working memory is one of memory types , and it allows transitory information to be actively held for several seconds on line .
several brain regions , such as medial prefrontal cortex , and hippocampus , are known to play critical roles in holding transitory memory .
testing working memory in the mouse has typically used the delayed non - matched to position ( dnmtp ) paradigm .
for example , during a working memory test in a t - maze , intervention by the experimenter is inevitable because the maze needs to be cleaned and the animal must be relocated to the start box after sampling .
therefore , there is a need to test working memory in operant - conditioning - based paradigm in which all experimental procedures will be scheduled automatically .
this touch screen paradigm has several advantages over traditional behavioral testing paradigms ( e.g. , morris water maze [ mwm ] , t - maze , and radial - arm maze ) .
first , testing can be scheduled almost automatically , permitting more experimental sessions to be conducted with high accuracy and flexibility .
second , the method greatly reduces the need for experimenter intervention during memory test trials , because the experimental procedure is fully automated .
third , it can provide the animal with multiple choice locations on the screen , which enables the capability of assessing the animal 's ability to discriminate between choice locations , such as pattern separation .
fourth , various visual stimuli ( e.g. , from simple white rectangles to complex figures ) can be presented on the screen .
this enables testing of complex cognition , which was not able to test in the traditional behavioral tests .
these advantages would advance research on working memory , if touch - screen - based technology were to be applied .
unfortunately , a touch - screen system for the working memory test paradigm for mice is not available yet .
therefore , in the present study , we developed a delayed matched to position behavioral assessment paradigm using a touch - screen testing system for evaluating working memory in mice .
animals were housed in groups ( four mice ) , maintained on a 12-h light / dark cycle , and food and water were provided ad libitum .
all animal procedures were conducted in accordance with the guidelines of the institutional animal care and use committee of seoul national university .
we used campden instruments bussey - saksida touchscreen chamber ( campden instruments ltd , uk ) for touch - screen testing .
food restriction started at age of 10 weeks , and their weights were maintained to about 80% of initial weight through all behavioral tasks . before mice learned the rules of the cue - response paradigm and dmtp , they were trained to respond to visual stimuli on an lcd monitor to earn a reward .
all experiments were conducted as described in the manufacturer 's guide and previous studies . briefly , on day 1 , mice were habituated to a touch - screen chamber for 10 min . on day 2 , mice were given liquid sweetened milk as a reward .
liquid sweetened milk was made by diluting condensed milk ( seoulmilk , korea ) with the same volume of tap water . in this phase ,
the animal 's nose poking to the reward magazine located behind the chamber resulted in delivery of reward . on day 3 , in initial touch phase ,
both nose poking to reward magazine and response to visual stimulus on the lcd monitor delivered the reward ( fig .
next , on days 4 through 6 , in the must touch phase , only when mice responded to the visual stimulus reward was delivered ( fig .
after the must touch phase , mice were trained to avoid responding to a blank window ; incorrect punishment ( fig .
when the mice responded to a blank window , the room light was illuminated as a punishment signal .
sixty trials were conducted within 60 min until mice reached the criterion ( 70% correct response in consecutive 2 days ) .
after incorrect punishment , mice were trained to associate central cue and correct location choice for a reward .
after central cue diminished , white squares were presented both in left and right windows .
guided by the central cues , mice could choose either the left or right location to receive a reward .
when the animal made an incorrect choice , the room light was illuminated , and additional correction trials were given until the animal made a correct choice . in cue - response training , 60 trials within 90 min were given for 5 sessions .
three touches to the left- or right - positioned sample / cue stimulus on the screen by the mouse resulted in presentation of a visual stimulus in a central window .
when the animal responded to the central visual stimulus , visual test stimuli were presented both on the left and right side of the screen .
as in cue - response training , an incorrect choice resulted in room light illumination and additional correction trials .
after successful dmtp rule learning , delay time between final response to the sample location cue stimulus and presentation of the central visual test stimulus was increased to either 3 s or 9 s.
animals were housed in groups ( four mice ) , maintained on a 12-h light / dark cycle , and food and water were provided ad libitum .
