Neurobiological Effects of Bisphenol A May Be Mediated by Somatostatin Subtype 3 Receptors in Some Regions of the Developing Rat Brain

Rosa Maria Facciolo*,1, Maria Madeo*, Raffaella Alò*, Marcello Canonaco* and Francesco Dessì-Fulgheri{dagger}

* Comparative Neuroanatomy Laboratory of Ecology Department, University of Calabria, 87030 Arcavacata di Rende-Cosenza, Italy; {dagger} Animal Biology Department, University of Firenze, 50100 Firenze, Italy

1 To whom correspondence should be addressed at Comparative Neuroanatomy Laboratory, Ecology Department, University of Calabria, Ponte Pietro Bucci, 87030 Arcavacata di Rende, Cosenza, Italy. Tel. +39-984 492973-4 Fax +39-984 492986. E-mail: rm.facciolo{at}unical.it.

Received July 6, 2005; accepted September 8, 2005


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Considerable attention has been focused on environmental disruptors such as the xenoestrogen bisphenol A, which influences reproductive, developmental, and cognitive activities through its interaction with specific neuromediating systems in an estrogen-like fashion. In the present study, the effects of this xenoestrogen proved to be preferentially directed toward hypothalamic and extrahypothalamic somatostatin receptor subtype 3, which displayed a higher binding affinity of its specific nonpeptide agonist L-796-778 than that of L-779–976 (subtype 2). One type of action, with respect to animals treated with vehicle alone, consisted of a very strong (p < 0.001) decrease of somatostatin receptor subtype 3 mRNA levels in layer V of the frontoparietal cortex of adult rats (Sprague-Dawley) after transplacental and lactational exposure to bisphenol A (400 µg/kg/day). Similarly, such treatment in 7-day-old rats was responsible for a very strong reduction of the subtype 3 mRNA levels in the hypothalamic periventricular nuclei and a strong (p < 0.01) increase of the subtype 3 mRNA levels in the ventromedial nuclei. Moreover, even greater upregulated and downregulated activities were reported when subtype 3 mRNA levels were determined in the presence of receptor agonists specific for distinct {alpha} GABAA receptor subunits ({alpha}1,5). The predominant effects of bisphenol A on somatostatin receptor subtype 3 mRNA levels occurring in an {alpha} GABAA subunit–dependent manner tend to suggest the early modulatory importance of this environmental disruptor on cross-talking mechanisms that are implicated in the plasticity of neural circuits, with consequential influence on neuroendocrine/sociosexual behaviors.

Key Words: nonpeptide agonists; GABAA receptor; xenoestrogens; cortex; hippocampus; hypothalamus.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific concern has begun to increase in response to alarming reports regarding the environmental, reproductive, and overall health hazards of endocrine-disrupting chemicals such as xenoestrogens. Of specific interest is the high-production-volume chemical bisphenol A (BPA), which is widely used in the manufacture of polycarbonate plastics and resins (Howdeshell et al., 2003Go). Because of its broad industrial applications, BPA can enter the body by ingestion or adsorption, and it may mimic estrogenic actions, despite its weaker bioactivity as compared to estradiol (Kitamura et al., 2005Go). Consequently, the estrogen-like actions of this xenoestrogen have accounted for potentially adverse effects, as displayed by both reproductive malformations of offspring (Ramos et al., 2003Go) and the incapacity of offspring to react to certain behavioral stimuli, such as fear-dependent locomotor responses (Negishi et al., 2004Go). As a matter of fact, administration of BPA to pregnant mice during both gestation and lactation, at a dose that is within the range of environmental exposure of humans, induced modifications of exploratory and sociosexual behaviors (Dessì-Fulgheri et al., 2002Go; Farabollini et al., 2002Go). Recently, the beneficial effect of this xenoestrogen via the interaction of estrogenic receptors has aroused particular attention, especially with reference to the promotion of dendritic morphological organization in some brain regions of the developing rat (Shikimi et al., 2004Go). Nonetheless other investigators have displayed the potential risks of spine synapse formation in hippocampal regions in animals exposed to BPA (MacLusky et al., 2005Go).

