2008.09.039 Get rights and content

Abstract

Mu opioid receptors (MOP-r) play an important role in the rewarding and locomotor stimulatory effects of heroin. The aim of the current study was to determine whether infusion of small interfering RNAs (siRNA) targeting MOP-r into the midbrain could knock down MOP-r mRNA and affect heroin-induced locomotor activity or heroin-induced conditioned place preference. Ten-week-old male C57BL/6J mice were surgically implanted bilaterally with guide cannulae directed between the substantia nigra and ventral tegmental area. After 4 days' recovery, mice were infused bilaterally with siRNAs that target the MOP-r (2 mM×0.75 μl/side/day for 3 days) or control siRNA. Seven days after the last infusion, a procedure for conditioned place preference was begun with four heroin (3 mg/kg i.p.) administration sessions alternating with four saline sessions. While heroin induced an increase in locomotor activity in all groups, siRNAs targeting specific regions of MOP-r significantly attenuated this effect. Of particular interest, mice infused with specific siRNAs targeting the MOP-r failed to develop and express conditioned place preference to heroin, or showed a significantly attenuated preference. These alterations in reward-related behaviors are likely due to the reduction in MOP-r mRNA and protein, shown in separate studies by in situ hybridization and autoradiography using the same MOP-r- siRNA infusions. Taken together, these studies demonstrate the utility of siRNA in the neurobiological study of specific components of the reward system and should contribute to the study of other complex behaviors.

Section snippets

Subjects

Male C57BL/6J mice (10-week, Jackson Laboratory, Bar Harbor, ME, USA) weighing 22–25 g were individually housed with free access to food and water in a light- (12-h light/dark cycle, lights on at 7:00 am) and temperature- (25 °C) controlled room. Animal care and experimental procedures were conducted according to the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources on Life Sciences, National Research Council, 1996). The experimental protocols used were

Cannulae placement

Fig. 2A is a photomicrograph of a tissue section from the brain of a representative mouse used in the study, showing cannulae tracks left in the striatum.

Fig. 2B is a photomicrograph of a tissue section from the brain of a representative mouse used in the study, showing cannulae tracks left in the SN/VTA. Of a total of 78 mice started in this study, 68 were used in the final analyses; 10 were not included due to misplacement of cannulae.

The ventral points of the cannulae showing the infusion

Discussion

In the current study, we found that infusion of siRNAs targeting mouse MOP-r in the midbrain, where mesolimbic and nigrostriatal dopaminergic neurons are located, attenuated heroin-induced increases in locomotor activity and blocked or attenuated conditioned place preference in adult C57BL/6J mice. In separate mice, delivery of the same siRNAs into the same brain region resulted in a decrease in MOP-r mRNA levels and receptor density in the area surrounding the site of infusion. Furthermore,

Conclusion

In conclusion, infusion of siRNAs targeting mouse MOP-r directly into the midbrain attenuated heroin-induced increases in locomotor activity and blocked or attenuated the formation of conditioned place preference in adult C57BL/6J mice. These behavioral changes are likely to be due to knockdown of MOP-r mRNA resulting in decreased receptor density at the infusion site, shown in our studies, demonstrating the utility of siRNA in the neurobiological study of specific components of the reward

Acknowledgments

This work was supported by NIH-NIDA P60 DA05130 to Dr. Mary Jeanne Kreek. We thank Dr. David Nielsen for his participation in the design of siRNAs and his critical reading of the manuscript. Disclosure/conflict of interest: The author(s) declares that, except for income received from my primary employer, no financial support or compensation has been received from any individual or corporate entity over the past 3 years for research or professional service and there are no personal financial

References (43)

A.A. Baumeister et al.
The antinociceptive and motivational effects of intranigral injection of opioid agonists
Neuropharmacology
(1993)
S.E. Boehnke et al.
Time course and effective spread of lidocaine and tetrodotoxin delivered via microdialysis: an electrophysiological study in cerebral cortex
J Neurosci Methods
(2001)
A. Contarino et al.
Lack of reward and locomotor stimulation induced by heroin in mu-opioid receptor-deficient mice
Eur J Pharmacol
(2002)
B.L. Kieffer
Opioids: first lessons from knockout mice
Trends Pharmacol Sci
(1999)
B.L. Kieffer et al.
Exploring the opioid system by gene knockout
Prog Neurobiol
(2002)
A.M. Mathieu-Kia et al.
Repeated administration of cocaine, nicotine and ethanol: effects on preprodynorphin, preprotachykinin A and preproenkephalin mRNA expression in the dorsal and the ventral striatum of the rat
Brain Res Mol Brain Res
(1998)
R.T. Matthews et al.
Electrophysiological evidence for excitation of rat ventral tegmental area dopamine neurons by morphine
Neuroscience
(1984)
M. Minami et al.
Molecular cloning and in situ hybridization histochemistry for rat mu-opioid receptor
Neurosci Res
(1994)
R.C. Pierce et al.
The mesolimbic dopamine system: the final common pathway for the reinforcing effect of drugs of abuse?
Neurosci Biobehav Rev
(2006)
O. Snove et al.
Designing effective siRNAs with off-target control
Biochem Biophys Res Commun
(2004)

