Neonatal exposure to estradiol in rats influences neuroactive steroid concentrations, GABAA receptor expression, and behavioral sensitivity to anxiolytic drugs
Version of Record online: 24 MAR 2010
© 2010 The Authors. Journal Compilation © 2010 International Society for Neurochemistry
Journal of Neurochemistry
Volume 113, Issue 5, pages 1285–1295, June 2010
How to Cite
Calza, A., Sogliano, C., Santoru, F., Marra, C., Angioni, M. M., Mostallino, M. C., Biggio, G. and Concas, A. (2010), Neonatal exposure to estradiol in rats influences neuroactive steroid concentrations, GABAA receptor expression, and behavioral sensitivity to anxiolytic drugs. Journal of Neurochemistry, 113: 1285–1295. doi: 10.1111/j.1471-4159.2010.06696.x
- Issue online: 4 MAY 2010
- Version of Record online: 24 MAR 2010
- Received December 16, 2009; revised manuscript received March 10, 2010; accepted March 16, 2010.
- cerebral cortex;
- GABAA receptor;
- neuroactive steroid
J. Neurochem. (2010) 113, 1285–1295.
Gonadal steroids, in particular estradiol, exert important actions during pre- and perinatal periods in the regulation of sexual dimorphism and development of the nervous system. We have now examined the effects of neonatal estradiol administration in female rats on brain concentrations of the neuroactive steroids allopregnanolone and tetrahydrodeoxycorticosterone, expression of GABAA receptor subunits, and behavioral sensitivity to benzodiazepines and allopregnanolone. Administration of β-estradiol 3-benzoate on the day of birth resulted in marked decreases in the concentrations of progesterone and allopregnanolone in the cerebral cortex at 21, 60, and 180 days after birth. The concentrations of tetrahydrodeoxycorticosterone, 17β-estradiol, and dehydroepiandrosterone in the brain at 60 days were not affected by such treatment. Neonatal administration of β-estradiol 3-benzoate also increased the cerebrocortical abundance of α1, α2, and γ2 subunits of the GABAA receptor without affecting that of α3, α4, α5, or δ subunits. Diazepam induced a greater reduction in locomotor activity as well as a more pronounced anxiolytic-like effect in the elevated plus-maze test in rats subjected to neonatal treatment with β-estradiol 3-benzoate than in vehicle-treated controls, while allopregnanolone induced a similar effect in both groups. These effects of estradiol suggest that it plays a major role in regulation both of GABAergic transmission and of the abundance of endogenous modulators of such transmission during development of the central nervous system.
GABA type A (GABAA) receptors consist of five subunits surrounding a central channel selective for chloride ions in the plasma membrane, with the precise subunit composition determining the physiologic and pharmacological properties of each receptor subtype (Olsen and Sieghart 2009). GABAA receptors manifest a high level of structural heterogeneity, much greater than that of any other ligand-gated ion channel, with the five subunits belonging to various subunit classes (α1 to α6, β1 to β4, γ1 to γ3, δ, ε, π, θ, ρ1 to ρ3). This variety of subunits, each with a specific pattern of expression within the brain, suggests the existence of many subtypes of GABAA receptor, each with different potential functions and sensitivities to various drugs. For example, receptors composed of α1, α2, or α3 subunits together with a β and γ subunit are sensitive to benzodiazepines, are located pre-dominantly at synapses, and mediate most phasic inhibition in the brain. By contrast, most receptors composed of α4 or α6 subunits together with a β and δ subunit are insensitive to benzodiazepines, are located extrasynaptically, and mediate tonic inhibition.
Long-term administration of sedative-hypnotic, anxiolytic, or anti-convulsant drugs or of certain drugs of abuse results in marked changes in the expression of specific GABAA receptor subunits that lead to the assembly of receptors with different subunit compositions and consequently different drug sensitivities and functions. Neuroactive steroids that are active at GABAA receptors also affect GABAA receptor gene expression and activity in various regions of the brain in rats (Concas et al. 1998; Biggio et al. 2007; Maguire and Mody 2007; Smith et al. 2007). In particular, the progesterone metabolite allopregnanolone, one of the most potent and efficient modulators of GABAA receptor function, induces pharmacological effects similar to those elicited by classical positive allosteric modulators such as benzodiazepines (Majewska 1992). Given that allopregnanolone is produced both in the periphery and in the brain from endogenous progesterone (Mellon and Griffin 2002), physiologic or pharmacologically induced fluctuations in the concentration of this gonadal steroid are paralleled by changes in the synaptic concentration of allopregnanolone, which contribute to the regulation of GABAA receptor plasticity. As GABAA receptors are implicated in a variety of neuropsychophysiologic phenomena, including anxiety, sleep, seizures, and depression, such fluctuations in the concentrations of neuroactive steroids may contribute to the cognitive and psychiatric manifestations of conditions characterized by marked changes in the hormonal milieu. Pregnancy, delivery, the estrous cycle, and inhibition of gonadal function are thus all associated with pronounced changes in the expression of specific GABAA receptor subunits in various regions of the brain and with consequent changes in receptor function (Concas et al. 1998; Follesa et al. 1998; Griffiths and Lovick 2005; Maguire and Mody 2007, 2008; Sanna et al. 2009).
