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NEUROPHARMACOLOGY

GABA_B(1) Receptor Subunit Isoforms Exert a Differential Influence on Baseline but Not GABA_B Receptor Agonist-Induced Changes in Mice

Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland (L.H.J., K.K., J.F.C.); and Institute of Physiology, Department of Clinical-Biological Sciences, Pharmazentrum, University of Basel, Basel, Switzerland (B.B.)

Received for publication August 7, 2006
Accepted September 20, 2006.

	Abstract

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GABA_B receptor agonists produce hypothermia and motor incoordination. Two GABA_B(1) receptor subunit isoforms exist, but because of lack of specific molecular or pharmacological tools, the relevance of these isoforms in controlling basal body temperature, locomotor activity, or in vivo responses to GABA_B receptor agonists has been unknown. Here, we used mice deficient in the GABA_B(1a) and GABA_B(1b) subunit isoforms to examine the influence of these isoforms on both baseline motor behavior and body temperature and on the motor-incoordinating and hypothermic responses to the GABA_B receptor agonists L-baclofen and -hydroxybutyrate (GHB). GABA_B(1b)^–/– mice were hyperactive in a novel environment and showed slower habituation than either GABA_B(1a)^–/– or wild-type mice. GABA_B(1b)^–/– mice were hyperactive throughout the circadian dark phase. Hypothermia in response to L-baclofen (6 and 12 mg/kg) or GHB (1 g/kg), baclofen-induced ataxia as determined on the fixed-speed Rotarod, and GHB-induced hypolocomotion were significantly, but for the most part similarly, attenuated in both GABA_B(1a)^–/– and GABA_B(1b)^–/– mice. We conclude that L-baclofen and GHB are nonselective for either GABA_B(1) receptor isoform in terms of in vivo responses. However, GABA_B(1) receptor isoforms have distinct and different roles in mediating locomotor behavioral responses to a novel environment. Therefore, GABA_B(1a) and GABA_B(1b) isoforms are functionally relevant molecular variants of the GABA_B(1) receptor subunit, which are differentially involved in specific neurophysiological processes and behaviors.

The Gabbr1 gene is transcribed from two different promoter sites to generate two predominant isoforms in the brain: GABA_B(1a) and GABA_B(1b) (Steiger et al., 2004), both of which can heterodimerize with the GABA_B(2) subunit to form functional receptors (Bettler et al., 2004). The two isoforms differ in sequence only by the inclusion of a pair of sushi domains (also called short consensus repeats) at the N terminus on the GABA_B(1a) isoform, which are absent in the GABA_B(1b) isoform (Blein et al., 2004). Other GABA_B(1) receptor variants have been reported in recombinant systems, although many of these variants are either not expressed in native tissues or are not evolutionarily conserved across different species, and as such the functional relevance of these variants remains controversial (Bettler et al., 2004; Cryan and Kaupmann, 2005). Therefore, the molecular diversity of native GABA_B receptors has been regarded as being relatively limited, which is at odds with the reported variability in the nature of responses to GABA_B receptor ligands (Marshall et al., 1999; Bettler et al., 2004; Huang, 2006).

In recombinant systems, many studies attest to a lack of pharmacological differences between the GABA_B(1a,2) and GABA_B(1b,2) isoforms (Kaupmann et al., 1998; Malitschek et al., 1998; Brauner-Osborne and Krogsgaard-Larsen, 1999; Green et al., 2000), although there are also a few findings to the contrary, that differences exist in the pharmacology of the receptor isoforms (see Bettler et al., 2004). However, to date, research tools with which to probe the in vivo pharmacology of GABA_B(1a) and GABA_B(1b) isoforms have not been available. The anatomical expression profile of GABA_B(1a) and GABA_B(1b) receptor isoforms diverges in many structures (Benke et al., 1999; Bischoff et al., 1999; Liang et al., 2000; Fritschy et al., 2004), which has given rise to speculation that the isoforms may have functional heterogeneity, possibly meditated by differential synaptic localization (Bettler et al., 2004). Furthermore, many studies have shown pharmacological differences between heteroreceptors and autoreceptors, and some GABA_B receptor ligands have been postulated to act preferentially at either presynaptic or postsynaptic locations (for reviews, see Bowery et al., 2002; Bettler et al., 2004). However, the possible contribution of these different isoforms to characteristic in vivo responses to GABA_B receptor activation, such as hypothermia and motor performance in response to baclofen (Gray et al., 1987; Jacobson and Cryan, 2005), are currently unknown.

Recently, mice deficient in the GABA_B(1a) and GABA_B(1b) subunit isoforms have been generated using a knock-in genetic approach (Vigot et al., 2006). Electron microscopy and electrophysiological characterization of the mice revealed that, at least in the hippocampus (Vigot et al., 2006) and in the lateral amygdala (Shaban et al., 2006), the GABA_B(1a) isoform was predominantly a presynaptic heteroreceptor, whereas the GABA_B(1b) isoform was mainly located postsynaptically, and both were autoreceptors. In addition, this distribution has been shown to generalize to layer 5 cortical neurons, where the GABA_B(1b) isoform was also shown to be predominantly postsynaptically located, whereas GABA_B(1a) was the main isoform represented at presynaptic terminals of local interneurons synapsing on the dendritic tuft (Perez-Garci et al., 2006). As such, GABA_B(1a)^–/– and GABA_B(1b)^–/– mice provide an ideal tool with which to investigate the roles of the two GABA_B(1) isoforms in the in vivo pharmacology of GABA_B receptor agonist-induced responses. Therefore, the aim of the present study was to determine the influence of the two GABA_B(1) isoforms on both the motor-incoordinating and hypothermic responses to the GABA_B receptor agonists baclofen and -hydroxybutyrate (GHB), as well as on baseline locomotor behavior and body temperature.

