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. Author manuscript; available in PMC: 2012 Nov 1.
Published in final edited form as: Pharmacol Biochem Behav. 2011 Jul 23;100(1):8–16. doi: 10.1016/j.pbb.2011.07.013

Regional mRNA expression of the endogenous opioid and dopaminergic systems in brains of C57BL/6J and 129P3/J mice: Strain and heroin effects

SD Schlussman 1, J Cassin 1, Y Zhang 1, O Levran 1, A Ho 1, MJ Kreek 1
PMCID: PMC3201798  NIHMSID: NIHMS319198  PMID: 21807019

Abstract

We have previously shown strain and dose differences in heroin-induced behavior, reward and regional expression of somatostatin receptor mRNAs in C57BL/6J and 129P3/J mice. Using Real Time PCR we examined the effects of five doses of heroin on the levels of the transcripts of endogenous opioid peptides and their receptors and dopaminergic receptors in the mesocorticolimbic and nigrostriatal pathways in these same mice. Compared to C57BL/6J animals, 129P3/J mice had higher mRNA levels of Oprk1 in the nucleus accumbens and of Oprd1 in the nucleus accumbens and a region containing both the substantia nigra and ventral tegmental area (SN/VTA). In the cortex of 129P3/J mice, lower levels of both Oprk1 and Oprd1 mRNAs were observed. Pdyn mRNA was also lower in the caudate putamen of 129P3/J mice. Strain differences were not found in the levels of Oprm1, Penk or Pomc mRNAs in any region examined. Within strains, complex patterns of heroin dose-dependent changes in the levels of Oprm1, Oprk1 and Oprd1 mRNAs were observed in the SN/VTA. Additionally, Oprd1 mRNA was dose-dependently elevated in the hypothalamus. Also in the hypothalamus, we found higher levels of Drd1a mRNA in C57BL/6J mice than in 129P3/J animals and higher levels of DAT (Slc6a3) mRNA in the caudate putamen of C57BL/6J animals than in 129P3/J counterparts. Heroin had dose-related effects on Drd1a mRNA in the hypothalamus and on Drd2 mRNA in the caudate putamen.

Keywords: Endogenous opioid system, Dopaminergic system, Heroin, mRNA

1: Introduction

The endogenous opioid system consists of the mu, kappa and delta opioid receptors and their ligands (β-endorphin, dynorphins and enkephalins respectively). The endogenous opioid system is widely dispersed throughout the CNS and the periphery (e.g. (Boublik et al. 1983; DePaoli et al. 1994; Dumont & Lemaire 1984; Jamensky & Gianoulakis 1997; Konturek 1980; Mansour et al. 1994; Mansour et al. 1988; Minami et al. 1993; Pert et al. 1975; Sanchez-Blazquez & Garzon 1985; Sharif & Hughes 1989)). In the CNS, opioid receptors and their ligands are found throughout the mesocorticolimbic and nigrostriatal dopaminergic systems, although the relative levels of each receptor and ligand show significant regional specificity (for review see e.g. (Mansour et al. 1988).

The mesocorticolimbic and nigrostriatal dopaminergic projections originate from cell bodies in the ventral tegmental area and the substantia nigra respectively, and project throughout the limbic system and to the striatum. A third dopaminergic system, the tuberoinfundibular system, originates from cell bodies in the hypothalamus and projects to the pituitary and the median eminence. Within these dopaminergic systems there are five distinct dopamine receptors (D1–5).

Dopamine receptors are known to colocalize with specific endogenous opioid peptides. The medium spiny neurons of the striatonigral projection contain the endogenous opioid peptide dynorphin and the dopamine D1 receptor, whereas those of the striatopallidal projection contain enkephalin and the dopamine D2 receptor (e.g. (Afifi 1994; Gerfen et al. 1990; Le Moine & Bloch 1995; McGinty 2007)).

Morphine, the biologically active metabolite of heroin (e.g.(Inturrisi et al. 1983; Selley et al. 2001)), binds to the mu opioid receptor on GABAergic interneurons in the substantia nigra and ventral tegmental area, which release GABAergic inhibition of dopamine release (e.g. (Johnson & North 1992)). This disinhibtion results in elevated levels of dopamine in the projection fields (e.g. (Di Chiara & Imperato 1988a, b; Spanagel et al. 1990)) where it activates pre-and post-synaptic dopaminergic receptors.

In vivo, endogenous opioid peptides and their receptors modulate extracellular dopamine levels in nucleus accumbens and caudate putamen. Activation of the mu opioid receptor increases extracellular dopamine levels (Di Chiara & Imperato 1988a; Spanagel et al. 1990, 1992). Conversely, acute stimulation of Oprk1 lowers dopamine levels (Di Chiara & Imperato 1988a; Spanagel et al. 1990, 1992; Zhang et al. 2003). Furthermore, administration of the selective Oprk1 antagonist nor-BNI into the nucleus accumbens increased extracellular dopamine levels in the same region, demonstrating an ongoing inhibitory tone at the kappa opioid receptor (Spanagel et al. 1992). Consistent with this, we found that locally administered dynorphin A(1–17) decreased dopamine dialysate levels in the caudate putamen in mice, and blocked cocaine-induced conditioned place preference (Zhang et al. 2004a). This further implicates the kappa opioid receptor system in the dorsal striatum (i.e., caudate putamen) as an important mediator of the acquisition of reward-related behaviors of cocaine (Everitt & Robbins 2005; Fagergren et al. 2003; Porrino et al. 2004). Thus, the mu and kappa opioid peptides and ligands countermodulate dopaminergic tone.

