Despite its pharmacological relevance, the mechanism of the development of tolerance to the action of benzodiazepines is essentially unknown. The acute sedative action of diazepam is mediated via α1-GABAA receptors. Therefore, we tested whether chronic activation of these receptors by diazepam is sufficient to induce tolerance to its sedative action. Knock-in mice, in which theα1-,α2-,α3-, orα5-GABAA receptors had been rendered insensitive to diazepam by histidine-arginine point mutation, were chronically treated with diazepam (8 d; 15 mg · kg-1 · d-1) and tested for motor activity. Wild-type, α2(H101R), and α3(H126R) mice showed a robust diminution of the motor-depressant drug action. In contrast, α5(H105R) mice failed to display any sedative tolerance. α1(H101R) mice showed no alteration of motor activity with chronic diazepam treatment. Autoradiography with [3H]flumazenil revealed no change in benzodiazepine binding sites. However, a decrease in α5-subunit radioligand binding was detected selectively in the dentate gyrus with specific ligands. This alteration was observed only in diazepam-tolerant animals, indicating that the manifestation of tolerance to the sedative action of diazepam is associated with a downregulation of α5-GABAA receptors in the dentate gyrus. Thus, the chronic activation of α5-GABAA receptors is crucial for the normal development of sedative tolerance to diazepam, which manifests itself in conjunction with α1-GABAA receptors.
Loss of sedative efficacy of diazepam with chronic treatment has been proposed to result from the development of adaptive processes counteracting the repeated enhancement by the benzodiazepine of GABAA receptor-mediated inhibitory neurotransmission (Steppuhn and Turski, 1993; File and Fernandes, 1994; Fernandes et al., 1996; Marin et al., 1996, 1999; Perez et al., 2003). Functional alterations of GABAA receptors have frequently been reported after various chronic treatment regimens with diazepam (Hutchinson et al., 1996; Itier et al., 1996; Primus et al., 1996; Ali and Olsen, 2001; Costa et al., 2001; Bateson, 2002). Uncoupling of the allosteric interaction between the benzodiazepine binding site and the GABA site, probably linked to GABAA receptor internalization, has been proposed as a correlate of diazepam tolerance (Hutchinson et al., 1996; Itier et al., 1996; Primus et al., 1996; Ali and Olsen, 2001; Costa et al., 2001). Furthermore, subtle changes in the expression of GABAA receptor subunits were described notably in the cerebral cortex and hippocampus (Wu et al., 1994; Impagnatiello et al., 1996; Pesold et al., 1997; Arnot et al., 2001). In particular, a selective decrease in the expression of genes encoding the α1- and γ2-subunits in dendrites and spines of cortical pyramidal cells has been associated with tolerance to the anticonvulsant action of diazepam (Costa et al., 2002). In addition, α5-GABAA receptors were affected, as shown by an increase in α5-subunit mRNA in the frontoparietal cortex or a reduced radioligand binding in the hippocampus after 2 or 3 weeks of diazepam administration (Impagnatiello et al., 1996; Pesold et al., 1997; Li et al., 2000).
The recognition of distinct pharmacological functions of GABAA receptor subtypes opened new avenues to investigate the mechanisms of tolerance. Using a histidine-to-arginine point mutation strategy that selectively abolishes diazepam binding to GABAA receptors containing the α1-, α2-, α3-, or α5-subunit in vivo, it has been shown that α1-GABAA receptors mediate the acute sedative (Rudolph et al., 1999; McKernan et al., 2000) and anticonvulsant (Rudolph et al., 1999) properties of diazepam, whereas α2-GABAA receptors are the substrate for anxiolytic (Löw et al., 2000) and muscle-relaxant activity, the latter also requiring α3- and α5-GABAA receptors (Crestani et al., 2001, 2002). In view of such functional receptor specificity, we tested whether tolerance to a particular effect of diazepam is mediated via the same receptor subtype involved in the acute effect or whether additional GABAA receptor subtypes are required for neuronal plasticity leading to the development of tolerance with chronic diazepam treatment.
In the present study, the contribution of specific GABAA receptor subtypes in the development of tolerance to the motor-depressant action of diazepam was examined using wild-type and histidine-arginine point-mutated mice. Potential changes in benzodiazepine binding sites were analyzed by autoradiography.
