the power tower is placed on a rectangular piece of plywood that is clamped to a table .
a 4.5 rpm gear motor from grainger rests on top of four 2"x6 " boards and one  inch plywood board .
the motor , an ac / dc speed control , a fuse , and an on / off switch are wired together into a juncture box . to cool the motor , a hole 3 inches in diameter
on one side , a 3-inch fan is attached to the plywood , and on the other , a 3-inch diameter metal cylinder is attached .
a custom - made rotating arm with a roller mounted on the end is connected to the motor .
the motor and attached arm rotate in a clockwise direction , pushing down one side of a bent  inch square tube stock that is attached to the plywood with a door hinge .
a wheel is attached to the top side of the tube stock below the platform to allow it to roll against the platform that is directly above it .
the arm pushes down on one end of the tube stock and lifts the opposite end , raising a two - level platform ( figure 1d ) . when the roller on the arm clears the tube stock it allows the arm to return to the starting position , dropping the platform back down ( figure 1f ) .
the platforms consist of racks that are placed on top of plywood and secured by bungee cords that are hooked onto screw eyes .
the platforms slide up and down along drawer sliders that are attached to the plywood with brackets .
vials are placed into the racks and secured by a square grid screen that is held in place with bungee cords .
a styrofoam cushion is placed below the platform to dampen the shock of the drop .
the power of the motor is adjusted such that a full rotational period is 15 seconds long .
flies are housed in a 25 c incubator with 50% humidity and a 12-hour light / dark cycle .
both exercise training and assessment of exercise capacity are conducted in a 25 c temperature - controlled room .
flies are collected and age - matched within 1 - 2 days of each other .
a minimum of 240 flies is required for longitudinal monitoring of endurance and negative geotaxis ( climbing ) ability , two physiological indicators that reflect the effects of exercise training .
longitudinal monitoring allows isolation of induced change relative to starting values for each cohort , without confounding effects from variation between cohorts .
after collection , flies are divided into 120 experimental flies , which will be subjected to the exercise regimen , and 120 control flies , which will not be exercised .
the vials of experimental flies are plugged with firm cellulose acetate flugs and the vials of control ( unexercised ) flies are plugged with soft sponge stoppers , which can be pushed down to immobilize the flies while on the power tower .
sponge is used for immobilization because its adaptive size allows easy placement near the bottom of the vial .
flies on the power tower are stored and exercised in vials that contain 5 ml of food , which provides them with a softer landing than flies that are exercised in empty vials .
the food is typically composed of 10% sucrose , 10% yeast , and 2% agar in water , although we do not observe flies to eat often during the course of a training session .
if empty vials are used during training , the percentage of flies that suffer injuries is dramatically increased .
the power tower is operated in a temperature - controlled room that is maintained at 25 c .
control flies are also placed on the power tower , but the sponge stopper is pushed down into the vial approximately 3 mm above the flies in order to limit movement .
this group serves as a control for any effects of the power tower that are unrelated to exercise .
although some room still exists for these flies to maneuver , we have repeatedly observed that flies under these conditions run very little and do not demonstrate a physiological response to exercise .
it may also be desirable to include an additional control group that is not placed on the power tower at all .
flies are exercised five consecutive days each week , utilizing a ramped schedule ( figure 2 ) . during week one
the flies are exercised for 2 hours per session , week two for 2.5 hours per session , and week three for 3 hours per session .
although other regimens have been tested , this protocol produces consistent results across genotypes and under divergent conditions .
this protocol is , however , subject to a wide array of possible variations that might suit a particular experiment .
time to fatigue during endurance exercise is a useful physiological indicator to confirm and/or quantitate exercise capacity of a given cohort .
fatigue assays can be performed longitudinally on cohorts that will be used in training experiments .
assays may be conducted either prior to training , after the completion of the training protocol , or both .
other intermediate time points can be added if charting incremental progress during training is desired .
the fatigue assay can also be used to measure exercise endurance as a response to treatments other than the training protocol , such as diet alteration ( figure 3b ) .
both the experimental and control flies are placed on the power tower in vials of 20 each and made to climb until fatigued .
fatigue is recognized as the failure to respond to a negative geotaxis stimulus with climbing behavior .
a vial of flies is considered " fatigued " when 5 or fewer flies are able to climb higher than 2 inches for four consecutive drops .