Gonadal steroids, in particular androgens and estradiol, play important roles in the regulation of sexual dimorphism as well as in the growth and development of the nervous system during both pre- and perinatal periods (McEwen 1983). A short exposure of developing females to androgens during the period of sexual differentiation, which in the rat begins in late embryonic life and extends postnatally for the first 7 to 10 days, thus induces long-lasting effects that “program” the female neuroendocrine axis to malfunction in adulthood (Foecking et al. 2008; McCarthy et al. 2008). Treatment of female neonates with testosterone or estrogen changes the responsiveness of the hypothalamus and pituitary gland to the feedback action of ovarian steroids, leading to dysregulation of the secretion of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH), in adulthood (Robinson 2006). This phenomenon results in adult females that exhibit persistent vaginal cornification, acycliticy and ovulatory failure (Handa et al. 1985). We have now evaluated whether the changes in the hormonal milieu induced by administration of estrogen to neonatal female rats might influence the concentrations of gonadal steroids and their neuroactive steroid metabolites, including allopregnanolone and tetrahydrodeoxycorticosterone (THDOC), in the adult brain. Moreover, given that both allopregnanolone and THDOC positively modulate GABAA receptor function (Majewska 1992) and that long-term physiologic (Fénelon and Herbison 1996; Concas et al. 1998; Maguire and Mody 2007; Sanna et al. 2009) or pharmacological (Smith et al. 1998; Follesa et al. 2000, 2002; Biggio et al. 2007) exposure to these steroid derivatives regulates the expression of specific GABAA receptor subunits, we also examined whether neonatal estrogen administration affects the expression of such subunits in the cerebral cortex. Finally, given that the hormonal milieu influences the pharmacological efficacy of benzodiazepines and allopregnanolone (Bitran et al. 1991; Fernández-Guasti and Picazo 1997, 1999; Laconi et al. 2001), we investigated whether the behavioral effects of these drugs in adulthood might be affected by neonatal administration of estrogen.
Materials and methods
Female Sprague-Dawley rats (Charles River, Como, Italy) were bred in our colony and maintained on an artificial 12-h-light, 12-h-dark cycle (light on from 08.00 to 20.00 h) at a constant temperature of 22° ± 2°C and a relative humidity of 65%. Food and water were available ad libitum. Animal care and handling throughout the experimental procedures were in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and were approved by the local ethics committee. Females were mated with males at regular intervals. On the day of birth (day 0), the male pups were removed from the litter and the female offspring were injected s.c. with 50 μL of sesame oil (controls) or with 10 μg of β-estradiol 3-benzoate in 50 μL of sesame oil (Rodriguez et al. 1993; Solum and Handa 2002). To avoid leakage of β-estradiol 3-benzoate pups were injected under hypothermia anesthesia. After injection, neonatal estradiol-treated females were randomly distributed among litters of the same age so that each mother had five to eight pups. All female pups within a litter received the same treatment. After weaning females rats were housed in groups (control and neonatal estradiol-treated) of six to eight per cage. They were killed on days 21, 60, or 180 for measurement of plasma or brain steroids and GABAA receptor subunit expression. The animals were subjected to behavioral tests between days 60 and 90. To avoid effects of estrous cycle status and the stress of vaginal smears on steroid concentrations, GABAA receptor subunit expression, and behavioral tests, we killed control animals in randomized phases of the estrous cycle.
Diazepam was dissolved with one drop of Tween 80 in distilled water and was injected i.p. at a dose of 0.5, 1, 2 or 6 mg per kilogram of body weight in a volume of 3 mL/kg. Allopregnanolone was dissolved in 20% 2-hydroxypropyl-β-cyclodextrin (Sigma-Aldrich, Milan, Italy) by exposure to ultrasound for 4 h and was injected s.c. at a dose of 5 or 8 mg/kg in a volume of 2 mL/kg. Control animals received the same volume of vehicle.