	Materials and Methods

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Animals and Housing. The generation (Vigot et al., 2006) and breeding strategy (Jacobson et al., 2006) of wild-type (WT), GABA_B(1a)^–/–, and GABA_B(1b)^–/– mice as used in the present studies has been described previously. In brief, mice were generated using a knock-in point mutation strategy, whereby GABA_B(1a) and GABA_B(1b) initiation codons were converted to stop codons by targeted insertion of a floxed neo-cassette. All the mutant and WT mice were maintained on a pure inbred BALB/c genetic background. GABA_B(1a)^–/– and GABA_B(1b)^–/– mice used in the present experiments were derived from homozygous breeding (F3–4) of siblings originating from the founding heterozygotic mice. Homozygous WT controls for the GABA_B(1) isoform mutant mice were derived from mating together WT siblings generated from GABA_B(1a)^+/– and GABA_B(1b)^+/– heterozygous breeding (F3–4). The breeding strategy was applied in accordance with the recommendations proposed by The Jackson Laboratory (Bar Harbor, ME) to obviate genetic drift and the formation of substrains (http://jaxmice.jax.org/geneticquality/guidelines.html).

Mice were singly housed in Macrolon cages with sawdust bedding, tissue paper nesting materials, and one red, triangular, polycarbonate Mouse House (Nalgene, Nalge Nunc International, Rochester, NY) per cage. Housing was at a constant room temperature of 22–24°C in a 12-h light/dark cycle with lights on at either 6:00 AM to 6:30 AM. Food pellets and tap water were available ad libitum (except during experimentation, unless stated). Separate cohorts of mice were used for each experiment. All the mice were drug-naive before experimentation. Male mice were used in all the experiments with the exception of GHB-induced hypolocomotion, for which only females were available. In certain experiments as indicated, experimental replication was also carried out in a cohort of female mice. All of the animal experiments were conducted during the light phase with the exception of continuous locomotor activity assessments, which were made during both the light and dark phases. All of the animal experiments were conducted in accordance with Swiss guidelines and approved by the Veterinary Authority of Basel-Stadt, Switzerland.

Drugs. All of the drug solutions were prepared freshly before use. L-Baclofen (Novartis, Basel, Switzerland) and GHB (Novartis) were dissolved in 0.5% methyl cellulose (vehicle) and applied p.o. in a volume of 10 ml/kg.

Primary Observation Test. A battery of behavioral and physiological observations were made as described previously (Cryan et al., 2003) to investigate whether GABA_B(1a)^–/– or GABA_B(1b)^–/– mice had any gross differences compared with WT mice. This was important to investigate because both GABA_B(1)- and GABA_B(2)-deficient mice have been shown previously to develop an enhanced susceptibility to seizures (Prosser et al., 2001; Schuler et al., 2001; Gassmann et al., 2004). Mice used in this experiment were singly housed WT, GABA_B(1a)^–/–, and GABA_B(1b)^–/– mice. The experiment was replicated separately in male and female mice. The mean age (±S.E.M.) of the mice was 20.0 ± 1.0 weeks for males and 23.1 ± 0.8 weeks for females (n = 11–12 for each genotype and gender). The observations quantified were the presence of twitches, tremor, convulsions, piloerection, stereotyped behavior, lacrimation, salivation, ptosis, catalepsy, passivity, falling convulsion, and ataxia. In addition, the frequency and quality of breathing were observed. Alterations in skin color, tail position, pelvic position, limb tonus, abdominal tonus, and pupil width were observed. The nature of locomotion, motility, and rearing in the home cage was quantified, as was overall flight and startle reactions. In addition, novelty behavior was observed and a series of reflexes checked, including pinna reflex, toepinch, tail-pinch, and provoked biting. Body temperature was also quantified. This battery of tests has been validated in our laboratories to detect stimulant and sedative effects in mice, in addition to other effects of pharmacological agents.

Locomotor Activity of GABA_B(1a)^–/–, GABA_B(1b)^–/–, and WT Mice in a Novel Environment. Locomotor activity of mutant and WT mice when placed in a novel environment was investigated in two separate experiments with male and female mice, respectively. Horizontal locomotor activity of the mice [n = 10–11 for each genotype and sex; mean age (±S.E.M.) was 17.5 (±0.5) and 14.5 (±0.4) weeks for males and females, respectively] was recorded for 1 h after individuals were placed into a novel enclosure (transparent Plexiglas boxes, 19 x 31 x 16 cm), with motion detection determined by infrared light beam interruptions along the x- and y-axes. Distance traveled was automatically calculated using a TSE Moti system (TSE, Bad Homburg, Germany).

Continuous 3-Day Locomotor Activity of Male GABA_B(1a)^–/–, GABA_B(1b)^–/–, and WT Mice. Horizontal locomotor activity of age-matched, singly housed male mice (WT, n = 9; GABA_B(1a)^–/–, n = 9; GABA_B(1b)^–/–, n = 6; mean age ± S.E.M., 15.6 ± 0.6 weeks) was continuously recorded over 67 h (TSE Moti system). Mice were transferred to new home cages at 1 PM in the afternoon. Food and water was provided ad libitum, as usual, although tissue paper nesting material and the Mouse House were removed. Motion detection was determined in the new home cages from the time immediately after rehousing over the following 3 dark and 2.5 light cycles by infrared light beam interruptions along the x- and y-axes. Distance traveled was automatically calculated using a TSE Moti system. The testing room in which the experiment took place was undisturbed during the course of the experiment.