Opiates affect mRNA levels within the striatum, including those of the endogenous opioid system (e.g. (Buzas et al. 1996; Garcia de Yebenes & Pelletier 1993; Georges et al. 1999; Romualdi et al. 1990, 1991; Trujillo et al. 1995; Yukhananov et al. 1993)) and the dopaminergic system (Spangler et al. 2003). In the nucleus accumbens and caudate putamen of rats, Penk mRNA levels were decreased by chronic administration of morphine (Basheer & Tempel 1993; Georges et al. 1999; Uhl et al. 1988). We have shown that acute intermittent morphine administration to rats produced elevations in Pdyn and Oprm1 mRNA in the whole brain, with no effect on Penk mRNA levels (Wang et al. 1999). Acute morphine withdrawal increased Penk mRNA expression in the hypothalamus and striatum (Fukunaga et al. 1996; Gudehithlu & Bhargava 1995; Harbuz et al. 1991; Lightman & Young 1987).

There is a large body of data demonstrating considerable individual differences in the vulnerability to develop addictive disease. One way to study these individual, and perhaps genetic, roots of vulnerability is to examine inbred strains of animals which are known to differ in their response to drugs of abuse (e.g. (Belknap et al. 1989; Kosten et al. 1994)). Numerous neurochemical and drug-induced behavioral differences have been described in C57BL/6J and DBA2 strains of mice (e.g.(Castellano et al. 1976; Crabbe et al. 1980; De Waele et al. 1992; Jamensky & Gianoulakis 1997; Phillips et al. 1994)). The C57BL6 and 129 strains of mice are frequently used in the generation of knockout animals (e.g (Crawley et al. 1997); however these strains of mice have been studied much less extensively (for review see (Crawley et al. 1997)). Mice of various 129 sub-strains have consistently been shown to be less responsive to the locomotor stimulating effects of cocaine (e.g. (Kuzmin & Johansson 2000; Kuzmin et al. 2000; Miner 1997; Schlussman et al. 1998; Schlussman et al. 2003a) or rewarding effects of heroin (e.g. (Schlussman et al. 2008; Szumlinski et al. 2005)) or cocaine (Miner 1997). Szumlinski et al showed that C57BL/6J mice developed conditioned place preference to heroin at a dose of 100 ug/kg, while 129/sVJ mice developed conditioned place aversion to the same dose (Szumlinski et al. 2005). We have shown significant differences in the development of heroin-induced conditioned place preference, in the same mice described in the study presented here, with C57BL/6J mice developing preference to relatively low doses of heroin and 129P3/J mice developing preference only to higher doses of heroin ((Schlussman et al. 2008). We suggested that this might represent a decreased sensitivity to the rewarding effects of heroin in 129P3/J mice relative to C57BL/6J animals.

Here we extend our behavioral studies by examining the relative expression of mRNAs of the endogenous opioid and dopaminergic systems in the same mice we found to differ in their behavioral response to heroin.

2: Materials and Methods

A total of 125 age-matched male mice (6 weeks old on arrival; Jackson Laboratory, Bar Harbor, ME), 55 C57BL/6J and 70 129P3/J, were studied. All animals were individually housed in an environmentally controlled room dedicated to this study. Food and water were available ad lib and animals were allowed two weeks to acclimate prior to the start of the experiments. Mice of each strain were randomly assigned to one of six groups, each administered a specific dose (0, 1.25, 2.5, 5, 10 or 20 mg/kg) of heroin (diacetyl-morphine HCl, obtained from NIH – NIDA). This study was approved by the Rockefeller University Institutional Animal Care and Use Committee and included provisions to minimize pain and discomfort.

Mice whose tissue was studied here were from a study of heroin-induced conditioned place preference which has been reported elsewhere (Schlussman et al. 2008). Animals in the 0 mg/kg group received i.p. injections of isotonic saline on all days of the study. Animals in the other groups received i.p. injections of heroin or saline on alternate days for a total of 8 days (for details see (Schlussman et al. 2008). Animals were sacrificed immediately following the testing session (24.5 hours following the last conditioning session) by decapitation following brief CO2 exposure (< 20 seconds), their brains rapidly removed, and slices were cut with a rodent brain matrix (ASI Intruments, Warren, MI). The hypothalamus, cortex (an area containing the cingulate cortex area 1, the primary and secondary motor cortices and the prelimbic cortex (Franklin & Paxinos 1997)), nucleus accumbens, the caudate putamen and a region containing both the substantia nigra and the ventral tegmental area (SN/VTA) were dissected and homogenized in guanidium thiocyanate as previously described (Branch et al. 1992). RNA was isolated from homogenates of the hypothalamus, cortex and caudate putamen with the RNAqueous system (Ambion [ABI], Austin TX) according to manufacturer’s instructions. RNA from the nucleus accumbens and SN/VTA was isolated using an acid phenolic extraction (Chomczynski & Sacchi 1987). Following RNA isolation, all samples were treated with DNase (Turbo DNA-free™, Ambion [ABI], Austin, TX). The quantity and quality of RNA in each extract was determined with the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA).

cDNA was synthesized from each sample using the Super Script™ III first strand synthesis kit (Invitrogen, Carlsbad, CA). Five hundred ng of RNA from the hypothalamus, cortex and caudate putamen was used for reverse transcription. The entire nucleus accumbens and SN/VTA were utilized for generation of cDNAs. cDNAs were diluted 1:10 for real time PCR analysis.

