Integrating optical neuroscience tools into touchscreen operant systems

Patrick T Piantadosi; Oren Princz-Lebel; Miguel Skirzewski; Julie R Dumont; Daniel Palmer; Sara Memar; Lisa M Saksida; Vania F Prado; Marco A M Prado; Tim J Bussey; Andrew Holmes

doi:10.1038/s41596-025-01143-x

. Author manuscript; available in PMC: 2025 Aug 7.

Published in final edited form as: Nat Protoc. 2025 May 23;20(9):2418–2452. doi: 10.1038/s41596-025-01143-x

Integrating optical neuroscience tools into touchscreen operant systems

Patrick T Piantadosi ^1,⁶, Oren Princz-Lebel ^2,^3,⁶, Miguel Skirzewski ^2,^4,⁶, Julie R Dumont ², Daniel Palmer ², Sara Memar ², Lisa M Saksida ^2,^3,^4,⁵, Vania F Prado ^2,^3,^4,⁵, Marco A M Prado ^2,^3,^4,⁵, Tim J Bussey ^2,^3,⁵, Andrew Holmes ^1,^✉

PMCID: PMC12329216 NIHMSID: NIHMS2098881 PMID: 40410621

Abstract

Unlocking the neural regulation of complex behavior is a foundational goal of brain science. Touchscreen-based assessments of behavior have been used extensively in the pursuit of this goal, with traditional pharmacological and neurochemical approaches being employed to provide key insights into underlying neural systems. So far, optically based approaches to measure and manipulate neural function, which have begun to revolutionize our understanding of relatively simple behaviors, have been less widely adopted for more complex cognitive functions of the type assessed with touchscreen-based behavioral tasks. Here we provide guidance and procedural descriptions to enable researchers to integrate optically based manipulation and measurement techniques into their touchscreen experimental systems. We focus primarily on three techniques, optogenetic manipulation, fiber photometry and microendoscopic imaging, describing experimental design adjustments that we have found to be critical to the successful integration of these approaches with extant touchscreen behavior pipelines. These include factors related to surgical procedures and timing, alterations to touchscreen operant environments and approaches to synchronizing light delivery and task design. A detailed protocol is included for each of the three techniques, covering their use from implementation through data analysis. The procedures in this protocol can be conducted in as short a time as a few days or over the course of weeks or months.

Introduction

Defining and understanding the neural computations that govern behavior remains one of the most important and difficult problems in neuroscience. Nonetheless, identifying the neural signatures of adaptive and maladaptive behavior holds considerable promise for the betterment of human health given the rising burden of disease from psychiatric and neurological disorders in the USA and globally^1–5. Animal models provide a key investigative tool in this regard, enabling the examination of tightly controlled, complex behaviors in conjunction with readouts and causal manipulation of neural function^6–8.

Over the last two decades, the advent and combination of novel optical and genetic techniques has greatly improved our ability to elucidate the neural principles underlying behavior. These techniques rely upon the genetic and/or viral introduction of optically sensitive molecules into an experimental system to manipulate or measure neural activity. Manipulation approaches employ actuators to alter cellular activity, while measurement approaches use indicators to visualize cellular activity^9–12. Actuators, also referred to as opsins, are a class of light-sensitive transmembrane proteins used to manipulate cellular excitability or modulate intracellular signaling cascades¹³. Actuators initiate their effects upon exposure to wavelengths of light within specific ranges and induce changes in cellular function with millisecond precision. Optically based measurement approaches rely upon indicators, which are genetically engineered protein complexes that report dynamic changes in levels of molecules related to neuronal activity (for example, intracellular calcium) or neurotransmitters/modulators (for example, dopamine (DA)^14,15. Indicators are typically composed of a modified neuromodulator receptor or calcium-binding domain fused with a circularly permutated fluorescent protein^16–19, with fluorescence changes serving as a proxy measure for underlying neural events in vivo when measured using techniques such as fiber photometry (FP)^20,21 or microendoscopic imaging^22–25.

While these optical tools offer unprecedented insight into neural circuit function, fully realizing their potential requires behavior to be assessed in an equally rich, rigorous and reproducible manner^26,27. In this regard, touchscreen-based cognitive assessment offers a flexible, programmable environment that allows for customizable presentation of operant or Pavlovian stimuli across multiple species^28–38. Yet, there are a number of challenges to integrating optical tools with touchscreen behavioral assessment. In particular, because optical tools require that animals be implanted with a skull-mounted optical interface, a variety of concerns related to the surgical preparation, touchscreen environment and experimental design must be addressed. While these issues are also relevant to other behavioral settings, their importance is compounded in touchscreen experiments that necessitate freely moving animals perform complex actions in an operant environment, often over extended, sometimes months-long, periods.

Here, we extend previous protocols that described rodent touchscreen-based behavioral assessments of motivation, memory and executive function^39–44 by now detailing the best practices for incorporating optical tools with touchscreen testing.

Overview

This protocol extension covers three techniques now widely used in neuroscience: (1) optogenetic manipulation, (2) FP and (3) microendoscopic imaging. Based on our ongoing experience^37,45–47, we describe how to establish and troubleshoot these approaches in touchscreen experiments. The protocol first highlights experimental considerations shared across the three optical techniques (‘Experimental design’ section). Each technique is then described in detail as a separate procedure, with Procedure 1 dedicated to optogenetic manipulation (Steps 1–39), Procedure 2 dedicated to FP (Steps 1–23) and Procedure 3 dedicated to microendoscopic imaging (Steps 1–41).

Applications of the protocol

Throughout, we focus on the mouse Bussey–Saksida Touch Screen System (Lafayette Instrument Company; Box 1 and Fig. 1), reflecting the popularity of this system among touchscreen platforms (>90% of users; PubScreen database, https://mousebytes.ca/pubScreen) and the use of the mouse as the model species of choice for experiments involving either optical tools or touchscreens (~62% of touchscreen publications; PubScreen dashboard, 15 August 2023).

BOX 1. General touchscreen system setup and preparation.

Briefly^40,42,43,48, each first-generation Bussey–Saksida Touch Screen System (Fig. 1) comprises a trapezoidal chamber containing a touch-sensitive digital interface (‘touchscreen’) onto which visual stimuli are displayed and can be responded at behind a black Plexiglas aperture mask configured to restrict responses to stimulus locations on a given task. In this configuration, a reward receptacle is located opposite the touchscreen, into which food pellets or liquid reinforcer can be delivered (with entries detected via infrared beams). These components are controlled and logged by an I/O box connected to a computer running software (for example, Animal Behavior Environment Test (ABET II Touch) and WhiskerServer). A screw-down terminal block within the I/O box allows for the addition and removal of external devices (for example, lasers for optogenetic manipulation or processors for FP). Light delivery/collection from the intracranial implant is made possible by an external fiber-optic patch or data cable suspended from above the chamber, which may be connected to a commutator. A digital camera can also be situated above the chamber to record behavior.

Setup

• TIMING 1 d

Ensure that the touchscreen controlling PC is turned of and that power is disconnected from the ABET 2G expander(s) associated with the chambers.
If a commutator is housed outside the sound-attenuating box, a hole must be drilled into the top of the sound-attenuating box. Using a handheld drill with a circular hole saw attachment (diameter ~3 inches), drill a hole above the chamber to ensure cables can enter without bending.

▲ CAUTION Drill and saw with care. The particle board within the sound-attenuating box will create a large amount of dust during drilling. Remove all components before modifying the chamber and thoroughly vacuum and clean the area of residual dust when complete. The resulting hole in the sound-attenuating chamber will lower its resistance to sound. To limit potential distraction from external sounds, a white noise machine can be placed near the chambers. Note that modifying components purchased from commercial sources may void vendor warranties or repair agreements.
If a commutator will be attached inside the sound-attenuating box, ensure that fiber-optic patch cables are strung through the prefabricated holes in the sides of the sound-attenuating box. Minimize pinch points where cables may bend excessively.
If conducting a behavior where aperture masks (if used) are too small for the size of the implanted optical interface, use a handheld drill and sanding attachment to widen apertures appropriately.

▲ CAUTION Drill and sand with care. Plastic should be thoroughly cleaned and sanded to eliminate sharp edges after cutting.
Attach the nonrecessed reward receptacle to arena and plug in the I/O cable.
Connect the TTL Breakout to power.

▲ CRITICAL STEP First-generation Bussey–Saksida touchscreen chambers have an I/O box with ‘open collector’ outputs, which send ‘Low True’ TTL signals. Because many commonly used optical components (e.g., lasers, DAQs, etc.) accept ‘High True’ TTL signals, a TTL Breakout box can be used to convert ‘Low True’ TTLs to ‘High True’ TTLs. This allows touchscreens to interact with external devices requiring this form of TTL input. If using an alternative system, ensure it can output a ‘High True’ TTL or implement a method to convert these signals.
Attach one end of a DB-25 cable to the TTL Breakout device and the other to the ABET II 2G Expander.

▲ CRITICAL STEP The TTL Breakout can be connected directly to the digital interface (ABET 2G Expander) corresponding to a set of chambers. To assign TTL Breakout outputs, users must edit the WhiskerServer device definition file used to establish I/O lines. These edits expand the number of outputs to allow external devices to be triggered by ABET II Touch. For example, to add a single output to each chamber (output #16), the text-based device definition file can be edited to include an additional array of outputs (30 total additional outputs available), with lines corresponding to chambers (interfaces) 1 (line 112), 2 (line 113), 3 (line 120) and 4 (line 121), with output named LINE_O_16 (note that the lines starting with a # are commented out and ignored by WhiskerServer). For additional support adding outputs or inputs via the TTL Expander, contact the vendor.
Plug in ABET 2G Expander(s) to power and turn on touchscreen-controlling PC.
Proceed with equipment setup and procedures for specific optical techniques.

Fig. 1 | — Cartoon depicting key components of the touchscreen testing apparatus. Note that some components are not depicted to scale and/or rendered semitransparent for viewing. Created in BioRender. Piantadosi, P. (2024) https://BioRender.com/f56i078.

Given that most touchscreen tasks have also been validated for use in rats^40,42,43,48, where implantation of optical tools is feasible, many of the procedures described herein could readily be adapted for this species. Many of the principles discussed (with exceptions noted in the relevant sections) are also applicable to other commercially available (for example, Med Associates Touchscreens) or open-source^49–52 systems. Indeed, researchers using non-touchscreen operant environments may also find this protocol relevant to their testing procedures.

Optogenetic manipulation

Optogenetic manipulation allows for precise light-evoked control over cellular activity to produce reversable loss or gain of function with high temporal resolution. Reversibility enables powerful within-subjects experimental designs, while temporal precision affords dissection of discrete cognitive constructs that often occur in a close temporal procession^53–56. For instance, we have shown that optogenetically silencing dorsolateral striatal neurons during touchscreen visual stimulus discrimination improved learning, while the same manipulation ~1–2 s later, during reward collection, was without affect⁴⁵. In another example, optogenetic stimulation of a cingulate-to-visual cortex projection enhanced discrimination following an error but not a correct response⁵⁷.

FP

By detecting changes in fluorescent signals arising from genetically encoded calcium indicators (GECIs) or sensors engineered to report the presence of specific neuroactive chemicals^{20,21,37,46,58–60}, FP measures neural activity on timescales relevant to the types of cognition assessed in touchscreen tasks⁶¹. FP typically entails tethering mice to a flexible, multimodal fiber-optic cable delivering modulated excitation light-emitting diode (LED) pulses at distinct wavelengths corresponding broadly to fluorescence signal (for example, 465 nm) or noise (for example, isosbestic 405 nm). The emitted fluorescence is collected via the same fiber-optic cable, detected and then transformed into an analog signal that can be demodulated, detrended and expressed as a signal-to-noise differential (ΔF/F₀). We have recently applied FP to describe fluctuations in acetylcholine and DA in the ventral striatum of mice performing a touchscreen-based reward prediction task³⁷.

