An Open-Source 3D-Printable Platform for Testing Head-Fixed Cognitive Flexibility in Rodents

Rig design and custom part schematics. A, Photograph of a completed rig. B, Rig schematic. C, Left, Arduino PCB shield wiring diagram. Right, Photograph of assembled PCB shield and sound card. D, Schematic top view and breakout views of 3D-printed components. P38, A set of adapters for mounting left and right lickspouts to a vertical translating post and an XY translating stage (lickSpoutPostHolder.stl); P70, A set of funnels for directing a concentrated low-frequency sound wave toward the vibrissae (speakerFunnel.stl); P9, An adapter for mounting the animal's head plate to a 3″ vertical post (headbar.stl); P28, An adapter for mounting air and water routing components, including a water bottle, a set of four odorant bottles, and a set of two 3-way stopcocks (airWaterHolder.stl); P48, A set of aluminum treadmill sprockets for supporting and rotating a conductive treadmill (treadmillSprockets.stl); P47, A base for holding the treadmill (treadmillBase.stl).

Interface between animal and hardware. A, Example trace of spontaneous treadmill running from a single animal. B, Treadmill running from the session shown in C, arranged as per-trial heat plots. Animals generally begin running in anticipation of trial onsets and stop running during lick responding and water consumption. C, Onset and offset of odorant concentration in the nose area, as measured with an MQ-3 sensor (10 sweeps). Latency between the odorant-on command signal and positive detection slope is 115–120 ms. D, Soundwave-mediated whisker vibration. Superimposed mean video frames from a 10 s baseline (whiskers in red) and 2 s whisker vibration (whiskers in green) recording. Stimulus presented was 155 Hz, and camera framerate was 30fps. Stimulus intensity was ∼80 dB at the whiskers. E, Maximum whisker displacement across a range of stimulus frequencies for the whiskers labeled in A.

Training of a cross-modal attentional set-shifting task in mice. A, Schematic of the head-fixed attentional set-shifting task. Mouse must periodically shift attention between compound whisker + odor stimuli to locate water. B, Positioning of lickspouts relative to the mouse's nose. C, Trial schematic. D, Raster plot of licks toward (blue) and away from (red) the animal's final choice (time 0). Stimuli begin 2.5 s before the onset of the 1.5 s response window. Anticipatory licking accelerates through the stimulus presentation epoch and takes on an 8–10 Hz oscillatory pattern (bottom). E, Response accuracy traces from the initial training sessions on simple whisker (left) or odor (right) discrimination, 20-trial moving window (N = 5 and N = 6, respectively). Criterion is 80% correct responses in a 20-trial moving window. Rank sum comparison for trials to criterion across modality, p = 0.2. F, Box plots of number of trials completed in the set-shifting task per session. N = 13 animals; 274 sessions; 123,482 trials; 1,108 trial blocks. G, Box plots of trials to criterion for whisker and odor discrimination blocks during set-shifting for the same 13 animals from F. Rank sum p = 0.1. H, Accuracy (percentage trials correct) for trials aligned to the rule switch (mean ± SD across the same 13 animals from F, G). I, Mean ± standard deviation, trial accuracy by modality rule, trial congruency, and trial timing relative to rule switch (within 10 trials pre- or postrule shift) for the sessions in F–H. Three-way ANOVA performed on the 20-trial periswitch window yielded significant effects of congruency (F = 1,323; p ≈ 0), trial timing (i.e., whether a trial was within the last 10 trials prior to a shift or the first 10 trials following a shift; F = 1,752; p ≈ 0), and the interaction of congruency × timing (F = 5.11; p = 0.02), but not for modality or any interactions with modality. Horizontal red lines indicate Bonferroni-corrected significance for group differences.

