Abstract:
A system for measuring memory and learning capabilities of a small animal, according to the present invention, includes:  
     a feed holder storing therein feed to be given to the small animal;  
     an observation field having a plurality of through holes opened into the feed holder, and holding the small animal therein;  
     an openable cover for opening and closing the respective through holes, the cover having breathability;  
     a dark chamber joined to the observation field;  
     an observation unit for measuring a position of the small animal in the observation field successively by using infrared rays; and  
     a computer for controlling timing of opening and closing each openable cover, and calculating the number of accessing times during each unit period, of the small animal to the through holes on the basis of an output from the observation unit.

Description:
BACKGROUND OF THE INVENTION  
         [0001]    This invention relates to techniques in the region of behavioral science in the medical physiological field, and more particularly to a method of and a system for measuring the memory and learning capabilities of small animals by utilizing the natural ingestion and/or drinking appetite of such animals.  
           [0002]    To measure the memory and learning functions of small animals, for example, a Morris&#39;s water maze, a cowering reaction of small animals against an electric shock, an elevated maze, a radial arm maze and the like are utilized. According to the techniques utilizing these, it is necessary that an experimenter transfers animals from a breeding cage to a specially provided experimental environment and conducts experiments in a light period (at an inactive phase of animals) in which the experimenter can observe the animals with the naked eye. To conduct experiments, the experimenter usually touches the animals so as to fasten them but touching the animals causes stresses on them in some cases due to the physical stimulus attended with the touching action. This results in occurrence of a scatter in the results of the experiments.  
         SUMMARY OF THE INVENTION  
         [0003]    An object of the present invention is to provide a system for measuring the memory and learning capabilities of a subject animal by utilizing the unaffected ingestion and/or drinking appetite thereof, capable of reducing the influence exerted by an experimenter, upon the results of the experiments. Further, the invention enables measuring the memory and learning capabilities of nocturnal animals by utilizing the unaffected ingestion and/or drinking appetite thereof even at an active phase (dark period) thereof.  
           [0004]    (1) The present invention provides a system for measuring the memory and learning capabilities of a small animal, including:  
           [0005]    a feed holder in which feed to be given to the small animal is stored,  
           [0006]    an observation field having a plurality of through holes opened into the feed holder, and holding therein the small animal,  
           [0007]    openable covers adapted to open and close the respective through holes, and having a ventilating function,  
           [0008]    a dark chamber joined to the observation field,  
           [0009]    an observation unit to measure the positions of the small animals in the observation field successively by using infrared rays, and  
           [0010]    a computer to control opening and closing timing of the openable covers and calculate the number of access during each unit time of the small animals to the respective through holes on the basis of an output from the observation unit.  
           [0011]    (2) The present invention also provides a system for measuring the memory and learning capabilities of a small animal, including:  
           [0012]    an observation field holding the small animal therein,  
           [0013]    a dark chamber joined to the observation field,  
           [0014]    a water supply unit having a plurality of nozzles inserted into the observation field, and adapted to supply water, which is to be given to the small animal to the nozzles,  
           [0015]    an observation unit adapted to measure the positions of the small animal successively by using infrared rays, and  
           [0016]    a computer for controlling timing of supplying water to the respective nozzles, and calculating the number of accessing times, during each unit time, of the small animal to the nozzles on the basis of an output from the observation unit. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0017]    [0017]FIG. 1 is a schematic construction diagram illustrating one embodiment of a measuring system according to the present invention;  
         [0018]    [0018]FIG. 2 is a perspective view of a measuring unit according to the embodiment of the present invention;  
         [0019]    [0019]FIG. 3 is a plan view of the measuring unit according to the embodiment of the present invention;  
         [0020]    [0020]FIG. 4 is a front view of the measuring unit according to the embodiment of the present invention;  
         [0021]    [0021]FIG. 5 is a schematic construction diagram illustrating the measuring system according to the embodiment of the present invention;  
         [0022]    [0022]FIG. 6 is a flow chart of a process carried out by the measuring system according to the embodiment of the present invention;  
         [0023]    [0023]FIG. 7 is a time chart showing behavioral patterns of normal mice outputted from the measuring system according to the embodiment of the present invention;  
         [0024]    [0024]FIG. 8 is a time chart showing behavioral patterns of hippocampus-damaged mice outputted from the measuring system according to the embodiment of the present invention; and  
         [0025]    [0025]FIGS. 9A and 9B are two kinds of graphs, outputted from the measuring system, showing manners in which normal mice learn changes of feeding stations according to the embodiment of the present invention. 
