Effect on the expression of drd2 and drd3 after neonatal lesion in the lymphocytes, nucleus accumbens, hippocampus and prefrontal cortex: comparative analysis between juvenile and adult Wistar rats

Neonatal lesion in the ventral hippocampus (NLVH) is a validated animal model to study schizophrenia from a neurodevelopmental perspective. This animal model is also used to investigate how neonatal lesions may alter the genetic expression of dopaminergic receptors. The present study compares mRNA expression levels of dopamine receptors (drd2 and drd3) in lymphocytes and brain of NLVH animals at two different age stages: young and adult. The NLVH procedure was performed on 20 male Wistar rats at postnatal days 5–7. The mRNA expression levels of drd2 and drd3 genes in lymphocytes, nucleus accumbens, hippocampus and prefrontal cortex were measured and analyzed at postnatal days 45 and 90. The results were compared and contrasted with respective sham groups. In lymphocytes, only in NLVH-adult group we observed drd2 mRNA expression, while drd2 mRNA expression was not observed in the NLVH-juvenile rats; on the other hand, the drd3 mRNA expression did not show significant statistical differences. In hippocampus no differences were observed between drd2 mRNA or drd3 mRNA expression when comparing juvenile/adult shams with NLVH groups. In the prefrontal area, a decrease in drd2 mRNA expression levels were observed in the NLVH-adult group (F(1,3) = 52.83, p = 0,005) in comparison to the sham-adult group. Finally, in the nucleus accumbens, a strong decrease of drd3 mRNA expression was observed in the NLVH-adult group in comparison to the sham-adult group (F(1,3) = 123,2, p < 0.001). Our results show that differences in drd2 and drd3 mRNA levels in NLVH-adults are patent when compared to the sham-adult group or with the NLVH-juvenile group. These findings suggest that the expression levels may be regulated during adulthood, leading to behavioral and neurochemical changes related to schizophrenia. Therefore, more studies are necessary to determine the role of dopamine receptors as possible molecular markers for neurodevelopmental changes associated with schizophrenia.


Background
Schizophrenia is a mental illness which causes a heavy economic burden to society; it affects 1 % of the world population, and it usually develops during the reproductive stage of the person and remains present throughout his/her life [1,2]. The difficulties to obtain adequate human brain-samples and the increasing necessity to study schizophrenia have led to the development of several animal models. These animal models can be divided into three categories: pharmacological models, genetic models (induced genetic mutations or deletions) and neurodevelopmental models (induced by a natural or neurotoxic injury or by environmental factors during neurodevelopment) [3][4][5].
The neonatal lesion in the ventral hippocampus (NLVH) model in animals is based on a neurodevelopmental hypothesis implicating that at an early age (probably during the prenatal stage) fundamental alterations may induce the occurrence of schizophrenia [6][7][8]. For instance, Tseng et al. (2009) suggest that NLVH disrupts the dorso-lateral cortical morphogenesis, since these authors observed that the dopaminergic pathway sets off from the ventral hippocampus to the dorso-lateral area in rat embryos [9]. This effect is more evident in young rats whereas during adolescence rats lose the drd2 modulation in cortical interneurons, as well as the onset of cortical maturation [10]. These findings increase the possibility that maturation of local inhibitory circuits within the PFC may be caused by NLVH [11]. In mental diseases, particularly those treated with antipsychotic medication (including schizophrenia), dopaminergic receptors are of particular interest [3,4]. The gene expression of dopamine receptors has been directly studied in the brain, and it has also been monitored in other cell populations such as lymphocytes [12]. Therefore, peripheral dopamine receptors may serve as representative markers for changes occurring within the central nervous system associated with neuropsychiatric syndromes such as depression and schizophrenia [13].
There are various hypotheses trying to explain the impairment in dopamine transmission [14]. This monoamine dysfunction may be due to immuneinflammatory or neuroendocrine causes or to neuroplasticity malfunctioning [15]. Some authors ascertain that dopamine can directly affect the immune system. They suggest that further research on animal models must address not only the effects of dopaminergic substances on peripheral expression of dopamine receptors, but also the correlation between the regulation of central and peripheral dopamine receptors [16,17]. To explore this possibility the present study compares mRNA expression of dopamine receptors (drd2 and drd3) in lymphocytes and in some areas of rat brain with NLVH at two different age periods: young and adult.

