SUPPLEMENTARY MATERIAL FOR
Fractionation of Spatial Memory in GRM2/3 (mGlu2/mGlu3) Double Knockout
Mice Reveals a Role for Group II mGluRs at the Interface between Arousal and
Cognition
Louisa Lyon, Philip WJ Burnet, James NC Kew, Corrado Corti, J. Nicholas P Rawlins,
Tracy Lane, Bianca De Filippis, Paul J Harrison, David M Bannerman
SUPPLEMENTARY MATERIAL
Subjects
Age-matched (> 2.5 months old), male, wild-type and GRM2/3-/- mice were
obtained from GlaxoSmithKline, Harlow, UK. GRM2-/- mice (Yokoi et al, 1996) were
crossed with GRM3-/- mice (Corti et al, 2007a) to generate GRM2+/-GRM3+/- double
heterozygous offspring. GRM2-/- mice had been backcrossed onto the C57Bl/6 line for 21
generations, and GRM3-/- mice backcrossed onto C57Bl/6 for 11 generations. The double
heterozygous animals were thus considered to be N11. Double heterozygous mice were
then crossed to generate 1:16 GRM2-/-GRM3-/- double knockout (‘GRM2/3-/-‘ mice, 1:16
GRM2+/+GRM3+/+ wild-type (wt) mice, and 14:16 mice that were heterozygous for
GRM2 and/ or GRM3. To avoid the prohibitive wastage of animals that would occur if
all double knockout and wild-type mice were derived from double heterozygous crosses,
separate lines of true breeding double knockout and wild-type mice were established.
Separate wild-type and knockout lines were bred for up to 6 generations. The knockout
line was then out-crossed onto C57Bl/6 in order to generate mice that were heterozygous
at both loci and these mice were then inter-crossed to produce new wild-type and double
knockout lines, and thus refresh the colony. The animals used in these behavioural
experiments were obtained from between the F2 - F6 generations of these separate lines
(for details, see below).
Animals were housed in groups of 2-4 and kept on a 12-hour light-dark cycle
(lights on at 07:00 and off at 19:00), with all testing conducted during the light phase. For
all appetitively motivated tasks, mice were maintained on a restricted feeding schedule at
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not less than 90% of their free-feeding weight. For several days prior to the start of each
appetitive test, mice were habituated to the maze and to drinking the sweetened
condensed milk (Carnation) (diluted 50:50 with water) that was used as a reward.
Habituation was conducted in a room other than the experimental test room. For all
aversively motivated tasks, mice were given ad libitum access to food and water.
Order of testing
Four separate cohorts of experimentally naïve mice were used. The first cohort
(N15;F2 generation) completed the 2 hr test of spontaneous locomotor activity, followed
by experiments 1-8. The order of testing for the cognitive tasks was as follows: appetitive
spatial reference memory (SRM) Y-maze (Expt 1), appetitive spatial working memory
(SWM) T-maze (Expt 2), appetitive visual discrimination (Expt 6), watermaze (Expt 4),
appetitive six-arm radial maze (Expt 3), swimming Y-maze (Expt 5), spontaneous spatial
novelty preference (Expt 7). The second cohort (N15;F5 generation) was used in
Experiment 5, to replicate the SRM swimming Y-maze and appetitive Y-maze with the
order of testing and the room in which testing was performed counterbalanced with
respect to cohort 1. Cohort 2 also completed experiment 9 (effects of injection stress on
appetitive T-maze SWM performance) and experiment 8 (amphetamine challenge – low
dose, 2.5 mg/kg). The third cohort (N16;F2 generation) was used to study the effects of a
high dose of amphetamine (10 mg/kg) on locomotor activity. Cohort 4 (N16;F6
generation) was used for the assessment of locomotor activity during the diurnal cycle
over 70 hr (Expt 8), followed by the replication of the T-maze spatial working memory
task with recording of latencies for sample and choice runs (Expt 2). All experiments
were conducted under the auspices of the UK Home Office project and personal licences
held by the authors.
Supplementary Methods
Genotyping
The absence of GRM2 mRNA in founding members of the GRM2-/- population
was confirmed by the use of in situ hybridisation histochemistry (Yokoi et al., 1996), as
was the absence of GRM3 mRNA in the founding members of the GRM3-/- population
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(Corti et al., 2007a). Genotypes of all animals were confirmed by the “Mouse genotyping
group” at GSK, Harlow, using separate PCRs for GRM2 and GRM3 mRNA.