Materials and Methods
Animals and drugs. Nine- to 11-week-old female wild-type, α1(H101R), α2(H101R), α3(H123R), and α5(H105R) mice (>10 backcrosses to 129/SvJ background) were used (Rudolph et al., 1999; Löw et al., 2000; Crestani et al., 2002). The sensitivity of GABAA receptors to diazepam is conferred by the presence of a histidine residue at a conserved position in the extracellular portions of the α subunits (α1-H101, α2-H101, α3-H126, α-H105). The histidine-arginine substitution in the GABAA receptor α1-, α2-, α3-, or α5-subunit results in a marked reduction of the binding affinity of the corresponding GABAA receptor subtype to diazepam, as shown on recombinant receptors (Benson et al., 1998). Thus, each point-mutated mouse line possesses a particular GABAA receptor subtype insensitive to allosteric modulation by diazepam, although its response to GABA is essentially preserved. Females were preferred to males because of the prominent intramale aggressive behavior inherent to the strain used as genetic background. They were reared in group-housed cages in the testing room under reversed 12 hr light/dark conditions. Treatments and behavioral testing were performed during the dark phase. The Cantonal Veterinary Office of Zürich approved all experimental procedures. Diazepam was from Hoffmann-La Roche (Basel, Switzerland).
Induction of sedative tolerance. Mice were subjected to daily injections of diazepam (10 mg/kg at 10 A.M. and 5 mg/kg at 4 P.M., i.p.) for 8 d in the home cage. On day 9, half of the mice were given vehicle (Diaz-Veh) and the other half were given the morning dose of 10 mg/kg diazepam as the test dose. Control animals, which received the vehicle (0.3% Tween 80-saline solution) as chronic treatment, were distributed in two groups. One group was treated with vehicle (Veh-Veh), and the other group was treated with 10 mg/kg diazepam. A fifth group of mice, which served as control for the effects of repeated injections, received a single diazepam (10 mg/kg) injection as the test dose. Animals were left undisturbed to experience the drug effects in the home cage for 30 min. At the end of this period, they were placed in individual circular alleys (Imetronic, Pessac, France) for motor-activity assessment, measured as the number of photocell interruptions during a 10 min period. The term test dose was used to indicate the association of the last diazepam morning injection of the chronic treatment with the behavioral assessment. The terms sedation and sedative, as defined by Katzung (1995), indicate the drug-induced decrease in the animal's spontaneous activity. Measurement of motor activity in rodents represents a standard behavioral assay for testing the sedative potential of drugs (Vogel, 2002).
Quantitative receptor autoradiography. A second series of animals received the same chronic treatment regimen with either vehicle or diazepam. On day 9, tolerance to the motor-depressant action of 10 mg/kg diazepam was tested, and the mice were killed by decapitation 5 hr thereafter. Binding assays using [3H]flumazenil (PerkinElmer, Boston, MA) or two ligands with a preferential affinity for the α5-subunit (Quirk et al., 1996; Skolnick et al., 1997), [3H]RY80 (PerkinElmer) and [3H]L655708 (Amersham Biosciences, Otelfingen, Switzerland), were performed on transverse 12 μm brain cryosections (Fritschy et al., 1997). Briefly, after a 1 hr incubation with 12 nm [3H]flumazenil or a 2 nm concentration of either [3H]RY80 or [3H]L655708 in 50 mm Tris-Cl at pH 7.5, the sections were exposed to a tritium-sensitive phosphor screen (Packard Cyclone Storage Phosphor System; Packard, Meridian, CT) for 2 or 8 d, respectively. Adding 10 μm clonazepam assessed unspecific labeling. The screens were digitized with a Packard Cyclone Scanner, and labeling intensities were measured in the motor cortex, striatum, nucleus accumbens, and hippocampal formation (CA1, CA3 stratum oriens-pyrami-dale, and dentate gyrus) of both hemispheres. At the concentrations used, both [3H]RY80 and [3H]L655708 have been reported to saturate α5-subunit binding sites with high affinity in hippocampal membranes (Quirk et al., 1996; Skolnick et al., 1997; Sur et al., 1999).
Statistics. Results, expressed as mean ± SE (or SD for binding studies), were analyzed using nonparametric Kruskal-Wallis analysis and, whenever appropriate, Mann-Whitney tests for post hoc mean comparisons.
