From mouse to man: the 5-HT3 receptor modulates physical dependence on opioid narcotics

Abstract

Objectives: Addiction to opioid narcotics represents a major public health challenge. Animal models of one component of addiction, physical dependence, show this trait to be highly heritable. The analysis of opioid dependence using contemporary in-silico techniques offers an approach to discover novel treatments for dependence and addiction.

Methods: In these experiments, opioid withdrawal behavior in 18 inbred strains of mice was assessed. Mice were treated for 4 days with escalating doses of morphine before the administration of naloxone allowing the quantification of opioid dependence. After haplotypic analysis, experiments were designed to evaluate the top gene candidate as a modulator of physical dependence. Behavioral studies as well as measurements of gene expression on the mRNA and protein levels were completed. Finally, a human model of opioid dependence was used to quantify the effects of the 5-HT3 antagonist ondansetron on signs and symptoms of withdrawal.

Results: The Htr3a gene corresponding to the 5-HT3 receptor emerged as the leading candidate. Pharmacological studies using the selective 5-HT3 antagonist ondansetron supported the link in mice. Morphine strongly regulated the expression of the Htr3a gene in various central nervous system regions including the amygdala, dorsal raphe, and periaqueductal gray nuclei, which have been linked to opioid dependence in previous studies. Using an acute morphine administration model, the role of 5-HT3 in controlling the objective signs of withdrawal in humans was confirmed.

Conclusion: These studies show the power of in-silico genetic mapping, and reveal a novel target for treating an important component of opioid addiction.

Fig. 1

Computational genetic analysis of interstrain differences in physical dependence on morphine. (a) Eighteen strains (eight mice per strain) were treated for 4 days with morphine to establish physical dependence. On the fifth day, the number of jumps made during the 15-min period after naloxone injection was measured as an indicator of the degree of opioid dependence. The data represent the mean number of jumps for each indicated strain ± SEM. (b) The morphine physical dependence data (mean number of jumps for each strain) was analyzed by computational genetic mapping. The 10 most strongly correlated haplotype blocks are shown. For each block, the chromosomal location (chr.), number of single nucleotide polymorphisms (SNPs) within a block and its gene symbol are listed. For each gene, the haplotypes are represented by a colored block, and the blocks are presented in the same rank order as the phenotypic data. Strains sharing the same haplotype have the same colored block. The calculated P value measures the probability that the strain groupings within a block would have the same degree of association with the phenotypic data by random chance. The genetic effect indicates the fraction of the interstrain variance that is potentially attributable to the haplotype. The letter ‘E’ is appropriate as an indicator of ‘exponent’.

Fig. 2

Diagram of the Htr3a gene and associated haplotypic blocks. (a) The intron/exon structure of the Htr3a gene along with the relative positions of the two associated haplotypic blocks are displayed. The two haplotype blocks whose pattern of genetic correlation best correlated with the severity of the naloxone-precipitated jumping response are located at the 5′ and 3′ regions of the Htr3a gene. Each of these haplotype blocks has three different haplotypes, and none of the 86 single nucleotide polymorphisms (SNPs) within these two blocks alters the predicted amino acid sequence of the 5-HT3a receptor (http://mouseSNP.roche.com). (b) There are seven haplotype blocks across the Htr3a gene. The two major blocks are the dependence correlated 3′ and 5′ blocks, each with 76 and 10 SNPs, respectively, covering most of Htr3a gene. The other five small blocks (not shown in the figure) are between the 5′ and 3′ blocks (between exons 2 and 4) and contain total 15 SNPs. The 5′ and 3′ blocks, which exhibited the highest correlation with physical dependence on morphine, had the same pattern of genetic variation across the 18 strains of mice used in our studies.

Fig. 3

Administration of a selective 5-HT3 receptor antagonist (ondansetron) decreases naloxone-precipitated withdrawal behavior. (a) Mice (C57BL/6J) were treated with morphine over a 4-day period, and then were treated with saline or the indicated dose of ondansetron on the fifth day before assessment of naloxone-precipitated jumping behavior. Ondansetron treatment induced a statistically significant and dose-dependent reduction in jumping behavior. (b) Mice (C57BL/6J) were treated with saline or ondansetron (1 mg/kg) at the time of each of the bi-daily morphine injections during the 4-day dependence building protocol. Eighteen hours after the final dose, naloxone-precipitated jumping behavior was assessed. (c) Mice (C57BL/6J) were treated with saline or the indicated dose of ondansetron intracerebroventricular (i.c.v.) before assessment of naloxone-precipitated jumping behavior. Ondansetron treatment induced a statistically significant and dose-dependent reduction in jumping behavior. For all experiments six to eight mice were used per group. Data represent mean values ± SEM. *P<0.05, **P<0.01, ***P<0.001.

