Generalized apparatus for behavioral assessment

Abstract

A fixation system comprising a base, a platform connected atop the base, one or more restraining arms positioned above the platform, a translational manipulator positioned adjacent the platform, a plurality of spouts connected to the translational manipulator, a liquid delivery system fluidly connected to at least two spouts of the one or more spouts, an air delivery system fluidly connected to at least one spout of the one or more spouts, and a controller communicatively connected to the liquid delivery system and the air delivery system, wherein the controller is configured to control the delivery of the one or more stimuli, wherein the liquid delivery system and the air delivery system are configured to provide one or more stimuli to a subject.

Description

Attorney Docket No : 206150-0011-00WOGENERALIZED APPARATUS FOR BEHAVIORAL ASSESSMENTCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 741,275 filed on January 2, 2025, the contents of which are incorporated by reference herein in its entirety.BACKGROUND

[0002] In 2023, over $1.4 billion in grant funding from the NIH, CDC, FDA, AHRQ, and ACF was allocated to mouse behavior research (NIH RePORTER). Mouse behavior studies are a standard approach to investigating neurological disorders, pharmacological interventions, genetic influences, environmental factors, and cognitive functions underlying behavioral processes. There are two common strategies to study cognitive processes like associative learning and motivation during mouse behavior - freely moving and head-fixed approaches (Guo et al. 2014, Juczewski et al. 2020). Metal head-bars are surgically implanted into the skull to head-fix mice and restrict brain movement during behavior. Head fixation offers significant advantages over a freely moving approach, including easy brain access, reduced noise and motion artifacts, and high temporal resolution during behavioral tasks. These benefits make head fixation a standard experimental approach used in conjunction with neuroscience techniques that allow one to precisely track and alter brain activity, such as optogenetics, calcium imaging, and electrophysiology (Matsumoto & Hikosaka et al. 2009, Menegas et al. 2017). Despite the benefits, head fixation incurs a high cost - restrained animal movement limits the ease and feasibility of implementing various associative learning and operant tasks (Nasr et al. 2022).

[0003] In basic research, head-fixed experimental systems are pivotal to implementing associative learning and operant conditioning tasks during neural activity recording in mice. While systems often overlap in utility, they accomplish these functions through different methodologies. Commercial head fixation structures alone can cost hundreds of dollarsAttorney Docket No : 206150-0011-00WO(Labeotech, Maze Engineers). Experiment-ready systems that use high-end parts to control environmental stimuli, record behavior, and orchestrate task protocols can cost upwards of $10,000 (Neurotar). In response to the high cost of these systems, some labs have produced custom-made systems that offset expenses while retaining function through cheap microcomputers and 3D-printed parts. However, even these custom systems are not fully optimized for cost-efficiency and can cost upwards of $10,000 (OHRBETS, B-CALM), with raw material costs of about $2000. Moreover, there are clear areas to improve design simplicity and functionality. For example, custom systems often rely on simple structural components purchased through commercial vendors rather than 3D-printing these parts. Custom systems also typically only include functions to deliver rewarding but not aversive stimuli. Many of the capabilities of existing systems leave substantial room for design improvement to enhance functionality and promote longevity.

[0004] Thus, there is a need in the art for cost-effective head-fixed experimental systems that use electronic components to control environmental stimuli and measure behavioral and neural readouts across various tasks.SUMMARY

[0005] Described herein is a fixation system comprising a base, a platform connected atop the base, one or more restraining arms positioned above the platform, a translational manipulator positioned adjacent the platform, a plurality of spouts connected to the translational manipulator; a liquid delivery system fluidly connected to at least two spouts of the one or more spouts, an air delivery system fluidly connected to at least one spout of the one or more spouts, anda controller communicatively connected to the liquid delivery system and the air delivery system, wherein the controller is configured to control the delivery of the one or more stimuli, wherein the liquid delivery system and the air delivery system are configured to provide one or more stimuli to a subject.

[0006] In some examples, the one or more stimuli may be chosen from the group consisting of: appetitive stimuli, aversive stimuli, visual stimuli, auditory stimuli, gustatory stimuli, olfactoryAttorney Docket No : 206150-0011-00WOstimuli, thermal stimuli, and combinations thereof. In some examples, the platform is freely rotatable. In some examples, the platform is attached to the base via a ball and socket joint, configured to angle the platform in a plane. In some examples, the platform is configured to allow the subject to stand or move freely. In some examples, the platform is configured to fix at least a portion of a body of the subject to the platform via the restraining arms.

[0007] In some examples, the liquid delivery system delivers appetitive or gustatory stimuli to the subject. In some examples, the liquid delivery system comprises one or more liquid reservoirs and one or more valves connected between the liquid reservoirs and the at least two spouts of the one or more spouts and wherein the one or more valves are communicatively connected to the controller. In some examples, the one or more liquid reservoirs comprise water, a mixture of liquids, a solution, a sucrose solution. In some examples, the air delivery system delivers aversive or tactile stimuli to the subject. In some examples, the air delivery system comprises an air reservoir, a pump, and a valve connected between the air reservoir and the at least one spout and wherein the valve is communicatively connected to the controller.

[0008] In some examples, the fixation system further comprises one or more camera holders configured to position one or more cameras to image behavioral responses from the subject. In some examples, the position of the one or more spouts is adjustable via the translational manipulator. In some examples, the one or more spouts are retractable. In some examples, the one or more spouts each comprise a capacitance sensor, configured to detect capacitance changes in the spout when the subject licks the spout. In some examples, the fixation system further comprises a light delivery device configured to deliver visual or thermal stimuli to the subject. In some examples, the fixation system further comprises a sound delivery device configured to deliver auditory stimuli to the subject. In some examples, the fixation system further comprises one or more sensors, communicatively connected to the controller, wherein the one or more sensors are configured to monitor stimuli intensity or measure physiological changes in the subject in response to the stimuli.

[0009] In some examples, the controller is configured to control a frequency, timing, and intensity of the delivery of the one or more stimuli. In some examples, the controller is communicatively connected to a computing system comprising a processor and a non-transitoryAttorney Docket No : 206150-0011-00WOcomputer readable medium, wherein the non-transitory computer-readable medium comprises instructions, which when executed by the processor positions the one or more spouts and delivers a set of stimuli to the subject via the spouts.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The foregoing purposes and features, as well as other purposes and features, will become apparent with reference to the description and accompanying figures below, which are included to provide an understanding of the invention and constitute a part of the specification, in which like numerals represent like elements.

[0011] Fig. 1 shows a perspective view of an exemplary head fixation system.

[0012] Figs. 2A through 2F show different views of an exemplary fixation platform. Fig. 2A shows a perspective view of an exemplary fixation platform. Fig. 2B shows a front view of an exemplary fixation platform. Fig. 2C shows a side view of an exemplary fixation platform. Fig.2D shows a top view of an exemplary fixation platform. Fig. 2E shows a bottom view of an exemplary fixation platform. Fig. 2F shows exemplary planes of rotation of the fixation platform.

[0013] Figs. 3A through 3F show multiple views of an exemplary translational manipulator. Fig.3A shows a perspective view of an exemplary translational manipulator. Fig. 3B shows a front view of an exemplary translational manipulator. Fig. 3C shows a side view of an exemplary translational manipulator. Fig. 3D shows a top view of an exemplary translational manipulator. Fig. 3E shows a bottom view of an exemplary translational manipulator. Fig. 3F shows exemplary axes of motion of the translational manipulator.

