Distinct Roles of Adenylyl Cyclases 1 and 8 in Opiate Dependence: Behavioral, Electrophysiological, and Molecular Studies

doi:10.1016/j.biopsych.2007.11.021

Biological Psychiatry

Volume 63, Issue 11, 1 June 2008, Pages 1013-1021

https://doi.org/10.1016/j.biopsych.2007.11.021 Get rights and content

Background

Opiate dependence is a result of adaptive changes in signal transduction networks in several brain regions. Noradrenergic neurons of the locus coeruleus (LC) have provided a useful model system in which to understand the molecular basis of these adaptive changes. One of most robust signaling adaptations to repeated morphine exposure in this brain region is upregulation of adenylyl cyclase (AC) activity. Earlier work revealed the selective induction of two calmodulin-dependent AC isoforms, AC1 and AC8, after chronic morphine, but their role in opiate dependence has remained unknown.

Methods

Whole cell recordings from LC slices, behavioral paradigms for dependence, and gene array technology have been used to dissect the role of AC1 and AC8 in chronic morphine responses.

Results

Both AC1 and AC8 knockout mice exhibit reduced opiate dependence on the basis of attenuated withdrawal; however, partially distinct withdrawal symptoms were affected in the two lines. Loss of AC1 or AC8 also attenuated the electrophysiological effects of morphine on LC neurons: knockout of either cyclase attenuated the chronic morphine-induced enhancement of baseline firing rates as well as of regulation of neuronal firing by forskolin (an activator of ACs). The DNA microarray analysis revealed that both AC1 and AC8 affect gene regulation in the LC by chronic morphine and, in addition to common genes, each cyclase influences the expression of a distinct subset of genes.

Conclusions

Together, these findings provide fundamentally new insight into the molecular and cellular basis of opiate dependence.

Section snippets

Animals

Mice derived from heterozygous matings of AC1 or AC8 lines, N7 generation onto C57Bl/6, were used in all behavioral tests. AC1−/− mice and AC8−/− mice backcrossed seven generations to C57Bl/6 were mated to produce individual mice heterozygous for both AC1 and AC8. These mice were then mated to generate individual mice mutant for both AC1 and AC8 as well as wildtype control animals.

For all behavioral, electrophysiological, and molecular studies, we used 2–4-month-old male mice. Animals were

Behavioral Responses to Morphine in AC1 and AC8 KO Mice

To determine the role of AC1 and AC8 in opiate dependence, we first monitored opiate withdrawal behavior in morphine-dependent AC1 KO mice and their respective wildtype littermate control subjects. Animals chronically treated with morphine received an injection of the opioid receptor antagonist naloxone (1 mg/kg SC), and withdrawal behavior was monitored for 25 min. The AC1 KO mice become less dependent on morphine, because several standard opiate withdrawal signs are dramatically decreased

Discussion

Results of the present study contribute to our understanding of the role of AC1 and AC8 in mediating the long-term effects of morphine on the LC. Loss of either enzyme decreases the ability of forskolin to excite LC neurons under control and morphine-treated conditions and the ability of chronic morphine to increase the baseline firing rate of LC neurons. Gene expression profiling supports the importance of both AC isoforms for normal genomic responses to chronic morphine, with roughly one-half

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Citation Excerpt :
As revealed in Fig. 3B, after treatment with 50 μM 8 and 50 μM FSK respectively, both remarkably increase the cAMP levels in a concentration-dependent manner with EC50 of 8.10 ± 0.04 μM for 8 and EC50 of 14.56 ± 1.21 μM for FSK. To investigate the selectivity of hit 8 on different AC isoforms, AC1,AC5 and AC8 were chosen as well based on the following observations: a) AC1 and AC2 are dominantly expressed in human lung over other subtypes AC3-10 [20]; b) AC5 is mainly expressed in the heart and its role in the heart is the most concerned [21], while AC8 is highly expressed in brain and is involved in the regulation of stress adaptation and mood, which plays a key role in neural plasticity [22–27]. Therefore, the selectivity testing of hit 8 on AC5 and AC8 along with AC1 and AC2 is also important to evaluate the possible side effects in heart disease or nervous system disease.
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Archival ReportDistinct Roles of Adenylyl Cyclases 1 and 8 in Opiate Dependence: Behavioral, Electrophysiological, and Molecular Studies

Background

Methods

Results

Conclusions

Section snippets

Animals

Behavioral Responses to Morphine in AC1 and AC8 KO Mice

Discussion

Trends Neurosci

Brain Res

Brain Res

Eur J Pharmacol

Eur J Pharmacol

Cell Signal

Eur J Pharmacol

J Biol Chem

J Biol Chem

J Biol Chem

Neuron

Neuron

Neuron

Brain Res

Neuropharmacology

Trends Pharmacol Sci

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

Nature

Clonidine infusions into the locus coeruleus attenuate behavioral and neurochemical changes associated with naloxone-precipitated withdrawal

Psychopharmacology

Noradrenaline in the ventral forebrain is critical for opiate withdrawal-induced aversion

Nature

Cellular and synaptic adaptations mediating opioid dependence

Physiol Rev

Acute and chronic opiate regulation of adenylate cyclase in brain: Specific effects in locus coeruleus

J Pharmacol Exp Ther

Opiate withdrawal and the rat locus coeruleus: Behavioral, electrophysiological, and biochemical correlates

J Neurosci

CREB (cAMP response element-binding protein) in the locus coeruleus: biochemical, physiological, and behavioral evidence for a role in opiate dependence

J Neurosci

Archival Report
Distinct Roles of Adenylyl Cyclases 1 and 8 in Opiate Dependence: Behavioral, Electrophysiological, and Molecular Studies