Original Article

The Pharmacogenomics Journal (2006) 6, 255–264. doi:10.1038/sj.tpj.6500375; published online 14 February 2006

Discrete opioid gene expression impairment in the human fetal brain associated with maternal marijuana use

X Wang1, D Dow-Edwards2, V Anderson3, H Minkoff4 and Y L Hurd1

  1. 1Department of Clinical Neuroscience, Psychiatry Section, Karolinska Institute, Stockholm, Sweden
  2. 2Department of Pharmacology, State University of New York, New York, NY, USA
  3. 3Department of Pathology, State University of New York, New York, NY, USA
  4. 4Department of Obstetrics and Gynecology, Maimonides Medical Center and SUNY Downstate Medical Center, Kings County Hospital, Brooklyn, NY, USA

Correspondence: Professor YL Hurd, Department of Clinical Neuroscience, Psychiatry Section, Karolinska University Hospital, Stockholm 17176, Sweden. E-mail: Yasmin.Hurd@cns.ki.se

Received 2 August 2005; Revised 5 December 2005; Accepted 21 December 2005; Published online 14 February 2006.

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Abstract

Fetal development is a period sensitive to environmental influences such as maternal drug use. The most commonly used illicit drug by pregnant women is marijuana. The present study investigated the effects of in utero marijuana exposure on expression levels of opioid-related genes in the human fetal forebrain in light of the strong interaction between the cannabinoid and opioid systems. The study group consisted of 42 midgestation fetuses from saline-induced voluntary abortions. The opioid peptide precursors (preprodynorphin and preproenkephalin (PENK)) and receptor (mu, kappa and delta) mRNA expression were assessed in distinct brain regions. The effect of prenatal cannabis exposure was analyzed by multiple regression controlling for confounding variables (maternal alcohol and cigarette use, fetal age, sex, growth measure and post-mortem interval). Prenatal cannabis exposure was significantly associated with increased mu receptor expression in the amygdala, reduced kappa receptor mRNA in mediodorsal thalamic nucleus and reduced preproenkephalin expression in the caudal putamen. Prenatal alcohol exposure primarily influenced the kappa receptor mRNA with reduced levels in the amygdala, claustrum, putamen and insula cortex. No significant effect of prenatal nicotine exposure could be discerned in the present study group. These results indicate that maternal cannabis and alcohol exposure during pregnancy differentially impair opioid-related genes in distinct brain circuits that may have long-term effects on cognitive and emotional behaviors.

Keywords:

cannabinoid, alcohol, in utero, limbic system, striatum

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Introduction

Marijuana (Cannabis sativa) is the most commonly used illicit drug in western countries1 and many pregnant women are active users of the drug. According to a recent study carried out in the US, 7% of the participating women used marijuana, and even after awareness of pregnancy, 3% continued taking the drug.2 The major psychoactive component in cannabis, delta9-tetrahydrocannabinol (THC), can readily pass the placenta and affect the fetus. Delta9-tetrahydrocannabino exerts its function through the type one cannabinoid receptor (CB1) in the brain. Functional CB1 receptors have been documented in the human fetal brain from week 193 and in the rat brain from gestational day 14,4 therefore, prenatal cannabis exposure could directly activate the CB1 during early neurodevelopment. The CB1 mainly couples to the G-protein of Gi/o family, which decreases cAMP levels and modifies Ca2+ and K+ channel function upon activation (for reviews see5, 6). The CB1 is one of the most abundant G-protein-coupled receptors in the adult brain with high expression levels in the basal ganglia, cerebellum, cerebral cortex and limbic structures.7 During human fetal development, the CB1 receptor is preferentially expressed in limbic-related structures such as the amygdala and hippocampus.8