Influence of L-Baclofen on Rotarod Endurance and Body Temperature of GABA_B(1a)^–/–, GABA_B(1b)^–/–, and WT Mice. The effect of the GABA_B receptor agonist, L-baclofen, on motor coordination and body temperature in GABA_B(1a)^–/–, GABA_B(1b)^–/–, and WT mice was investigated in two experiments: one with male and one with female mice, respectively. Each experiment was conducted with two cohorts of mice. In the first cohort, 0 and 12 mg/kg L-baclofen was examined in age-matched mutant and WT mice (male mice, n = 19–20 per genotype; mean age ± S.E.M., 15.2 ± 0.3 weeks; female mice, n = 18–21 per genotype; mean age ± S.E.M., 18 ± 0.3 weeks). In the second cohort, 0 and 6 mg/kg L-baclofen was examined (male mice, n = 9–10 per genotype; mean age ± S.E.M., 15.2 ± 0.3 weeks; female mice, n = 8–10 per genotype; mean age ± S.E.M., 25.2 ± 0.5 weeks).

In the experimental protocol, performance on the fixed-speed Rotarod (Dunham and Miya, 1957) was combined with the evaluation of rectal temperature within each animal, as described previously (Cryan et al., 2004; Jacobson and Cryan, 2005). The Rotarod apparatus consisted of a 280-mm diameter rod of approximately 3000-mm length, which was partitioned into five lanes, 580 mm wide, to accommodate individual mice. The rod was positioned 30 cm above a surface and rotated at a constant speed of 12 rpm. Each day the rod lanes were tightly lined with fresh paper towels. Rectal temperature was recorded (thermistor probe; ELLAB Instruments, Copenhagen, Denmark) from individual mice while hand-held near the base of the tail against the wall of the home cage. The probe was left in place until steady readings were obtained (approximately 15 s).

Two days before testing, the Mouse House and tissue paper nesting materials were removed from each cage to reduce possible intercage variations in mouse body temperatures on the day of testing. On the day before testing, mice were acclimatized to the rectal thermistor probe by performing a single body temperature measurement and were then trained to walk on the Rotarod for 300 s. Rotarod training was performed in two to four sessions, depending on the innate ability of each animal. The number of falls during training was recorded. Mice were returned to the home cage for an interval of approximately 30 min between training sessions.

On the day of testing, mice were moved to the experimental laboratory at least 2 h before dosing. One hour before dosing, rectal temperature was taken. At time 0 h, rectal temperature was taken, and then the mice were placed on the Rotarod for 300 s to reestablish training and to provide an experimental baseline. Each mouse was then immediately dosed with its allocated treatment on completing 300 s on the Rotarod. Rectal temperature and endurance on the Rotarod were recorded 1, 2, and 4 h thereafter.

An index of the degree of hypothermia (summed T) was calculated by totaling the differences between control (predose) rectal temperature and the temperatures at 1, 2, and 4 h post-treatment, respectively, within each animal. An index of the degree of ataxia was calculated by determining the difference between cumulative total time on the Rotarod 1, 2, and 4 h after drug or vehicle application as a proportion of the total possible (i.e., percent reduction from 1200 s).

Effect of GHB on Body Temperature of Male GABA_B(1a)^–/–, GABA_B(1b)^–/–, and WT Mice. The effect of GHB (1 g/kg) on body temperature has been shown to be GABA_B(1) receptor subunit-dependent (Kaupmann et al., 2003). Therefore, we assessed whether either receptor subunit isoform conferred susceptibility to GHB-induced hypothermia. This dose was selected based on our dose-response data obtained in BALB/c mice (Kaupmann et al., 2003), the background strain for the GABA_B(1) isoform mutant and WT mice. Rectal temperature (ELLAB Instruments thermistor probe, as described above) of singly housed, age-matched male mice (WT, n = 7; GABA_B(1a)^–/–, n = 9; GABA_B(1b)^–/–, n = 13; mean age ± S.E.M., 27.0 ± 1.7 weeks) was recorded 1 h before, immediately before, and 30, 60, 120, and 240 min after p.o. administration of GHB [1 g/kg in methyl cellulose (0.5%)] in a volume of 10 ml/kg.

Effect of GHB on Locomotor Activity of Female GABA_B(1a)^–/–, GABA_B(1b)^–/–, and WT Mice. Previous studies have shown that the motor-impairing effects of GHB (1g/kg) are GABA_B(1) receptor subunit-mediated (Kaupmann et al., 2003). Therefore, it was important to assess whether either of the GABA_B(1) receptor subunit isoforms conferred susceptibility to GHB-induced hypoactivity. Singly housed, age-matched female mice (WT, n = 23; GABA_B(1a)^–/–, n = 16; GABA_B(1b)^–/–, n = 34; mean age ± S.E.M., 26.7 ± 0.6 weeks) were given GHB (1 g/kg) or vehicle (0.5% methyl cellulose) p.o. in a volume of 10 ml/kg. The dose of GHB was selected based on previous dose-response studies showing maximal effects at this dose in this background mouse strain (Kaupmann et al., 2003). One hour after dosing, mice were placed in a transparent Plexiglas box (19 x 31 x 16 cm), and motion detection was determined over the following hour by infrared light beam interruptions along the x- and y-axes. Distance traveled was automatically calculated using a TSE Moti system.

Statistical Analyses. Because studies that were carried out in both male and female mice were conducted in independent experiments, data for each sex were analyzed separately. Live-weight and rectal temperature as determined in the primary observation test (POT) were analyzed with one-way analysis of variance (ANOVA). Locomotor activity in a novel environment and continuous 3-day locomotor activity were analyzed for the effects of genotype and time using two-way repeated measures ANOVA. Summed mean distance traveled during the first 2 and the full 12 h of the three complete dark cycles and during the 12 h of the two complete light cycles was analyzed for the effect of genotype using one-way ANOVA. Basal body temperature (at time points –1 and 0 h in the combined Rotarod-temperature experiment) was analyzed for the effect of replicate, genotype, and time using three-way ANOVA. Fisher's least significant difference post hoc comparisons were made when indicated by significant ANOVA factors. Falls during Rotarod training and body temperature responses to GHB were analyzed for the effect of genotype in a pairwise fashion using the Mann-Whitney rank sum method. All the variables from the POT, with the exception of live-weight and rectal temperature (see above), summed Rotarod data (within each baclofen dose), and distance traveled after vehicle or GHB treatment (within treatment and time point) were analyzed for the effect of genotype using Kruskal-Wallis one-way ANOVA on ranks. Dunn's method post hoc comparisons were made when indicated by significant Kruskal-Wallis ANOVA factors, with the exception of the influence of GHB on locomotor activity, for which pairwise Mann-Whitney rank sum comparison was used as a post hoc comparison method when appropriate.