[0014] Figs. 4A through 4F show multiple views of exemplary liquid-delivery spouts. Fig. 4A shows a perspective view of the exemplary liquid-delivery spouts. Fig. 4B shows a front view of the exemplary liquid-delivery spouts. Fig. 4C shows a side view of the exemplary liquid-delivery spouts. Fig. 4D shows a top view of the exemplary liquid-delivery spouts. Fig. 4E shows a bottom view of the exemplary liquid-delivery spouts. Fig. 4F is an image showing exemplary liquid-delivery spouts in retracted and non-retracted configurations.Attorney Docket No : 206150-0011-00WO

[0015] Figs. 5A through 5E show multiple views of an exemplary liquid-delivery system. Fig.5A shows a perspective view of the exemplary liquid-delivery system. Fig. 5B shows a front view of the exemplary liquid-delivery system. Fig. 5C shows a side view of the exemplary liquid-delivery system. Fig. 5D shows a top view of the exemplary liquid-delivery system. Fig.5E shows a bottom view of the exemplary liquid-delivery system.

[0016] Figs. 6A through 6E show multiple views of an exemplary air-delivery system. Fig. 6A shows a perspective view of the exemplary air-delivery system. Fig. 6B shows a front view of the exemplary air-delivery system. Fig. 6C shows a side view of the exemplary air-delivery system. Fig. 6D shows a top view of the exemplary air-delivery system. Fig. 6E shows a bottom view of the exemplary air-delivery system.

[0017] Figs. 7A through 7G show multiple views of an exemplary camera holder. Fig. 7A shows a perspective view of the exemplary camera holder. Fig. 7B shows a front view of the exemplary camera holder. Fig. 7C shows a side view of the exemplary camera holder. Fig. 7D shows a top view of the exemplary camera holder. Fig. 6E shows a bottom view of the exemplary camera holder. Fig. 7F shows an exemplary image taken from a camera of the fixation system. Fig. 7G shows an exemplary image taken from a camera of the fixation system.

[0018] Figs. 8A through 8G show multiple views of controller. Fig. 8A shows a perspective view of the exemplary controller. Fig. 8B shows a front view of the exemplary controller. Fig.8C shows a side view of the exemplary controller. Fig. 8D shows a top view of the exemplary controller. Fig. 8E shows a bottom view of the exemplary controller.

[0019] Fig. 9 depicts the electronic components of the controller.

[0020] Fig. 10 depicts an exemplary computing environment in which aspects of the present disclosure may be practiced.

[0021] Figs. 11A through 1 IF show the results of proof-of-principle experiments using fiber photometry. Fig. 11A shows a schematic showing viral injection into dorsal striatum (DS) and optic fiber implantation in the same region. Fig. 1 IB shows exemplary slice of a mouse showing virus expression (left) and optic fiber implantation in dorsal striatum and a schematic of dorsal striatum based on the Allen Brain Atlas Image P56 (right). Fig. 11C shows a schematic of rewardAttorney Docket No : 206150-0011-00WOand air puff delivery in a head-fixed mouse. Fig. 1 ID shows a plot showing the group average (n=3 mice) of lick rate during reward delivery. Fig. 1 IE shows a plot showing the group average (n=3 mice) of dopamine transients in DS during air puff delivery. Fig 1 IF shows a plot showing the group average (n=3 mice) of dopamine transients during reward delivery.

[0022] Figs. 12A through 12D show the results of proof-of-principle experiments using fiber photometry. Fig. 12A shows a schematic of auditory cue and reward delivery onto left or right spout alternating every 20 to 30 trials. Fig. 12B depicts a plot showing the group average (n=10) of licks during cued reward delivery. Fig. 12C depicts a plot showing the group average (n=10 mice) of dopamine transients in the dorsal striatum during cued reward delivery. Fig. 12D depicts plots showing the group average (n=10 mice) of anticipatory licks onto both spouts per trial across two reversals of the reward-spout contingency after 10 training days.

[0023] Figs. 13A through 13D show the results of a proof of principle experiments using fiber photometry. Fig. 13A depicts a schematic showing spout choice during cue that leads to reward or omission, with a contingency reversal after 30 to 50 trials. Fig. 13B depicts a plot showing the group average (n=7 mice) of error rate across trials aligned to the reward-spout contingency reversal. Fig. 13C depicts a plot showing the group average (n=7 mice) traces of dopamine response in the dorsal striatum during correct and incorrect choice trials. Fig. 13D depicts a plot showing the paired t-test (n=7 mice) comparing dopamine AUC during correct vs. incorrect trials between choice and outcome.

[0024] Fig. 14 depicts images showing the results of histology experiments validating the results of the experiments of Figs. 1 IE and 1 IF.

[0025] Fig. 15 depicts images showing the results of histology experiments validating the results of the experiment of Fig. 12C.

[0026] Fig. 16 depicts images showing the results of histology experiments validating the results of the experiments of Figs. 13C and 13D.DETAILED DESCRIPTIONAttorney Docket No : 206150-0011-00WO

[0027] The following discussion omits or only briefly describes conventional features of head fixation devices that are apparent to those skilled in the art. Those of ordinary skill in the pertinent arts may thus recognize that other elements may be desirable and / or necessary to implement the devices, systems, and / or methods described herein. It is noted that various examples are described in detail with reference to the drawings. Reference to these various examples does not limit the scope of the claims attached hereto. Additionally, any examples set forth in this specification are intended to be non-limiting and merely set forth some of the many possible implementations for the appended claims. Further, particular features described herein can be used in combination with other described features in each of the various possible combinations and permutations. As such, it is understood that this detailed description is exemplary and explanatory only and is not restrictive of the broad inventive concepts upon which the examples disclosed herein are based.

[0028] Unless otherwise specifically defined herein, all terms are to be given their broadest reasonable interpretation. This includes meanings implied from the specification as well as meanings understood by those skilled in the art and / or as defined in dictionaries, treatises, etc.

[0029] It is noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless otherwise specified. The term “includes” and / or “including,” when used in this specification, specify the presence of stated features, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0030] Relative terms such as “horizontal,” “vertical,” “up,” “down,” “top,” and “bottom” as well as derivatives thereof (e.g., “horizontally,” “downwardly,” “upwardly,” etc.) should be construed to refer to the orientation as then-described or as shown in the drawing figure under discussion. These relative terms are for convenience of description and normally are not intended to require a particular orientation in actuality. Terms including “inwardly” versus “outwardly,” “longitudinal” versus “lateral,” and the like are to be interpreted relative to one another or relative to an axis of elongation, or an axis or center of rotation, as appropriate. Terms concerning attachments, coupling and the like, such as “connected” and “interconnected,” refer to a relationship wherein structures are secured or attached to one another either directly orAttorney Docket No : 206150-0011-00WOindirectly through intervening structures, as well as both movable or rigid attachments or relationships, unless expressly described otherwise. The phrases “operatively” or “operably connected” indicates such an attachment, coupling, or connection that allows the pertinent structures to operate as intended by virtue of that relationship.

[0031] Reference throughout the specification to “exemplary,” “one example,” “an example,” or “some examples” means that a particular feature, structure, or characteristic described in connection with at least one example of the subject matter is included in at least one example of the subject matter disclosed. Thus, the appearance of the phrases “in one example,” “in an example,” or “in some examples” in various places throughout the specification is not necessarily referring to the same example. Further, the particular features, structures, or characteristics of “one example,” “an example,” or “some examples” may be combined in any suitable manner with each other to form additional examples of such combinations. It is intended that examples of the disclosed subject matter cover modifications and variations thereof. Terms such as “first,” “second,” “third,” etc., merely identify one of a number of portions, components, steps, operations, functions, and / or points of reference as disclosed herein, and likewise to not necessarily limit examples of the present disclosure to any particular configuration or orientation.

