Benzodiazepine-induced anxiolysis and reduction of conditioned fear are mediated by distinct GABAA receptor subtypes in mice
Highlights
► We used H–R point mutated mice to study benzodiazepine effects on anxiety and fear. ► α2-subunits are required for benzodiazepine-induced anxiolysis in elevated plus maze. ► α1- and α2-subunits are required for benzodiazepine-induced reduction of conditioned fear. ► Pharmacological interventions for specific anxiety disorders can be differentially tailored.
Introduction
Anxiety disorders are the most common psychiatric disorders with a lifetime prevalence of approximately 25% (Kessler et al., 1994). While these disorders affect millions of people, the molecular basis for the development of these disorders remains largely unknown. However, both experimental and clinical data support a role of the GABAergic system and specifically of GABAA receptors in anxiety- and depression-related pathologies (Crestani et al., 1999; Kalueff and Nutt, 2007; Luscher et al., 2011; Smith and Rudolph, 2011).
GABAA receptors represent a highly diverse group of receptors with 19 known GABAA receptor subunits (α1-6, β1-3, γ1-3, δ, ɛ, π, ρ1-3, θ); see (Mohler et al., 2002; Olsen and Sieghart, 2009 for a review). Benzodiazepines (BZs) such as diazepam bind to a specific site (BZ site) between the α and γ subunits and allosterically modulate GABAA receptors containing the α1, α2, α3 and/or α5 subunits. These receptors modulate processes as diverse as sleep, anxiety, learning and memory, seizures, depressive symptomatology, and reward (Rudolph and Knoflach, 2011). GABAA receptor modulating drugs such as benzodiazepines have been used to treat anxiety disorders for five decades (Shader and Greenblatt, 1993).
BZ-sensitive GABAA receptors can be rendered insensitive to this class of drugs through the replacement (i.e., point mutation) of a conserved histidine residue by arginine in the drug-binding site (Benson et al., 1998; Wieland et al., 1992). Using point mutated mice carrying diazepam-insensitive α1, α2, α3, or α5 subunits, the anxiolytic-like and sedative actions of diazepam were shown to be pharmacologically separable. Specifically, the sedative action of diazepam was blocked in α1(H101R) mice demonstrating that this property is mediated by α1-containing GABAA receptors (McKernan et al., 2000; Rudolph et al., 1999). In contrast, the anxiolytic-like actions of diazepam were absent in α2(H101R) mice when tested in unconditioned anxiety paradigms such as the elevated plus maze or the light/dark box; thus, α2-containing GABAA receptors are required for diazepam-induced anxiolysis (Low et al., 2000). Furthermore, the anxiolytic-like effects of diazepam in these unconditioned paradigms remained intact in α1-, α3- and α5-point mutant [α1(H101R), α3(H126R) and α5(H105R), respectively] mice signifying that these subunits are not necessary for the anxiolytic effect of diazepam in mice (Crestani et al., 2002; Low et al., 2000; Rudolph et al., 1999).
While the involvement of the α2-containing GABAA receptors in the modulation of anxiety by benzodiazepines is now undisputed, α3-containing GABAA receptors have also been postulated to mediate anxiolytic-like actions. In rats, TP003, a highly α3-selective agonist in vitro, displays an anxiolytic-like action in the elevated plus maze and in the stress-induced hyperthermia test. This compound also disinhibited responding in a conditioned emotional response paradigm in squirrel monkeys, suggesting that TP003 is also active in conditioned fear models (Dias et al., 2005). However, selectivity of TP003 for α3-containing GABAA receptors has not been demonstrated in vivo, and it is important to note that TP003-induced anxiolysis was observed only at high receptor occupancy levels (75%), whereas the BZ chlordiazepoxide already exerts its anxiolytic-like action at a receptor occupancy of 25% (Dias et al., 2005). Combined with the aforementioned evidence from studies with point mutant mice, this suggests that at commonly-used therapeutic doses non-selective BZs exert their anxiolytic-like effects via α2-containing GABAA receptors. The precise role of α3-containing GABAA receptors in anxiolysis remains unclear.
It also remains unknown whether the effects of BZs on unconditioned anxiety and conditioned fear2 are mediated by the same GABAA receptor subtypes when these compounds are administered at therapeutically-relevant doses, since e.g., not only the α2 subunit, but also the α1 and α3 subunits are expressed in the lateral and basolateral amygdala (Marowsky et al., 2004), regions which have been implicated in both conditioned fear and unconditioned anxiety. The α1 subunit has also been shown to play a critical role in amygdalar plasticity and fear learning (Wiltgen et al., 2009). While previous research suggests that this subunit is not involved in the anxiolytic effects of benzodiazepines in models of unconditioned anxiety (McKernan et al., 2000; Rudolph et al., 1999), the possible contribution of this subunit to the fear-reducing effects of benzodiazepines in conditioned fear paradigms has not been examined.
