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Benzodiazepine (BZ) site ligands affect vigilance, anxiety, memory processes, muscle tone and epileptogenic propensity through modulation of neurotransmission at GABAA receptors containing α1, α2, α3 or α5 subunits, and may have numerous experimental and clinical applications. The ability of nonselective BZ site inverse agonists to enhance cognition, documented in animal models and human studies, is clinically not feasible due to potentially unacceptable psychomotor effects. Most investigations to date have proposed the α1 and/or α5 subunit-containing GABAA receptors as comprising the memory-modulating population of these receptors. The novel ligand PWZ-029, which we synthesised and characterized electrophysiologically, possesses in vitro binding selectivity and moderate inverse agonist functional selectivity at α5-containing GABAA receptors. This ligand has also been examined in rats in the passive and active avoidance, spontaneous locomotor activity, elevated plus maze and grip strength tests, primarily predictive of the effects on the memory acquisition, basal locomotor activity, anxiety level and muscle tone, respectively. The improvement of task learning was detected at the dose of 5 mg/kg in the passive, but not active avoidance test. The inverse agonist PWZ-029 had no effect on anxiety or muscle tone, whereas at higher doses (10 and 20 mg/kg) it decreased locomotor activity. This effect was antagonized by flumazenil and also by the lower (but not the higher) dose of an agonist (SH-053-R-CH3-2’F) selective for GABAA receptors containing the α5 subunit. The hypolocomotor effect of PWZ-029 was not antagonized by the antagonist β-CCt exhibiting a preferential affinity for α1-subunit containing receptors. These data suggest that moderate negative modulation at GABAA receptors containing the α5 subunit is a sufficient condition for eliciting enhanced encoding/consolidation of declarative memory, while the influence of higher doses of modulators at these receptors on motor activity shows an intricate pattern whose relevance and mechanism await to be defined.
The majority of fast inhibitory neurotransmission in the mammalian central nervous system is mediated by GABAA receptors. They are, as a whole, profoundly involved in the regulation of vigilance, anxiety, memory processes, muscle tone and epileptogenic propensity (Rudolph and Möhler, 2004). Beside the multiplicity of subunits comprising the GABAA receptor pentamer (19 subunits have been identified to the present; Simon et al., 2004), the variety of possibilities for fine tuning of neurotransmission stems from a number of allosteric modulatory sites. It is well established that ligands of the benzodiazepine (BZ) binding site may exert their effects through modulation of four distinct populations of GABAA receptors, containing the α1, α2, α3 or α5 subunit in addition to the γ2 and a β subunit (Sieghart and Sperk, 2002; Rudolph and Möhler, 2004). The recent genetic studies with mice carrying a point mutation of histidine to arginine in α1, α2, α3 or α5 subunits, rendering the respective GABAA receptors selectively insensitive to effects of BZ site ligands, suggested a specific contribution of individual receptor subtypes to the spectrum of behavioral actions of these compounds (Rudolph and Möhler, 2004). These genetic advances have encouraged synthesis of new, selective BZ site ligands, that possess affinity and/or efficacy profiles which enable separation of wanted from unwanted effects (Sieghart and Ernst, 2005).