Animals
The cohort of animals in this report is the same used in a previous study [18]. Twenty male Wistar rats were used in this study; all animals came from pregnant rats in our laboratory. They were individually housed in breeding cages under a 12-h light/12-h dark cycle. The sample was divided into four groups: 1) NLVH-juvenile group, 2) shamjuvenile group, 3) NLVH-adult group and 4) sham-adult group. Each group was housed in a cage and kept under an inverted 12-h light/12-h dark cycle (lights off at 10:00 a.m.) and controlled temperature (22.2°C). Testing was routinely performed between 10:00 a.m. and 3:00 p.m.; animals had free access to food and water throughout the experiment [5]. The local committee of ethics on animal experimentation approved the experimental procedures, which complied with the regulations established in the Mexican official norm for the use and care of laboratory animals, "NOM-062-ZOO-1999", as well as the regulations of the Ethics Committee of the International Association for the Study of Pain [19].

Neonatal ventral hippocampus lesion
Bilateral NLVH was performed in neonatal specimens as described by Lipska et al. [20]. Litters of four to eight male pups were grouped and submitted to surgical alteration on postnatal days five to seven (PD 5-7) (weight 10-13 g). Pups were randomly assigned to lesion or sham status and anesthetized with hypothermia by placing the animals on ice for 10-15 min. Pups were then taped to a platform fixed to a stereotaxic Kopf instrument and skin incisions overlying the skull were made. Bilateral ventral hippocampus lesions were performed by infusing 0.15 μl/min (over a period of 2 min) of ibotenic acid (Sigma, St Louis, MO, USA) (lesion) or PBS solution (for the sham groups) through a Hamilton needle in the following coordinates relative to Bregma: anteriorposterior (AP) -2.0; medium lateral (ML) ±2.5, and dorsum-ventral (DV) -3.3 in relation to Bregma [21]. We had successfully established the rat NLVH model as reported elsewhere [18].

RNA extraction
The animals were sacrificed by rapid decapitation at 45 and 95 days old. Following this procedure, their brains were dissected and the following regions were selected: prefrontal cortex, nucleus accumbens and hippocampus. Trunk blood was collected and lymphocytes were isolated by differential centrifugation using a Ficoll density gradient centrifugation and stored at −70°C until use. Subsequently, RNA extraction for each brain region was performed following the Trizol-method (Life Technologies) according to the manufacturer's instructions. The RNA obtained was quantified by plaque fluorometry utilizing fluorescent dyes to determine the concentration of nucleic acids in a simple using the Quant-IT Ribo-Green RNA assay (Invitrogen) according to the manufacturer's instructions, as well as a Fluoroskan Ascent equipment (Thermo Electron Corporation, Vantaa, Finland). The cDNA was synthesized using Super Script III First-Strand Synthesis Supermix (Invitrogen) according to the manufacturer's instructions. The quantification of drd2 and drd3 expression was performed through TaqMan Gene Expression Assays using the Applied Biosystems 7900HT Fast Real-Time PCR Systems. Relative expression was evaluated through 18S rRNA gene as the endogenous control. The comparative C T method (2ΔΔCT) was used to calculate drd2 and drd3 expression levels in the brain samples and lymphocytes.
drd2 and drd3 expression assays Gene expression of drd2 and drd3 was determined by real time PCR, running the cDNA reactions mixtures on a 4900 Real Time PCR System (Applied Biosystems). The initial cDNA quantity was 25 ng, although increasing concentrations were also used (50, 100 and 200 ng). We utilized TaqMan® rat probes drd2-10 FAM (Rn00561126-m1) and drd3 FAM (Rn00691132-m1) (Applied Biosystems), in multiple assays (by triplicate), using ribosomal RNA as endogenous gene (TaqMan Ribosomal RNA Control Reagents VIC® dye; Applied Biosystems). Data were analyzed with the C T comparative method (2ΔΔCT). All the experiments were run in triplicate. Ventral hippocampus lesions were verified by means of histological stained sections for both juvenile and adult stages.