Real time PCR analysis of the relative mRNA expression levels of Oprm1, Oprk1, Oprd1, Penk, proopiomelanocortin (Pomc; hypothalamus, caudate putamen only), Pdyn, tyrosine hydroxylase (Th; hypothalamus and SN/VTA only), DAT (Slc6a3; caudate putamen, nucleus accumbens and cortex) dopamine D1 receptor (Drd1a; hypothalamus, nucleus accumbens and caudate putamen), dopamine D2 receptor (Drd2; hypothalamus, nucleus accumbens and caudate putamen) and dopamine D3 receptor (Drd3; nucleus accumbens and caudate putamen) was conducted using commercially available primers and master mix (RT2qPCR™ primer assays and RT2 Real Time™ SYBR® Green PCR Master Mix; Qiagen, Valencia, CA) according to manufacturer’s directions in an ABI Prism 7900 HT Sequence Detection System (Applied Biosystems, Forster City, CA). Water controls for each primer set were included in every assay. Any sample with a cycle threshold (Ct) greater than that of the water control or a Ct greater than or equal to 35 was not included in the analysis. All data were normalized to Gapdh and reported as 2-ΔCt where ΔCt is the cycle threshold of the mRNA of interest minus the cycle threshold of Gapdh.

Data for each mRNA of interest was analyzed by two-way ANOVA, Strain X Dose, followed by Newman-Keuls post hoc analysis where appropriate. Due to missing data points in certain C57BL/6J mice, resulting in an incomplete ANOVA design, significant main effects of Strain, in the cortex and nucleus accumbens were also analyzed in the saline treated animals only, using two-tailed t or Mann-Whitney U tests as appropriate. Any sample that was ≥ 2.5 standard deviations from the strain mean was considered an outlier and dropped from the analysis.

3: Results

We only present data which reached or neared statistical significance. All our findings are shown in supplemental Figures 1–11.

3.1 Endogenous Opioid Strain Effects

We observed significant region-specific strain differences in the mRNA levels of several components of the endogenous opioid system.

3.1.1 SN/VTA

In the region containing the substantia nigra and ventral tegmental area, the relative levels of Oprd1 mRNA were significantly higher in 129P3/J mice compared to C57BL/6J counterparts (F(1,87) = 7.32, p < 0.01; Fig. 1A).

Figure 1.

Figure 1

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Endogenous Opioid System; Strain Effects. Regionally-specific strain differences in the relative expression levels of mRNA for components of the endogenous opioid system were found. Solid bars represent statistically significant differences; stippled bars represent strain differences which did not reach the 0.05 level of statistical significance.

3.1.2 Caudate putamen

In the caudate putamen, 129P3/J mice had lower levels of Pdyn mRNA than did C57BL/6J mice (F(1,109) = 7.67, p < 0.01; Fig. 1B).

3.1.3 Nucleus accumbens

Several strain differences in the relative mRNA expression levels were observed in the nucleus accumbens.

Levels of Oprm1 mRNA were greater in the nucleus accumbens of 129P3/J mice, relative to C57BL/6J mice, although this just missed statistical significance (F(1,58) = 3.96, p = 0.051). However, when Oprm1 mRNA levels in saline controls were compared, this effect was significant (U = 1.00, P < 0.005; Figure 1C).

There were apparently higher levels of Oprk1 mRNA in the nucleus accumbens of 129P3/J mice compared to C57BL/6J animals, however, this did not reach statistical significance (F(1,57) = 2.87 p = 0.096; Fig. 1D). Such a trend was also observed in saline controls (t = 1.74 p = 0.096).

In the nucleus accumbens, Oprd1 mRNA levels were significantly higher in 129P3/J mice than in C57BL/6J animals (F(1,38) = 4.61, p < 0.05; Fig. 1E). This significant difference was also observed between saline controls (U = 6.00, p < 0.05).

A significant main effect of Strain on Penk mRNA levels was not observed, however there was a trend for higher levels of expression in 129P3/J mice when saline controls were examined (t = 1.91 p = 0.072; Fig. 1F).

3.1.4 Cortex and Hypothalamus

Strain differences in the relative levels of mRNA for any component of the endogenous system were not found in either region.

3.2 Dopaminergic System Strain Effects

We observed regional strain differences in the relative expression levels of mRNAs of the dopaminergic system.

3.2.1 SN/VTA

In the SN/VTA there was a trend toward higher levels of Th mRNA expression in C57BL/6J mice relative to 129P3/J animals (F(1,99) = 3.11, p = 0.08; Fig. 2A).

Figure 2.

Figure 2

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Dopaminergic System; Strain Effects. Regionally-specific strain differences in the relative expression levels of mRNA for components of the dopaminergic systems were found. Solid bars represent statistically significant differences; stippled bars represent strain differences that did not reach the 0.05 level of statistical significance.