     
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0026]    An embodiment of the present invention will now be described with reference to the attached drawings.  
         [0027]    First, the configuration of this embodiment of the measuring system will be described with reference to FIG. 1 to FIG. 5.  
         [0028]    [0028]FIG. 1 is a schematic general construction diagram showing one embodiment of the measuring system, and FIG. 2 and FIG. 3 a perspective view and a plan view of a measuring unit included in the embodiment of the measuring system. In each of the drawings, a rectangular coordinate system having as an X-Y plane a horizontal plane on which the measuring unit is set, is defined for the convenience of giving the following description.  
         [0029]    As shown in FIG. 1, this measuring system has a measuring unit  100  to make measurement concerning a small animal (rats, mice and the like), and a computer (personal computer)  200  to control the measuring unit  100  and analyze the behaviors of the small animal on the basis of the results of the measurement conducted by the measuring unit  100 .  
         [0030]    The measuring unit  100  has as shown in FIG. 1, FIG. 2 and FIG. 3, a residence box  20  housing therein the small animal, a subject of the experiment, a water supply unit  60  provided in the residence box  20  so as to supply drinking water to the small animal, a rotary feeder  70  for storing feed, such as powdered feed and the like, which are to be given to the small animals, in the residence box  20 , a support base  90  for positioning the residence box  20  on the rotary feeder  70 , and an observation unit  80  for tracing the movements of the small animals in a predetermined region in the residence box  20 . The details of each portion of the measuring unit  100  are as follows.  
         [0031]    As shown in FIG. 2 and FIG. 3, the residence box  20  is provided in the interior thereof with an observation field  30  in which the condition of the animals is observed by an observation unit  80 , and a dark resting chamber (which will hereinafter be referred to as a nest)  40  narrower than the observation field  30 .  
         [0032]    As shown in FIG. 2, the nest  40  is provided so as to contact the whole of an inner surface of one side-wall-member  22 A 2  out of four side wall members  22 A 1 ,  22 A 2 ,  22 A 3 ,  22 A 4  which constitute the residence box  20 , and has a gate  40   a  facing the observation field  30 .  
         [0033]    The observation field  30  is provided so as to be positioned in a substantially central portion of the measuring unit  100 . Although the observation field  30  may have any shape, the observation field in this embodiment has a square shape in horizontal section. The floor of this observation field  30  has a double structure as shown in FIG. 1 which is formed by a stainless steel screen  31  of such meshes that do not permit the feet of the small animals to be sunken into the eyes of the screen, and a detachable plate member  32  provided under the stainless steel screen  31 . The feces and urine excreted by the small animals in the observation field fall from the eyes of the stainless steel screen  31  and are collected on the detachable plate type member  32 . This prevents the interior of the observation field  30  from being soiled with the feces and urine.  
