GENETIC MANIPULATION OF CATECHOL-O-METHYLTRANSFERASE (COMT) IN MICE AFFECTS SPECIFIC COGNITIVE PROCESSES

BACKGROUND

In humans, a functional single nucleotide polymorphism in the coding region of the COMT gene produces two COMT variants: Val and Met, with higher and lower enzyme activity, respectively1, 2. Moreover, at least three common haplotypes of the human COMT gene have been recently associated with different levels of COMT enzymatic activity, which parallel protein levels3. In humans and rodents, COMT plays a crucial role in the catabolism of cortical dopamine (DA) but not cortical norepinephrine nor striatal DA4, 5.COMT enzyme activity has undergone progressive evolutionary attenuation across several animal species1, maybe due to evolutionary pressure towards higher cortical function. In fact genetic variations in human COMT has been associated with prefrontal cortex (PFC) physiological functions6, 7, and behavioral phenotypes related to PFC and hippocampal information processing, including cognition5, 8-10. However, the data remain controversial11, 12 probably due to the complexity of human behavior and/or to complex set of genetic variants within the human COMT gene13.

AIM OF THE STUDY

Using genetically engineered mice lacking functional COMT and transgenic mice overexpressing the human COMT Val variant in a neuron-specific manner (COMT Val-tg), we studied the impact of a life-long decrease and an increase in COMT enzyme activity on cognitive processes to elucidate the role of COMT and the neurobiological basis of the behavioral associations in humans. Genetically altered mice provide a level of molecular specificity that is not possible in human association studies, where genetic background is uncontrollable.

SUMMARY OF RESULTS

1. COMT Val-tg mice showed impaired recognition memory. Cognitive performance in this task involves PFC and perirhinal cortex regions19, 20.2. Amphetamine restored recognition memory performance of COMT Val-tg mice, but deteriorated it in control mice (suggesting an inverted U-shaped relationship between cognitive performance and DA levels, and its modulation by COMT).3. Mice were able to acquire the attentional set shifting test in only 3-7 days. COMT Val-tg mice displayed a selective impairment of the EDS, but no alteration in acquisition or reversal learning.4. COMT-/- mice acquired a clinically relevant discrete paired-trial alternation T-maze task faster than +/+ and +/- mice. In contrast, COMT Val-tg mice required more days than their control mice to acquire it.5. Despite the crucial role of the COMT genotype on working memory processes, COMT Val-tg mice showed normal performance in a continuous delayed alternation T-maze task. This task involve a regularly repeated sequence of events rather than trial-specific experience, and shown not to depend on working memory16, 17.

IMPLICATIONS OF THE STUDY

• These results indicate a causal link between functional polymorphisms in the COMT gene and human cognition and establish the importance of COMT in diverse aspects of cortical information processing.• Extend to the mice the evidence that an inverted U-shaped relationship exists between DA levels and performance on PFC-dependent cognitive tasks.• Our results highlight the COMT gene as a critical hot spot in the regulation of executive and working memory processes, which are critically dependent on DA pathways in the PFC and not on general cognitive abilities and reference-memory processes.• These findings may be relevant to the development of new therapeutic strategies for cognitive deficits associated with several psychiatric illnesses.

Materials and Methods

Subjects.All procedures were approved by the National Institute of Mental Health Animal Care and Use Committee and following the NIH Guidelines “Using Animals in Intramural Research.” COMT Val-tg mice were mated with control littermates. COMT knockout mice were bred by heterozygote mating. Testing was conducted in male mice, 3-7 months old, during the light phase. Experimenters were blind to the genotype during behavioral testing.

Two different groups of naïve control and COMT Val-tg mice were tested in a new object recognition task and a continuous delayed alternation T-maze task as described previously16, 17, 24, 25.

Discrete paired-trial variable-delay T-maze task.A new cohort of naïve control and COMT Val-tg mice and a group of naïve COMT+/+, +/-, and -/- mice were tested in a discrete paired-trial variable-delay T-maze task, as previously described18, 24. In this task, animals were presented with a sequence of randomly chosen forced runs, each followed by a choice run so that they were required to integrate information held online (the forced run) with the learned rule (non-match to sample).