Steroid extraction and assay
Animals were killed with a guillotine, the brain was rapidly (< 1 min) removed, and the cerebral cortex was dissected and frozen at −20°C until steroid extraction. Blood was collected from the trunk into heparinized tubes and centrifuged at 1000 g for 15 min, after which the plasma supernatant was frozen until assayed for steroids. All steroids were assayed in the same tissue sample. Steroids present in cerebral cortical homogenates (400 mg of tissue in 4 mL of phosphate-buffered saline) were extracted three times with an equal volume of ethyl acetate. The combined organic phases were dried under vacuum, the resulting residue was dissolved in 5 mL of n-hexane and applied to a Seppak silica cartridge (Waters, Milan, Italy), and residue components were eluted with a mixture of n-hexane and 2-propanol (7 : 3, v/v). Steroids were further purified by HPLC on a 5-μm Lichrosorb-diol column (250 by 4 mm; Chemtek Analytica, Bologna, Italy) with a gradient of 2-propanol in n-hexane. Given that cholesterol, which coelutes from the Lichrosorb-diol column with progesterone, was found to reduce the sensitivity of the radioimmunoassay for progesterone, this latter steroid was separated from cholesterol by washing the corresponding dried column fractions twice with 200 μL of dimethylsulfoxide and once with 400 μL of water. Progesterone was extracted from the aqueous phase twice with 1.5 mL of n-hexane. The recovery of each steroid through the extraction-purification procedures (70 to 80%) was monitored by the addition of trace amounts (4000–6000 cpm) of 3H-labeled standards to the brain tissue homogenate. Steroids were quantified by radioimmunoassay as described (Porcu et al. 2003). Steroid concentrations in plasma (1 mL) were measured after extraction three times with 1.5 mL of ethyl acetate.
GABAA receptor subunits were estimated in cerebrocortical membranes as previously described (Follesa et al. 2002). Cerebral cortex was homogenized in a solution containing 10 mM Tris-HCl (pH 7.4), 0.32 M sucrose, 5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, bacitracin (200 μg/mL), and aprotinin (1 μg/mL), and the homogenate was centrifuged at 1000 g for 20 min at 4°C. The resulting supernatant was then centrifuged at 17 000 g for 20 min at 4°C, and the pellet so obtained was washed three times with homogenization buffer and then stored at −20°C until use. The protein concentration was measured, and equal amounts of protein (20–40 μg) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 4–12% minigels (NuPAGE Novex; Invitrogen, Milan, Italy). The separated proteins were transferred electrophoretically to a polyvinylidene difluoride membrane and then subjected to immunoblot analysis with antibodies to α1, α2, or γ2 subunits of the GABAA receptor (each at a 1 : 300 dilution; Sigma-Aldrich), to the α3 subunit (1 : 300; Millipore, Milan, Italy), or to α4, α5, or δ subunits (1 : 800; Phosphosolutions, Milan, Italy). Immune complexes were detected with the use of an ECL Plus detection kit (Amersham, Little Chalfont, UK). The amounts of each subunit were quantitated by measurement of the intensity of the immunoreactive bands with the use of a Geliance Imaging System (Perkin Elmer, Monza, Italy), and they were normalized by the corresponding amount of glyceraldehyde-3-phosphate dehydrogenase.
Elevated plus-maze test
The plus-maze was constructed of black polyvinyl chloride and comprised two open and two closed arms (12 by 60 cm) connected by a central square (12 by 12 cm) that served as the start point. The apparatus was mounted 50 cm above the floor of a quiet, dimly lit room. On the day of the test, rats were allowed to adapt to the experimental room for 1 h before the test. Each rat was tested only once. The animal was placed at the start point of the maze facing an open arm. During the 5-min test, the number of entries into open and closed arms and the time spent in each type of arm were monitored for each rat by two observers unaware of the treatment group; arm entry was defined as the presence of all four feet of the animal in the arm. Rats were tested in a randomized order between 09.00 and 14.00 h beginning 30 min after the administration of diazepam or 15 min after injection of allopregnanolone. The maze was cleaned thoroughly at the end of each test.