The CB1 receptors have been strongly coupled to endogenous opioid systems especially in relation to reward and addictive behaviors.9, 10, 11, 12, 13, 14 The opioid system (endogenous ligands and receptors) is involved in a wide variety of neuronal functions including pain perception, cognition, emotional regulation and addictive behaviors (for a review see15). There are two major groups of endogenous opioid neuropeptides, which are strongly involved in drug-dependent processes, enkephalins and dynorphins. These peptides are generated by enzymatic processing from separate precursors, proenkephalin and prodynorphin, which are encoded by preproenkephalin (PENK) and preprodynorphin (PDYN) genes (for a review see16). The endogenous opioids act through specific receptors mu, kappa and delta. Enkephalins bind preferably to mu and delta receptors and dynorphins have the highest affinity to the kappa receptor.17 Similar to the CB1, opioid receptors are primarily coupled to Gi/o G-protein with common signal transduction pathways.18

The consequence of in utero cannabis exposure in the fetus is far from clear. This issue has been investigated in different settings both in animals (mostly rodents) and humans. In rat studies, perinatal exposure to cannabis has been linked to long-term behavioral and cognitive disturbances.19 In humans, longitudinal studies have documented motor, social and cognitive disturbances in offspring who were exposed to cannabis prenatally.20, 21 Despite documented behavioral impairments, there is presently limited information about specific neurobiological consequences of prenatal cannabis exposure in humans. Recently, we observed altered amygdala dopamine D2 receptor mRNA expression in human fetuses with prenatal cannabis exposure.22 Other neurotransmitters aside from dopamine have been implicated in the neural actions of THC. Considering the tight anatomical and functional interactions between the cannabinoid and opioid systems, the current study was designed to investigate the effects of prenatal cannabis exposure on opioid system gene expression in the human fetal brain. To this end, the mRNA expression of the PDYN, PENK and three opioid receptor subtypes was assessed in the midgestation human fetal brain using in situ hybridization histochemistry.

In addition to cannabis, alcohol and cigarettes are the two major licit drugs used by many pregnant women. Both alcohol and cigarette exposure in utero are acknowledged to affect fetal development (for a review see23). Both animal and human studies have shown that cigarette use during pregnancy can lead to neurobehavioral deficits in the offspring (for a review see24). Heavy prenatal alcohol exposure, a well-established teratogen is associated with fetal alcohol syndrome, which is characterized by pre- and postnatal growth retardation, facial anomalies and mental retardation.25 Light or moderate alcohol exposure has also been shown to have a strong influence on behavioral and cognitive functions in the offspring.26, 27 On the basis of the documented influence of maternal alcohol and cigarette use on fetal development, the potential impact of these substances on opioid gene expression was also evaluated in the current sample population.

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Results

The 42 brain samples in the present study were from singleton abortion. The characteristics of the fetal samples are shown in Table 1. The fetuses were at the midgestation stage of development (20.26plusminus0.18 weeks). The mothers were predominantly young African-American women with an average age of 22.90plusminus0.68 years. Our investigation on the effects of the in utero cannabis-exposure on mRNA expression was focused at the caudal level of the fetal forebrain in which the most intense CB1 expression was located.28


