Central myelin gene expression during postnatal development in rats exposed to nicotine gestationally

Abstract

Abnormal myelin gene expression in the central nervous system (CNS) is associated with many mental illnesses, including psychiatric disorders and drug addiction. We have previously shown that prenatal exposure to nicotine, the major psychoactive component in cigarette smoke, alters myelin gene expression in the CNS of adolescent rats. To examine whether this effect is specific for adolescents, we examined myelin gene expression in the CNS of juveniles and adults. Pregnant Sprague-Dawley rats were treated with nicotine (3 mg/kg/day; GN) or saline (GS) via osmotic mini pumps from gestational days 4 to 18. Both male and female offspring were sacrificed at postnatal day P20-21 (juveniles), P35-36 (adolescents), or P59-60 (adults). Three limbic brain regions, the prefrontal cortex (PFC), caudate putamen (CPu), and nucleus accumbens (NAc), were dissected. The expression of genes encoding major myelin components was evaluated using quantitative RT-PCR. We found that GN altered myelin gene expression in juveniles with brain region and sex differences. The pattern of alteration was different from that observed in adolescents. Although these genes were expressed normally in male adults, we observed decreased expression in GN-treated female adults, especially in the CPu. Thus, GN altered myelin gene expression throughout postnatal development and adulthood. The effect on adolescents was quite different from that at other ages, which correlated with the unique symptoms of many psychiatric disorders during adolescence.

Introduction

Maternal smoking during pregnancy (MSDP) has been associated with many neurobehavioral problems in the offspring. Those whose mothers smoke during pregnancy are more likely to show reduced cognitive abilities [10] and to develop neuropsychiatric disorders such as attention deficit hyperactivity disorder, conduct disorder, depression, autism, and drug addiction [24].

To evaluate the underlying mechanisms, we established a rat model of gestational exposure to a moderate dose of nicotine (GN) [35], the major psychoactive component of tobacco. Our previous studies focusing on adolescents showed that GN altered behavioral responses to addictive substances [16, 17], cell death/survival pathways [45], and expression of cell adhesion molecules in the central nervous system (CNS) [7]. Recently, we found that central myelin gene expression was also changed during adolescence by GN treatment in a brain region- and sex-dependent manner [8]. These studies suggest that nicotine replacement therapy during pregnancy may carry many of the same risks to the offspring as maternal smoking.

Myelin is a membrane structure produced by oligodendrocytes (OLGs) in the CNS and consists of many specific proteins and large amounts of glycolipids and cholesterol [3, 12]. Myelin basic protein (MBP) and proteolipid protein (PLP) contribute approximately 85% of the protein content of myelin. The remaining 15% includes 2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNP), myelin oligodendrocyte glycoprotein (MOG), myelin-associated oligodendrocytic basic protein (Mobp), and T-cell differentiation protein (Mal) [3]. Deficits in these major myelin components lead not only to abnormal myelin structure but also to axonal degeneration [3].

The functional acknowledgment of the OLG–myelin complex has greatly advanced in the past decades. Myelin structure not only increases the conduction velocity of action potentials [22], but also interacts with axons to support neuronal survival and modulate neurotransmission [13, 34, 39]. OLGs also synthesize neurotrophic factors to promote neuronal survival and axonal growth [46]. Recently, OLG-myelin complex has attracted new interest because of its apparent involvement in drug addiction and various psychiatric disorders such as schizophrenia, bipolar disorder, and major depression [6, 15].

The process of myelination involves OLG-precursor migration, proliferation, and differentiation into OLGs followed by maturation and formation of a myelin sheath around axons [3]. Myelination initiates during embryonic development, continues into adolescence, and still exhibits great plasticity in the adult nervous system [2, 4, 31]. As the major resource for cholesterol production in the brain and high energy demands for producing and maintaining the massive membrane structure, the process of myelination is vulnerable to environmental challenges [2]. Our recent study suggested that prenatal nicotine exposure affects myelination in the adolescent brain. However, it is not clear whether this effect is specific to adolescents. Therefore, we examined major myelin gene expression in GN-treated rats at different ages, including juveniles and young adults.