Locomotor activity assay
Locomotor activity of rats was assessed with the use of a Digiscan Animal Activity Analyzer (AccuScan Instruments, Columbus, OH, USA). The test chamber consists of a cubicle made of clear Perspex (48 by 50 cm) and with 50-cm-high walls. Two facing blocks containing an infrared array record horizontal activity, and a similar system assesses vertical activity. Each animal was gently placed at the center of the chamber and allowed to explore the apparatus in an illuminated and quiet room. Locomotor activity was recorded in the light cycle between 09.00 and 14.00 h beginning 15 min after the administration of diazepam. Rats were tested in a randomized order, and locomotor activity was assessed for each animal individually during a 10-min period. The total distance travelled (cm) by each animal was accumulated over consecutive 2-min time windows, and the number of movements was recorded. The arena was cleaned thoroughly at the end of each test.
Allopregnanolone and THDOC were synthesized by R. H.Purdy (Department of Psychiatry, University of California, San Diego, CA, USA). β-Estradiol 3-benzoate, progesterone, dehydroepiandrosterone (DHEA), 17β-estradiol, and corticosterone were obtained from Sigma-Aldrich. Antisera to allopregnanolone or to THDOC were generated and characterized as described (Purdy et al. 1990). Antisera to progesterone, to 17β-estradiol, to DHEA, or to corticosterone were obtained from ICN (Costa Mesa, CA, USA). Diazepam was obtained from Fabbrica Italiana Sintetici (Vicenza, Italy). All other chemicals were of the best available quality from commercial sources.
Data are presented as means ± SEM. The statistical significance of differences was assessed by one- or two-way anova with post hoc analysis by Newman–Keuls test. A p value of < 0.05 was considered statistically significant.
Effects of neonatal administration of β-estradiol 3-benzoate on neuroactive steroid concentrations in rat cerebral cortex and plasma
The administration of β-estradiol 3-benzoate (10 μg, s.c.) on the day of birth resulted in significant decreases in the concentrations of progesterone (–58%) and allopregnanolone (–85%) in the cerebral cortex of female rats killed 21 days after birth (Fig. 1). These effects were maximal 60 days after birth (−86 and −92%, respectively) and were still apparent at 6 months (−61 and −78%, respectively). In contrast, the same treatment had no significant effect on the cerebrocortical concentrations of THDOC (−16%p = 0.087), DHEA (−19%p = 0.33), or 17β-estradiol (+3%p = 0.86) measured 60 days after birth (Fig. 2). The neonatal administration of β-estradiol 3-benzoate also resulted in significant decreases in the plasma concentrations of progesterone and allopregnanolone apparent at 60 days after birth (Table 1), although these effects (−73 and −58%, respectively) were less pronounced than those observed in the cerebral cortex. In contrast to the lack of effect of such treatment on the cerebrocortical concentration of THDOC, it reduced the plasma level of THDOC by 44% at 6 months, although the plasma concentrations of 17β-estradiol, DHEA, and corticosterone were not affected.
|Progesterone||10.14 ± 0.68||2.77 ± 0.44*|
|Allopregnanolone||18.45 ± 0.88||7.8 ± 0.51*|
|THDOC||2.83 ± 0.23||1.60 ± 0.19*|
|17β-Estradiol||0.067 ± 0.007||0.081 ± 0.009|
|DHEA||0.37 ± 0.03||0.37 ± 0.07|
|Corticosterone||170 ± 39||158 ± 32|
Effects of neonatal administration of β-estradiol 3-benzoate on expression of GABAA receptor subunits in rat cerebral cortex
We next evaluated the effects of neonatal administration of β-estradiol 3-benzoate on the abundance of GABAA receptor subunits as measured by immunoblot analysis of a cerebrocortical membrane fraction. Such treatment induced significant increases in the amounts of α1 (+26%) and α2 (+22%) subunits but had no effect on the expression of α3, α4, or α5 subunits (Fig. 3). Furthermore, the neonatal administration of β-estradiol 3-benzoate induced a marked increase (+58%) in the abundance of the γ2 subunit in the cerebral cortex without affecting that of the δ subunit (Fig. 4).
Effects of neonatal administration of β-estradiol 3-benzoate on the behavior of rats in the elevated plus-maze test
Neonatal administration of β-estradiol 3-benzoate did not affect the behavior of adult female rats in the elevated plus-maze test. The percentage time spent in the open arms and the percentage open-arm entries thus did not differ between control (13.2 ± 2.7 and 17.1 ± 2.9, respectively) and β-estradiol 3-benzoate–treated (11.2 ± 2.5 and 13.4 ±2.9, respectively) rats.
