Galanin negatively modulates opiate withdrawal via galanin receptor 1

Abstract

Rationale: The neuropeptide galanin has been shown to modulate opiate dependence and withdrawal. These effects could be mediated via activation of one or more of the three distinct G protein-coupled receptors, namely galanin receptors 1 (GalR1), 2 (GalR2), and 3 (GalR3).

Objectives: In this study, we used several transgenic mouse lines to further define the mechanisms underlying the role played by galanin and its receptors in the modulation of morphine dependence. First, transgenic mice expressing β-galactosidase under the control of the galanin promoter were used to assess the regulation of galanin expression in response to chronic morphine administration and withdrawal. Next, the behavioral responses to chronic morphine administration and withdrawal were tested in mice that over-express galanin, lack the GalR1 gene, or lack the GalR2 gene.

Methods: Transgenic and matched wild-type mice were given increasing doses of morphine followed by precipitation of withdrawal by naloxone and behavioral responses to withdrawal were assessed.

Results: Both morphine administration and withdrawal increased galanin gene transcription in the locus coeruleus (LC). Increasing galanin levels in the brain reduced signs of opiate withdrawal. Mice lacking GalR1 undergo more severe opiate withdrawal, whereas mice lacking GalR2 show no significant difference in withdrawal signs, compare with matched wild-type controls.

Conclusions: Opiate administration and withdrawal increase galanin expression in the LC. Galanin opposes the actions of morphine which leads to opiate dependence and withdrawal, an effect that is mediated via GalR1.

Fig. 1. Regulation of galanin promoter activity by chronic morphine and morphine withdrawal

Transgenic mice expressing beta-galactosidase (LacZ) under the control of the galanin promoter were injected with increasing doses of morphine for 3 days, as described in the Methods, and withdrawal was precipitated using naloxone. Animals received saline (a) or morphine (b and c) and on the third day received a saline (b) or naloxone (a, c) injection 3 hrs after the last morphine injection to precipitate withdrawal. Quantitation of the LacZ positive neurons in the LC following saline-naloxone (SAL-NLX), morphine (MOR), and morphine-naloxone (MOR-NLX) treatment (d) there was a significant increase in the number of LacZ positive nuclei after morphine-naloxone treatment (n=4–5 per group, **p<0.01, one way ANOVA followed by Dunnett post hoc test. LacZ expression in the LC was monitored using immunofluorescence. Micrographs were taken at 20X.

Fig. 2. Galanin overexpression in the locus coeruleus leads to a milder withdrawal syndrome

Mice over-expressing galanin (GalOE) and their wild-type (WT) controls (derived from the homozygous offspring of heterozygous breeding pairs) received increasing doses of morphine for 3 days, as described in the Methods, and withdrawal was precipitated using naloxone. Withdrawal signs (Jumps, wet dog shakes, tremor, ptosis, paw tremor, mastication (Mast.), backward walking, diarrhea and weight loss) were monitored for 30 min after naloxone injection. Data are expressed as mean ± SEM (n = 8/group). *p < 0.05, **p < 0.01, two way ANOVA followed by Bonferroni correction for multiple comparisons.

Fig. 3. GalR1 modulates morphine withdrawal behaviors

GalR1 mutant mice (GalR1 mut) and their wild-type (WT) controls (derived from the homozygous offspring of heterozygous breeding pairs) were injected with increasing doses of morphine for 3 days, as described in the Methods, and withdrawal was precipitated using naloxone. Withdrawal signs were monitored for 30 min after naloxone injection. Knockout of the GalR1 gene significantly exacerbated several signs of morphine withdrawal, including jumps, tremor, ptosis, paw tremor and diarrhea. Data are expressed as mean ± SEM (n = 7 per group). *p < 0.05, **p<0.01, two way ANOVA followed by Bonferroni correction for multiple comparisons.

Fig. 4. Absence of GalR2 does not affect opiate withdrawal behavior

GalR2 mutant mice (GalR2 mut) and their wild-type controls (GalR2 WT) (derived from the homozygous offspring of heterozygous breeding pairs) were injected with increasing doses of morphine for 3 days, as described in the Methods, and withdrawal was precipitated using naloxone. Withdrawal signs were monitored for 30 min after naloxone injection. Lack of the GalR2 gene had no significant effect on morphine withdrawal signs. Data are expressed as mean ± SEM (n=8 per group).

Fig. 5. Absence of GalR1 or GalR2 does not affect the initial locomotor activating actions of morphine

GalR1 and GalR2 mutant mice (GalR1 mut, GalR2 mut) and their respective matched wild-type controls (WT) were injected with morphine and their locomotion was measured over thirty minutes. All mice showed comparable beam breaks regardless of genotype. Data are expressed as a percent of average saline beam breaks ± SEM.