         [0034]    In each corner of the floor of the observation field  30 , feed stations  50   a,    50   b,    50   c,    50   d  for use in givin feed stored on the rotary feeder  70  to the small animals are provided as shown in FIG. 3. To be concrete, a feeding through hole  51  facing a surface of the feed on the rotary feeder  70  is made open in each corner of the floor of the observation field  30  as shown in FIG. 1, and escape preventing metal nets (not shown) having two holes of a diameter (around 15 mm in case where the small animal is a mouse) slightly smaller than that of a scull of the small animal are respectively spread over these feeding through holes  51 . The small animal in the observation field  30  can eat the feed on the rotary feeder  70  by inserting their muffles from through holes for feeding through the mesh of the escape preventing metal nets. Each of the four feed stations  50   a,    50   b,    50   c,    40   d  is provided with an openable cover  52  for closing and opening the feeding through holes  51 , and two fence-like frames  53  standing up on the floor so as to separate the feeding through holes  51  from a region of the stainless steel screen  31 . In this embodiment, for example, a slide shutter reciprocatingly movable in an X-axis direction under the escape preventing metal nets  31  can be used as the openable cover  52 . Although the openable cover  52  is provided so as to control the time for feeding the small animal, the openable cover is formed of a material having ventilation properties, for example, mesh or a lattice so that the smell of the feed on the rotary feeder  70  reaches the interior of the observation field  30  even when the feeding through holes  51  are closed. The fence-like frames  53  are provided so as to prevent the powdered feed and the like from scattering in the interior of the observation field  30 , and the feces and urine of the animal from being mixed in the feed on the rotary feeder  70 . However, it is necessary that the fence-like frames  53  be formed in such a height that the small animal can bring its head from the outside of the fence-like frames  53  to the feeding through holes  51  on the inner side of the fence-like frames  53 .  
         [0035]    As shown in FIG. 2 and FIG. 3, among the side-wall-members  22 A 1 ,  22 A 2    22 A 3 , and  22 A 4 constituting the residence box  20 , the three side-wall-members  22 A 1 ,  22 A 3 , and  22 A 4  facing the interior of the observation field  30  each have a through hole  21  for water supply formed at a height h to which the small animal on the stainless steel screen  31  can reach with it muffle. It is preferable that the water supply through holes  21  are each formed at a position of a half width, D X /2 of a width D X  with respect to the X-direction of the observation field  30  and at a position of a half width, D Y /2 of a width Dy with respect to the Y-direction.  
         [0036]    When the small animal housed in the residence box  20  having the above construction, takes feed or drink water, they stay in the observation field  30 . When the small animal neither eat feed nor drink water, they stay in the shaded nest  40 .  
         [0037]    As shown in FIG. 1, water supply units  60  each have a hose  62  formed of silicon tube or the like with a diameter of about  2  mm, a water supply tank (not shown) joined to one end of the hose  62  via a connector, a water supply nozzle  61  fixed to the other end of the hose  62 , and an electromagnetic valve  63  provided in the hose  62 . The water supply tank is provided at a position higher than the three water supply through holes  21  of the residence box  20 . The three water supply nozzles  61  are each inserted through the three feed water through holes  21  of the residence box  20  into the observation field  30 , and fixed to the water supply through hole  21  by a metal member (not shown). Each of the water supply nozzles  61  used in this embodiment opens a nozzle port when a force is applied from the outside to a free end thereof, and closes the nozzle port when the applied force to the free end is removed. According to such a water supply unit  60 , when a small animal makes a contacting action against the free end of the water supply nozzle  61 , in other words, when a small animal pushes the free end of the water supply nozzle  61  with its nose, mouth, etc., in a water supply time zone during which the electromagnetic valve  63  is opened, the water is discharged from the water tank through the nozzle port of the water supply nozzle  61  via the hose  62 .  