One desirable property in this regard is the pro-mnesic activity of BZ site inverse agonists, repeatedly reported in animal models (e.g. Venault et al., 1986; Jensen et al., 1987), as well as in human volunteers (e.g. Dorow et al., 1983; Duka et al., 1996). However, this desirable effect is confounded by different concomitant psychomotor effects (increased vigilance, anxiogenic and/or proconvulsant state), some of which have been described in memory studies with non-selective inverse agonists in humans, urging their early termination (Dorow et al., 1983). Point mutated mice could not be used to identify the receptor subtypes mediating the promnesic activity of inverse agonists because an unexplained switch to the agonist mode of action occurs when an inverse agonist at wild type diazepam-sensitive recombinant GABAA receptors is tested at the respective point-mutated receptors (Benson et al., 1998; Crestani et al., 2002a). Thus, inverse agonists exerted agonistic-like sedative and anticonvulsant effects in mice with the point-mutated α1 subunits (Crestani et al., 2002a), while corresponding experiments in models of learning and memory were not performed. Nevertheless, behavioral examination of genetically modified animals conducted to date has indicated the α1 and α5 subunit-containing GABAA receptors comprise the ‘memory-modulating’ population of these receptors (Rudolph et al., 1999; Collinson et al., 2002; Crestani et al., 2002b). It is notable that GABAA receptors containing the α5 subunit are abundantly expressed in the hippocampus (Pirker et al., 2000; Sieghart and Sperk, 2002), the structure substantially involved in memory formation (Izquierdo and Medina, 1997). Recent evidence from animal studies with affinity-selective (Atack et al., 2006a) or efficacy-selective ligands (Chambers et al., 2003; Dawson et al., 2006, Collinson et al., 2006) has confirmed that the α5 subunit was significantly involved in cognition enhancement mediated by the negative modulation of GABAA receptor functions. Moreover, it was shown in humans that pre-treatment with an α5 efficacy-selective inverse agonist significantly reduces the amnesic effect of alcohol on learning a word list (Nutt et al., 2007). However, the affinity- or efficacy-selectivity of the ligands, as well as the diversity of the behavioral tasks used in their characterization to date, were of limited extent, which necessitates screening of newer BZ site negative ligands, to determine the putative therapeutic role of such compounds in various disorders with diminished cognitive capabilities in humans (Maubach, 2003).
In this regard, the BZ site ligand PWZ-029 was synthesized (Figure 1a). Its efficacy profile was examined by two-electrode voltage clamp experiments in Xenopus oocytes expressing recombinant GABAA receptor subtypes. The behavioral effects on adult male Wistar rats were evaluated in the passive and active avoidance, spontaneous locomotor activity, elevated plus maze and grip strength tests, primarily predictive of detecting the changes in the memory acquisition, basal locomotor activity, anxiety level and muscle tone, respectively.
In vitro data for PWZ-029 (Figure 1b) demonstrated that at concentrations up to 1 µM this ligand engendered a significant partial inverse agonist efficacy at the α5-containing GABAA receptors (reduction of control current by 20%), whereas its activity at the other three types of receptors tended to be weakly and for α1- and α3-containing receptors significantly agonistic. Similarly, at 10 µM concentrations PWZ-029 exhibited a weak but significant partial agonistic effect at α1-and α3-containing receptors. The lower potencies of PWZ-029 for α1-, α2- and α3-, relative to the α5-containing GABAA receptors, deduced from the respective efficacy curves, conform with the explicit binding affinity data (Table 1). Hence, PWZ-029 shows a distinct affinity-, potency-, as well as efficacy- selectivity for the α5-containing GABAA receptors. PWZ-029 exerted negligible activity in 42 other receptor and enzyme assays (B. Roth et al., NIMH Psychoactive Drug Screening Program, UNC, unpublished results, available at https://kidbdev.med.unc.edu/pdsp). The in vitro concentration-effects curves for SH-053-R-CH3-2’F, the ligand used for interaction study in the locomotor activity test, showed it is a high-efficacy agonist at the α5-containing GABAA receptors, with very low efficacies at α1, α2 and α3-containing subtypes (Rowlett, Furtmüller, Cook, unpublished data); the profile was comparable with that published for its close congener SH-053-R-CH3 (Savić et al., 2008), except for greater potencies and α5-efficacy of SH-053-R-CH3-2’F.
The acquisition session latencies did not differ significantly irrespective of the treatment administered 15 min before the session (data not shown). The influence of the pre-acquisition treatment on the retention trial latency was significant [H(4) =11.45, p=0.022] (Figure2). Subsequent Dunn's test indicated that DMCM (0.2 mg/kg) and PWZ-029 at 5 mg/kg, administered before the acquisition session, significantly increased retention session latency relative to the control group.