In order to determine whether the same or different GABAA receptor subtypes are necessary for the anxiolytic-like action of classical benzodiazepines in unconditioned anxiety and conditioned fear models, we investigated the role of different GABAA receptor subtypes by challenging wild type, α1(H101R), α2(H101R) and α3(H126R) mice bred on the C57BL/6J background with non-sedating doses of either diazepam or chlordiazepoxide in the elevated plus maze and the fear-potentiated startle paradigms. Activity responses in a novel open field test were also measured after drug administration to determine whether non-specific locomotor effects could impact behavior in the other two tests, particularly the elevated plus maze which is a locomotor-dependent task (Reynolds et al., 2001; but see Crestani et al., 2001).
Section snippets
Animals
Adult male wild type, α1(H101R), α2(H101R), and α3(H126R) point mutant mice were used in our experiments. These mice were generated by the introduction of a histidine to arginine point mutation in the normally benzodiazepine-sensitive α1, α2, and α3 subunits of the GABAA receptor as previously described (Low et al., 2000; Rudolph et al., 1999) and were bred as homozygous pairings. All subjects were bred on the C57BL/6J background (original stock from Jackson Laboratory, Bar Harbor, ME) at
Elevated plus maze
The effects of diazepam dose and genotype on the percentage of time spent in the open arms and percentage of open arm entries were examined by 4 × 3 two-way (genotype × drug dose) ANOVAs. Percent open arm time was significantly affected by genotype (F(3,145) = 5.55, p < 0.01), drug dose (F(2,145) = 9.61, p < 0.01) and the genotype × drug dose interaction (F(6, 145) = 2.13, p < 0.05) (Fig. 1A). Post hoc Dunnett tests revealed that the 2 mg/kg dose of diazepam increased percent open arm time in
Discussion
The above findings provide evidence for a differential role of GABAA receptor subtypes in mediating the effects of benzodiazepines on unconditioned anxiety and conditioned fear responses at the same drug doses. In the elevated plus maze, both diazepam and chlordiazepoxide significantly increased the amount of open arm exploration in wild type, α1(H101R) and α3(H126R) mice but this effect was abolished in α2(H101R) mice when either drug was administered. These results are similar to studies
Disclosure/Conflict of interest
UR is a consultant for Sunovion and for Concert Pharmaceuticals.
Acknowledgments
The project described was supported by Award Number R01MH080006 of the National Institute of Mental Health to UR. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Mental Health or the National Institutes of Health.
References (28)
- et al.
Pharmacology of recombinant gamma-aminobutyric acidA receptors rendered diazepam-insensitive by point-mutated alpha-subunits
FEBS Letters
(1998) - et al.
Anxiolytic-like action of diazepam: mediated by GABAA receptors containing the alpha2 subunit
Trends in Pharmacological Sciences
(2001) - et al.
The effects of intra-cerebral drug infusions on animals' unconditioned fear reactions: a systematic review
Progress in Neuro-Psychopharmacology and Biological Psychiatry
(2008) - et al.
GABAA receptors: subtypes provide diversity of function and pharmacology
Neuropharmacology
(2009) - et al.
GABA(A) receptors: immunocytochemical distribution of 13 subunits in the adult rat brain
Neuroscience
(2000) - et al.
A single histidine in GABAA receptors is essential for benzodiazepine agonist binding
Journal of Biological Chemistry
(1992) - et al.
TPA023 [7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine], an agonist selective for alpha2- and alpha3-containing GABAA receptors, is a nonsedating anxiolytic in rodents and primates
Journal of Pharmacology and Experimental Therapeutics
(2006) - et al.
Circadian modulation of the rat acoustic startle response
Behavioral Neuroscience
(1992) - et al.
Trace fear conditioning involves hippocampal alpha5 GABA(A) receptors
Proceedings of the National Academy of Sciences of the United States of America
(2002) - et al.
Decreased GABAA-receptor clustering results in enhanced anxiety and a bias for threat cues
Nature Neuroscience
(1999)
Resolving differences in GABAA receptor mutant mouse studies
Nature Neuroscience
Evidence for a significant role of alpha 3-containing GABAA receptors in mediating the anxiolytic effects of benzodiazepines
Journal of Neuroscience
GABAA-receptor heterogeneity in the adult rat brain: differential regional and cellular distribution of seven major subunits
Journal of Comparative Neurology
Role of GABA in anxiety and depression
Depression and Anxiety
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KSS and EE contributed equally to this work.