all animal procedures were conducted in accordance with the guidelines of the institutional animal care and use committee of seoul national university .
we used campden instruments bussey - saksida touchscreen chamber ( campden instruments ltd , uk ) for touch - screen testing .
food restriction started at age of 10 weeks , and their weights were maintained to about 80% of initial weight through all behavioral tasks . before mice learned the rules of the cue - response paradigm and dmtp , they were trained to respond to visual stimuli on an lcd monitor to earn a reward .
briefly , on day 1 , mice were habituated to a touch - screen chamber for 10 min . on day 2 , mice were given liquid sweetened milk as a reward .
liquid sweetened milk was made by diluting condensed milk ( seoulmilk , korea ) with the same volume of tap water . in this phase ,
the animal 's nose poking to the reward magazine located behind the chamber resulted in delivery of reward . on day 3 , in initial touch phase , both nose poking to reward magazine and response to visual stimulus on the lcd monitor delivered the reward ( fig .
next , on days 4 through 6 , in the must touch phase , only when mice responded to the visual stimulus reward was delivered ( fig .
after the must touch phase , mice were trained to avoid responding to a blank window ; incorrect punishment ( fig .
to train this avoidance , when the mice responded to a blank window , the room light was illuminated as a punishment signal .
sixty trials were conducted within 60 min until mice reached the criterion ( 70% correct response in consecutive 2 days ) .
after incorrect punishment , mice were trained to associate central cue and correct location choice for a reward .
after central cue diminished , white squares were presented both in left and right windows .
guided by the central cues , mice could choose either the left or right location to receive a reward .
when the animal made an incorrect choice , the room light was illuminated , and additional correction trials were given until the animal made a correct choice . in cue - response training , 60 trials within 90 min were given for 5 sessions .
three touches to the left- or right - positioned sample / cue stimulus on the screen by the mouse resulted in presentation of a visual stimulus in a central window .
when the animal responded to the central visual stimulus , visual test stimuli were presented both on the left and right side of the screen .
as in cue - response training , an incorrect choice resulted in room light illumination and additional correction trials .
after successful dmtp rule learning , delay time between final response to the sample location cue stimulus and presentation of the central visual test stimulus was increased to either 3 s or 9 s.
first , in the " cue - delay - response " approach , the experimental animal makes a correct response guided by various cues presented at the start of the test trial .
after the cue is diminished , the animal should hold a memory of the cue during the delay and up until the animal 's choice moment is coming . to use this system
, it is necessary to create a reference memory by which the cues can guide the animal to make a correct choice .
second , in the " delayed matched to position ( dmtp ) " or " non - matched to position ( dnmtp ) " paradigms , the animal should first learn the basic rule of the delayed matched or non - matched position to make a correct choice response .
after the sampling location , a temporal delay is instituted before the animal is allowed to choose the matching ( or non - matching ) location . to test working memory in a touch - screen system , we first tested whether mice can learn a reference memory in which they choose between locations on the left or right , following presentation of a visual cue presented in the center ( fig .
this task also has been used to test rodent 's visuo - motor response . on a given trial , to receive a reward , the mouse must touch a visual cue presented in the center location on the screen , and then choose either the left or right location of subsequent visual choice stimuli , in accordance with the visual cues ( fig .
2a ) that have different colors and shapes . when choice moments were given 1 second after touching the center cue , however , mice performed poorly this task ( fig .
their failure to learn the reference rule indicates that using the cue - delay - response method in the touch - screen paradigm is not an appropriate way to test working memory in mice .
we next employed a dmtp paradigm in which working memory of mice can be assessed in a lever - pressing operant chamber , to determine whether mice can learn a dmtp rule ( fig .
3 ) . to do sampling , the mouse was required to touch the visual stimulus presented in either the left or right position of the screen three times .
one second later , the mouse was allowed to make a final sample touch of the visual cue presented in the center of the screen .
after the mice made this final touch of the center position , they were then presented with two alternative visual stimuli that were positioned in both the left and right position .
when mice chose a position that matched the cue 's position , a sweet milk reward was delivered ( fig .
when mice made an incorrect choice , correction trials were given until they made a correct choice .
as was true in the lever - pressing operant chamber , mice successfully learned the matched to position rule ( fig .