Figure 3a shows that DMCM (0.1 mg/kg) and PWZ-029 (2–10 mg/kg) did not affect acquisition of avoidance responses on the training day [F(4, 35) = 0.19, p=0.98]. Retention AA performance on the second day was significantly altered by the treatment before the acquisition session [F(4, 35) = 2.82, p=0.039]; however, the DMCM (0.1 mg/kg), but not the PWZ-029, was effective (Dunnett’s test) (Figure 3b). Neither of the motor parameters measured (habituation crossings, intertrial crossings) differed significantly on the training or testing day (data not shown).
An ANOVA showed a significant effect of treatment on total distance travelled during 30 min of monitoring (F(4,43) = 4.06, p = 0.007) (Figure 4, whole bars). According to Dunnett’s test, the two higher doses of PWZ-029 (10 and 20 mg/kg) exerted the activity-decreasing effect related to solvent. When the ANOVA with post hoc analysis was applied on the 5-min intervals of travelled distance (Figure 5), it turned out that locomotor activity was significantly depressed in the time periods 0–10 min (DMCM 2 mg/kg and PWZ-029 10 and 20 mg/kg), 10–15 min and 20–25 (PWZ-029 20 mg/kg). It is notable that in the periods 15–20 min, 20–25 min and 25–30 min the rats treated with DMCM (2 mg/kg) travelled on average for 23%, 21% and 96% greater distance, respectively, than control animals; the difference did not reach statistical significance at either of the periods (Student’s t-test).
An ANOVA showed a significant effect of treatment on distance travelled in the arbitrarily set central parts of the chamber during 30 min of monitoring (F(4,43) = 4.35, p=0.005) (Figure 4, hatched bars); the effects of PWZ-029 10 and 20 mg/kg were statistically significant. According to ANOVA, the influence of treatment on activity in peripheral parts of the chamber (Figure 4, open bars) was similar: F(4,43) = 2.91, p=0.033; PWZ-029 20 mg/kg significantly decreased activity (Dunnett’s test).
Both β-CCt (30 mg/kg) and flumazenil (10 mg/kg), the α1-affinity selective and non-selective antagonist of BZ site, respectively, failed to affect on their own the total distance travelled in the whole chamber, whereas only flumazenil reversed the suppression of locomotion induced by PWZ-029. The α5-selective agonist SH-053-R-CH3-2’F shows a dose-dependent hypolocomotor influence on rats in the spontaneous locomotor activity assay, with the effective dose of 30 mg/kg (Savić, Furtmüller, Cook, unpublished data). When combined with PWZ-029 20 mg/kg, the dose of 10 mg/kg, but not 20 mg/kg, of SH-053-R-CH3-2’F antagonized the effect of the inverse agonist. There were no significant differences among groups in distances travelled in the central zone of the activity chamber.
The influence of treatment on closed arm entries (F(4,35) = 0.23, p=0.922), total arm entries (F(4,35) = 1.54, p=0.213) and total distance travelled (F(4,35) = 1.44, p=0.241) did not reach statistical significance.
The overall influence of treatment on distance travelled in open arms (Figure 7a) was not significant (F(4,35) = 2.04, p=0.11). There were significant effects of treatment on the percentage of open arm entries (F(4,35) = 4.21, p=0.007) (Figure 7b) and percentage of time spent on open arms (F(4,35) = 3.13, p=0.026) (Figure 7c). In both cases, the effective treatment was DMCM 1.25 mg/kg.
According to ANOVA, the overall influence of treatment on grip strength was not significant (F(4,35) = 2.31, p=0.077). The grip strength (mean ± SEM) of rats treated with solvent, DMCM 1.25 mg/kg, PWZ-029 5 mg/kg, PWZ-029 10 mg/kg and PWZ-029 20 mg/kg was 505.0 ± 11.4 g, 561.1 ± 29.3 g, 546.5 ± 42.7 g, 468.0 ± 23.3 g, and 467.2 ± 27.1 g, respectively.
