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Keywords:

  • allopregnanolone;
  • ethanol;
  • γ-aminobutyric acidA receptor;
  • isoniazid;
  • miniature inhibitory postsynaptic current;
  • social isolation

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Previously we have demonstrated that social isolation of rats reduces both the cerebrocortical and plasma concentrations of 3α-hydroxy-5α-pregnan-20-one (3α,5α-TH PROG), and potentiates the positive effects of acute ethanol administration on the concentrations of this neurosteroid. We now show that the ethanol-induced increase in 3α,5α-TH PROG is more pronounced in the brain than in the plasma of isolated rats. The ability of ethanol to inhibit isoniazid-induced convulsions is greater in isolated rats than in group-housed animals and this effect is prevented by treatment with finasteride. Social isolation modified the effects of ethanol on the amounts of steroidogenic regulatory protein mRNA and protein in the brain. Moreover, ethanol increased the amplitude of GABAA receptor-mediated miniature inhibitory postsynaptic currents recorded from CA1 pyramidal neurones with greater potency in hippocampal slices prepared from socially isolated rats than in those from group-housed rats, an effect inhibited by finasteride. The amounts of the α4 and δ subunits of the GABAA receptor in the hippocampus were increased in isolated rats as were GABAA receptor-mediated tonic inhibitory currents in granule cells of the dentate gyrus. These results suggest that social isolation results in changes in GABAA receptor expression in the brain, and in an enhancement of the stimulatory effect of ethanol on brain steroidogenesis, GABAA receptor function and associated behaviour.

Abbreviations used
ACSF

artificial cerebrospinal fluid

3α,5α-TH PROG or AP

3α-hydroxy-5α-pregnan-20-one

bic

bicuculline

DG

dentate gyrus

eIPSC

synaptically evoked miniature inhibitory postsynaptic current

EtOH

ethanol

GAPDH

glyceraldehyde-3-phopshate dehydrogenase

HPA

hypothalamic–pituitary–adrenal

mIPSC

miniature inhibitory postsynaptic current

PBR

peripheral benzodiazepine receptor

PBS

phosphate-buffered saline

PBS-T

PBS containing 0.2% Triton X-100

StAR

steroidogenic regulatory protein

Social isolation of rats after weaning results in a decrease in the brain and plasma concentrations of neuroactive steroids such as 3α-hydroxy-5α-pregnan-20-one (allopregnanolone, 3α,5α-TH PROG) and 3α,5α-tetrahydrodeoxycorticosterone (Serra et al. 2000). The molecular mechanisms that underlie this effect remain unclear. However, consistent with the hypothesis that ethanol (EtOH) activates the hypothalamic–pituitary–adrenal (HPA) axis (Ellis 1966; Rivier et al. 1984; Rivier 1996), we have previously shown that the increases in both the activity of the HPA axis and in the plasma and brain concentrations of neuroactive steroids induced by an acute injection of EtOH are potentiated by social isolation (Serra et al. 2003). EtOH increases the abundance of 3α,5α-TH PROG and 3α,5α-tetrahydrodeoxycorticosterone in the brain and plasma of control rats (Barbaccia et al. 1999; Van Doren et al. 2000), an effect thought to be dependent predominantly on stimulation of the HPA axis, given that it is largely abolished after adrenalectomy (O'Dell et al. 2004).

We have shown recently that EtOH promotes brain steroidogenesis by a local action independent of the HPA axis. EtOH increased both the amount of 3α,5α-TH PROG in isolated hippocampal tissue of control rats and the amplitude of GABAA receptor-mediated spontaneous miniature inhibitory postsynaptic currents (mIPSCs) recorded from CA1 pyramidal neurones, two effects prevented by finasteride (Sanna et al. 2004). These observations are consistent with evidence that the steroidogenic machinery, including steroidogenic regulatory protein (StAR), is present in specific regions of the brain (Furukawa et al. 1998; King et al. 2002; Sierra 2004). StAR is responsible for the delivery of cholesterol to the cytochrome P450 side-chain cleavage enzyme (P450scc), which catalyses the conversion of cholesterol to pregnenolone in mitochondria.

To examine the mechanism responsible for the reduction in the basal concentrations of neuroactive steroids and the increased sensitivity of the production of these steroids to EtOH induced by social isolation, we have now investigated possible changes in the expression of StAR in the cerebral cortex of isolated rats under basal conditions and after administration of EtOH. To investigate further whether the greater increase in brain concentration of 3α,5α-TH PROG induced by EtOH in isolated rats is of functional significance in terms of GABAA receptor activity, we also examined the effects of EtOH both on isoniazid-induced seizures in group-housed and isolated rats as well as on mIPSCs in hippocampal slices prepared from such animals. Moreover, given that fluctuations in brain and plasma concentrations of neuroactive steroids associated with physiological conditions are functionally correlated with changes in the expression of genes for specific GABAA receptor subunits (Concas et al. 1998; Herbison 2001; Koksma et al. 2005; Maguire et al. 2005), the effect of social isolation on the abundance of α4 and δ subunits of the GABAA receptor was also studied. Our results provide insight into the functional link between 3α,5α-TH PROG and GABAA receptor expression and function, and the relevance of this system to the pharmacology of EtOH.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Animals

Male Sprague–Dawley CD (caesarian derived) rats at 30 days of age, immediately after weaning, were housed for 30 days either in groups of six to eight per cage or individually in smaller cages. They were maintained under an artificial 12-h light, 12-h dark cycle at a constant temperature of 23 ± 2°C and 65% humidity. Food and water were freely available at all times. Animal care and handling throughout the experimental procedures were in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC).

