INTRODUCTION

Naturally occurring variations in maternal behavior in the rat directly influence the development of individual differences in stress reactivity (Liu et al, 1997; Caldji et al, 1998; Francis et al, 1999). Under conditions of stress, the adult offspring of mothers who showed an increased frequency of pup licking/grooming and arched-back nursing (high LG-ABN mothers), exhibit more modest pituitary–adrenal responses and decreased fearfulness compared with the offspring of low LG-ABN dams. Importantly, cross-fostering offspring from low-to-high LG-ABN mothers reverses the pattern of stress reactivity that is characteristic of the normal offspring of low LG-ABN mothers, and the reverse is also true (Francis et al, 1999). The increased stress reactivity of the adult offspring of low LG-ABN mothers is associated with elevated corticotropin-releasing factor (CRF) gene expression in both the paraventricular nucleus of the hypothalamus and the central nucleus of the amygdala (Diorio, Francis and Meaney, unpublished). CRF has been established as a mediator of behavioral and endocrine responses to stress, and this effect is, in part, mediated by CRF action at the level of the noradrenergic cell bodies in the locus coeruleus and the nucleus of the solitary tract (Butler et al, 1990; Valentino et al, 1998; Bakshi et al, 2000).

Maternal care alters benzodiazepine (BZ) receptor binding in the amygdala and locus coeruleus of the offspring such that among adult animals, BZ receptor levels in the central nucleus of the amygdala are highly correlated (r=+0.87) with the frequency of maternal licking/grooming in infancy (Caldji et al, 1998). The gamma-aminobutyric acidA (GABAA) receptor system inhibits CRF activity, particularly at the level of the amygdala and locus coeruleus (Owens et al, 1991; Skelton et al, 2000). Predictably, behavioral responses to stress are inhibited by BZs, which exert their potent anxiolytic effect by enhancing GABA-mediated Cl− currents through GABAA receptors (Barnard et al, 1988; Sieghart, 1995; McKernan and Whiting, 1997; Mehta and Ticku, 1999). BZ receptor agonists exert anxiolytic effects via their actions at a number of limbic areas, depending upon the test conditions. However, to date, the evidence is perhaps strongest for BZ effects at the level of the basolateral complex of the amygdala, comprising the lateral, basal and anterior basal nuclei (Pitkanen et al, 1997), and the central nucleus of the amygdala. Direct administration of BZs into the basolateral or central regions of the amygdala yields an anxiolytic effect (Hodges et al, 1987; Pesold and Treit, 1995; Gonzalez et al, 1996). Previous studies have found that the offspring of low LG-ABN mothers exhibit increased fearfulness in comparison to those of high LG-ABN dams (Caldji et al, 1998). In the current studies, we provide evidence for a profound influence of maternal behavior on GABAA receptor subunit gene expression that is most apparent in the basolateral and central nuclei of the amygdala, regions that are crucial for behavioral and autonomic expressions of fear (Schafe et al, 2001).

MATERIALS AND METHODS

Animals

The mothers were Long-Evans hooded rats obtained from Charles River Canada (St Constant, Qué bec) and housed in 46 cm × 18 cm × 30 cm ‘Plexiglass’ cages. Food and water were provided ad libitum. The colony was maintained on a 12 : 12 light : dark schedule with lights on at 08.00 h. The animals underwent routine cage maintenance beginning on Day 12, but were otherwise unmanipulated. All procedures were performed according to guidelines developed by the Canadian Council on Animal Care with protocols approved by the McGill Committee on Animal Care.