Tolerance to the sedative action of diazepam in point-mutated mice
To identify the diazepam-sensitive GABAA receptor subtypes implicated in sedative tolerance, we examined the potential of histidine-arginine point-mutated mice to develop tolerance against the motor-depressant action of diazepam during the course of a chronic drug-treatment regimen. We focused on this behavioral effect for two main reasons. First, the effectiveness of diazepam in decreasing motor activity in rodents is considered as a valid behavioral manifestation of its sedative properties (Vogel, 2002). Second, this drug effect is exclusively mediated by α1-GABAA receptors, because it is abolished in α1(H101R) mice but unaltered in α2(H101R), α3(H126R), and α5(H105R) mice (Rudolph et al., 1999; Löw et al., 2000; McKernan et al., 2000; Crestani et al., 2002).
Sedative tolerance to diazepam (i.e., the diminution of its motor-depressant action during chronic treatment) was first analyzed using wild-type and α1(H101R) mice. The different treatment conditions affected motor activity differently in wild-type mice (H = 22.958; p < 0.001; n = 6-7 mice per group). In wild-type mice chronically treated with vehicle, the administration of 10 mg/kg diazepam was followed by a marked decrease in motor activity (p < 0.01 compared with Veh-Veh) (Fig. 1a). This effect was comparable with that seen in mice that received a single acute injection of diazepam. Mice chronically treated with diazepam did not show a reduction in motor activity in response to the test dose of diazepam and were indistinguishable from mice challenged with vehicle or from mice chronically treated with vehicle only (Fig. 1a). In α1(H101R) mice, neither a single acute injection of diazepam (10 mg/kg) nor the chronic diazepam treatment altered the level of motor activity compared with the chronic vehicle treatment, as revealed by the lack of statistical significance of the overall analysis of the effects of the different drug-treatment conditions (H = 5.914; not significant; n = 6 mice per group) (Fig. 1b). The increased motor activity seen in response to diazepam in α1(H101R) mice chronically treated with vehicle achieved significance only when compared, in a separate two-mean comparison, with the effect of the vehicle test injection (p < 0.01; Mann-Whitney test) but not when compared with that induced by a single acute diazepam injection (Fig. 1b).
To characterize the role of diazepam-sensitive GABAA receptor subtypes other than those containing the α1-subunit in sedative tolerance, α2(H101R), α3(H126R), and α5(H105R) mice were subjected to the same chronic diazepam treatment regimen. There was a significant overall effect of the different treatment conditions on motor activity in all three mutant lines [α2(H101R), H = 14.942, p < 0.001, n = 8 mice per group; α3(H126R), H = 12.194, p < 0.002, n = 8 mice per group; α5(H105R), H = 12.005, p < 0.002, n = 8 mice per group]. Diazepam (10 mg/kg) failed to decrease motor activity levels in α2(H101R) and α3(H126R) mice chronically treated with diazepam, but it induced sedation in those mutants chronically treated with vehicle (p < 0.01 compared with the respective Veh-Veh groups) (Fig. 1c). However, in α5(H105R) mice, the same test dose of diazepam was equally effective in depressing motor activity in animals chronically treated with either vehicle or diazepam (p < 0.01 compared with Veh-Veh) (Fig. 1c). Thus, tolerance to the motor-depressant action of diazepam developed to the same extent in wild-type, α2(H101R), and α3(H126R) mice within 8 d of chronic drug administration, but was not seen in α5(H105R) mice. Furthermore, the same chronic diazepam treatment regimen did not alter motor activity in α1(H101R) mice.
Brain autoradiography of α5-GABAA receptor binding sites
To assess whether the expression of sedative tolerance to diazepam would be associated with alterations in GABAA receptor binding sites, we analyzed benzodiazepine binding autoradiographically with [3H]flumazenil in wild-type, α1(H101R), and α2(H101R) mice. The levels of [3H]flumazenil binding differed across genotypes as a result of the presence of the respective point mutation (Fig. 2). However, no alteration in relation to the chronic drug-treatment regimen and the behavioral testing was detected, in particular in forebrain regions involved in the control of motor activity, including the primary motor cortex, striatum, and nucleus accumbens, as well as in the hippocampal formation (Fig. 2). Thus, there was no evidence for a general GABAA receptor downregulation.