Treatments

Isoniazid (isonicotinic acid hydrazide; Sigma, Milan, Italy) was dissolved in physiological saline and administered s.c. (300 mg per kg body mass). EtOH (20% v/v in saline) was administered i.p. (1 g/kg). Finasteride was extracted from tablets of Prostide (Trapani et al. 2002) and dissolved in 20% 2-hydroxypropyl-β-cyclodextrin by ultrasonic treatment for 4 h; it was injected s.c. (25 mg/kg) 48 and 24 h before injection of EtOH. Rats were observed for 3 h to monitor the development of tonic–clonic seizures.

Extraction and assay of steroids

Rats were killed either with a guillotine (for measurement of plasma steroids) or by focused microwave irradiation (70 W/cm2 for 4 s) of the head (for measurement of brain steroids). The cerebral cortices were dissected and frozen at −20°C until steroid extraction. 3α,5α-TH PROG was extracted and purified as described previously (Serra et al. 2000). The extract residue was dissolved in 5 mL n-hexane and applied to a SepPak silica cartridge (Waters Corporation, Milford, MA, USA), and eluted components were separated and further purified by HPLC on a 5-µm Lichrosorb-diol column (250 × 4 mm; Phenomenex, Torrance, CA, USA) with a discontinuous gradient of 2-propanol (0–30%) in n-hexane. The recovery (70–80%) of 3α,5α-TH PROG through the extraction and purification procedures was monitored by addition of a trace amount (6000–8000 cpm; 20–80 Ci/mmol) of 3H-labelled standard to the brain homogenate.

Blood was collected from the trunk of killed rats into heparinized tubes and centrifuged at 900 g for 20 min at room temperature (25°C). The resulting plasma was frozen at − 80°C pending steroid assay. 3α,5α-TH PROG was extracted from plasma three times with 1.5 mL ethyl acetate and, in a similar manner to that purified from brain tissue, was quantified by radioimmunoassay with specific antibodies generated in sheep (Purdy et al. 1990; Serra et al. 2000).

RT–PCR

Total RNA was extracted from rat brain and subjected to RT with MultiScribe reverse transcriptase (GeneAmp Gold RNA PCR reagent kit; Applied Biosystems, Foster City, CA, USA) in the presence of oligo(dT). The resulting cDNA (1–10 ng) was amplified by PCR with Taq DNA polymerase (2.5 U) in a 50-µL reaction mixture (Master Mix kit; Eppendorf, Hamburg, Germany) containing 1 µm each of sense (5′-CATCCAGCAAGGAGAGGAAG-3′, corresponding to nucleotide positions 297–316) and antisense (5′-CGTGAGTTTGGTCTTTGAGG-3′, corresponding to nucleotide positions 792–773) primers specific for rat StAR cDNA (National Center for Biotechnology Information, accession no. AB001349). The reaction was performed in a thermal cycler (Eppendorf) for 30 cycles of 1 min at 94°C, 1 min at 55°C and 2 min at 72°C, followed by a final extension for 15 min at 72°C. The PCR product (496 bp) was cloned into the pCRII-TOPO cloning vector (Invitrogen, Carlsbad, CA, USA) and used to generate [α32-P]CTP-labelled cRNA probes for Rnase protection assay.

RNA extraction and quantitation of StAR mRNA

Total RNA was extracted from the cerebral cortex by the guanidine isothiocyanate method and the amount of StAR mRNA was determined with an RNA protection assay. For each reaction, 50 µg total RNA was dissolved in 20 µL hybridization solution containing 150 000 cpm 32P-labelled StAR cRNA probe (specific activity 6 × 107−7 × 107 cpm/µg) and 15 000 cpm 32P-labelled cyclophilin cRNA (1 × 106 cpm/µg); cyclophilin mRNA was used as an internal standard. The reaction mixture was incubated overnight at 50°C and then subjected to digestion with Rnase, after which RNA–RNA hybrids were detected by electrophoresis (on a sequencing gel containing 5% polyacrylamide and urea) and autoradiography. The autoradiogram was subjected to digital image analysis with a densitometer (GS-700; Bio-Rad, Hercules, CA, USA).

Immunoblot analysis

Tissue was dissolved directly in 2% sodium dodecyl sulphate sample buffer by incubation for 5 min at 95°C. Protein samples (20 µg in 20 µL) were then fractionated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis on 10% minigels (Mini Protean II; Bio-Rad). The separated proteins were transferred to a polyvinylidene difluoride membrane and subjected to immunoblot analysis with rabbit polyclonal antibodies to StAR (Calbiochem, San Diego, CA, USA), horseradish peroxidase-conjugated secondary antibody and chemiluminescence reagents (ECL; Amersham Biosciences, Little Chalfont, UK). The amount of StAR was quantified by analysis of the corresponding band on the autoradiogram with a densitometer (GS-700). Data were normalized by dividing the optical density of the band corresponding to StAR by that of the band for glyceraldehyde-3-phosphate (GAPDH) (internal standard), which was revealed by re-probing the membrane with a mouse monoclonal antibody to this protein (Chemicon).

Hippocampal slice preparation

Rats were anaesthetized with chloroform and the brain was rapidly removed into ice-cold cutting solution (220 mm sucrose, 2 mm KCl, 1.3 mm NaH2PO4, 12 mm MgSO4, 0.2 mm CaCl2, 10 mm glucose, and 2.6 mm NaHCO3, pH 7.3, equilibrated with 95% O2 and 5% CO2). Coronal slices (thickness 300 µm) of hippocampus were cut with a Vibratome 1000 Plus (Vibratome, St Louis, MO, USA) and then incubated in artificial cerebrospinal fluid (ACSF) (126 mm NaCl, 3 mm KCl, 1.25 mm NaH2PO4, 1 mm MgSO4, 2 mm CaCl2, 10 mm glucose, and 26 mm NaHCO3, pH 7.3, equilibrated with 95% O2 and 5% CO2) first for 40 min at 34°C and then for 30 min at room temperature before experiments.