Assembly instructions (Steps 1 through 24). (1) Cover a 6″ × 6″ optical breadboard (Part P1; Table 1) with self-adhesive polytetrafluoroethylene (PTFE, P3) and cut through-holes for 1/4-20 screws. (2) Mount two 3″ × 1/2″ optical posts (P2). Insert adapters for mounting Arduino (P4) and set and cap screw for treadmill base. (3) Fasten Arduino case using a 4-40 button-head screw. (4) Fasten Arduino using a 4-40 cap screw. (5) Fasten flowmeter (P11) to air/water holder (P27) via an M5 cap screw (P12). Fasten NPT elbow barbs (P19) to flowmeter. (6) Attach Luer three-way stopcocks (P13) to an air/water holder. (7) Assemble connection from air intake stopcock to flowmeter by inserting a 6″ length of 1/16″ O.D. Teflon tubing (P20) into a 1/2″ length of 1/32″ I.D. silicon tubing (P26) at each end. At the stopcock end, couple the tubing to a 3/32″ male Luer barb (P16). The Teflon tubing should go inside the barb, silicon tubing outside the barb. (8) Thread male Luer barb onto a bottom female port on the air intake stopcock. (9) Couple Teflon/silicon tubing assemble to the barb on the flowmeter intake port. (10) Assemble connection from water intake stopcock to water bottle by inserting an 8″ length of 1/16″ O.D. Teflon tubing (P20) into a 1/2″ length of 1/32″ I.D. silicon tubing (P26) at each end. Move the silicon segment on the bottle side of the assembly to the middle of the assembly. Couple the stopcock side of the assembly to a 3/32″ female Luer barb (P15). (11) Insert the bottle end of the Teflon tubing through a Luer T-splitter (F/F/M, P14) at a right angle, so that the tubing exits the splitter through the bottom port. Attach a Luer male thread piece (P18) to the male port of the T-splitter. (12) Insert the free end of the Teflon tubing from the assembly into a 2 oz Luer-coupled water bottle (P25) and couple the bottle to the T-splitter. (13) Slot the water bottle onto the air/water holder and couple the T-splitter assembly from the water bottle to the air intake stopcock. (14) Fasten the air/water holder assembly to the base using low-profile 1/4-20 cap screws (P6). (15) Attach a pair of two-way 12 V solenoid valves to the solenoid face mount (P51) using three 2-56 cap screws, 1/2″ in length (P53). Note: the use of face-mount solenoids here, which requires a custom-machine face mount (P51), is optional. This design was favored as it enables the use of threaded ferrules for coupling the water and air tubing, which provides stability and security for these connections. Free-standing solenoids, which couple directly to tubing, are available from the same manufacturer. Attach a set of four three-way 12 V solenoid valves to the solenoid face mount. (16) Couple the water intake port on the solenoid face mount (side port) to the water intake stopcock. Create a tubing assembly by coupling a 12″ length of Teflon tubing (P20) to a 6-40 threaded ferrule (P23) on one end and a 3/32″ male Luer barb (P16) on the other end. (17) Create tubing assemblies for left and right lickspouts by coupling 2 8″ lengths of Teflon tubing (P20) to 6-40 threaded ferrules (P23) on the one end and F/M Luer elbow connectors (P17) on the other. Screw the ferrules from these assemblies into the left and right water output ports on the front of the solenoid face mount. (18) Attach the lickspout holder (P38) to the vertical translation post (P31) using a plastic 8-32 thumb screw (P35). Note: the use of plastic here is essential, to prevent current conduction between the left and right lickspouts. (19) Attach the vertical translation post to the post holder piece using a 3/4″-long 2-56 screw and nut. (20) Attach the post holder to the XY translation stage (P32) using a 1/4″-long 8-32 cap screw (P39). (21) Slide the XY translation stage to the base of the lickspout holder. (22) Attach the lickspout holder assemble to the base using 1/4-20 cap screws. (23) Assemble the treadmill by first inserting the steel axle bearings (P41) into the front and rear bearing slots on the treadmill holder base. (24) Secure the bearings to the treadmill holder base using 5/16″-long 4-40 screws and nuts (P43).