	Results

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POT. Unlike GABA_B(1)- and GABA_B(2)-deficient mice, in which spontaneous seizures were observed (Schuler et al., 2001; Gassmann et al., 2004), this phenotype was not behaviorally apparent in GABA_B(1a)^–/– or GABA_B(1b)^–/– mice. To assess whether ablation of GABA_B(1a) or GABA_B(1b) receptor isoforms had any other effects on gross behavior and physiology, male and female WT, GABA_B(1a)^–/–, or GABA_B(1b)^–/– mice were subjected to an extensive POT battery.

Within the male mice, GABA_B(1b)^–/– mice were significantly heavier than either WT (P < 0.01) or GABA_B(1a)^–/– (P < 0.001) mice, and WT mice were heavier than GABA_B(1a)^–/– mice (P < 0.001). Mean (±S.E.M.) weights of male mice were WT, 32.5 (±0.6) g; GABA_B(1a)^–/–, 29.2 (±0.5) g; and GABA_B(1b)^–/–, 34.8 (±0.6) g (genotype F_2,34 = 23.73, P < 0.001). Female GABA_B(1b)^–/– mice were slightly, but significantly, heavier than female WT mice (P < 0.01; mean ± S.E.M. weights were 27.6 ± 0.7 and 24.5 ± 0.7 g, respectively). However, there were no differences between the weights of female GABA_B(1a)^–/– mice (25.9 ± 0.8 g) and either WT or GABA_B(1b)^–/– female mice (genotype F_2,35 = 4.20, P < 0.05). Otherwise, the POT battery revealed no significant differences between GABA_B(1a)^–/–, GABA_B(1b)^–/–, and WT mice in any of the other 31 parameters assessed (see Materials and Methods). This included body temperature as assessed in the home cage, where mean rectal temperature for both the male and female mice was not significantly influenced by genotype, respectively (males: mean rectal temperature ± S.E.M., 35.9 ± 0.13°C; genotype F_2,34 = 0.36, P = 0.70; females: mean rectal temperature, 36.2 ± 0.15°C; genotype F_2,35 = 1.20, P = 0.32).

Locomotor Activity of GABA_B(1a)^–/–, GABA_B(1b)^–/–, and WT Mice in a Novel Environment. Data from one male and one female GABA_B(1a)^–/– mouse were excluded from analysis as statistical outliers. The male mouse traveled 158 m in 1 h compared with the mean for the remaining nine male GABA_B(1a)^–/– mice of 79.6 ± 5.5. The female mouse traveled 208 m in 1 h compared with the mean for the remaining 10 female mice of 54.1 ± 3.6 m. Both of these animals showed intermittent periods of repetitive, stereotypic circling behavior in the home cage, which might have increased locomotor activity during the test. No other signs of stereotyped behavior were observed in any genotype across all the other experiments.

Genotype influenced the pattern of locomotor activity in male mice (time F_11,347 = 15.39, P < 0.001; genotype F_2,347 = 1.67, P > 0.05; interaction F_22,347 = 1.708, P < 0.05) (Fig. 1a). Post hoc analysis revealed the male GABA_B(1b)^–/– mice were more active than WT and GABA_B(1a)^–/– male mice within the first 5 min and at other time points within the first 20 min of the experiment (Fig. 1a).

View larger version (21K): [in this window] [in a new window]   Fig. 1. GABAB(1b)–/– mice were hyperactive in a novel environment. Distance traveled during spontaneous locomotor activity in a novel enclosure by male (a) and female (b) WT, GABAB(1a)–/– (1a–/–), and GABAB(1b)–/– (1b–/–) mice. *, P < 0.05 versus WT; #, P < 0.05 versus 1a–/–; ##, P < 0.01 versus 1a–/–.] denotes ANOVA main effects for genotype and time.

Continuous 3-Day Locomotor Activity of GABA_B(1a)^–/–, GABA_B(1b)^–/–, and WT Mice. Male GABA_B(1b)^–/– mice traveled significantly greater distances than either WT or GABA_B(1a)^–/– mice during the 1st h of habituation to a new enclosure and during the following three dark phases, whereas GABA_B(1a)^–/– mice traveled similar distances to that of WT mice during the dark phases (genotype F_2,1607 = 2.62, P = 0.096; time F_66,1607 = 34.08, P < 0.001; interaction F_132,1607 = 1.75, P < 0.001) (Fig. 2, a and b). The hyperactivity of the GABA_B(1b)^–/– mice was particularly prevalent during the first 2 h of the dark phase, as the distance traveled (averaged over the three dark phases and expressed as a proportion the WT mean) during this time was 154% that of the WT controls (genotype F_2,23 = 6.697, P < 0.01) (Fig. 2c). This pattern of behavior was confirmed when assessing data as mean summed distance (within animal) in the dark, as GABA_B(1b)^–/– mice traveled a greater mean distance during the dark phase than either WT or GABA_B(1a)^–/– mice (P < 0.05, respectively; genotype F_2,23 = 3.81, P < 0.05) (Fig. 2d). In comparison, during the two complete light phases, the GABA_B(1a)^–/– mice traveled a greater mean distance per light phase than the WT mice (genotype F_2,23 = 4.15, P < 0.05) (Fig. 2e).