In contrast, neonatal administration of β-estradiol 3-benzoate increased the sensitivity of rats to the effects of diazepam in the test (Fig. 5). Consistent with previous observations (Bitran et al. 1991), diazepam (0.5 to 2 mg/kg, i.p.) induced an anxiolytic-like action in control rats subjected to the elevated plus-maze test. However, this action was more pronounced in rats that had been treated with β-estradiol 3-benzoate. The percentage increases in the time spent in the open arms and in the number of entries into the open arms induced by diazepam were thus greater in β-estradiol 3-benzoate–treated rats than in control animals (at the diazepam dose of 2 mg/kg: open-arm time, +675 vs. +139%; open-arm entries, +256% vs. +134%). In relation to the open arm time two way anova revealed a significant main effect of neonatal treatment [F(1, 64) = 42.4911; p < 0.001], a significant main effect of drug treatment [F(3, 64) = 11.1610; p < 0.001], and a significant interaction between factors [F(3, 64) = 5.4172; p < 0.01]. In relation to the open arm entries, two way anova revealed a significant main effect of neonatal treatment [F(1, 64) = 9.8640; p < 0.01], a significant main effect of drug treatment [F(3, 64) = 17.3721; p < 0.001], and no significant interaction between factors [F(3, 64) = 2.7165; p = 0.0519].
As expected (Finn et al. 1997), the administration of allopregnanolone (5–8 mg/kg, s.c.) induced a anxiolytic-like action in both groups of rats subjected to the elevated plus-maze test (Fig. 6). The time spent in the open arms and the number of open-arm entries were thus increased by allopregnanolone to similar extents in both β-estradiol 3-benzoate–treated and control animals (at the allopregnanolone dose of 8 mg/kg: open-arm time, +192 vs. +196%, respectively; open-arm entries, +205 vs. +151%, respectively).
Effects of neonatal administration of β-estradiol 3-benzoate on locomotor activity
Neonatal administration of β-estradiol 3-benzoate did not affect the total distance travelled and the total number of movements of adult female rats (Fig. 7).
As expected (Savićet al. 2009), administration of diazepam (6 mg/kg, i.p.) resulted in a significant decrease in the total distance travelled and in the number of movements relative to those apparent for vehicle-treated animals in both control and β-estradiol 3-benzoate–treated rats (Fig. 7). However, in rats treated neonatally with β-estradiol 3-benzoate, diazepam induced a more pronounced effect on the total distance travelled (−99% vs. −70%) and on the number of movements (−96% vs. −64%).
We have shown that neonatal administration of β-estradiol 3-benzoate in female rats resulted in marked decreases in the cerebrocortical and plasma concentrations of progesterone and allopregnanolone that persisted into adulthood. These effects were accompanied by changes both in the expression of specific GABAA receptor subunits in the cerebral cortex as well as in the behavioral sensitivity of the animals to diazepam.
In addition to its previously demonstrated effect on peripheral steroidogenic tissue (Rodriguez et al. 1993), we have now shown that the administration of β-estradiol 3-benzoate to female neonates induced pronounced decreases in the concentrations of progesterone and its metabolite allopregnanolone in the cerebral cortex that were apparent in both juvenile (3 weeks) and adult (2–6 months) animals. In contrast, the brain and plasma levels of 17β-estradiol measured in adult rats were not affected by the neonatal administration of β-estradiol 3-benzoate, although a marked increase in estradiol plasma levels 24 h after injection of estradiol benzoate, that remained higher 10 days after injection has been previously observed (Rodriguez et al. 1993; Amateau et al. 2004). Prenatal exposure of female rats to exogenous androgen was previously found not to affect the serum concentration of estradiol in adulthood (Foecking et al. 2005). Neonatal exposure to exogenous estrogen is thought to program hormonal cyclicity in adulthood, at least in part by promoting the development of neuronal circuits that are resistant to the positive feedback actions of estrogen (Robinson 2006). This effect of estrogen impairs the ability of this steroid to stimulate the GnRH surge that is required to trigger the LH surge and subsequent ovulation. Exposure of female rat pups to testosterone or estrogen on postnatal days 1–10 thus renders them permanently incapable of undergoing the LH surge (Korenbrot et al. 1975) and consequently results in reduced ovarian and plasma concentrations of progesterone (Handa et al. 1985; Rodriguez et al. 1993). As a result of such impaired production of progesterone by the gonads, we have now found that the brain and plasma concentrations of its neuroactive steroid metabolite allopregnanolone are also reduced. Treatment with oral contraceptives, which reduce both the basal and stimulated serum concentrations of LH in rats, also results in decreased brain and plasma concentrations of progesterone and allopregnanolone in these animals (Follesa et al. 2002) as well as prevents the increase in the serum concentrations of these steroids in women that normally occur in the luteal phase of the menstrual cycle (Rapkin et al. 2006).