[0032] Moreover, throughout this disclosure, various aspects can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, 6, and any whole and partial increments therebetween. This applies regardless of the breadth of the range. As used herein, the term “about” in reference to a measurable value, such as an amount, a temporal duration, and the like, is meant to encompass the specified value and / or variations of plus or minus 20%, plus or minus 10%, plus or minus 5%, plus or minus 1%, and plus or minus 0.1% of the specified value, as such variations are appropriate and fit within the confines of a functional system.Attorney Docket No : 206150-0011-00WO

[0033] The terms “proximal,” “distal,” “anterior,” “posterior,” “medial,” “lateral,” “superior,” and “inferior” are defined by their standard usage indicating a directional term of reference. For example, “proximal” refers to a position that is situated nearer to the center of a body or point of attachment or interest. In another example, “anterior” refers to the front of a body or structure, while “posterior” refers to the rear of a body or structure, in relation to a relative viewpoint. In another example, “medial” refers to the direction towards the midline of a body or structure, and “lateral” refers to the direction away from the midline of a body or structure. In some examples, “lateral” or “laterally” may refer to any sideways direction. In another example, “superior” refers to the top of a body or structure, while “inferior” refers to the bottom of a body or structure. It should be understood, however, that the directional term of reference may be interpreted within the context of a specific body or structure, such that a directional term referring to a location in the context of the reference body or structure may remain consistent as the orientation of the body or structure changes.

[0034] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein and refer to any human, animal, or other living organism amenable to the systems, devices, and methods described herein.

[0035] Described herein is a fixation system configured for use with a subject, such as a mouse or other small animal, such that the subject is wholly or partially held in a fixed position during experiments. In some examples, the fixation system is configured to administer one or more stimuli to the object. In some examples, the fixation system is capable of imaging a subject’s behavioral responses to the administered stimuli.

[0036] Referring now to Fig. 1, shown is a perspective view of the fixation system 100.Generally, the fixation system 100 includes one or more of a fixation platform 102, a translational manipulator 104, a spout attachment 106, a liquid delivery system 108, an air puff delivery system 110, a camera holder attachment 112, and a controller 114. The fixation system 100 may be configured to at least partially fix a subject’s body on the fixation platform 102. In some examples, the fixation system 100 is configured to provide one or more stimuli to the subject. The one or more stimuli may be chosen from: appetitive stimuli, aversive stimuli, tactileAttorney Docket No : 206150-0011-00WOstimuli, visual stimuli (e.g. rotational motion of the spouts or static / fl ashing light stimuli), gustative stimuli, auditory stimuli, olfactory stimuli, and combinations thereof.

[0037] Referring now to Figs. 2A - 2E, shown are multiple views of the fixation platform 102. The fixation platform 102 generally comprises one or more of a base 202, a platform 204 connected atop the base 202, and a first and second restraining arms 206 and 208 positioned on two opposing sides of the platform 204. In some examples, the platform 204 may be a running wheel platform, configured such that the platform 204 may rotate freely about a vertical axis of rotation 210 at the center of the platform (see Fig. 2B). In some examples, the platform 204 is connected to the base 202 via a ball and socket mechanism, thereby allowing the base 202 to be freely oriented in any desired plane as depicted in Fig. 2F. The base 202 may be configured to be clamped to a flat surface and may have an adjustable height. In some examples, the platform 204 may have a circular, ovular, square, rectangular, polygonal, or irregular shape. In some examples, the platform 204 may have a length or diameter ranging between about 5 cm and 50 cm, and a thickness ranging between about 0.1 cm and 1 cm. In some examples, the sizes of the base 202, and the restraining arms 206 and 208 may be adjusted based on the size of the platform, the size of the subject, and / or experimental needs.

[0038] In some examples, the first and second restraining arms 206 and 208 each comprise a horizontal bar 212 and a vertical post 214 oriented perpendicular to each other and attached via a coupler 216. In some examples, the horizontal bar 212 may have a length ranging between about 5 cm and 10 cm, and a thickness or diameter ranging between 0.1 cm and 2 cm. In some examples, the vertical post 214 may have a height ranging between 1 cm and 10 cm. The coupler 216 may further comprise a screw 218 configured to clamp the horizontal bar 212 and thereby secure at least part of subject’s body to the platform 204. In some examples, the vertical posts 214 may be clamped to a flat surface and may have an adjustable height. In some examples, one or more components of the fixation platform 102 may be 3D-printed. In some examples, the fixation platform 102 may comprise any material known to one of skill in the art. For example, plastics, metals, metal alloys polymers, and any combinations thereof may be utilized. In some examples, the fixation apparatus 102 is configured to fit a subject, such as a mouse or other small animal, and provides a surface on which the subject may freely stand, walk, or run. In someAttorney Docket No : 206150-0011-00WOexamples, the fixation apparatus 102 is configured to fix at least a part of the body of the subject on the platform 204 via the restraining arms 206, 208.

[0039] Referring now to Figs. 3 A - 3E, shown are multiple views of a translational manipulator 104. The translational manipulator 104 generally comprises a frame 302, a first connector 304, a second connector 306, and a third connector 308. In some examples, the first, second and third connectors 304, 306, and 308 are configured to be separably movable. In some examples, the first connector 304 is attached to the frame 302 via a screw 310. In some examples, a hex nut is embedded within the first connector 304 and the screw 310 passes through the grooves of the hex nut which serves the minimize wear on the connector 204 upon rotation of the screw 310. Screw 310 is fixedly attached at two opposite ends within the frame 302 and passes through the first connector 304. Screw 310 is configured such that rotating the screw 308 allows the first connector 304 to move along the x-axis (or in a left to right direction) as depicted in Fig. 3F. The second connector 306 is oriented perpendicular to the first connector 304 and is attached to the first connector 304 via a second screw 312. In some examples, a hex nut is embedded within the second connector 306 and the screw 312 passes through the grooves of the hex nut which serves to minimize wear on the second connector 306 upon rotation of the screw 312. The screw 312 is fixedly attached at two opposing ends within the first connector 304 and passes through the second connector 306. Screw 312 is configured such that rotating the screw 312 allows the second connector to move along the y-axis (or in a forwards and backwards direction) as depicted in Fig. 3F. The second connector 306 may comprise a third screw 314 fixedly attached to two opposing ends within the second connector 306 and passing through the third connector 308. In some examples, a hex nut is embedded within the third connector 308 and the screw 314 passes through the grooves of the hex nut which serves to minimize wear of the third connector 308 upon rotation of the screw 314. Screw 314 is configured such that rotating screw 314 causes the third connector 308 to move along the z-axis (or in an up and down motion), as depicted in Fig. 3F. In some examples, each screw 310, 312, and 314 may be attached to a dial, such as dials 311, 313, and 315, to facilitate the rotation of the screws. In some examples, the translational manipulator may comprise one or more motors, which may be configured to move the connectors 304, 306, and 308 along their respective axes.Attorney Docket No : 206150-0011-00WO

[0040] In some examples, the third connector 308 further comprises a holder 316 configured to attach one or more lick spouts to the translational manipulator 104. In some examples, the translational manipulator 104 allows for precise positioning and adjustment of the one or more lick spouts via rotating the screws 310, 312, and 314. In some examples, the frame 302 further comprises a camera holder 318 configured to fixedly attach and orient a camera such that a subject’s snout may be imaged from below the platform 204. In some examples, the translational manipulator 104 is positioned adjacent to a subject’s head while the subject is secured to the fixation platform 102. In some examples, any combination of the components of the translational manipulator 104 may be 3D-printed, and each component may comprise any suitable material known to one of skill in the art. In some examples, the may be configured to acquire one or more images of the subject. In some examples, the camera may be configured to acquire a live video stream fo the subject.

