Neurochem Res (2011) 36:1740–1749 DOI 10.1007/s11064-011-0489-z
ORIGINAL PAPER
Prenatal Amphetamine Exposure Effects on Dopaminergic Receptors and Transporter in Postnatal Rats Gonzalo Flores • Marı´a de Jesu´s Go´mez-Villalobos Leonardo Rodrı´guez-Sosa
•
Accepted: 26 April 2011 / Published online: 25 May 2011 Ó Springer Science+Business Media, LLC 2011
Abstract We investigated the influence of prenatal amphetamine exposure (PAE) on dopamine (DA) receptors, and dopamine transporter (DAT) in various striatal and limbic subregions and locomotor activity induced by novel environmental conditions and amphetamine at two postnatal ages, 35 days old (prepubertal) and 60 days old (postpubertal). Experiments were carried out on pregnant female Sprague–Dawley rats, which were daily injected with either d-amphetamine sulfate (1 mg/kg) or saline solution (0.9%) for 11 days, from gestation day 11–21. In PAE rats compared to control we found the following: at pre-pubertal age, an enhancement of DA D1 in the dorsolateral area of the caudate-putamen (CPu), CPu-ventral and shell of the nucleus accumbens (NAcc) with a decrement of the DA D3 receptors in NAcc, olfactory tubercle (OT), and the islands of Calleja (IoC); whereas at postpubertal age, an increase in the levels of DAT in the NAcc and fundus of the CPu, and OT along with a decrease in the expression of DA D2 receptors only in the NAcc shell were found in PAE rats compared to control. In addition, amphetamine induces a marked decrease in locomotor activity at postpubertal age in rats with PAE. These results suggest a differential effect of amphetamines on the DAT mechanism of the nervous system during embryonic development of animals with implications in behavior and drug addictions at adulthood age. G. Flores (&) M. de Jesu´s Go´mez-Villalobos Instituto de Fisiologı´a, Universidad Auto´noma de Puebla, 14 Sur 6301, San Manuel, 72570 Puebla, CP, Me´xico e-mail:
[email protected];
[email protected] L. Rodrı´guez-Sosa Departamento de Fisiologı´a, Facultad de Medicina, Universidad Nacional Auto´noma de Me´xico, Av. Universidad No. 3000. Ciudad Universitaria, 04510 Me´xico, DF, Me´xico
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Keywords Amphetamine Prenatal Dopamine receptors Dopamine transporter Locomotor activity Postnatal Prepubertal Postpubertal
Introduction Amphetamines are psychostimulants with a target in the monoaminergic system. These drugs reverse the action of monoamine transporters and enhanced lifetime of dopamine (DA) after release as well as norepinephrine and 5-Hydroxytriptamine (5-HT, serotonin) into the synaptic cleft, increasing their availability to act upon post-synaptic receptors. Re-uptake blocking and degradation of these neurotransmitters increases their concentrations and lifetime in the synaptic cleft. Amphetamines are routinely ingested, injected and smoked. The frequency of the use of amphetamine in many regions of the United States has been reported [1]. Studies on the effects of the use of amphetamines during pregnancy, suggest that newborn babies and children between 4 and 8 years of age present persistent behavioral abnormalities; for example, aggressive behavior, poor adjustment and low values of weight and length, which correlate with socio-environmental factors [2, 3]. Whereas at peripubertal age, children born to women who used amphetamines during pregnancy showed one grade lower at school with changes in weight and height compared to their peers; for instance, girls are shorter and lighter [4]. Surprisingly, there is no further information at adult age. There is more information about the effects of other psychostimulants such as cocaine and methamphetamine in children and pre-pubertal and adult animals exposed to these drugs during prenatal development. For example, children exposed to methamphetamine