The present results demonstrated that PWZ-029, a novel BZ site ligand, possesses in vitro binding selectivity as well as moderate inverse agonist functional selectivity for the α5-containing GABAA receptors, which has translated into the pro-mnesic effect detected in the PA, but not AA, learning task in rats. It has been shown that the non-selective inverse agonist DMCM enhances retention performance when administered at a small dose before the acquisition session of the active (Savić et al., 2005a), or passive (File and Pellow, 1988; Savić et al., 2005b) avoidance test, and these findings were replicated as a positive control in the present study. The interactions between DMCM and the antagonists flumazenil and β-CCt have suggested that the α1-subunit is substantially involved in processing of both of these memory tasks, whereas the interactions between agonists (midazolam and zolpidem) and antagonists pointed to the importance of some other α-subunit(s), in addition to the α1-subunit, in the passive, but not active avoidance task (Savić et al., 2005a; 2005b). The latter agrees well with the memory-enhancing effect of the α5-selective inverse agonist PWZ-029, a ligand supposedly largely inactive at α1-containing GABAA receptors, in the PA task only.
Although seemingly similar, these two tests differ in some important characteristics, including the type of memory assessed and the brain regions involved in memory processing. Namely, the one-trial step-through PA test assesses declarative memory (to the point that terms such as ‘declarative’ or ‘explicit’ can be applied to rodent experiments) (Izquierdo et al., 1999), and for the retention of the task requires the preserved hippocampus (Izquierdo et al., 1997). On the other hand, the two-way AA test is an implicit, i.e. procedural (Squire, 1992), hippocampal-independent (Gray and McNaughton, 1983; Collinson et al., 2002) memory task. However, as aversively motivated tests, both ‘measure’ memory of emotionally arousing experiences, and critically involve activity of the amygdala, especially the basolateral amygdala (McGaugh, 2004) in which the α1 subunit is the most abundant of the six α subunits (Pirker et al., 2000; Fujimura et al., 2005). It is felt that this fact at least in part explains the uniform involvement of the α1 subunit-containing GABAA receptors in modulation of memory acquisition, found for the present (Rudolph et al., 1999; Savić et al., 2005a; 2005b) as well as similar, aversively motivated, memory tasks, such as the water-lick suppression test (Rudolph et al., 1999).
Receptors containing the α5 subunit constitute a modest fraction of BZ-sensitive GABAA receptors (approximately 5%), but are substantially expressed in several brain regions, most notably the hippocampus, the olfactory bulb and layers V and VI of the neocortex (Pirker et al., 2000; Sieghart and Sperk, 2002). Studies with untreated mice with the point mutation at the α5 subunit, which unexpectedly displayed a partial loss, restricted to the hippocampus, of the expression of this subunit, have indicated the significance of the α5 subunit for learning of certain aversively- as well as appetitively- motivated memory tasks which require temporal integration (Crestani et al., 2002b; Yee et al., 2004). On the other hand, in line with the present findings, mice with the deleted α5 subunit did not differ in comparison to the wild-type controls in acquisition of the two-way AA task (Collinson et al., 2002). The results with PWZ-029 in the present memory tests suggest that moderate negative modulation of GABA-ergic transmission mediated by GABAA receptors containing the α5 subunit is a sufficient, but possibly not prerequisite condition for eliciting enhanced encoding/consolidation of declarative memory assessed by the PA test.