From the precise correlation between behavioral and neuronal activities, many neurotransmitter systems such as somatostatin and GABA have been shown to be linked to the predominant estrogen-like BPA effects (Facciolo et al., 2002bGo). In mammals, somatostatin has been proven to exert a major inhibitory role on growth hormone (GH) secretion, as well as to regulate various neuroendocrine and cognitive functions such as body temperature, satiety, and memory (Lahlou et al., 2004Go). At present, five different somatostatin receptor subtypes (sst1-5) have been cloned, and all possess a heptahelical architecture that is typical of the G protein–coupled receptors (Csaba and Dournaud, 2001Go). Of the above-mentioned somatostatin receptor subtypes, sst3, which is widely distributed throughout the brain, appears relatively early during development and remains stable throughout adult life; as a consequence, it exerts a key influence on the organization of the central nervous system (CNS) (Thoss et al., 1995Go). In this context, the interference of BPA with protein G–coupled receptors (Ishido et al., 2005Go) through the interaction of estrogen receptors (Quesada et al., 2002Go), as well as the upregulating effects of estrogens on mRNA expression of sst2,3 (Djordjijevic et al., 1998Go) and the co-localization of such somatostatin receptor subtypes to both estrogen and GABA receptors (Arancibia et al., 1997Go; Saha et al., 2002Go), point to sst3 as a major neuromediating target of BPA-dependent actions.

GABA, the other component co-localized with somatostatin receptor subtypes and estrogen receptors, is also considered an important neuronal receptor capable of mediating the bulk of rapid inhibitory synaptic transmission in the CNS. The functional features of GABAA receptors are determined by heteromeric combinations of subunits encoded by 20 genes: {alpha}(1–6), ß(1–4), {gamma}(1–4), {delta}, {varepsilon}, {pi}, {theta}, and {rho}(1–2). Recombination of these subunits forms different binding sites that display various affinity states for drugs such as benzodiazepines, barbiturates, convulsants, and neurosteroids (Barnard et al., 1998Go). In particular, the {alpha} subunit is involved in the assembly of the other subunits, as well as the overall biophysical and pharmacological properties of the GABAA receptor complex (Rudolph et al., 1999Go). Because somatostatin receptor subtypes are tightly coupled to this complex, the above {alpha} subunit becomes a determing cross-talking element in specific BPA-dependent neurophysiological risks, an event that seems to rely, in an estrogenic fashion, on alterations evoked during early developmental stages in the rat (Facciolo et al., 2002bGo).

Thus, it was our intention to determine the type of relationship that is explicated by BPA toward the formation of sst3 mRNA and to understand whether this effect requires the participation of the {alpha} GABAA receptor subunits. A relationship that was previously demonstrated for the mRNA formation of other important endocrine-disrupting targets such as the arylhydrocarbon receptor, which is involved in the mediation of stressor activities in some GABAergic neurons (Hays et al., 2002Go). Moreover, it was interesting to establish a regional specificity of BPA–sst3 interactions in specific female brain regions, with particular attention to hypothalamic sites, because somatostatin receptor subtype effects have been reported in areas such as the hypothalamic periventricular nucleus (Simonian et al., 1998Go), which in females is highly estrogen-enriched (McEwen, 2002Go). In addition, the extrahypothalamic areas, such as the frontoparietal cortex (layer III and V) and the hippocampus, have also been considered in this study because of their elevated densities of estrogen receptors plus their somatostatin receptor subtype–dependent role in memory and spatial tasks, as well as sociosexual and neurovegetative functions (Csaba and Dournaud, 2001Go).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals
For the present study, 32 sexually mature Sprague-Dawley female rats (200–250 g; Charles River, Como, Italy) were maintained on a 12-h dark/light schedule (lights on 1400–0200 h). Subsequently, they were subdivided into groups of four and housed in eight cages with stainless-steel wire lids (vivarium of the Cell Biology Department, University of Calabria) for 3 days for acclimation purposes, as described in previous studies (Facciolo et al., 2002bGo). Of the 32 female rats, 12 received, orally with a pipette, a dose of 400 µg/kg/day of BPA (BPA400; Sigma Chemical, Milan) dissolved in arachis oil at a concentration of 10 µg/ml. Another 12 rats received a lower dose of 40 µg/kg/day of BPA (BPA40) dissolved in the same vehicle, and the remaining 8 animals received only vehicle (OIL = control). Treatment began 8 days before mating and continued throughout mating (5 days), pregnancy, and lactation (42 days). The BPA doses used in this study are similar to those responsible for neuroanatomical morphological—and as a consequence functional—variations (MacLusky et al., 2005Go). These doses are also based on the U.S. Environmental Protection Agency calculated reference dose, which was arrived at by dividing the Lowest-Observed-Adverse-Effect-Level (LOAEL, 50 mg/kg body weight per day) from an earlier chronic toxicity study by an uncertainty factor of 1000. Applying that same uncertainty factor to the No-Observed-Adverse-Effect-Level (NOAEL, 50 mg/kg body weight per day) from a three-generation study in rats (Tyl et al., 2002Go) confirms the safety of the reference dose, 0.05 mg BPA per kilogram body weight per day.