fatigued vials are removed from the power tower while it is still operating , and the time of removal is recorded .
times of removal can be plotted as a nested histogram , or as a " time - to - failure " plot .
an example of data plotted as a " time - to - failure " plot is shown in figure 3b .
vials are monitored at 10 minute intervals for a maximum of 10 hours ( figure 3b ) . since this training protocol relies on induced running behavior , a primary assay for quantitating effects of training
is to longitudinally measure change in average running speed during training . during the course of training , 120-fly cohorts of exercised and unexercised flies are tested daily for their response to a negative geotaxis stimulus . the ring technique described in gargano et al .
briefly , this technique measures average height climbed by a vial of flies during a defined time after induction of negative geotaxis .
measurements are performed before placing flies on the machine for daily training so as to avoid complications from fatigue after the day 's run . in our standard protocol
, flies are exercised for a total of three weeks and climbing ability is examined for a total of five weeks : three weeks during exercise - training and two weeks after cessation of exercise ( figure 2 ) .
this allows the experimenter to chart whether effects of exercise on mobility persist after cessation of the program .
four photographs of each cohort 's daily ring performance are used for the analysis of climbing ability .
negative geotaxis is induced , and using a timed camera , a photograph is taken after a defined number of seconds .
we typically use two seconds , although this can be varied depending on experimental conditions . for every photograph , each vial is divided into four quadrants of equal height , and image processing software is used to assign each fly a score based on the quadrant it reached within the allotted time period . flies that climb to the topmost quadrant receive a score of 4 , flies in the next highest quadrant receive a 3 , flies in the second highest quadrant receive a 2 , and flies in the lowest quadrant receive a 1 .
flies that do not climb off of the bottom at all are given a score of 0 .
for each vial in a given photograph , a " climbing index " is generated by averaging the scores of all the flies in that vial .
each vial 's indices from the four photographs are then averaged to give a final climbing index for that day .
results are normalized to the initial " pre - exercise " score in order to reduce the effect of cohort variation in climbing ability and highlight the relative longitudinal change in a cohort 's climbing capacity as the result of exercise training .
the initial score is determined by the average of the first three days ' climbing indices and is assigned a value of 1 .
wild - type flies respond to an endurance protocol with a diminished age - related decrease in climbing ability that persists following the end of training , as reflected in longitudinal ring assays across five weeks of age ( figure 3a ) .
this delayed decline in negative geotaxis is a standard phenotypic response that can serve as a positive control to ensure that wild - type exercise response is occurring normally .
this data set is presented as an example of how the induction of exercise by the power tower program can be used as a behavioral input . the ability of various genetic or environmental factors to modulate this effect can then be assessed .
conversely , the power tower can also be used as an output in various experimental designs .
, the effect of these variations on exercise physiology can be tested using the power tower . here
, we show an example of this approach . when flies with a varying percentage of sucrose in their diets were tested for time to fatigue , increased sucrose content correlated with increased endurance capacity ( figure 3b ) .
( a - c )   the motorized bent arm with attached roller rotates clockwise until it comes into contact with the bent square tube .  
( d , e ) the arm pushes down on the bent square tube , causing the platform that is laden with vials of flies to lift .  
( f ) as the arm clears the tube the platform is allowed to fall back down , forcing the flies to return to the bottom of the vial .
flies subjected to the training protocol are made to exercise for five days each week under a three - week long ramped regimen that progressively increases the duration of exercise from the initial 2 hours by 30 minutes each week .
standard analyses include fatigue assays before and following the exercise program and ring assays from week 1 through week 5 .
all assays are performed in duplicate on an equal number of unexercised flies as a negative control .
( a ) ring assays performed longitudinally across ages in a single pair of male yw cohorts .
age - matched , genetically identical exercised and unexercised control flies were measured daily for average climbing speed .
results are expressed in terms of a climbing index that is normalized to the average climbing height across the first three days of measurement .
exercise - trained flies displayed a diminished age - related decline in negative geotaxis ability compared to age - matched unexercised siblings across ages ( 2-way anova , p < 0.005 ) .