View larger version (32K): [in this window] [in a new window]   Fig. 2. GABAB(1b)–/– mice were hyperactive during the circadian dark phase. Distance traveled during 67 h of continuously monitored locomotor activity by male WT and GABAB(1a)–/– (1a–/–) mice (a) and of the WT versus GABAB(1b)–/– (1b–/–) mice (b). Mean (+S.E.M.) summed distance traveled by WT, 1a–/–, and 1b–/– mice in the 2 h immediately after the start of the dark phase, averaged over the three dark phases and expressed as a proportion of the WT mean (c). Mean (+S.E.M.) summed distance traveled per 12 h for male WT, 1a–/–, and 1b–/– mice during the three complete dark phases (d) and two complete light phases (e). *, P < 0.05, **, P < 0.01, ***, P < 0.001 versus WT; #, P < 0.05, ##, P < 0.01 versus 1a–/–.

Influence of L-Baclofen on Rotarod Endurance and Body Temperature of GABA_B(1a)^–/–, GABA_B(1b)^–/–, and WT Mice. Temperature data from one female GABA_B(1a)^–/– mouse in the baclofen 12-mg/kg dataset were eliminated as a statistical outlier (the two predrug body temperature measurements on the day of the experiment were less than 35.0°C). Rotarod data from one male GABA_B(1a)^–/– mouse in the vehicle-treated dataset were eliminated because of repeated voluntary jumping from the Rotarod. Data from one female GABA_B(1a)^–/– mouse at a time point 4 h after vehicle dosing were lost because of repeated voluntary jumping from the Rotarod.

Body temperature at times –1 and 0 h, as well as Rotarod training performance and duration on the Rotarod at time 0 h of the experiment, did not differ between the two experimental cohorts for either of the experiments involving male or female mice (temperature: males, cohort F_1,175 = 1.399, P = 0.24; females, cohort F_1,171 = 0.47, P = 0.50; all the mice walked for 300 s on the Rotarod at time point 0); therefore, temperature and Rotarod data for the two cohorts were pooled (within experiments).

Mean body temperature before drug treatment was slightly, but significantly, lower in female GABA_B(1a)^–/– mice than either the WT (P < 0.05) or GABA_B(1b)^–/– mice (P < 0.05), although the GABA_B(1a)^–/– and WT mice were not different from each other [P > 0.05; genotype F_2,171 = 3.37, P < 0.05; mean temperature ± S.E.M. (°C): WT, 35.87 ± 0.08; GABA_B(1a)^–/–, 35.60 ± 0.08; GABA_B(1b)^–/–, 35.85 ± 0.06].

In contrast, in the experiment with male mice, GABA_B(1b)^–/– mice had a slightly higher mean basal body temperature than either the WT (P < 0.05) or GABA_B(1a)^–/– mice (P < 0.05), although the GABA_B(1a)^–/– and WT mice were not significantly different from each other [P > 0.05; genotype F_2,175 = 5.11, P < 0.01; mean temperature ± S.E.M. (°C): WT, 35.94 ± 0.10; GABA_B(1a)^–/–, 36.09 ± 0.11; GABA_B(1b)^–/–, 36.40 ± 0.10].

Baclofen produced profound, long-lasting hypothermia in male WT mice (male WT: baclofen dose F_2,149 = 13.74, P < 0.001; time F_4,149 = 13.99, P < 0.001; interaction F_8,149 = 16.36, P < 0.001) (Fig. 3a). Baclofen at 12 mg/kg, but not 6 mg/kg, also induced hypothermia in male GABA_B(1b)^–/– mice (male GABA_B(1b)^–/–: baclofen dose F_2,149 = 5.95, P < 0.01; time F_4,149 = 9.03, P < 0.001; interaction F_8,149 = 2.95, P < 0.01) (Fig. 3c). In contrast, neither the 6nor 12-mg/kg doses of baclofen induced hypothermia in the male GABA_B(1a)^–/– mice, relative to vehicle-treated GABA_B(1a)^–/– males, at the three postdrug time points measured (male GABA_B(1a)^–/–: baclofen dose F_2,139 = 2.78, P = 0.08; time F_4,139 = 1.73, P = 0.149; interaction F_8,139 = 0.61, P = 0.76) (Fig. 3b). When examining the total hypothermic response to baclofen over the duration of the experiment, both GABA_B(1a)^–/– and GABA_B(1b)^–/– male mice showed an attenuated summed T in response to 12 mg/kg baclofen compared with the WT mice, although neither of the mutant mice strains differed from each other in this regard (males summed T: genotype F_2,87 = 3.26, P < 0.05; baclofen dose F_2,87 = 10.64, P < 0.001; interaction F_4,87 = 4.66, P < 0.01) (Fig. 3d).

View larger version (20K): [in this window] [in a new window]   Fig. 3. Hypothermic responses to baclofen were attenuated in GABAB(1a)–/– and GABAB(1b)–/– mice. Mean (±S.E.M.) body temperature of male (a–c) and female (e–g) WT, GABAB(1a)–/– (1a–/–), and GABAB(1b)–/– (1b–/–) mice following vehicle or L-baclofen (6 and 12 mg/kg) administration. Data are also presented as mean (±S.E.M.) summed area under the curve (T, calculated within animal) for male (d) and female (h) WT, 1a–/–, and 1b–/– mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus WT.

It is interesting to note that in the experiments with both male and female GABA_B(1b)^–/– mice, the lower dose of baclofen seemed to induce hyperthermia 1 h after baclofen administration (Fig. 3, c and g). In the female mice, post hoc analysis within the 6-mg/kg group showed significant increase in body temperature from the –1 to the 0 h time point (P < 0.01) and a tendency to further increase from the 0 to the 1 h time point (P = 0.077). In male mice, post hoc analysis within the 6-mg/kg baclofen treatment, similar to the females, showed a significant increase in body temperature from the –1 to the 0 h time point (P < 0.05). However, mean body temperature at time points 0 and 1 h were not significantly different from each other (P = 0.144).