Eight days after the beginning of treatment, a different sexually mature Sprague-Dawley male was assigned to each of the eight cages of female rats for 8 days. Pregnant females that subsequently gave birth to all pups successfully were isolated in single stainless-steel cages where they continued to receive the same BPA treatment until weaning, i.e., postnatal day (PND) 23. To minimize litter effects, at birth one female pup per litter was randomly assigned to a new cage (n = 8 pups of the same age) containing a dam of the same BPA treatment group, as follows: higher dose (BPA400) and lower dose (BPA40). Another 16 pups were culled for the OIL group. For this part of the study and throughout, all experimental data refer to the female offspring at PND 7 (n = 12) and at 55 days of age (adults n = 16) of all treatment groups. The animals were decapitated and their brains were quickly removed for in situ hybridization histochemistry and for in vitro quantitative autoradiography studies.

Animal maintenance and all experimental procedures were carried out in accordance with the Guide for Care and Use of Laboratory Animals issued by the European Communities Council Directive of 24 November 1986 (86/609/EEC). Efforts were made to minimize animal suffering and to minimize the number of specimens used.

BPA and sst3 mRNA Levels
In vitro quantitative autoradiography.
In a preliminary phase of the histochemistry, competition autoradiography approaches for sst3 were carried out on some coronal brain sections (two sections per slide; 12-µm-thick) of adult females (n = 4) from the OIL group. For this phase, animals were decapitated and brains were rapidly removed and frozen using powdered dry ice, after which they were stored at –40°C until sectioning at the cryostat and thaw-mounting onto gelatin-coated slides, as previously described (Facciolo et al., 2002bGo), for further biochemical analyses. Subsequently, brain sections were incubated for 1 h at room temperature in 50 mM Tris HCl, pH 7.4, containing 0.5% bovine serum albumin and 25 pM of 125I-Tyr1-ss-14 (81.4 TBq/mmol; NEN Division) in the presence of different concentrations (1 µM–1 pM) of ss-14 and highly specific nonpeptide agonists (L-779,976 and L-796,778) selective for sst2 and sst3 (Rohrer et al., 1998Go), respectively, ±1 µM cold ss-14 for nonspecific binding. A somewhat low concentration of the radioligand was preferred because most somatostatinergic actions are mediated via specific membrane-bound high-affinity somatostatin receptor subtypes (Reubi et al., 2000Go). After drying, slides were apposed to a 125I-sensitive Hyperfilm (Amersham, Milan-Italy) for 18 days; the film was then developed and autoradiograms were captured via a Panasonic Telecamera (Canon Objective Lens FD 50 mm, 1:3.5), and densitometric quantification was accomplished with a computer-assisted image analyzer system (VIDAS-Zeiss, Germany) running National Institutes of Health Image software. Values for binding (pmol/mg wet tissue weight; means ± SEM) were determined by comparison of two-dimensional images of a tissue section with variable quenching and consistency to a plastic standard of known radioactivity, as reported elsewhere (Facciolo et al., 2002bGo).