Statistical analysis
Data are presented as mean ± SEM. For each region: lymphocytes, hippocampus, prefrontal area and nucleus accumbens a Two-Way ANOVAs was performed to compare the sham and the NLVH groups. To compare juveniles against adults, a two-way ANOVA was performed followed by Bonferroni test for post hoc. The level of statistical significance was established at p < 0.05.

Results
drd2 and drd3 mRNA expression levels in lymphocytes of juvenile and adult groups Gene expression and quantification of drd2 mRNA in lymphocytes were higher in NLVH-adult rats than in the sham-adults (where no drd2 mRNA expression was observed). This means that the expression of this marker was only present when the lesion procedure was performed (Fig. 1). In the sham-adult group, only the endogenous expression of the 18S rRNA gene was detected. In the juvenile group no expression was observed. In consequence, the expression of the drd2 mRNA in the NLVH-group adults showed major expression that in NLVH-juvenile groups.

drd2 and drd3 mRNA expression levels in prefrontal area of juvenile and adult groups
In the prefrontal cortex (Fig. 3), a difference was observed when comparing the adults, as the sham group showed higher levels of drd2 mRNA expression than the NLVH group (F(1,3) = 52.83, p = 0.005). However, no differences of drd2 mRNA expression levels were observed in shamjuveniles when compared with the NLVH-juveniles (F(1,3) = 5,5, p = 0.1). When a comparison between juvenile and adult groups was performed, we did not observe differences between the NLVH groups (mean diff. 6.53 CI diff:-1.12--14.19, p > 0.05) or between the sham groups (mean diff. 2.64 CI diff:-4.96 -10.35, p > 0.05).
When the drd3 mRNA expression levels were evaluated in adults, a marked decrease of drd3 mRNA was observed in NLVH-adults when compared with sham-adults (F (1,3) = 123.2, p < 0.001) (Fig. 4). Between the juvenile groups no differences were observed (F (1,3) = 0.074, p = 0.45). Finally, the expression of drd3 mRNA in the sham-adult group was higher than in the sham-juvenile group (mean diff. -5.25, p < 0.05); whereas the comparison between NLVH-juveniles and NLVH-adults showed a lower expression of drd3 in the adult group (mean diff. 12.48, p < 0.01).