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show smaller caudated putamen (CPu) and hippocampus volumes with a reduced amount of D2 dopamine receptors, as well as a decreased dopamine transporter (DAT) density [5, 6]. They also obtain lower scores in different neurocognitive tests such as sustained attention, long-term spatial and verbal memory, as well as visual motor integration [5, 6]. In contrast, prenatal rats exposed to methamphetamines exhibit enlarged striatal volumes with lower levels of dopamine and serotonin transporters and reduced dopamine D2 receptors in the CPu [6]. In contrast to prenatal amphetamine exposure (PAE) in rats, there is more information about the effect of the neonatal rats exposed to amphetamine. A recent report suggests that amphetamine produced a dose-related decrease in body weight without effect on brain weight [7]. In addition, stereology analysis of the hippocampus indicated that neonatal amphetamine exposure did not alter cell number in this structure [8]. However, low levels of tyrosine hydroxylase enzyme in the Cpu and nucleus accumbens (NAcc) with changes in the DA receptors have been reported in rats with neonatal amphetamine exposure [9]. In addition, locomotor activity in some open-field testing parameters are lower with high doses of amphetamine injected at neonatal age [10]. The first aim of the present study was to assess whether prenatal amphetamine exposure (PAE) produces longlasting effects on locomotor activity induced by novel environmental conditions and amphetamine at two critical ages, pre-pubertal- (posnatal day 35, PD35) and postpubertal-rats (PD60). The second objective was to assess the effect of prenatal amphetamine exposure at prepubertal and post-pubertal age on DA receptors and DAT in the striatum and limbic sub-regions by using autoradiograpy. Our findings suggest that PAE may modify the levels of DAT, and those of locomotor activity induced by amphetamine at post-pubertal age (PD60).
Experimental Procedure Animals Fourteen pregnant Sprague–Dawley rats were obtained at gestational day 10 from our facilities (University of Puebla). Rats were individually housed in controlled temperature (20–23°C), humidity (40–50%), and environment on a 12–13 h light–dark cycle with free access to food and water. All experimental procedures described in the present study were in agreement with the ‘‘Guide for Care and Use of Laboratory Animals’’ of the Mexican Council for Animal Care (Norma Oficial Mexicana NOM-062-ZOO-1999) and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
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Amphetamine Administration The procedure began on day 11 of pregnancy; dams were randomly kept under two different conditions. Each animal received a subcutaneous (s.c.) injection of either vehicle (1 ml/kg saline), or d-amphetamine (1 mg/kg), once daily from pregnancy day 11 to pregnancy day 21. Offspring Development After birth, ten offspring per mother were kept in their home cages until weaning at PD 21. Subsequently, only male rats were kept and left undisturbed until PD35 or PD60. The locomotor activity in a novel environment and the autoradiographic studies were then assessed at two postnatal ages (PD35 and PD60) as described below. Therefore, four groups were formed: control rats of 35 days born to vehicle mothers (prenatal-vehicle exposure, PVE35), control rats of 60 days born to vehicle mothers (PVE60), experimental rats of 35-days born to amphetamine-exposed mothers (prenatal-amphetamine exposure, PAE, PAE35) and experimental rats of 60-days born to amphetamine-exposed mothers (PAE60). Locomotor Activity Locomotor activity was tested between eight and twelve o’clock in the morning and was monitored in 16 individual cages (20 9 40 9 30 cm). Each one was equipped with 8-photo-beam detectors connected to a computer counter (Tecnologı´a Digital, Me´xico). At pre-pubertal or postpubertal age (PD35 or PD60, n = 7–8 animals per group), each male rats was assessed with the following protocol: (1) after exposure to a novel environment: unacclimatized rats were placed in an activity box for 60 min, and the locomotor activity score was recorded. (2) After exposure to vehicle effect, animals were injected first with 1 ml/kg 0.9% NaCl, s.c., and moved to an activity box for 60 more minutes, and (3) after d-amphetamine injection: animals were injected with a 1 mg/ml solution of d-amphetamine sulfate (Sigma, St. Louis, MO, USA) dissolved in 0.9% NaCl (1 mg/kg free base, s.c.). The locomotor activity was recorded for the next 120 min. All movements were quantified and analyzed with the statistics-software package Graph Pad 4.0. Receptor Autoradiography Another cohort of rats was used to study DA D1-like, D2-like and D3 receptors and DAT binding. At PD35 and PD60, rats from prenatal amphetamine or vehicle exposed (n = 4–6 per group) were sacrificed by decapitation and the brains were rapidly removed, frozen in isopentane,