sst3 In situ hybridization histochemistry.
For the next phase of the study, the brains of other animals were quickly removed from the skull, rapidly frozen using powdered dry ice for a very brief period, and then immediately used for in situ hybridization histochemistry assay, as suggested by other investigators (Siegel, 1998Go). Coronal frozen-fresh brain sections (at a thickness of 12 µm; two sections per slide) from PND 7 (n = 4) and adult (n = 4) rats were used for all treatment groups (BPA400; BPA40; OIL) and processed for in situ hybridization. Hybridization was performed on brain sections fixed for 5 min in 4% formaldehyde using oligonucleotide sst3 probes (Genosys, UK) of 40 bases in length according to previously described methods (Facciolo et al., 2002aGo), with some modifications. The probes, which were the same as (sense) or complementary to (antisense) sst3 of mammalian mRNA sequences (Thoss et al., 1995Go), were labeled to a specific activity of 2.5 x 108 cpm/µg on the 3' end with terminal deoxynucleotide transferase (Boehringer) and radiolabeled 35S-deoxyadenosine 5'({alpha}-thio)triphosphate (NEN). Hybridization solution containing 4 x 105 cpm of either 35S-labeled oligonucleotide antisense or sense (control) probes to sst3 mRNA was used. After hybridization, the labeled tissue was extensively washed in different SSC buffers, dipped in distilled water, air-dried, and subsequently exposed to a 125I-sensitive Hyperfilm for a period of 15 days. Next, the developed films were analyzed in the same manner as for the autoradiography receptor study, applying a standard curve generated with 14C standards (Amersham), and values (pmol/mg wet tissue weight; means ± SEM) for BPA-treated groups were expressed as a percentage with respect to the OIL group. For histological verification of some hypothalamic and extrahypothalamic areas, the cytoarchitectonic study was performed on counterstained (cresyl violet stained with 0.5% acetate) sections, and brain areas were reported in the exact manner as they are identified in the Paxinos and Watson atlas (Paxionos and Watson, 1986Go), except that the stratum radiatum hippocampal CA1 field and the lacunosum moleculare CA1 field of the hippocampus in the present work were indicated as a single area, the RAD.

BPA and sst3-{alpha} GABAA subunit interaction.
To evaluate interaction of sst3 with GABAA receptor {alpha} subunits, adjacent frozen-fresh brain sections (two sections per slide) of the same adult and PND 7 rats of the OIL group used for in situ hybridization histochemistry were incubated in a similar fashion as previously described. However, before proceeding with fixation procedure, brain sections were preincubated for 6–8 h in Tris HCl buffer, pH 7.4, in the presence of different concentrations (1 nM–100 µM) of highly selective agonists of some {alpha} GABAA receptor subunits: the imidazopyridine zolpidem ({alpha}1; Synthelabo Recherche, Bagneux France), the benzodiazepine flunitrazepam ({alpha}2/3)/imidazobenzodiazepinone Ro 15–4513 ({alpha}4; Hoffmann-LaRoche, Basel), and the imidazobenzodiazepine RY 080 ({alpha}5; kindly provided by Dr. J. M. Cook, University of Wisconsin-Milwaukee). The effects of the higher dose of BPA on interaction of sst3 with {alpha}1 and {alpha}5 subunits were evaluated on adjacent frozen-fresh brain sections of the same adult and PND 7 rats used for in situ hybridization histochemistry, in the presence or absence of the different concentrations (5–500 nM) of two of the above selective agonists (zolpidem or RY080) that induced selectively greater mRNA levels. The relative sst3 mRNA levels were expressed as a ratio (BPA ± zolpidem or RY 080 with respect to OIL ± selective agonists).

Statistical analysis.
For the competition study, binding values (pmol/mg wet tissue weight; means ± SEM) were based on a nonlinear least-squares regression analysis. In all cases, Ki values were calculated using the Cheng and Prusoff equation: Ki = IC50/[1 + ( C/Kd)], where C is the concentration of 125I-Tyr1-ss-14, and Kd is the dissociation constant of the radioligand. The effects of the two BPA doses on sst3 mRNA levels with respect to controls were evaluated using a one-way analysis of variance (ANOVA). If the ANOVA was significant, each treatment condition was evaluated by Dunnett's test ({alpha} = 0.05). The statistical significance of the role of the GABAA receptor {alpha} subunit agonists on sst3 mRNA levels of all treatment groups was evaluated using a one-way ANOVA, followed, when a significant p value was equal to or less than 0.05, by the Neuman-Keuls multiple range post-hoc test.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The evaluations of this study demonstrate how the early influences of the xenoestrogen BPA accounted for altered neuronal functions in some brain regions of the developing rat through the preferential interaction of sst3. In the preliminary phases of this study, the displacement curve (Fig. 1) of 125I-Tyr1-ss-14 in the presence of the highly specific nonpeptide agonist L-796,778 (sst3) was shifted to the left, in a similar manner to that of ss-14 with respect to L-779,976 (sst2), a binding activity that emphasizes the predominance of the former somatostatin receptor subtype in most brain areas examined. From the highly distinct and heterogeneous pattern of sst3 mRNA levels observed in representative autoradiograms of both hypothalamic and extra-hypothalamic regions, it seems that this neuronal expression is conserved above all in the presence of the higher BPA dose in rats of both ages (Fig. 2).