3.2.2 Caudate putamen

In the caudate putamen there were lower levels of Slc6a3 mRNA in 129P3/J mice but this difference just missed statistical significance (F(1,106) = 3.90, p = 0.051; Fig. 2B).

3.2.3 Nucleus accumbens

In the nucleus accumbens of 129P3/J mice, we found a trend toward lower levels of Slc6a3 mRNA compared to C57BL/6J mice, (F(1,65) = 3.07, p = 0.084) In the saline controls, this difference was statistically significant (U = 15.00, p < 0.05; Fig. 2C).

3.2.4 Cortex

Strain differences in the relative levels of mRNA of the dopaminergic system were not found.

3.2.5 Hypothalamus

In the hypothalamus, 129P3/J mice had lower levels of Drd1a mRNA compared to C57BL/6J mice (F(1,95) = 5.49, p < 0.05; Fig. 2D). In the hypothalamus, Drd2 mRNA levels appeared higher in 129P3/J mice than in C57BL/6J mice, although this difference did not reach statistical significance (F(1,79) = 3.35, p = 0.071; Fig 2E).

3.3 Endogenous Opioid System; Heroin Dose Effects

Within strains, heroin had a complex pattern of effects on expression of several mRNAs. Interestingly, most of these were observed in the SN/VTA.

3.3.1 SN/VTA

A significant heroin Dose effect was observed, in this region, on the relative expression levels of mRNAs for all three endogenous opioid receptor genes (Oprm1, F(5,101) = 5.31, p < 0.0005; Oprk1 F(5,97) = 3.59, p < 0.01; Oprd1, F(5,87) = 2.95, p < 0.05). Oprm1 mRNA levels were significantly reduced by heroin at doses of 5 and 10 mg/kg (p < 0.05; Fig. 3A). Oprm1 mRNA levels were also lower following 20 mg/kg of heroin, although this did not reach significance (p = 0.08). Lower doses of heroin (1.25 and 2.5 mg/kg) had no effect on levels of Oprm1 mRNA. Oprk1 mRNA levels were significantly increased by 2.5 mg/kg of heroin (p < 0.05; Fig. 3B). Oprk1 mRNA levels appeared to increase following 1.25 mg/kg, but this did not reach statistical significance (p = 0.07). The higher doses of heroin had no effect on Oprk1 mRNA. Oprd1 mRNA levels in the SN/VTA were significantly elevated by the lowest dose of heroin (p < 0.05; Fig. 3D). We also observed a Strain by Dose interaction in the level of Oprd1 mRNA in the SN/VTA (F(5,87) = 3.44, p < 0.01). Newman-Keuls post hoc tests showed that the effect of the 1.25 mg/kg dose on expression levels of Oprd1 mRNA was due to an elevation of Oprd1 mRNA levels only in 129P3/J mice (data not shown). A significant main effect of heroin Dose on Penk mRNA expression was found in the SN/VTA (F(5,104) = 3.25, p <0.01; Fig. 3D). Newman-Kuels post hoc tests indicated an apparent lower level of Penk expression following the 10 mg/kg dose of heroin (p = 0.08).

Figure 3.

Figure 3

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Endogenous Opioid System. Dose Effects within strains, a statistically significant main effect of Heroin Dose on relative mRNA levels of components of the endogenous opioid system was found in several brain regions. * represents Newman-Kuels post-hoc test p < 0.05.

3.3.2 Hypothalamus

In the hypothalamus, we observed a significant main effect of heroin Dose on relative levels of Oprd1 mRNA (F(5,78) = 6.27, p < 0.0001; Fig. 3E). Newman-Kuels post hoc tests showed that Oprd1 mRNA levels were significantly elevated by the 10 mg/kg dose (p < 0.05). A significant Strain by Dose interaction was also observed (F(1,78) = 2.62, p < 0.05). Newman-Kuels post hoc tests indicated that the main effect of Dose was due to an elevation of Oprd1 mRNA by 10 mg/kg of heroin in C57BL/6J mice, but not in 129P3/J animals (data not shown).

3.4 Dopaminergic System

Heroin Dose Effects: A significant effect of heroin Dose on mRNA levels of the dopaminergic system was found in the caudate putamen and the hypothalamus.

Caudate putamen

In the caudate putamen, there was a significant main effect of Dose on the relative levels of Drd2 mRNA (F(5,111) = 4.07, p < 0.005; Fig. 4A). Newman-Kuels post hoc tests showed that heroin significantly elevated Drd2 mRNA levels at the 2.5 mg/kg dose.

Figure 4.

Figure 4

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Dopaminergic System; Dose Effects. Within strains, a statistically significant main effect of Heroin Dose was found for the relative expression of Drd2 mRNA in the caudate putamen and on Drd1a mRNA in the hypothalamus. * represents Newman-Kuels post-hoc test p < 0.05.

Hypothalamus

We observed a significant main effect of heroin Dose on Drd1a mRNA (F(5,95) = 2.76, p < 0.05l; Fig. 4B). Newman-Kuels post hoc tests showed that heroin significantly elevated Drd1a mRNA levels at the 10 mg/kg dose.

Heroin dose effects on mRNAs of the dopaminergic system were not observed in any other region examined.