PWZ-029 had no effect on anxiety-related parameters in the EPM, in agreement with results with the other α5-selective inverse agonist tested in this model (Dawson et al., 2006); the α5 affinity-selective inverse agonist L-655,708 exerted an unusual U-shaped anxiogenic effect in the murine EPM, but with the use of a non-standard statistical analysis (Navarro et al., 2002). The effect of PWZ-029 on muscle tension measured by the grip strength test also was absent. Hence, its hypolocomotor effect seen in the locomotor activity assay could not be ascribed to any muscle relaxation. Further, there was no hint of convulsive activity in any of the rats treated with PWZ-029 throughout the behavioral studies. Decreases of locomotor activity may also reflect an increase in emotional reactivity, i.e. anxiety (cf. Atack et al., 2005). However, such an activity was not seen with PWZ-029 in the EPM test, nor was its hypolocomotor effect disproportionately more pronounced in the central, more exposed, and hence ‘emotional’, zone of the chamber, but was notably different from standard patterns of activity of a nonselective inverse agonist (i.e. DMCM). Namely, as a rule, when rodents explore a novel environment under the influence of a negative modulator of GABAA receptors, locomotor activity is depressed, but usually in a relatively short period of habituation (e.g. Jaskiw et al., 2003); afterwards, a type of rebound hyperlocomotion may ensue (Savić et al., 2006). However, as we have concluded from the experiment with DMCM and the α1-subunit selective antagonist β-CCt, hyperlocomotion in habituated animals may depend primarily on the negative modulation at α1 subunit-containing GABAA receptors (Savić et al., 2006), and this was not observed with PWZ-029. Instead, the temporal pattern of the extended motor depression with PWZ-029 (20 mg/kg) was more similar to activity of an agonist, rather than an inverse agonist, and the most parsimonious explanation would be that, at the higher doses, PWZ-029 binds to enough α1-containing GABAA receptors to manifest its slight partial agonistic activity. Nevertheless, the effect of PWZ-029 was mediated by BZ site(s) (antagonism of the effect by flumazenil) other than those at the α1-containing GABAA receptors (lack of antagonism by β-CCt). Comparative analysis of in vitro affinity and efficacy data of flumazenil and β-CCt (Cox et al., 1995; June et al., 2003; Furtmüller and Sieghart, unpublished data) suggests that the principal difference between them lies in activity at α5 subunit-containing GABAA receptors. Namely, flumazenil and β-CCt show qualitatively similar mixed antagonist/agonist efficacy profiles, with the main distinction being at α5 subunit-containing GABAA receptors at which β-CCt is a weak inverse agonist, and flumazenil an antagonist/weak agonist. Moreover, a 150-fold lower affinity of β-CCt for the α5 over the α1 subtype of GABAA receptors (Cox et al., 1995) should plausibly preclude its in vivo binding at α5 subunit-containing GABAA receptors and influencing any effect mediated by them. Hence, it is hardly conceivable that β-CCt may antagonize the effects of PWZ-029 at these receptors. Similarly, the slight positive modulation at α2 or α3 subunit-containing GABAA receptors, also observed at high doses of PWZ-029, could hardly have contributed to its hypolocomotor activity (cf. Low et al., 2000; Atack et al., 2006b).