Imaging

Microendoscopic imaging⁶² measures the in vivo activity of cellular assemblies with single-cell resolution in freely moving animals using miniaturized, head-mounted microscopes^{23,24,63–65}. Microendoscopes utilize relatively inexpensive LEDs to deliver one-photon excitation to GECIs or sensors expressed in brain via a chronically implanted gradient refractive index (GRIN) lens. The emitted fluorescence is detected by a metal oxide semiconductor (CMOS) sensor that provides a digital readout of fluorescence which can be used to manually or algorithmically identify tens to hundreds of individual brain cells (neurons and non-neuronal types)^66–68. Importantly, these individual cells can be tracked across multiple trials and even across multiple days (sessions)⁶⁹. We have used this technology to understand the role of cortical and amygdalar neurons in emotional memory^70,71, and others have demonstrated hippocampal⁷² and nucleus accumbens (NAc)⁶² contributions to touchscreen-based behavior.

Comparison with alternative available methods

A major motivation to integrate touchscreen environments with cutting-edge optically based approaches relates to the advantages afforded by touchscreen testing, as compared with other forms of behavioral assessment^30,38. One key distinguishing feature of touchscreen testing systems is that they offer a high degree of translational (and reverse-translational) capability, with common clinical neurological assessments (for example, the Automated Neuropsychological Assessment Metrics or the Cambridge Neuropsychological Test Automated Battery) being conducted in a computerized, touchscreen-based fashion^29,35,44. Thus, it is feasible to assess cognitive functions identically across species, which has been cited as a powerful approach with which to narrow the bench-to-bedside gap that exists for neuropsychiatric illness^73,74. Indeed, comparable deficits in touchscreen-based behavior have been reported in both mice and humans that carry genetic mutations relevant to neuropsychiatric or neurodegenerative disorders^75,76, as well as between chronic stroke survivors and photothrombotic stroke model mice⁷⁷.

Another advantage is that commercially available touchscreen chambers and the tasks associated with them are largely standardized, ensuring that experimental conditions are uniform within and across laboratories. This consistency has allowed for the development of initiatives such as MouseBytes^28,78 and the McGill-Mouse-Miniscope platform³⁴ that aim to collect, compare, disseminate and store open-source data generated using touchscreen tasks. Such initiatives will probably be critical to parsing the complex datasets generated when combining touchscreen cognition with optically based manipulation or measurement techniques.

Optogenetic manipulation

Researchers interested in perturbing neural function during touchscreen behavior have relied on brain lesions^79–83, intracranial electrical manipulation⁸⁴, pharmacological approaches^36,85,86, genetic manipulation^87–91 and, most recently, chemogenetics^92–95 to achieve their experimental goals. Although these techniques have led to valuable insights, optogenetic manipulation offers several advantages that make this approach ideal for probing causal questions during touchscreen testing.

Unlike the aforementioned chronic or sustained manipulations, optogenetic actuators can be used to precisely affect cellular activity⁹⁶ or neural oscillations^97,98, enabling reversable loss or gain of function with high temporal resolution. The fact that opsin-based effects are largely reversible in the absence of illumination allows for powerful within-subjects testing that is impossible with lesions and more difficult with pharmacological or chemogenetic approaches that require repeated administration of exogenous substances via intracranial infusion or systemic injection. The marked improvement in temporal precision afforded by optogenetic manipulation approaches is also of paramount importance to dissecting the cognitive functions that can be readily tested within touchscreen chambers, as these functions can often be decomposed into distinct constructs occurring in temporal procession^53–56.

Optogenetic actuators can also be targeted using increasingly specific genetic tools^99–101. Many questions in neuroscience, including in touchscreen research, revolve around understanding the function of a population of cells that exists within a morass of other populations. Nearly all commonly used manipulation approaches are unable to disentangle the function of such intermingled cell populations, but the viral-genetic targeting tools afforded by optogenetic approaches allow researchers to express opsins specifically in their population of interest. Similarly, while classic genetic manipulation techniques (for example, gene knockouts) often produce changes that are present throughout an organism’s development, opsins can be introduced at essentially any developmental timepoint, allowing an organism to develop normally before any causal insult.

FP

FP offers several advantages over other methods that have been used to index neural events during touchscreen-based behavioral testing, such as electrophysiology⁸⁸ and fast-scan cyclic voltammetry (FSCV)⁴⁷. Similar to optogenetic manipulation, one major factor that sets FP apart from alternative approaches is the ability to target specific neural populations or pathways using viral-genetic techniques. Although electrophysiological approaches can achieve some degree of pathway or cell-type specificity via opto-tagging and waveform or pharmacological analysis^102,103, these approaches are subject to various forms of interpretational ambiguity. Similarly, although microdialysis and FSCV allow for neurochemical identification, they are limited in their temporal resolution (in the case of microdialysis) and in their neurochemical specificity and sensitivity (in the case of FSCV). Improvements in the temporal resolution afforded by FP are of critical importance, as the ability to accurately describe brain-behavior relationships necessitates resolving neural activity on fast timescales. We also know that the behaving brain is awash in neurochemicals and other signaling molecules that can affect neural activity. Ongoing and rapid advances in indicator and sensor development have made it feasible to monitor the dynamics of many distinct neurochemicals¹⁰⁴, as well as intracellular signaling molecules such as cyclic adenosine monophosphate (cAMP^105,106).

In addition, unlike microdialysis and FSCV, whose probe size and neurochemical specificity (respectively) limit their brain-wide utility, FP can be employed to measure neural signals across multiple brain regions within the same behaving animal¹⁰⁷. Finally, FP is amenable to multiplexing with optogenetic manipulation, allowing investigators to optically perturb the function of neural processes while simultaneously measuring changes in sensor fluorescence corresponding to neural events^108,109.

Microendoscopic imaging

Advantages associated with imaging during touchscreen behavior are largely driven by the single-cell resolution afforded by one-photon microendoscopes. For example, the ability to image at single-cell resolution enables researchers not only to identify ensembles composed of individual cells that are relevant to specific behavioral events but to potentially track the development of ensembles across behavioral sessions⁶⁹. This ability to faithfully track cells with a high degree of accuracy across many experimental sessions sets imaging apart from other approaches capable of single-neuron resolution (for example, in vivo electrophysiology). This approach can be extended in many interesting ways, including to measure multiple populations of cells within the same brain region simultaneously by combining a cell-type specific static fluorophore with a dynamic calcium indicator¹¹⁰ or by multiplexing optogenetic manipulation of neurons or neuronal processes within the field of view during imaging^111,112. It is also becoming possible to acquire images with one-photon illumination at multiple depths^113,114 or over wide swaths of cortex^115–117, advances that will enable researchers to increase their experimental yield and more conclusively answer questions related to the spatial organization of task-related activity.

Limitations

The application of this protocol is curtailed by the specific limitations inherent to each optically based technique.

Optogenetic manipulation

Minutes-long periods of optogenetic illumination can cause certain opsins to alter the local pH and ionic gradients, resulting in paradoxical increases in calcium influx, spontaneous transmitter release and synaptically evoked spiking^118,119. Prolonged illumination can also cause photobleaching or brain tissue heating that adversely affects opsin viability or tissue health^120–122. These concerns are particularly relevant for touchscreen experiments where cognitively relevant epochs of interest span periods of many seconds and occur repeatedly during prolonged testing sessions. It is possible to partially mitigate these effects by implementing pulsed or sinusoidal light stimulation instead of continuous illumination, by minimizing the intensity and duration of the photoinhibition or by using a wavelength of illumination that is as red-shifted as possible given the actuator used^123,124. It is also critical to include appropriate opsin-negative controls in each experimental cohort to confirm light-mediated changes in behavior are driven by opsin expression.

FP

Although the temporal resolution afforded by FP is superior to neurochemical approaches such as microdialysis, the kinetics of fluorescent protein-based sensors are slower than electrophysiological recordings. Thus, because events of interest in touchscreen tasks can occur in concatenated chains (for example, a conditioned stimulus (CS+) presentation, followed by touchscreen press, followed by reward collection), it can be difficult to disambiguate fluorescent signals associated with specific events. One solution to this issue is to modify the task design to allow for better separation of key events. An alternative is to maintain task-design and apply, post hoc, regression-based encoding models to the analysis to parse events^125,126.

Microendoscopic imaging

Commercially available microendoscope systems (for example, Inscopix or Doric Lenses) are more expensive than FP systems, owing to hardware and software requirements and customer support guarantees. Open-source microendoscope options can be substantially cheaper (for example, miniscope.org⁶³), though the reagents required (for example, implantable GRIN lenses, baseplate) are generally more numerous and expensive than for FP. Another difference with FP is that the size of the head-mounted interface required for imaging limits the ability to record from multiple brain regions simultaneously (although techniques to enable this are currently being developed¹²⁷). In addition, the surgical procedures required for imaging are more rigorous, due to size of the implant and the need to ensure a clear imaging plane¹²⁸. In general, microendoscopic imaging can be technically challenging, with a variety of potential issues arising from hardware or software failures (for example, image banding, dropped imaging frames and so on). Users should make use of troubleshooting resources, such as Inscopix customer support and/or user groups for open-source options (for example, for UCLA miniscopes, https://groups.google.com/g/miniscope). Lastly, data storage solutions and computational approaches must be carefully considered at the outset of imaging experiments. In our experience, a typical touchscreen imaging experiment generates many terabytes of data—orders of magnitude greater than in a typical FP experiment.

Experimental design

Surgical considerations relevant to touchscreen testing

The use of optical tools typically requires intracranial surgery to (1) express virally packaged genetic material encoding light-sensitive proteins (unless transgenic mice are used that express the relevant actuator/indicator) and (2) introduce an externally accessible, chronically implanted optical interface (for example, fiber-optic ferrule or microendoscope baseplate secured directly to the skull). These surgeries require expertise and care, as previously described in protocols for optogenetic manipulation^12,129–131, FP¹³² and microendoscopic imaging^133–135. Here, we highlight several considerations pertaining to intracranial surgeries performed for the purpose of touchscreen experiments.

One key issue is the timing of surgeries relative to testing. Intracranial injections/implants are usually completed in one to two surgical sessions, after which animals recover over a monitoring period determined by an animal care and use regulatory body. Time must also be allowed for actuator or indicator proteins to be produced, trafficked and expressed in cell membranes^136–138. In general, researchers should anticipate a postsurgical interval of 4–6 weeks before starting optical manipulation or measurement. The length of touchscreen training required before the start of optical application can itself vary from several days to many weeks, depending on the task, experimental question and model being tested (for example, different mouse strains attain performance competency at different rates³²).

For experiments involving relatively few touchscreen training sessions (for example, Pavlovian autoshaping³⁷), we recommend conducting surgery before any touchscreen testing—such that the postsurgical interval serves as a natural bridge between experimental viability (for example, adequate virus expression) and behavioral testing. When more extensive training is required (for example, trial-unique delayed nonmatching to location^139,140), we suggest training to a predetermined criterion level of performance before surgery and then, after recovery, re-establishing performance to the desired level before testing using an optical intervention. This sequence will reduce the interval between surgery and intervention, thereby lessening the chance that the integrity of the implanted interface will fail during the remainder of what may still be a protracted period of testing. If conducting surgeries following training, it is critical to assign animals to experimental conditions (for example, opsin versus control for optogenetic manipulation) such that task proficiency (defined by the experimenter) is balanced across the groups.