The extents of the decreases in the plasma concentrations of progesterone and allopregnanolone, especially that of the latter, induced by neonatal administration of β-estradiol 3-benzoate were less pronounced than were those in the cerebrocortical concentrations, suggesting that the neonatal treatment might also directly affect the synthesis and accumulation of these steroid hormones in the brain. Differential regulation of neuroactive steroid concentrations in the plasma and those in the brain has also been suggested on the basis of the effects of various pharmacological treatments (Concas et al. 2000; Barbaccia et al. 2001; Follesa et al. 2002) as well as in certain physiologic conditions (Concas et al. 1998). Moreover, de novo synthesis of neuroactive steroids in the brain in a manner independent of the periphery has been demonstrated (Sanna et al. 2004).
The decreases in the brain allopregnanolone and progesterone concentrations apparent in β-estradiol 3-benzoate–treated rats appear to be specific, given that the concentrations of other neuroactive steroids, including THDOC and DHEA, were not similarly affected. Although the molecular mechanisms responsible for this difference remain to be determined, our results are consistent with previous data showing that the brain concentrations of these various neuroactive steroids in rats are differentially affected by pharmacological treatments (Concas et al. 2000; Ströhle et al. 2000; Porcu et al. 2003) as well as by various stressors (Barbaccia et al. 1996). Unlike allopregnanolone, which has multiple sources including the gonads and de novo synthesis in the brain, THDOC is derived almost exclusively from the adrenal gland (Mellon and Griffin 2002) and is thus probably not influenced by the specific effects of estradiol on the gonadal axis. Moreover, DHEA is synthesized via a distinct steroidogenic pathway, being derived directly from pregnenolone without the involvement of progesterone. The activity or expression of enzymes that catalyze the biosynthesis of DHEA specifically may thus not be altered by neonatal administration of β-estradiol 3-benzoate.
The persistent reduction in the brain concentrations of progesterone and allopregnanolone elicited by neonatal exposure to β-estradiol 3-benzoate was associated with changes in the expression of specific GABAA receptor subunits in the cerebral cortex. In particular, immunoblot analysis revealed increases in the abundance of α1, α2, and γ2 subunits, whereas the amounts of α3, α4, α5, and δ subunits were not affected. The decrease in neuroactive steroid concentrations induced by long-term treatment with oral contraceptives was previously shown to be accompanied by a specific increase in the amounts of γ2 subunit mRNA and protein in the rat cerebral cortex (Follesa et al. 2002). Moreover, the long-lasting increase in the brain concentrations of neuroactive steroids associated with pregnancy is accompanied by a decrease in the abundance of γ2 subunit mRNA and protein in the rat cerebral cortex (Concas et al. 1998; Follesa et al. 1998), an effect that is reversed by the abrupt fall in the brain concentrations of progesterone and allopregnanolone that precedes parturition or that is prevented by finasteride, a specific inhibitor of 5α-reductase (Concas et al. 1998). Moreover, a substantial increase in the abundance of the γ2 subunit mRNA was observed in the hypothalamus in association with low brain concentrations of neuroactive steroids during lactation in rats (Fénelon and Herbison 1996), whereas a decrease in the expression of this subunit in the hippocampus was also observed during pregnancy (Concas et al. 1998; Maguire and Mody 2008; Sanna et al. 2009). Our present observations thus further support the notion that marked fluctuations in the brain concentrations of progesterone and allopregnanolone have a pronounced influence on expression of the γ2 subunit of the GABAA receptor.
Our finding that neonatal administration of β-estradiol 3-benzoate resulted in significant increases in the cerebrocortical levels of α1 and α2 subunits of the GABAA receptor is consistent with the previous observation that long-term exposure to allopregnanolone induced specific decreases in the amounts of α1 and α2 subunit mRNAs in cultured cerebrocortical or cerebellar neurons (Yu et al. 1996; Follesa et al. 2000). In contrast, in parturient animals, in which the brain allopregnanolone concentration is low (Concas et al. 1998), the abundance of the α1 subunit mRNA is reduced in specific hypothalamic nuclei but not in the cerebral cortex (Fénelon and Herbison 1996; Concas et al. 1998; Follesa et al. 1998). The discrepancies between the apparent effects of allopregnanolone on α subunit expression may be attributable to differences in regulatory mechanisms among brain regions. It is thus possible that neonatal treatment with β-estradiol 3-benzoate targets only specific neurons in the cerebral cortex that express specific populations of GABAA receptors.