Standard tests of locomotor activity were not usually employed in reported studies with α5 subunit-selective ligands (Atack et al., 2006a; Chambers et al., 2003; Collinson et al., 2006; Dawson et al., 2006). Based on the results with three novel BZ site agonists functionally silent at the α1 subunit, we recently set out the hypothesis that positive modulation at the GABAA receptors containing α5 subunits may contribute to sedative properties of BZ site agonists (Savić et al., 2008). The finding that SH-053-R-CH3-2’F at the lower (10 mg/kg), but not higher dose (20 mg/kg), antagonized the hypolocomotor effect of PWZ-029 adds to the notion that there may exist certain discontinuous ‘effective windows’ of the modulation of locomotion exerted by neurons expressing the α5-subunit containing GABAA receptors. Namely, the maximal level of receptor inhibition obtained with PWZ (80% of the basal GABA effects sustained by the α5-subunit containing receptors) is clearly different from that present in mice lacking this subunit at all (0% of the basal GABA effects at the affected receptors; Collinson et al., 2002) or in mice harboring a point mutation at the α5 subunits (approximately 70% of the basal GABA effects in the hippocampus related to the α5-subunit containing receptors; Crestani et al., 2002b; Hauser et al., 2005). Nevertheless, the respective locomotor outputs (a decrease with higher doses of PWZ-029, no changes in knockout mice, stimulation/no changes in mice with a point mutation at the α5 subunits) were apparently random in relation to the level of receptor modulation, but certainly dependent on the stress level and experimental settings (cf. an increase of spontaneous locomotor activity in the mice with point-mutated α5 GABAA receptor subunits in Hauser et al., 2005, vs. lack of changes in Crestani et al., 2002). The present hypothesis of the protean influences of the α5-subunit containing receptors on locomotion, possibly related to the intricacy of the different processes which are concomitantly modulated by these receptors (such as sensorimotor gating; Hauser et al., 2005, and firing of the layer V pyramidal neurons of the neocortex; Yamada et al., 2006), should be checked through various locomotor tests employing a range of doses of different α5 subunit-selective ligands, both as single treatments and in combination.
In conclusion, examination of the present results suggests that the negative modulation engendered by the novel BZ site ligand PWZ-029, which demonstrates binding and functional selectivity for the α5-containing GABAA receptors, may be sufficient to effect enhanced formation of declarative memory. The sedative-like effects, seen at doses higher than those affecting memory formation, reduce the probability of generation of anxiogenic or proconvulsant states. The molecular and neuronal substrates in regard to the role of α5-subunit containing GABAA receptors in control of vigilance, including the proposed phenomenon of the ‘on/off switch’, may be numerous and await to be further elucidated.
PWZ-029 (methyl(8-chloro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-α][1,4]benzodiazepin-3-yl)methyl ether), SH-053-R-CH3-2’F (the (R) stereoisomer of 8-ethynyl-6-(2-fluorophenyl)-4-methyl-4H-2,5,10b-triaza-benzo[e]azulene-3-carboxylic acid ethyl ester) and the preferential α1-subunit selective antagonist β-carboline-3-carboxylate-t-butyl ester (β-CCt) were synthesized at the Department of Chemistry and Biochemistry, University of Wisconsin - Milwaukee. The non-selective antagonist flumazenil was donated from F. Hoffman-La Roche (Basel, Switzerland) and the non-selective inverse agonist DMCM was purchased from Research Biochemicals Incorporated (Natick, MA, USA).
Cloning of GABAA receptor subunits α1, β3 and γ2 into pCDM8 expression vectors (Invitrogen, CA) has been described elsewhere (Fuchs et al., 1995). cDNAs for subunits α2, α3 and α5 were gifts from P. Malherbe and were subcloned into pCI-vector. After linearizing the cDNA vectors with appropriate restriction endonucleases, capped transcripts were produced using the mMessage mMachine T7 transcription kit (Ambion, TX). The capped transcripts were polyadenylated using yeast poly(A) polymerase (USB, OH) and were diluted and stored in diethylpyrocarbonate-treated water at −70°C.
The methods used for isolating, culturing, injecting and defolliculating of the oocytes were identical as described previously (Sigel, 1987; Sigel et al., 1990). Briefly, mature female Xenopus laevis (Nasco, WI) were anaesthetized in a bath of ice-cold 0.17 % Tricain (Ethyl-m-aminobenzoat, Sigma, MO) before decapitation and removal of the frogs ovary. Stage 5 to 6 oocytes with the follicle cell layer around them were singled out of the ovary using a platinum wire loop. Oocytes were stored and incubated at 18°C in modified Barths' Medium (MB, containing 88 mM NaCl, 10 mM HEPES-NaOH (pH 7.4), 2.4 mM NaHCO3, 1 mM KCl, 0.82 mM MgSO4, 0.41 mM CaCl2, 0.34 mM Ca(NO3)2) that was supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin. Oocytes with follicle cell layers still around them were injected with a total of 2.25 ng of cRNA. cRNA ratio used was 1:1:5 for the α subunits, β3 and γ2, respectively. After injection of cRNA, oocytes were incubated for at least 36 hours before the enveloping follicle cell layers were removed. To this end, oocytes were incubated for 20 min at 37°C in MB that contained 1 mg/ml collagenase type IA and 0.1 mg/ml trypsin inhibitor I-S (both Sigma). This was followed by osmotic shrinkage of the oocytes in doubly concentrated MB medium supplied with 4 mM Na-EGTA and manually removing of the follicle cell layer. After peeling off the follicle cell layer, the cells were allowed to recover overnight before being used in electrophysiological experiments.