The behavior of each dam was observed for eight, 60-min observation periods daily for the first 8 days postpartum (Myers et al, 1989; Liu et al, 1997; Caldji et al, 1998; Francis et al, 1999). Observers were trained, using video tapes and still photography, to a high level of inter-rater reliability (ie >0.90). Observations were performed at six periods during the light phase (08.00, 10.00, 11.00, 14.30, 16.00, and 18.00 h) and two periods during the dark phase of the L : D cycle (20.00 and 06.00 h). Within each observation period, the behavior of each mother was scored every 4 min (15 observations/period × eight periods per day=120 observations/mother/day) for the following behaviors: mother off pups, mother carrying pup, mother licking and grooming any pup, mother nursing pups in either an arched-back posture, a ‘blanket’ posture in which the mother lays over the pups, or a passive posture in which the mother is lying either on her back or side while the pups nurse (see Myers et al, 1989; Liu et al, 1997; Caldji et al, 1998; Francis et al, 1999 for a description of behaviors).

The frequency of maternal licking/grooming and arched-back nursing across a large number of mothers is normally and not bi-modally distributed (Champagne et al, in press). Hence, the high and low LG-ABN mothers represent two ends of a continuum, rather than distinct populations. In order to define these populations for the current study, we observed the maternal behavior in cohorts of mothers, generally 30–40 dams with their pups, and devised the group mean and standard deviation for each behavior over the first 8 days of life as previously described (Liu et al, 1997; Caldji et al, 1998; Francis et al, 1999). High LG-ABN mothers were defined as females whose frequency scores for both licking/grooming and arched-back nursing were greater than 1 SD above the mean. Low LG-ABN mothers were defined as females whose frequency scores for both licking/grooming and arched-back nursing were greater than 1 SD below the mean.

At the time of weaning on Day 22 of life, the male offspring were housed in same-sex, same litter groups of three to four animals per cage until Day 45 of life, and two animals per cage from this point until the time of testing, which occurred no earlier than 100 days of age. All experiments were performed by individuals who were blind to the developmental history of the animals.

Adoption Study

The adoption study was performed using a limited cross-fostering design (McCarty and Lee, 1996; Francis et al, 1999) in which only two pups from each litter, one male and one female, were fostered onto other mothers in order to maintain the original gender distribution. The intention here was to avoid disturbances in maternal behavior than are known to be associated with the wholesale fostering of entire litters (Maccari et al, 1995). Within 6 h following the birth of the last pup, one male and one female were selected at random and placed into the host litter. The entire cross-fostering procedure was completed within 15 min of having originally disturbed the litters. Pups born to mothers previously characterized as high or low LG-ABN dams were used on the basis of previous findings showing that the frequency of maternal licking/grooming towards pups of the first litter is highly correlated (r=+0.84) with that directed towards pups of the second litter (Champagne et al, in press). Pups born to high LG-ABN mothers were then cross-fostered onto either another high LG-ABN mothers (high–high) or low LG-ABN mothers (high–low). Likewise, pups born to low LG-ABN mothers were then cross-fostered onto either other high LG-ABN mothers (low–high) or other low LG-ABN mothers (low–low). The results of a previous adoption study showed that the limited fostering does not alter the maternal behavior of the dam (Francis et al, 1999).