In the midgestational fetus, high PENK and PDYN mRNA expression levels were found primarily in the striatum; higher expression in the patch than the matrix compartment (Figure 1a and b). Preprodynorphin also had intense expression levels in the subthalamic nucleus (Figure 1b). The mRNA distribution pattern for the three opioid receptors showed distinct anatomical patterns. The kappa receptor mRNA was the most abundantly expressed of the three opioid receptors (Figure 1e) and was found in the thalamus, cerebral cortex (at a caudal striatal level, representing the parietal, temporal and insula), claustrum, striatum, amygdala and hippocampus. Heterogeneous expression was evident in the thalamus with intense levels in the dorsomedial and ventrolateral nuclei. The mRNA expression of the kappa receptor in the claustrum and amygdala was moderate, whereas the striatal expression was quite weak. The mu receptor expression was primarily found in the thalamus and amygdala (Figure 1d) with a heterogeneous thalamic pattern (high levels in the dorsomedial and reticular nuclei, moderate in the ventral nuclei, and weak in the lateral nuclei). The mu receptor expression in amygdaloid was predominantly in the basal nucleus. The delta expression was the most weakly expressed of the opioid receptor mRNAs (Figure 1f). The delta receptor mRNA was evident in the striatum, cerebral cortices, amygdala and hippocampus.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Distribution of mRNA expression in coronal sections of the human fetal brain at the caudal striatal level. The ISHH images for each mRNA species were from a 21-week male subject. They showed distinct expression pattern for PENK: (a) PDYN (b) mu receptor (d) kappa receptor (e) and delta receptor (f). (c) Schematic drawing of various brain regions. Abbreviations used in (c) were: Am, amygdala; Cc, corpus callosum; Cl, claustrum; Cn, caudate nucleus; Gl, germinal layer; Hi, hippocampus; Hy, hypothalamus; I, insula cortex; Lv, lateral ventricle; MD, dorsomedial nucleus of thalamus; Pu, putamen; Re, reticular nucleus of thalamus; St, subthalamic nucleus; VL, ventrolateral nucleus of thalamus. The scale bar in 1C was 1 cm.

Full figure and legend (202K)

Prenatal drug exposure was associated with significant alterations of the mRNA expression levels in distinct brain regions (Tables 2 and 3). Prenatal cannabis exposure had a significant impact on PENK as well as the opioid receptor mu and kappa expression levels. The PENK mRNA levels in the putamen were reduced significantly in the cannabis-exposed group (57.3plusminus5.3 percent of control value, meanplusminuss.e.m., P=0.028; Figures 2 and 3). The patch and matrix compartments showed similar levels of reduction in the cannabis-exposed samples. The reduction of the PENK mRNA expression levels was significantly correlated with the amount of maternal cannabis intake during pregnancy (r=-0.49, P=0.003). In contrast to PENK, the mu opioid receptor expression was significantly increased in the amygdala association with cannabis exposure (126.7plusminus14.0% of controls, P=0.017; Figure 4). The increase was dependent on the amount of reported maternal cannabis intake (r=0.40, P=0.044). The kappa receptor expression in the mediodorsal thalamic nucleus was significantly reduced in association to prenatal cannabis exposure (56.9plusminus11.1% of controls, P=0.034; Figure 4).

Figure 2.
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The opioid peptide PENK and PDYN mRNA expression levels in the cannabis-exposed group (gray bar) expressed as a percentage of the control (white bar) in distinct brain regions. The unit for mRNA expression is dpm/mg. *P<0.05.

Full figure and legend (66K)

Figure 3.
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Representative PENK and PDYN mRNA expression images from a normal and a cannabis-exposed brain sample are shown.

Full figure and legend (78K)

Figure 4.
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The opioid receptors mRNA expression levels in the in the cannabis-exposed group (gray bar) expressed as a percentage of the control (white bar) in distinct brain regions. *P<0.05; **P<0.01.

Full figure and legend (121K)



The PDYN mRNA expression in putamen was not significantly altered in association with prenatal cannabis exposure; neither the patch nor the matrix compartment showed any significant alteration (Figures 2 and 3 and Table 2). No significant effects of prenatal cannabis exposure were evident for PDYN expression in the subthalamic nucleus, which had the most intense PDYN expression levels. The mRNA expression of kappa and delta opioid receptor did not show a significant association with prenatal cannabis exposure.

In contrast to cannabis, prenatal alcohol exposure was significantly associated with decreased mu receptor expression in the amygdala (76.3plusminus11.5% of controls, P<0.001; Figure 5). Prenatal alcohol exposure also had a strong negative impact on the kappa receptor expression in the amygdala (36.8plusminus5.6% of controls, P<0.001), claustrum (40.3plusminus10.5% of controls, P<0.001), putamen (74.4plusminus9.6% of controls, P=0.006) and insular cortex (34.0plusminus6.2% of controls, P<0.001). The reduction of the kappa receptor was significantly correlated to the amount of maternal alcohol intake in the amygdala (r=-0.63, P<0.001), insula (r=-0.70, P<0.001) and claustrum (r=-0.51, P=0.003), whereas in the putamen the correlation was not significant but had a strong trend (P=0.065). Prenatal alcohol exposure did not significantly affect the neuropeptide PENK and PDYN or the delta receptor mRNA expression levels in the brain regions studied.