For electrophysiological recordings, oocytes were placed on a nylon-grid in a bath of Xenopus Ringer solution (XR, containing 90 mM NaCl, 5 mM HEPES-NaOH (pH 7.4), 1 mM MgCl2, 1 mM KCl and 1 mM CaCl2). The oocytes were constantly washed by a flow of 6 ml/min XR which could be switched to XR containing GABA and/or drugs. Drugs were diluted into XR from DMSO-solutions resulting in a final concentration of 0.1 % DMSO perfusing the oocytes. Drugs were preapplied for 30 sec before the addition of GABA, which was coapplied with the drugs until a peak response was observed. Between two applications, oocytes were washed in XR for up to 15 min to ensure full recovery from desensitization. For current measurements the oocytes were impaled with two microelectrodes (2–3 mΩ) which were filled with 2 mM KCl. All recordings were performed at room temperature at a holding potential of −60 mV using a Warner OC-725C two-electrode voltage clamp (Warner Instruments, Hamden, CT). Data were digitised, recorded and measured using a Digidata 1322A data acquisition system (Axon Instruments, Union City, CA). Results of concentration response experiments were graphed using GraphPad Prism 4.00 (GraphPad Software, San Diego, CA). Data were graphed as mean ± SEM of at least four oocytes from at least two batches.
Experiments were carried out on male Wistar rats (Military Farm, Belgrade, Serbia), weighing 220–250 g. All procedures in the study conformed to EEC Directive 86/609 and were approved by the Ethical Committee on Animal Experimentation of the Medical Faculty in Belgrade. The rats were housed in transparent plastic cages, six animals per cage, and had free access to pelleted food and tap water. The temperature of the animal room was 22±1°C, the relative humidity 40–70%, the illumination 120 lux, and the 12/12 h light/dark period (light on at 6:00 h). All handling and testing took place during the light phase of the diurnal cycle. Throughout the study the animals were used only once, with exception of the grip strength measurement, which was done immediately after the tracking of behavior on the elevated plus maze. Spontaneous locomotor activity and elevated plus maze behavior were analyzed by the ANY-maze Video Tracking System software (Stoelting Co., Wood Dale, IL, USA). All drugs were dissolved/suspended with the aid of sonication in a solvent containing 85% distilled water, 14% propylene glycol, and 1% Tween 80, and were administered intraperitoneally in a volume of 1 ml/kg, 15 min before behavioral testing (for active and passive avoidance test, before the acquisition session). Time of administration, as well as the doses of DMCM in various tests were chosen based on our previous studies (Savić et al., 2005a; 2005b; 2006). For interaction studies in the locomotor activity assay, the first treatment indicated in combination was administered into the lower left quadrant of the peritoneum 20 min before testing, and the second treatment 5 min later into the lower right quadrant of the peritoneum.