         [0038]    As shown in FIG. 1 and FIG. 3, the rotary feeder  70  has a disk type feed holder  72  provided so that the axis of the disk type feed holder extends along a Z-axis, and a motor  71  for turning the feed holder  72  around the Z-axis. On the inner side of the feed holder  72 , an inner annular wall  72 b is formed along an outer wall  72   a  as shown in FIG. 5. The feed for the small animals is stored in a belt-like region  72   c  formed between the outer wall  72   a  and inner wall  72   b.  In order that the small animals in the observation field  30  can eat the feed via the feeding through holes  51  of the feed stations  50   a,    50   b,    50   c,    50   d,  this belt-like region  72   c  passes lower portions of the four feed stations  50   a,    50   b,    50   c,    50   d  by the rotation of the motor  71 . Namely, since the feed stations  50   a,    50   b,    50   c,    50   d  are provided at the corners of the square observation field  30  in this embodiment, the feed holder  72  having the outer wall  72   a  the inner diameter of which is substantially equal to the length of a diagonal line of the observation field  30 , and the inner wall  72   b  the inner diameter of which is substantially equal to a distance between the feed stations which are in diagonally opposite positions is provided so that a rotary shaft of the feed holder passes an intersection of the diagonal lines of the observation field  30 .  
         [0039]    According to such a rotary feeder  70 , the positions of the feed stations  50   a,    50   b,    50   c,    50   d  with respect to the surface of the feed in the belt-like region  72   c  can be changed by turning the feed holder  72 . This can prevent the surface of the feed in the feed holder  72  from being partially recessed due to the ingestion of the feed by the small animals. Therefore, it can prevent the occurrence of a region, in which the small animals are difficult to eat the feed, on the surface of the feed in the feed holder  72 .  
         [0040]    The support base  90  has a top plate  93  on which the residence box  20  is fixed, two plate type support legs  91   a,    91   b,  for supporting both ends of the top plate  93  and height adjusting caps  92  fixed to lower ends of the two plate type support legs  91   a,    91   b.  By this support base  90 , the residence box  20  is held on an upper portion of the rotary feeder  70 .  
         [0041]    As shown in FIG. 1, the observation unit  80  has an infrared motion picture camera  81  while enables shooting even in a dark period, a behavioral period of the small animal and a processor  82  for calculating a barycentric position of an image of the small animal taken as shielding bodies by the infrared motion picture camera  81 . The infrared motion picture camera  81  is provided at an upper part of the observation field  30  so that a lens thereof faces the interior of the observation field  30 . The processor  82  calculates a barycentric position of an image of the small animal at suitable time intervals (for example, at intervals of 0.5 seconds or 1 second), and output the results of the calculation successively to an output unit (printer) and a computer  200 .  
         [0042]    Although the observation unit  80  having the infrared motion picture camera  81  is used in this embodiment, a position detector having an infrared ray detector other than the infrared motion picture camera  81  may also be used. For example, an infrared ray sensor including an infrared projector and an infrared ray receiver may be used instead of the infrared motion picture camera  81 . When the infrared ray sensor is used, it is necessary to provide the floor of the nest  40  higher than that of the observation field  30 , and form sensor set chambers  94  enclosing four sides of a space in the vicinity of the stainless steel screen  32  of the observation field  30  as shown in FIG. 4 and FIG. 5. It is necessary to provide a transparent glass window  95  between sensor set chambers  94  and the observation field  30 . Paired infrared ray projector  83   a   x , and infrared ray receiver  83   b   x  opposed to each other with respect to the Y-direction and paired infrared ray projector  83   a   y  and infrared ray receiver  83   b   y  opposed to each other with respect to the X-direction are provided in the interior of the sensor set chamber  94 . One paired infrared ray projector  83   a   x  and infrared ray receiver  83   b   x  are used to detect the X-coordinates of the small animals in the observation field  30 , while the other paired infrared ray projector  83   a   y  and infrared ray receiver  83   b   y  are used to detect the Y-coordinates of the small animals in the observation field  30 .  
         [0043]    The computer  200  not only controls the opening and closing of the openable covers  52 , the opening and closing of the electromagnetic valves  63  and the rotation of the motor  71  in accordance with a program executed thereby, but also analyzes an output from the position detector. By the processing operation of the computer  200 , the memory and learning capabilities of the small animal are measured. A method of measuring the memory and learning capabilities of the small animal utilizing this mode of embodiment of the measuring system inclusive of a process executed by the computer  200  will now be described with reference to FIG. 6. Inside the residence box  20  of the measuring unit  100 , the small animal, a subject of the measurement of memory and learning capabilities thereof shall be held in advance.  