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maintained at -40°C for 15 s, and stored at -80°C until they were used. Frozen brains were sectioned at 16-lm thickness coronally using a Leica CM1100 cryostat. Sections were collected on cleaned, gelatin-coated microscope slides (4 sections/slide), thaw-mounted, vacuum-dried at 4°C overnight and then stored at -80°C until the day of the experiment. Coronal brain sections taken at the level of CPu and NAcc (plates 10–12 of Paxinos and Watson Atlas, [11]) were used as follows: For D1-like receptor binding, sections were first incubated for 10 min at room temperature in a buffer containing 50 mM Tris–HCl pH 7.4, 154 mM NaCl, 1 mM EDTA, and 0.1% bovine serum albumin. Sections were then incubated for 90 min at room temperature in the same buffer with the addition of 1 nM 3H-SCH23390 (85 Ci/mmol) and 30 nM ketanserin (to block possible binding of the ligand to serotonergic 5-HT2 sites). Nonspecific binding was determined on adjacent brain sections by adding 1 lM (?)-butaclamol to the buffer. Incubations were stopped by dipping the slides into the icecold buffer and rinsing twice for 10 min in buffer. After a final dip in ice-cold distilled water, slides were dried at room temperature and exposed to Kodak-BioMax MR Film (No. 891-2590) for 7 days alongside microscale-calibrated tritium standards. The same protocol was used for D2-like receptor binding, except that 1 nM 3H-spiperone (99 Ci/ mmol) was added to the buffer and the slides were exposed for 12 days. 3 H-7-OH-DPAT binding to D3 receptor was assessed as previously described [12, 13]. Tissue sections were first pre-incubated for 30 min in 50 mM Tris–HCl pH 7.4 containing 100 mM NaCl and 300 lM GTP. Sections were then incubated for 2 h at room temperature with 2 nM {3H}-7OH-DPAT, 50 mM Tris–HCl, pH 7.4, 100 mM NaCl, 300 lM GTP and 10 lM 1,3-di(2-5-tolyl) guanidine (to block binding to sigma site). Dopamine (1 lM) was used to determine non-specific labeling. Incubations were terminated by washing the brain sections twice for 10 min each in ice-cold 50 mM Tris–HCl, pH 7.4. After a brief dipping in ice-cold distilled water, brain sections were rapidly dried and exposed to Kodak- BioMax MR Film (No. 891-2590) for 8 weeks. DAT binding autoradiography was assayed in accordance with previous protocols [13]. Tissue sections were first pre-incubated for 20 min at 4°C in 50 mM Tris–HCl pH 7.4 containing 100 mM NaCl, the slides were incubated at 4°C for 2 h in the same buffer containing 10 nM {3H}WIN-35428 (84 Ci/mmol). Non-specific binding was determined on adjacent brain sections by adding 1 lM GBR-12909 to the binding buffer. Incubations were terminated by washing the brain sections twice for 1 min each in ice-cold 50 mM Tris–HCl, pH 7.4. After a brief dipping
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in ice-cold distilled water, brain sections were rapidly dried and exposed to Kodak- BioMax MR Film (No. 891-2590) for 8 weeks. For data analysis, the films from the autoradiography assays were analyzed using a computerized image-analysis system (NIH ImageJ software version 1.44; http://rsb. info.nih.gov/ij/). The binding data was analyzed in caudateputamen (CPu) and NAcc subregions and the olfactory tubercle (OT) according to Paxinos and Watson [11], namely the dorsolateral, dorsomedial, ventral, and fundus of the CPu (CPu-DL, CPu-Dm, CPu-V and CPu-fundus), the shell and core of NAcc, and OT are expressed as fmol/ mg wet tissue weight. Statistical Analysis Data from locomotor activity and autoradiographic analysis were analyzed using a two-way ANOVA analysis, followed by Tukey tests for post-hoc comparisons, with prenatal amphetamine exposure effect and age as independent factors (P \ 0.05 was considered significant).