4: Discussion

We identified region-specific strain differences in the relative expression levels of mRNA for important components of the endogenous opioid and the dopaminergic systems. The C57BL/6J and 129 strains of mice are known to differ in their behavioral response to cocaine (e.g. (Miner 1997; Schlussman et al. 1998; Schlussman et al. 2003a; Szumlinski et al. 2005; Unterwald & Cuntapay 2000)) and to heroin (e.g. (Schlussman et al. 2008; Szumlinski et al. 2005)). C57BL/6J mice have been shown to be more sensitive to thermal stimulation than 129/J (now 129P3/J) mice but less sensitive to morphine-induced antinociception (Mogil & Wilson 1997). 129/J mice have also been reported to be relatively resistant to developing dependence to morphine (Kest et al. 2002). Strains of 129 mice are consistently hyporesponsive to the behaviorally stimulating effects of cocaine, showing little or no horizontal activity in response to cocaine (e.g.(Kuzmin et al. 2000; Miner 1997; Schlussman et al. 1998; Schlussman et al. 2003a)). Additionally, 129Ola/Hsd mice failed to acquire cocaine self-administration while C57BL/6J and DBA mice did (Kuzmin & Johansson 2000). We have previously shown, in the same animals studied in the present report, that C57BL/6J and 129P 3/J mice differ in the locomotor-stimulating effect of heroin and in the establishment of heroin-induced conditioned place preference (Schlussman et al. 2008). Contrary to findings with cocaine, 129P3/J mice had a robust, dose-dependent locomotor response to heroin. The 129P3/J mice also developed conditioned place preference to heroin, although only to relatively high doses, to which C57BL/6J mice did not form a preference (Schlussman et al. 2008). Another substrain of 129 mice, 129X1/sVJ developed conditioned place aversion to a lower dose of heroin than we used (Szumlinski et al. 2005). Interestingly, this same sub-strain of mice was shown to require both contextual and drug cues in order to express conditioned place preference to morphine and it was suggested that higher relative levels of anxiety in the 129 strain of mice may explain these differences (Dockstader & van der Kooy 2001). We have also reported that these same mice whose tissues were used in the present study show significant differences in the expression of specific somatostatin receptor mRNAs within discrete regions of the central nervous system (Schlussman et al. 2010b).

In the region containing the substantia nigra and ventral tegmental area, and in the nucleus accumbens, we observed significantly higher levels of Oprd1 mRNA in 129P3/J mice, and in the nucleus accumbens we report a trend toward higher levels of Penk mRNA in 129P3/J mice. Delta opioid receptors have been implicated in mediating anxiety-like behaviors. For instance, mice with life-long deletion of Oprd1 show increased levels of anxiety (Filliol et al. 2000). Interestingly, 129/J mice have been shown to exhibit lower levels of anxiety, as measured in the open field, when compared to C57BL/6J mice (Montkowski et al. 1997). This may be related to a higher relative expression of Oprd1 mRNA. We have reported that in the mice whose tissue was studied here, 129P3/J animals have higher levels of somatostatin receptor-2 mRNA in the caudate putamen (Schlussman et al. 2010a). Somatostatin receptor-2 knockout mice have increased anxiety-like behavior (Viollet et al. 2000) which suggest that higher levels of Somatostatin receptor 2 mRNA might be associated with decreased levels of anxiety. However, although we did not examine sstr-2 mRNA levels in brain regions other than the caudate putamen. However, another group has suggested that 129/SvJ mice have high levels of anxiety during acute opiate withdrawal, in a conditioned place preference paradigm (Dockstader & van der Kooy 2001).

In addition to Oprd1 mRNA, we also observed significantly higher levels of Oprm1 mRNA, and a trend to higher levels of Oprk1 mRNA in the nucleus accumbens of 129P3/J mice compared to that of C57BL/6J animals. The nucleus accumbens is an integral part of the reward pathway and is a major target of the mesocorticolimbic dopaminergic projection. We also found a small but statistically significant negative correlation between Oprk1 mRNA levels in the nucleus accumbens of 129P3/J, but not in C57BL/6J, mice. The mu and kappa opioid receptors are thought to act in a countermodulatory manner to mediate dopaminergic tone (e.g. (Zhang et al. 2004a, b; Zhang et al. 2009)). Therefore it is somewhat counterintuitive to find elevated levels of mRNA for both of these receptors in this important dopaminergic terminal field, although it must be remembered that mRNA levels may not directly reflect protein levels, and Oprm1 and Oprk1 densities have not yet been compared in these two strains. A recent study has shown that DBA/2J mice are more sensitive to the rewarding effects of heroin (as measured by conditioned place preference) than are C57BL/6J animals (Bailey et al. 2010). These authors did not report basal differences in MOP-r binding densities in the nucleus accumbens between these two strains but they did report that MOP-r density was decreased by chronic heroin administration in many brain regions of the C57BL/6J animals, including the nucleus accumbens shell, while MOP-r density was not altered in any region of the DBA/2J brain (Bailey et al. 2010). Interestingly, significant strain differences in the DAMGO stimulated [35S] GTPγ were reported with higher levels of binding in the nucleus accumbens (core and shell) of C57BL/6J mice than in DBA/2J animals (Bailey et al. 2010).