I. Sora et al.

Mu opiate receptor gene dose effects on different morphine actions: evidence for differential in vivo mu receptor reserve

Neuropsychopharmacology

(2001)

C.K. Surratt et al.

−Mu opiate receptorCharged transmembrane domain amino acids are critical for agonist recognition and intrinsic activity

J Biol Chem

(1994)

R.C. Thompson et al.

Cloning and pharmacological characterization of a rat mu opioid receptor

Neuron

(1993)

E.M. Unterwald et al.

Characterization of opioid receptors in rat nucleus accumbens following mesolimbic dopaminergic lesions

Brain Res

(1989)

G.K. Aghajanian

Tolerance of locus coeruleus neurones to morphine and suppression of withdrawal response by clonidine

Nature

(1978)

R. Bals-Kubik et al.

Neuroanatomical sites mediating the motivational effects of opioids as mapped by the conditioned place preference paradigm in rats

J Pharmacol Exp Ther

(1993)

A. Becker et al.

Morphine self-administration in mu-opioid receptor-deficient mice

Naunyn Schmiedebergs Arch Pharmacol

(2000)

A. Becker et al.

Rewarding effects of ethanol and cocaine in mu opioid receptor-deficient mice

Naunyn Schmiedebergs Arch Pharmacol

(2002)

V. David et al.

Differential effects of the dopamine D2/D3 receptor antagonist sulpiride on self-administration of morphine into the ventral tegmental area or the nucleus accumbens

Psychopharmacology

(2002)

V. David et al.

Brain regional fos expression elicited by the activation of mu- but not delta-opioid receptors of the ventral tegmental area: evidence for an implication of the ventral thalamus in opiate reward

Neuropsychopharmacology

(2008)

D.P. Devine et al.

Self-administration of morphine, DAMGO, and DPDPE into the ventral tegmental area of rats

J Neurosci

(1994)

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Behavioural NeuroscienceMu opioid receptor knockdown in the substantia nigra/ventral tegmental area by synthetic small interfering RNA blocks the rewarding and locomotor effects of heroin

Abstract

Section snippets

Subjects

Cannulae placement

Discussion

Conclusion

Acknowledgments

Neuropharmacology

J Neurosci Methods

Eur J Pharmacol

Trends Pharmacol Sci

Prog Neurobiol

Brain Res Mol Brain Res

Neuroscience

Neurosci Res

Neurosci Biobehav Rev

Biochem Biophys Res Commun

Neuropsychopharmacology

J Biol Chem

Neuron

Brain Res

Tolerance of locus coeruleus neurones to morphine and suppression of withdrawal response by clonidine

Nature

Neuroanatomical sites mediating the motivational effects of opioids as mapped by the conditioned place preference paradigm in rats

J Pharmacol Exp Ther

Morphine self-administration in mu-opioid receptor-deficient mice

Naunyn Schmiedebergs Arch Pharmacol

Rewarding effects of ethanol and cocaine in mu opioid receptor-deficient mice

Naunyn Schmiedebergs Arch Pharmacol

Differential effects of the dopamine D2/D3 receptor antagonist sulpiride on self-administration of morphine into the ventral tegmental area or the nucleus accumbens

Psychopharmacology

Brain regional fos expression elicited by the activation of mu- but not delta-opioid receptors of the ventral tegmental area: evidence for an implication of the ventral thalamus in opiate reward

Neuropsychopharmacology

Self-administration of morphine, DAMGO, and DPDPE into the ventral tegmental area of rats

J Neurosci

Behavioural Neuroscience
Mu opioid receptor knockdown in the substantia nigra/ventral tegmental area by synthetic small interfering RNA blocks the rewarding and locomotor effects of heroin