GRM2 fragments were amplified using forward oligonucleotide primers (CTG
TCT CTC TAT CTC TCT GC) and reverse primers (TGT GTG TGT GTA ACA TGA
TGG). PCRs were performed with a denaturing step at 95 °C (15 mins) then 94 °C (30 s),
followed by annealing at 60 °C (90 s) and extension at 72 °C (1 min). After 35 cycles, the
reaction was maintained at 72 °C for a further 10 mins. PCR product was then resolved
onto a 2% agarose gel. The wildtype product was a single 900 bp band, and the ko
product a 450 bp band.
GRM3 genotyping yielded a wildtype product that was 2 kbp long, and a
knockout product that was 500 bp. The large disparity in size prevented the two
fragments from being amplified in a single multiplex PCR. Two separate PCRs were
therefore conducted, one for the wildtype product (forward primer: GTT TCT AGG ACT
TCC TAT GG; reverse primer: AAC GAT GCT CTG ACA AAC TCC) and a second for
the knockout product (forward primer: CGT ACG TCG GTT GCT ATG G; reverse
primer: GTC AGA TAT AGT GAG AGC AGG). Both PCRs were performed with a
denaturing step at 95 °C (15 mins) then 94 °C (30 s), followed by annealing at 56 °C (90
s) and extension at 72 °C (150 s). After 35 cycles, the reaction was maintained at 72 °C
for 10 mins. PCR product was resolved onto a 2% agarose gel.
Spatial memory tests
Hippocampus-dependent, spatial learning was assessed using a battery of
appetitively and aversively motivated spatial reference memory (SRM) and spatial
working memory (SWM) tasks. SRM tasks are those in which the correct spatial response
remains constant from trial to trial. SWM tasks, by contrast, are characterised by their
flexible stimulus response requirements with different spatial responses being variably
correct and incorrect.
Experiment 1: SRM on the elevated Y-maze
The elevated Y-maze consisted of three identical wooden arms, each 50 cm long
by 9 cm wide, with a low wall (0.5 cm), connected by a central polygonal platform (14
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cm diameter). A food well was positioned at the end of each arm. Each mouse was
assigned a goal arm, defined by its position relative to extramaze spatial cues, which was
baited with 0.1 ml sweetened condensed milk on all trials. On each trial, the mouse was
placed at the end of one of the two non-baited arms (the “start arm”), facing the
experimenter; 50% of trials began from the arm to the right of the goal arm, and 50%
from the arm to the left. Neither arm was used as the start arm for more than three
consecutive trials. Allocation of start and goal arms was counterbalanced across groups.
Having been placed at the end of the start arm, the mouse was allowed to choose one of
the remaining arms. If it chose the goal arm, it was allowed to consume the milk reward
before being returned to the home cage. Mice that chose incorrectly were returned to the
home cage immediately. Previous work in this laboratory, using the same maze in the
same room with the same spatial cues, has demonstrated that this task is hippocampusdependent (Deacon et al., 2002). To prevent the use of intra-maze cues, the entire maze
was rotated periodically (approximately every 5 trials). Mice received ten trials per day
for nine days, with an inter-trial interval (ITI) of approximately five minutes. The last
block of ten trials was conducted using post-choice reinforcement: the condensed milk
reward was added to the food well only after the mouse had made a choice, to ensure that
the animals were not locating the milk by virtue of its odor.
Experiment 2: SWM on the elevated T-maze
The T-maze consisted of a wooden start arm (47 x 10 cm) and two identical goal
arms (35 x 10 cm), surrounded by a 10 cm high wall. A food well was positioned 3 cm
from the end of each goal arm, and the whole maze was surrounded by prominent distal
extramaze cues. Mice received five trials per day for ten days, with an ITI of
approximately ten minutes. Each trial consisted of a sample run followed by a choice run.
On the sample run, mice were forced either left or right (chosen pseudorandomly with
equal numbers of left and right turns, and no more than three consecutive turns in any
direction) by the presence of a large wooden block, closing off one of the goal arms. At
the end of the goal arm the mouse collected a reward of 0.1 ml sweetened condensed
milk. The block was then removed and the mouse placed back in the start arm, facing the
experimenter, for the choice run. The mouse could now select either goal arm but was
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rewarded only for choosing the arm that had not been visited on the sample run, i.e., it
was rewarded for alternating (non-matching to place). The interval between the sample
run and the choice run was approximately 5 seconds. SWM performance on the T-maze
is also dependent on the hippocampus (Deacon et al., 2002).