To specifically analyze the role of α5-GABAA receptors, two selective α5-subunit ligands, [3H]RY80 and [3H]L655708, were used in the autoradiographic analysis. In wild-type mice, the levels of [3H]RY80 binding were differentially affected by the three treatment conditions in the dentate gyrus (H = 14.769; p < 0.001; n = 7-8 mice per group) (Fig. 3a). A significantly lower [3H]RY80 binding level (-13.6% relative to Veh-Veh) was observed in diazepam-tolerant animals (p < 0.01 compared with Veh-Veh and Veh-Diaz). A similar effect of the chronic diazepam treatment was confirmed using [3H]L655708 as the radioligand (H = 10.903; p < 0.004; n = 6-7 mice per group) (Fig. 3b). The [3H]L655708 binding level was significantly reduced (14.7% relative to Veh-Veh) only in tolerant mice (p < 0.01 compared with Veh-Veh and Veh-Diaz). In contrast, the effects of the three treatment conditions on [3H]RY80 and [3H]L655708 binding levels were comparable in the hippocampal CA1 area ([3H]RY80, H = 1.563, not significant, n = 5-6 mice per group; [3H]L655708, H = 3.661, not significant, n = 6-7 mice per group) as well as in the CA3 area ([3H]RY80, H = 4.662, not significant, n = 5-6 mice per group; [3H]L655708, H = 1.988, not significant, n = 6-7 mice per group) (data not shown).
[3H]RY80 binding was also analyzed in α1(H101R) and α2(H101R) mice subjected to the same chronic drug treatment and behavioral testing. In α2(H101R) mice, as in wild-type mice, the chronic administration of diazepam was accompanied by sedative tolerance (data not shown) and by a decrease in [3H]RY80 binding levels in the dentate gyrus (-12.5% relative to Veh-Veh; p < 0.01) (H = 9.231; p < 0.009; n = 5-6 mice per group) (Fig. 3c). The α1(H101R) mice displayed similar levels of [3H]RY80 binding regardless of the treatment conditions (H = 2.788; not significant; n = 5-6 mice per group) (Fig. 3d). Thus, the manifestation of tolerance to the motor-depressant action of diazepam was accompanied by an apparent reduction of α5-GABAA receptor binding selectively in the dentate gyrus, as shown for wild-type and α2(H101R) mice. This reduction of α5-subunit binding did not occur in α1(H101R) mice subjected to the same chronic diazepam treatment.
The present results point to a critical role of α5-GABAA receptors, in conjunction with α1-GABAA receptors, in the development of tolerance against the sedative action of diazepam. Interaction of diazepam with α5-GABAA receptors appears to be a prerequisite for the normal development of tolerance against its motor-depressant action. This is shown by the retained capacity of diazepam (10 mg/kg) to efficiently reduce motor activity in α5(H105R) mice, which possess diazepam-insensitive α5-GABAA receptors, at least within the limits of our chronic drug-treatment regimen (Fig. 1c). This motor effect cannot be attributed to a retained myorelaxant action of diazepam, because we have shown that these mutants, as well as the α2(H101R) mice, do not express the acute myorelaxant action of diazepam in the range of doses used here for the chronic treatment (Crestani et al., 2001, 2002). However, α2(H101R) and α3(H126R) mice developed sedative tolerance to diazepam to the same extent as wild-type mice (Fig. 1c), indicating that diazepam-sensitive α2- or α3-GABAA receptors are not essential for this effect. In rodents, tolerance to the motor-depressant action of diazepam develops rapidly, within 3-5 d of chronic administration (Steppuhn and Turski, 1993; File and Fernandes, 1994; Marin et al., 1996). We designed a chronic treatment protocol, using high doses of diazepam over a period of 9 d, to induce a robust sedative tolerance in our control wild-type mice. Nevertheless, we cannot rule out the possibility of a delayed occurrence of sedative tolerance (i.e., after a longer chronic diazepam treatment) in α5(H105R) mice. Conversely, the absence of a motor-depressant drug effect in wild-type, α2(H101R), and α3(H126R) mice with chronic treatment could reflect a rightward shift in the dose-response curve of diazepam, with higher doses of diazepam (>10 mg/kg) producing sedation. However, this appears improbable, especially with regard to our test conditions. Motor activity was measured 30 min after the last diazepam injection of the chronic treatment and not after a period of drug withdrawal, as is often the case in the literature, to assess the retained sedative drug efficacy at a particular time of the chronic diazepam treatment regimen. To our knowledge, only the study by Perrault et al. (1993), using the anticonvulsant action of diazepam as endpoint, described a rightward and downward shift in the dose-response curve of diazepam, but only when the test dose was given 42 hr after termination of a 10 d chronic treatment regimen (5 mg/kg, p.o., twice daily). This shift was attributed to the rapid development of a hypersensitivity of the mice to the convulsant drug in relation to the diazepam withdrawal experience. A significant residual anticonvulsant action of diazepam was seen in chronically treated animals when the convulsant drug was administered 6 hr after the last diazepam dose (Perrault et al., 1993).