Materials and Methods

Animals and tissue collection

Sprague-Dawley rats were maintained in a temperature (21°C)- and humidity (50%)-controlled room on a 12-h light–dark cycle (lights on 0700–1900) with unlimited access to food and water. Pregnant rats (Harlan, San Diego, CA) were treated with either nicotine at a concentration of 3 mg/kg/day (calculated free base) or saline via osmotic minipumps (Alzet Model 2002) as described previously [35]. The minipump has a 14-day delivery period and was implanted subcutaneously on the back of each dam on gestational day 4. Blood concentrations resulting from this dose of nicotine are equivalent to those found in humans who smoke about 1½ packs of cigarettes per day [29]. After birth, pups were cross-fostered on drug-naive dams to minimize the effects of abnormal maternal behaviors or milk output attributable to nicotine treatment. As previously reported [16], GN treatment did not influence dam weight gain, litter size, or pup weight gain during postnatal development. Pups were weaned on postnatal day (P) 21, and for each sex at each age, non-sibling animals were used. Brain tissues from the prefrontal cortex (PFC), dorsal caudate putamen (CPu), and nucleus accumbens (NAc) were collected from pups at P20-21 (juveniles), P35-36 (adolescents), or P59-60 (adults) and stored at −80°C until being used for quantitative real-time polymerase chain reaction (qRT-PCR) assay (N = 5 or 6). The tissues were excised using a brain punch tissue set (Stoelting, WI) and rat brain matrices (Kent Scientific, CT) according to coordinates from Paxinos and Watson [37]. All experiments were carried out in accordance with the Institutional Animal Care and Use Committees at the University of California, Irvine, and University of Virginia and were consistent with Federal guidelines.

Quantitative real-time PCR array

We examined eight genes encoding major myelin components, namely, myelin basic protein (Mbp), proteolipid protein 1 (Plp1), 2′,3′-cyclic nucleotide 3′ phosphodiesterase (Cnp), T-cell differentiation protein (Mal), gap junction protein, gamma 3 (Gjc3), myelin-associated oligodendrocytic basic protein (Mobp), myelin oligodendrocyte glycoprotein (Mog), and aspartoacylase (Aspa) using qRT-PCR. The primers were designed using Primer Express (v. 3.0) software (Applied Biosystems) and spanned introns to avoid amplifying genomic DNA. The amplicon sequences were subjected to a BLAST search to ensure the specificity of the primers for the target genes and synthesized by Fisher Scientific (Pittsburgh, PA). All the primers were tested for their specificity by checking cycle number and the dissociation curve prior to inclusion in the qRT-PCR array. The primer sequences are listed in Supplementary Table 1.

The RNA was isolated from each brain region using TRIZol reagent (Invitrogen) according to the manufacturer's instructions and amplified as described previously for adequate cDNA [7]. The qRT-PCR was conducted as described previously [21, 27]. Briefly, the RT product was amplified in a volume of 10 μl containing 5 μl of 2× Power SYBR® Green PCR Master Mix (Applied Biosystems) and combined sense and antisense primers (3 μl; final concentration 250 nM) in a 384-well plate using the 7900HT Sequence Detection System (Applied Biosystems). Expressions of all genes were normalized to the expression of actin and GAPDH and then analyzed using a comparative C_t method [47]. Because data normalized to GAPDH yielded results similar to those normalized by actin, only the results normalized by actin are provided in this report.

Data analysis

Instead of comparing gene expression directly across ages and sexes, we calculated the ratios of gene expression in the GN group versus the corresponding GS group at each age and for each sex. This eliminates the potential impact of age and sex differences on brain structure and tissue collection.

Data were analyzed by mixed-design ANOVA with between-subjects factors (Age, Sex, Brain Region, and Drug) and within-subject factor (Gene). Significant main effects and interactions were further analyzed by appropriate ANOVA and post-hoc analysis with Bonferroni correction for multiple comparisons. Significant alteration in mRNA expression was defined as a fold change > 20% with a p value < 0.05.

Results

ANOVA analysis revealed the expression levels of myelin genes were altered by GN treatment with a significant interaction of treatment with brain region, sex, and age (F_{4, 122} = 6.665, p < 0.0001).

