shape a platform from the lid of 35 mm falcon dish by melting the sidewall and carving it to form an appropriate angle and thickness ( figure 1a ; figure 2 ) . drill a hole in the platform to accept the fly head ( figure 1a , b ) , such that the mouthparts are freely exposed to the outside of the chamber ( figure 1a ) . cut the end of a pipetman tip , with the size matching the fly to be observed . glue the tip to the platform as shown in figure 1a to complete the flies ( figure 2 ) . starve an adult fly for 24 hours at 25 c prior to the experiment by placing it in a vial with only a wet paper towel , if starvation is necessary . anesthetize the fly by placing it in a 15ml plastic tube standing on ice . using forceps , insert a fly into the chamber of the flies , and gently push the fly in until it is unable to move . then , insert a plug to retain the fly . seal the surrounding parts of the proximal proboscis ( rostrum ) to the inner edge of the hole ; apply a small amount of light - curing glue ( tetric evoflow ) to the sides and above the rostrum from the outside ( arrows in figure 3 ) . also apply the glue from the inside of the flies to the space between the dorsal part of the head and the inner edge of the hole ( figure 1b ) . to allow the rostrum to move freely , care should be taken to prevent any glue from touching the rostrum by using an eyelash ( or something comparable ) to spread the glue . finally , cure the glue with the weakest illumination of blue light sufficient for curing to avoid damaging the fly from excessive heat . remove the plug . a thread to hold the rostrum partially lifted ( arrow in figure 3 ) may be employed to stabilize the fly 's head by avoiding bump of the pharyngeal part to sog and to expose the ventral part of the sog , especially for imaging presynaptic terminals of gr5a neurons , which are covered by part of the pharynx when the proboscis is fully retracted . fill the surface of the platform with a sugar - free saline containing ( in mm ) : nacl , 140 ; kcl , 2 ; mgcl2 , 4.5 ; cacl2 , 1.5 ; and hepes - naoh , 5 with a ph of 7.1 . this sucrose - free saline has similar tonicity to those of commonly - used sucrose - containing salines such as hl3.1 . open the head capsule using a tungsten blade and forceps with sharpened tips like scissors , which is a custom - made device and originally designed by dr . kageyuki yamaoka , japan , to clip the cuticle and trachea to expose the sog ( figure 4 ) . the ventral edge of the head cuticle was cut as ventral as possible whereas the dorsal edge cut was not as critical . antennae and antennal nerves should be cut out by the sharpened forceps . in order to avoid movement artifacts during recordings , the brain must be stabilized through the removal of certain parts of the fly . cut the oesophagus at the end toward the pharynx and at the end going into the sog to remove the connection between the pharynx and the brain . then pull out muscle 16 , and finally , detach any trachea connecting the brain and the cuticle . we set our flies on the stage of an olympus bx51wi microscope connected to a spinning disk confocal microscope system ( improvision in perkinelmer ) ( figure 5 ) . a water immersion lens , e.g. 40x/0.8 n.a , was immersed into the bath ( figure 6 ) . take a single optical section at 4hz with an exposure time of 122ms using 491 nm excitation laser , focusing at the region of interest ( a cell body of a motor neuron , or an interneuron , or presynaptic terminals of gustatory sensory neurons ) with velocity software , ver . aspirate 100 mm aqueous sucrose solution into a hypodermic needle with a small wick ( made from japanese washi paper , see table of specific reagents ) protruding from the tip ( figure 7 ) . discharge a small drop of sucrose solution onto the wick , then , aspirate it , immediately before applying the soaked washi paper wick to the tip of the proboscis . the washi paper wick should only be applied for an instant to avoid satiation ( figure 8) . monitor the proboscis extension response ( per ) behavior using a ccd camera attached to a dissection microscope ( figure 5 ) simultaneously with ca imaging . data must be taken within one hour after starting the dissection because the per response is not maintained more than one hour under these conditions . motor neurons of the rostrum protractor , a pair of major muscles responsible for lifting the rostrum for proboscis extension , have been reported to show robust responses to sweet stimuli . figure 9 shows ca signals through gcamp3.0 expressed by a gal 4 driver , e49 , from a cell body of the motor neuron . as shown in the video , a robust per a , the flies is built such that the angle of the wall allows for the fly head to come out straight . three segments of the fly proboscis are color - coded ; the rostrum is magenta . the fly is