Whole-cell patch-clamp recording

Hippocampal tissue slices were transferred to a chamber perfused with ACSF at a rate of ∼ 2 mL/min at room temperature. Whole-cell patch-clamp electrophysiological recordings from CA1 pyramidal neurones and dentate gyrus (DG) granule cells were performed with an Axopatch 200-B amplifier (Axon Instruments, Union City, CA, USA) and an infrared differential interference contrast microscope. Patch microelectrodes (borosilicate capillaries containing a filament; outer diameter 1.5 µm) (Sutter Instruments, Novato, CA, USA) were prepared with a two-step vertical puller (Sutter Instruments) and had a resistance of 4–6 MΩ. GABAA receptor-mediated currents were recorded at a holding potential of − 60 mV with an internal solution containing 140 mm CsCl, 2 mm MgCl2, 1 mm CaCl2, 10 mm EGTA, 10 mm Hepes-CsOH (pH 7.3), 2 mm ATP (disodium salt) and 5 mm QX-314 (lidocaine N-ethyl bromide). In all experiments, kynurenic acid, a the broad-spectrum antagonist of ionotropic glutamate receptors, was added to the ACSF at a concentration of 3 mm to pharmacologically isolate GABAA receptor-mediated currents. Access resistance varied between 20 and 40 MΩ; if it changed by > 20% during an experiment, the recording was discarded. Currents through the patch-clamp amplifier were filtered at 2 kHz and digitized at 5.5 kHz with commercial software (pClamp 8.2; Axon Instruments).

Spontaneous mIPSCs were recorded in CA1 pyramidal neurones at a holding potential of − 60 mV in ACSF containing 500 μM lidocaine. The recording protocol and analysis have been described previously (Sanna et al. 2004). The mIPSCs were analysed with MiniAnalysis 5.4.17 software (Synaptosoft, Decatur, GA, USA). Each event identified was confirmed by visual inspection in each experiment. The effects of the various drugs on the different mIPSC kinetic parameters in individual neurones were evaluated by cumulative probability analysis, with statistical significance determined with the Kolmogorov–Smirnov non-parametric two-sample test.

In another set of experiments, we recorded synaptically evoked GABAA receptor-mediated IPSCs (eIPSCs) from CA1 pyramidal neurones at − 65 mV in ACSF containing 3 mm kynurenic acid. The eIPSCs were evoked at 20-s intervals with a concentric bipolar stimulating electrode placed in the stratum radiatum (current stimulation with 100–500 μA for 100 μs). They were continuously recorded both before (for ∼ 10 min) and after the onset of perfusion with 3α,5α-TH PROG (1 or 3 μM). The eIPSCs were analysed using Clampfit 8.2 software (Axon Instruments).

In a third set of experiments, we recorded tonic GABAA receptor-mediated currents from DG granule cells at − 65 mV in ACSF containing 3 mm kynurenic acid. Analysis of tonic GABAergic currents was performed with MiniAnalysis software by selecting epochs of 3 s every 30 s of continuous recording. Changes in noise of the tonic current during pharmacological treatment with respect to the control period were measured. We visually excluded synaptic IPSCs from the analysis of tonic currents.

Immunohistochemistry

Rats were anaesthetized by i.p. injection of Equithesin (1 g sodium pentobarbital, 4.251 g choral hydrate, 2.125 g MgSO4, 12 mL EtOH, 43.6 mL propylene glycol, adjusted to a total volume of 100 mL with distilled water) at 0.3 mL/g and were perfused through the ascending aorta with 4% paraformaldehyde in phosphate-buffered saline (PBS). The brain was removed, exposed to the same fixative for 1 h, and then transferred to 20% (w/v) sucrose. Sagittal sections (thickness 50 µm) were cut with a vibratome and maintained in antifreeze solution (30% ethylene glycol, 20% glycerol and 50% 0.05 m phosphate buffer v/v) at − 20°C. Indirect immunohistochemistry was performed with free-floating sections. The sections were washed with PBS, incubated for 30 min at room temperature with 0.1% phenylhydrazine, permeabilized for 1 h with 0.2% Triton X-100 in PBS (PBS-T), and incubated for 1 h with 10% normal donkey serum (Jackson ImmunoResearch, West Grove, PA, USA) in PBS-T. They were then incubated with goat antibodies to the α4 or δ subunits of the GABAA receptor (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1 : 500 in PBS-T containing 10% normal donkey serum. After several washes, the sections were incubated for 2 h with biotinylated donkey antibodies to goat IgG (Jackson ImmunoResearch) diluted 1 : 1000 in PBS-T. Immune complexes were detected by incubation first for 2 h with horseradish peroxidase–conjugated streptavidin (Jackson ImmunoResearch) at 2 µg/mL and then for 6–10 min with 0.4 mm 3,3′-diaminobenzidine (Sigma) and 0.01% H2O2. After washing, the sections were mounted on gelatin-coated slides, dehydrated and covered. Control sections not exposed to primary antibodies did not yield positive staining.

Sections were examined with an Olympus BX-41 (Hamburg, Germany) microscope and photographed with an F-View charge-coupled device camera. Representative images obtained with a Plan 2 × objective (numerical aperture, 0.05) or with a UPlan FI 20 × objective (numerical aperture, 0.50; for inserts) are shown. Semiquantitative analysis of the images was performed with AnalySIS 3.2 software (Soft Imaging System, Münster, Germany). Different areas of the hippocampus (plates 48–50; Paxinos and Watson 1986) were selected for each image, and the intensity of grey values was measured for each region of interest.