( b ) fatigue assays conducted for 8 hours on age - matched female canton s flies show a significant effect of dietary sucrose content on time to fatigue ( log - rank , p < 0.0001 ) .
prior to experiment , flies were fed a yeast / sucrose / agar diet , with 10% weight / volume yeast concentration , and a varying percentage of dietary sucrose .
graph displays how many vials still have five or more flies running at a given time point .
these results can be treated statistically and graphically as a survival ( or time to failure ) curve , with " failure " for a vial being defined as a point in time when less than five flies continue to respond to negative geotaxis stimulus .
note that many other possible study designs and statistical treatments are possible , and data treatment and measurement should be tailored to fit individual purposes .
the power tower is placed on a rectangular piece of plywood that is clamped to a table .
a 4.5 rpm gear motor from grainger rests on top of four 2"x6 " boards and one  inch plywood board .
the motor , an ac / dc speed control , a fuse , and an on / off switch are wired together into a juncture box . to cool the motor , a hole 3 inches in diameter
on one side , a 3-inch fan is attached to the plywood , and on the other , a 3-inch diameter metal cylinder is attached .
a custom - made rotating arm with a roller mounted on the end is connected to the motor .
the motor and attached arm rotate in a clockwise direction , pushing down one side of a bent  inch square tube stock that is attached to the plywood with a door hinge .
a wheel is attached to the top side of the tube stock below the platform to allow it to roll against the platform that is directly above it .
the arm pushes down on one end of the tube stock and lifts the opposite end , raising a two - level platform ( figure 1d ) . when the roller on the arm clears the tube stock it allows the arm to return to the starting position , dropping the platform back down ( figure 1f ) .
the platforms consist of racks that are placed on top of plywood and secured by bungee cords that are hooked onto screw eyes .
the platforms slide up and down along drawer sliders that are attached to the plywood with brackets .
vials are placed into the racks and secured by a square grid screen that is held in place with bungee cords .
a styrofoam cushion is placed below the platform to dampen the shock of the drop .
the power of the motor is adjusted such that a full rotational period is 15 seconds long .
flies are housed in a 25 c incubator with 50% humidity and a 12-hour light / dark cycle .
both exercise training and assessment of exercise capacity are conducted in a 25 c temperature - controlled room .
flies are collected and age - matched within 1 - 2 days of each other .
a minimum of 240 flies is required for longitudinal monitoring of endurance and negative geotaxis ( climbing ) ability , two physiological indicators that reflect the effects of exercise training .
longitudinal monitoring allows isolation of induced change relative to starting values for each cohort , without confounding effects from variation between cohorts .
after collection , flies are divided into 120 experimental flies , which will be subjected to the exercise regimen , and 120 control flies , which will not be exercised .
the vials of experimental flies are plugged with firm cellulose acetate flugs and the vials of control ( unexercised ) flies are plugged with soft sponge stoppers , which can be pushed down to immobilize the flies while on the power tower .
sponge is used for immobilization because its adaptive size allows easy placement near the bottom of the vial .
flies on the power tower are stored and exercised in vials that contain 5 ml of food , which provides them with a softer landing than flies that are exercised in empty vials .
the food is typically composed of 10% sucrose , 10% yeast , and 2% agar in water , although we do not observe flies to eat often during the course of a training session .
if empty vials are used during training , the percentage of flies that suffer injuries is dramatically increased .
the power tower is operated in a temperature - controlled room that is maintained at 25 c .
control flies are also placed on the power tower , but the sponge stopper is pushed down into the vial approximately 3 mm above the flies in order to limit movement .
this group serves as a control for any effects of the power tower that are unrelated to exercise .
although some room still exists for these flies to maneuver , we have repeatedly observed that flies under these conditions run very little and do not demonstrate a physiological response to exercise .
it may also be desirable to include an additional control group that is not placed on the power tower at all .
flies are exercised five consecutive days each week , utilizing a ramped schedule ( figure 2 ) . during week one
the flies are exercised for 2 hours per session , week two for 2.5 hours per session , and week three for 3 hours per session .
although other regimens have been tested , this protocol produces consistent results across genotypes and under divergent conditions .
this protocol is , however , subject to a wide array of possible variations that might suit a particular experiment .
time to fatigue during endurance exercise is a useful physiological indicator to confirm and/or quantitate exercise capacity of a given cohort .
fatigue assays can be performed longitudinally on cohorts that will be used in training experiments .
assays may be conducted either prior to training , after the completion of the training protocol , or both .