Figure 5.
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The opioid receptors mRNA expression levels in the in the alcohol-exposed group (gray bar) expressed as a percentage of the control (white bar) in distinct brain regions. **P<0.01.

Full figure and legend (111K)

There was no significant association between prenatal cigarette exposure and gene expression in the brain regions examined. In addition to prenatal drug exposure, several variables were found to influence some of the mRNA expression levels. Fetal weight had a significant positive association with PENK expression in the putamen (P=0.005). Moreover, a significant influence of fetal sex was also evident for the kappa receptor gene expression levels in ventrolateral thalamic nucleus. Male fetuses had higher expression levels than females (328.84plusminus30.51 vs 206.98plusminus29.76, P=0.008). Post-mortem interval (PMI) showed a significant association with delta receptor expression in the insula (P<0.001). Post-mortem interval was also significantly associated with PDYN expression in the putamen (P=0.039), which was more related to the matrix compartment (P=0.028) but less to the patch (P=0.073).

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Discussion

The present data provides evidence for impairments of the endogenous opioid system in association with in utero cannabis exposure in the human fetal brain. Maternal cannabis use was significantly associated with reduced mRNA expression levels for PENK in the putamen (both patch and matrix compartments), increased mu expression in the amygdala and decreased kappa expression in the mediodorsal thalamic nucleus.

The results from the present study are unique and important since they offer direct insight into molecular alterations of the human fetal brain exposed to cannabis during embryogenesis. After controlling for various confounding factors, the data revealed specific associations between cannabis exposure during pregnancy and opioid gene expression. A number of the present human results are consistent with observations obtained with animal models. Rat studies have documented that fetal animals exposed to THC in utero have decreased PENK mRNA expression levels in the caudate-putamen.29 Preproenkephalin mRNA levels in the caudate-putamen is also significantly reduced when perinatally exposed rats are studied at an adult age.30 However, the effects of prenatal THC treatment on PENK expression are strongly dependent on sex. Significant alterations were observed only in fetal male and adult female rats. Alterations of PENK expression levels are also evident in rats that receive chronic THC administration during adulthood.31, 32 Taken together, it is apparent that disturbances of PENK gene expression is a common consequence of the repeated exposure to THC in rat models though the exact mRNA changes are dependent on the pattern of drug administration, dose and time course of drug administration, brain regions examined, age when the rat is studied and the sex. Our result with PDYN gene expression is also in line with rat studies. Fetal rats exposed to THC in utero also failed to show a significant effect on PDYN expression levels in the caudate-putamen even when examined at two gestation ages.33 No animal report currently exist about the effect of prenatal cannabinoid exposure on mu opioid receptor expression during fetal development. However, prenatal THC exposure has been shown to induce sex-dependent changes of the mu opioid receptor binding in several brain regions including the amygdala and caudate-putamen in the adult brain.34 In the adult rat, repeated administration of THC also results in increased mu opioid receptor binding in the amygdala and caudate-putamen in addition to the hippocampus and hindbrain regions. The onset, duration and magnitude of the effects varied between the brain regions examined.35 Overall, the fact that the present human investigation also identified mu and PENK alterations in prenatal cannabis-exposed fetuses similar to THC-treated rodents strengthens the reliability of our study since human post-mortem samples have many confounds due in part to, for example, limited drug history information as compared to well-controlled animal experiments.