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FIG. 1. Displacement curves of 125 I-Tyr1-ss-14 (mean % of total binding ± SEM) showing the binding activity in the hypothalamic arcuate nucleus (Arc) of female rats treated with arachis oil (OIL = controls). Competition study was carried out in the presence of different concentrations (1 µM-1 pM) of ss-14 (triangle) and of some nonpeptide agonists (L-779,976, dark/white square; L-796,778, dark circle), which are highly specific for the sst2 and sst3 receptor subtypes, respectively, as described in Materials and Methods. Each point represents the mean of five separate tests.

 


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FIG. 2. Representative autoradiograms (a–d) and relative controls (sense probes; a'–d') of sst3 mRNA levels in some hypothalamic and extrahypothalamic areas of (a, b) PND 7 and (c, d) adult rats treated with (a, c) arachis oil (OIL = controls) or (b, d) BPA400 as described in Materials and Methods. Bar = 4 mm.

 
A first type of influence consisted of a very strong reduction (p < 0.001) of sst3 mRNA percentages in layer V of the frontoparietal cortex (COR V) and RAD of BPA400-treated adults (Fig. 3a) with respect to those that received arachis oil alone. On the other hand, adults receiving the lower BPA dose (BPA40) only showed a strong enhancement (p < 0.01) and a moderate reduction (p < 0.05) of mRNA percentages in some hypothalamic and extrahypothalamic areas such as the ventromedial hypothalamic nucleus (VMN) and RAD, respectively (Fig. 3a). However, it was still the higher dose that accounted for a very strong increase of mRNA percentage values in this same hypothalamic nucleus, as well as a strong enhancement in another hypothalamic area, i.e., the hypothalamic arcuate nucleus (Arc). In addition, other extrahypothalamic regions responded in a similar fashion, as shown by moderately enhanced percentage values (Fig. 3a) in layer III of the frontoparietal cortex (COR III). Interestingly, while the higher BPA dose induced very strong decrease of sst3 mRNA percentages in the hypothalamic periventricular nucleus (Pe) and RAD of PND 7 rats, only strongly enhanced mRNA percentages were obtained for COR III and VMN of the same age group (Fig. 3b). This early biological age did not seem to be influenced by the lower BPA dose apart from RAD, in which moderately diminished percentages were obtained (Fig. 3b).



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FIG. 3. Bisphenol A effect on sst3 mRNA levels was reported as a percentage of sst3 mRNA levels (pmol/mg wet tissue weight; mean % ± SEM) in the presence of BPA40 (white square) and BPA400 (dark square) with respect to the arachis oil–treated animals (OIL = controls) in both the (a) adult and (b) PND 7 groups of rats. Values in each region of the same treatment group were analyzed by ANOVA followed by a Dunnett's test. ap < 0.05; bp < 0.01; cp < 0.001.

 
When the sst3 mRNA values were tested in the presence of selective agonists specific for {alpha} subunits of GABAA receptor (zolpidem, RY 080, flunitrazepam, and Ro 15–4513), a greater percentage variation of sst3 mRNA levels was reported in most of the above-mentioned areas of both adult (Fig. 4a) and PND 7 (Fig. 4b) controls, especially for the first two agonists. Zolpidem (Fig. 5a) and RY 080 (Fig. 5b), which produced comparably heterogeneous hybridization signals to those reported in Figure 2, generated greater effects on animals treated with BPA400 and thus permitted us to characterize the optimal concentrations of these two selective agonists on sst3 mRNA values in the same regions of both the adult (45 nM) and the PND 7 (70 nM) rats. Application of these concentrations did in fact account for even greater upregulating and downregulating activities in the presence of the higher BPA dose, as shown by a very strong RY 080–dependent decrease of mRNA levels in RAD, COR V, and VMN of adults as compared to the very strong increase and moderate reduction in COR III and RAD, respectively, in the presence of zolpidem (Fig. 6a). Surprisingly, an even greater effect of these agonists was obtained in PND 7 animals, especially in the presence of RY 080. In particular, Arc displayed strongly enhanced mRNA levels, whereas very strongly reduced densities were reported for Pe, RAD, and the stratum oriens and pyramidale of the CA1 field of the hippocampus (Or-Py), under the influence of this last selective agonist (Fig. 6b). Moreover, the hypothalamic sites, especially at such an early age, did not prove to be a main target for zolpidem-dependent BPA effects on sst3 mRNA levels, because only RY 080 displayed a very strong inhibiting effects of BPA400 in VMN. Rather, it was the few telencephalic areas in which zolpidem potentiated the blocking effects of BPA, as shown by strongly reduced mRNA levels in Or-Py (Fig. 6b).