         [0044]    The computer  200  drives the motor  71  so that the feed holder  72  can be turned intermittently by predetermined angles (for example, 22.5° at intervals of 1 hour) at predetermined time intervals. The computer  200  opens the three electromagnetic valves  63  one by one in order at predetermined time intervals so that only any one of the three electromagnetic valves  63  can be always opened. The computer  200  also opens the four openable covers  52  one by one in order at predetermined time intervals so that only any one of the four openable covers  52  can be always opened. As a result, out of the plural feed water nozzles  61  and plural feed stations  50   a,    50   b,    50   c,    50   d,  the feed water nozzle from which the small animals can obtain the water and the feed station from which the small animals can obtain the feed come to change sequentially as the feed holder  72  is turned (Step  60 ).  
         [0045]    The observation unit  80  calculates successively the coordinates (X, Y) of the positions of the small animal placed under such a circumstance, and successively outputs the results of calculation to the computer  200  (Step  61 ).  
         [0046]    The computer  200  detects based on an output from the observation unit  80 , the number of visits of the small animal to each feed water nozzle and the number of visits of the small animal to each feed station (Step  62 ). To be concrete, when the coordinates which the observation unit  80  successively outputs continue to indicate for a predetermined period of time (for example, 2 seconds) a position in an ambit region of any feed water nozzle, the computer  200  counts this condition as one visit of the small animal to the relative feed water nozzle, and retains the counted value along with the time at which the counted value is obtained. When the coordinates which the observation unit  80  successively outputs continue to indicate for a predetermined period of time (for example, 4 seconds) a position in an ambit region of any feed station, the computer  200  also counts this condition as one visit of the small animal to the relative feed station, and retains the counted value along with the time at which the counted value is obtained. Hereinbefore, the ambit regions of each water supply nozzle and that of each feed station are predetermined as regions of interest around each water supply nozzle and each feed station.  
         [0047]    During such measurement period, the computer  200  judges whether a predetermined period of experiment time (for example, 7 days) has passed or not (S 63 ). When the results of judgment show that experiment time has passed, the computer  200  analyzes the data obtained as the results of the operations repeatedly carried out in S 62 , and outputs the results of the analysis (S 64 ). Out of these results of analysis, concrete examples of the above analytical results concerning the feeding behaviors of the small animal are shown in FIG. 7, FIG. 8 and FIG. 9.  
         [0048]    [0048]FIG. 7 is a time chart showing behavioral patterns of four normal mice A, B, C, and D in a case where the feed available station is shifted in the order of from  50   a,    50   b,    50   c,  to  50   d  at unit time intervals (in this embodiment, at 1 hour intervals). The lapse of time from the experiment starting time is taken along the lateral axis, and each time zone of a unit period of time on the chart is given a color having a certain density in accordance with the number (number/time) of visits of the small animal to each feed station. On this time chart, a color of a higher density is used to represent a larger number of visits of the small animal to the feed stations. It was ascertained from this time chart that the behavioral patterns of the normal mice A, B, C, and D were fitted to the feed station changing order of from  50   a,    50   b,    50   c,  to  50   d  with the lapse of time from the experiment starting time.  
         [0049]    [0049]FIG. 8 is a time chart showing behavioral patterns of four hippocampus damaged mice a, b, c, and d in a case where the feed available station is shifted in the order of from  50   a,    50   b,    50   c,  to  50   d  at unit time intervals. The lapse of time from the experiment starting time is expressed by the lateral axis, and each time zone of a unit period of time on the chart is given a color having a certain density in accordance with the number (number/time) of visits of the small animals to each feed station. On this time chart as well, a color of a higher density is used to represent a larger number of visits of the small animal to the feed stations. It was ascertained from this time chart that the behavioral patterns of the hippocampus damaged mice a, b, c, and d were not fitted to the feed station changing order of from  50   a,    50   b,    50   c,  and  50   d  irrespective of the lapse of time from the experiment starting time.  