Results Locomotor Activity The effect of PAE on spontaneous locomotor activity in a novel environment at PD35 and PD60 is illustrated in Fig. 1. All animals (PVE and PAE) initially showed increases in locomotion, reflecting an active exploratory behavior in a novel environment. Then, the locomotor activity gradually declined in 40–60 min to a stable level (Fig. 1a). A two-way ANOVA analysis of the total locomotor activity data for the entire 60 min revealed that the locomotion was significantly affected by interaction of PAE with age (F1,26 = 4.46, P = 0.04). Post-hoc tests showed that at postpubertal age, rats with PAE only present a trend to increase the spontaneous locomotor activity compared to its corresponding control group (P = 0.08). Amphetamine injection caused a marked decrease in locomotor activity at PD60 in rats with PAE (Fig. 1c). A two-way ANOVA analysis revealed the significance of the interaction between PAE and age (F1,26 = 9.2, P \ 0.01) (Fig. 1c). Post-hoc analyses revealed that rats with PAE at postpubertal age were less active after d-amphetamine injection compared to their corresponding PVE-control groups (P \ 0.05) (Fig. 1c). No significant effect of vehicle injection (two-way ANOVA, PAE; F1,26 = 2.5, P = 0.12, age; F1,26 = 0.03, P = 0.85, and interaction of PAE with age; F1,26 = 0.09, P \ 0.76) was observed in these groups (Fig. 1b).
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Fig. 1 Locomotor activity (mean number of beam interruptions ± SEM; n = 8–10 per group) in a novel environment (a), after vehicle (saline) (b) and d-amphetamine administration (1 mg/kg, s.c) (c) of prenatal amphetamine exposure (PAE) or prenatal vehicle exposure (PVE) control animals tested either at PD35 or PD60. Locomotor activity was determined as described in Materials and Methods. At PD35, PAE animals did not differ from PVE-control animals at any testing interval. In contrast, at PD60, PAE rats were less active than PVE-control rats after d-amphetamine administration
Receptor Autoradiography D1-like receptors as measured by [3H] SCH23390 binding are distributed throughout the dorsal and the ventral part of the striatum (Fig. 2). PAE animals showed no significant difference in D1 receptor binding at PD60 when compared with PVE60 animals. However, when both groups were compared at PD35, a significant increase in D1 receptor density was observed in the CPu-DL (2-way ANOVA, PAE; F1,14 = 5.81, P \ 0.05, Age; F1,14 = 59, P \ 0.01; post-hoc test P \ 0.05) and CPu-V (2-way ANOVA, PAE; F1,14 = 6.8, P \ 0.05, Age; F1,14 = 54, P \ 0.01; PAE
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and Age interaction; F1,14 = 5.8, P \ 0.05; post-hoc test P \ 0.01), as well as NAcc shell (2-way ANOVA, PAE; F1,14 = 5.3, P \ 0.05, Age; F1,14 = 40, P \ 0.01; posthoc test P \ 0.01) of PAE animals (Fig. 2b, d and g). [3H] spiperone labels DA receptor subtypes belonging to the D2- like family (D2, D3, and D4). D2-like receptor density shows a dorsoventral density gradient maximal in the CPu region (100–150 fmol/mg). The distribution and density of D2-like binding sites do not differ significantly between PVE and PAE animals at prepubertal age. However, at PD60, a small (20%) but significant decrease in D2 receptor density was observed in the NAcc shell (2-way ANOVA PAE; F1,16 = 8.04, P \ 0.05, post-hoc test P \ 0.05) of PAE animals (Fig. 3g). DA D3 receptors, measured by [3H]-7-OH-DPAT binding, were found mainly in the IoC, OT and the shell and core of the NAcc (Fig. 4). A low level of D3 receptor expression was also detected in the CPu. D3 receptor levels of the shell and core of the NAcc were significantly higher in PD60 than PD35 in PAE animals (P \ 0.01). Compared with PVE controls, PAE35 animals showed a significant decrease in D3 receptor levels in the shell (2-way ANOVA, PAE; F1,16 = 7.8, P = 0.01; Age; F1,16 = 5.6, P \ 0.05, post-hoc test P \ 0.05) and core (2-way ANOVA, PAE; F1,16 = 6.3, P \ 0.05; Age; F1,16 = 7, P = 0.01, post-hoc test P \ 0.05) of