The diminution of motor activity seen in α5(H105R) mice with chronic diazepam treatment also reveals that α1-GABAA receptors remain responsive during the course of the chronic diazepam treatment, recurrently mediating sedation in these mutants. Therefore, sedative tolerance does not appear to be attributable to a reduction of the motor-depressant efficacy of diazepam, thus confirming a previous report (Bourin et al., 1992). Rather, its manifestation might be secondary to the development of an α5-GABAA receptor-dependent response to chronic diazepam that would oppose its motor-depressant action. In α1(H101R) mice, which do not display the sedative drug action, chronic interaction of diazepam with GABAA receptors other than those containing the α1 subunit was not associated with a change in motor activity (Fig. 1b). This result argues against an oppositional α1-GABAA receptor-independent mechanism, which would counterbalance and thus mask the sedative drug action. α1- and α5-GABAA receptors appear to be the specific molecular substrates contributing in a competitive manner to the chronic effects of diazepam on motor activity. Chronic interaction of diazepam with α1-GABAA receptors results in a recurrent motor-depressant action while the concurrent interaction with α5-GABAA receptors is essential for the behavioral manifestation of tolerance to this effect. This result is in line with reports indicating that benzodiazepine site ligands that do not interact with α5-GABAA receptors, such as zolpidem, show little or no evidence for sedative tolerance in rodents and fail to alter α5-subunit levels (Zivkovic et al., 1994; Costa and Guidotti, 1996; Holt et al., 1997).
The α5-GABAA receptors of the dentate gyrus appear to be a specific target for adaptive changes associated with sedative tolerance to diazepam. Indeed, a downregulation of α5-GABAA receptors, as assessed with two selective α5-subunit radioligands used at saturating concentrations, was observed only in wild-type and α2(H101R) mice tolerant to the motor-depressant action of diazepam (Fig. 3a-c). This reduction of receptors was restricted to the dentate gyrus. No change in α5-subunit radioligand binding levels was detected in the CA1 and CA3 areas of these animals. Within the limits of statistical power, a decreased binding of α5-subunit-specific radioligands in diazepam-tolerant animals could be resolved only in the hippocampal formation. We admit that the chronic diazepam treatment regimen used here can give rise to alterations in α5-GABAA receptors in other brain regions. However, the unchanged levels of [3H]flumazenil binding in forebrain regions involved in control of motor activity argues against a general downregulation of GABAA receptors and against a regionally specific loss of another major GABAA receptor subtype after chronic diazepam treatment. This observation is in concordance with other reports that flumazenil binding sites remain unaltered after chronic benzodiazepine treatment (Hutchinson et al., 1996; Costa et al., 2001; Bateson, 2002). The absence of alteration in α5-subunit radioligand binding in α1(H101R) mice chronically treated with diazepam (Fig. 3d) further indicates that occurrence of α5-GABAA receptor downregulation in the dentate gyrus closely depends on the chronic interaction of the drug with α1-GABAA receptors but not with GABAA receptors containing the α2-, α3-, or α5-subunit.