other intermediate time points can be added if charting incremental progress during training is desired .
the fatigue assay can also be used to measure exercise endurance as a response to treatments other than the training protocol , such as diet alteration ( figure 3b ) .
both the experimental and control flies are placed on the power tower in vials of 20 each and made to climb until fatigued .
fatigue is recognized as the failure to respond to a negative geotaxis stimulus with climbing behavior .
a vial of flies is considered " fatigued " when 5 or fewer flies are able to climb higher than 2 inches for four consecutive drops .
fatigued vials are removed from the power tower while it is still operating , and the time of removal is recorded .
times of removal can be plotted as a nested histogram , or as a " time - to - failure " plot .
an example of data plotted as a " time - to - failure " plot is shown in figure 3b .
vials are monitored at 10 minute intervals for a maximum of 10 hours ( figure 3b ) .
since this training protocol relies on induced running behavior , a primary assay for quantitating effects of training is to longitudinally measure change in average running speed during training . during the course of training , 120-fly cohorts of exercised and unexercised flies are tested daily for their response to a negative geotaxis stimulus . the ring technique described in gargano et al .
briefly , this technique measures average height climbed by a vial of flies during a defined time after induction of negative geotaxis .
measurements are performed before placing flies on the machine for daily training so as to avoid complications from fatigue after the day 's run . in our standard protocol ,
flies are exercised for a total of three weeks and climbing ability is examined for a total of five weeks : three weeks during exercise - training and two weeks after cessation of exercise ( figure 2 ) .
this allows the experimenter to chart whether effects of exercise on mobility persist after cessation of the program .
four photographs of each cohort 's daily ring performance are used for the analysis of climbing ability .
negative geotaxis is induced , and using a timed camera , a photograph is taken after a defined number of seconds .
we typically use two seconds , although this can be varied depending on experimental conditions . for every photograph , each vial is divided into four quadrants of equal height , and image processing software is used to assign each fly a score based on the quadrant it reached within the allotted time period . flies that climb to the topmost quadrant receive a score of 4 , flies in the next highest quadrant receive a 3 , flies in the second highest quadrant receive a 2 , and flies in the lowest quadrant receive a 1 .
flies that do not climb off of the bottom at all are given a score of 0 .
for each vial in a given photograph , a " climbing index " is generated by averaging the scores of all the flies in that vial .
each vial 's indices from the four photographs are then averaged to give a final climbing index for that day .
results are normalized to the initial " pre - exercise " score in order to reduce the effect of cohort variation in climbing ability and highlight the relative longitudinal change in a cohort 's climbing capacity as the result of exercise training .
the initial score is determined by the average of the first three days ' climbing indices and is assigned a value of 1 .
wild - type flies respond to an endurance protocol with a diminished age - related decrease in climbing ability that persists following the end of training , as reflected in longitudinal ring assays across five weeks of age ( figure 3a ) .
this delayed decline in negative geotaxis is a standard phenotypic response that can serve as a positive control to ensure that wild - type exercise response is occurring normally .
this data set is presented as an example of how the induction of exercise by the power tower program can be used as a behavioral input . the ability of various genetic or environmental factors to modulate this effect can then be assessed .
conversely , the power tower can also be used as an output in various experimental designs .
then , the effect of these variations on exercise physiology can be tested using the power tower . here
when flies with a varying percentage of sucrose in their diets were tested for time to fatigue , increased sucrose content correlated with increased endurance capacity ( figure 3b ) .
( a - c )   the motorized bent arm with attached roller rotates clockwise until it comes into contact with the bent square tube .  
( d , e ) the arm pushes down on the bent square tube , causing the platform that is laden with vials of flies to lift .  
( f ) as the arm clears the tube the platform is allowed to fall back down , forcing the flies to return to the bottom of the vial .
flies subjected to the training protocol are made to exercise for five days each week under a three - week long ramped regimen that progressively increases the duration of exercise from the initial 2 hours by 30 minutes each week .
standard analyses include fatigue assays before and following the exercise program and ring assays from week 1 through week 5 .
all assays are performed in duplicate on an equal number of unexercised flies as a negative control .