[0041] Referring now to Figs. 4A - 4E, shown are multiple views of a spout attachment 106. Spout attachment 106 generally comprises one or more lick spouts 402, a base 404, one or more holders 406, each attached to a lick spout 402, and a coupler 408. The coupler 408 is configured to be secured to the third connector 308 of the translational manipulator 104 such that the spout attachment is movable via adjustment of the translational manipulator 104. In some examples, the spout attachment 106 may comprise any number of lick spouts 402. For example, the spout attachment 106 may comprise at least 1, at least 2, at least 3, at least 4, or at least 5 lick spouts. In some examples, the holders 406 are rotatable about axes of rotation 410 and 412. This configuration allows each lick spout 402 to rotate about each axis such that each lick spout 402 may be angled closer to or further from subject’s mouth / snout and may have a retracted or nonretracted configuration (as depicted in Fig. 4F). Each lick spout 402 may be rotated about an angle ranging between 0° and 45°. In some examples, the spout attachment 106 further comprises one or more motors (not pictured) connected to the holders 406 and configured to rotate the one or more lick spouts 402 such that the lick spouts may be moved from the nonretracted configuration to the retracted configuration, or vice versa. In some examples, the one or more motors may control the speed of rotation and rotational angle of the one or more lick spouts 402. In some examples, the one or more motors may be communicatively connected to the controller 114, wherein instructions received via the controller 114 cause the motor to adjust the positioning of the lick spouts 402. In some examples, the lick spouts 402 each further comprise aAttorney Docket No : 206150-0011-00WOcapacitance sensor configured to detect capacitance changes in the lick spouts 402 when a subject licks the spout. In some examples, the one or more lick spouts 402 may be retracted at a timescale sufficient to prevent erroneous licking behavior during liquid consumption tasks. That is, the one or more lick spouts 402 may detect a lick and rapidly retract before a second lick occurs, which is crucial for obtaining precise data during decision making tasks. In some examples, the retraction time of each spout of the one or more spouts 402 may be less than 10 ms, less than 20 ms, less than 30 ms, less than 40 ms, or less than 50 ms.

[0042] Referring now to Fig. 5 A - 5E, shown are multiple views of a liquid delivery system 108 for use with the head fixation system 100. In some examples, the liquid delivery system 108 comprises one or more reservoirs 502, wherein each reservoir 502 is fluidly connected to the spout attachment 106 via tubing 504. In some examples, and referring back to Fig. 4A, the spout attachment 106 connects tubing 504 from each reservoir 502 to each lick spout 402 via openings 409 in the spout attachment 106. The reservoirs 502 may be configured to hold any suitable liquid, for example water, a mixture of liquids, a solution, a sucrose solution, a saline solution, an alcohol solution, a quinine solution, a non-nutritive flavorant, and any combinations thereof. In some examples, each reservoir 502 may hold the same liquid, or may hold different liquids. In some examples, the reservoirs 502 may be syringes that are mounted on a 3D-printed holder, wherein the syringes are each fluidly connected to each lick spout 402 via tubing 504. In some examples, valves 506 may be connected between the reservoirs 502 and the lick spout 402. The valves 506 may be solenoid valves configured to control the amount and timing of the liquid delivery to the lick spouts 402. In some examples, the valve 506 may be communicatively connected to the controller 114, wherein the controller 114 is configured to actuate the opening / closing of each valve 506. For example, the liquid delivery system 108 may be configured to deliver a first amount of solution to a first lick spout 402 for a first duration, and a second amount of solution to a second lick spout 402 for a second duration. In other examples, the liquid delivery system 108 may be configured to deliver a solution to each lick spout 402 at a predetermined rate of droplets / min, where the rate of delivery to each lick spout 402 may be the same or may be different. In some examples, the liquid delivery system 108 is configured to deliver one or more appetitive or gustative stimuli to the subject.Attorney Docket No : 206150-0011-00WO

[0043] Referring now to Figs. 6A - 6E, shown are multiple views of an air puff delivery system 110. In some examples, the air puff delivery system 110 may comprise one or more of an air reservoir or air pump (not pictured), an air spout 602 fluidly connected to the air reservoir, and a valve 604 connected between the air reservoir and the air spout 602. In some examples, the spout 602 may be attached to a 3D-printed holder 606, wherein the holder 606 comprises a base 608, a flexible stem 610 and a spout attachment 612. The holder 606 may be configured to be fixed to a flat surface and the position of the air spout 602 may be adjusted via the flexible stem 610. In some examples, the valve 604 may be a solenoid valve, and may be communicatively connected to the controller 114, such that the controller actuates the opening / closing of the valve 604. In some examples, an air pressure regulator may be connected between the air reservoir and the air spout 602, configured to adjust the air pressure. In some examples, air or gas may be delivered at a pressure ranging between 5 and 40 PSI. In some examples, the air puff delivery system 110 may have two modes of pressurized air pumping. In some examples, the valve 604 may be connected to two different sources of airflow. For example, the valve 604 may be connected to a motorized pump, or to an external positive air supply. In some examples, the air puff delivery system 110 may be configured to deliver an aversive stimulus to the subject’s eye or snout. In some examples, the air puff delivery system 110 may be configured to deliver a tactile stimulus to the subject.

[0044] Referring now to Figs. 7A - 7E, shown is a camera holder attachment 112, configured to be positioned adjacent to the fixation platform 102 and / or the translational manipulator 104. In some examples, the camera holder attachment 112 may generally comprise a base 702, a stem 704 attached to the base 702, and a frame 706 attached to the stem 704. The frame 706 may be configured to fit a camera 708 or any other imaging device known to one of skill in the art. In some examples, the stem 704 may be flexible such that the position and orientation of the frame 706, and therefore the camera, may be adjusted. In some examples, the camera may be communicatively connected to the controller 114, and / or a display configured to display the camera feed in real-time. In some examples, the live feed may be utilized for the proper positioning of the lick spouts, or to observe subjects during behavioral experiments. Figs. 7F and 7G depict exemplary images of a subject on the head fixation system 100 taken by the camera 708.Attorney Docket No : 206150-0011-00WO

[0045] In some examples, the head fixation system 100 may further comprise a sound delivery device (e.g. a speaker) or a light delivery device (e.g. an LED or a laser) to provide auditory or visual stimulation to the subject. In some examples, the head fixation system 100 may further comprise one or more sensors to monitor stimuli intensity, or to measure physiological changes in the subject in response to the one or more stimuli. The one or more sensors may include, but are not limited to, biosensors, thermal sensors, pressure sensors and diode laser systems. In some examples, the diode laser system may be configured for timed manipulation of cellular activity via genetic technologies (optogenetics) based on behavioral or physiological feedback.

[0046] Referring now to Figs. 8A - 8E, shown is the controller 114 for use with the head fixation system 100. The controller 114 may be communicatively connected to one or more of: the translational manipulator 104, the spout attachment 106, the liquid delivery system 108, the air delivery system 110, the light delivery device, the sound delivery device, or the one or more cameras. In some examples, instructions may be provided to the controller 114 to deliver one or more stimuli, or one or more sets of stimuli. In some examples, instructions may be provided to the controller 114 such that the head fixation system 100 automatically delivers a predetermined set of stimuli for a predetermined amount of time. In some examples, the controller 114 may be communicatively connected to one or more sensors to monitor stimulation intensity or physiological changes in a subject in response to the stimuli. In some examples, the controller 114 may comprise an Arduino, one or more transistors, and a power supply module. In some examples, the controller 114 may be communicatively connected to a computing system (for example computer 2000 depicted in Fig. 10). The electronic components of the controller 114 are depicted in Fig. 9.