In a separate cohort of mice we replicated this study but, in addition, we recorded
latencies for both the sample runs and the choice runs. We recorded the latency of the
mice to run (i) from the beginning of the start arm to the food well on the sample trial,
and (ii) from the beginning of the start arm until making a choice into one of the goal
arms on the choice trial. Mice received five trials per day for twelve days, with an ITI of
approximately ten minutes.
Experiment 3: SRM and SWM in the six-arm radial maze
The radial maze consisted of six arms (60 cm x 7 cm) radiating out from a central
platform. Each arm was surrounded by a 1 cm raised edge and contained a food well
located at the end. The central platform was surrounded by a transparent Perspex cylinder
(18 cm diameter, 30 cm high). At the entrance to each arm was a Perspex door (6 cm
wide, 7 cm high), which could be controlled manually by the experimenter using a series
of strings. The maze was positioned 80 cm above the floor, and was surrounded by
prominent distal extramaze cues. The radial arm maze can be used to assess SRM and
SWM separately using a two-stage, within-subjects and within-task design (Schmitt et al.,
2003).
SRM acquisition: in the initial acquisition phase, three of the six arms were baited
with 0.1 ml sweetened condensed milk. The three baited arms were allocated such that
two were adjacent, and the third was between two unbaited arms. Combinations of baited
arms were counterbalanced across all mice with respect to genotype. At the start of each
trial, the mouse was placed on the central platform and given a free choice of arms. After
visiting the food well in the chosen arm and consuming the milk reward/ discovering that
the arm was not baited, the mouse would return to the central platform. The door to the
visited arm was then closed and remained so for all subsequent choices on that trial,
preventing the mouse from re-entering that arm (see Schmitt et al., 2003). After 10
seconds, all other doors were opened and the mouse allowed a second choice. This was
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repeated, with five doors open for the second choice, four for the third, three for the
fourth, and so on, until the mouse had visited all three baited arms. Each entry into an
unbaited arm was scored as a SRM error (maximum number of errors = 3 per trial). SRM
acquisition on the radial maze is prevented by cytotoxic hippocampal lesions in mice
(Schmitt et al., 2003).
Simultaneous assessment of SRM and SWM: When all mice had successfully
acquired the SRM task, the SWM component was introduced. Entries into previously
visited arms were now no longer prevented but the food rewards were not replaced within
a trial. The doors were now used only to retain the mouse on the central platform for 10
seconds in between choices. Three types of error were scored (Jarrard, 1993), including
spatial reference memory (SRM) errors in which the mouse visited a never-baited arm,
and spatial working memory (SWM) errors in which the mouse visited a baited arm that
had already been visited on that trial. If second and subsequent visits to a never-baited
arm occurred, these were scored separately as spatial reference memory repeat (SRM-R)
errors, because these might be regarded as involving failure in both SRM and SWM. In
practice, there were very few SRM-R errors made by either group.
Experiment 4: SRM in the watermaze
Mice received no swim pretraining prior to this task. Testing was conducted in a
large circular tank (diameter 2.0 m, depth 0.6 m) containing water at 20 + 1°C to a depth
of 0.3 m. To escape from the water, mice had to locate a platform (diameter 21 cm,
covered in wire mesh) hidden approximately 1 cm below the surface. The water was
made opaque by the addition of 2 litres of whole milk to prevent the mice from seeing the
platform. The pool was surrounded by prominent distal extramaze cues that could be used
as landmarks (shelves, table, posters on walls, etc). Swim paths were tracked by a video
camera mounted in the ceiling, and relayed to a computer for image analysis using
specialised software (HVS Image Analyse, Hampton, UK; Archimedes hardware). Mice
were assigned to one of four platform positions (NE, NW, SE, SW), with the platform
located in the centre of the quadrant. Platform assignation was counterbalanced according
to genotype. For each individual mouse, however, the platform remained in the same
position across all trials. Each animal was given four trials per day (1 block) for nine
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days. Mice were placed into the pool facing the sidewall at one of eight starting positions
(N, S, E, W, NW, NE, SW, SE) that varied pseudorandomly across trials. On each trial,
the mouse was allowed to swim in the pool until it found the platform, or for a maximum
of 90 seconds. Mice that failed to find the platform after this time were placed there by
the experimenter. All mice remained on the platform for 30 seconds before beginning the
next trial. On day 7 (24 hours after training trial 24) and day 11 (24 hours after training
trial 36), transfer (probe) tests were conducted. The platform was removed from the pool
and the mouse allowed to swim freely for 60 seconds. The percentage of time spent in
each quadrant was recorded, together with the number of times the mouse swam across
the former location of the platform. We have previously shown using this watermaze, in
this laboratory, with the same spatial cues, that acquisition of this fixed location, hidden
platform task is prevented by cytotoxic hippocampal lesions (Deacon et al., 2002).