the NAcc, OT (2-way ANOVA, PAE; F1,16 = 8.3, P = 0.01; Age; F1,16 = 4.8, P \ 0.05; PAE and Age interaction; F1,16 = 5, P \ 0.05; post-hoc test P \ 0.01) and the IoC (2-way ANOVA, PAE; F1,16 = 8.7, P = 0.01; post hoc test P \ 0.05) (Fig. 4c, d, e and f). The DAT, as measured using [3H]-WIN-35428, are distributed throughout the dorsal and ventral part of the striatum. The distribution and level of DAT binding sites do not differ significantly between the PVE-control and PAE animals at PD35 (Fig. 5). At PD60, however, the level of DAT binding sites was significantly increased (34%) in the CPufundus (2-way ANOVA, PAS; F1,16 = 7.9, P = 0.01; Age; F1,16 = 5, P \ 0.05; PAE and Age interaction; F1,16 = 5.6, P \ 0.05; post-hoc test P \ 0.01), (30%) in the core (2-way ANOVA, PAE; F1,16 = 6, P \ 0.05; Age; F1,16 = 4.5, P \ 0.05; post-hoc test P \ 0.05) and (29%) in the shell of the NAcc (2-way ANOVA, PAE; F1,16 = 4.8, P \ 0.05; post-hoc test P \ 0.05), and (53%) in the OT (2-way ANOVA, PAE; F1,16 = 5.7, P \ 0.05; PAE and Age interaction; F1,16 = 10, P \ 0.01; post-hoc test P \ 0.01) in PAE animals (Fig. 5e, f, g and h).
Discussion The aim of the present study was to investigate the effects of PAE on locomotor activity, dopamine receptors and transporter at two ages, pre-pubertal (PD35) and
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Fig. 2 Quantitative autoradiographic analysis of [3H]- SCH-23390/ dopamine (DA) D1-like receptor binding in prenatal amphetamine exposure (PAE)- and prenatal vehicle exposure (PVE)-rats of the nucleus accumbens (NAcc), caudate–putamen (CPu) and olfactory tubercle (OT). Each value represents the mean ± SEM of 4–5
animals per group. Pre-pubertal (PD35) animals with PAE displayed an enhanced DA D1 in CPu-DL, CPu-V and shell of the NAcc. Finally, at post-pubertal age, PAE animals did not differ from PVEcontrol animals in any region studied
post-pubertal (PD60). Interestingly, at pre-pubertal age, PAE caused DA receptor changes enhancing DA D1 in CPu-dorsolateral, CPu-ventral and shell of the NAcc with a decrement of DA D3 receptors in ventral striatum, OT and IoC. On the other hand, at postpubertal age, we observed an increase in the levels of the DAT in the regions that received DA innervations from the VTA [14], together with a decrease in the expression of the DA D2 receptors only in the NAcc shell. In accordance with this data, PAE animals at post-pubertal age have also shown less locomotion in response to 1 mg/kg of the d-amphetamine. Rodents are born at an earlier stage of brain maturation than humans [15, 16]. The gestation period of the rat is only 22 days and is roughly equivalent to the first 2 trimesters of human brain development [17]. During this prenatal period in both humans and rodents, most neuronal cell groups are born and early synaptogenesis occurs [15]. In our present report, we started to administer amphetamine at the gestational day 11, because in rats the central
nervous system begins to form around this gestational day as the neuroepithelium, composed of a ventricle surrounded by actively proliferating cells. This generates waves of neurons and glia that ultimately form the cell populations of the more mature central nervous system [17]. After neurons migrate, they begin to form synaptic connections and chemical transmission soon follows. However, DA and other monoamine neurons are an exception in that the cell bodies and axons can express neurotransmitter before their target neurons are fully differentiated [18]. Locomotor activity induced by psychostimulants such as amphetamine is the result of increases lifetime DA in the synaptic cleft by blocking or reversing the direction of DAT [19–21], which in turn acts on postsynaptic receptors. The DAT is expressed exclusively by DA neurons and are found extrasynaptically on DA axons in CPu and Nacc [22]. In addition, DA receptors are also predominantly extrasynaptic [23–26]. Extracellular DA concentration and