[0047] In some examples, the head fixation system 100 is capable of rapid internal feedback between the one or more sensors and the stimulus delivery systems (e.g. the liquid delivery system 108, or the air puff delivery system 110), thereby enabling closed-looped experimental control. In some examples, behavioral data is generated in real-time to drive the output of stimuli at any specified timepoint. In some examples, the head fixation system 100 is capable of adaptive behavioral experiments. For example, the system can respond to animal behavior with millisecond precision to adjust parameters, such as task difficulty level, reward delivery thresholds, spout placement, air puffintensity.Attorney Docket No : 206150-0011-00WO

[0048] In some aspects of the present invention, software executing the instructions provided herein may be stored on a non-transitory computer-readable medium, wherein the software performs some or all of the steps of the present invention when executed on a processor.

[0049] Aspects of the invention relate to algorithms executed in computer software. Though certain embodiments may be described as written in particular programming languages, or executed on particular operating systems or computing platforms, it is understood that the system and method of the present invention is not limited to any particular computing language, platform, or combination thereof. Software executing the algorithms described herein may be written in any programming language known in the art, compiled or interpreted, including but not limited to C, C++, C#, Objective-C, Java, JavaScript, MATLAB, Python, PHP, Perl, Ruby, or Visual Basic. It is further understood that elements of the present invention may be executed on any acceptable computing platform, including but not limited to a server, a cloud instance, a workstation, a thin client, a mobile device, an embedded microcontroller, a television, or any other suitable computing device known in the art.

[0050] Parts of this invention are described as software running on a computing device. Though software described herein may be disclosed as operating on one particular computing device (e.g. a dedicated server or a workstation), it is understood in the art that software is intrinsically portable and that most software running on a dedicated server may also be run, for the purposes of the present invention, on any of a wide range of devices including desktop or mobile devices, laptops, tablets, smartphones, watches, wearable electronics or other wireless digital / cellular phones, televisions, cloud instances, embedded microcontrollers, thin client devices, or any other suitable computing device known in the art.

[0051] Similarly, parts of this invention are described as communicating over a variety of wireless or wired computer networks. For the purposes of this invention, the words “network”, “networked”, and “networking” are understood to encompass wired Ethernet, fiber optic connections, wireless connections including any of the various 802.11 standards, cellular WAN infrastructures such as 3G, 4G / LTE, or 5G networks, Bluetooth®, Bluetooth® Low Energy (BLE) or Zigbee® communication links, or any other method by which one electronic device is capable of communicating with another. In some embodiments, elements of the networked portion of the invention may be implemented over a Virtual Private Network (VPN).Attorney Docket No : 206150-0011-00WO

[0052] Fig. 10 and the following discussion are intended to provide a brief, general description of a suitable computing environment in which the invention may be implemented. While the invention is described above in the general context of program modules that execute in conjunction with an application program that runs on an operating system on a computer, those skilled in the art will recognize that the invention may also be implemented in combination with other program modules.

[0053] Generally, program modules include routines, programs, components, data structures, and other types of structures that perform particular tasks or implement particular abstract data types. Moreover, those skilled in the art will appreciate that the invention may be practiced with other computer system configurations, including hand-held devices, multiprocessor systems, microprocessor-based or programmable consumer electronics, minicomputers, mainframe computers, and the like. The invention may also be practiced in distributed computing environments where tasks are performed by remote processing devices that are linked through a communications network. In a distributed computing environment, program modules may be located in both local and remote memory storage devices.

[0054] Fig. 10 depicts an illustrative computer architecture for a computer 2000 for practicing the various embodiments of the invention. The computer architecture shown in Fig. 10 illustrates a conventional personal computer, including a central processing unit 2050 (“CPU”), a system memory 2005, including a random-access memory 2010 (“RAM”) and a read-only memory (“ROM”) 2015, and a system bus 2035 that couples the system memory 2005 to the CPU 2050. A basic input / output system containing the basic routines that help to transfer information between elements within the computer, such as during startup, is stored in the ROM 2015. The computer 2000 further includes a storage device 2020 for storing an operating system 2025, application / program 2030, and data.

[0055] The storage device 2020 is connected to the CPU 2050 through a storage controller (not shown) connected to the bus 2035. The storage device 2020 and its associated computer-readable media, provide non-volatile storage for the computer 2000. Although the description of computer-readable media contained herein refers to a storage device, such as a hard disk or CD-ROM drive, it should be appreciated by those skilled in the art that computer-readable media can be any available media that can be accessed by the computer 2000.Attorney Docket No : 206150-0011-00WO

[0056] By way of example, and not to be limiting, computer-readable media may comprise computer storage media. Computer storage media includes volatile and non-volatile, removable and non-removable media implemented in any method or technology for storage of information such as computer-readable instructions, data structures, program modules or other data.Computer storage media includes, but is not limited to, RAM, ROM, EPROM, EEPROM, flash memory or other solid state memory technology, CD-ROM, DVD, or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to store the desired information and which can be accessed by the computer.

[0057] According to various embodiments of the invention, the computer 2000 may operate in a networked environment using logical connections to remote computers through a network 2040, such as TCP / IP network such as the Internet or an intranet. The computer 2000 may connect to the network 2040 through a network interface unit 2045 connected to the bus 2035. It should be appreciated that the network interface unit 2045 may also be utilized to connect to other types of networks and remote computer systems.

[0058] The computer 2000 may also include an input / output controller 2055 for receiving and processing input from a number of input / output devices 2060, including a keyboard, a mouse, a touchscreen, a camera, a microphone, a controller, a joystick, or other type of input device. Similarly, the input / output controller 2055 may provide output to a display screen, a printer, a speaker, or other type of output device. The computer 2000 can connect to the input / output device 2060 via a wired connection including, but not limited to, fiber optic, ethernet, or copper wire or wireless means including, but not limited to, Bluetooth, Near-Field Communication (NFC), infrared, or other suitable wired or wireless connections.

[0059] As mentioned briefly above, a number of program modules and data files may be stored in the storage device 2020 and RAM 2010 of the computer 2000, including an operating system 2025 suitable for controlling the operation of a networked computer. The storage device 2020 and RAM 2010 may also store one or more applications / programs 2030. In particular, the storage device 2020 and RAM 2010 may store an application / program 2030 for providing a variety of functionalities to a user. For instance, the application / program 2030 may comprise many types of programs such as a word processing application, a spreadsheet application, aAttorney Docket No : 206150-0011-00WOdesktop publishing application, a database application, a gaming application, internet browsing application, electronic mail application, messaging application, and the like. According to an embodiment of the present invention, the application / program 2030 comprises a multiple functionality software application for providing word processing functionality, slide presentation functionality, spreadsheet functionality, database functionality and the like.