Another important surgery-related consideration relates to the length and diameter of the optical medium (for example, fiber-optic implant) implanted at the targeted brain locus. The implant size depends on idiosyncratic experimental factors (for example, size of targeted locus, desired optical coverage and so on) but will, regardless of size, inevitably damage the brain above the target. Given that the purpose of the touchscreen system is to evaluate complex cognitive functions that might be especially sensitive to brain damage, we recommend using the smallest implant possible to minimize damage^135,141. The consequences of damage can be gauged by comparing data collected from implanted animals to nonimplanted controls, using in-house data or open-source repositories^28,78 (for example, https://mousebytes.ca/home). If damage is found to interfere with performance, the use of smaller implants (for example, 0.6 mm diameter GRIN lens instead of 1 mm diameter), when feasible, will be particularly important.

Once implanted, ensuring the long-term integrity of the optical interface is critical to the success of touchscreen experiments wherein repeated, often daily, connection/disconnection from optical devices are necessary. To improve stability, we recommend affixing one or more stainless-steel cranial screws to strengthen the bond between the interface, the interface-securing acrylic (or other substance) and the skull (as detailed in refs. 20,129,132,135). Importantly, a properly affixed interface will also reduce the potential for light to leak from the connection with the skull and interfere with the perception of visual stimuli presented on the touchscreen. This will also reduce the possibility of light from the touchscreen penetrating the skull and producing optical artifacts that could be misinterpreted as neural signal. To further minimize both issues, we recommend encasing the optical interface in a light-resistant substance (for example, black dental acrylic or acrylic painted with nail polish) and ensuring all other optical components, including the cabling connecting the interface, are well-shielded. Control experiments should also be considered to exclude confounds arising from light from the touchscreen, for example, testing for touchscreen light-evoked measurement artifacts in animals not expressing a genetically encoded activity indicator.

Addressing tether-related artifacts during touchscreen testing

During behavior, the light necessary for optical approaches is usually delivered through a cable connected to the skull-mounted optical interface, resulting in a tethered preparation. Although wireless, tether-free systems are becoming more common^131,142, tethering remains the most cost-effective and commonly used interfacing method. If not properly instituted, however, tethering can produce a variety of experimental problems.

One set of issues stems from the choice of implanted optical interface and the method used to secure the interface to the tether during testing. This is particularly relevant to optogenetic manipulation and FP, for which there are numerous fiber-optic interface options. The most popular among these are chronically implanted stainless-steel or zirconia ferrules housing an optic fiber, which can be connected to external devices by a fiber-optic patch cable via a mating sleeve. As many touchscreen tasks require repeated movement between the touchscreen and reward receptacle, often located at opposite ends of the apparatus, rotation and separation between the mating sleeve, the patch cable and the implanted ferrule can occur. Separation can compromise the light-penetration and efficacy of optogenetic manipulation and introduce recording artifacts and reduce signal-to-noise in FP recordings. Note that most microendoscope designs use a locking screw to secure the miniaturized microscope to the implanted baseplate during imaging, making this technique less susceptible to potential movement-induced separation.

For optogenetic manipulation or FP, we recommend coating the plastic or ceramic sleeves in heat shrink tubing to insulate them (Fig. 2a) or purchasing metal sleeves that can be pinched slightly to narrow their inner diameter. Both approaches increase the friction seal between the sleeve and the implanted ferrule, minimizing the possibility of separation or disconnection. The use of heat shrink tubing or metal sleeves also effectively limits light leakage; leakage which, as noted above, could detrimentally impact behavior by acting as a visual cue^143,144. Another option is to use an interconnect device that provides a secure seal between the implant and patch cable, although this device is bulkier and can be difficult to accommodate bilaterally.

Tethers can also complicate behavioral testing by restricting movement due to the weight and torque of the attached equipment¹⁴⁵. This is of particular concern to touchscreen tasks, which often entail long experimental sessions (~0.5–1.5 h) during which the mouse repeatedly rotates. Such rotation will cause the tether to tangle or kink, resulting in mobility impairment that can interfere with data interpretation and make it difficult to compare task performance to that of untethered mice across experiments and laboratories (for example, if using repositories such as MouseBytes.ca). Similarly, although touchscreen testing involves less experimenter handling than other cognitive assays (for example, water maze), repeated testing and associated food-restriction can still alter stress-hormone levels^146–148. These effects could be compounded by forceful tethering in naive animals. It is, therefore, crucial to habituate mice to the handling technique required to attach the tether and to being tethered in the chamber. We recommend habituating mice, for at least 1 week before testing, to daily scruffing (Fig. 2b, left) or gentle restraint of the area around the neural implant (Fig. 2b, right). Because touchscreen behavior is often conducted daily for a period of weeks or months, we find gentle restraint to be well tolerated. Touchscreen training sessions that precede optical intervention can be a good opportunity to habituate mice to the tether.

An effective method for further mitigating tether-related mobility issues is to connect the cable to a rotary joint or commutator. Commutators are either passive (Fig. 2c), rotating in accordance with the mouse’s movement, or active, containing or interfacing with a sensor that detects rotational movement and engages a motor to counteract it. We have found that commutators greatly improve motor freedom in touchscreen experiments. For example, tethered mice performing a touchscreen decision-making task¹⁴⁹ have significantly shorter response latencies and complete testing in significantly less time when connected to an active commutator than when not (Fig. 2d). Active commutators are most beneficial when the head-mounted cable is rigid, heavy or large in diameter—as in microendoscopic imaging, whereas a passive commutator usually suffices when a thin-diameter, lightweight cable can be used—as in optogenetic manipulation or FP. For example, we have found that mice tethered to a passive commutator for optogenetic manipulation perform similarly to nonimplanted, tether-free mice in the aforementioned decision-making task (Fig. 2e).

Irrespective of the type of commutator used, it is important to minimize slack in the cable to prevent kinking that can impede light transmission and allow mice to access and chew the cable (which would be particularly costly if expensive cables used for FP and imaging are damaged). To further limit the potential for damage, nonfunctional ‘dummy’ cables or microendoscopes can be used as a substitute during sessions where light delivery is not required. Finally, if a commutator is not available, it may be possible to adjust the experimental design to collect data from tethered animals. In this case, we suggest keeping experimental sessions as short as possible and using extralong fiber-optic or data cables (with most of their length strung above the chamber) to circumvent tangling.

Recommended touchscreen chamber alterations

It can be difficult for tethered animals to access certain key areas within operant chambers, including those with touchscreens. In a typical chamber design, the reward receptacle is recessed outside of the chamber to limit entry with the snout only (Fig. 3a, left). This can leave implanted mice unable to access the reward or force them to adopt contorted positions that may impact performance (for example, reduced motivation or increased reward-collection latency) or damage equipment or affect light transmission, resulting in technical artifacts and difficulty interpreting results. This problem can be addressed by carefully modifying the receptacle to make it higher and wider (Fig. 3a, center). Note that some mice will climb into an excessively expanded receptacle, and this can lead to potential distraction, avoidance of aversive stimuli (for example, footshock) or difficulty determining the intentionality of receptacle entries.

Fig. 3 | — a, Cartoon depicting three common touchscreen reward-receptacle styles. Left: the standard recessed receptacles can be difficult to access for mice with implanted optical interfaces (bilateral fiber-optic implants depicted here) and are not recommended. Middle: expanded receptacles are more accessible for mice with large optical implants but should not be so large that mice can enter with their entire body. Right: nonrecessed receptacles protrude into the chamber slightly, allowing easy access for mice with large implants. b, Using nonrecessed receptacles (NR), reward collection latencies are similar between mice that were tethered (NR + T, n = 8) or tether free (NR + TF, n = 8; unpaired samples t-test, t(14) = 0.838, P = 0.416). c, Touchscreen mask before modification (top) and post-modification (bottom). d, Left: touchscreen computer running behavioral control software interfaces with an I/O box. Middle: example of a class II laser for optogenetic manipulation, connected directly to the touchscreen I/O box for TTL control. Right: example FP system and acquisition computer, interfacing directly with the touchscreen I/O box for synchronization via a TTL signal. Created in BioRender. Piantadosi, P. (2023) https://BioRender.com/z79f687.

Using a nonrecessed receptacle (as some systems do^49,50)—for example, a trough protruding into the chamber—is another option to facilitate reward collection in implanted mice (Fig. 3a, right). Our experience is that this design produces performance in tethered mice that is comparable with untethered animals (Fig. 3b). Although this type of receptacle can have the same drawbacks as an expanded recessed receptable, most commercially available options are designed to limit the ability of mice to climb into/on top of them.

In addition to issues with accessing the reward receptacle, mice may exhibit reluctance to interact with the touchscreen while tethered. Such reluctance may be related to the use of touchscreen window aperture masks used in some touchscreen systems (including the Bussey–Saksida system) to limit touchscreen access to only areas where visual stimuli will appear (Fig. 3c, top). By design, openings in the masks are small, which may not allow mice with implants to easily access the touchscreen itself. In this case, we recommend increasing mask size (Fig. 3c, bottom) or forgoing the mask altogether (as some touchscreen variants do^49,50), with the caveat that this can affect task performance by increasing stimulus-noncontingent and accidental screen touches.

Synchronizing data streams during touchscreen testing

Touchscreen experiments generate a primary data stream that is a time-stamped record of input/output (I/O) from the touchscreen chamber (for example, time of stimulus presentation, time of response and so on) (Fig. 3d, left). When adding optically based tools to this typical touchscreen experiment, additional data streams must be harmonized with touchscreen I/O data. For optogenetic manipulations, touchscreen I/O can be used to control the onset and duration of light delivery (for example, synchronize light on/off with touchscreen stimulus presentation). This is achieved by enabling communication directly between the touchscreen I/O (via transistor–transistor logic (TTL) signals) and the light-generating laser, LED or intermediate pulse generating device (Fig. 3d, center).

In the case of optical measurement, FP or microendoscopic imaging systems are usually controlled by a standalone computer to avoid taxing computing resources, which could reduce the accuracy of I/O or, where incorporated, video timestamping. This computer is, in turn, connected to intermediate hardware, processors or data acquisition systems (DAQs). Digital data recorded by multiple devices must, therefore, be synchronized for subsequent analysis. In its simplest form, this involves directly connecting the relevant external hardware (for example, FP processor or microendoscopic DAQ) to the touchscreen I/O to allow for the delivery of timestamped TTL signals related to behavioral events of interest (Fig. 3d, right). Although effective, this can be cumbersome in practice when there are many I/O signals to analyze, as is common for complex touchscreen cognition assessments. Furthermore, this approach requires researchers to predetermine which behaviors are relevant before data collection. A more flexible alternative is to send TTL signals from the touchscreen I/O at defined periods (for example, session or trial start) that are logged by processors or DAQs. This allows offline synchronization of each data stream around the shared timestamps, providing a temporal reference to align optical data around any event or epoch of interest.

Task-dependent experimental considerations

Touchscreen tasks differ in whether events occur contingently or independently of the mouse’s behavior, with implications for the application and analysis of data collected using all three optical techniques described in the protocol. For example, event timing in the Pavlovian autoshaping task^37,150 is controlled by the experimenter (Fig. 4a). Here, a CS precedes delivery of an unconditioned reinforcer (US reward) and CS onset, offset and reward delivery are triggered at preprogrammed, experimenter-controlled intervals (for example, 10 s CS onset–offset interval). In the case of such ‘animal-independent’ events, light delivery can be precisely targeted to specific experimental epochs (for example, CS onset), without trial-to-trial variability in the duration of illumination (Fig. 4b). This has the benefit of minimizing potential photobleaching or tissue heating with long illumination-duration intervals, particularly if conducting optogenetic manipulation using lasers as a light source^120–122. Pulsed (for example, 20 Hz) or intermittent (for example, 5 s on and 5 s off) illumination protocols can also help in this regard.