In Situ Hybridization

Brains were obtained by rapid decapitation shortly (<20 s) following the removal of the animal from the home cage. Brains were frozen in isopentane maintained on dry ice and 15 μM coronal sections were prepared under RNase-free conditions and stored at −80°C. In preparation for the hybridization experiments, sections were prefixed in a 4% paraformaldehyde solution for 10 min. The sections were then washed in 2 × SSC buffer (2 × 5 min) and in 0.25% acetic anhydride and 0.1 M triethanolamine solution (pH 8.0; 1 × 10 min). They were then dehydrated using a 50–100% ethanol gradient, placed in chloroform for 10 min, followed by rehydration in 95% ethanol. The sections were then incubated overnight at 37°C with 75 μl/section of hybridization buffer containing 50% dionized formamide, 10 mM dithiothreitol, 10 mM Tris (pH 7.5), 600 mM sodium chloride, 1 mM EDTA, 10% dextran sulfate, 1 × Denhardt's solution, 100 μg salmon sperm DNA, 100 μg/ml yeast tRNA, with 1 × 106 CPM [35S]ddATP-labeled oligonucleotide probe. Oligonucleotide probes (see Table 1) were synthesized (Beckman 1000 DNA Synthesizer, Beckman, USA) and labeled using a DNA 3′-end labeling kit (Boehringer Mannheim, USA). Note that the γ2 oligonucleotide sequence used in this study recognizes both γ2L and γ2S variants of the γ2 subunit. Preliminary studies using a scrambled versions of the α1, α2, and γ2 probes yielded no specific signal on brain sections (data not shown). Following hybridization, slides were washed 4 × 30 min in 1 × SSC at 55°C, rinsed briefly in water, dried, and apposed to Hyperfilm for 21 days along with sections of 35S-labeled standards prepared with known amounts of 35S in a brain paste. The hybridization signal within various brain regions was quantified using densitometry with an image analysis system (MCID, St Catherines, Ontario). The data are presented as arbitrary optical density (absorbance) units following correction for background and are the averages drawn from three sections per brain region/per animal. The anatomical level of analysis was verified using the rat brain atlas of Paxinos and Watson (1982) with Nissl-staining of sections following autoradiography.

Table 1 List of Antisense Oligonucleotide Probes used in the In Situ Hybridization Studies and the Sources for the Sequences
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Western Blotting

Following rapid decapitation, brains were removed and placed on ice. The amygdala were dissected, snap-frozen on dry ice, and stored at −80°C. Frozen samples were placed into a microcentrifuge tube containing ∼5 volume of ice-cold sucrose buffer (320 mM sucrose/4 mM Hepes, pH 7.4) and homogenized in the same buffer using a Teflon-glass homogenizer. The homogenate was centrifuged for 10 min at 1100g and the supernatant was centrifuged for 15 min at 9200g. The resulting pellet was then resuspended in sucrose buffer (with 9 vol of ice-cold dH2O), and homogenized with a Dounce homogenizer. Hepes (1 M, pH 7.4) was added to a final concentration (7.5 mM) and incubated on ice (30 min). The homogenate was centrifuged for 20 min at 25 500g and the pellet was discarded. The supernatant was centrifuged (48 000 rpm, 2 h), and the resulting pellet resuspended in 1 ml of buffer (sucrose (30 mM)/Hepes (4 mM), pH 7.4), and homogenized using a 25-gauge needle. Aliquots of the homogenates were taken to determine the levels of protein using the method of Bradford (1976) and ranged from 1.5 to 2.5 μg/μl.

Protein samples (25 μg) were mixed with an equal volume of 2 × sample buffer (0.25 M Tris-HCl, 20% glycerol, 4% SDS, 0.005% bromoethanol blue, and 5% β-mercaptoethanol) and subjected to denaturing and reducing conditions prior to separation using electrophoresis with Novex 4–12% Tris-glycine PAGE precast gels (Helixx Technologies, Scarborough, Ontario, Canada) with stained molecular markers loaded for reference (SeeBlue, Santa Cruz Biotech, CA, USA). Proteins were electrophoretically transferred according to the method of Towbin onto nitrocellulose membranes (Amersham, Oakville, Ontario) and air-dried overnight. The membranes were blocked for 1 h at room temperature with 5% Carnation dried milk in TBS-T (Tris, NaCl, 0.1% Tween-20, pH 7.6), washed briefly in TBS-T and incubated overnight at 4°C with antibodies to the α1, α2, or γ2 subunits of the GABAA receptor. All antibodies were generously provided by Dr Ruth McKernan (Merck Sharpe Dohme, Harlow, UK) and used at concentrations of 1 : 1000 (McKernan et al, 1991). Membranes were washed for 20 min with TBS-T and incubated with secondary horseradish peroxidase-labeled antibody (anti-rabbit Igg, Amersham Inc.) diluted 1 : 5000 in TBS-T for 1 h at room temperature. After 4 × 15 min washes in TBS-T, the membranes were then exposed using an ECL kit (Amersham) and apposed to film (ECL Hyperfilm, Amersham). In order to verify the accuracy of sample loading, membranes were stripped and reprobed with an α-tubulin monoclonal antibody (Biodesign International, Kennebunkport, ME) diluted at 1 : 5000. Optical density readings for bands were determined using a computer-assisted densitometry system (MCID Systems; St Catherines, Ontario).