In the caudate putamen we found a significantly lower level of Pdyn mRNA in 129P3/J mice than in C57BL/6J counterparts. This was unexpected. In another study, using solution hybridization / RNase protection, we did not find a difference in Pdyn mRNA in the caudate putamen of the two strains (Schlussman et al. 2003b). Additionally, dynorphin peptide lowers dopaminergic tone through its action on the kappa opioid receptor (e.g. (Zhang et al. 2004a)) and we did not observe differences in the basal levels of dopamine in the caudate putamen of these two strains (Zhang et al. 2001). Dynorphin, and other kappa opioid receptor agonists block the formation of cocaine-induced conditioned place preference (e.g.(Zhang et al. 2004a, b, 2005)). However we have reported that these same 129P3/J mice develop conditioned place preference to heroin, but only at relatively high doses (Schlussman et al. 2008). Previous studies have shown differences in the rewarding effects of opiates in C57BL/6J and DBA/2J mice, with often contradictory results (e.g. see (Bailey et al. 2010; Cunningham et al. 1992; Orsini et al. 2005; Semenova et al. 1995). Similar to the DBA mice, another strain, SWR, has also been shown to be less sensitive to the rewarding effects of opiates than C57BL/6J animals (Solecki et al. 2009). A recent study reported lower levels of Pyn and Penk mRNA in the dorsolateral striatum of SWR/J relative to either C57BL/6J or DBA/2J animals (Gieryk et al. 2010). In the nucleus accumbens we showed a trend toward higher levels of Penk mRNA levels in 129P3/J mice than in C57BL/6J animals. While we found that 129P3/J mice only develop conditioned place preference to relatively high doses of heroin (Schlussman et al. 2008), in the present study we did not find a statistically significant effect of heroin dose on Penk mRNA levels in this region. In an earlier report, Gieryk et al reported that C57BL/6J mice had higher levels of Penk mRNA and lower levels of Pdyn mRNA in the nucleus accumbens than did either DBA/2J or SWR/J mice (Gieryk et al. 2010). Interestingly, this groups had previously shown that C57BL/6J have a high sensitivity to morphine reward (as measured by conditioned place preference) while both DBA/2Jand SWR/J mice showed lower levels of morphine reward (Solecki et al. 2009) which led them to suggest that sensitivity to morphine reward may be related to basal levels of nucleus accumbal Penk and Pdyn mRNA (Gieryk et al. 2010). In the present study, we did not observe differences in levels of either Penk or Pdyn mRNA in the nucleus accumbens, despite having shown strain differences in the sensitivity to heroin-induced conditioned place preference (Schlussman et al. 2008). In the absence of a direct comparison of C57BL/6J, DBA/2J, SWR/J and 129P3/J, it is difficult to interpret these data.

In the present study we found lower levels of mRNA for Th, the rate limiting biosynthetic enzyme for dopamine, in the region containing the substantia nigra and ventral tegmental area, which may suggest reduced dopamine biosynthesis. We also observed lower levels of mRNA for the dopamine transporter (Slc6a3) in both the caudate putamen and the nucleus accumbens of 129P3/J mice compared to C57BL/6J animals.. Interestingly, rats that are more susceptible to acquisition of psychostimulant self-administration show lower levels of DAT binding sites in these regions (e.g. (Flores et al. 1998)) which led us to expect lower DAT mRNA levels in striatal regions of C57BL/6J mice. We did not observe an effect of heroin on SSlc6a3 mRNA levels. A previous report demonstrated higher levels of DAT binding in the nucleus accumbens, caudate putamen and olfactory tubercle of DBA/2J mice, compared to C57BL/6J animals, following chronic heroin administration (Bailey et al. 2010). These authors suggested that strain differences in heroin-induced levels of DAT binding might be partially responsible for the strain differences in heroin-induced locomotor activity in the strains (Bailey et al. 2010). However, contrary to the findings with DBA/2J mice, we reported that both the 129P3/J and C57BL/6J mice utilized in the present study showed a robust locomotor response to heroin (Schlussman et al. 2008). Nonetheless, it would be of interest to examine DAT binding and mRNA levels in C57BL/6J and 129P3/J mice following chronic administration of heroin.

In the hypothalamus, we reported opposite strain differences on levels of mRNA for the Drd1a and Drd2. 129P3/J mice had lower levels of Drd1a mRNA, and trended toward higher levels of Drd2 mRNA in this region. Activation of the stress responsive hypothalamic-pituitary-adrenal axis is, at least partially, regulated by dopaminergic Drd1a and Drd2 (e.g. (Borowsky & Kuhn 1992; Eaton et al. 1996)) and we have shown that Drd2 exerts tonic inhibition on hypothalamic Pomc mRNA expression (Zhou et al. 2004). We have not yet examined HPA function, in depth, in these two strains of mice, but in the present report we did not find strain differences in hypothalamic Pomc mRNA levels.