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FIG. 4. The role of the different {alpha} GABAA receptor subunits on sst3 mRNA levels (pmol/mg wet tissue weight ± SEM) was represented as a percentage (mean %) in the Or-Py of (a) adult and Pe of (b) PND 7 rats treated with arachis oil in the presence of the different concentrations (1 nM–100 µM) of the {alpha} GABAA receptor agonists (zolpidem, white square; flunitrazepam, dark circle; Ro 15–4513, triangle; and RY 080, dark square) with respect to those in the absence of the selective GABAA agonists (controls). These selective agonists are highly specific for {alpha}1, {alpha}2,3, {alpha}4, and {alpha}5, respectively, as described in Materials and Methods. Each point represents the mean of five separate tests.

 


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FIG. 5. The effect of the higher BPA dose (BPA400) on sst3 mRNA levels (pmol/mg wet tissue weight; mean % ± SEM) under the influence of only {alpha}1,5 GABAA receptor subunits (5–500 nM of zolpidem and RY 080, respectively) was evaluated in a similar manner as in Figure 4 for (a) RAD and (b) COR III of adult (triangle) and PND 7 (dark square) rats. Evaluation of this assay supplied similar results in five separate tests.

 


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FIG. 6. The regional distribution pattern of {alpha}1,5-dependent BPA400 influences on sst3 mRNA levels (expressed as a ratio) in (a) adult and (b) PND 7 rats was analyzed using an optimal concentration of the selective agonists specific for the two {alpha} subunits (zolpidem and RY 080), as described in Materials and Methods. The ratios (mean % ± SEM; BPA ± zolpidem or RY 080 with respect to control ± selective agonists) were compared by ANOVA, and the differences where necessary were obtained by the Neuman-Keuls multiple range post-hoc test. ap < 0.05; bp < 0.01; cp < 0.001. Dark square = BPA/OIL; white square = BPA + zolpidem/OIL + zolpidem; gray square = BPA + RY 080/OIL + RY 080.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, the modifications of sst3 mRNA levels in some hypothalamic and extrahypothalamic areas by the environmental estrogen BPA provided, for the first time, evidence of a key influence of this xenoestrogen on ss-dependent neurobiological events in the developing rat. To achieve this purpose, it was necessary to use the highly powerful selective agonists L-779,976 and L-796,778 (Rohrer et al., 1998Go), which have gained wide recognition not only for their pharmacological therapeutical properties (Helyes et al., 2001Go) but also because their application was essential for the characterization of the somatostatin receptor subtype family (Reubi et al., 2000Go). In this context, the establishment of sst3 as the major subtype in the above brain areas enabled us to suggest that such a deglycosylated somatostatin receptor subtype is involved in BPA effects through the interaction of either classical or membrane estrogen receptor mechanisms (Quesada et al., 2002Go). A finding that is in good agreement with recent results showing that gene expression of G protein–coupled receptors appears to be a major target of several endocrine disruptors (Ishido et al., 2005Go).

From the comparison of the effects induced by the two BPA doses, it was possible to demonstrate, in a similar manner for the other major subtype-sst2 (Facciolo et al., 2002bGo), that the higher dose was responsible for evident heterogeneous sst3 mRNA levels. Indeed, such a dose accounted for early sst3 mRNA modifications in those hypothalamic and extrahypothalamic areas that are considered to be main targets of BPA-dependent differentiation events, at least in PND 7 rats. In particular, the greater sst3 transcript variations detected in hypothalamic areas such as Pe and Arc tend to emphasize the neuroendocrine importance of these major female estrogen-enriched hypothalamic targets (McEwen, 2002Go), especially under the influence of the higher BPA dose. This is particularly significant for the very low sst3 mRNA levels observed in Pe of the developing rat, a condition that appears to be contrary to the high estrogen-dependent somatostatin production and, as a consequence reduced GH release levels that are typical of such a biological period (Simonian et al., 1998Go). It would be interesting at this point to establish whether the early actions of BPA toward the acceleration of puberty (Howdeshell et al., 1999Go) may be consistent with elevated GH secretion.