Statistical Analysis

For each experiment, there were between one and two pups per litter representing at least three to a maximum of five litters per group. In order to control for any potential ‘litter’ effects, we analyzed the data both by subject and by litter. There were no differences between these two methods of analyses in the statistical outcomes for any of the experiments. For the sake of brevity, we provide the results of the analysis by subject. The in situ hybridization data were analyzed using a two-way (Group × Region) analysis of variance with Tukey post hoc tests where appropriate. The results of the Western blots were analyzed using t-tests for simple main effects of maternal care.

RESULTS

α Subunit mRNA Expression

There was a significant Group (F=13.22; df=1,9; P<0.01) effect for α1 mRNA expression (Figure 1) reflecting the widespread differences across multiple regions. Post hoc analysis revealed increased α1 mRNA levels in the offspring of the high LG-ABN mothers in the prefrontal cortex, the CA2 and CA4 regions of the hippocampus, the central, lateral and basolateral nuclei of the amygdala, and the locus coeruleus. Analysis of the α2 mRNA data revealed neither a significant group nor a Group × Regions interaction effect. In general, levels of α2 mRNA expression were comparable between groups. There was a significant Group × Region interaction (F=5.73; df=8,72; P<0.0001) for α3 subunit mRNA expression. In contrast with the α1 expression pattern, α3 mRNA levels were significantly increased in the offspring of low LG-ABN mothers in the central and especially the basolateral nucleus of the amygdala. Likewise, there was a Group × Region interaction that approached significance (F=1.96; df=8,72; P<0.10) for α4 subunit mRNA expression and post hoc analysis showed increased levels of the mRNA levels for the α4 subunit in the central and basolateral nuclei of the amygdala, as well as in the prefrontal cortex of the offspring of low LG-ABN mothers. There was only a marginally significant Group × Region effect for levels of α5 subunit mRNA (F=1.73; df=8,72; P<0.15), although mRNA levels were significantly higher in the central and basolateral nuclei of the amygdala in the offspring of high compared with low LG-ABN mothers. Owing to the absence of the interaction effects, these latter findings should be viewed with caution. No other comparisons approached significance. There were no group difference in mRNA levels in the parietal cortex, hypothalamus, or thalamus for any of the α subunits.

Figure 1
figure 1

Mean±SEM levels of mRNA for various α subunits of the GABAA receptor in various brain regions expressed as optical density units from autoradiograms from in situ hybridization studies with a 35S-labeled oligonucleotide probes (see Table 1; n=5 animals per group). Abbreviations: medial prefrontal cortex (mPFC), dentate gyrus (DG), hippocampal Ammon's horn (CA1-4), basolateral nucleus of the amygdala (BLA), lateral nucleus of the amygdala (LA), central nucleus of the amygdala (CA), locus coeruleus (LC), nucleus of the solitary tract (NTS), thalamus (Thal), parietal cortex (PCtx). ***P<0.01; **P<0.01; *P<0.05.