( a ) ring assays performed longitudinally across ages in a single pair of male yw cohorts .
age - matched , genetically identical exercised and unexercised control flies were measured daily for average climbing speed .
results are expressed in terms of a climbing index that is normalized to the average climbing height across the first three days of measurement .
exercise - trained flies displayed a diminished age - related decline in negative geotaxis ability compared to age - matched unexercised siblings across ages ( 2-way anova , p < 0.005 ) .
( b ) fatigue assays conducted for 8 hours on age - matched female canton s flies show a significant effect of dietary sucrose content on time to fatigue ( log - rank , p < 0.0001 ) . prior to experiment , flies were fed a yeast / sucrose / agar diet , with 10% weight / volume yeast concentration , and a varying percentage of dietary sucrose .
graph displays how many vials still have five or more flies running at a given time point .
these results can be treated statistically and graphically as a survival ( or time to failure ) curve , with " failure " for a vial being defined as a point in time when less than five flies continue to respond to negative geotaxis stimulus .
note that many other possible study designs and statistical treatments are possible , and data treatment and measurement should be tailored to fit individual purposes .
however , several areas in this protocol are subject to modification to fit particular experimental needs . for example
, the length of training and number of bouts could potentially be varied to make the program more or less challenging , as desired .
the height of the container in which negative geotaxis ability is measured could be altered to increase the available area for improvement to be documented .
various methods of automating quantitation of climbing speed may also be applicable . in principle , any software program capable of distinguishing a fly from background can be used to speed the data gathering process .
for example , preliminary experiments strongly indicate that at least one day of rest per week tends to facilitate greater improvement than relentless daily exercise .
additionally , circadian rhythms and temperature are known to affect the movement of cold - blooded animals .
the time of day that training takes place can be varied , but should always be consistent within particular groups under comparison , in order to avoid the possibility of confounding effects of circadian rhythms .
temperature control is also essential , and we recommend a dedicated room at constant temperature to house exercise equipment .
lastly , males and females must be reared and measured separately , in order to avoid the potential of confounding effects of fertility and sex differences in exercise capacity .
potential applications of this methodology are limited only by the imagination of the researcher . in preliminary work
, we have utilized this methodology in three broad applications : to measure the effect of endurance exercise on various aspects of wild type biology across ages . to measure the effect of endurance exercise on specific mutant phenotypes .
based on our preliminary experience , mutant phenotypes tend to vary with exercise level as much as they vary with diet . the use of invertebrate models to better understand the relationship between diet , exercise , and aging physiology is perhaps the most important general application of this protocol
 one of the most pressing problems facing modern medical researchers is the surging levels of obesity , with the consequent increase in associated disorders such as diabetes and cardiovascular disease 1 - 3 . 
 an important topic of research into these associated health problems involves the role of endurance exercise as a beneficial intervention.exercise training is an inexpensive , non - invasive intervention with several beneficial results , including reduction in excess body fat 4 , increased insulin sensitivity in skeletal muscle 5 , increased anti - inflammatory and antioxidative responses 6 , and improved contractile capacity in cardiomyocytes 7 . 
 low intensity exercise is known to increase mitochondrial activity and biogenesis in humans 8 and mice , with the transcriptional coactivator pgc1- as an important intermediate 9,10 . despite the importance of exercise as a tool for combating several important age - related diseases , 
 extensive longitudinal genetic studies have been impeded by the lack of an endurance training protocol for a short - lived genetic model species . 
 the variety of genetic tools available for use with drosophila , together with its short lifespan and inexpensive maintenance , make it an appealing model for further study of these genetic mechanisms . with this in mind 
 we have developed a novel apparatus , known as the power tower , for large scale exercise - training in drosophila melanogaster 
 11 . 
 the power tower utilizes the flies ' instinctive negative geotaxis behavior to repetitively induce rapid climbing . 
 each time the machine lifts , then drops , the platform of flies , the flies are induced to climb . 
 flies continue to respond as long as the machine is in operation or until they become too fatigued to respond . 
 thus , the researcher can use this machine to provide simultaneous training to large numbers of age - matched and genetically identical flies . 
 additionally , we describe associated assays useful to track longitudinal progress of fly cohorts during training . 
Protocol
1. Power Tower Setup and Operation
2. Exercise Protocol
3. Exercise and Locomotor Fatigue
4. Exercise, Age, and Locomotor Ability
5. Representative Results
Discussion
Disclosures