inserted into the chamber with forceps , and a piece cut from the end of a pipetman tip is plugged into the back of the chamber to block the fly from escaping . the remaining gaps are sealed with light - curing glue as indicated to ensure that there are no leaks . after the glue has cured , the pipetman tip as a plug is removed . saline is poured into the flies such that the surface of the brain is completely covered . it should be ensured that there is no saline leaking out onto the anterior end of the fly head . the shaded area surrounded by the dashed line in panel b shows the part to be dissected out . the crescent shaped wall of glue ( from a glue gun ) is made to control the flow of saline solution during perfusion , and to reduce the amount of saline used . this image shows how a fly is set in the flies , and how the light - curing glue is applied around the head of the fly ( arrows ) to create a saline - proof seal . if any saline leaks through to the proboscis of the fly , then the experiment will fail the thread ( arrowheads ) tethered to the stage is to hold the proboscis in position , so that it is not completely retracted . otherwise it would obstruct the more ventral portion of the sog and cause movement artifacts . the esophagus , muscle 16 and tracheae connected to the brain , as well as selected air - sacks have also been removed to prevent them from disturbing the brain , which can lead to artifacts in the calcium signals . this apparatus is assembled from an olympus bx51wi microscope attached to a spinning disk confocal microscope system ( improvision ) , and a side - mounted nikon smz800 microscope . photographs to show the side - view of the flies .   the thin layer of saline solution is sandwiched between the 40x water - immersion lens and the fly . gampi - washi paper is a traditional japanese parchment that is extremely thin and smooth ( haibara , japan ) . the washi paper must be cut into a trapezoid with very specific dimensions ; 0.3 mm and 0.7 mm in width , and 4 - 5 mm in length ( a ) . this trapezoid is then inserted from the 0.7 mm side into the tip of a 23 g hypodermic needle and can also be stabilized through the insertion of an insect pin , which is bent to a v - shape ( b ) . c , the washi paper becomes transparent following exposure to a liquid , making it ideal for this experiment . the washi paper wick at the tip of a hypodermic needle is applied for an instant using a joystick manipulator with one hand . the needle is connected through a flexible tube to an injector manipulated with the other hand , for aspirating and discharging sucrose solution . frontal views of a starved fly with an extended proboscis ( arrows ) before the stimulation , at the stimulation , and after the stimulation with a wick containing 100 mm sucrose aqueous solution ( arrowhead ) . b , sucrose - induced gcamp 3.0 response in the motor neuron for the protractor of rostrum muscle in the starved state . the top panel , before the stimulation ; the bottom panel , immediately after the stimulation . the motor neuron responds to a sucrose stimulus by a sharp increase in fluorescence followed by a restoration phase of several seconds to the base line . shape a platform from the lid of 35 mm falcon dish by melting the sidewall and carving it to form an appropriate angle and thickness ( figure 1a ; figure 2 ) . drill a hole in the platform to accept the fly head ( figure 1a , b ) , such that the mouthparts are freely exposed to the outside of the chamber ( figure 1a ) . cut the end of a pipetman tip , with the size matching the fly to be observed . glue the tip to the platform as shown in figure 1a to complete the flies ( figure 2 ) . starve an adult fly for 24 hours at 25 c prior to the experiment by placing it in a vial with only a wet paper towel , if starvation is necessary . anesthetize the fly by placing it in a 15ml plastic tube standing on ice . using forceps , insert a fly into the chamber of the flies , and gently push the fly in until it is unable to move . then , insert a plug to retain the fly . seal the surrounding parts of the proximal proboscis ( rostrum ) to the inner edge of the hole ; apply a small amount of light - curing glue ( tetric evoflow ) to the sides and above the rostrum from the outside ( arrows in figure 3 ) . also apply the glue from the inside of the flies to the space between the dorsal part of the head and the inner edge of the hole ( figure 1b ) . to allow the rostrum to move freely , care should be taken to prevent any glue from touching the rostrum by using an eyelash ( or something comparable ) to spread the glue . finally , cure the glue with the weakest illumination of blue light sufficient for curing to avoid damaging the fly from excessive heat . remove the plug . a thread to hold the rostrum partially lifted ( arrow in figure 3 ) may be