Statistical analysis

Data are expressed as mean ± SEM. The amounts of 3α,5α-TH PROG were compared by two-way anova; individual means were compared by Newman–Keuls post hoc test. Behavioural data were analysed by Fisher's exact probability test or Student's t-test. Comparisons of pooled electrophysiological data were performed by Student's t-test or one-way anova followed by Scheffe's post hoc test. Protein data are derived from seven animals in each experimental group, and mRNA data from two independent experiments, each with at least three animals in each experimental group, and are expressed as percentage change relative to control values; they were subjected to one- or two-way anova followed by Scheffe's post hoc test. In all instances, p < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Effects of EtOH on the abundance of 3α,5α-TH PROG and the expression of StAR in socially isolated rats

Consistent with our previous data (Serra et al. 2000), social isolation for 30 days in the absence of any other stressor induced significant decreases in the cerebrocortical and hippocampal concentrations of 3α,5α-TH PROG (−42% and – 49% respectively) compared with the corresponding values for group-housed animals (data not shown). Also consistent with previous observations (Barbaccia et al. 1999; Van Doren et al. 2000), a single injection of EtOH markedly increased both the cerebrocortical and plasma concentrations of 3α,5α-TH PROG in both group-housed and isolated rats. However, as we showed previously (Serra et al. 2003), the percentage increases in the brain and plasma concentrations of 3α,5α-TH PROG induced by EtOH were much greater in isolated rats (+ 273% and + 138% respectively) than in group-housed rats (+ 101% and + 68% respectively) (Fig. 1). The effect of EtOH on the level of 3α,5α-TH PROG in isolated rats was also significantly greater (F1,20 = 5.75, p = 0.03) for the brain than for plasma. anova revealed a significant effect of social isolation (F1,41 = 13.862, p = 0.006) and interaction between factors (F1,41 = 6.673, p = 0.012).

image

Figure 1.  Potentiation by social isolation of the effects of EtOH on the cerebrocortical and plasma concentrations of 3α,5α-TH PROG. Group-housed or socially isolated animals were killed 20 min after injection of EtOH (1 g/kg, 20% v/v, i.p.) or saline. Data are expressed as percentage increase induced by EtOH relative to the respective control (saline) value and are mean ± SEM of about 12 animals per group. *P < 0.05, **P < 0.01 versus respective control value; †P < 0.05, ††P < 0.01 versus corresponding group-housed value; ‡P < 0.01 versus corresponding value for plasma (two-way anova followed by Newman-Keuls post hoc test).

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To examine the molecular mechanism responsible for both the reduction in the basal concentrations of neuroactive steroids and for the greater steroidogenic effect of EtOH in the brain of socially isolated rats, we investigated possible changes in the abundance of StAR mRNA and protein. The 37-kDa precursor form of StAR is produced in the cytoplasm and is converted to a 32-kDa form during its incorporation into the inner mitochondrial membrane (Lin et al. 1995). The amounts of both StAR mRNA and the two forms of the protein (37 and 32 kDa) in the cerebral cortex or hippocampus did not differ between isolated and group-housed animals under basal conditions (data not shown). In contrast, 40 min after administration of EtOH, the amount of StAR mRNA was increased in the brain of both groups of rats, but the percentage increase in the cerebral cortex (Table 1) or hippocampus (data not shown) was significantly greater for isolated than for group-housed rats. anova revealed a significant effect of EtOH treatment (F1,17 = 67.084, p < 0.001) and interaction between factors (F1,17 = 7.650, p = 0.013).

Table 1.   EtOH-induced increase in the amount of StAR mRNA in the cerebral cortex
 Group-housedIsolated
  1. Animals were killed 40 min after administration of EtOH or saline Data are expressed as percentage relative to group-housed rats treated with saline (control) or with EtOH and are mean ± SEM of values from about six animals per group. *p < 0.05, **p < 0.01 versus control; p < 0.05 versus group-housed rats treated with EtOH (one-way anova followed by Scheffe's post hoc test).

Saline100 ± 2.08104 ± 4.51
Ethanol124 ± 10.3*138 ± 12.7**,

Moreover, EtOH reduced the amount of the 37-kDa StAR protein and increased that of the 32-kDa form in the cerebral cortex of socially isolated rats, whereas it increased the abundance of both forms of StAR in the cortex of group-housed animals (Fig. 2).

image

Figure 2.  Effects of EtOH on the abundance of StAR protein in the cerebral cortex of isolated and group-housed rats. (a) Animals were killed 40 min after the administration of EtOH or saline. Data are expressed as percentage change in the amounts of the 37- or 32-kDa forms of StAR relative to the values for group-housed rats treated with saline (control) and are mean ± SEM from about seven animals per group. *P < 0.01 versus control value (two-way anova followed by Scheffe's post hoc test). (b) Representative immunoblots for StAR and GAPDH (internal standard).