         [0050]    [0050]FIGS. 9A and 9B are two kinds of graphs showing learning process of four normal mice A, B, C, and D. FIG. 9B shows long-term learning effects of the four normal mice A, B, C, and D, while FIG. 9A shows short-term learning effects of the four normal mice A, B, C, D.  
         [0051]    [0051]FIG. 9A in which the lapse of time from the experiment starting time is expressed by the lateral axis shows the transition of a correct response rate (%)  303 , and that of a ratio (%)  304  of the number of visits of the mice to a feed station in which the ingestion of the feed was possible 1 hour before to the number of visits of the mice to a feed station in which the ingestion of the feed is impossible. It was understood from this graph that the correct response rate increased with the lapse of time from the experiment starting time, and that the number of erroneous visits of the mice to a feed station in which the ingestion of the feed was possible 1 hour before decreased. This proved that the normal mice learned the feed station changing order in a short period of time.  
         [0052]    [0052]FIG. 9B in which the lapse of time from the experiment starting date is taken along the lateral axis shows the transition of the number of visits (number of correct visits)  301  of the mice during one day to feed stations in which the ingestion of the feed is possible, and that of the number of visits (number of searching visits)  300  of the mice during one day to all the feed stations. The right-hand graph further shows the transition during one day of a correct response rate (number of correct visits/number of searching visits×100%)  302 . It was understood from this graph that the number of visits of the mice to each feed station for making a search for a feed station in which the ingestion of the feed was possible decreased with a correct response rate increased with the lapse of time from the experiment starting date. This proved that the normal mice learned the feed station changing order continuously for a long period of time, and that the learning effect improved steadily.  
         [0053]    Although FIG. 7 and FIG. 8 show only the results of an analysis of the behavioral patterns of the mice concerning the ingestion of the feed, the behavioral patterns of the mice concerning the drinking of water are also analyzed in a similar manner. FIG. 9 shows only the results of an analysis of the learning patterns of normal mice concerning the ingestion of the feed, the learning patterns of normal mice concerning the drinking of water and the learning patterns of hippocampus damaged mice concerning the ingestion of the feed and drinking of water are also analyzed.  
         [0054]    In the foregoing, when the measuring system according to one embodiment of the present invention is used, a computer controls supply of feed, water or the like to the small animal, thereby avoiding experimenter&#39;s contact to the small animal to as great an extent as possible. Then, it becomes possible to reduce stress the small animal receives. This can prevent dispersion (variations) in experiment results, depending on experimenters, and highly reliable experiment results can be obtained.  
         [0055]    Further, all-the-time observation on the behaviors of the small animal by the observation unit  80 , as well as computer-controlled feed and water supply to the small animal, allow long-term continuation of experiments without any change of the experimental conditions. Furthermore, since the infrared ray detector is employed in the observation unit  80 , the positions of the small animal can be determined both in the light period and in the dark period (both in the daytime and in the nighttime).  
         [0056]    The present invention is not limited to the above-described modes of embodiment but various kinds of modifications can be made within the scope of the invention which does not depart from the spirits of the invention. For example, the memory and learning capabilities of the small animal concerning only the ingestion of the feed may be analyzed by the measuring system according to the present invention. In such a case, it is not necessary to provide the water supply unit  60  in the measuring unit  100 . Conversely, the memory and learning capabilities of the small animal concerning only the drinking of water may be analyzed by the measuring system according to the present invention. In this case, it is not necessary to provide the feed stations  50   a,    50   b,    50   c,  and  50   d  in the measuring unit  100 .