Fig. 4 | — a, In tasks structured around animal-independent events, for example, Pavlovian autoshaping, events are triggered based on programmed, experimenter-controlled durations, regardless of an animal’s behavior. b, Optogenetic manipulation parameters are identical across trials when triggered by animal-independent events. c, Neural data collected using FP or microendoscopic imaging can be concatenated over trials and averaged when examining animal-independent events. d, In tasks involving animal-controlled events (for example, pairwise visual discrimination), mice control the pacing and sequence of events. e, The timing and duration of optogenetic manipulation varies across trials and mice for animal-controlled events, as they are initiated and terminated by each animal’s behavior. f, Neural data can be uniformly aligned to animal-controlled events to examine activity in a predetermined time window around an event (for example, from −2 to 2 s around stimulus choice). Created in BioRender. Lab, T. (2023) https://BioRender.com/c59b060.

In terms of analysis, neural activity measured via FP or microendoscopy that is related to animal-independent events is easily time locked to task events, such as CS onset or US delivery. Data can then be concatenated or averaged across stimulus iterations for straightforward statistical comparison (Fig. 4c). Behavioral or task events (for example, CS onset) that are of consistent length also makes it relatively straightforward to apply statistical approaches to analyze and visualize high-dimensional neural data in low-dimensional space (for example, principal component analysis or Gaussian process factor analysis)¹⁵¹.

There are, however, drawbacks to analyzing data from animal-independent events. One issue is that the mouse may not be familiar with the timing of experimenter-controlled task features, particularly early in training. Hence, the interval to collect a reward or the precise period when a stimulus is being attended to, for example, will vary across trials and stages of training. This variability adds an unknown factor that must be considered when interpreting data. Another limitation is that imposing the timing of task events limits the ability to capture the natural dynamics of behavior as compared with tasks wherein the mouse has more control over the pacing and sequence of events.

An example of a touchscreen task featuring self-paced, ‘animal-controlled’ events is the pairwise visual discrimination task^40,87,152 (Fig. 4d). Here, the mouse is trained to discriminate between two visual stimuli, only one of which, when selected, delivers reward^40,153 (Fig. 4d). Each discrimination trial is initiated by a reward-receptacle entry and is followed by an indeterminate period (though this may be capped by the experimenter), during which a stimulus choice is made and then the reward is or is not delivered and collected. The time to initiate a trial, make a choice and collect the reward, therefore, varies across trials which, in turn, means that the timing of optogenetic manipulation or neural activity measurement around these events also varies—with technical (for example, photobleaching) and analytical (for example, inconsistent event duration) implications (Fig. 4e,f). Restricting light delivery to a maximum period from the start of an event^139,154 or during a subset of trials or task phases can limit photobleaching/tissue heating. In terms of analyses, when concatenating data across trials is not possible due to variable event length (Fig. 4f, left), the fluorescent trace can be linearly interpolated around events to stretch/contract the data into temporal uniformity^125,155. Alternatively, cross-trial data can be aligned to a defined period to or from event onset that is deemed pertinent by the experimenter (Fig. 4f, right).

Materials

Biological materials

Male or female C57BL/6J (The Jackson Laboratory) mice

▲ CAUTION Experiments involving live rodents must comply with institutional and governmental regulations.

▲ CRITICAL Unless conducting an aging or developmental study, mice should be between 2 and 5 months of age at the start of training.

Reagents

Liquid reinforcers (for example, milkshake): Neilson Dairy strawberry milkshake or Nestle Nesquik strawberry flavored low-fat milk (Amazon (https://www.amazon.com), cat. no. B002WWLEWM)

▲ CRITICAL The availability of specific liquid reinforcers may be restricted by geographic region. Although milkshake produces higher rates of responding than comparable alternatives (for example, saccharin)¹⁵⁶, substituting reinforcers with other options (for example, 10% condensed milk) should produce comparable results, particularly if caloric content is matched¹⁵⁷.
Chamber cleaning solution. Ethanol (FisherScientific, cat. no. BP82011) or quatricide (FisherScientific, cat. no. NC0282465)

▲ CAUTION Selection of a cleaning solution may be dependent on animal care and use regulations. Between mice, a diluted disinfectant (for example, Vimoba 128 or Quatricide) or EtOH solution should be applied to a paper towel, which is then used to clean the interior of the touchscreen chamber. Low concentrations of EtOH solution (for example, 10–20%) are preferable when cleaning touchscreen chambers between mice. Higher concentrations (for example, 70% or above) may leave a strong odor that can be aversive. A higher concentration can be used to clean the chambers more thoroughly, provided that time is allowed for lingering odor to dissipate.

Equipment

General touchscreen equipment

First generation Bussey–Saksida Touch System for mice (Lafayette Instrument Company, cat. no. 80614–20)

▲ CRITICAL If using the Bussey–Saksida Touch System, parts can be purchased from Lafayette Instrument Company (https://lafayetteinstrument.com/), unless otherwise noted. These chambers can be purchased with many of the components depicted in Fig. 1, including a sound-attenuating box, touchscreens and aperture masks, infrared (IR) detectors for locomotion, an integrated video camera and a touchscreen-controlling PC running a contemporary Windows operating system with ABET II Touch software for creating and running experiments. Note that each system comprises four separate chambers.
Touchscreen-controlling PC with Windows 10 (or higher), a 2 GHz processor (or better), 8 GB of rapid-access memory (RAM) (or higher) and 100 GB disk space (or more)
ABET II Video Touch Software (version 21.02.26)
WhiskerServer (Cambridge University Technical Services Ltd., version 4.7)
Nonrecessed reward receptacle (Lafayette Instrument Company, cat. no. 80614A-07–10)

▲ CRITICAL Nonrecessed reward receptacles are recommended for optically based techniques due to their accessibility to animals with a variety of implanted optical interfaces.
TTL Breakout (Lafayette Instrument Company, cat. no. 81510)

Drill supplies for altering sound-attenuating box and aperture mask (as necessary)

Handheld drill (DeWalt Dcd991P2 cordless drill/driver) (McMaster-Carr, cat. no. 29835A311)
Circular hole saw (3-inch diameter hole saw with built-in arbor) (McMaster-Carr, cat. no. 4008A693)
Drum for sanding sleeves (1/4-inch diameter shank, 1-inch diameter, 1–1/2 inch long) (McMaster-Carr, cat. no. 4650A24)
Sanding sleeve (for aluminum, non- and soft metal, for rough finish, 1-inch ID, 1.5-inch long) (McMaster-Carr, cat. no. 4756A196)

Optogenetic manipulation

▲ CRITICAL Materials and procedures described are based on prior publications^45,47 using class II lasers for bilateral optogenetic manipulation within touchscreen chambers. Alternative optogenetic systems and components (for example, LEDs) are available from a variety of vendors that can be installed with relatively minor alterations to the protocol. Note, LEDs may lack the maximal output power needed for in vivo manipulation using certain opsin variants.

Uncleaved fiber-optic cannula 1.25 mm, ceramic ferrule 200 μm (Thorlabs, cat. no. CFMLC12U-20)
Ceramic zirconia (Doric Lenses, cat. no. SLEEVE_ZR_1.25)

▲ CRITICAL Heat shrink tubing can be added in-house or purchased preassembled.
Bronze split sleeve (can adjust tightness with hemostat or similar) (Doric Lenses, cat. no. SLEEVE_BR_1.25)
Curved micro-mosquito hemostat (Fine Science Tools, cat. no. 13011–12)

▲ CRITICAL Hemostat can be used to gently pinch bronze sleeve to increase friction.
Quick-release interconnect device (for 1.25 mm ferrules) (Thorlabs, cat. no. ADAL3)
Heat shrink tubing for insulating sleeve (DigiKey, cat. no. Q2-Z-3/64–01-QB6IN-40)
Weller heatgun for shrinking tubing (DigiKey, cat. no. 6966C-ND)
1 × 2 Fiber-optic rotary joint—intensity division (Doric Lenses, cat. no. FRJ_1×2i_FC-2FC_0.22)
Holder FRJ Large (Doric Lenses, cat. no. Holder_FRJ_large)
316 Stainless Steel Washer (McMaster-Carr, cat. no. 91525A124)
18–8 Stainless steel hex head screw, 1/4-inch-20 thread size, 5-inch long (McMaster-Carr, cat. no. 92240A563)
18–8 Stainless steel wing nut, 1/4-inch-20 thread size (McMaster-Carr, cat. no. 92001A321)
18–8 Stainless steel hex nut, 1/4-inch-20 thread size, ASTM F594, (McMaster-Carr, cat. no. 92673A113)
Mono fiber-optic patch cord (Doric Lenses, cat. no. MFP_200/220/900–0.22_0.5m_FC-ZF1.25(F)

▲ CRITICAL Fiber-optic patch cable to run from commutator to mouse; the length of the cable can vary depending on commutator distance.
Mono fiber-optic patch cord (Doric Lenses, cat. no. MFP_200/240/LWMJ-0.22_1m_FC-FC)

▲ CRITICAL Fiber-optic patch cable to run from light source to commutator; the length of the cable can vary depending on distance between laser light source and commutator.
Solid state 473 nm laser (Opto Engine LLC, cat. no. MBL-III-473)

▲ CRITICAL Note that the illumination wavelength of the laser light source will vary depending on opsin in use.
Solid State 532 nm laser (Opto Engine LLC, cat. no. MGL-III-432)

▲ CRITICAL Note that (illumination wavelength of laser light source will vary depending on opsin in use).
Coupler to connect fiber-optic patch cable to laser (Opto Engine LLC, cat. no. FC/PC Coupler)
Fiber optic power meter (Thorlabs, cat. no. PM20A)
Laser safety glasses, universal style (Thorlabs, cat. no. LG2)

▲ CRITICAL The desired wavelength to be filtered must match laser light source.
Pulse Pal v2 (Sanworks.io, cat. no. 1102)
Static-control precise-control screwdriver for connecting leads to screwdown terminals in touchscreen I/O box (McMaster-Carr, cat. no. 7440A41)
- BNC (Bayonet Neill–Concelman; female) lead for connecting to screwdown terminals in touchscreen I/O box and BNC to wire leads (DigiKey, cat. no. 314–1394-ND)
BNC (male–male) to connect touchscreen I/O or TTL Breakout box to laser (DigiKey, cat. no. BKCT2942–100-ND)

▲ CRITICAL The length of the cable can vary depending on distance between laser and TTL source.
BNC to BNC (DigiKey, cat. no. BKCT2942–100-ND)
70% EtOH (Fisher Scientific, cat. no. BP82011)
Lens paper (Fisher Scientific, cat. no. 11–996)

▲ CRITICAL For cleaning implanted baseplate and microendoscope.
Fiber cleaning fluid (Thorlabs, cat no. FCS3)

▲ CRITICAL Clean the fiber-optic implant and fiber patch cable before attaching to mouse.

FP

▲ CRITICAL The materials described here are based on experiments in which FP was used to record DA, acetylcholine and calcium dynamics in NAc during touchscreen testing, using FP components and software from Doric Lenses^37,46. Other commercial and open-source alternatives can be used in much the same manner.