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Prevention by finasteride of the effect of EtOH on isoniazid-induced convulsions in socially isolated rats

We next examined the functional relevance of the greater steroidogenic effect of EtOH in the brain of socially isolated rats by evaluating the ability of EtOH to antagonize convulsions elicited by isoniazid, an inhibitor of GABA synthesis, in isolated and group-housed animals. EtOH inhibited isoniazid-induced convulsions in isolated rats, as revealed by a greater delay in the onset of convulsions and by a reduced percentage of animals manifesting convulsions, whereas it had no effect on the pattern of convulsions induced by isoniazid in group-housed animals (Fig. 3a). Systemic administration of the 5α-reductase inhibitor finasteride, which inhibits the EtOH-induced increase in the amount of 3α,5α-TH PROG in rat cerebral cortex (Van Doren et al. 2000), prevented the effect of EtOH on isoniazid-induced seizures in isolated rats (Fig. 3b). It is worth noting that in the group-housed rats finasteride decreased the onset of convulsions by 20 min (p < 0.05) but it had no effect in isolated animals (Fig. 3b).

image

Figure 3.  Prevention by finasteride of the protective effect of EtOH on isoniazid-induced convulsions in socially isolated rats. (a) Group-housed or socially isolated animals were injected with EtOH (1 g/kg, 20% v/v, i.p.) or saline 5 min before injection with isoniazid (300 mg/kg, s.c.). Rats were observed for 3 h to determine the time of onset of tonic–clonic seizures (left) and the number of animals manifesting convulsions (right). Data are mean ± SEM from 24 animals per group. *P < 0.05, **P < 0.01 versus the respective isoniazid-treated group. (b) Group-housed or socially isolated animals were injected with finasteride (25 mg/kg, s.c.) both 48 and 24 h before injection with EtOH (or saline) and isoniazid as in (a). Rats were observed for 3 h to determine the time of onset of tonic–clonic seizures. Data are mean ± SEM from 24 animals per group. †P < 0.05 versus respective group treated with isoniazid plus EtOH; **P < 0.01 versus respective isoniazid-treated group.

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Role of 3α,5α-TH PROG in the enhanced effect of EtOH on GABAA receptor-mediated mIPSCs in CA1 neurones of socially isolated rats

We next examined changes in GABAA receptor function in CA1 pyramidal neurones present in fresh hippocampal slices prepared from group-housed or socially isolated rats. Spontaneous mIPSCs were recorded in the whole-cell voltage-clamp mode (holding potential, − 60 mV) in the presence of lidocaine (500 µm) and kynurenic acid (3 mm), a broad-spectrum antagonist of ionotropic glutamate receptors. Inward mIPSCs were completely abolished by the GABAA receptor antagonist bicuculline methiodide at 20 µm (data not shown). The kinetic characteristics of mIPSCs under basal conditions did not differ significantly between group-housed and isolated rats (Table 2).

Table 2.   Characteristics of mIPSCs recorded in CA1 pyramidal neurones in hippocampal slices from group-housed or isolated rats
 Group-housedIsolated
  1. Values are mean ± SEM.

Peak amplitude (pA)25.2 ± 1.424.7 ± 0.9
Frequency (Hz)1.68 ± 0.221.45 ± 0.15
Decay time (ms)34.5 ± 3.328.6 ± 3.1

Consistent with previous observations (Sanna et al. 2004), bath application of EtOH (25–100 mm) for up to 30 min increased the amplitude of mIPSCs recorded from cells of both group-housed and isolated rats in a time- and concentration-dependent manner (Figs 4a–d). However, the potency of EtOH was markedly greater in hippocampal slices from isolated rats than in those from group-housed rats. Indeed, EtOH increased mIPSC amplitude only at a concentration of 100 mm in neurones from group-housed rats, whereas it proved similarly effective at concentrations as low as 25 mm in those from isolated animals. The efficacy of EtOH at 100 mm did not differ between isolated and group-housed rats, however. Furthermore, consistent with previous results (Sanna et al. 2004), the effect of EtOH on mIPSC amplitude appeared biphasic, comprising an immediate (time 0, initial 3 min of perfusion) and a delayed (onset at ∼ 10–20 min after the beginning of perfusion) phase in both group-housed (apparent only at 100 mm EtOH) and isolated (apparent at all EtOH concentrations tested) rats. Pretreatment of hippocampal slices with 1 µm finasteride for 10 min, which per se failed to affect mIPSC amplitude (Sanna et al. 2004), completely prevented the delayed, but not the immediate, enhancement of mIPSC amplitude by EtOH (Figs 4e and f), suggesting that this delayed action might be mediated by an increased production and local release of neurosteroids.

image

Figure 4.  Increased potency of EtOH in enhancement of GABAA receptor-mediated mIPSCs in CA1 pyramidal neurones from socially isolated rats. (a, b) Averaged mIPSC traces recorded at various times during bath application of 25 mm EtOH for 30 min as well as 10 min after washout in neurones from group-housed (a) or isolated (b) rats. Arrows indicate traces obtained in the presence of EtOH. (c, d) Concentration–response relationship for the effects of EtOH on mIPSC amplitude in CA1 pyramidal neurones from group-housed (c) or isolated (d) rats. Values are percentage change in mIPSC amplitude induced by bath application of the indicated concentrations of EtOH and are mean ± SEM from eight to 12 (group-housed rats) or 10–16 (isolated rats) cells. *P < 0.05, **P < 0.01 versus control. (e, f) Percentage change in mean mIPSC amplitude induced by bath application of 100 mm EtOH in the absence or presence of 1 µm finasteride (applied 10 min before EtOH) in neurones from group-housed (e) or isolated (f) rats. Values are mean ± SEM of values from six to eight (group-housed rats) or seven to 10 (isolated rats) cells. *P < 0.05, **P < 0.01 versus control. Control values represent the mean response before drug application.

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Effects of 3α,5α-TH PROG on eIPSCs in CA1 pyramidal neurones of group-housed and socially isolated rats

To determine whether the increased sensitivity to EtOH of neurones from isolated rats might be due to a change in postsynaptic GABAA receptor responsiveness to neurosteroids, we measured the acute modulatory effect of 3α,5α-TH PROG on eIPSCs recorded from CA1 pyramidal neurones in the presence of 3 mm kynurenic acid. At concentrations of 1 and 3 µm, 3α,5α-TH PROG had no significant effect on mean IPSC amplitude but increased the total current associated with each IPSC (as reflected by the area under the current trace) to a similar extent in both group-housed and isolated rats (Fig. 5).

image

Figure 5.  Effects of 3α,5α-TH PROG on eIPSCs in CA1 pyramidal neurones of group-housed and isolated rats. Data are expressed as percentage change in eIPSC amplitude (a) or in the area of the corresponding current trace (b) induced by bath application of 1 or 3 µm 3α,5α-TH PROG. Values are mean ± SEM from four to nine (group-housed rats) or six to12 (isolated rats) cells. *P < 0.05 versus corresponding control response. Control values represent the mean response before drug application.