During training for the Rotarod experiment, the mean number of falls from the Rotarod during training for male mice was not significantly affected by genotype (P > 0.05 for all the pairings; mean ± S.E.M.: WT, 0.73 ± 0.24; GABA_B(1a)^–/–, 0.43 ± 0.17; GABA_B(1b)^–/–, 0.67 ± 0.19). In comparison, female GABA_B(1a)^–/– mice fell from the Rotarod more often during training than either WT or GABA_B(1b)^–/– mice (P < 0.05, for each respective pairing; mean ± S.E.M.: WT, 0.15 ± 0.07; GABA_B(1a)^–/–, 0.94 ± 0.25; GABA_B(1b)^–/–, 0.13 ± 0.06). On the day of testing, however, all the mice walked on the Rotarod for the allocated 300 s on the first experimental time point (immediately before dosing), thus indicating they had learned the task adequately.

Baclofen significantly impaired Rotarod endurance in male WT mice in a time- and dose-dependent manner (male WT: baclofen dose F_2,119 = 6.99, P < 0.01; time F_3,119 = 17.06, P < 0.001; interaction F_6,119 = 7.64, P < 0.001) (Fig. 4a). Baclofen similarly reduced Rotarod endurance in male GABA_B(1a)^–/– and GABA_B(1b)^–/– mice by 1 h after dosing, although the duration of impairment was shorter than that of the WT mice in both mutant strains of mice (GABA_B(1a)^–/–: baclofen dose F_2,111 = 2.44, P = 0.107; time F_3,111 = 9.63, P < 0.001; interaction F_6,111 = 3.72, P < 0.01; GABA_B(1b)^–/–: baclofen dose F_2,119 = 5.82, P < 0.01; time F_3,119 = 5.60, P < 0.01; interaction F_6,119 = 5.63, P < 0.001) (Fig. 4, b and c). Overall, when examining total summed endurance on the Rotarod, male GABA_B(1a)^–/– and GABA_B(1b)^–/– mice performed similarly to WT mice at 0-, 6-, and 12-mg/kg doses of baclofen (vehicle, H = 3.30, P = 0.19; baclofen 6 mg/kg, H = 4.51, P = 0.162; baclofen 12 mg/kg, H = 1.20, P = 0.55) (Fig. 4d).

View larger version (21K): [in this window] [in a new window]   Fig. 4. Ataxic responses to baclofen were attenuated in GABAB(1a)–/– and GABAB(1b)–/– mice. Mean (±S.E.M.) endurance on a fixed-speed Rotarod (12 rpm, maximum allowed was 300 s per time point) for male (a–c) and female (e–g). WT, GABAB(1a)–/– (1a–/–), and GABAB(1b)–/– (1b–/–) mice following vehicle or L-baclofen (6 and 12 mg/kg) administration. Data are also presented as a proportion of the total time available (1200 s), which was spent walking on the Rotarod by male (d) and female (h) WT, 1a–/–, and 1b–/– mice. *, P < 0.05, **, P < 0.01, ***, P < 0.001 versus WT.

Effect of GHB on Body Temperature of GABA_B(1a)^–/–, GABA_B(1b)^–/–, and WT Mice. All of the mice responded to GHB with hypothermia, relative to predrug application body temperature. However, the degree of hypothermia was influenced by genotype. Thirty minutes after GHB application, GABA_B(1a)^–/– mice had a significantly lower mean reduction in body temperature than the WT mice (P < 0.05) (Fig. 5). One hour after p.o. application, both GABA_B(1a)^–/– and GABA_B(1b)^–/– mice had attenuated hypothermic responses to the GHB compared with WT mice (P < 0.05 for GABA_B(1a)^–/– and GABA_B(1b)^–/– mice versus WT, respectively) (Fig. 5). Body temperature of the mice before drug administration was not influenced by genotype (P > 0.05).

View larger version (16K): [in this window] [in a new window]   Fig. 5. Hypothermic responses to GHB were attenuated in GABAB(1a)–/– and GABAB(1b)–/– mice. Mean (±S.E.M.) body temperature of male WT, GABAB(1a)–/– (1a–/–), and GABAB(1b)–/– (1b) mice following administration of GHB (1 g/kg). *, P < 0.05 1a–/– versus WT; #, P < 0.05 1b–/– versus WT.

Effect of GHB on Locomotor Activity of GABA_B(1a)^–/–, GABA_B(1b)^–/–, and WT Mice. Interestingly, the genotype did not affect the distance traveled for vehicle-treated mice at any of the postdrug administration time intervals investigated (P > 0.05). In contrast, in the GHB-treated mice, genotype influenced the median distance traveled in the 15 to 20 min (H = 6.02, P < 0.05), 20 to 25 min (H = 6.25, P < 0.05), 25 to 30 min (H = 9.01, P < 0.05), 30 to 35 min (H = 6.93, P < 0.05), and 40 to 45 min intervals (H = 6.61, P < 0.05). Post hoc comparison revealed that the GABA_B(1b)^–/– mice tended to be more active than WT mice during the 15 to 20 min interval (P < 0.056). However, at the other time intervals, GABA_B(1a)^–/– mice traveled a greater distance than the WT mice (20–25 min, P < 0.05; 25–30 min, P < 0.01; 30–35 min, P < 0.05; 40–45 min, P < 0.01) (Fig. 6). This indicated that both GABA_B(1a)^–/– and GABA_B(1b)^–/– mice were less sensitive to the locomotor suppressing effects of GHB.