Fiber-optic implants; 400 μm outer diameter (OD) core, 5 mm length, 0.48 NA, metal 1.25 μm ferrule (Doric Lenses, cat. no. B280–4419-5)

▲ CRITICAL Width, numerical aperture, length of fiber, ferrule size and ferrule type are customizable.
Pigtailed 1 × 1 fiber-optic rotary joint, lengths and connectors customizable (Doric Lenses, cat. no. FRJ_1×1_PT_400–0.57_2m_FCM_0.12m_FCM)
Fiber-optic commutator holder (Doric Lenses, cat. no. Holder_FRJ_Small)
Mating adapter for connecting pigtailed rotary joint to fiber-optic patch cable to mouse (Doric Lenses, cat. no. ADAPTER_FC)
Mono fiber-optic patchcord, from mating adapter to mouse, customizable (Doric Lenses, cat. no. MFP_400/430/LWMJ-0.57_0.5m_FCM-MF1.25(F))
Cable M5 (Doric Lenses, cat. No. Cable_M5-M5)
Connectorized LED 405 nm (Doric Lenses, cat. no. CLED_505)
Connectorized LED 465 nm (Doric Lenses, cat. no. CLED_465)
Connectorized LED 560 nm (Doric Lenses, cat. no. CLED_560)
Four-channel LED driver (Doric Lenses, cat. no. LEDD_4)

▲ CRITICAL The choice of LED wavelength depends on experimental design. For standard FP recordings using biosensors with a 470 nm excitation wavelength (for example, GCaMP7), LEDs with 405 nm and 465 nm wavelengths are sufficient. If experiments use red-shifted biosensors (for example, jRCaMP1) alone or in combination with GCaMP-based sensors, a 560 nm LED is required.
Branching fiber-optic patch cable, to connect CLED_405 and CLED_465 to minicube (Doric Lenses, cat. no. BFP(2)_400/430/LWMJ-0.48_1m_FCM-2xFCM)
Branching fiber-optic cable with attenuator T5%, to connect CLED_560 to minicube (Doric Lenses, cat. no. BFP(2)_400/430/LWMJ-0.48_1m_FCM-2xFCM_T0.05)

▲ CRITICAL Branched fiber-optic cables enable the use of a single FP setup to simultaneously record from two mice. Alternatively, branched cables can be used to record simultaneously from two brain regions in the same mouse.
FP console (Doric Lenses, cat. no. FPC)
Doric Neuroscience Studio (Doric Lenses, version 6)
Six-port fluorescence mini cube with integrated photodetector/amplifier head (Doric Lenses, cat. no. iFMC6_IE(400–410)_E1(460–490)_F1(500–540)_E2(555–570)_F2(580–680)_S)

▲ CRITICAL The port number and wavelength assignments are customizable to experimental needs.
Four-port fluorescence mini cube with integrated photodetector/amplifier head (Doric Lenses, cat. no. iFMC4_AE(405)_E(460–490)_F(500–550)_S)

▲ CRITICAL The port number and wavelength assignments are customizable to experimental needs.
Data acquisition computer: no hardware specifications but must have 1 USB2.0 or 3.0 slot to plug FP console and be able to run Windows
External hard drive for data extraction: WD My Book 18TB USB 3.0 (newegg.com, cat. no. N82E16822234461)
Data analysis computer with a minimum of 64 GB of RAM, a 1 TB solid-state drive and a central processing unit (CPU) with a minimum of eight cores and a speed of 3.00 GHz or greater, running any contemporary operating system

Microendoscopic imaging

▲ CRITICAL Reagents and procedural descriptions are based on an experiment imaging NAc neuronal activity using an integrated lens (baseplate attached) connected to an Inscopix nVista microendoscopic system. Alternative microendoscopic imaging systems are implemented similarly and could be adapted with minor modifications. For this protocol, all reagents required for imaging can be purchased from Inscopix Inc. (https://www.inscopix.com/), except where noted.

Implantable GRIN lenses (dimensions largely customizable): ProView Integrated Lens 0.6 mm diameter, 7.3 mm length (cat. no. 1050–004413)
Baseplate cover (cat. no. 1050–004639)
Baseplate cap screw (cat. no. 1050–004221)
ProView and cap, hex driver, screwdriver (cat. no. 1050–004195)
nVista 3.0 imaging system for in vivo research (cat. no. 1000–004329)
Dummy microscope, threaded (cat. no. 1050–003762)
Dummy microscope cable, threaded (cat. no. 1050–003767)
Inscopix commutator system, including optional metal perch for attaching data cable (cat. no. https://www.inscopix.com/)
M4 nuts for securing commutator (MiSUMi, cat. no. HNTTSN5–4)
316 Stainless steel washer (McMaster-Carr, cat. no. 91525A124)
Dust Off compressed gas, for cleaning implanted baseplate and microendoscope (Office Depot, cat. no. 1381978)
6–100 Dessing forcep, for baseplate cover removal (Henry Schein, cat. no. 9538661)
Test lead BNC (male) for interfacing with Bussey–Saksida touchscreen I/O (DigiKey, cat. no. 501–1031-ND)
Coaxial connector adapter for extending male BNCs, if necessary; BNC to BNC, female to female (DigiKey, cat. no. 501–1132-ND)
Data acquisition computer: no requirements on hardware specifications but must have one ethernet port and multiple USB2.0 or 3.0 slots and be able to run latest version of Google Chrome
Inscopix Data Acquisition Software (IDAS; version v2.0.0 or higher)
Inscopix Data Processing Software (version 1.9.1 or higher)
Google Chrome (Google, version 116.0.5845.141 or higher)
External hard drive for data extraction; WD My Book 18TB USB 3.0 (newegg.com, cat. no. N82E16822234461)
Data analysis computer with a minimum of 64 GB of RAM, a 1 TB solid-state drive and a CPU with a minimum of eight cores and a speed of 3.00 GHz or greater, running Windows 10/11, macOS 10.11–10.15 or Linux CentOS 7, Ubuntu 18.04–20.04
MATLAB (https://www.mathworks.com/products/matlab.html)

Equipment setup

General touchscreen system setup and preparation (Box 1)

Procedure 1: combining touchscreen testing with optogenetic manipulation

▲ CRITICAL To illustrate this procedure, an experiment is described involving bilateral inhibition of ventral hippocampal (vHPC) fibers in the NAc. Mice were injected with a virus to express the inhibitory opsin archaerhodopsin (ArchT) or an equivalent control (enhanced yellow fluorescent protein, eYFP) in vHPC and fiber-optic implants were positioned above NAc (Fig. 5a). Mice performed a risky decision-making task¹⁴⁹, during which square pulse illumination of a 532 nm laser was triggered via a TTL signal timed to the prechoice period (Fig. 5b). Experiments requiring other illumination wavelengths or pulsed stimulation can be conducted as below, with slight alterations.

Fig. 5 | — a, Cartoon depicting a single fiber-optic implant targeting ArchT-expressing terminals in the NAc. b, A touchscreen chamber outfitted with a commutator for bilateral optogenetic manipulation using a 532 nm laser. Note that some components are not to scale, and the specific laser wavelength will be dependent on the opsin used. Created in BioRender. Piantadosi, P. (2023) https://BioRender.com/f81h248.

Day 1: set up touchscreen equipment and optogenetic manipulation hardware

● TIMING ~1 d

1
Power off the touchscreen system (remove power from ABET 2G expander(s) and controlling PC).
2
Remove the DB-25 data cable from touchscreen I/O box and unscrew box cover (Fig. 6a).
3
Using a precise-control screwdriver, unscrew a ground terminal and the terminal corresponding to the output to be assigned to the laser.
4
Plug the prestripped black (ground) lead from the female BNC into the ground terminal slot and secure screw using precise-control screwdriver. Plug the prestripped red lead from the female BNC into the output slot and secure (Fig. 6b).
5
Replace the I/O box cover and DB-25 cable.
6
Power the touchscreen system back on.
7
Connect the laser to power and place on or near sound-attenuating chamber.
8
Connect one end of a male BNC cable to the newly installed female BNC arising from the I/O box (Fig. 6c) and the other male BNC end to the female BNC port on the laser.
9
Within ABET II Touch, navigate to the ‘Environment Designer’ panel corresponding to the chamber and add the new output as ‘Laser 1’ (or similar; Fig. 6d).
10
If an experiment requires constant (for example, square pulse) laser illumination during a particular phase of a touchscreen behavior, edit the behavioral schedule in the ‘Schedule Designer’ tab of ABET II Touch, such that the ‘Laser #1’ output is activated during the relevant phase (Fig. 6e).
11
If an experiment requires pulsed illumination at a specific frequency (for example, 20 Hz), set up Pulse Pal v2 and TTL Breakout.

▲ CRITICAL STEP Pulse Pal v2 requires ‘High True’ TTL input, which can be provided by the TTL Breakout box (but not the touchscreen I/O itself, which is limited to ‘Low True’).
12
Connect Pulse Pal v2 to power via USB cable.
13
Use the joystick to navigate the Pulse Pal v2 interface. Set up stimulation parameters appropriate for your given experiment. Here, we will select a pulse frequency of 20 Hz (5 ms on, 45 ms off).

▲ CRITICAL STEP The laser stimulation parameters should be selected carefully. The factors to be considered include the native activity of the targeted cell type/region, the opsin’s kinetics and potential photobleaching/tissue heating related to stimulation power/duration.
14
Select channel 1 (output channels) and set up the following parameters:

Phase1 voltage: 5.000 V

Phase 1 duration: 0.005 s

Pulse interval: 0.045 s

Set all other duration options to 0.00 s

Link trigger 1: On

Custom train#: 0

Custom target: pulses

Resting voltage: 0.00 V

Trigger Now → Continuous → On
15
Next, select channel 1 (trigger channels) and set up the following parameters: Trigger mode: pulse gated.
16
Use the ‘Save Settings’ menu to save these settings for future use.

▲ CAUTION The ‘Continuous → On’ option must be toggled back on every time power is reconnected to the Pulse Pal v2, even when using the ‘Load Settings’ option.
17
Ensure that DB-25 cable from the TTL Breakout is connected to the ABET 2G Expander appropriately.
18
Connect one end of a male BNC to the female BNC port on the TTL Breakout that corresponds to the desired output and the other end to the female BNC channel 1 (triggers) port on the Pulse Pal v2.
19
Connect one end of the male BNC to the female BNC channel 1 (output channels) port on the Pulse Pal v2 and the other end to the female BNC port on the laser.
20
Within ABET II Touch, navigate to the ‘Environment Designer’ panel corresponding to the chamber and add the new TTL Breakout output as ‘Laser 2’ (or similar; Fig. 6d).
21
In the ‘Schedule Designer’ tab, open the touchscreen behavioral program to be used for pulsed optogenetic manipulation. Ensure that ‘Laser #2’ (connection from the TTL Breakout to the laser for frequency pulse delivery) is included during the relevant behavioral epochs.
22
Mount commutator to wire rack.
23
Screw wing nut on to 5-inch screw, followed by two sandwiched washers and three nuts. Slide the washers over wire rack such that they sandwich the wire racks, top wing nut and one hex nut into place to secure the upper portion of the mount (Fig. 7a).
24
Place the holder and commutator in between the two hex nuts on each screw, center over the hole in sound-attenuating box and screw the hex nuts into place to secure the commutator and holder (Fig. 7b).
25
Connect two fiber-optic patch cables (0.5 m) to the commutator fiber channel (FC) connector.

▲ CAUTION The length of the patch cable should be adjusted to allow the mouse to freely move within the chamber while avoiding excess fiber-optic patch cable slack, which increases the likelihood of tangling.

▲ CAUTION Thin (200 nm) patch cables can be damaged or destroyed if mice can access and chew them (for example, if there is too much slack in the system or if the sleeve disconnects from the fiber-optic implant). To prevent this, use the cables of an appropriate length or purchase patch cables with protective jacketing, each outfitted with tightly fitting sleeves.
26
Connect one fiber-optic patch cable (1 m; length customizable on the basis of needs) to the top FC connector on the commutator and the other end to the FC connector on the laser.
27
Place secure mating sleeves (Fig. 2; or metal sleeves or interconnect device) on the end of each fiber-optic patch cable to allow for stable connection to mouse.

Fig. 6 | — a, Cartoon of touchscreen I/O box and DB-25 cable. b, Open I/O box, output #9 will be used to control a laser after connecting a female BNC via two leads (red and black) using a precise-control screwdriver to tighten terminals. c, Finished female BNC connected to a male BNC going to a 532 nm laser. d, ‘Environment Designer’ panel in ABET II Touch. The relevant components are highlighted in red, including the chamber and output lines 9 and 10. The green check mark (outlined with red dashed border) allows users to test-trigger outputs. e, ‘Schedule Designer’ panel in ABET II Touch allowing creation of behavioral programs (schedules). The sample program in which laser turns on during stimulus presentation and off following a touchscreen response. Created in BioRender. Piantadosi, P. (2023) https://BioRender.com/v03c172.