We did not observe a strain difference on the expression level of mRNA for Drd1a, Drd2 or Drd3 in either the nucleus accumbens or the caudate putamen. These findings for Drd1a mRNA support our earlier finding that, within the entire nucleus accumbens or caudate putamen there was no difference in the dopamine D1 receptor binding density in these strains (Schlussman et al. 2003a). Inbred strains of rats which have been reported to be more susceptible to acquisition of psychostimulant self-administration have fewer Drd2 binding sites in the caudate putamen or nucleus accumbens (e.g. (Hooks et al. 1994)) and lower levels of Drd2 mRNA in the nucleus accumbens (e.g.(Dalley et al. 2007; Hooks et al. 1994)). Similarly, inbred strains of rats (e.g. F344 and Lewis) which are known to differ in their acquisition of drug self-administration (e.g. (Guitart et al. 1992; Kosten et al. 2007)) show differences in the density of Drd2 and Drd3. Specifically, Lewis rats, which have a higher propensity to self administer drugs of abuse, had lower levels of Drd2 binding in the nucleus accumbens and caudate putamen than did F344 rats. Lewis rats also have lower levels of dopamine D3 receptor binding in the nucleus accumbens (Flores et al. 1998). Based on our previous behavioral studies in these two strains of mice, we had hypothesized that C57BL/6J mice would have lower levels of Drd2 and Drd3 mRNA in the caudate putamen and nucleus accumbens than do 129P3/J mice.

In the region containing the substantia nigra and ventral tegmental area, heroin had a dose-dependent effect on the relative mRNA levels of all three endogenous opioid receptors. Oprm1 mRNA was lowered by the three highest doses of heroin (5, 10 and 20 mg/kg). Heroin doses of 1.25 or 2.5 mg/kg did not alter Oprm1 mRNA levels. In contrast, Oprk1 mRNA levels were increased by the 1.25 and 2.5 mg/kg dose, while the higher doses had no effect. This may be reflective of the countermodulation of dopamine by Oprm1 and Oprk1.

Few other heroin effects were observed in the present study. However, it must be noted that in the context of the original conditioned place preference study, these animals were sacrificed either 24 or 48 hours following the last of four single injections of heroin. It is likely that additional effects would be observed following longer exposure to heroin. Additionally, real time PCR, like all PCR techniques, is based on an exponential function and, as a result, can only detect relatively large changes in mRNA levels. It is likely that additional heroin-induced effects on the mRNAs examined in the present report would be uncovered using techniques such as in situ hybridization or solution hybridization / RNase protection. Also, while we did not analyze the efficiency of amplification, it is possible that some of the strain effects that we report here may be due to strain differences in the efficiency of amplification. However, since the direction of strain-related differences in relative mRNA levels was gene-specific, we consider this possibility remote.

In summary, this report describes significant strain differences in the relative expression levels of important components of the endogenous opioid and dopaminergic systems. The previously-reported behavioral differences between these two strains may be due, in part, to these strain differences in the endogenous opioid and dopaminergic systems.

Supplementary Material

01. Supplemental Figure 1.

Relative expression of Oprm1 mRNA. Figures A–E represent Strain effects of relative levels of Oprm1 mRNA in individual brain regions. Figures F–J represent Heroin Dose effects on relative levels of Oprm1 mRNA in individual brain regions. * = p < 0.05

02. Supplemental Figure 2.

Relative expression of Oprk1 mRNA. Figures A–E represent Strain effects of relative levels of Oprk1 mRNA in individual brain regions. Figures F–J represent Heroin Dose effects on relative levels of Oprk1 mRNA in individual brain regions. * = p < 0.05

03. Supplemental Figure 3.

Relative expression of Oprd1 mRNA. Figures A–E represent Strain effects of relative levels of Oprd1 mRNA in individual brain regions. Figures F–J represent Heroin Dose effects on relative levels of Oprd1 mRNA in individual brain regions. * = p < 0.05

04. Supplemental Figure 4.

Relative expression of Penk mRNA. Figures A–E represent Strain effects of relative levels of Penk mRNA in individual brain regions. Figures F–J represent Heroin Dose effects on relative levels of Penk mRNA in individual brain regions. * = p < 0.05

05. Supplemental Figure 5.

Relative expression of Pdyn mRNA. Figures A–E represent Strain effects of relative levels of Pdyn mRNA in individual brain regions. Figures F–J represent Heroin Dose effects on relative levels of Pdyn mRNA in individual brain regions. * = p < 0.05

06. Supplemental Figure 6.

Relative expression of Pomc mRNA. Figures A and B represent Strain effects of relative levels of Pomc mRNA in hypothalamus (A) and caudate putamen (B). Figures C and D represent Heroin Dose effects on relative levels of Pomc mRNA in these same brain regions. * = p < 0.05

07. Supplemental Figure 7.

Relative expression of Drd1a mRNA. Figures A–D represent Strain effects of relative levels of Drd1a mRNA in individual brain regions. Figures E–H represent Heroin Dose effects on relative levels of Drd1a mRNA in individual brain regions. * = p < 0.05

08. Supplemental Figure 8.

Relative expression of Drd2 mRNA. Figures A–D represent Strain effects of relative levels of Drd2 mRNA in individual brain regions. Figures E–H represent Heroin Dose effects on relative levels of Drd2 mRNA in individual brain regions. * = p < 0.05

09. Supplemental Figure 9.

Relative expression of Drd3 mRNA. Figures A–C represent Strain effects of relative levels of Drd3 mRNA in the cortex, nucleus accumbens and caudate putamen. Figures D–F represent Heroin Dose effects on relative levels of Drd3 mRNA in these same regions.