Prefrontal cortex

In the PFC, myelin gene expression was altered by GN treatment with a significant interaction of treatment with Sex and Age (F_{2, 37} = 7.487; p = 0.002). All examined genes showed similar patterns of alteration within each age and sex, as indicated by an insignificant Gene effect and insignificant interactions of Gene with other factors (Fig.1). In juveniles, these genes generally showed decreased expression in GN-treated animals compared with GS controls in both males (p < 0.05 for Mbp, Plp1, Mal, Gjc3, Mobp, Mog, and Aspa) and females (p < 0.05 for Mbp, Mobp, Mog, and Aspa; p < 0.01 for Cnp). In adolescents, these genes were upregulated in GN-treated males (p < 0.05 for Mbp, Plp1, Gjc3, Mobp; p < 0.001 for Aspa), whereas they were downregulated in GN-treated females (p < 0.05 for Plp1, Gjc3, and Mobp; p < 0.01 for Mbp, Cnp, Mal, and Mog). These genes were expressed normally in both GN-treated male and female adults.

Figure 1.

Fold change in mRNA expression of eight myelin genes in the PFC in juvenile, adolescent, and adult rats exposed to gestational nicotine (GN; solid lines) or saline (GS; dotted lines). Data from males and females are presented separately. Data are expressed as means ± SEM (N = 4 or 5). *p <0.05; **p <0.01; ***p <0.001 significantly different from GS controls. Abbreviations for Figures 1–3: Aspa = aspartoacylase; Cnp = 2′,3′-cyclic nucleotide 3′ phosphodiesterase; Gjc3 = gap junction protein, gamma 3; Mal = T-cell differentiation protein; Mbp = myelin basic protein; Mobp = myelin-associated oligodendrocytic basic protein; Mog = myelin oligodendrocyte glycoprotein; and Plp1 = proteolipid protein 1.

Caudate putamen

In the CPu, GN altered myelin gene expression with a significant interaction of treatment with Sex and Age (F_{2, 40} = 6.007, p = 0.005) (Fig. 2). In male juveniles, the expression of Plp1 was significantly increased in the GN group compared with GS controls (p < 0.05), whereas all other genes were expressed normally. In contrast, these genes were generally upregulated in GN females (p < 0.05 for Cnp; p < 0.01 for Mbp, Plp1, Mal, and Mobp). In adolescents, the expression of these genes was generally increased in GN males (p < 0.05 for Mbp, Plp1, Cnp, Mobp, Mog, and Aspa; p < 0.01 for Mal), whereas they were expressed normally in GN females. In adults, these genes were expressed normally in GN males while generally having decreased expression in GN females compared with GS controls (p < 0.05 for Mbp, Plp1, Mal, and Mog; p < 0.01 for Gjc3).

Figure 2.

Fold change in mRNA expression of eight myelin genes in the CPu in juvenile, adolescent and adult rats exposed to gestational nicotine (GN; solid lines) or saline (GS; dotted lines). Data for males and females are presented separately. Data are expressed as means ± SEM (N = 4 or 5). *p <0.05; **p <0.01; ***p <0.001 significantly different from GS controls.

Nucleus accumbens

In the NAc, GN altered myelin gene expression with a significant interaction of treatment with Sex and Age (F_{2, 45} = 15.275, p = 0.001). All examined genes showed similar patterns of alterations within each age and sex, as indicated by an insignificant Gene effect and insignificant interactions of Gene with other factors (Fig. 3). In juveniles, these genes showed decreased expression in GN-treated males (p < 0.05 for Gjc3 and Aspa; p < 0.01 for Mbp, Mal, Mobp, and Mog; p < 0.001 for Cnp) and increased expression in GN-treated females compared with GS controls (p < 0.05 for Mbp, Plp1, Cnp, Mal, Gjc3, Mobp, and Mog). These genes generally showed upregulation in adolescent males (p < 0.05 for Mbp, Plp1, Cnp, Mobp, and Mog), whereas only Mbp (p < 0.05) and Plp1 (p < 0.05) were upregulated in GN-treated adolescent females. These genes were expressed normally in both adult males and females.

Figure 3.

Fold change in mRNA expression of eight myelin genes in the NAc in juvenile, adolescent, and adult rats exposed to gestational nicotine (GN; solid lines) or saline (GS; dotted lines). Data from males and females are presented separately. Data are expressed as means ± SEM (N = 4 or 5). *p <0.05; **p <0.01; ***p <0.001 significantly different from GS controls.