Fig. 4

Ondansetron treatment reduces morphine dependence-related hyperalgesia. The effect of the selective 5-HT3 antagonist ondansetron was evaluated in mice that were made dependent on morphine over a 4-day period. As a measure of physical dependence on morphine, mechanical nociceptive sensitization was measured 18 h after administration of the last dose of morphine, the point of maximal nociceptive sensitization. Mice were treated with saline (control) or the indicated dose of ondansetron 30 min before threshold assessment. Ondansetron dose dependently reversed the mechanical nociceptive sensitization caused by morphine withdrawal, but did not alter baseline nociceptive thresholds in control animals, even when doses of ondansetron higher than those effectively reversing hyperalgesia were administered. Six mice per group were used in these experiments, and the data represent mean values ± SEM. **P<0.01.

Fig. 5

Ondansetron blocks the conditioned place preference associated with morphine administration. In this figure the mean percent time spent in the assigned drug associated chamber in the conditioned place preference assessments before and after conditioning is displayed. Three groups of mice were used: vehicle (n = 15); morphine 5 mg/kg (n = 14); ondansetron 1 mg/kg and morphine 5 mg/kg (n = 10). ***P<0.001.

Fig. 6

Morphine treatment has a differential and brain region-specific effect on Htr3a mRNA expression. (a) Mice with a high (C57BL/6J) or low propensity (129/SvlmJ) to develop morphine dependence were exposed to saline or morphine for 4 days. On the fifth day the mice were killed, the indicated brain regions were dissected, and level of Htr3a mRNA expression was measured using real-time quantitative PCR. Data represent mean values±SEM from duplicate measurements made on at least six mice per group. (b) Morphine induces changes in Htr3a mRNA expression in selected brainstem nuclei. Mice (C57BL/6J) were treated with saline or morphine as above. The mice were then killed, the indicated brainstem nuclei were isolated by laser capture microdissection, and Htr3a mRNA expression in the brainstem nuclei was analyzed. The Htr3a mRNA levels in morphine-treated mice were normalized relative to those in saline-treated animals. The data are displayed as the mean normalized value for tissues from n = 6 mice per group ± SEM. CNS, central nervous system; PAG, periaqueductal gray. **P<0.01.

Fig. 7

The regulation of central nervous system expression for several genes by morphine. Brain tissue was harvested after 4 days of saline versus morphine treatment for both high (C57BL/6J) and low (129/SvlmJ) dependence developing strains. In this survey of genes only expression of the Htr3a gene coding for the 5-HT3 serotonin receptor was opioid regulated (P < 0.05). Importantly, expression of the adjacent Htr3b gene coding for an alternate form of the 5-HT3 receptor was not altered by morphine treatment. Five mice per group were used in these experiments, and the displayed data represent mean values ± SEM. *P<0.05 (difference between strains).

Fig. 8

The regulation of brainstem 5-HT3 protein levels by morphine. Brain tissue was harvested after 4 days of saline versus morphine treatment for both high (C57BL/6J) and low (129/SvlmJ) dependence developing strains. Only in tissue from the C57BL/6J strain showed morphine-induced 5-HT3 changes. Five mice per group were used in these experiments, and the displayed data represent mean values±SEM. Average control mouse expression was set to 1. ***P<0.001 (difference between control and morphine-treated groups).

Fig. 9

The effect of ondansetron pretreatment on the acute, naloxone-precipitated withdrawal response in human volunteers. Ondansetron 8 mg intravenous (i.v.) or placebo (normal saline) i.v. was administered 30 min before morphine (10 mg/70 kg) i.v. administration in eight volunteers. Naloxone-precipitated (10 mg/70 kg) withdrawal was then induced 120 min after morphine administration. (a) The composite OOWS scores for each volunteer after naloxone-precipitated opioid withdrawal after saline or ondansetron pretreatment are displayed. A P value=0.0313 was calculated based on signed-rank test of the difference in OOWS score after pretreatment with ondansetron and placebo. (b) The Objective Opioid Withdrawal scale (OOWS) subcategory responses to ondansetron pretreatment are displayed. The OOWS scale is composed of thirteen physically observable signs, which are rated as present (1) or absent (0) during the observation period. The percent of the volunteers who experienced each indicated naloxone-precipitated withdrawal sign after ondansetron or placebo pretreatment is shown.

Fig. 10

The effect of ondansetron pretreatment on the acute, naloxone-precipitated Subjective Opiate Withdrawal scale (SOWS) response in human volunteers. Ondansetron 8 mg intravenous (i.v.) or placebo (normal saline) i.v. was administered 30 min before morphine (10 mg/70 kg) i.v. administration in eight volunteers. Naloxone-precipitated (10 mg/70 kg) opioid withdrawal was then induced 120 min after morphine administration. (a) The composite SOWS scores for each volunteer after naloxone-precipitated opioid withdrawal for volunteers receiving saline or ondansetron pretreatment are displayed. A P value=0.5625 was calculated based on signed-rank test of the difference in SOWS score after pretreatment with ondansetron and placebo. (b) The SOWS subcategory responses to ondansetron pretreatment in humans are displayed. The SOWS score is composed of 16 subjective symptoms rated on a scale of 0−4 (0=not at all, 1=a little, 2=moderately, 3=quite a bit, 4=extremely) based on what volunteers were experiencing at the time of testing. The mean score for volunteers who experienced each indicated naloxone-precipitated withdrawal symptom after ondansetron or placebo pretreatment is shown.