10. Supplemental Figure 10.

Relative expression of Slc6a3 mRNA. Figures A–C represent Strain effects of relative levels of Slc6a3 mRNA in the cortex, nucleus accumbens and caudate putamen. Figures D–F represent Heroin Dose effects on relative levels of Slc6a3mRNA in these same regions. * = p < 0.05

11. Supplemental Figure 11.

Relative expression of Th mRNA. Figures A and B represent Strain effects of relative levels of Th mRNA in hypothalamus (A) and the region containing the SN and VTA (B). Figures C and D represent Heroin Dose effects on relative levels of Th mRNA in these same brain regions.

Highlights.

  1. C57-129 mouse strain differences in mRNA expression in the brain were found.

  2. Heroin has a region specific effect on the EOS and DAergic system.

Acknowledgments

The authors would like to thank Dr. Eduardo Butelman for helpful discussions. 3, 6 diacetyl-morphine HCl was generously provided by NIH-NIDA Division of Drug Supply and Analytical Services. This work was supported by grants from NIH-NIDA (DA05130), the Arcadia Charitable Trust and The Carson Family Charitable Trust to MJK.

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01. Supplemental Figure 1.

Relative expression of Oprm1 mRNA. Figures A–E represent Strain effects of relative levels of Oprm1 mRNA in individual brain regions. Figures F–J represent Heroin Dose effects on relative levels of Oprm1 mRNA in individual brain regions. * = p < 0.05

NIHMS319198-supplement-01.jpg (340.5KB, jpg)
02. Supplemental Figure 2.

Relative expression of Oprk1 mRNA. Figures A–E represent Strain effects of relative levels of Oprk1 mRNA in individual brain regions. Figures F–J represent Heroin Dose effects on relative levels of Oprk1 mRNA in individual brain regions. * = p < 0.05

NIHMS319198-supplement-02.jpg (357.6KB, jpg)
03. Supplemental Figure 3.

Relative expression of Oprd1 mRNA. Figures A–E represent Strain effects of relative levels of Oprd1 mRNA in individual brain regions. Figures F–J represent Heroin Dose effects on relative levels of Oprd1 mRNA in individual brain regions. * = p < 0.05

NIHMS319198-supplement-03.jpg (353.8KB, jpg)
04. Supplemental Figure 4.

Relative expression of Penk mRNA. Figures A–E represent Strain effects of relative levels of Penk mRNA in individual brain regions. Figures F–J represent Heroin Dose effects on relative levels of Penk mRNA in individual brain regions. * = p < 0.05

NIHMS319198-supplement-04.jpg (353.7KB, jpg)
05. Supplemental Figure 5.

Relative expression of Pdyn mRNA. Figures A–E represent Strain effects of relative levels of Pdyn mRNA in individual brain regions. Figures F–J represent Heroin Dose effects on relative levels of Pdyn mRNA in individual brain regions. * = p < 0.05

NIHMS319198-supplement-05.jpg (356.1KB, jpg)
06. Supplemental Figure 6.

Relative expression of Pomc mRNA. Figures A and B represent Strain effects of relative levels of Pomc mRNA in hypothalamus (A) and caudate putamen (B). Figures C and D represent Heroin Dose effects on relative levels of Pomc mRNA in these same brain regions. * = p < 0.05

NIHMS319198-supplement-06.jpg (241.3KB, jpg)
07. Supplemental Figure 7.

Relative expression of Drd1a mRNA. Figures A–D represent Strain effects of relative levels of Drd1a mRNA in individual brain regions. Figures E–H represent Heroin Dose effects on relative levels of Drd1a mRNA in individual brain regions. * = p < 0.05

NIHMS319198-supplement-07.jpg (387.1KB, jpg)
08. Supplemental Figure 8.

Relative expression of Drd2 mRNA. Figures A–D represent Strain effects of relative levels of Drd2 mRNA in individual brain regions. Figures E–H represent Heroin Dose effects on relative levels of Drd2 mRNA in individual brain regions. * = p < 0.05

NIHMS319198-supplement-08.jpg (386.5KB, jpg)
09. Supplemental Figure 9.

Relative expression of Drd3 mRNA. Figures A–C represent Strain effects of relative levels of Drd3 mRNA in the cortex, nucleus accumbens and caudate putamen. Figures D–F represent Heroin Dose effects on relative levels of Drd3 mRNA in these same regions.

NIHMS319198-supplement-09.jpg (324.2KB, jpg)
10. Supplemental Figure 10.

Relative expression of Slc6a3 mRNA. Figures A–C represent Strain effects of relative levels of Slc6a3 mRNA in the cortex, nucleus accumbens and caudate putamen. Figures D–F represent Heroin Dose effects on relative levels of Slc6a3mRNA in these same regions. * = p < 0.05

NIHMS319198-supplement-10.jpg (332.6KB, jpg)
11. Supplemental Figure 11.

Relative expression of Th mRNA. Figures A and B represent Strain effects of relative levels of Th mRNA in hypothalamus (A) and the region containing the SN and VTA (B). Figures C and D represent Heroin Dose effects on relative levels of Th mRNA in these same brain regions.

NIHMS319198-supplement-11.jpg (239.6KB, jpg)

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