View larger version (30K): [in this window] [in a new window]   Fig. 6. Hypolocomotor effects of GHB were attenuated in GABAB(1) isoform-deficient mice. Mean (±S.E.M.) distance traveled by female WT, GABAB(1a)–/– (1a–/–), and GABAB(1b)–/– (1b–/–) mice following administration of vehicle or GHB (1 g/kg). Note: analysis performed with nonparametric testing. +, P < 0.10 GHB-treated 1b–/– versus WT; *, P < 0.05, **, P < 0.01, GHB-treated 1a–/– versus WT.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The Gabbr1 gene is predominantly transcribed into two differentially expressed isoforms, GABA_B(1a) and GABA_B(1b), which differ in sequence primarily by the inclusion of a pair of evolutionary conserved sushi repeats (also known as short consensus repeats) in the GABA_B(1a) N terminus (Bettler et al., 2004). The recent generation of GABA_B(1a)^–/– and GABA_B(1b)^–/– mice (Vigot et al., 2006) has opened up new possibilities for understanding the functions of these sushi domains. Here we show that GABA_B(1a)^–/– and GABA_B(1b)^–/– mice possessed normal overt behavioral responses as observed in the primary observation test, as well as in basic righting and motor function, as indicated by unimpaired baseline performance on the Rotarod. In addition, unlike the full GABA_B(1)^–/– mice (Prosser et al., 2001; Schuler et al., 2001), basic observations of the mice suggested that neither GABA_B(1a)^–/– nor GABA_B(1b)^–/– mice had spontaneous seizures. However, the characteristic hypothermia and motor impairment in response to the GABA_B receptor agonists L-baclofen or GHB were markedly attenuated and, interestingly, to a relatively similar degree in both GABA_B(1a)^–/– and GABA_B(1b)^–/– mice compared with WT controls. Moreover, the two mutant lines of mice diverged significantly in their baseline behavior. The GABA_B(1b)^–/– mice were hyperactive in a novel environment and habituated more slowly than either the WT or GABA_B(1a)^–/– mice. Furthermore, GABA_B(1b)^–/– mice were more active throughout the dark phase than either the GABA_B(1a)^–/– or WT mice. Finally, the findings of the lack of overt phenotype in the POT, similar attenuation of baclofen-induced hypothermia and ataxia in both mutants, and hyperlocomotor responses to a novel environment by GABA_B(1b)^–/– mice were replicated in separate experiments with both male and female mice, attesting to the reproducibility of these phenotypes.

GABA_B receptors play a crucial role in mediating normal motor responses (Jacobson and Cryan, 2005). Furthermore, deletion of the GABA_B(1) receptor subunit results in a complex locomotor response (Mombereau et al., 2004; Vacher et al., 2006). This includes marked hyperlocomotion when exposed to a novel environment, whereas in a familiar environment, GABA_B(1)^–/– mice display an altered pattern of circadian activity but no hyperlocomotion (Vacher et al., 2006). Therefore, the baseline motor hyperactivity of GABA_B(1b)^–/– mice in a novel environment was not entirely surprising. Although the effect of GABA_B(1b) isoform deletion on locomotor activity seemed somewhat modest, the magnitude of the effect is more apparent when examined as a relative proportion of the WT controls. For example, male GABA_B(1b)^–/– mice traveled approximately 140% of the distance of WT controls in the first 5 min of exposure to a novel environment and 154% of that of WT mice in the first 2 h of the dark cycle. Furthermore, hyperlocomotor responses were shown on four occasions with three different cohorts of mice: novelty-induced hyperlocomotion was replicated in a separate cohort of female mice; in another cohort of male mice in the 3-day locomotor experiment during the 1st h in a new home cage; and hyperlocomotion of GABA_B(1b)^–/– in the dark phase was subsequently shown during the following three dark cycles. Together, these data suggest that the loss of the GABA_B(1b) isoform may contribute to a significant degree to the aforementioned hyperlocomotion of GABA_B(1)^–/– mice.

Although the locomotor behavior of GABA_B(1a)^–/– mice was similar to WT controls in the present investigation, in other test systems differences to WT and GABA_B(1b)^–/– mice have been identified. We have previously shown that GABA_B(1a)^–/– mice were impaired in an object recognition task (Vigot et al., 2006), whereas the GABA_B(1b)^–/– mice were not. Furthermore, GABA_B(1a)^–/– mice had deficits in the acquisition of conditioned taste aversion (Jacobson et al., 2006) and in the generalization of fear conditioning-induced freezing (Shaban et al., 2006). Correspondingly, the GABA_B(1a)^–/–, but not the GABA_B(1b)^–/–, mice were also deficient in hippocampal (Vigot et al., 2006) and amygdala long-term potentiation (Shaban et al., 2006). In addition, Perez-Garci et al. (2006) have also shown specific and differential roles for the GABA_B(1a) and GABA_B(1b) isoforms in mediating different components of GABA_B receptor-induced inhibition of layer 5 cortical neurons. Together with the hyperactive phenotype in locomotor activity of the GABA_B(1b)^–/– mice shown in the present study, these data show that GABA_B(1) receptor isoforms have divergent and functionally relevant influences on behavioral output and the underlying neurophysiology.

Given the phenotypic differences between these two isoform-deficient lines of mice, it was interesting to note that although both GABA_B(1a)^–/– and GABA_B(1b)^–/– mice both showed attenuated responses to the GABA_B receptor agonists baclofen, the degree of attenuation was largely similar in both mutant strains of mice. In GABA_B(1)^–/– mice, baclofen does not induce the hypothermic or ataxic effects normally seen in WT mice (Schuler et al., 2001; Queva et al., 2003). This indicated that the full GABA_B receptor heterodimer is necessary for the actions of these agonists and, likewise, that the actions of these agonists are specific to the GABA_B receptor. Results of the present study, however, suggest that neither the GABA_B(1a) nor GABA_B(1b) isoforms are solely responsible for the hypothermic or ataxic actions of baclofen. The data also show that neither isoform can fully compensate for the loss of the other with regard to agonist-induced responses. To further confirm this, we investigated the effects of another GABA_B receptor agonist, GHB. We have previously shown that the hypothermic and motor-impairing effects of GHB (1 g/kg) are completely absent in mice lacking the GABA_B(1) receptor (Kaupmann et al., 2003). Here we show that the effects of GHB were modestly but significantly attenuated in mice lacking either of the two GABA_B(1) isoforms. Thus, we can conclude, as in the case of baclofen, that neither the GABA_B(1a) nor GABA_B(1b) isoform is solely responsible for the hypothermic or ataxic actions of GHB. These in vivo data are in support of a number of recombinant studies demonstrating that neither baclofen nor GHB show specificity for either of the two predominant GABA_B(1) subunit isoforms (see Bettler et al., 2004).