Our findings that neonatal administration of β-estradiol 3-benzoate did not affect the cerebrocortical abundance of α3, α4, α5, or δ subunits of the GABAA receptor also contrasts with the results of previous in vitro or in vivo studies showing changes in the amounts of the corresponding subunit mRNAs or proteins in other brain regions or in cultured neurons after short-term exposure to or subsequent withdrawal of progesterone or allopregnanolone (Yu et al. 1996; Smith et al. 1998; Follesa et al. 2000). Progesterone withdrawal was thus found to induce a transient increase in expression of the α4 subunit in cultured cerebrocortical or cerebellar granule neurons (Follesa et al. 2000, 2001). Withdrawal from a prolonged period of systemic dosing with progesterone or allopregnanolone was also shown to up-regulate expression of the α4 subunit in the hippocampus (Smith et al. 1998), amygdala (Gulinello et al. 2003), or periaqueductal gray matter (Griffiths and Lovick 2005) of rats. In contrast, short-term exposure to progesterone or allopregnanolone resulted in an increase in the levels of both α4 and δ subunits in the hippocampus (Smith et al. 2007). Moreover, the physiologic increases in the brain and plasma levels of these steroids during pregnancy are associated with a marked increase in the abundance of the δ subunit in specific areas of the hippocampus, whereas the expression of both α4 and δ subunits in this brain region decreases greatly after delivery (Sanna et al. 2009). Steroid-associated plasticity in the expression of GABAA receptor subunits therefore appears to be dependent on neuronal cell type and brain region.
The functional relevance of the changes in α1, α2, and γ2 subunit expression in the cerebral cortex induced by neonatal treatment with β-estradiol 3-benzoate remains to be determined. Given that the presence of an α subunit, a β subunit, and the γ2 subunit is required for sensitivity of GABAA receptors to benzodiazepines (Pritchett et al. 1989), these molecular changes might be expected to affect the pharmacology of GABAA receptor–mediated neurotransmission. Indeed, we found that the observed changes in receptor subunit expression were associated with changes in behavioral responses to diazepam. Diazepam thus induced a more pronounced anxiolytic-like effect in the elevated plus-maze test and a greater reduction in locomotor activity in rats subjected to neonatal administration of β-estradiol 3-benzoate than in those subjected to similar administration of vehicle. A similar increased sensitivity to the anxiolytic effect of diazepam was previously observed in female rats subjected to neonatal androgen treatment (Fernández-Guasti and Picazo 1997). Given that the sedative effect of benzodiazepines requires the α1 subunit of the GABAA receptor (Rudolph et al. 1999) whereas the α2 subunit appears to be a major determinant of the anxiolytic effect of these drugs (Löw et al. 2000), the enhancement of the locomotion-suppressing and anxiolytic effects of diazepam observed in rats subjected to neonatal administration of β-estradiol 3-benzoate may be attributable to the increased expression of the α1 and α2 subunits, respectively, in the cerebral cortex. Expression of the γ2 subunit also appears to be a determinant of the action of benzodiazepines at GABAA receptors, given that the binding site for these compounds is located at the interface between α and γ2 subunits (Olsen and Sieghart 2009). Diazepam thus fails to induce sedation or loss of the righting reflex in mice with targeted disruption of the γ2 subunit gene (Günther et al. 1995). In addition, transgenic mice that express reduced levels of the γ2 subunit exhibit enhanced anxiety-like behavior in the elevated plus-maze and forced novel exploratory tests (Crestani et al. 1999). Our observations thus suggest that the increased expression of α1, α2, and γ2 subunits in the brain of adult female rats subjected to neonatal administration of β-estradiol 3-benzoate results in the increased assembly of GABAA receptors that contain these specific subunits and which therefore exhibit an increased sensitivity to benzodiazepines.