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β Subunit mRNA Expression

There was no significant Group × Region interaction for β1 subunit mRNA expression and no pattern of group differences was evident (Figure 2). β1 mRNA levels were significantly higher in the thalamus in the offspring of low LG-ABN mothers (data not shown). This was the only instance in which significant group differences in the mRNA levels for the GABAA receptor subunits were found in this region. There were no significant differences in the hippocampal/amygdaloid regions. Differences in β2 mRNA levels were regionally very specific, generally mapping onto the differences observed for the α1 subunit mRNA, and yielding a significant Group × Region interaction effect (F=7.72; df=8,72; P<0.001). β2 mRNA levels in the offspring of high LG-ABN mothers were significantly higher in the basolateral and central nuclei of the amygdala as well as the locus coeruleus (Figure 2). Levels of β3 mRNA were consistently higher in the offspring of high LG-ABN mothers yielding significant Group and Group × Region interaction effects (F=2.52; df=8,72; P<0.05). Post hoc analyses revealed that in the several regions of the hippocampus and amygdala, β3 mRNA levels were significantly in the offspring of high LG-ABN mothers. There were no group differences in mRNA levels in the parietal cortex or hypothalamus for any of the β subunits.

Figure 2
figure 2

Mean±SEM levels of levels of mRNA for various β subunits of the GABAA receptor in various brain regions expressed as optical density units from autoradiograms from in situ hybridization studies with a 35S-labeled oligonucleotide probes (see Table 1; n=5 animals per group). Abbreviations described in the caption to Figure 1. ***P<0.01; **P<0.01; *P<0.05. Note that the signal in the mPFC was too low for reliable quantification.

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γ Subunit mRNA Expression

The pattern of group differences in the expression of the γ subunits was strikingly unique to the amygdala. Thus, there were significant Group × Region interaction effects in mRNA levels for both the γ1 (F=235; df=8,72; P<0.05) and γ2 (F=2.69; df=8,72; P<0.01) subunits (see Figures 3a). Post hoc analysis revealed significantly increased levels of γ1 and γ2 mRNA levels in the central, basolateral, and lateral nuclei of the amygdala in the offspring of high compared with low LG-ABN mothers. In no other region were these differences significant. Figure 3b provides a photomicrograph of an autoradiogram revealing the differences in γ2 mRNA levels in the central, basolateral, and lateral nuclei of the amygdala as a function of postnatal maternal care. Note the increased density of the signal in these regions in comparison to the neighboring corticomedial areas. There were no group differences in mRNA levels in the parietal cortex, hypothalamus, or thalamus for any of the γ subunits.

Figure 3
figure 3

(a) Mean±SEM levels of mRNA for various γ subunits of the GABAA receptor in various brain regions expressed as optical density units from autoradiograms from in situ hybridization studies with a 35S-labeled oligonucleotide probes (see Table 1; n=5 animals per group). Abbreviations described in the caption to Figure 1. ***P<0.01; **P<0.01; *P<0.05. (b) Photomicrograph of a representative autoradiogram from the in situ hybridization study comparing adult offspring of high and low LG-ABN mothers. The photo is focused on the amygdaloid region. Note the higher density signal in the lateral, nasolateral, and central regions of the amygdala in the high LG-ABN offspring. Abbreviations as described in the caption to Figure 1.

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Western Blotting

In order to at least partially verify the functional significance of the mRNA studies, we compared GABAA receptor subunit-like immunoreactivity in amygdaloid samples from adult offspring of high or low LG-ABN mothers using Western blotting (see Figure 4). We focused on two subunits that provided the most reliable differences in mRNA, α1 and γ2 subunits, as well as a negative control, the α2 subunit. The results reveal significant group differences in α1- and γ2-like immunoreactivity (t=4.10; P<0.01; t=5.69; P<0.005, respectively), with no group difference in the levels of α2-like immunoreactivity. Bearing in mind the limitations with such comparisons, the group differences at the level of protein (∼100–150%) exceeded those for mRNA (∼30–80% differences depending upon the region).

Figure 4
figure 4

Mean±SEM levels of α1, α2, and γ2 immunoreactivities on Western blots in amygdala samples from adult offspring high or low LG-ABN mothers (n=3–4/group). *P<0.01. The left-hand side panels depict representatives blots for each subunit (top bands) along with bands for β-actin immunoreactivity from the same membranes used to control for loading errors.