Paradoxically, at the lower dose of 6 mg/kg, baclofen seemed to increase body temperature in GABA_B(1b)^–/– mice. Examination of post hoc statistical comparisons indicated that the bulk of the temperature increase of mice in this treatment group occurred between time –1 and 0 h, both of which preceded baclofen injection. Therefore, it seems likely that handling stress may have induced the apparent hyperthermic responses in these mice. However, it should be noted that GHB at low doses has been previously reported to induce hyperthermia as opposed to hypothermia (Kaufman et al., 1990). Therefore, it remains possible that some influence of residual GABA_B(1) activity in the GABA_B(1b)^–/– mice may have contributed to a hyperthermic response to baclofen in these mice.

Control of body temperature and motor activity is anatomically and neurochemically heterogeneous (Cryan et al., 1999, 2000; Jacobson and Cryan, 2005). Both GABA_B(1a) and GABA_B(1b) isoforms are abundantly expressed throughout the brain, although their expression level relative to each other and distribution profile within structures diverge in many regions, including in many of those involved in the control of body temperature or motor activity and coordination (Benke et al., 1999; Bischoff et al., 1999; Liang et al., 2000; Fritschy et al., 2004). Therefore, it may seem reasonable to have expected that, irrespective of a lack of specificity of the isoforms for baclofen or GHB, responses to the agonists may yet have varied between the two mutant mouse lines. In the present study, however, this was not the case. Previously we have shown that genetic background (in the form of different mouse strains) can greatly influence hypothermic and ataxic responses to baclofen (Jacobson and Cryan, 2005). The present study may indicate that this is not necessarily because of overall variations in the relative expression of the GABA_B(1) subunit isoforms. Indeed, given that baseline body temperatures of the GABA_B(1a)^–/– and GABA_B(1b)^–/– mice showed negligible differences, it may be that normal homeostasis of body temperature is not specifically controlled by one or another of the GABA_B(1) subunit isoforms. This is in contrast to the full GABA_B(1)^–/– mice, in which basal body temperature was shown to be approximately 1°C less than that of the WT controls (Kaupmann et al., 2003; Queva et al., 2003).

The lack of differential responses to baclofen and GHB between the GABA_B(1) isoform-deficient mice may also be in part the result of the complex neural control of body temperature and motor activity. For example, when strongly activated by pharmacological means, loss of either presynaptic or postsynaptic inhibition in a complex multisynaptic system may ultimately appear similar downstream (i.e., appearing as an increase in excitability in a convergent output). With regard to the contribution of different cellular components in these systems, the prospective roles of interneurons in GABA_B receptor agonist-induced hypothermia and ataxia are unknown at the present time. Hippocampal and lateral amygdala autoreceptor function was preserved in both GABA_B(1a)^–/– and GABA_B(1b)^–/– mice (Shaban et al., 2006; Vigot et al., 2006) but was completely absent in the GABA_B(1)^–/– mice (Prosser et al., 2001; Schuler et al., 2001). This indicated that in these structures, both isoforms can act as autoreceptors. However, GABAergic interneurons synapsing on distal dendrites of layer 5 cortical neurons appeared to preferentially express the GABA_B(1a) isoform but not the GABA_B(1b) isoform (Perez-Garci et al., 2006). This shows that different interneuron populations may variably express the two GABA_B(1) subunit isoforms. Clearly, further studies are needed to evaluate the expression profile and roles of GABA_B(1) isoform autoreceptors in GABA_B receptor-mediated hypothermia and ataxia.

In conclusion, GABA_B(1a) and GABA_B(1b) isoforms are functionally relevant molecular variants of the GABA_B(1) receptor subunit, which are differentially involved in specific neurophysiological processes and behaviors. It is evident from the present study, however, that the GABA_B receptor agonists baclofen and GHB were unable to pharmacologically discriminate these differences, at least with regard to body temperature and motor coordination. As the sequence of the GABA_B(1a) and GABA_B(1b) isoforms differ primarily in the N terminus, and not in the region coding the ligand binding domain (Kaupmann et al., 1997; Bettler et al., 2004), future studies should focus on strategies to uncover novel interaction sites at either receptor isoform to enable specific pharmaceutical intervention.

	Acknowledgements

 
We thank Conrad Gentsch for his valuable input in the 3-day locomotor activity study. We also thank Christine Hunn, Roland Mayer, and Hugo Bürki for excellent technical assistance.

	Footnotes

 
This work was supported by the National Institutes of Mental Health/National Institute on Drug Abuse Grant U01 MH69062 (J.F.C., L.H.J., K.K.) and the Swiss Science Foundation (3100-067100.01) (B.B.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.111971.

ABBREVIATIONS: GHB, -hydroxybutyrate; WT, wild-type; POT, primary observation test; ANOVA, analysis of variance.

¹ Current affiliation: School of Pharmacy, Department of Pharmacology and Therapeutics, University College Cork, Cork City, Ireland.

Address correspondence to: John F. Cryan, Senior Lecturer in Pharmacology, School of Pharmacy, Department of Pharmacology and Therapeutics, University College Cork, Cork, Ireland. E-mail: j.cryan{at}ucc.ie

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