Neonatal treatment with β-estradiol 3-benzoate did not affect the anxiolytic-like action of allopregnanolone in the elevated plus-maze test. Neonatally androgenized or ovariectomized female rats were previously shown to be insensitive to the anxiolytic action of allopregnanolone in the elevated plus-maze test (Fernández-Guasti and Picazo 1999; Laconi et al. 2001). The lack of a difference in sensitivity to the anxiolytic effect of allopregnanolone between rats subjected to neonatal treatment with β-estradiol 3-benzoate or with vehicle in the present study might be explained by the failure of β-estradiol 3-benzoate to affect expression of the α4 and δ subunits of the GABAA receptor. Receptors that contain these subunits are located at extrasynaptic sites in various brain regions, mediate tonic inhibition of neuronal activity, and are highly sensitive to modulation by allopregnanolone but not by benzodiazepines (Smith et al. 2007). Ablation of the δ subunit influences the behavioral profile of neuroactive steroids in mice, with the anxiolytic-like effect in the elevated plus-maze test induced by allopregnanolone or ganaxolone being reduced in animals lacking the δ subunit (Mihalek et al. 1999).
The precise mechanisms by which a single administration of β-estradiol 3-benzoate in neonatal females induces the observed neuroendocrine, molecular, and behavioral changes in adulthood remain to be elucidated. Exposure of developing females to estrogen affects the organization of all levels of the reproductive axis from the GnRH neuronal network to the gonads (Robinson 2006; McCarthy et al. 2008). These effects occur during a specific time window during development that spans late gestation to the early postnatal period. Estradiol has been shown to affect GABAergic transmission, especially during early development (McCarthy et al. 2008), when GABA acts as an excitatory neurotransmitter that induces neuronal depolarization and increases the cytosolic Ca2+ concentration (Chen et al. 1996). Exposure to estradiol thus prolongs the depolarizing action of GABA in developing hypothalamic (Perrot-Sinal et al. 2001) and hippocampal (Nuñez et al. 2005) neurons. Moreover, long-term treatment with estradiol affects synaptic and tonic GABAergic currents in the developing hippocampus both in vitro and in vivo (Pytel et al. 2007; Wójtowicz et al. 2008). The surge in estradiol levels in the neonatal female brain induced by a single administration of β-estradiol 3-benzoate might therefore delay processes underlying development of the GABAergic system and thereby prolong neuronal excitation and result in permanent changes in synaptic patterning of specific regions of the brain. Given the important role of GABAergic transmission in regulation of GnRH neurons, such effects may account for the impaired function of the reproductive axis in females exposed to β-estradiol 3-benzoate as neonates.
However, whereas such a mechanism might be responsible for the altered endocrine profile of female rats subjected to neonatal administration of β-estradiol 3-benzoate, it is unlikely to account for the changes in GABAA receptor expression and drug sensitivity apparent in adulthood. Indeed, we found that the neonatal treatment with β-estradiol 3-benzoate resulted in marked decreases in the concentrations of progesterone and allopregnanolone in adulthood, whereas 17β-estradiol levels remained unchanged in the brain and plasma of the adult animals. Allopregnanolone levels in the cerebral cortex of rats have previously been shown to undergo dynamic changes during development, with maximal concentrations apparent around the second postnatal week (Grobin and Morrow 2001). Given that allopregnanolone exposure alters GABAA receptor subunit expression, these changes were suggested to have implications for the structure and function of GABAA receptors during development. Indeed, perinatal administration of allopregnanolone was found to influence the localization of GABAergic interneurons in the prefrontal cortex of adult rats (Grobin et al. 2003). In the present study, we found that neonatal administration of β-estradiol 3-benzoate induced a marked decrease in the concentration of allopregnanolone in the cerebral cortex of rats at 21 days of age. Such treatment might thus prevent the surge in allopregnanolone levels during the first weeks of life and thereby affect the developmental plasticity of GABAA receptors.
In conclusion, our results showing that the brain concentrations of progesterone and allopregnanolone, expression of GABAA receptor subunits, and behavioral sensitivity to GABAA receptor–targeting drugs in adulthood are dependent on neonatal estradiol levels suggest that estradiol plays a major role in regulation both of GABAergic transmission and of the abundance of endogenous modulators of such transmission during development of the central nervous system. Moreover, neonatal estradiol treatment might represent a useful experimental model with which to investigate further the physiologic role of neuroactive steroids in the modulation of GABAergic transmission.
This work was supported by grants from the PRIN 2003057334 from the Ministry of Instruction, Universities, and Research, Italy; the Sardinian Department of Health and Welfare, and the Gioventu’ in Armonia (GIO I A) Foundation (Pisa, Italy). The authors declare no conflict of interest.
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