REFERENCES1)Chen, J., et al. Am J Hum Genet 75, 807-821 (2004). 2)Lachman, H.M., et al. Pharmacogenetics 6, 243-250 (1996). 3)Nackley, A.G., et al. Science 314, 1930-1933 (2006). 4)Gogos, J.A., et al. Proc Natl Acad Sci U S A 95, 9991-9996 (1998). 5)Tunbridge, E.M., Bannerman, D.M., Sharp, T. & Harrison, P.J. J Neurosci 24, 5331-5335 (2004). 6)Egan, M.F., et al. Proc Natl Acad Sci U S A 98, 6917-6922 (2001). 7)Winterer, G., et al. Biol Psychiatry 60, 578-584 (2006). 8)Bertolino, A., et al. Am J Psychiatry 161, 1798-1805 (2004). 9)Blasi, G., et al. J Neurosci 25, 5038-5045 (2005). 10)Goldberg, T.E., et al. Arch Gen Psychiatry 60, 889-896 (2003). 11)Ho, B.C., Wassink, T.H., O'Leary, D.S., Sheffield, V.C. & Andreasen, N.C. Mol Psychiatry 10, 229, 287-298 (2005). 12)McGrath, M., et al. Am J Psychiatry 161, 1703-1705 (2004). 13)Tunbridge, E.M., Harrison, P.J. & Weinberger, D.R. Biol Psychiatry 60, 141-151 (2006). 14)Birrell, J.M. & Brown, V.J. J Neurosci 20, 4320-4324 (2000). 15)Garner, J.P., Thogerson, C.M., Wurbel, H., Murray, J.D. & Mench, J.A. Behav Brain Res 173, 53-61 (2006). 16)Brito, L.S., Yamasaki, E.N., Paumgartten, F.J. & Brito, G.N. Braz J Med Biol Res 20, 125-135 (1987). 17)Green, R.J. & Stanton, M.E. Behav Neurosci 103, 98-105 (1989). 18)Kellendonk, C., et al. Neuron 49, 603-615 (2006). 19)Morrow, B.A., Roth, R.H. & Elsworth, J.D. Brain Res Bull 52, 519-523 (2000). 20)Mumby, D.G. Behav Brain Res 127, 159-181 (2001). 21)Robbins, T.W. Philos Trans R Soc Lond B Biol Sci 362, 917-932 (2007). 22)Vijayraghavan, S., Wang, M., Birnbaum, S.G., Williams, G.V. & Arnsten, A.F. Nat Neurosci 10, 376-384 (2007). 23)Owen, A.M., et al. Brain 116 ( Pt 5), 1159-1175 (1993). 24)Lipska, B.K., Aultman, J.M., Verma, A., Weinberger, D.R. & Moghaddam, B. Neuropsychopharmacology 27, 47-54 (2002). 25)Nagai, T., et al. Learn Mem 14, 117-125 (2007).

ACKNOWLEDGMENTSWe thank Payal Patnaik and Carla Bes for technical assistance with behavioral tests and Guangping Liu for mice genotyping. We thank Drs. Maria Karayiorgou and Joseph A. Gogos (The Rockefeller University, New York, NY) for generously donating the COMT knockout mice breeders. This research was supported by the Intramural Program of the NIH, NIMH.

Francesco Papaleo1*, Jacqueline N. Crawley2, Daniel R. Weinberger1, Jingshan Chen1

741.20/ZZ15

GENETIC MANIPULATION OF CATECHOL-O-METHYLTRANSFERASE (COMT) IN MICE AFFECTS SPECIFIC COGNITIVE PROCESSES

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1Clinical Brain Disorders Branch, National Institute of Mental Health, NIH, 10 Center Drive, Bldg. 10, Bethesda, MD, USA. 2Laboratory of Behavioral Neuroscience, National Institute of Mental Health, Bethesda, MD USA; *papaleof@mail.nih.gov

Attentional Set Shifting Test.Metal bowls were used to hold the digging medium. The outer surfaces of the bowls were covered with a texture, and the bowls were filled with a digging medium, which could be scented. Thus, the bowls could be varied by their odor, the texture of their outer surface, or the digging medium in which the food bait was hidden. The bait was a 14 mg food pellet (5TUL, dustfree purified rodent tablets, TestDiet, Richmond, IN). After a week of singled housing, mice were food restricted through the experiment to maintain 85% of their ad libitum body weight. Two days of habituation followed. During the first day, animals were placed in the apparatus and exposed for 15 min to two baited bowls that did not contain a medium. Then, for 30 min, the bowls were filled with sawdust and baited again. On the second day of habituation, the mice were exposed for 30 min to two sawdust filled bowls that were rebaited every 2min. When the mouse was reliably digging to retrieve the rewards, it was trained on a series of two simple discriminations (SDs) between digging media to a criterion of 8 out of 10 consecutive trials. These exemplar scents were not used again. On the 3rd day the testing paradigm started. Each trial was initiated by raising the two dividers to give the mouse access to the two digging bowls, only one of which was baited. We took care to raise the doors simultaneously and only when the mouse was not sniffing at or facing a door. The first four trials were discovery trials: the mouse was permitted to dig in both of the bowls, but only one was baited. An error was recorded if the mouse dug first in the unbaited bowl. On subsequent trials, if the mouse started to dig in the unbaited bowl, an error was recorded, and the trial was terminated. The mice had to reach a criterion of 8 correct choices out of 10 consecutive trials in order to complete each stage. The time to finish each stage was also recorded. If a mouse made 3 consecutive incomplete trials (no dig after 2 min) the session was terminated and the test was continued the next day. The sequence of stages to complete comprised a SD, compound discrimination (CD), compound discrimination reversal (CDRe), intradimensional shift (IDS), intradimensional shift reversal (IDSRe), intradimensional shift 2 (IDS2), intradimensional shift reversal 2 (IDSRe2), extradimensional shift (EDS), and extradimensional reversal (EDSRe). The mice were exposed to the tasks in this order so that they could develop a set, or bias, towards discriminating between the baited bowls. In the SD, the mice were introduced to a dimension that was relevant throughout the tasks until the EDS, in which the mouse had to find the bait following a new stimulus dimension and the previously relevant dimension became now irrelevant. The order of the discriminations was always the same, but the dimensions and the pairs of exemplars were equally represented within groups and counterbalanced between groups.