Discussion

Myelination in the CNS continues into adolescence and exhibits large plasticity in adulthood [2, 4, 31]. By examining major myelin gene expression, our study suggested that central myelination is disturbed by GN treatment in both developing and mature brains. This effect was complicated and depended on sex and the brain region examined.

In this study, we selectively examined a few major myelin genes that play important roles in the CNS. MBP is the major protein maintaining myelin structure [28], and ASPA is an important enzyme for myelin lipid synthesis [44]. Although we only reported mRNA expression in this study, our previous study has shown that the alteration in protein expression of MBP is consistent with that of mRNA in adolescent brains [8]. The membrane-associated proteins such as MAL and GJE3 interact with neighbor cells to support myelin structure and function [11, 40]. Other proteins, such as PLP1 and CNP, are less involved in myelination but help shape the underlying axon and also support axon function [19, 26]. Both MOBP and MOG are located selectively on the outside surface of myelin sheaths and have been related to the autoimmune response [30, 33]. Abnormal expression of these genes therefore can lead to abnormal myelin structure and deficits in neuronal function.

We have previously shown opposite sex differences in GN-induced alterations in myelin gene expression, which is specifically observed in the PFC of adolescents, with increased expression in GN males but decreased expression in GN females [8]. In comparing gene expression in juveniles, our data further suggest that the opposite sex differences also exist in the striatum during adolescent development, as these genes in GN-treated males were changed from downregulation in juveniles to upregulation in adolescents, whereas they were altered from upregulation in juvenile females to normal expression in adolescent females. Together, our data suggest that GN increases central myelination in adolescent males but decreases it in the females in all the brain regions examined. The opposite sex differences may result from the surge of gonadal hormones during puberty, because gonadal hormones stimulate OLG development and myelination [23, 43]. Gestational nicotine exposure delays the onset of puberty in female animals [32], whereas early onset of puberty in males has been linked to MSDP [18].

In contrast to those seen in the adolescents, the sex differences in juveniles were observed mainly in the striatum, with opposite sex differences especially in the NAc. The sex differences in the juvenile striatum were opposite to those in adolescents, which were characterized by either downregulation or normal myelin gene expression in GN-treated males but upregulation in GN females. The observation of sex differences in juveniles suggests a mechanism independent of pubertal gonadal hormone surges. Many studies have suggest that sexual dimorphism in the brain results not only from gonadal hormones but also from sex chromosomes (see [1] for a review). Plp1, one of the major myelin protein genes, is an X chromosome-linked gene [20], although it is still not clear whether it is related to observed sex differences. Heterogeneity in brain development may contribute to the brain region differences, as PFC has prolonged postnatal development and matures relatively later than other brain regions [25, 42].

By examining young adults, our data showed that GN's effect continued into adulthood, especially in adult females, suggesting that females are more vulnerable to GN's effects than are males. A decrease in myelin gene expression in the cortex and striatum has been observed in drug abusers and patients with schizophrenia, bipolar disorder, and major depressive disorder [41]. The downregulation of myelin genes in GN-treated female adults implies that females are susceptible to these disorders as a result of a long-term effect of prenatal exposure to smoking. Given the role of gonadal hormones in myelination [9], a close monitor on the estrous cycle and a further study on later ages will help to understand whether the GN's effects on adults are related to hormone changes.

Together, our data show that GN induced abnormal myelin gene expression throughout postnatal development and adulthood. Myelin deficits are observed in many psychiatric disorders and drug addiction [14]. These disorders are evidenced by unique symptoms during adolescence. For example, the onset of major depression, bipolar illness, and schizophrenia often is observed in adolescents [36]. Other syndromes with a childhood onset, such as attention deficit hyperactivity disorder and Tourette's syndrome, frequently either remit or change symptomatology during the adolescent period [5, 38]. Although abnormal myelin gene expression was observed in juveniles and adults, the effects on adolescents were quite different, which correlates with the unique symptoms of psychiatric disorders in adolescents.

Highlights.

• Postnatal myelin gene expression was examined in rats exposed to gestational nicotine (GN).

• Myelin gene expression in the brain was altered in GN-treated juveniles, adolescents, and adults.

• Age, brain region, and sex differences were observed in GN's effect on myelin gene expression.

• The myelin gene expression response to GN in adolescence is unique.

• Long-term effects on myelin gene expression were observed in female but not male adults.