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Up-regulation of the α4 and δ subunits of the GABAA receptor by social isolation

We next examined expression of the α4 and δ subunits of the GABAA receptor in the hippocampus by immunohistochemistry with specific antibodies that were generated in response to extracellular epitopes of these proteins. We previously showed that these antibodies recognized single proteins of ∼ 70 and 54 kDa respectively in immunoblot analysis of a crude membrane fraction prepared from hippocampal neurones (Sanna et al. 2003; Follesa et al. 2005). A diffuse moderate level of immunolabelling of the α4 subunit was distributed throughout the hippocampal formation of group-housed animals, with labelling slightly more concentrated in the granule cell layer of the DG and in pyramidal cells of CA1 and CA3 (Fig. 6a). A low level of immunoreactivity corresponding to the δ subunit was also apparent throughout the granule cell layer of the DG and the pyramidal cell layer of CA1 and CA3 in group-housed animals (Fig. 7a). In socially isolated rats, the level of α4 immunoreactivity was increased throughout the hippocampus compared with that in group-housed rats (Fig. 6b); the increase was significant in the strata oriens and radiatum of CA1 and CA3, in the stratum lacunosum moleculare of CA1, in the molecular layer of the DG and in CA4, and it was most pronounced in granule cells of the DG (+ 164%) and in the pyramidal cell layers of CA1 and CA3 (+ 183 and + 178% respectively) (Figs 6bi–iii and c). Social isolation also induced small increases in the level of δ subunit labelling (Fig. 7b), which were statistically significant in the pyramidal cells of CA1 and CA3 (+ 17 and + 26% respectively), in the stratum oriens of CA3 (+ 14%), and in the molecular layer and granular cells of the DG (+ 32 and + 16% respectively) (Figs 7bi–iii and c).

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Figure 6.  Changes in immunoreactivity for the α4 subunit of the GABAA receptor in the hippocampal formation induced by social isolation. (a, b) Representative immunohistochemical analysis of the distribution of the α4 subunit in hippocampal slices from group-housed (a) or isolated (b) rats. Scale bar 1 mm. (ai–iii, bi–iii) Quantification of changes in the level of α4 subunit immunoreactivity in the various regions of the hippocampus in group-housed (ai–iii) and isolated (bi–iii) rats. Scale bar 50 µm. (ai, bi) Pyramidal cell layer of CA3; (aii, bii) pyramidal cell layer of CA1; (aiii, biii) granule cell layer of DG. Values are percentage change in grey intensity relative to the corresponding value for group-housed rats and are mean ± SEM from six animals in each experimental group. †P < 0.05, †††P < 0.001 versus corresponding value for group-housed rats (one-way anova followed by Scheffe's post hoc test).

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Figure 7.  Changes in immunoreactivity for the δ subunit of the GABAA receptor in the hippocampal formation induced by social isolation. (a, b) Representative immunohistochemical analysis of the distribution of the δ subunit in hippocampal slices from group-housed (a) or isolated (b) rats. Scale bar 1 mm. (ai–iii, bi–iii) Quantification of changes in the level of δ subunit immunoreactivity in the various regions of the hippocampus in group-housed (ai–iii) and isolated (bi–iii) rats. Scale bar 50 µm. (ai, bi) Pyramidal cell layer of CA3; (aii, bii) pyramidal cell layer of CA1; (aiii, biii) granule cell layer of DG. Values are percentage change in grey intensity relative to the corresponding value for group-housed rats and are mean ± SEM from six animals for each experimental group. †P < 0.05, ††P < 0.001 versus corresponding value for group-housed rats (one-way anova followed by Scheffe's post hoc test).

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Social isolation and tonic inhibitory currents in granule cells of the DG

Functional changes associated with increased expression of the δ subunit of the GABAA receptor induced by social isolation were assessed by recording GABAA receptor-mediated tonic inhibitory currents in granule cells of the DG. The reduction in tonic current noise induced by bath application of 20 μm bicuculline was significantly greater in hippocampal slices from isolated rats than in those from group-housed animals (p = 0.035) (Figs 8a and b). In addition, the enhancement of tonic current noise induced by 3α,5α-TH PROG (3 μm) was markedly greater in granule cells of the DG from isolated rats (+ 104 ± 24%) than in those from group-housed animals (+ 53 ± 12%) (p = 0.048) (Figs 8c and d). Tonic currents were also recorded from CA1 pyramidal neurones, but there was no significant difference in the effect of bicuculline between the two groups (Fig. 8b).

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Figure 8.  Social isolation alters tonic inhibitory currents in granule cells of the DG. (a) Representative traces of tonic currents recorded in the whole-cell voltage-clamp mode from granule cells of the DG in hippocampal slices from group-housed or isolated rats. Currents were recorded both before (Control) and during the bath application of 20 μm bicuculline (Bic). (b) Quantification of the percentage change in tonic current noise induced by bicuculline in granule cells of the DG and in CA1 pyramidal neurones. Values are mean ± SEM from five to 11 different cells. †p < 0.05 versus group-housed rats. (c) Representative traces of tonic currents in granule cells of the DG from group-housed and isolated rats recorded before (Control) and during bath application of 3 μM 3α,5α-TH PROG (AP) either alone or together with 20 μm bicuculline. (d) Quantification of the percentage change in tonic current noise induced by 3α,5α-TH PROG in granule cells of the dentate gyrus. Values are mean ± SEM from 12 different cells. †P < 0.05 versus group-housed rats.