Statistical analysis.Results are expressed as mean ± standard error mean (S.E.M.) throughout. Student’s t test was used to compare COMT Val-tg versus control littermates on the days required to reach the criteria in the T-maze, the time spent exploring the two copies of the same object during the acquisition session of the object recognition task. Two-tailed Fisher exact analyses were used to compare genotypes for the number of mice reaching the learning criteria of the T-maze tasks. Two-Way analysis of variance (ANOVA) with genotype (control or COMT Val-tg) as between subjects factors, and within-session 5-min intervals as a repeated measure within-subject factor was used to analyze the total distance performed in the empty open field arena during the object recognition test. Two-Way ANOVA with genotype and treatment (vehicle or amphetamine) as independent variables were used to examine the exploration time during the acquisition session, and the new object exploration during the retention session, of the new object recognition task. In the attentional set shifting test, a Two-Way ANOVA with genotype (control versus COMT Val-tg) as a between subjects factor and the different stages (SD, CD, CDRe, IDS, IDSRe, IDS2, IDRe2, EDS, and EDRe) as a within-subject factor were used to examine the number of trials to reach the criteria and timing needed to complete each stage of this task. Comparison of the COMT +/+, +/-, and -/- employed One-Way ANOVA to examine the days needed to reach the criteria in the T-maze task. A Two-Way ANOVA with genotype (+/+, +/-, and -/-) and different intra-trial delay (4, 30, 60, or 240 sec) as independent variables was used to examine the percentage of correct choices made during the T-maze. Post-hoc analyses for individual group comparisons employed Newman-Keuls analyses. The accepted value for significance was P<0.05.

We tested COMT Val-tg mice in an attentional set shifting “stuck-in-set” paradigm, adapted and improved from previous studies14, 15 and modeled after the human WCST and the IEDS subtest of the CANTAB. Each mouse was trained to perform a series of discriminations, including reversals, two intradimensional shifts, and an extradimensional shift. The analysis of the number of trials required to reach the criteria revealed a significant genotype x discrimination interaction effect (F8,104=3.07; P<0.005), attributable entirely to impaired performance in COMT Val-tg mice on the extradimensional shift (**P<0.0005). Moreover, COMT Val-tg mice required more time to solve the EDS stage compared to all the other discriminations and to the time required by controls (**P<0.0005).

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During the retention trial, in contrast to their control littermates, vehicle-treated COMT Val-tg mice failed to spend more time exploring the new object (*P<0.05 vs vehicle-treated control mice), indicating impaired recognition memory. Whereas amphetamine treatment tended to reduce new object exploration in control mice (P=0.07), amphetamine increased it in COMT Val-tg (#P<0.05 vs vehicle-treated COMT Val-tg).

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COMT Val-tg mice required more days to acquire the PFC-dependent discrete paired-trial T-maze task (t=-2.33, df=14, *P<0.05 vs control), indicating impaired acquisition of this working memory task. An equal number of COMT Val-tg and control mice acquired this task (P=0.91).

COMT Val-tg and control mice required the same number of days to reach criterion in a continuous delayed alternation T-maze task (t=0.17, df=21, P=0.86). An equal number of COMT Val-tg and control mice acquired this task (P=0.73). Thus, COMT Val-tg mice manifest specific deficits in working memory functions that are not due to alterations in reference memory, spatial learning, motivation, food-reward associations, or non-specific cognitive deficit.

COMT-/- acquired this task faster than +/- and +/+ (*P<0.05 vs +/+). An equal number of COMT-/-, +/- and +/+ reached the criteria (P=0.93;). Thus, disruption of the COMT gene resulted in improved working memory, opposite to the impaired working memory seen in the COMT Val-tg mice, and in mice with lesions of the mPFC18.

Mice were further tested in the discrete paired-trial T-maze paradigm but with four different intra-trial delays, and decreasing the inter-trial delay to 20 sec. All groups displayed delay-dependent performance, but the COMT+/- mice showed a consistent improvement of their performance compared to +/+ and -/- (*P<0.05).