The experiments were performed in an adapted Automatic reflex conditioner (Ugo Basile, Milan, Italy, Model 7051), as described earlier (Savić et al., 2005b). In short, the apparatus consisted of a shuttle-box, equipped with a grid floor and divided with a sliding door into a lit and a dark compartment, and a programming unit. The animals were submitted to two, 24 h – separated sessions. The acquisition session started by placing individual subjects in the illuminated compartment. After 30 s, the entrance to the dark compartment was opened, and as soon as the rat had entered it with all four paws, the footshock (2 s, 0.3 mA) was delivered. Immediately afterwards, the rat was returned to its home cage. The same procedure was repeated 24 h later (retention session), without footshock. A cut-off time of 180 s was used on the training day, whereas, on the retention trial, a ceiling of 300 s was imposed.
The AA test was performed in automated two-way shuttle-boxes (Campden Instruments, Sileby, UK), as described earlier (Savić et al., 2005a). In short, the animals were submitted to two, 24-h–separated sessions. Training and test sessions were procedurally identical. Animals were placed singly into the shuttle box and left to freely explore for 15 min, and habituation crossings were automatically counted. Afterward, they received 30 tone foot shock trials. During the first 5 s of each trial, a sound signal was presented (broadband noise of 69 dB), allowing the animal to avoid shocks by moving to the other compartment (avoidance response). If the animal did not respond within this period, a foot shock of 0.5 mA (7-s duration) was applied. Crossing to the adjacent compartment during the shock discontinued its delivery. The animal could move freely in the apparatus between trials (18-s intertrial intervals), and the intertrial crossings were automatically counted.
Activity of single rats in a clear Plexiglas chamber (40 × 25 × 35 cm) under dim red light (20 lux) was recorded for a total of 30 min, without any habituation period, using ANY-maze software (as described above). For purposes of improving data analysis, the central 20% of the chamber (200 cm2) was virtually set as a central zone. The minimum percentage of animal that must have been in the zone for an entry to occur was set at 70%, and 50% of the animal must have remained in the zone for an exit not to occur.
The apparatus consisted of two open (50 × 10 cm, with a ledge of 0.3 cm) and two enclosed arms (50 × 10 × 40 cm), connected by a junction area (10 × 10 cm). The illumination in the experimental room was 10 lux on the surface of the arms. At the beginning of the experiment, single rats were placed in the center of the maze, facing one of the enclosed arms, and their behavior was recorded for 5 min. An entry into an open or closed arm was scored when 90% of the animal crossed the virtual line separating the central square of the maze from the arm, whereas an exit occurred when more than 90 % of the animal left the respective arm. After each trial, the maze was cleaned with dry and wet towels.
Muscle strength was assessed by the grip strength meter (Ugo Basile, Milan, Italy, model 47105). When pulled by the tail, the rat grasps the trapeze connected to a force transducer, and the apparatus measures the peak force of experimenter’s pull (in g) necessary to overcome the strength of the animal’s forelimbs grip. Each animal was given three consecutive trials, and the median value was used for further statistics.
All numerical data presented in the figures were given as the mean ± SEM, except for results from PA test (median latency with 25th, 75th interquartile range; data were non-parametric because the procedure involved a cut-off). For electrophysiological data Student’s t-test was used for statistical analysis. Data from PA test were assessed by a Kruskal-Wallis non-parametric ANOVA, with post-hoc comparison relative to solvent control by a Dunn's test (α=0.05). Data from the AA, EPM, grip strength and activity assay were assessed by a one-way ANOVA. If the ANOVA was significant, each treatment condition was compared with control by a Dunnett's test (α=0.05). Where appropriate, the assessment of the antagonist influence on the inverse agonist effect was conducted by a Student’s t-test. Statistical analyses were performed with ANY-maze Video Tracking System software (Stoelting Co., Wood Dale, IL, USA) and SigmaStat 2.0 (SPSS, Inc., Chicago, IL, USA).
This work was supported in part by NIMH 46851 (JMC) and by The Ministry of Science, R. Serbia – Grant No. 145022B (MMS).
We acknowledge the support of this work by the Research Growth Initiative of the University of Wisconsin-Milwaukee. The authors wish to thank Dr. Ruth McKernan (MSD, Harlow, UK) for the binding affinity of PWZ-029.
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