Discussion
The present study provides important evidence about the role of drd2 and drd3 genes in lymphocytes and in some brain areas (hippocampus, prefrontal cortex and nucleus accumbens) in NLVH rats at two different age periods: young and adult. Our study showed that the drd3 mRNA expression levels in lymphocytes were detected from postnatal day 45. In contrast, drd2 expression was only detected in the NLVH-adult group (90 days of age); which may indicate late neurodevelopmental effects. In rats, synaptic pruning in drd1 and drd2 receptors occurs during the peri-adolescent period (postnatal days 42-44). It has been described that after this pruning, the onset of schizophrenia commonly occurs [22,23]. In the juvenile stage no expression changes were found in any of the tissues studied. Previously, Flores and colleagues conducted a study of dopamine receptors expression using the NLHV model and autoradiography at 41 days of age and found no changes in the expression of drd1, drd2 and drd3 receptors [24]. El-Rawas et al. also evaluated drd2 expression by immunohistochemistry in several brain regions and only encountered changes in the putamen [25]. In our evaluation, only changes in adult rats were detected (Figs. 1, 3 and  4). These findings suggest that the expression levels of some dopamine receptors are regulated during adulthood, leading to behavioral and neurochemical changes related to schizophrenia.
Regarding the group of NLHV-adult rats, a decreasing trend for drd3 and a clear decrease in drd2 mRNA expression was observed in the prefrontal cortex compared to sham-adults (Fig. 3). Flores et al. used autoradiography and reported no changes in expression observed in this molecule in animals at 62 days of age [24,26]. Possibly, the drd2 expression may be observed at later ages, since we detected these differences only after 90 days. The regulation of drd2 expression in the prefrontal cortex of the adult rats group may indicate late neurodevelopmental effects. Cognitive deficits in humans have been correlated with an imbalance of D1 and D2 receptors in the prefrontal cortex [27,28]. The prefrontal cortex, hippocampus and the developmentally dependent synaptic pruning in the juvenile stage may contribute to the present results [22]. In rats, neuronal pruning of drd1 and drd2 receptors occurs during the peri-adolescent period (42-44 days). In humans, after this pruning takes place, it is possible to observe the symptoms of schizophrenia [22,23]. Furthermore, the development of the prefrontal cortex in the early gestation stage and in adolescence represent two critical periods of vulnerability for schizophrenia in humans [29].
In the case of the mRNA expression in the hippocampus, no changes were observed for drd2 or drd3. However, with regard to the accumbens core, a clear decrease in the drd2 transcript was observed in adults against juveniles (Fig. 4), possibly due to neuronal pruning, since the dopamine hypothesis suggests a disturbed and hyperactive dopaminergic signal transduction; the evidence indicates that dopamine receptors are produced in excess before puberty (day 40). Moreover, Teicher et al. reported that at 40 days of age there is a slight decrease in drd2 receptors in the nucleus accumbens [22]. Also, a decrease of thedrd3 expression was observed in the NLVH-adults, as reported by Flores et al. when they analyzed rats at 63 days of age using autoradiography [24]. Several reports of reduced drd3 mRNA in schizophrenic brains further support the potential implication of this receptor subtype in this disorder [30,31].
We consider that when our rats reached adulthood, the drd2 was expressed only as a response to the lesion performed when neonates. In addition, we observed a reduction of the drd2 expression in the prefrontal cortex in NLVH-adult rats. Conversely, in lymphocytes we encountered high drd2 expression levels also in NLVH-adults. These preliminary results appear contradictory, which may indicate that blood changes do not reflect brain changes in the expression of the message for this receptor. Therefore, it is important to further investigate expression profiles in other brain areas represented in schizophrenia. On the other hand, it was not possible to detect drd2 expression in the sham groups; this is consistent with other reports, which indicates that although all five dopaminergic receptors are present in lymphocytes of peripheral blood, the drd2 expression is not observed in healthy animals without NLVH manipulation (sham groups) [16]. Therefore, drd2 expression may be directly related to NLVH in this study.
To date, there are few reports on dopaminergic receptors using the NLVH model. Using microarray technology, drd2 over-expression has been observed in patients; hence this dopamine receptor has been proposed as a peripheral blood biomarker [32,33]. Another study showed that drd2 mRNA levels in lymphocytes of peripheral blood were correlated with positive symptoms in acute schizophrenia patients [34]. The analysis of dopamine receptors in lymphocytes of peripheral blood could be a useful tool for assessing the properties of dopaminergic function in psychopathological traits of schizophrenia [35,36]. Finally, further studies that allow the analysis of behavioral alterations in rats are needed to confirm the findings of this study.

Conclusions
We encountered significant differences in the effect of NLVH on drd2 and drd3 mRNA expression levels among the brain regions analyzed in rat adulthood. These findings suggest that expression levels of some dopamine receptors may be regulated during adulthood, leading to behavioral and neurochemical changes associated with schizophrenia. However, more studies are necessary to establish whether changes in drd2 mRNA expression levels could represent a molecular marker for neurodevelopmental changes related to schizophrenia.