Fig. 7 | — a, The procedure for mounting commutator to wire rack. The washers are held in place by a wing nut and hex nut, attached to a 5 inch screw. The washer size is dependent on the separation distance on wire rack. b, A view of drilled commutator hole, commutator holder and commutator. The holder is attached by two hex nuts to stabilize the commutator holder.

Day 2+: conduct training and testing using optogenetic manipulation

● TIMING ≥1 d

28
Set laser power meter to the wavelength to be used (for example, 532 nm).
29
Power laser(s) on, turn key to unlock and use TTL switch on rear to activate illumination.

▲ CAUTION Follow all the institutional guidelines and safety precautions when lasers are in use. This may include creating and posting signage outside the experimental room and using laser safety goggles when adjusting laser intensities.
30
While wearing laser safety glasses, remove the sleeves from the ends of the fiber-optic patch cable (to mouse) and place one end of the patch cable ferrule in laser power meter.
31
Turn the laser output on and adjust the output intensity such that the laser power meter reads the desired value (for example, 10 mW). Repeat for the other fiber-optic patch cable.

▲ CRITICAL STEP The light power (milliwatts) should account for the light transmission efficiency of the fiber-optic implant, which can vary based on how the fiber-optic implant was fashioned. Adjust the intensity of light to the desired power based on the efficiency of the fiber-optic implants. For example, if fiber-optic implants were 80% efficient when fashioned and the desired illumination to the brain is 10 mW, adjust the output of the fiber-optic patch cable to 12.5 mW, which can be calculated with the formula (desired power/[% efficiency/100] = required power).

◆ TROUBLESHOOTING
32
Ensure the laser is triggered with activation of the ‘Laser #1’ output by selecting the green checkmark in the ABET ‘Environment Designer’ panel (Fig. 6d, red highlighted box) and toggling on the ‘Laser #1’ output.

◆ TROUBLESHOOTING
33
Restrain mouse and gently clean implanted fiber-optic ferrules with a small amount of fiber-optic cleaner or EtOH solution (70%).

▲ CRITICAL STEP Cleaning detritus from implanted ferrules will ensure light reaches the target region unobstructed.
34
Carefully connect mouse to each fiber-optic patch cable and place it in touchscreen chamber.
35
Start the behavioral program within ABET II Touch.
36
Monitor mouse via video camera feed to ensure the commutator is working effectively and optogenetic stimulation occurs at the expected behavioral epochs.

◆ TROUBLESHOOTING
37
Following session completion, carefully disconnect the mouse and return it to the home-cage.

▲ CAUTION The fiber-optic patch cable should be removed from the implanted ferrule by gently twisting upwards on each sleeve.
38
Extract behavioral data to an external hard drive by using the ABET II Touch Data Viewer panel.

▲ CRITICAL STEP The data can be extracted on the basis of raw experimental timestamps arising from the I/O data or can be extracted within ABET II Touch by using the ‘Analysis Set Designer’ panel to aggregate predefined events.

Day 3+: optogenetic data analysis

39
Analyze resulting behavioral data according to experimental needs, using variables embedded within the ABET behavioral program to aggregate events of interest or to extract ancillary measures. Within the ABET II program, a variable segregates trials into three trial blocks. Each trial contains timestamps associated with trial events (for example, trial start, choice and reward collection), as well as indices related to trial outcomes (for example, large or small choice and punishment delivered or not). Use these records to extract aggregate data, such as the total number of small reward choices and choice-latencies, for example, by designing an ABET Analysis Set to group these data or by using custom-written scripts in coding languages such as MATLAB or Python. Once variables have been identified and extracted, compare them across opsin and control conditions (‘Anticipated results’ section).

Procedure 2: combining touchscreen testing with FP

▲ CRITICAL We describe the setup of an experiment in which the DA biosensor GRABDA2m⁶⁰ was virally expressed unilaterally in NAc and a fiber-optic implanted above the injection site³⁷ (Fig. 8a). The mice were then trained on Pavlovian autoshaping, a conditioning paradigm measuring approach toward stimuli predicting rewards¹⁵⁰.

Fig. 8 | — a, Cartoon depicting a fiber-optic implant targeting NAc neurons expressing the sensor GRABDA2m. b, Touchscreen setup for FP using a laptop computer running Doric Neuroscience Studio software connected to a console. The console and LED driver control excitation light delivery, which goes through the minicube, optical rotary joint, fiber-optic path and fiber-optic implant to the mouse brain. Bulk fluorescence emission from indicator returns through the same optic-fiber path and is diverted to the photodetector through the minicube. For synchronization, the TTL Breakout box connects the touchscreen system directly to the console via BNC input. Created in BioRender. Lab, T. (2024) https://BioRender.com/k87w878.

Day 1: set up FP hardware and software

● TIMING 1 d

1
Place the FP components (for example, console, LED driver and minicubes) near the touchscreens (Fig. 8b). Install Doric Neuroscience Studio and select the FP controller.

▲ CAUTION The minicubes are sensitive to motion and vibration. Ensure that minicubes are placed on a secure surface with minimal potential for disruption.
2
Set up your acquisition protocol as follows. Select designated analog input channel. LED 405 nm: lock-in mode, voltage_max 0.8 V, voltage_min 0.2 V, frequency 208.616 Hz LED 465 nm: lock-in mode, voltage_max 0.8 V, voltage_min 0.2 V, frequency 572.205 Hz LED 560 nm: lock-in mode, voltage_max 0.8 V, voltage_min 0.2 V, frequency 333.786 Hz. This LED is only used with compatible red-shifted biosensors.

▲ CAUTION Doric Neuroscience Studio allows users to set up ‘standard’ FP acquisition conditions, including modulated or continuous waves, and accepts TTL input to synchronize neural data with behavioral events arising from the ABET program.

▲ CAUTION When possible, the light power (milliamperes) specified in the controller software/hardware should be optimized to account for the light transmission efficiency of the fiber implant, LED and patch cable.
3
Add TTL control to each touchscreen operant chambers output lines in ABET II Touch.
4
Add TTL signals as outputs to the ABET II Touch schedule file.

▲ CRITICAL STEP TTL signals for FP can be set up within the touchscreen hardware and software in largely the same manner as for optogenetic manipulation. TTL signals can be triggered to timestamp key behavioral events repeated during the session.
5
Install fiber-optic commutator above chamber (Fig. 7a,b).
6
Connect fiber-optic patch cable (0.5 m length) to commutator.

▲ CAUTION Patch cable length should allow the mouse to move freely throughout the chamber without excess slack.

▲ CAUTION Light bleed through can occur with uncoated patch cables or sleeves, which may impact behavior. Patch cables with black rubberized coating reduce this risk.
7
Plug commutator cable into the minicube.
8
Plug the M5 cable connecting LED from minicube to LED driver output.
9
Connect the BNC cable output from minicube to analog input channel in console.
10
Connect the BNC cable output from TTL Breakout box to analog input channel in console.
11
Connect the BNC cable from console analog output channel to analog input of LED driver. Repeat this with each LED channel.

Day 2: collect FP data during touchscreen testing

● TIMING ≥1 d

12
Turn on the FP console, LED driver and minicube(s).

◆ TROUBLESHOOTING
13
Load recording protocol within the Doric Neuroscience Studio software.

▲ CRITICAL STEP The recording protocol will depend on your experimental design and cell population of interest.

▲ CAUTION When using GECIs (for example, GCaMPs), optical artifacts can be corrected using the emitted fluorescence from the isosbestic (for example, 405 nm wavelength) channel. Choice of isosbestic wavelength is dependent on the properties of the indicator and should be empirically defined. Null mutant ‘control’ sensors with minimal activity-dependent fluorescence, delivered in the same viral packaging as the functional sensor, can be used to report and control for artifactual changes in fluorescence^19,158.
14
In ABET II Touch software, load the touchscreen behavioral program that has been modified to include TTL triggers.
15
Before testing, adjust the power output of the LEDs to account for the efficiency of the implanted probes. The power output can be tested using a Thorlabs digital power meter (PM100D) with a Thorlabs integrating sphere detector.

▲ CRITICAL STEP The output power measured at the end of the mono-optic fiber patch should be ~20–25 μW for all wavelengths. However, desired LED power may differ based on experimental conditions and the biosensor used.
16
Ensure that the touchscreen is on and place the appropriate mask (if used) in front of touchscreen.
17
Carefully connect the mouse to the optical patch cable and place in chamber.
18
Start recording program in Doric Neuroscience Studio.
19
Start behavioral program in ABET II Touch.

◆ TROUBLESHOOTING
20
Following session completion, carefully disconnect the mouse from the patch cable and return it to the home cage. Recap the optic fibers from the fiber patch cable and the mouse head to prevent accumulation of dust and debris. Raw FP data will be automatically saved as a *.doric output file in the FP acquisition computer. Default files from Doric Neuroscience (.doric) are hdf5 format, and they contain metadata from the collection window and information from the acquisition channels.

◆ TROUBLESHOOTING

FP data analysis

▲ CRITICAL Analysis of FP data can be conducted in four basic steps: (1) filter the recording channels, (2) conduct a least squares regression to fit control channel (for example, fluorescence from 405 nm wavelength) to the signal channel, (3) calculate $Δ F / F_{0}$ and (4) align Z-scored $Δ F / F_{0}$ data to behavioral events. These steps may be conducted in any common scientific programming language (for example, MATLAB, Python and R) or using a variety of freely available software packages^159–161.

21
Process data offline. First, filter data from the isosbestic control channel (for example, 405 nm) and the signal channel (for example, 465 or 560 nm) to remove artifacts, which typically occur within the high-frequency range. One common approach for removing high-frequency noise is low-pass filtering (for example, Butterworth). A second- or third-order filter can also be used in conjunction with a cutoff (set based on the kinetics of the sensor in use) to remove noise.
22
Following signal filtering, fit data from the isosbestic channel to the active channel using a least squares regression to fit signal data to a linear function. Alternatively, data can be fit to a curvilinear function to reflect uneven changes in signal due to bleaching. Once isosbestic data have been fit to the active channel, generate the primary FP measure ( $Δ F / F_{0}$ ) using the formula
$Δ F / F_{0} = \frac{(F_{t} - F_{0})}{F_{0}},$
where $F_{t}$ is the active channel data and $F_{0}$ is the fitted isosbestic data.
23
Following generation of the $Δ F / F_{0}$ signal, align behavioral data based on TTL data or the synchronized behavioral event data. Once behavioral events are defined, data can be extracted around each event using a predefined analysis window (for example, −5 s before the event to 5 s after the event) that is pertinent to the behavior of interest and the task structure.
24
We recommend analyzing behaviorally aligned $Δ F / F_{0}$ data in a Z-score normalized format. Z-scoring typically involves normalizing each sample of the time-series $Δ F / F_{0}$ data by subtracting the mean of a predefined ‘baseline’ period and dividing this value by a measure of this baseline’s variance. The baseline could correspond to the entire event epoch (or even session) or a period judged to be behaviorally irrelevant (for example, intertrial period), depending on the experimental specifics, including whether the event of interest is animal independent or animal controlled. There are ‘standard’ or ‘robust’ Z-score functions; robust functions are designed to produce a more stable Z-score in datasets where distributions are skewed¹⁵⁹.

The standard Z-score formula is:
$z = \frac{(x - \overline{X})}{s.d.},$
where $x$ is the sample data, $\overline{X}$ is the sample mean and s.d. is the standard deviation.

The robust Z-score formula is:
$z = \frac{(x - \tilde{X})}{M A D} .$
where MAD refers to the median absolute deviation.

Procedure 3: combining touchscreen testing with microendoscopic imaging

▲ CRITICAL To illustrate this protocol, reagents and procedural descriptions focus on an experiment where GCaMP6m-mediated fluorescence from neurons in the mouse NAc are imaged using an integrated lens (baseplate attached) connected to an Inscopix nVista microendoscopic system (Fig. 9a). This system is integrated with the touchscreen chamber to allow for the relationship between NAc activity and decision-making to be analyzed (Fig. 9b).