[0060] The computer 2000 in some embodiments can include a variety of sensors 2065 for monitoring the environment surrounding and the environment internal to the computer 2000. These sensors 2065 can include a Global Positioning System (GPS) sensor, a photosensitive sensor, a gyroscope, a magnetometer, thermometer, a proximity sensor, an accelerometer, a microphone, biometric sensor, barometer, humidity sensor, radiation sensor, or any other suitable sensor.EXPERIMENTAL EXAMPLES

[0061] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0062] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the system and method of the present invention. The following working examples therefore, specifically point out the exemplary embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.Example 1 : Liquid Delivery System

[0063] The primary function of the liquid delivery system (e.g., the liquid delivery system illustrated in Figs. 5A - 5E) is to precisely deliver sucrose liquid droplets from two different spouts in a time-dependent manner. The system includes of two 50mL syringes that hold liquidAttorney Docket No : 206150-0011-00WOmounted to a 3D-printed holder that attaches to the floor. 3D-printed connectors attach the syringes to tubing that leads to a two-way, normally closed solenoid valve that gates liquid delivery. After the gating mechanism, the tubing connects to a blunted 20-gauge needle lick spout using a 3D-printed adapter. The lick spout (e.g., the liquid-delivery spouts of Figs. 4A -4E) fits into a 3D-printed coupler that mount directly onto an MG90 Servo motor that controls spout retraction. The two retractable lick spouts are fitted into a 3D-printed holder that can be clamped into the translational manipulator (e.g., the translational manipulator of Figs. 3A - 3E). The liquid delivery system offers control over the amount of liquid delivered, which can be controlled based on the duration of opening of the two-way solenoid valve. The system also detects when mice lick the spouts. This is accomplished through modified jumper wires that clamp onto the lick spout and connect to the electronic circuit board to measure changes in capacitance. The liquid delivery system supports either one or two spout tasks, which opens the door to several variations of experiments. For example, a reversal -learning task was tested, in which mice adjust spout associations based on rewards. However, one could adapt the same system to a preference test by varying the sucrose concentrations or droplet sizes on one of the spouts. The system could feasibly be adapted to tactile simulation tasks by adapting the spout retraction mechanism and spout position to brush the mouse whiskers (Hao et al., 2021; Guo et al., 2014).Example 2: Air Puff Delivery System

[0064] The air puff delivery system (e.g., the air puff delivery system of Figs. 6A - 6E) controls the time-dependent release of pressurized air into the mouse eye. The primary structure uses a 3D-printed adapter to connect a blunted 20-gauge needle spout to a gooseneck arm that allows one to freely adjust the spout position. The gooseneck arm is fixed to the floor using a 3D-printed adapter. Airflow is gated by a 5V air solenoid valve. The tubing is further connected to an oxygen regulator which allows for adjustment of air pressure. Moreover, air puff duration can be controlled via the duration of the solenoid valve opening. The system supports two modes of pressurized air pumping. Most labs contain a positive air supply that can easily attach to tubing. Still, a fully self-sufficient system was desired, so a cheap bike pump capable of generating the desired target of 5 - 40 PSI air pressure was tested. The aversive capability of the air puff delivery system was demonstrated by measuring the inhibition of dorsal striatal dopamineAttorney Docket No : 206150-0011-00WOrelease, which is typically associated with punishment (Menegas et al., 2018). Alternatively, the air puff system could be adapted to deliver a tactile whisker stimulus by reducing air pressure with the oxygen regulator and changing spout positioning to the side of the mouse snout (Hao et al., 2021; Guo et al., 2014).Example 3 : Head Fixation System

[0065] The head fixation system is largely 3D-printed and serves to restrain the mouse’s head and provide a platform from which the mouse can freely stand, walk, or run. The system includes two head-fixing components to clamp either side of the mouse head bar, as well as a running wheel platform (e.g., the fixation platform of Figs. 2A - 2E). The head-fixing component includes a horizontal post that on one end contains a hole where a small (screw size) screw tightens the mouse head-bar. The opposite side of the horizontal post friction fits into a 3D-printed coupler that attaches to a vertical post. The base of the vertical post is then fixed in place on the floor using a 3D-printed clamp and screw. The running wheel platform includes a 3D-printed running wheel that fits into a ball bearing, which attaches to a 3D-printed base that is fixed to the floor with a 3D-printed clamp. The base can be loosened using a 3D-printed turn dial, which allows the running wheel to be freely rotated to the desired angle (Fig. 2F). Overall, the head fixation system both restrains mouse movement and provided a platform for the mouse to run.Example 4: Translational Manipulator

[0066] The translational manipulator (e.g., the translational manipulator of Figs. 3A - 3E) enables precise control over the placement of lick spouts and facilitates tasks involving liquid consumption and decision-making. The manipulator includes three 3D-printed bases that each control the X (e.g., left and right), Y (e.g., forward and backward), or Z (e.g., up and down) axis positions of the lick spouts respectively (Fig. 3F). The bases are layered on top of one another and contain a lead screw that passes through another base. For example, the X-axis base contains a lead screw that can be rotated to move the Y-axis base left or right. The Z-axis base position can be similarly adjusted forward or backward by turning the Y-axis screw. Lastly, the spout holder connects to the Z-axis base using a clamp and can be adjusted up and down by turning the Z-axis screw. Each screw is fixed in placed in its base by gluing an insert lock hex nut to one endAttorney Docket No : 206150-0011-00WOand can be rotated using 3D-printed turn dials that slot onto the hex nut. Additionally, the conversion of rotational to linear motion is facilitated by a hex nut that is embedded into each base. While the bases can still move without these hex nuts, the metal grooves of the hex nut compared to the grooves of the 3D-printed base extend the longevity of the component. Overall, the dial turning enables precise control over the spout position around the mouse face. To facilitate positioning, a 3D-printed camera holder attaches to the X-axis base, which provides a video feed of the spouts and the mouse snout.Example 5: Electronics

[0067] A 3D-printed base includes an Arduino Mega 2560 Rev3, a breadboard, three Mosfet modules, and a 5 V power supply module (e.g., the controller of Figs. 8A - 8E). The Arduino controls task timing and executes commands to deliver stimuli or collects sensor data sent to a computer or an external neural recording apparatus. The Arduino connects to a speaker to deliver auditory cues, an LED to provide visual cues, three resistors that detect changes in capacitance on the lick spouts, two Servo motors that control spout retraction, and three Mosfet modules that control solenoid valve opening (Fig. 9).

[0068] Three solenoid valves are used outside the housing unit to control liquid and air puff delivery. Precise liquid delivery is best controlled via research-rated 12V solenoid valves, whereas air puff delivery can be controlled through cheaper 5 V air solenoid valves. If experimenters require additional precision for air puff delivery, the same type of 12V solenoid valve used in the liquid delivery system can be fitted for the air puff system.

[0069] Two MG90 Servo motors are used to control spout retraction. The advantage of the retraction design is that these Servos directly control retraction via the rotation of the 3D-printed lick spout coupler, rather than indirectly through a rack and pinion. The precise speed and amount of retraction can be adjusted in the Arduino code. This flexibility is useful during retraction habituation and opens the door to adapting the retractable spouts for whisker stimulation. The Servos are powered via a 5V power supply module.

[0070] The system also contains two cameras which allow simultaneous monitoring of different angles of behavior. The ‘snout-view’ camera angle is advantageous during spout positioning,Attorney Docket No : 206150-0011-00WOparticularly in decision-making tasks. The snout-view camera facilitates precise control over the placement of the dual spouts to the mouse snout, using the crease in the mouse snout as a marker (Figs. 7A - 7G). The snout-view camera also helps to verify spout retraction and mouse licking during behavior. The ‘adjustable-view’ camera facilitates control over the placement of the air puff spout around the mouse’s eye. The adjustable-view camera may assess anticipatory blinking during air puff delivery similarly to anticipatory licking during sucrose droplet delivery (Siegel et al., 2015). Moreover, the adjustable-view camera may empower the analysis of mouse facial expressions (Dolensek et al., 2020).Example 6: Experimental Validation

[0071] The Generalized Apparatus for Behavioral Assessment (GABA) was tested on three different behavioral tasks to validate various apparatus functions. Delivery of sucrose droplets, air puff delivery, and lick detection systems were tested in mice. 12-week-old male mice (n = 3) were habituated to the head-fixation unit for 3 days while they were provided with free access to sucrose droplets, with the air puff spout inactive at the side of the mouse face (Fig. 11 A). The position of the lick spout was precisely controlled using the video fed under the mouse snout (Fig. 7G), in combination with the translational manipulator. The position of the air putt spout was also assessed using the adjustable view camera. After habituation, a session was conducted with 40 sucrose droplet trials and 20 air puff trials in a pseudo-random order with a 15-second inter-trial interval. It was found that mice reliably licked in response to sucrose droplet delivery, but no licking occurred for an air puff (Fig. 1 ID). To validate the integration of GABA with fiber photometry, dopamine release was recorded in the dorsal striatum, and it was observed that sucrose droplets reliably increased dopamine release (Fig. 1 IF). In contrast, air puff delivery reduced dopamine release (Fig. 1 IE). These release dynamics correspond with existing literature that suggests the respective rewarding and aversive properties of the sucrose droplets and air puffs (Bari et al., 2019; Menegas et al., 2018). The results of this experiment demonstrate that GABA can reliably deliver rewarding and aversive stimuli to mice and record licking behavior with high temporal precision.