Interestingly, the greater BPA-dependent sst3 mRNA densities occurred in the presence of the selective {alpha} GABAA receptor agonists zolpidem and RY 080. A relationship that not only corroborates the importance of this GABAA receptor subunit in BPA-dependent activities (Aoshima et al., 2001Go) but also underlies the specificity of {alpha}1 and {alpha}5 isoforms toward the promotion of somatostatin functions through the inhibition of GABAergic interneurons (Bassant et al., 2005Go). Even in this case, it was the early postnatal period that displayed greater GABAA-dependent BPA effects, which tend to further strengthen the importance of such a biological period during the onset of estrogen-dependent neural plasticity events.

It is exactly during this biological period that the formation of axo-somatic contacts and dendritic spines occur (Murphy and Segal, 1996Go; Wooley and McEwen, 1996Go), as does early assembly of the GABAA receptor complex (Scotti and Reuter, 2001Go). However, some hypothalamic areas did not respond to the early GABAA-dependent BPA effects, a condition that was notable for VMN, a major hypothalamic nucleus implicated in reproductive and feeding behaviors, in which no sst3 transcript variations occurred after exposure to zolpidem. This result suggests that, during the early postnatal age, VMN is probably not a preferential target of zolpidem-dependent BPA estrogen-like actions, a finding that coincides with the {alpha}1 subunit of the GABAA receptor not being fully assembled during the same period in the rat (Davies and McCarthy, 2000Go).

It is worthwhile to note that sst3 mRNA variations were not only typical of the early biological stages, as demonstrated by changes resulting in some extrahypothalamic areas of adults. In fact, the greater post-pubertal sst3 mRNA levels appear to rely on the two {alpha} subunits of the GABAA receptor, a relationship that tends to overlap the early BPA effects occurring predominantly outside the hypothalamus and that responds to elevated quantities of estrogen receptors being located in these and other extrahypothalamic areas after prolonged exposure to BPA (Aloisi et al., 2001Go). Consequently, the high densities of both {alpha} and ß estrogen receptor subunits promoting estrogen-like BPA actions (Ramos et al., 2003Go) appear to be determining factors responsible for the inhibition of higher cerebral activities such as learning, memory, and sensory functions (Negishi et al., 2003Go), despite the evident modulatory actions of different neurotransmitters (Segarra et al., 1998Go) that strongly depend on these estrogen subunits.

Taken together, these results provide direct evidence that BPA is capable of altering early neurobiological activities via the regulation of sst3 mRNA levels. The persistent exposure to the higher doses of this xenoestrogen during pregnancy and lactation appears to be related to the modifications of a variety of physiological and behavioral parameters (Dessì-Fulgheri et al., 2002Go; Negishi et al., 2004Go), probably as a consequence of the regionally specific GABAA {alpha}1,5–dependent variations of sst3 mRNA levels. However, the influence of BPA on the developing rat brain is probably not accomplished through modulation of estrogen responses alone, but may also require subtle developmental changes of other neuronal and endocrine systems. It is obvious that we are still at the beginning, but knowledge of BPA estrogen-like activities might bring us closer to an understanding of molecular cross-talking mechanisms of this somatostatin receptor subtype under both stressful and beneficial neural conditions such as pain and nociception (Aloisi et al., 2002Go) or in the shaping and organization of dendritic formation during rat neonatal life, even after a 100-fold greater BPA concentration than that of estradiol (Shikimi et al., 2004Go).


    ACKNOWLEDGMENTS
 
We are grateful to Synthélabo Recherche (Bagneux, France) for providing zolpidem, Hoffmann-La Roche (Basel, Switzerland) for flunitrazepam and Ro 15–4513, Dr. J. M. Cook (University of Wisconsin-Milwaukee) for RY 080, and Dr. S. P. Rohrer of the Merck Research Laboratories (Pennsylvania) for L-779,976 and L-796,778. This study was supported in part by the contract grant sponsor COFIN (F.D.F.) and MEMO-BIOMAR (M.C.) Project of MIUR (Italy).


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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