employed to stabilize the fly 's head by avoiding bump of the pharyngeal part to sog and to expose the ventral part of the sog , especially for imaging presynaptic terminals of gr5a neurons , which are covered by part of the pharynx when the proboscis is fully retracted . fill the surface of the platform with a sugar - free saline containing ( in mm ) : nacl , 140 ; kcl , 2 ; mgcl2 , 4.5 ; cacl2 , 1.5 ; and hepes - naoh , 5 with a ph of 7.1 . this sucrose - free saline has similar tonicity to those of commonly - used sucrose - containing salines such as hl3.1 . open the head capsule using a tungsten blade and forceps with sharpened tips like scissors , which is a custom - made device and originally designed by dr . kageyuki yamaoka , japan , to clip the cuticle and trachea to expose the sog ( figure 4 ) . the ventral edge of the head cuticle was cut as ventral as possible whereas the dorsal edge cut was not as critical . first , make an incision through the posterior edge using a tungsten blade . then , cut through the side and the anterior edges using the sharpened forceps . antennae and antennal nerves should be cut out by the sharpened forceps . in order to avoid movement artifacts during recordings , the brain must be stabilized through the removal of certain parts of the fly . cut the oesophagus at the end toward the pharynx and at the end going into the sog to remove the connection between the pharynx and the brain . then pull out muscle 16 , and finally , detach any trachea connecting the brain and the cuticle . we set our flies on the stage of an olympus bx51wi microscope connected to a spinning disk confocal microscope system ( improvision in perkinelmer ) ( figure 5 ) . a water immersion lens , e.g. 40x/0.8 n.a , was immersed into the bath ( figure 6 ) . take a single optical section at 4hz with an exposure time of 122ms using 491 nm excitation laser , focusing at the region of interest ( a cell body of a motor neuron , or an interneuron , or presynaptic terminals of gustatory sensory neurons ) with velocity software , ver . aspirate 100 mm aqueous sucrose solution into a hypodermic needle with a small wick ( made from japanese washi paper , see table of specific reagents ) protruding from the tip ( figure 7 ) . discharge a small drop of sucrose solution onto the wick , then , aspirate it , immediately before applying the soaked washi paper wick to the tip of the proboscis . the washi paper wick should only be applied for an instant to avoid satiation ( figure 8) . monitor the proboscis extension response ( per ) behavior using a ccd camera attached to a dissection microscope ( figure 5 ) simultaneously with ca imaging . data must be taken within one hour after starting the dissection because the per response is not maintained more than one hour under these conditions . motor neurons of the rostrum protractor , a pair of major muscles responsible for lifting the rostrum for proboscis extension , have been reported to show robust responses to sweet stimuli . figure 9 shows ca signals through gcamp3.0 expressed by a gal 4 driver , e49 , from a cell body of the motor neuron . as shown in the video , a robust per a , the flies is built such that the angle of the wall allows for the fly head to come out straight . three segments of the fly proboscis are color - coded ; the rostrum is magenta . the fly is inserted into the chamber with forceps , and a piece cut from the end of a pipetman tip is plugged into the back of the chamber to block the fly from escaping . the remaining gaps are sealed with light - curing glue as indicated to ensure that there are no leaks . saline is poured into the flies such that the surface of the brain is completely covered . it should be ensured that there is no saline leaking out onto the anterior end of the fly head . the shaded area surrounded by the dashed line in panel b shows the part to be dissected out . the crescent shaped wall of glue ( from a glue gun ) is made to control the flow of saline solution during perfusion , and to reduce the amount of saline used . this image shows how a fly is set in the flies , and how the light - curing glue is applied around the head of the fly ( arrows ) to create a saline - proof seal . if any saline leaks through to the proboscis of the fly , then the experiment will fail . the thread ( arrowheads ) tethered to the stage is to hold the proboscis in position , so that it is not completely retracted . otherwise it would obstruct the more ventral portion of the sog and cause movement artifacts . the esophagus , muscle 16 and tracheae connected to the brain , as well as selected air - sacks have also been removed to prevent them from disturbing the brain , which can lead to artifacts in the calcium signals . this apparatus is assembled from an olympus bx51wi microscope attached to a spinning disk confocal microscope