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Social isolation and GABAA receptor sensitivity to Ro15–4513

Finally, to evaluate the functional relevance of the increased expression of the α4 subunit of the GABAA receptor in the hippocampus of isolated rats, we tested the modulatory effect of Ro15-4513 on GABAA receptor-mediated mIPSCs in CA1 pyramidal neurones. This compound acts as an inverse agonist at the benzodiazepine-binding site of GABAA receptors containing either the α1, α2, α3 or α5 subunit together with the γ2 subunit (Barnard et al. 1998), but it is able to bind and positively modulate receptors comprising α4, β and γ2 subunits (Knoflach et al. 1996; Wafford et al. 1996). In hippocampal slices obtained from group-housed rats, bath application of 3 μm Ro15-4513 decreased (−15 ± 0.4%) the time constant of mIPSC decay. In contrast, in CA1 pyramidal neurones of isolated rats, Ro15-4513 slightly increased (+ 18 ± 7%) the time constant of mIPSC decay, although the difference in the effects of this drug between the two groups did not reach statistical significance. The frequency and amplitude of mIPSCs were not influenced by Ro15-4513 (data not shown). The effects of Ro15-4513 on GABAA receptor-mediated mIPSCs in granule cells of the DG did not differ significantly between isolated and group-housed animals (data not shown).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We have shown that social isolation results in changes in GABAA receptor expression in the brain as well as in enhancement of the stimulatory effect of EtOH on brain steroidogenesis, GABAA receptor function and associated behaviour. The increases in the abundance of 3α,5α-TH PROG in brain and plasma induced by systemic injection of EtOH were greater in isolated rats than in group-housed animals. Acute administration of EtOH to socially isolated rats increased the concentration of 3α,5α-TH PROG to a markedly greater extent in the cerebral cortex than in plasma (Fig. 1) (Serra et al. 2003). This finding, which is consistent with our previous observation that EtOH increases local neurosteroid synthesis in the brain independently of the HPA axis (Sanna et al. 2004), suggests that a hyper-responsiveness of the HPA axis due to chronic stress (Akana et al. 1992) is not the only mechanism responsible for the enhanced effect of EtOH on neuroactive steroid concentrations in the brain of isolated rats. This greater efficacy of EtOH in isolated rats might also be attributable to functional changes induced by social isolation in the brain steroidogenic machinery, including enzymes, peripheral benzodiazepine receptors (PBRs) and StAR.

We previously showed that, although social isolation had no significant effect on the binding kinetics of PBRs in the cerebral cortex, this housing condition increased the steroidogenic response to the selective PBR agonist CB34, a response more pronounced in the cerebral cortex than in the plasma of isolated rats (Serra et al. 2004). Given that EtOH is not known to interact with PBRs, it is likely that the greater effects of EtOH and CB34 on the cerebrocortical versus plasma concentrations of neuroactive steroids in isolated rats are due to a change in another component of the neuronal steroidogenic system induced by isolation.

The present data show that the acute administration of EtOH induced a greater increase in the amount of StAR mRNA in the cerebral cortex of isolated rats than in group-housed rats. EtOH treatment reduced the amount of the 37-kDa form and increased that of the 32-kDa form in the cerebral cortex of isolated rats, whereas the abundance of both StAR forms was increased by EtOH in the cortex of group-housed rats. Given that the molecular mechanism of StAR function remains unclear (Sierra 2004), it is not yet possible to explain the opposite effects of EtOH on the abundance of the two forms of the protein in the cerebral cortex of isolated rats. An increase in the amount of the 32- (or 30-) kDa form of StAR has previously been shown to be accompanied by a decrease in the amount of the 37-kDa form (Kimoto et al. 2001; Shibuya et al. 2003). Our data suggest that the rate of proteolytic conversion of the full-length StAR protein to the 32-kDa form may be increased by EtOH in the cerebral cortex of isolated rats, although the levels of StAR mRNA in isolated animals was increased to an greater extent than in group-housed animals.

The increase in the amount of 3α,5α-TH PROG in the brain induced by EtOH is thought to contribute to its anticonvulsant effect (Van Doren et al. 2000). We have now shown that the greater efficacy of EtOH in increasing 3α,5α-TH PROG synthesis in the brain of socially isolated rats is associated with an increase in the ability of EtOH to antagonize convulsions induced by isoniazid, an effect prevented by pretreatment with finasteride, a 5α-reductase inhibitor that blocks the synthesis of 3α,5α-TH PROG. Finasteride reduced the delay in the onset of isoniazid-induced seizures in group-housed animals but not in isolated rats, consistent with their reduced basal level of 3α,5α-TH PROG in the brain. The reduction in the brain level of 3α,5α-TH PROG induced by social isolation is thus probably responsible for the increased vulnerability to seizures apparent in isolated animals. Accordingly, isolated mice were more susceptible to picrotoxin-induced seizures (Matsumoto et al. 2003).

The enhanced anticonvulsant action of EtOH in isolated rats is consistent with our finding that EtOH induced a concentration-dependent increase in the amplitude of GABAA receptor-mediated mIPSCs in CA1 pyramidal neurones with a greater potency in socially isolated rats than in group-housed animals. Moreover, the observation that the effect of EtOH on mIPSC amplitude was inhibited by finasteride supports the idea that this action of EtOH is mediated by an increased production of 3α,5α-TH PROG. This hypothesis is also consistent with the acute modulatory effect of 3α,5α-TH PROG on eGABAA receptor-mediated IPSCs recorded from CA1 pyramidal neurones. The lack of a difference in this effect of 3α,5α-TH PROG between isolated and group-housed rats suggests that the greater potency of EtOH with regard to increasing the amplitude of GABAA receptor-mediated mIPSCs in the hippocampus of isolated rats might be due to the greater production of 3α,5α-TH PROG induced by EtOH in these animals.