[0072] Next, cue delivery and the dual-spout design were tested using a two-spout Pavlovian reversal learning task. Male and female 20-week-old C57BI / 6J mice (n = 14) were habituated forAttorney Docket No : 206150-0011-00WOthree days while providing free access to sucrose droplets from both spouts, positioned at the left and right side of the mouse snout (Fig. 12A). Importantly, the snout-view camera and translational manipulator were used to precisely control spout positions. Mice were then trained for three days on a single-spout, Pavlovian task in which a 1 -second white noise delivered by a speaker preceded sucrose delivery. On the third day, the number of licks during each trial was measured and it was found that mice reliably produced anticipatory licks during the cue period before reward delivery (Fig. 12B). Dopamine release in the dorsal striatum was also recorded and showed that dopamine activity precisely tracked licking behavior during both cue and reward periods (Fig. 12C). These results suggest that the mice successfully associated the auditory cue with reward. After cue conditioning, the mice were returned to the dual-spout setup to record 10 days of the Pavlovian reversal learning task where white noise cued reward delivery from one of two spouts, with the rewarded spout alternating after 20 to 30 trials. It was found that mice reliably adapted their anticipatory licking toward the rewarded spout across two contingency reversals (Fig. 12C). These results suggest that GABA enables mice to engage in associative learning and value-based discrimination.

[0073] Lastly, the spout retraction design was tested in an operant reversal learning task. Male and female 12-week-old C57BI / 6J mice (n = 7) were trained in the same scheme used in the Pavlovian reversal learning experiment, except the Pavlovian reversal learning training was truncated to three days. In addition, mice were habituated to spout retractions for three days by delivering free sucrose droplets from alternating spouts across 60 trials. Importantly, the snoutview camera allowed for precise adjustment of spout positioning during training to prevent spout-side bias formation. Because of this, there was no need to conduct bias untraining sessions that are often necessary for this type of decision-making task. After habituation and training, mice were transitioned to the operant reversal learning task, where an auditory cue signaled mice to select either the left or right spout. After spout selection, the unchosen spout was retracted, and a rewarding sucrose droplet was either delivered or omitted to the chosen spout if it was the correct option (Fig. 13 A). It was found that mice reliably selected the rewarded spout and adapted their choices after a contingency reversal (Fig. 13B). Importantly, the retraction mechanism ensured that mice could only pick a single spout during the cue period as spout selection triggered immediate retraction of the unchosen spout. Fiber photometry was also used to record striatal dopamine release during the operant reversal learning task (Fig. 13C). It wasAttorney Docket No : 206150-0011-00WOfound that dopamine release during the choice period is significantly higher when the mouse performs a correct trial versus an incorrect trial (Fig. 13D). These results underscore the functionality of GABA in modulating behavior and neural activity during decision-making tasks.

[0074] Referring now to Fig. 14, shown are images depicting the results from histology experiments. Mice received 400 nl of concentrated (~1012infectious units per ml) solution of AAV2 / 9-hSyn-GRAB_DA3m (Brian VTAZBiohippo) and were implanted with optical fibers (400 pm, numerical aperture = 0.48; Doric Lenses) in the medial region of the dorsal striatum (DMS) (bregma, +0.74 mm; lateral + / -1.3 mm; ventral -2.6 mm) and the lateral region of the dorsal striatum (DLS) (bregma +0.74 mm; lateral + / - 2.2 mm; ventral -2.6 mm).

[0075] Referring now to Fig. 15, shown are images depicting results from histology experiments. Mice received 400 nl of concentrated (-1012 infectious units per ml) solution of AAV2 / 9-hSyn-GRAB_DA3m (Brian VTA / Biohippo) and were implanted with optical fibers (400 pm, numerical aperture = 0.48; Doric Lenses) in the medial region of the dorsal striatum (DMS) (bregma, +0.74 mm; lateral, + / - 1.3 mm; ventral, -2.6 mm) and the lateral region of the dorsal striatum (DLS) (bregma, +0.74 mm; lateral, + / - 2.2 mm; ventral, -2.6 mm).

[0076] Referring now to Fig. 16, shown are images depicting results from histology experiments. Mice received 400 nl of concentrated (-1012 infectious units per ml) solution of pAAV9-hSyn-GRAB_rDAlm (Addgene) and were implanted with optical fibers (400 pm, numerical aperture = 0.48; Doric Lenses) in the dorsal striatum (bregma, +0.74 mm; lateral, + / - 2.2 mm; ventral, -2.6 mm).Example 7: Materials and Methods

[0077] Subjects: C57BL / 6J (Jackson Laboratory, stock: 00664) male and female mice between 12 -24 weeks old and 20 - 24 g were used for experiments. Mice were maintained on a 12-hour light / 12-hour dark cycle (lights on at 07:00) with food ad libitum and room temperature of 22 -25 °C and 55% humidity. All procedures adhered to animal care standards set by NationalAttomey Docket No : 206150-0011-00WOInstitutes of Health and received approval from the University of California Berkeley’s Administrative Panel on Laboratory Animal Care.

[0078] Stereotaxic Surgeries: Surgeries were conducted under general ketaminedexmedetomidine anesthesia using a stereotaxic instrument (Kopf Instruments, model 1900). Mice in Figs. 11 and 12 were injected with 400 nL of concentrated (~1012infectious units per mb) AAV2 / 9-hSyn-GRAB_DA3m (Brian VTABiohippo) into the DLS (bregma, +0.74 ml; lateral, + / - 2.2 mm; ventral, -2.6 mm) orDMS (bregma, +0.74 ml; lateral + / - 1.3 mm; ventral, -2.6 mm) using a syringe pump (Harvard Apparatus) at a rate of 150 nL / min. Mice in Fig. 13 were injected with 400 nL of concentrated (~1012infectious units per mL) pAAV9-hSyn-GRAB rDAlm) (Addgene) into the DLS. The injection needles were withdrawn 10 minutes after infusion. The incision was closed with a suture and tissue adhesive (Vetbond; 3M) and the animals were kept on a heating pad until they recovered from anesthesia. After 4 weeks, mice were placed under the same anesthesia and implanted with optical fibers (400 pm, numerical aperture (NA) = 0.48; Doric Lenses) targeted to either the DMS or DLS (counterbalanced across animals). Headplates were attached to the skull for head fixation. The fiber and headplates were secured to the skull using one layer of adhesive cement (C&B Metabond; Parkell) followed by acrylic (Jet Denture Repair; Lang Dental).

[0079] Behavioral Assays - Randomized Reward and Air Puff Delivery: This refers to the assay shown in Fig. 11. Before the experiment, mice were placed on water restriction (1 mL per day for 1 week) and habituated (1-hour sessions for 3 days with intermittent sucrose droplet delivery to lick spout) to GABA inside a sound-attenuating chamber (55.9 x 38.1 x 40.6 cm, Med Associates). Each session consisted of 40 trials in which sucrose droplets (10%, 4 pL) were disposed on a single lick spout and 20 air puff trials during which air (50 ms, 10 PSI) was blown into the mouse eye. Trials occurred in pseudorandom order with a 15-second inter-trial interval.