Assembly instructions (Steps 25 through 48). (25) To the left front bearing screw, couple a 2″ length of wire and a 3 mm banana plug (P46). (26) Insert 2″ posts (P42) into treadmill sprockets (P48) to serve as axles. (27) Insert sprocket axles into front and rear bearings, and mount treads (P40) around front and rear sprockets. 28) Slot the completed treadmill assembly onto the base using the set screw and cap screw that protrude from the base. Adjust the height of the rear cap screw so that the treadmill base slots securely beneath it. (29) Separate and twist the ends of a seven-wire ribbon cable (P68), and insert three of the wires into the screw terminals corresponding with the 3.3 V, A0, and A5 channels on the PCB board. (30) Couple the other end of the ribbon cable (∼10″ in length) to the base of the left head post using a brass lug or bare wire. (31) Screw the left head post back onto the base. (32) Solder the wires corresponding with channels A0 and A5 to the left and right lickspout gavage needles (P30), respectively. Secure gavage needles to the lickpost holder using the plastic 8-32 thumb screw. (33) Insert the banana plug into the side hole near the top of the left head post. Current of 3.3 V will now flow as follows: Arduino→ribbon wire→head post→banana plug→4-40 cap screw→steel bearing→axle and sprocket→treadmill→animal→gavage needle→ribbon wire→Arduino analog input channels. (34) Connect the four remaining wires from the ribbon wire bundle to the left +/− and right +/− screw terminals of the sound board (P54). (35) Insert the header pins from the sound board to the corresponding sockets on the PCB board. The sound board can be loaded with .wav files by connecting it to a computer via USB connection. (36) Assemble speaker unit pieces. (37) Thread left +/− and right +/− wires from the ribbon wire bundle through the ball-and-socket swivel mount (P66) and speaker backing piece, then soldering to the +/− contacts on the speaker (P65). (38) Set the speaker backing piece into the swivel mount, followed by the speaker. Fasten the swivel mount to the base by screwing an 8-32 to 1/4-20 thread adapter (P68) into the bottom of the swivel mount. Loosen the ball to allow it to rotate ad libitum, then screw it to the base, and re-tighten. (39) Fasten the speaker funnel (P70) to the front of the speaker assemble using a retaining ring. (40) Create three tubing-ferrule assemblies, using 2.5″ of 1/16″ O.D. Teflon tubing for one and 2″ of the same tubing for the other two assemblies. Couple to 6-40 threaded ferrules by feeding the tubing through the ferrule so that it is flush with the ferrule tip, then screwing tightly into a 6-40 threaded socket. Wrap ferrule threads in a Teflon tape to ensure tight seals. (41) Connect the tubing assemblies from Step 40 to the solenoid face mount. For the four three-way solenoids used to direct air flow for odorant delivery, the middle port on each solenoid is the intake. With the solenoid is in an “off” state (0 V, the bottom port is the exhaust, and with 12 V applied across the solenoid pins, the top port is the exhaust. The leftmost 3-way solenoid (when viewed from the tubing side of the face mount) directs clean air directly to the animal when off, and toward the middle/intake port of solenoid 3 (third from left) when activated. Solenoid 3 routes air to odorants A and B when off and to odorants C and D when activated. Solenoid 2 routes air to odorant C when off and D when activated. Solenoid 4 routes air to odorant A when off and to odorant B when activated. (42) Create a tubing assembly to direct air from the flowmeter to the odorant solenoids. Use a 10″ length of Teflon tubing (P20). On the flowmeter/intake side, couple the tubing to the top/exhaust port of the flowmeter using a 1/2″ length of 1/32″ I.D. silicon tubing (P26). Couple the exhaust side to a 6-40 threaded ferrule (P23) and screw into the solenoid face mount. (43) Create an odorant bypass line using an 8″ length of Teflon tubing (P20), coupled to 6-40 threaded ferrules at both ends (P23). Screw one end of the assembly into the odorant bypass port (bottom port of the leftmost solenoid), and screw the other end into the odorant manifold (P29). Note: the use of a custom-milled aluminum manifold here was adopted in order to keep a small footprint on the rig and to ensure a secure connection. Those wishing to avoid the need for this custom piece can substitute a barbed or Luer-style manifold. (44) Create four odorant intake tubes to connect the odorant solenoids to the odorant bottles. For each, use an 8″ length of Teflon tubing (P20), coupled to a 6-40 threaded ferrule on the one side (P23) and a barbed Luer check valve on the other (P21). The use of check valves on both sides of each odorant tube prevents backdrifting and comingling of odorants. (45) Screw the tubing assemblies into the solenoid face mount according to the mapping shown in Step 41. (46) To route the odorant intake air to the odorant within the bottle, insert a 1–1.5″ length of Teflon tubing (P20) into a 1/4″ length of 1/8″ O.D. 1/16″ I.D. (P27) silicon tubing. (47) Mount this tubing assembly into the tip of the male intake Luer check valve, and guide the tip of the Teflon tubing through the Luer T-splitter at a right angle, so that it protrudes from the other female Luer port. (48) Insert the protruding Teflon tubing into the opening of a Luer-threaded 0.25 oz bottle (P24), and screw the Luer connector securely.