The slight increase in susceptibility to seizures apparent in isolated rats might also reflect changes in GABAA receptor gene expression induced by the persistent decrease in the brain concentration of neuroactive steroids. Both in vivo and in vitro studies have shown that fluctuations in the concentration of 3α,5α-TH PROG result in selective changes in the abundance of specific GABAA receptor subunits as well as in consequent changes in GABAA receptor function (Concas et al. 1998; Smith et al. 1998; Follesa et al. 2000, 2002; Herbison 2001Maguire et al. 2005). Withdrawal of progesterone or EtOH thus resulted in increased expression of the α4 subunit as well as a decreased seizure threshold and increased anxiogenic behaviour in rats (Mahmoudi et al. 1997; Smith et al. 1998; Reddy and Rogawski 2000; Gulinello et al. 2001). We have now shown that social isolation is associated with increased expression of α4 and δ subunits in hippocampal neurones. An increase in transcription of the gene for the α4 subunit in the hippocampus of isolated rats elicited by the decrease in level of 3α,5α-TH PROG might thus contribute to the increase in susceptibility to seizures as well as to anxiogenic behaviour in such animals (Serra et al. 2000). Increased expression of the α4 subunit is associated with many seizure-prone states (Brooks-Kayal et al. 1998; Peng et al. 2004). Consistent with the notion that GABAA receptors containing the α4 subunit possess high affinity for the benzodiazepine receptor antagonist flumazenil and the partial inverse agonist Ro15-4513 (Wafford et al. 1996; Whittemore et al. 1996; Sanna et al. 2003), we found that Ro15-4513 increased the time constant for mIPSC decay in CA1 pyramidal neurones from isolated rats whereas it reduced this parameter in group-housed rats, although this difference in effect did not achieve statistical significance.

Social isolation also increased the expression of the δ subunit in the rat hippocampus, probably resulting in the formation of GABAA receptors that contain both α4 and δ subunits. Similar results were obtained in rats after progesterone withdrawal (Sundstrom-Poromaa et al. 2002). The putative increase in the number of GABAA receptors containing the δ subunit as well as α4 may explain the limited potentiation of mIPSCs by Ro15-4513 in CA1 neurones of isolated rats. In fact, receptors containing both α4 and δ subunits manifest low affinity for benzodiazepines (Saxena and Macdonald 1994, 1996).

Given that δ substitutes for γ2 and that the latter subunit is essential for synaptic localization of GABAA receptors (Essrich et al. 1998), receptors containing α4 and δ would be expected to be extrasynaptic. Extrasynaptic GABAA receptors are responsible for tonic inhibition in granule cells of the cerebellum and DG (Nusser et al. 1998; Nusser and Mody 2002; Stell and Mody 2002). Our data show that GABAA receptor-mediated tonic inhibitory currents in granule cells of the DG were significantly enhanced in hippocampal slices from socially isolated rats. Consistent with the notion that tonic inhibition in CA1 pyramidal cells is not mediated by δ subunit-containing GABAA receptors, but most likely by extrasynaptic α5γ2-containing receptors (Crestani et al. 2002; Stell et al. 2003; Caraiscos et al. 2004), tonic currents recorded from these cells did not differ between isolated and group-housed rats. In agreement with the marked effect of neuroactive steroids on tonic inhibition mediated by δ subunit-containing GABAA receptors (Stell et al. 2003), we found that the current noise variation induced by 3α,5α-TH PROG in granule cells of the DG was greater for isolated rats than for group-housed animals. GABAA receptors containing the δ subunit play an important role in the regulation of neuronal excitability. Mice that lack this subunit develop epilepsy and show other signs of hyperexcitability (Mihalek et al. 1999; Spigelman et al. 2002), and expression of this subunit is decreased in the hippocampal formation in a mouse model of temporal lobe epilepsy (Peng et al. 2004) as well as in the rat kindling model of alcohol dependence (Cagetti et al. 2003). It should also be mentioned that the increased expression of GABAA receptors containing both the α4 and δ subunit associated with social isolation might also be expected to have important consequences for the ability of EtOH to directly modulate GABAergic inhibitory transmission. In fact, recombinant GABAA receptors comprising the α4 and δ subunits were shown to be selectively sensitive to extremely low concentrations of EtOH (Sundstrom-Poromaa et al. 2002; Wallner et al. 2003). In line with these data, it was also reported that the tonic inhibitory currents in DG granule cells, mediated by GABAA receptors containing the α4 and δ subunits, was potentiated by 30 mm EtOH (Wei et al. 2004). However, a recent paper (Borghese et al. 2006) failed to reproduce these observations both in recombinant receptors and in DG granule cells. Thus, the role of GABAA receptors containing α4 and δ subunits in the modulatory effects of EtOH is still controversial and requires further investigation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by grant CE000042735 (Project of Center of Excellence for the Neurobiology of Dependence D.M. 21 January 2001), Progetti di Ricerca di Interesse Nazionale (PRIN) grant 2003052254-001 from the Ministry of Instruction, University and Research of Italy, National institute on Alcohol Abuse and Alcoholism grant U01AA13641, the Sardinian Health Ministry (n°363, 12.04.05, c. 12076/00) and GIO.I.A. (Gioventù in Allegria) Foundation (Pisa, Italy).

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  3. Materials and methods
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  6. Acknowledgements
  7. References
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