system ( improvision ) , and a side - mounted nikon smz800 microscope . the thin layer of saline solution is sandwiched between the 40x water - immersion lens and the fly . gampi - washi paper is a traditional japanese parchment that is extremely thin and smooth ( haibara , japan ) . it is also very strong and can retain a large amount of liquid . the washi paper must be cut into a trapezoid with very specific dimensions ; 0.3 mm and 0.7 mm in width , and 4 - 5 mm in length ( a ) . this trapezoid is then inserted from the 0.7 mm side into the tip of a 23 g hypodermic needle and can also be stabilized through the insertion of an insect pin , which is bent to a v - shape ( b ) . c , the washi paper becomes transparent following exposure to a liquid , making it ideal for this experiment . the washi paper wick at the tip of a hypodermic needle is applied for an instant using a joystick manipulator with one hand . the needle is connected through a flexible tube to an injector manipulated with the other hand , for aspirating and discharging sucrose solution . frontal views of a starved fly with an extended proboscis ( arrows ) before the stimulation , at the stimulation , and after the stimulation with a wick containing 100 mm sucrose aqueous solution ( arrowhead ) . b , sucrose - induced gcamp 3.0 response in the motor neuron for the protractor of rostrum muscle in the starved state . the top panel , before the stimulation ; the bottom panel , immediately after the stimulation . c , quantification of the sucrose - induced gcamp 3.0 response . the motor neuron responds to a sucrose stimulus by a sharp increase in fluorescence followed by a restoration phase of several seconds to the base line . the flies allows for simultaneous recording of ca signals and per behavior . even with the brain exposed to saline normal per behavior was observed . using the gampi - washi wick instead of a kimwipe wick used in the original capillary method7 facilitates a highly reproducible and stable per behavior and avoids the need to become skilled in making and choosing a good kimwipe wick . the experimental tips stated above allowed us to successfully avoid disturbances by movement of the proboscis , leading to a very stable recording of ca signals with low noise . occasionally , tissue removal was not sufficient to expose cells or suppress movement adequately , leading to poor results . however , once we were skilled , more than 80 % of preparations produced good results . this methodology is not only for ca imaging , but can also be adapted to any live imaging while observing per behavior . for example , we can access any cell through the use of an electrode to directly record activity . combined with two - photon excitation microscopy , we are able to perform time lapse imaging of synaptic structure , which can be related to behavioral changes . therefore , this method of brain imaging with behavioral observation is not only valuable for the functional dissection of the neuronal network , but could be used as a powerful tool to correlate synaptic plasticity to the mechanisms behind memory .
to study neuronal networks in terms of their function in behavior , we must analyze how neurons operate when each behavioral pattern is generated . 
 thus , simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior . for such behavioral analyses , the fruit fly , drosophila melanogaster , 
 allows us to incorporate genetically encoded calcium indicators such as gcamp1 , to monitor neuronal activity , and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2 - 5 . 
 use of a thermogenetic technique has led us to find critical neurons for feeding behavior ( flood et al . , under revision ) . as a main part of feeding behavior 
 , a drosophila adult extends its proboscis for feeding6 ( proboscis extension response ; per ) , responding to a sweet stimulus from sensory cells on its proboscis or tarsi . combining the protocol for per7 with a calcium imaging technique8 using gcamp3.01 , 9 , 
 i have established an experimental system , where we can monitor activity of neurons in the feeding center  the suboesophageal ganglion ( sog ) , simultaneously with behavioral observation of the proboscis . 
 i have designed an apparatus ( " fly brain live imaging and electrophysiology stage " : " flies " ) to accommodate a drosophila adult , allowing its proboscis to freely move while its brain is exposed to the bath for ca2 + imaging through a water immersion lens . 
 the flies is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology . because the results from live imaging can be directly correlated with the simultaneous per behavior , this methodology can provide an excellent experimental system to study information processing of neuronal networks , and how this cellular activity is coupled to plastic processes and memory .