Fig. 9 | — a, Cartoon depicting lens implant and microendoscope containing an LED to pass a narrow wavelength of light to excite GCaMP6m expressed in NAc neurons via a GRIN lens, which will emit photons to be collected by a CMOS sensor. b, Touchscreen setup for imaging using a laptop computer running Inscopix software, connected to a DAQ. The microendoscope cable is suspended over the chamber and attached to a commutator, allowing the mouse to move freely. For synchronization, the TTL Breakout box connects the touchscreen system directly to the DAQ via BNC input. Created in BioRender. Piantadosi, P. (2023) https://BioRender.com/q55m820.

Day 1: set up microendoscope and DAQ

● TIMING 1 d

1
Plug the nVista DAQ into power source.
2
Connect one end of ethernet cable to nVista DAQ and the other end to the data acquisition computer.
3
Install commutator above chamber (Fig. 7a,b, substitute M4 nuts and M4 screw).
4
Attach an M4 nut to each M4 mounting screw, followed by two washers and a final M4 nut. Attach each M4 mounting screw to the commutator, and gently maneuver the washers to sandwich the wire rack before screwing the nuts down to secure the commutator in place (refer to the Inscopix Commutator User Manual for other mounting possibilities).

Setting up the ‘dummy’ microendoscope to habituate mice to the tether

5
Connect the commutator cable to the nVista DAQ.
6
Connect the dummy microendoscope to the threaded dummy cable.
7
String the dummy cable through the chamber and up through the hole in the top of the sound-attenuating box. Attach the dummy cable ensuring only a small amount of slack and no impediments to the free rotation of the commutator.

Setting up the functional microendoscope

8
Connect the commutator cable to the nVista DAQ.
9
String the microendoscope and attach the data cable to the commutator as in Step 7 (Fig. 10a). Plug the end of the data cable into the commutator’s microendoscope port.

▲ CAUTION When stringing the microendoscope through the chamber to attach to the commutator, leave the protective lens cap on to avoid potential damage. Ensure that enough slack is in the system to allow the mouse to move freely while connected to the commutator but not so much slack that the mouse is able to chew and damage the cable.
10
Connect one end of a male–male BNC cable to the TTL Breakout port corresponding to the chamber, and the other end to the nVista DAQ GPIO port I02.

▲ CRITICAL STEP The Inscopix DAQ requires ‘High True’ TTL input, which can be provided by the TTL Breakout box (but not the touchscreen I/O itself, which is limited to ‘Low True’).
11
Turn on DAQ and acquisition computer, connect to the wireless network associated with the DAQ (name and password found beneath the DAQ).
12
Open browser (Google Chrome) and type the IP address found on the bottom of the DAQ into the address bar to access the IDAS interface.
13
Create a wired connection by going to ‘System’ → ‘Network Settings’, selecting ‘DAQ Connected to PC’ and hitting ‘Apply’. Copy the new IP address created for a wired connection.

▲ CAUTION Although data visualization in IDAS can be streamed from the DAQ wirelessly, a wired connection is less susceptible to network disruption. If connecting wirelessly, ensure the physical distance between the DAQ and the acquisition computer is minimal to avoid connectivity issues, which may cause dropped frames.
14
Disconnect from the wireless connection and paste the new IP address into the browser address bar to access IDAS using the new wired connection.
15
In the IDAS I/O settings panel, select GPIO-2 as an ‘Input’ and type the description ‘ABET’ into the text location (Fig. 10b). This will allow ABET to send digital input regarding behavioral events to the DAQ.
16
In ABET II Touch, go to the ‘Environment Designer’ to ensure that ‘TTL #1’ (which corresponds to the TTL Breakout output assigned to the touchscreen chamber) is selected (Fig. 10c).
17
In the ‘Schedule Designer’ tab, open the touchscreen behavioral program to be used for imaging. Ensure TTL #1 (connected to the Inscopix DAQ) is pulsed (minimum duration 1 ms) at behavior start (Fig. 10d, top) and at the start of each behavioral trial (Fig. 10d, bottom).

▲ CRITICAL STEP The choice of behavioral event(s) to timestamp via TTL is made on the basis of experimental requirements. Here, we timestamp ‘Behavior start’ to align imaging data and behavioral events from the touchscreen I/O (Fig. 10d).

Fig. 10 | — a, A completed commutator mounted above the chamber, with wire perch visible and adequate data cable loop entering chamber. b, IDAS interface with I/O settings panel highlighted. Ensure that GPIO-2 is marked as an input. c, ABET II Touch ‘Environment Designer’ panel with the chamber and TTL output (line 16), highlighted in red. d, The ‘Schedule Designer’ tab in ABET II Touch, displaying program altered to send a TTL to mark ‘Behavior start’ and ‘Trial start’, with relevant regions highlighted in red.

Day 2+: conduct microendoscopic imaging during touchscreen behavior

● TIMING ≥1 d

18
Ensure that DAQ and acquisition computer are turned on and connected.
19
Open IDAS, input session variables (for example, ‘Mouse ID’, ‘Mouse Sex’, ‘Experimental Session’ and so on) as desired.
20
In IDAS, turn on the excitation LED (for GCaMP, select ‘Green Channel’).
21
Remove protective cover from microendoscope.

▲ CRITICAL STEP Carefully wipe clean the microendoscope lens using dry lens paper or lens paper with a small amount of 70% EtOH. Ensure that the lens is free of debris by visual inspection and/or by visualizing the lens within the IDAS.
22
Hold the mouse securely and use forceps to carefully remove protective cover from implanted baseplate.

▲ CRITICAL STEP Mice should be properly habituated to the restraint and handling procedure (Fig. 2b).
23
Ensure that implanted lens and baseplate are free from debris.

▲ CRITICAL STEP Use small burst(s) of compressed air dislodge any dust and/or carefully clean with lens paper twisted to a point.

▲ CAUTION If using compressed air, ensure that the can is always held upright so that no moisture is propelled onto the baseplate.
24
With excitation LED on, carefully plug microendoscope into baseplate and screw down the set screw using hex driver.

▲ CRITICAL STEP Plugging the mouse in while the excitation LED is on allows the user to visualize the live field of view (FOV) within IDAS, which should be consistent across days and free of debris.

▲ CAUTION Avoid overtightening the locking set screw, which can damage the microendoscope over time.
25
Within IDAS, turn commutator to ‘Automatic Mode’.

▲ CAUTION If conducting touchscreen sessions to habituate mice using the ‘dummy’ microendoscope, the IDAS interface will only allow access to the commutator panel. Behavior can be started (advance directly to Step 30) as soon as the mouse is plugged in to the ‘dummy’ microendoscope.
26
If this is the first time the mouse is being connected to the microendoscope, within IDAS, adjust the following setting and record these settings for future use, as necessary:
- Adjust the focus based on raw image
- Adjust the frame rate to accommodate kinetics of the GECI or sensor in use. For example, GCaMP6 varieties (‘slow’, ‘medium’ and ‘fast’) allow for frame rates of 10, 15 and 20 Hz, respectively
- Adjust the LED power
- Adjust the gain, which will increase or decrease the signal within the bit range
  
  ▲ CRITICAL STEP With parameters adjusted and the excitation LED on (and without on-line filters such as dF/F, if applicable), take an image or screenshot of the IDAS window or FOV directly. This image can be used to ensure the imaging plane is consistent during subsequent imaging sessions (that is, visual comparison between the ‘live’ image and the snapshot from an earlier session will help ensure the FOV is free of dust/debris and the microendoscope is seated above the GRIN lens properly). If debris or obstructions are observed, return to Step 21 before proceeding.
  
  ▲ CRITICAL STEP Excitation LED power should be as low as possible, while maintaining signal-to-noise ratio (SNR) and dynamic range. Excessive excitation LED power will produce photobleaching of the indicator and may negatively impact SNR.
  
  ▲ CAUTION Increasing gain may result in clipping, where data beyond the bit range are lost. IDAS provides a histogram (0–255 bit depth) and recommendations for histogram curves corresponding to under or over exposure.
  
  ◆ TROUBLESHOOTING
27
Within IDAS, turn off excitation LED.
28
Ensure that desired settings related to the triggering of the microendoscope imaging are defined.
29
Manually start recording data in IDAS.
30
Issue the touchscreen behavioral program in ABET II Touch.

◆ TROUBLESHOOTING
31
Once the behavioral program is complete, manually turn off the microendoscope recording within IDAS.
32
Carefully restrain the mouse, remove the microendoscope and replace the baseplate cover using forceps. Tighten the locking set screw using the hex driver.
33
Extract data from Inscopix DAQ on to an external hard drive.

▲ CRITICAL STEP The data can be extracted by connecting an external hard drive via USB to the rear of the DAQ or via a wired connection to the acquisition PC. Do not transfer the data while data are being acquired by the DAQ (may cause dropped frames).
34
Extract raw I/O data from ABET II Touch on to an external hard drive.

Day 3+: analyze microendoscopic imaging data

35
Conduct data processing by using Inscopix Data Processing Software (IDPS), Python or MATLAB scripts to access the Inscopix API or by using open-source tools^162–165.
36
Downsample the raw imaging video spatially (spatial downsample factor of 4) and temporally (temporal downsample factor of 2) to increase the data processing speed.

▲ CRITICAL STEP Spatial downsampling removes pixels and should be kept at a level that does not distort the FOV. Temporal downsampling removes samples, meaning the filter should be set based on the acquisition sampling rate and calcium indicator kinetics.
37
Motion correct the down-sampled data.

▲ CRITICAL STEP Each video frame is algorithmically transformed according to translations around a reference frame. The motion corrected data can further be spatially filtered to remove fluorescence that does not correspond to single cell activity.
38
Using motion corrected data, identify the putative single cells.

▲ CRITICAL STEP Although a variety of manual and algorithmic approaches exist to accomplish this^66,166, constrained non-negative matrix factorization for endoscopic imaging (CNMFe⁶⁸) is recommended.
39
Align processed single-cell data to events from touchscreen chamber. If the touchscreen behavioral program is scripted to send a TTL signal directly to the Inscopix DAQ (Fig. 10d), adjust each row of the ABET I/O data by the time that the TTL was received by the Inscopix DAQ.
40
In the example experiment outlined here (imaging the NAc during performance of a touchscreen-based risky decision-making task), the mouse occasionally receives a footshock after responding at one of the touchscreen stimuli. Extract the data corresponding to ‘Shock’ events (included in the ABET behavioral program) in a time window around footshock delivery, for example, from −5 to 5 s.
41
Z-score the event-aligned data separately for each cell. The period used as the baseline for this calculation will depend upon the task structure. For animal-controlled events, where it is difficult to define a period when the mouse is not engaged in pre- or post-choice cognitive processing (as in the decision-making example used here), use the mean and standard deviation of the calcium data across the entire session or trial. Use normalized traces to analyze and visualize the relationship between neural activity and task events.

Troubleshooting

Troubleshooting advice can be found in Table 1.

Table 1 |.

