Diapositive 1

1
GENETIC MANIPULATION OF CATECHOL-O-METHYLTRANSFERA
SE (COMT) IN MICE AFFECTS SPECIFIC COGNITIVE
PROCESSES
GENETIC MANIPULATION OF CATECHOL-O-METHYLTRANSFERA
SE (COMT) IN MICE AFFECTS SPECIFIC COGNITIVE
PROCESSES
Francesco Papaleo1, Jacqueline N. Crawley2,
Daniel R. Weinberger1, Jingshan Chen1
741.20/ZZ15
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_at_mail.nih.gov
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.
Materials and Methods

Stage Correct Discrimination 1 Discrimination 1 Discrimination 1 Discrimination 2 Discrimination 2 Discrimination 2
SD smooth plastic smooth plastic vs bubblewrap
CD smooth plastic smooth plastic cumin vs bubblewrap ginger smooth plastic ginger vs bubblewrap cumin
CDRe bubblewrap  smooth plastic cumin vs bubblewrap ginger smooth plastic ginger vs bubblewrap cumin
IDS coarse sandpaper  fine sandpaper corriander vs   coarse sandpaper sage fine sandpaper sage vs coarse sandpaper corriander
IDRe  fine sandpaper fine sandpaper corriander vs coarse sandpaper sage fine sandpaper sage vs coarse sandpaper corriander
IDS2 smooth cardboard smooth cardboard pepper vs ridged cardboard cinnamon smooth cardboard cinnamon vs ridged cardboard pepper
IDSRe2 ridged cardboard smooth cardboard pepper vs ridged cardboard cinnamon smooth cardboard cinnamon vs ridged cardboard pepper
EDS Moss Moss aluminum foil vs Kay-kob pink cloth Moss pink cloth vs Kay-kob aluminum foil
EDSRe Kay-kob Moss aluminum foil vs Kay-kob pink cloth Moss pink cloth vs Kay-kob aluminum foil
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.
Dimension Exemplar pairs Exemplar pairs Exemplar pairs Exemplar pairs Exemplar pairs
Outer texture A) Smooth plastic B) Bubblewrap A) Smooth cardboard B) Ridged cardboard A) Alluminium foil B) Pink cloth A) Coarse sandpaper B) Fine sandpaper A) Regular paper B) Waxed paper
Digging medium 1) Aspen bedding 2) Aquarium gravel 1) Moss 2) Kay-kob 1) Repti bark 2) Alpha-dri 1) Care fresh 2) Ultra white care fresh 1) Crystalit 2) PaperChip
Odor ) Sage ?) Coriander ) Pepper ?)Cinnamon ) Cumin ?) Ginger ) Thyme ?) Oregano ) Nutmeg ?) Cloves

• 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.

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).
Statistical analysis. Results are expressed as
mean standard error mean (S.E.M.) throughout.
Students 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 Plt0.05.
ACKNOWLEDGMENTS We 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.
REFERENCES 1)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).