Experiment 5: Aversively motivated spatial reference memory in the Y-maze
The Y-maze was made from transparent Perspex, and consisted of three 30 cm
long, 8 cm wide arms with 20 cm high walls, connected by a central junction. The maze
was filled with water (temperature 21 ºC ± 1 ºC) to a depth of approximately 12 cm
which obliged the mice to swim. Mice could escape from the water by climbing onto a
platform (8 cm by 8 cm) hidden approximately 1.5 cm below the water surface. Milk was
added to the water to prevent the mice from seeing the platform. Mice received five trials
per day in this deep water escape Y-maze for six days. On each trial the mouse was
allowed 90 seconds to find the platform; any that failed to do so were guided there by the
experimenter. Mice were allowed to rest on the platform for 30 seconds before being
transferred to a heated cage. On day seven (24 hours after training trial 30), a transfer test
was performed, analogous to that used in the watermaze, in order to assess the extent of
any spatial memory for the platform location. The platform was removed from the maze
and the mouse allowed to swim freely for 30 seconds. Time spent searching in each arm
was recorded.
Experiment 6: appetitively motivated visual discrimination
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This experiment compared visual discrimination learning for a food reward. The
T-shaped maze consisted of three arms (30 x 10 x 29 cm): a light gray start arm plus two
removable goal arms. The walls and floor of one of the goal arms were painted with
black and white stripes, while those of the other goal arm were plain dark gray. Food
wells were positioned 3 cm from the end of each goal arm. Mice received ten trials per
day for five days. Each mouse was assigned a particular goal arm (“black/ white stripes”
or “gray”), which was baited with 0.1 ml sweetened condensed milk on all trials.
Allocation of goal arms was counterbalanced with respect to genotype. On 50% of trials,
the rewarded goal arm was positioned to the right of the start arm, and on the remaining
50% it was positioned to the left. The rewarded goal arm did not occupy the same
physical location for more than three consecutive trials. On each trial, the mouse was
placed at the end of the start arm facing the experimenter and allowed to choose one of
the goal arms. If the mouse chose the correct arm (defined by its visual appearance), it
was allowed to consume the milk reward before being returned to the home cage. Mice
that chose incorrectly were returned to the home cage immediately without reward. Postchoice reinforcement was used in block 5.
Experiment 7: spontaneous spatial novelty preference task
GRM2/3-/- and wild-type mice were also compared on a spontaneous, spatial
novelty preference task in which behavior is driven, not by an overt unconditioned
stimulus (US; e.g. a food reward), but instead relies upon animals’ natural exploratory
drive. This task therefore provides a non-aversive experimental context but performance
does not rely on the motivating or rewarding effects of food.
The apparatus used was identical to the paddling and swimming Y-mazes, but
without the water. Instead, a thin layer of sawdust covered the floor of the maze. Each
mouse was assigned two arms (the “start arm” and the “other arm”) to which they were
exposed during the first phase of the task (the “exposure phase”). Allocation of arms to
specific spatial locations was counterbalanced within each genotype. During the 5-minute
“exposure” phase, the entrance to the third, “novel”, arm was closed off by the presence
of a large Perspex block. The mouse was placed at the end of the start arm, facing the
experimenter, and allowed to explore the start arm and the other arm freely for five
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minutes, beginning as soon as the mouse left the start arm. The number of entries into
each arm and the length of time spent there were recorded. At the end of the five minutes,
the mouse was removed from the maze and returned to the home cage for one minute.
During this time, the Perspex block closing off the novel arm was removed and the
sawdust redistributed throughout the maze to minimise the use of odor cues. The mouse
was then returned immediately to the start arm, facing the experimenter, for the 2-minute
test phase. This consisted of two minutes free exploration during which the mouse could
enter all 3 arms, beginning as soon as the mouse left the start arm.
The amount of time that the mouse spent in each arm, and the number of entries
into each arm, were recorded, during both the exposure and the test phase. For the test
phase, a discrimination ratio [(novel arm/ (novel + other arm)] was calculated both for
number of arm entries and time spent in each arm. Previous work in this laboratory has
demonstrated that wild-type mice display a marked preference for the novel arm during
the test phase, and that this preference relies on extramaze cues, whereas preference for
the novel arm is abolished in mice with cytotoxic hippocampal lesions (Sanderson et al.,
2007).
