Functional Observation Battery
There were no notable genotype differences on various measures of empty cage behavior, physical health, sensory, and neurological function ().
Genotypes were Similar in a Functional Observation Battery Assessing Physical Health, Sensory Reflexes, Neurological Functions, and Empty Cage Behaviors
Novelty-Induced Locomotor Activity
There was a significant effect of genotype (F2,25 = 8.36, P = 0.0017), and time (F11,275 = 15.00, P <0.0001) but no significant genotype × time interaction for total distance traveled (). Post hoc tests collapsed across the session showed that KO mice were significantly more active than WT (). There was no significant effect of genotype on percent time spent or frequency to enter the center of the open field for either the whole session or during the first 5 min when the anxiety response is considered to be the strongest (data not shown). When sex was added to the model, there was no significant effect of sex or genotype× sex interaction but there was a significant time × genotype × sex interaction (F22,242 = 2.19, P = 0.0020) for total distance traveled. Post hoc analysis revealed a faster habituation to the open field in female GLAST HET mice compared to male KOs.
Figure 1 GLAST KO showed locomotor hyperactivity in a novel open field. GLAST KO mice show increased locomotor activity compared to WT controls (a). Over the session as a whole, GLAST KO were more active than WT (b). n = 10–19 per genotype. Data are mean (more ...)
Sociability and Social Novelty Preference
There was a significant effect of genotype (F2,98 = 6.62, P = 0.0020) and stimulus (F1,98 = 53.21, P <0.0001), as well as a significant genotype × stimulus interaction (F2,98 = 5.19, P = 0.0072) for time spent sniffing during the sociability test. Post hoc tests showed WT and HET spent more time sniffing the cage containing the mouse than the empty cage. However, KO spent significantly less time sniffing the social stimulus than WT (). There was a significant effect of stimulus (F1,98=12.41, P = 0.0007) but no genotype or genotype × stimulus interaction for frequency to sniff during the sociability test (data not shown). When sex was included in the analysis, there was a significant sex_stimulus interaction on time (F1,92 = 4.38, P = 0.039) and frequency (F1,92 = 7.75, P = 0.0065), because of male mice sniffing the inanimate, but not social, stimulus more than females there was no significant main effect or interactions between sex and genotype. For mean sniffing time there was no significant effect of genotype, but a significant effect of stimulus (F1,98 = 32.68, P <0.0001) and genotype × stimulus interaction (F2,98 = 3.29, P = 0.041). Post hoc analysis revealed that GLAST KO mice spent on average less time sniffing the social stimulus mouse (mean duration sniffing, inanimate stimulus: WT = 1.36 ± 0.39 s, HET = 1.47 ± 0.08, KO = 1.44 ± 0.12, social stimulus: WT = 2.82 ± 0.39, HET = 2.39 ± 0.17, KO = 1.87 ± 0.15). There were no significant difference in transitions between the two stimuli (WT = 20.25 ± 1.78, HET = 21.47 ± 1.22, KO = 19.59 ± 1.49). There was no significant effect of genotype on latency to start sniffing either the empty or social stimulus cage (data now shown). Sex had no significant main effect or interaction on any of the parameters when incorporated into the analysis.
Figure 2 Decreased sociability and social novelty preference, but normal free social interaction in GLAST KO mice. WT and HET, but not KO, spent more time sniffing a cage containing a mouse than an empty cage, and KO spent less time sniffing the mouse-containing (more ...)