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Adoption Study

In the adoption study, we analyzed the expression of mRNAs for the α1 and γ2 subunits focusing on the basolateral, central, and lateral regions of the amygdala. The logic here was to again focus on those subunits that provided the most reliable differences in the in situ hybridization study. In both cases, the results revealed highly significant effects for rearing mothering, but not biological mothering (see Figure 5). This pattern was striking for both the α1 (rearing mother: F=31.60; 1,12; P<0.0001. biological mother: F<1.0; NS) and γ2 (rearing mother: F=15.88; 1,12; P<0.002. biological mother: F<1.0; NS) subunits in all regions, save for the γ2 subunit mRNA levels in the lateral nucleus of the amygdala. Even here, however, the effect of the rearing mother was significant. There was no significant main effect of the biological mother for either subunit in any region examined. Thus, expression of the mRNAs for both the α1 and γ2 subunits in animals born to low LG-ABn mothers, but reared by high LG-ABN dams was indistinguishable from that of the normal offspring of high LG-ABN mothers. The reverse was also true for pups born to high LG-ABN dams, but reared by low LG-ABn mothers.

Figure 5
figure 5

Mean±SEM levels of mRNA for α1 and γ2 subunits of the GABAA receptor in the amygdala expressed as optical density units from autoradiograms from in situ hybridization studies with a 35S-labeled oligonucleotide probes (n=4–5 animals per group) in the biological offspring of high LG-ABN mothers cross-fostered to either high (high–high) or low (high–low) LG-ABN mothers and the biological offspring of low LG-ABN mothers cross-fostered to either high (low–high) or low (low–low) LG-ABN mothers. Abbreviations as described in the caption to Figure 1. ***P<0.01; **P<0.01; *P<0.05.

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DISCUSSION

Neuronal inhibition is mediated through GABA-gated Cl− channels, collectively known as GABAA receptors. The GABAA receptor complex in the rat brain, which often includes a ‘BZ’ binding site, is most commonly arranged in a pentameric structure comprised of α, β, and γ subunits, in the form of two α, two β, and one γ subunit or of two α, one β, and two γ subunits (Barnard et al, 1988; Sieghart, 1995; McKernan and Whiting, 1997; Mehta and Ticku, 1999). The α subunit forms the GABA-binding site and the interface between the α and γ subunits appears to form the BZ receptor site (Khan et al, 1996). GABAA receptor activity, defined by Cl− influx and changes in neuronal potential, is allosterically regulated by compounds acting at BZ receptor sites (Braestrup et al, 1984; Im and Blakeman, 1991; Maksay, 1993; Haefely, 1994). Interestingly, dynamic variations in GABAA receptor function often occur as a result of such allosteric modulation of the GABAA receptor. Hence it seems reasonable to assume that the source of stable individual differences in GABAA receptor function might lie in variations in the expression of subunits that define BZ receptor binding.

Our results suggest that variations in maternal care permanently alter the subunit composition of the GABAA receptor complex in the offspring. Importantly, the results of the adoption study suggest that differences in GABAA receptor subunit expression were directly related to variations in maternal care (Figure 5). We found significantly increased levels of the mRNAs for the γ1 and γ2 subunits in the offspring of high compared with low LG-ABN mothers and the effect was strikingly unique to the amygdala. Differences in α subunit expression were more promiscuous. Levels of α1 mRNA were significantly higher in the amygdala, prefrontal cortex, hippocampus, and locus coeruleus of high compared with low LG-ABN offspring. The results of Western blotting studies for the α1 and γ2 subunits suggest that the effects at the level of mRNA expression are reflected in differences in protein levels. Likewise, there were no group differences in α2 subunit expression in the amygdala at either the level of mRNA or protein. Interestingly, the adult offspring of the low LG-ABN mothers show increased expression of the mRNAs for the α3 and, to a lesser extent, the α4 subunit. As with the γ subunits, such differences were largely limited to regions of the amygdala. The only substantial difference in the levels of mRNA encoding for the β subunits was in β2 mRNA in the amygdala and in β3 subunit mRNA levels that was more broadly apparent. What makes this finding intriguing is that in native GABAA receptor complexes, the β2 subunit is primarily associated with the α1 and γ2 subunits and each of these subunits is encoded by genes located on chromosome 5 (5q34–q35) (Wilcox et al, 1992).