Experiment 8: spontaneous and amphetamine-induced locomotor activity
Spontaneous locomotor activity was measured during a two-hour period in the
light phase (12pm – 2pm). All mice were placed singly into a transparent plastic cage (26
cm x 16 cm x 17 cm) with a ventilated lid. Two infrared photocell beams crossed the
cage 1.5 cm above the floor, with each beam 7 cm from the centre of the cage. Mice were
left in a quiet room with the lights on for 2 hours. The number of beam breaks made by
each mouse was recorded in 24 bins of 5 minutes.
A separate cohort of mice were then tested in a threshold activity monitoring
system for spontaneous locomotor activity over a 70 hr period (1 hr bins). Threshold
pressure pads were used to measure the activity of mice across the diurnal cycle. The
Threshold system (Version 3, Med Associates) converts changes in pressure on pads into
changes in voltage. Dedicated software records these changes in voltage and allows
threshold values to be set to give sensitive readings of locomotor activity. Mice were
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placed in cages of 22.5x12.5x13cm at approximately 3 p.m. (4 hours before lights off),
and left with ad libitum food and water (Supplementary Material, Figure S2).
A further, separate cohort of drug-naïve mice, with no previous experience of the
photocell activity boxes, was used for the amphetamine challenge experiments. Mice
were habituated to the activity boxes for 20 minutes prior to receiving an i.p. injection of
either saline or 2.5 mg/kg amphetamine (injection volume 10 ml/kg body weight). A latin
square within-subjects design was used, in which half of the animals received an injection
of amphetamine followed 3 days later by an injection of saline, and vice versa for the
other half. Mice were left undisturbed and their activity levels (number of beam breaks)
measured for 2 hours.
Higher doses of amphetamine are known to induce stereotypy, which reduces
locomotor activity. An additional cohort of drug-naïve mice was habituated to the activity
boxes for 20 minutes prior to receiving an i.p. injection of either saline or 10 mg/kg
amphetamine (10 ml/kg body weight), using a between-subjects design. Mice were
returned immediately to the activity boxes and their activity levels measured for 7 hours.
Every 10 minutes throughout the first 4 hours, stereotypy was scored for each animal by
an experimenter, using an established rating scale (Creese & Iversen, 1974)
(Supplementary Material, Figure S3).
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Supplementary Figure Legends
Fig. S1. GRM2/3-/- mice did not differ from wild-type mice in the watermaze SRM task.
A, Mean number of annulus crossings (i.e., swims across the former location of the
platform, or the equivalent location in the other quadrants) (± sem) in probe test 1 (day
7). B, Mean number of annulus crossings (± sem) in probe test 2 (day 11).
Fig. S2. GRM2/3-/- mice are hypoactive across an extended testing period. Mean
locomotor activity (arbitrary units) in wild-type and GRM2/3-/- mice across a 70 hr test
session. Error bars are not shown for clarity. There was a significant group by time block
interaction (F(69, 2553) = 2.09; p < 0.0001), and analysis of simple main effects revealed
that
the
activity
levels
of
the
groups
differed
significantly
in
blocks
2,3,4,9,11,12,32,33,34,36,38,56,58 and 62 (all p < 0.05). In all but block 36 the wildtypes were more active than the GRM2/3-/- mice.
Fig. S3. 10 mg/kg amphetamine induced stereotypy in both wt (n = 5) and GRM2/3 dko
(n = 6) mice, relative to saline-injected controls (n = 6 wt; n = 6 dko). Stereotypy began
to decrease in amphetamine-treated GRM2/3 dko mice approximately 140 minutes postinjection, and in wt mice approximately 190 minutes post-injection. Data shown are
median stereotypy score as rated by an experimenter using an established scale
(maximum = 5) (± IQR).
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Supplementary References
Corti C, Battaglia G, Molinaro G, Riozzi B, Pittaluga A, Corsi M, et al (2007a). The use
of knock-out mice unravels distinct roles for mGlu2 and mGlu3 metabotropic glutamate
receptors in mechanisms of neurodegeneration/neuroprotection. J Neurosci 27: 82978308.
Yokoi M, Kobayashi K, Manabe T, Takahashi T, Sakaguchi I, Katsuura G, et al (1996).
Impairment of hippocampal mossy fiber LTD in mice lacking mGluR2. Science 273:
645-647.
Other references cited in Supplementary Materials can be found in the main reference
list.
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