For the social novelty preference test, there was a significant effect of genotype (F2,98 = 4.46, P = 0.014) and stimulus (F1,98 = 72.86, P = 0.0000) and a significant genotype × stimulus interaction (F2,98 = 3.19, P = 0.045) for time spent sniffing. Post hoc analysis showed that all genotypes spent more time sniffing the cage containing the novel mouse than the familiar mouse. However, KO spent significantly less time sniffing both the novel and the familiar mouse compared to WT (). There was a significant effect of stimulus (F1,98 = 56.98, P <0.0001) but no genotype or genotype × stimulus interaction for frequency to sniff during social novelty preference test (data not shown). For average time sniffing there was a significant effect of genotype (F2,98 = 11.10, P <0.0001), stimulus (F1,98 = 16.58, P = 0.0001), and genotype × stimulus interaction (F2,98 = 4.17, P = 0.011). Post hoc test showed GLAST KO spent significantly less time sniffing the familiar mouse and both GLAST HET and KO mice spent less time sniffing the novel stimulus mouse compared to WT controls (mean duration sniffing, familiar stimulus: WT = 1.82 ± 0.10 s, HET = 2.08 ± 0.20, KO = 1.37 ± 0.17, novel stimulus: WT = 2.83 ± 0.14, HET = 2.13 ± 0.16, KO = 1.86 ± 0.13). There was no significant difference in transitions between the familiar and novel stimuli (WT = 14.69 ± 2.84, HET = 11.35 ± 1.18, KO = 10.24 ± 1.25). No significant differences between genotype on latency to start sniffing either the familiar or novel stimulus cage were seen (data now WT HET KO shown). Sex had no significant main effect or interaction on any of the parameters when incorporated into the analysis.
Free Social Interaction
There was no significant effect of genotype on overall social interaction () or duration of anogenital, non-anogenital or aggressive interactions (). There was a significant main effect of sex, (but no interactions with genotype), on overall social interaction (F1,53 = 7.74, P = 0.0075) as well as nonanogenital interaction (F1,53 = 8.54, P = 0.0051) because of males engaging in more interaction than females.
Hidden Food Retrieval Test for Olfaction
One WT and one KO failed to find the cookie within the time limit and were excluded. There was no effect of genotype on the latency to find the buried food (WT = 189 ± 52 s, HET = 113 ± 23, KO = 153 ± 43). There was a significant main effect of sex (F1,49 = 4.39, P = 0.041; n = 16–20), but no interaction with genotype, because of a longer latency to find the cookie in males than females.
There was a significant difference between genotypes in nesting behavior scores (H2,53 = 6.05, P = 0.048). Post hoc analysis showed that KO made poorer nests than both WT and HET mice (). There was no main effect or interaction of sex with genotype on this measure.
Poor Nesting Behavior in GLAST KO
There was no effect of genotype or sucrose concentration, or an interaction between the two, for sucrose preference (). Total fluid intake was also unaffected by genotype and sucrose concentration. When sex was added to the statistical model, there was no main effect of sex or genotype × sex interaction for preference, although there was a significant effect of sex (F1,36 = 10.79, P = 0.0041) and a significant sex × sucrose concentration interaction (F2,36 = 6.64, P = 0.0035) for total intake because of females consuming more total fluid than males.
GLAST KO Mice Showed Normal Preference for Sucrose
Acoustic Startle and Sensorimotor Gating
There was a significant effect of prepulse intensity (F2,104 = 81.27, P <0.0001) but not of genotype and no prepulse intensity × genotype interaction for percent PPI. Post hoc tests collapsed across genotype showed a progressive increase in percent PPI with increasing prepulse intensities ().
Figure 3 GLAST KO showed a reduced acoustic startle response but normal sensorimotor gating. Prepulse inhibition of the startle response was no different between genotypes regardless of prepulse intensity (a). GLAST KO showed a lesser acoustic startle response (more ...)
There was a significant effect of genotype for acoustic startle amplitude (F2,52 = 5.47, P = 0.0070). Post hoc tests revealed significantly lower startle amplitude in KO than WT (). Baseline movement did not differ between genotypes (data not shown). Sex had no significant main effect or interactions with genotype on either PPI or startle.
Auditory Brainstem Responses
There is no significant difference in the ABR thresholds between KO (28.3 ± 4.0 dB SPL, n = 6) and WT (29.2 ± 2.0 dB SPL, n = 6).
Instrumental Learning and Extinction
Genotypes did not differ in trials to instrumental learning criteria () or trials to extinction criterion (). There was no significant effect of sex and no genotype × sex interaction for these measures.