3 ) . after four training sessions , mice scored high in accuracy compared to their first session , and the number of correction trials also diminished ( fig .
3c ) . after mice learned the matched - to - position rule , to test working memory , we increased delay time between final sample touch and central visual stimulus presentation ( fig .
when delay time was increased to 3 s , there was a slight , but significant , decrease in the rate of correct choice to matched position ( fig .
4a ) , compared to the last session of the rule learning . when we further trained mice in a 3-s delay paradigm by twice , animals made correct choices comparable to those of the last session of rule learning ( fig .
finally , a 9-s delay resulted in a correct matched - to - position learning rate was substantially reduced compared to that in the last session of 3-s delay paradigm ( fig .
prolonged delay time between final sample touch and central visual stimulus presentation reduced the rate of animal 's correct matched - to - sample responses . for example , increasing delay time from 1 s to 3 s slightly reduced correct choice rate ( fig .
as more training sessions were given , however , mice made comparable correct choices to those in the 1-s delay condition .
this may indicate , in our experimental condition , that a 3-s delay is not long enough to test the working memory of mice .
when delay time was increased to 9 s , mice showed substantially reduced correct choice rate , which indicates that their rate of correct choice of matched position is affected by delay time between sampling and choice moment .
as clearly mentioned in the previous report , trial - unique , delayed nonmatching - to - location ( tunl ) , the only known working memory testing paradigm using touch screen , failed to test working memory in mice .
there are several differences between tunl task and our current paradigm , which successfully assessed working memory of mice .
firstly , we employed dmtp paradigm , whereas tunl task used dnmtp paradigm even though it is not clear how this difference affected cognitive performance .
secondly , we increased the number of sampling touch to 3 times as in lever pressing dmtp paradigm   whereas animals were allowed only one sampling touch in tunl task .
we suppose that this multiple sampling may increase retention of transitory memory during delay period .
thirdly , in our paradigm , mice were not required to do any specific task to initiate choice phase at the end of delay period , whereas in tunl task , animals are required to poke reward magazine at the end of delay paradigm to initiate choice phase .
this may also make our paradigm easier for mice to learn the rule of dmtp .
our present approach to assess working memory in mice using touch - screen test could be used to characterize several features of working memory .
first , how long mice can retain the memory of a sample position could be tested .
although not well investigated , 10 s is commonly used as the time limit in current t - maze - based experiments assessing working memory in mice . using a similar testing protocol in our present study
, mice could make correct matched - to - sample choices with as long as a 9-s delay .
therefore , with our highly flexible and accurate system , the duration of transitory memory during dmtp tasks could be determined for mice .
second , the effect of pattern separation on working memory could be studied in mice .
the hippocampal ca1 region is required to make non - matched choices between highly separated choices but not between close choices in the radial - arm maze task .
there is no study that assesses whether mice also have hippocampal ca1-dependent temporal separation memory . using our present paradigm
, it could be examined whether mice have temporal separation memory , and , if they do , which brain regions are involved in temporal separation memory .
 assessing the working memory of the rodent by using a touch - screen system has several advantages ( e.g. , allowing highly accurate data collection and flexibility in memory task design ) . 
 however , there is currently no available testing paradigm utilizing touch - screen systems that can assess working memory in the mouse . in this study 
 , we developed a touch - screen testing paradigm in which mice were trained to choose a location that is matched to a sample location after a time delay . 
 consistent with previous studies , this study showed that mice could not only learn the rule in the delayed matched to position ( dmtp ) , but also could retain a transitory memory of the sample position during delay . 
 this indicates that a touch - screen system can provide a dmtp testing platform to assess working memory in the mouse . 
INTRODUCTION
MATERIALS AND METHODS
Animal
Touch-screen testing
RESULTS AND DISCUSSION