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Fig. 3 Quantitative autoradiographic analysis of [3H]-spiperone/ dopamine (DA) D2-like receptor binding in prenatal amphetamine exposure (PAE)- and prenatal vehicle exposure (PVE)-rats of the nucleus accumbens (NAcc), caudate–putamen (CPu) and olfactory tubercle (OT). Each value represents the mean ± SEM of five
animals. At prepubertal age (PD35), PAE rats did not differ from PVE-control animals in any region studied. Postpubertal (PD60) animals with PAE showed a decreased of DA D2-like receptors in the shell of the NAcc
lifetime after release would be regulated by diffusion, dilution as well as uptake [21]. DA itself can regulate DAT via its interaction with the transporter or presynaptic autoreceptors [27]. Mice lacking DAT exhibit spontaneous hyperlocomotion and are unresponsive to amphetamine [28]. Recent reports suggest that DAT, but not the serotonin transport (SERT), is critical in mediating the reinforcing effects of cocaine. In addition, mice lacking DAT generally failed to acquire and maintain cocaine selfadministration [29] compared to wild-type, SERT-/- mice. Therefore, DAT inhibition may play a role in mediating the long-lasting neural changes associated with drug addiction [30, 31]. The mechanism(s) by which a prenatal amphetamine exposure induces a decrease in the level of DA D2 receptors with an increase in the DAT at postpubertal age is not clear at this time. However, several reports support the hypothesis that DA D2 receptor and DAT are involved in reinforcing the effects of drug abuse [32]. Clinical reports
by using imaging studies show a significant reduction of the DA D2 receptors levels in drugs abuser [33–35]. In addition, human imaging studies link DAT and D2 receptor occupancy with cocaine and amphetamine-induced euphoria [36, 37]. Interestingly, subjects with a positive family history of alcoholism—a predisposing factor to developing the disease—that have higher DA D2 receptor levels, are protected from developing drug dependence [38]. Imaging studies in nonhuman primates have indicated a role for DA D2 receptors in determining individual differences in intravenous cocaine self-administration [39, 40]. Specifically, low D2 receptor availability in the striatum inversely predicts subsequent levels of intravenous cocaine self-administration in rhesus monkeys [40], a result apparently similar to that seen in studies of human cocaine abusers [41]. Furthermore, imaging studies using positron emission tomography in drug-naive rats show that low levels of the D2 receptors in the NAcc predicts impulsivity and greater drug self-administration behavior [42]. More
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Fig. 4 Quantitative autoradiographic analysis of [3H]7-OHDPAT/ dopamine (DA) D3 receptor binding in prenatal amphetamine exposure (PAE)- and prenatal vehicle exposure (PVE)-rats of the nucleus accumbens (NAcc), caudate–putamen (CPu), olfactory tubercle (OT) and the island of Calleja (IoC). Each value represents
the mean ± SEM of five animals. Prepubertal (PD35) animals with PAE displayed a decrease of the DA D3 in the shell and core of NAcc, OT and IoC. Whereas, at postpubertal age (PD60) no differences between PAE- and PVE-rats were found
mutant mice lacking D2-like receptors show high rates of intravenous cocaine self-administration when high doses of cocaine are made available [43]. Therefore, our results are consistent with previous reports that associated low levels of DA D2 receptors in the NAcc with a drug abuse. In comparison, prenatal cocaine or prenatal stress also result in altered postnatal dopaminergic function [44–46]. However, the results are inconsistent: one study states that adult mice with prenatal cocaine exposure (GD 13–21) showed a reduction of DAT in the striatum [44], whereas other reports suggest that prenatal cocaine exposure (GD 18–21) resulted in a transient decrease in DA transporter binding in the dorsal lateral striatum at PD10 [45], with no change at adult age. Leslie et al. [47] found