Figure 4 GLAST KO showed normal instrumental learning and extinction. Genotypes did not differ in trials to instrumental learning criterion (a). Genotypes did not differ in trials to instrumental extinction criterion (b). n = 7–9 per genotype. Data are (more ...)
Pairwise Discrimination Learning and Reversal
Genotypes did not differ in sessions to criterion on any phase of pretraining (). For discrimination learning, there was a significant effect of genotype for total trials (F2,21 = 56.8, P <0.0001) and incorrect responses (F2,21 = 72.14, P <0.0001) committed. Post hoc tests showed that KO committed significantly more trials () and made significantly more incorrect responses () over the course of the discrimination problem than WT. There was no significant effect of genotype for trials omitted or in any of the auxiliary measures (), although KO mice tended to omit less (WT = 41.1 ± 24.6, HET = 21.3 ± 9.4, KO = 8.4 ± 2.5) and respond to stimuli, retrieve rewards and complete sessions more quickly than WT. There was no significant effect of sex and no genotype_sex interaction for these measures.
Genotypes did not Differ in Rate to Progress Through Pretraining Phases
Figure 5 GLAST KO showed impaired pairwise discrimination learning. GLAST KO committed (a) more trials and (b) incorrect responses than WT over a maximum of 60 discrimination sessions. (c) Survival analysis showed that all WT and HET had attained the 85% correct (more ...)
Auxiliary Measures of Task Performance During Discrimination Learning in GLAST KO Mice
Survival analysis revealed a significant effect of genotype (χ2 = 14.51, df = 1, P <0.0001) WT and HET had attained the 85% correct criterion by 30–40 sessions, whereas nearly all KO had failed to reach this criterion after the 60-session cutoff (). However, analysis of the percentage of each genotype attaining increasing performance thresholds revealed that nearly all KO were able to attain a 80% correct criterion level that was well above statistical chance (ie, >70% correct; ).
Because KO failed to attain the preset 85% criterion for discrimination learning, they were not tested for reversal. WT and HET did not differ in the number of trials committed (WT = 583 ± 205, HET = 604 ± 127) or incorrect responses (WT = 336 ± 81, HET = 348 ± 56) to attain reversal criterion. Trials omitted, stimulus reaction time, reward retrieval latency, and session completion time were also no different between WT and HET (data not shown). Sex had no significant main effect or interaction with genotype on these analyses.
Effects of LY379268 on Discrimination
All WT attained the 85% correct discrimination criterion within 22 sessions. There was a significant effect of genotype but not treatment and no genotype × treatment interaction for trials committed (F1,25 = 29.3, P = 0.0003) and incorrect responses (F1,25 = 67.3, P <0.0001) committed during these sessions. Post hoc analysis on data collapsed across treatment showed that KO committed significantly more trials () and incorrect responses () than WT. Survival analysis showed that all but one KO had failed to reach criterion over the 22 sessions, whether vehicle (χ2 = 8.63, df = 1, P <0.0001) or drug treated (χ2 = 8.92, df = 1, P <0.0001; data not shown). For the 22 sessions when all mice were tested, 67% of the saline treated KO mice attained the 70% correct performance level (ie, significantly above chance) and drug treatment did not improve this rate (57%). Sex produced no significant main effect or interaction with genotype or drug when incorporated into the analysis.
Figure 6 Treatment with the mGlu2/3 receptor agonist LY379268 did not rescue impaired pairwise discrimination learning in GLAST KO. Regardless of drug treatment, GLAST KO committed (a) more trials and (b) incorrect responses than WT over the 22 discrimination (more ...)
Because previous studies have shown that tolerance develops to LY379268’s effects on motor coordination and nicotine self-administration (Cartmell et al, 2000
; Liechti et al, 2007
), although not the drug’s effects on PCP-induced hyperactivity (Cartmell et al, 2000
), we separately analyzed the first six sessions of discrimination for significant drug effects that might have waned with the more prolonged treatment. There was no indication that LY379268 produced effects on behavior early in training that were lost with repeated treatment, regardless of genotype (data not shown).