The differences in subunit expression are likely to be of considerable importance for GABAA receptor pharmacology. Indeed, the adult offspring of high and low LG-ABN mothers differ in BZ receptor binding in the amygdala (Caldji et al, 1998). In this study, the correlation between maternal licking/grooming over the first 8 days of life and the adult level of BZ receptor binding in the central nucleus of the amygdala was +0.87. The inclusion of a γ1 or γ2 subunit in the GABAA receptor complex appears to define the presence of a BZ receptor site. Point mutations have been identified within both subunits that are sufficient to eliminate BZ receptor binding (Amin et al, 1997; Buhr and Sigel, 1997; Buhr et al, 1997; Wingrove et al, 1997) and animals bearing a null mutation of the γ2 subunit show approximately an 85% loss of [3H]flunitrazepam binding (Gunther et al, 1995). Thus, the effect of maternal care on the expression of the γ subunits might well contribute to the previously observed difference in BZ receptor-binding capacity between high and low LG-ABN mothers (Caldji et al, 1998). Interestingly, in this earlier study differences in [3H]flunitrazepam binding were largely unique to the amygdala, a finding that is similar to that for the γ1 or γ2 subunit mRNAs.

While the γ subunits define the presence of a BZ receptor, it is the α subunits that define the nature of the BZ receptor subtype (Barnard et al, 1988; Pritchett and Seeburg, 1991; Hadingham et al, 1993; McKernan and Whiting, 1997; Rudolph et al, 1999; Griebel et al, 2000). Receptors containing the α1 subunit exhibit BZ1 receptor pharmacology, with a high affinity for classical BZ agonists, such as diazepam and alprazolam, and also for the atypical BZ agonists, CL218872 and zolpidem. Transfection studies of recombinant GABAA receptors comprised of α1, β, γ1, or γ2 subunits exhibit BZ1 receptor pharmacology, with high-affinity binding for zolpidem (Barnard et al, 1988; Mehta and Ticku, 1999), which binds BZ1 receptors with an affinity (kd∼7 nM) that is at least 20 times greater than for BZ2 receptors (Arbilla et al, 1986). Receptors composed of the α2, α3, or α5 subunits show BZ2 receptor profiles, with high affinity for classical BZ agonists, but greatly reduced affinity for zolpidem. Thus, GABAA receptors composed of the α1, α2, α3, and α5 subunits are indistinguishable in their affinity for ligands, such as flunitrazepam, which bind with equal affinity to BZ1 and BZ2 receptors (see Barnard et al, 1988; Hadingham et al, 1993; McKernan and Whiting, 1997; Mehta and Ticku, 1999 for reviews). GABAA receptors comprised of any of these subunits or with the α4 subunit are also indistinguishable in their affinity for the partial inverse agonist RO15-4513; note, however, receptors bearing the α4 subunit do not bind BZ agonists (Yang et al, 1995). Moreover, GABAA receptors comprised of the α4 subunit show an increased affinity for full inverse agonists, such as the β-carbolines (Yang et al, 1995). Finally, there is also a modest contribution of the β subunits to BZ receptor pharmacology: GABAA receptors comprised of either the β2 or β3 subunit show a two-fold higher affinity for zolpidem compared with those bearing a β1 subunit. The mRNA levels for both the β2 and β3 subunit were higher in the offspring of high LG-ABN mothers, but again, only within the amygdala. These findings further underscore the apparent shift towards BZ1 receptor expression in the amygdala in the offspring of high LG-ABN mothers. Prolonged periods of maternal separation in early life decreased the levels of both α1 (Caldji, Diorio, Plotsky and Meaney, unpublished) and γ2 (Caldji et al, 2000) subunit mRNA. These effects on subunit expression were associated with differences in flunitrazepam binding, but such differences were substantially less impressive than those obtained using [3H]zolpidem, a selective ligand for the BZ1 receptor (Caldji et al, 2000).