Similar to our previous study on the dopamine D1 and D2 receptors,22 the present result also documented a significant impact of prenatal alcohol exposure on gene expression. Of the opioid markers examined, the kappa receptor expression was the most profoundly altered with reduced mRNA levels evident in several brain regions including the amygdala, insular cortex, claustrum and putamen in alcohol-exposed fetuses. Even though there is no report regarding the effects of prenatal alcohol exposure on kappa mRNA level in fetal animal models, experimental animal data have documented significant interactions between the kappa opioid receptor and alcohol use in adulthood.36, 37, 38, 39 Prenatal alcohol exposure in humans has in fact been shown to be most predictive of adolescent alcohol use even after adjusting for family history and other prenatal and environmental variables,40 which suggests that modification of the kappa system by prenatal alcohol exposure could have a long-lasting effect on behavior that enhances the vulnerability to alcoholism. It is interesting to note that alcohol-related changes on the kappa opioid receptor mRNA were not mimicked by alterations in the PDYN mRNA expression. These findings would also appear to be in line with the animal literature since rodent studies have failed to observe significant alterations of the PDYN mRNA expression following alcohol use in adult models.41, 42 The current findings also emphasize that there are specific prenatal neural responses to exposure to different classes of drugs. In addition to the more widespread effects of alcohol compared to cannabis, the mu opioid receptor expression was reduced in relation to alcohol exposure, but increased in association with maternal cannabis use. Alcohol-induced decrease of mu is not unexpected and elevation of the mu in amygdala is also consistent with animal studies.34 It is important to emphasize that many cannabis subjects were also exposed to alcohol, nevertheless the cannabis-induced elevation of the mu expression was still evident in the human fetal amygdala. The effects of prenatal cannabis and alcohol exposure are expected to have, for example, different dose range and time course of their effects. Thus, animal studies are needed to help tease out the distinct neural mechanism of each drug and their combinational effects.

Cigarette smoking was quite common in the present study population. However, we did not find any specific effect of prenatal cigarette exposure on gene expression levels. The opioid system is known to play an important role in smoking reinforcement (for a review see43). Nicotine exposure in adult rats has been associated with alterations in the opioid system, both increased and decreased expression levels have been observed which appear to depend on the time course of drug treatment and time point at which animals are studied.44 The apparent lack of maternal cigarette use on the fetal opioid genes in the present study could be due to differences in the time course of cigarette smoking between the women during pregnancy. Another reason for the apparent lack of cigarette effects could be related to the interaction of maternal cigarette and cannabis smoking since many cigarette smokers also used cannabis. The high coregistry of cannabis and cigarette use made it difficult to dissociate the effects of cannabis from cigarette. However, there was no dose-dependent effect for the reported maternal cigarette intake, whereas maternal cannabis use was significantly correlated with the gene expression levels of PENK and the mu opioid receptor. This finding suggests that the effect of maternal cannabis use on the genes studied is stronger than that of cigarette in this cohort, but we cannot completely exclude a contribution of cigarette. Future studies in well-dissociated populations are required to conclusively evaluate the independent influence of cigarette and cannabis.

The specific alterations observed for the opioid gene expression in the fetal brain in association with drug exposure are of significant relevance since the opioid system plays a critical role in the regulation of emotions, reinforcement, cognition, motor function and nociception.45 What is remarkable thus far in studies of the human fetal brain is the strong limbic impairment in cannabis-exposed fetuses (mediodorsal thalamic kappa and amygdaloid mu receptor expression in the present study as well as amygdaloid dopamine D2 expression in previous study22). The mediodorsal thalamus is a key component of the limbic system connecting subcortical structures such as the nucleus accumbens and amygdala with the prefrontal cortex. The amygdala is a critical limbic brain structure for the regulation and expression of emotions. The amygdala also affects higher cognitive functions through its reciprocal connections with the prefrontal cortex (decision-making, working memory) and hippocampus (memory consolidation) (for a review see46). Moreover, the amygdala has strong connections with the ventral tegmental area, the origin of the mesocorticolimbic system, and the nucleus accumbens which are central to the reward behaviour.47 Thus, disturbances of amygdala dopamine- and opioid-related function during development could influence cortical and limbic system functions. Longitudinal human studies have documented that prenatal marijuana-exposed children and young adults exhibit a variety of emotional, cognitive and behavioral deficits, such as deficit executive function, depression, anxiety, inattention and delinquency.20, 27, 48, 49, 50, 51, 52 Alterations of the PENK gene also suggest specific cannabis effects on discrete neural systems in the caudal striatum that could influence motor behavior. Preproenkephalin is primarily expressed in striatal medium spiny projection neurons which comprise the indirect pathway innervating the globus pallidus, whereas PDYN is primarily expressed in the direct pathway to the substantia nigra.53 Imbalance of these striatal output circuits is normally related to motor disturbances. Although prenatal marijuana exposure has been associated with increased tremors in newborns, this impairment disappears with increasing age (for a review see52). The PENK striatal alterations evident in the cannabis-exposed fetuses might thus be relevant to the motor impairments apparent in early life since persistent alterations in striatal PENK mRNA expression into adulthood have been observed in animal models of prenatal THC exposure.30, 32 The long-term influence of early PENK disturbance requires further study.