that prenatal cocaine exposure (GD10–21) caused an increase in DAT density from PD1 to PD5; however after PD14 they observed a reduction in DAT. Different prenatal periods of exposure and doses may explain these differences. In the same way, adult mice with prenatal stress also showed a reduction of DAT expression in the striatum [48]. Children with prenatal methamphetamine exposure showed reduced DAT, serotonin transporter density, and DA D2 receptors in the striatum measured by positron emission tomography (PET) [6]. In this respect, our results suggest that adult rats with PAE exhibited an enhanced DAT binding in all of the NAcc (ventral striatum) with a reduction of DA D2 receptors in the NAcc shell, regions innervated by VTA [14]. On the other hand, it has been found that prenatal cocaine from GD8–21, results in an enhancing of DA D2 receptor (27.1%) in NAcc with an increase of DA D3
receptor binding in NAcc (75.2%) and CPu (33.5%) [49]. Whereas, adults rats with prenatal stress also exhibit changes in DA receptors with a reduction of DA D3 receptor binding in both the shell (-16%) and the core (-26%) of the NAcc with an increase (?24%) in DA D2 receptor binding in the NAcc [50]. In addition, neonatal quinquirole (a dopamine D2/D3 agonist) treatment in rats results in a significant increase of DA D2 receptor sensitivity that persists throughout the animal’s lifetime, which is a phenomenon that has also been referred to as ‘‘D2 priming’’ [51–53]. According to our results, neonatal ventral hippocampal lesion, an animal model widely used as a neurodevelopment model that mimics schizophrenialike behaviors, also showed a reduction in DA D3 receptor binding at pre-pubertal age, without changes in amphetamine-induced locomotion compared to sham rats [12]. Cocaine and amphetamine may induce neural changes, including an increase in the density of spines on neuron dendrites in the NAcc [54] with locomotor sensitization [55]. More recently it has been suggested that cocaineinduced dendritic spine changes are correlated with the presence of DAT, because mice lacking DAT did not show an increase in dendritic spine density in the NAcc [30]. In addition, the stereotypy induced by cocaine is also absent in this transgenic mouse [56]. Therefore, DAT enhanced by PAE may have been participating in the greater risk of addictions reported in the children with prenatal psychostimulant drugs exposure [29, 31, 57]. In conclusion, PAE results in altered postnatal dopaminergic function suggesting that at post-pubertal age, behavioral, DAT and DA D2 receptors changes imply
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Fig. 5 Quantitative autoradiographic analysis of [3H] WIN-35428/ dopamine transporter (DAT) binding in prenatal amphetamine exposure (PAE)- and prenatal vehicle exposure (PVE)-rats of the nucleus accumbens (NAcc), caudate–putamen (CPu) and olfactory tubercle (OT). Each value represents the mean ± SEM of five
animals. At pre-pubertal age (PD35), no differences between PAEand vehicle-rats were found, whereas at post-pubertal age (PD60), PAE animals showed an increase of the CPu-fundus, NAcc (shell and core) and OT
limbic structures related to drug addictions. Moreover, PAE has a potent and specific effect on DAT and DA receptors on the NAcc at pre-pubertal and post-pubertal age. Therefore, at adult age, PAE alters DAT and DA D2 receptors with implications in behavior and drug addictions.
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Acknowledgments This study was supported by VIEP-BUAP grant (No. FLAG/SAL11/G) and CONACYT grant (No.138663) to G Flores. We also want to thank Dr. Carlos Escamilla for his help in the care of animals. Leonardo Rodrı´guez-Sosa, Ma. de Jesu´s Go´mezVillalobos and Gonzalo Flores are members of the Researcher National System of Mexico. Thanks to Miss. Annie McDermott and Mr. Jose´ Luis Pe´rez Garcı´a for reviewing the correct usage of English in this manuscript.
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