[0080] Paylovian Reversal Learning: This refers to the assay shown in Fig. 12. The experimental setup was identical to Fig. 11 except droplets were delivered to the two spouts. The snout-view camera and translational manipulator were used to calibrate the spout position before experimentation. Mice were trained for three days on a single-spout task where a 1 -second white noise preceded 4 pL sucrose droplet delivery for 60 trials. Mice were then placed in the dual-Attorney Docket No : 206150-0011-00WOspout setup, and 10 days of the main task were recorded where white noise preceded droplet delivery to one of the two spouts, alternating after 20 to 30 trials. The session concluded after 2 reversals of the spout-reward contingency.

[0081] Operant Reversal Learning: This refers to the assay shown in Fig. 13. The experiment setup and training were identical to Fig. 12 except Pavlovian reversal training occurred for 3 days, and mice were habituated to spout retraction for another 3 training days where sucrose droplets were dispensed onto alternating spouts. Mice were then transitioned to the main task for a single day. In the main task, white noise played for at least 1 second and stopped when the mouse licked on one of pouts or 5 seconds had elapsed. A 2-second ‘no-lick’ window was imposed before cue onset where the mouse needed to refrain from licking both spouts to prevent premature spout selection). No-choice trials in which the mouse did not lick a spout during the cue period seldom occurred (<1%). After the spout choice, the unchosen spout was retracted, and a reward was either delivered or omitted on the chosen spout depending on whether the choice was correct. After 30 to 50 trials, the spout-reward contingencies were reversed with no explicit cue indicating the change. The session consisted of a single reversal.

[0082] Fiber Photometry: Dopamine transients were recorded using a custom-built fiber photometry rig as described in previous work (de Jong et al., 2019). Fluorescent signals were collected using a 470 nm LED (20 pW at fiber tip) (Figs. 11 and 12) or a 565 nm LED (Fig. 13). Light emission was recorded using a complementary metal oxide semiconductor camera (Photometries Prime or Point Grey Blackfly), which acquired video frames containing the fiber bundle (two fibers, 1 m in length, NA = 0.48, 400 pm core; Doric Lenses) at the same frequency. Fluorescent signals were acquired using custom acquisition code written in MATLAB.Experimental time stamps were derived from the logic pulses generated by the Arduino Mega 2560 Rev3 sent to the acquisition device. Fluorescent signals were standardized for each trial according to the mean and standard deviation during the ITI (13 to 3 seconds before the trial outcome) to account for photobleaching during the recording session.

[0083] Statistical Analysis: Both lick events and fluorescence during fiber photometry recording for each trial were placed in 50 ms bins and averaged across all trials in the session. Peri-eventAttorney Docket No : 206150-0011-00WOtime histograms represent the group average across mice of these average trial traces. For Fig. 13D, the mean values of the area under the curve in the ‘correct choice’ were compared to the ‘incorrect choice’ trial traces during the period between choice and outcome using a paired t-test. Statistical significance was *P<0.05, **P<0.01, ***P<0.001. All data are presented as mean + s.e.m. unless otherwise specified.

[0084] Histology: Mice were injected with sodium pentobarbital and, after the absence of tail pinch reflex, underwent transcardial perfusion with IX PBS and then 4% paraformaldehyde (pH 7.4). Brains were extracted and post-fixed in 4% paraformaldehyde for 24 hours at 4°C. Brains were then transferred to 15% sucrose / PBS solution, until they sank, and then placed in 30% sucrose for 24 hours. 75 pm Coronal brain sections were collected using a vibrating blade microtome (VTS1000S, Leica Biosystems, Deer Park IL). To enhance visualization of viral-mediated gene expression, slices in Figs. 11 and 12 were stained with chicken anti-GFP (1:1000, Abeam: abl3970) followed by Alexa Fluor 488 goat anti-chicken (1:750, Abeam: abl50169). Slices in Fig. 13 were stained with rabbit anti-dsRed (1:1000, Takara: ab2534093). Brains were imaged using DAPI and 488 or 546 fluorescence channels on an upright widefield fluorescence microscope (Axiolmager M2, Zeiss, Dublin, CA) using a 2.5X objective (Figs. 14 - 16).

[0085] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

1. Attorney Docket No : 206150-0011-OOWOCLAIMSWhat is claimed is:

1. A fixation system comprising:a base;a platform connected atop the base;one or more restraining arms positioned above the platform;a translational manipulator positioned adjacent the platform;a plurality of spouts connected to the translational manipulator;a liquid delivery system fluidly connected to at least two spouts of the one or more spouts;an air delivery system fluidly connected to at least one spout of the one or more spouts; anda controller communicatively connected to the liquid delivery system and the air delivery system, wherein the controller is configured to control the delivery of the one or more stimuli;wherein the liquid delivery system and the air delivery system are configured to provide one or more stimuli to a subject.

2. The system of claim 1, wherein the one or more stimuli may be chosen from the group consisting of: appetitive stimuli, aversive stimuli, visual stimuli, auditory stimuli, gustatory stimuli, olfactory stimuli, thermal stimuli, and combinations thereof.

3. The system of claim 1, wherein the platform is freely rotatable.

4. The system of claim 1, wherein the platform is attached to the base via a ball and socket joint, configured to angle the platform in a plane.

5. The system of claim 1, wherein the platform is configured to allow the subject to stand or move freely.

6. The system of claim 1, wherein the platform is configured to fix at least a portion of a body of the subject to the platform via the restraining arms.Attorney Docket No : 206150-0011-00WO7. The system of claim 1, wherein the liquid delivery system delivers appetitive or gustatory stimuli to the subject.

8. The system of claim 1, wherein the liquid delivery system comprises one or more liquid reservoirs and one or more valves connected between the liquid reservoirs and the at least two spouts of the one or more spouts; andwherein the one or more valves are communicatively connected to the controller.

9. The system of claim 8, wherein the one or more liquid reservoirs comprise water, a mixture of liquids, a solution, a sucrose solution.

10. The system of claim 1, wherein the air delivery system delivers aversive or tactile stimuli to the subject.

11. The system of claim 1, wherein the air delivery system comprises an air reservoir, a pump, and a valve connected between the air reservoir and the at least one spout; and wherein the valve is communicatively connected to the controller.

12. The system of claim 1, further comprising one or more camera holders configured to position one or more cameras to image behavioral responses from the subject.

13. The system of claim 1, wherein the position of the one or more spouts is adjustable via the translational manipulator.

14. The system of claim 1, wherein the one or more spouts are retractable.

15. The system of claim 1, wherein the one or more spouts each comprise a capacitance sensor, configured to detect capacitance changes in the spout when the subject licks the spout.

16. The system of claim 1, further comprising a light delivery device configured to deliver visual or thermal stimuli to the subject.

17. The system of claim 1, further comprising a sound delivery device configured to deliver auditory stimuli to the subject.Attorney Docket No : 206150-0011-00WO18. The system of claim 1 , further comprising one or more sensors, communicatively connected to the controller,wherein the one or more sensors are configured to monitor stimuli intensity or measure physiological changes in the subject in response to the stimuli.

19. The system of claim 1, wherein the controller is configured to control a frequency, timing, and intensity of the delivery of the one or more stimuli.

20. The system of claim 1, wherein the controller is communicatively connected to a computing system comprising a processor and a non-transitory computer readable medium, wherein the non-transitory computer-readable medium comprises instructions, which when executed by the processor positions the one or more spouts anddelivers a set of stimuli to the subject via the spouts.

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