α5-GABAA receptors constitute a minor population of diazepam-sensitive GABAA receptors. They are found mainly in the hippocampal formation, olfactory bulb granule cell layer, and spinal cord dorsal horn and in lower amounts in the cerebral cortex and hypothalamus (Crestani et al., 2002). We have reported previously that the H105R point mutation in the α5 subunit gene resulted in a reduction of α5-GABAA receptors in the dendritic layers of the hippocampal CA1 and CA3 areas with no change in the dentate gyrus (Crestani et al., 2002). Although this specific deficit of receptors in α5(H105R) mice mimics to some extent the molecular changes seen in the dentate gyrus of diazepam-tolerant animals, it does not interfere with the expression of the motor-depressant action of diazepam when given acutely (Crestani et al., 2002) or chronically (Fig. 1c). This is in keeping with the lack of alteration in the expression pattern of the other major GABAA receptor subunits, notably the α1-subunit, in α5(H105R) mice (Crestani et al., 2002) as well as in mice with a complete loss of hippocampal α5-GABAA receptors (Collinson et al., 2002). Thus, it is not a reduction of α5-GABAA receptors per se, but rather the selective localization in the dentate gyrus, which appears to be associated with the expression of sedative tolerance to diazepam. Likewise, the induction of long-term potentiation in hippocampal pyramidal cells is unaltered in α5(H105R) mice (Crestani et al., 2002), whereas tolerance to the motor-depressant action of diazepam is associated with an increased synaptic plasticity in the rat dentate gyrus (Marin et al., 1996). Therefore, the failure of α5(H105R) mice to manifest sedative tolerance, despite their partial deficit in hippocampal α5-GABAA receptors, strengthens the hypothesis that diazepam binding to α5-GABAA receptors is a key mechanism underlying the robust diminution of its sedative efficacy with chronic treatment. This is striking, because these receptors are mainly extrasynaptic and mediate tonic inhibition in hippocampal CA1 pyramidal cells (Crestani et al., 2002; Caraiscos et al., 2004). The association of changes in α5-subunit binding sites, mRNA, or protein levels with tolerance to diazepam has been reported previously concerning its anticonvulsant properties (Wu et al., 1994; Impagnatiello et al., 1996; Pesold et al., 1997; Li et al., 2000). Here, we demonstrate that the decrease in α5-subunit binding in the dentate gyrus depends on the chronic activation by diazepam of α1-GABAA receptors, which primarily produce phasic inhibition in the brain. This is in keeping with the reported high plasticity in the expression of hippocampal extrasynaptic α5-GABAA receptors in response to intense synaptic activity (Houser and Esclapez, 2003).
In conclusion, we propose that the manifestation of tolerance to the motor-depressant action of diazepam depends on the chronic activation of two competitive mechanisms orchestrated by α1- and α5-GABAA receptors, respectively. Chronic drug interaction with α1-GABAA receptors results in a persistent augmentation of phasic inhibition in the forebrain areas involved in motor control, mediating motor depression. This recurrent increased phasic signaling would alter the weight of the tonic inhibition produced by the simultaneous drug activation of extrasynaptic α5-GABAA receptors in the hippocampal formation. The α1-GABAA receptor-dependent change in inhibitory efficacy, which occurs during the course of the chronic diazepam treatment, is reflected by the 15% diminution of α5-GABAA receptors in the dentate gyrus of tolerant animals. This is in keeping with the potential of a small reduction in the efficacy of GABAA receptor-mediated inhibition (∼10%) to markedly increase cortical excitation (Chagnac-Amitai and Connors, 1989). Tolerance against the motor-depressant action of diazepam has been associated with an enhancement of both hippocampal synaptic efficacy and NMDA receptor subunit mRNA expression in dentate gyrus (Marin et al., 1996; Perez et al., 2003). Moreover, it has been reported that acute application of diazepam on hippocampal slices prevents the long-term potentiation of population spikes, whereas zolpidem has no effect, suggesting a possible α5-GABAA receptor-dependent mechanism (Higashima et al., 1998). We do not exclude the possibility of a compensatory alteration in diazepam-insensitive tonic inhibition mediated by δ-subunit-containing GABAA receptors in the dentate granule cells, which could contribute to the increase in hippocampal excitability seen with chronic diazepam treatment (Nusser and Mody, 2002). However, the relatively high abundance of diazepam-sensitive α5-GABAA receptors in the hippocampal formation positions this structure as a key player for development of the sedative tolerance phenotype by means of its widespread efferent connections to brain areas involved in the regulation of motor control.
This work was supported by the Swiss National Science Foundation. We thank K. Löw for the generation of the α2(H101R) mice, D. Benke for help with autoradiography, and C. Sidler, H. Pocchetti, and E. Calame for technical assistance.
Correspondence should be addressed to Florence Crestani, Institute of Pharmacology and Toxicology, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland. E-mail:.
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↵* C.v.R. and M.T. contributed equally to this work.