There is very limited information regarding the potential mechanisms by which receptor expression could be altered as a consequence of cannabis exposure. We have previously studied the CB1 expression in human fetal forebrain and found no significant association between CB1 expression and prenatal cannabis exposure.22 However, THC is known to activate multiple transcription factor genes including c-fos and CREB important for the regulation of the genes currently studied.54, 55 Moreover, the CB1 mRNA has a high expression in the amygdala28 and functional CB1 binding sites are present in the putamen3 of the human fetal brain. As such activation of the CB1 signal transduction systems could regulate the expression levels of opioid-related genes within several structures observed in the present investigation.

In conclusion, our study provides biological data of significant relevance to a current problem since recent surveys continue to emphasize a high prevalence of marijuana use (usually together with alcohol and cigarette) during pregnancy. Study of the human fetal brain, though challenging, has helped to provide significant insights into the development of the normal fetal brain and about the influence of environmental insults on discrete neuronal populations. Identifying molecular alterations in discrete neuronal populations is a critical first step in understanding the brain circuits that are most vulnerable to prenatal cannabis exposure. The current data identified differential patterns of impairments of opioid genes in association with maternal cannabis and alcohol use in the midgestation human fetus. These findings emphasize the specific effects of drugs on the developing nervous system. The apparent vulnerability of the amygdala to prenatal drug exposure suggests that there is altered limbic function in cannabis-exposed offspring, which may have long-term impact on emotional and social behaviors.

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Materials and methods

Fetal brain sample collection and preparation

Fetal brain samples were collected after saline-induced voluntary abortion as described previously.22 Pregnant women at midgestation (18–22 weeks) who planned to carry out voluntary abortion were recruited for the study. After informed consent, the women were interviewed with a detailed questionnaire in order to obtain demographic, medical and substance usage information. Exclusion criteria for all the samples included: evidence of congenital abnormality, family history of mental disorder, use of other illicit drugs other than cannabis (such as psychostimulants, heroin and morphine). Fetal gestational age was determined by ultrasound and/or maternal physical examination. Fetal brain specimens were collected within a 24-h PMI after abortion under guidelines approved by the IRB committee at the SUNY Downstate Medical Center, Kings County hospital, Brooklyn, New York. The fetal brain samples were lightly fixed with 1% paraformaldehyde for 72 h at 4°C and then frozen in isopentane. Maternal urine and fetal meconium were collected and assayed for cannabis, stimulants, opiates and their respective metabolites. Samples positive for stimulants or opiates were excluded from the present study. Samples included in the cannabis-exposed group were based on positive maternal self-report and/or maternal urine that tested positive for THC, and/or fetal meconium positive for THC. The remaining samples were assigned to the control group.