BZ receptor agonists exert anxiolytic effects via their actions at a number of limbic areas, including the amygdala (Hodges et al, 1987; Pesold and Treit, 1995; Gonzalez et al, 1996). More recent studies have focused on the effects of intra-amygdaloid infusion of either GABAA or BZ receptor agonists or antagonists on fear conditioning. The results demonstrate potent effects of such treatments on the acquisition (amnesic effects) and expression (anxiolytic effects) of conditioned fear responses and underscore the importance of the amygdala as a critical target for the effects of GABAA and BZ receptor agonists (Brioni et al, 1989; Tomaz et al, 1993; Helmstetter and Bellgowan, 1994; Dickinson-Anson and McGaugh, 1997; Muller et al, 1997; Da Cunha et al, 1999). While maternal care altered subunit expression in the hippocampus and prefrontal cortex, the most consistent effects were clearly at the level of the basolateral and central regions of the amygdala. Predictably, the adult offspring of low LG-ABN mothers consistently show evidence for increased fearfulness in comparison to those of high LG-ABN mothers (Caldji et al, 1998; Francis et al, 1999), and these differences are reversed with cross-fostering (Francis et al, 1999).

The effect of maternal care on GABAA receptor subunit expression may provide a mechanism for the well-established relationship between early life events and vulnerability for anxiety disorders. Surprisingly, only a minority (∼30%) of humans subjected to even profound trauma develop PTSD (Ressnick et al, 1993). The quality of early family life serves as a highly significant predictor of vulnerability to PTSD (Udwin et al, 2000) and significantly influences the risk for anxiety disorders (Tweed et al, 1989; Stein et al, 1996; Pruessner et al, 2000). Emotionally adverse stimuli activate the human amygdala (Cahill and McGaugh, 1998). Indeed, the degree of amygdaloid activation is highly correlated (r=+0.93) with recall of emotionally disturbing, but not neutral material. With high temporal resolution fMRI techniques, LaBar et al (1998) found increased amygdala activity during the acquisition phase of fear conditioning, and patients with amygdala damage show profound deficits in fear conditioning (Bechara et al, 1995; LaBar et al, 1995). Interestingly, individual differences in the degree of amygdaloid activation occurring during fear conditioning was highly correlated with the strength of a conditioned autonomic fear response (Furmark et al, 1997).

Studies in humans support the idea that alterations in the GABAA/BZ receptor complex might form the basis of a vulnerability for anxiety disorders (Gorman et al, 2000). Unmedicated patients with a history of panic disorder show a significant decrease in labeling of the BZ receptor antagonist [11C]flumazenil in the orbitoprefrontal cortex and amygdala/hippocampal region in PET studies (Malizia et al, 1998). The findings are consistent with those of pharmacological measures of BZ receptor sensitivity. Subjects high on measures of neuroticism show reduced sensitivity to the BZ receptor agonist, midazolam (Glue et al, 1995). Roy-Byrne et al (1990), (1996) found reduced sensitivity to diazepam in patients with panic disorders, and proposed that the reduced BZ receptor sensitivity was related to anxiety. Patients with panic attacks or high levels of general anxiety show decreased sensitivity to BZ-induced amnesia, sedation, and dampening of noradrenergic function compared with controls (Melo de Paula, 1977; Oblowitz and Robins, 1983). These findings suggest that early life events might alter the development of the GABAA receptor system in brain regions that mediate stress reactivity, and thus contribute to individual differences in vulnerability to anxiety disorders (Gorman et al, 2000).