Forty-two post-mortem fetal brain samples were included in the present study with similar number of subjects for each sex in the cannabis-exposed and control groups (Table 1). Of those reporting marijuana use during pregnancy, six subjects indicated heavy marijuana intake (average daily joints greater than or equal to0.89 joints/day; the cut-off values for classifying the degree of marijuana use are based on published criteria49), four moderate intake (average daily joints greater than or equal to0.4 and <0.89 joints/day), and eight light intake (average daily joints <0.4 joints/day). Three subjects in the cannabis group did not have reported drug use but were identified by a THC-positive in meconium toxicology test. The amount of maternal alcohol and cigarette use was based on maternal report of average daily alcohol volume and number of cigarettes smoked daily. The physical development of the fetuses was determined by measurement of body weight, body length, head circumference and foot length. The fetal brains were sectioned in the coronal plane at 20 mum, thaw-mounted onto Superfrost Plus slides (Brain Research Laboratories, Newton, MA, USA), and stored at -30°C until processed.

In situ hybridization histochemistry

35S-labeled riboprobes complementary to the human PDYN, PENK and receptor genes were prepared by in vitro transcription. The target region for PDYN probe was located in the 5' untranslated region (from 9 to 148 in human PDYN gene sequence ID: NM_024411). The PENK probe had a sequence complimentary to the whole coding region of the PENK gene (from 77 to 880 in sequence accession ID: NM_006211). The probes for opioid receptors were targeted to a part of the coding region of each gene. The mu opioid receptor probe was 600 bp in length (from 366 to 966 in the human mu receptor gene, accession ID: AY521028). The length of the kappa receptor probe was 635 bp (from number 706 to 1341, accession ID: NM_000912). The delta receptor probe was 470 bp (from number 532 to 1002, gene accession ID: NM_000911). The in situ hybridization histochemistry techniques were similar to published protocols.56 Briefly, the labeled probe was applied to the brain sections in a concentration of 2 times 103 cpm/mm2 of coverslip area. Two adjacent sections from each subject were studied. Hybridization was carried out overnight at 55°C in a humidified chamber. After in situ hybridization, the slides were apposed to film with a 14C standard (ARC-146, ARC Inc., St Louis, MO, USA). The mRNA expression level was estimated by densitometric readings within specific brain structures of interest as identified by the use of histological landmarks in conjunction with the human fetal brain atlas.57 The values obtained from duplicate brain sections for each subject were averaged. The PENK and PDYN gene expression had been documented to have a differential expression in the striatal patch and matrix compartment; both genes have a higher expression in the patch vs matrix.53 Measurements were made separately on the patch (high expressing islands) and matrix compartment (low expressing surrounding area) by setting a threshold in optical density. The region with optical density above threshold was measured as patch and below as matrix compartment. Optical density values were converted to dpm/mg values by calibration to the coexposed 14C standard.

Statistics

The mRNA expression data were first tested for normality. Natural log-transformation was applied if the data were not normally distributed. Univariate statistical analyses were applied to test the influence of each independent variable (e.g. fetal age, sex, PMI, fetal development measure) on the mRNA expression levels. Variables which showed a P-value less than 0.25 were included in the multiple regression model, and a stepwise regression was performed. Cannabis and alcohol were always included in the final model, whereas the effect of cigarette was tested by univariate statistics due to a strong colinearity between cannabis and cigarette use. The final model contained variables which P-values were less than 0.10. The dose-dependent effects of prenatal drug exposure on the gene expression was analyzed by nonparametric correlation (Spearman ranked R). To minimize the influence of particular cases on the general result, outliers identified by residual analysis were excluded. P<0.05 was considered statistically significant.

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Notes

Duality of interest

None declared.

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Acknowledgements

This study was supported by Grants from the National Institutes of Health (NIDA DA12030) and the Swedish Scientific Council (11252). We thank Dr Diane Ashton for help with access to the Obstetrics and Gynecology clinic. We also thank Alexandra Guilliume and Dionne Dunkley for helping with maternal interviews and fetal brain collection and Alexandra Tylec for invaluable technical assistance with in situ hybridization experiments.