Troubleshooting table

Step	Problem	Possible reason	Solution
Procedure 1: optogenetic manipulation
31	Laser illumination power is too low	Laser light source is failing	Increase power or replace laser delivery device
		Laser coupler is misaligned	Realign according to manufacturer’s specifications
		Patch cable is faulty	Replace patch cables
		Debris build-up on fiber-optic implant or patch cable	Clean with compressed air and/or 70% EtOH. When possible, keep probes covered with dust caps
32	Laser is not being triggered by ABET behavioral program	Output or condition and action in ABET have not been properly assigned	Within the ABET ‘Environment Designer’, test laser output. Ensure all experimental contingencies have been accounted for in the ABET ‘Schedule Designer’
36	Tethered mouse is not performing normally	Implanted probe size or angle is interfering with touchscreen access	Check that touchscreen aperture masks are correctly sized and alter if necessary
		Reward receptacle is recessed and/or too narrow	Alter receptacle size or adopt a nonrecessed receptacle
		Commutator has not been added or is malfunctioning	Add commutator to experimental setup. Check that commutator is properly connected and functional
		Head-mounted interface is failing	Consult animal care protocol and veterinary staff
Procedure 2: FP
12	Doric Neuroscience Studio cannot detect the photometry console	Console power is off	Turn console power on
		USB port disconnected	Make sure both ends of USB cable are properly plugged in
		Firmware outdated or incompatible with installed Doric Neuroscience Studio version	Consult software requirements and update software/firmware accordingly
19	TTL pulse that triggers start of behavior is not registered by the FP system	Loose BNC cable	Adjust BNC cable to TTL breakout block and console
19		TTL pulse is not included in experimental schedule in ABET	Add TTL pulse at the start of experimental schedule in ABET
19	Tethered mouse not performing well	See ‘Troubleshooting’ section for Procedure 1	See ‘Troubleshooting’ section for Procedure 1
20	Emitted fluorescence signal is weak	Power output of LEDs is incorrectly set	Adjust power output
		Expression levels of fluorescence sensor is absent/weak	Check whether the adeno-associated virus and construct is suitable to use in the desired tissue/cell type
		Location of fiber probe tip is away from expressed fluorescent sensor	Revisit surgery and injection practices, revise stereotaxic coordinates
	Isosbestic signal is unstable	Optical rotary joint of fiber-optic path is defective	Test light path and replace damaged items as necessary
		Sleeve or interconnect device is not holding fiber-optic cable securely	Ensure that heat shrink tubing has been applied to sleeve (Fig. 2a) or use metal sleeve or interconnect device
		Head implant may be loose	Consult animal care protocol and veterinary staff
Procedure 3: microendoscopic imaging
26	FOV is inconsistent across days and/or excessive motion can be observed in FOV	IDAS settings are not consistent across sessions	Ensure that the correct IDAS settings (for example, focus, exposure time and so on) are applied before each recording
		Microendoscope is not seated in baseplate properly	Remove microendoscope from baseplate and plug back in, while viewing screenshot taken during first recording session
		Debris obscuring baseplate or microendoscope	Clean with compressed air and/or 70% EtOH and lens paper
		Head mounted interface is failing	Consult animal care protocol and veterinary staff
		Recording is being conducted in a brain region that is susceptible to pulsatile motion (for example, brainstem)	Alter recording approach and/or use wire to stabilize GRIN lens in brain tissue^159,160
30	Behavior start TTL from touchscreens is not registered by Inscopix DAQ	Cables (for example, power, BNC and DB-25) are disconnected	Check and label all cables to ensure that they are connected securely
		ABET program has not been edited to include TTL output	Edit ABET schedule
		TTL Breakout integration with WhiskerServer has not been correctly addressed	Check whisker device definition file
		ABET input to DAQ has not been correctly labeled in IDAS	Check that the I/O Settings panel in IDAS has ‘ABET’ listed as an Input in the correct GPIO slot
		Behavior was manually started before starting to record in IDAS	Check order of operations
30	Behavior with head-mounted microendoscopes deviates from those mice without	Commutator is not functioning	Ensure that commutator cables are connected and that commutator is set to ‘Automatic Mode’ in IDAS
		Commutator is not functioning	With ‘Automatic Mode’ selected and no mouse connected, manually twist wire perch to ensure that commutator swivels correctly
		The microendoscope size is interfering with touchscreen access	Check that touchscreen aperture masks are correctly sized and alter if necessary
		The reward receptacle is recessed and/or narrow	Alter receptacle size or adopt a nonrecessed receptacle

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Timing

Procedure 1:

Steps 1–27, optogenetic experiment set-up: 1 d

Steps 28–38, daily behavioral testing using optogenetic hardware: ≥1 d, often conducted daily for a period of weeks or months

Step 39, analyze data collected using optogenetics: ~1 d

Procedure 2:

Steps 1–11, FP experiment set-up: 1 d

Steps 12–20, daily behavioral testing using FP hardware: ≥1 d, often conducted daily for a period of weeks or months.

Steps 21–24, analyze data collected using FP: ≥1 d, depending on the number and length of FP sessions, the processing speed of the analysis computer and so on

Procedure 3:

Steps 1–17, imaging experiment set-up: 1 d

Steps 18–34, daily behavioral testing using imaging hardware: ≥1 d, often conducted daily for a period of weeks or months.

Step 35–41, analyze data collected using imaging: ≥1 d, depending on the number and length of imaging sessions, the processing speed of the analysis computer and so on

Anticipated results

Procedure 1: optogenetic manipulation

Once variables have been identified and extracted, they can be compared across opsin and control conditions. In the experiment described in Procedure 1, for example, primary measures are percent choice of the large (potentially punished) and small (safe) reward across trial-blocks. Secondarily, the latency to make choices across trial blocks can be taken as an estimate of punishment-induced indecision. In this example, vHPC-NAc inhibition using the opsin ArchT had no effect on choice-type or latency (Fig. 11a).

Fig. 11 | — a, The data from an optogenetic experiment in which vHPC-NAc neurons were silenced during risky decision-making. ArchT (n = 11) and eYFP (n = 10) groups did not differ in terms of percent risky (left) or safe (middle) choices or choice latencies (right; two-way analysis of variance, with Trial Block (0, 50, 75) as a within-subjects factor and Treatment (ArchT versus eYFP) as a between-subjects factor; all P values >0.05). b, FP traces represent individual trials (gray) collected during presentation of a reward-preceding stimulus (CS+, n = 20) and rewarded-unrelated stimulus (CS−, n = 20). Trial average during CS+ (red trace) and CS− (blue trace). The bar indicates 10 s CS presentation. Left: the arrow indicates reward delivery. Right: the mean amplitude of DA dynamics was larger on CS+ than CS− trials (n = 20; paired samples t-test, t(19) = 5.896, P < 0.0001). Experimental data in b was collected in agreement with the Canadian Council of Animal Care guidelines and the animal protocols approved by the Animal Care and Veterinary Services from Western University (protocols no. 2020–162 and 2020–163). c, Mean intensity projection image of FOV produced by microendoscopic imaging following preprocessing (left) and with CNMFe-identified putative neuron footprints overlaid in green (right). d, A heat map with the activity (dF/F) of D1 neurons in the NAc aligned to footshock punishment. The neurons are ordered based on mean activity during and immediately following punishment (0–2 s). Experimental data in a, c and d are unpublished and were collected in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the local National Institute on Alcohol Abuse and Alcoholism Animal Care and Use Committee. Created in BioRender. Piantadosi, P. (2024) https://BioRender.com/a71t309. Panel b reproduced from ref. 37, under a Creative Commons licence CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/).

Procedure 2: FP

In the case of the NAc-DA recordings described in Procedure 2, TTL timestamps for each CS+ and negative (CS−) trial were used to extract behaviorally aligned FP data. Following filtration, fitting and normalization of ΔF/F₀ data, the NAc-DA signal was found to change dynamically during CS+ and CS− trials, as demonstrated at the level of single and averaged trial DA dynamics in representative mice (Fig. 11b, left) and across mice (Fig. 11b, right).

Procedure 3: microendoscopic imaging

An example of a microendoscope FOV from neurons recorded in the NAc, with a mean-intensity projected image, is shown on the left of Fig. 11c, and CNMFe identified footprints corresponding to putative single neurons are shown on the right of Fig. 11c. Imaging of NAc revealed a complex regulation of neural activity by footshock punishment, with neurons increasing, decreasing or remaining unchanged (Fig. 11d).

Supplementary Material

Reporting Summary

NIHMS2098881-supplement-Reporting_Summary.pdf^{(1.7MB, pdf)}

Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41596-025-01143-x.

Key points.

This protocol extension enables users to integrate optically based manipulation and measurement techniques, such as optogenetic manipulation, fiber photometry and microendoscopic imaging, into their touchscreen experimental systems.
This builds on the authors’ previous protocols that described rodent touchscreen-based behavioral assessments of motivation, memory and executive function, now detailing best practices for incorporating optical tools with touchscreen testing.

Acknowledgements

P.T.P. and A.H. are supported by the NIAAA Intramural Research Program, including a Center on Compulsive Behaviors Fellowship to P.T.P. L.M.S., T.J.B., V.F.P. and M.A.M.P. received support from the Canadian Institutes of Health Research (CIHR, PJT 162431, PJT 159781, PJT 426966), Natural Science and Engineering Research Council of Canada (06577-2018 RGPIN; 03592-2021 RGPIN, 2019-06102 RGPIN and 2019-06087 RGPIN) and a BrainsCAN Canada First Research Excellence Fund Accelerator Awards, Initiative for Translational Neuroscience, Brain Canada Platform Support Grant, as well as support from New Frontiers Research Fund (NFRF-TRIDENT). M.A.M.P. is a Tier I Canada Research Chair in Neurochemistry of Dementia. L.M.S. is a Tier I Canada Research Chair in Translational Cognitive Neuroscience. T.J.B. is a Western Research Chair. Additional thanks to M. Croxall for technical support that was critical to establishing the protocols outlined here.

Footnotes

Competing interests

T.J.B. and L.M.S. have established a series of targeted cognitive tests for animals, administered via touchscreen within a custom environment known as the ‘Bussey–Saksida touchscreen chamber’. Cambridge Enterprise, the technology transfer office of the University of Cambridge, supported commercialization of the Bussey–Saksida chamber, culminating in a license to Campden Instruments. Any financial compensation received from commercialization of the technology is fully invested in further touchscreen development and/or maintenance.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Raw data corresponding to the data in Figs. 2d,e, 3b and 11a,d are available at Zenodo via https://doi.org/10.5281/zenodo.14487150 (ref. 167).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Reporting Summary

NIHMS2098881-supplement-Reporting_Summary.pdf^{(1.7MB, pdf)}

Data Availability Statement

Raw data corresponding to the data in Figs. 2d,e, 3b and 11a,d are available at Zenodo via https://doi.org/10.5281/zenodo.14487150 (ref. 167).

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PERMALINK

Integrating optical neuroscience tools into touchscreen operant systems

Patrick T Piantadosi

Oren Princz-Lebel

Miguel Skirzewski

Julie R Dumont

Daniel Palmer

Sara Memar

Lisa M Saksida

Vania F Prado

Marco A M Prado

Tim J Bussey

Andrew Holmes

Abstract

Introduction

Overview

Applications of the protocol

BOX 1. General touchscreen system setup and preparation.

Setup

• TIMING 1 d

Fig. 1 |. Touchscreen setup.

Optogenetic manipulation

FP

Imaging

Comparison with alternative available methods

Optogenetic manipulation

FP

Microendoscopic imaging

Limitations

Optogenetic manipulation

FP

Microendoscopic imaging

Experimental design

Surgical considerations relevant to touchscreen testing

Addressing tether-related artifacts during touchscreen testing

Fig. 2 |. Procedures for alleviating tether-related issues.

Recommended touchscreen chamber alterations

Fig. 3 |. Alleviating movement impediments and synchronizing data streams.

Synchronizing data streams during touchscreen testing

Task-dependent experimental considerations

Fig. 4 |. Animal-independent and animal-controlled task events.

Materials

Biological materials

Reagents

Equipment

General touchscreen equipment

Drill supplies for altering sound-attenuating box and aperture mask (as necessary)

Optogenetic manipulation

FP

Microendoscopic imaging

Equipment setup

Procedure 1: combining touchscreen testing with optogenetic manipulation

Fig. 5 |. Equipping touchscreen chambers for optogenetic manipulation.

Day 1: set up touchscreen equipment and optogenetic manipulation hardware

● TIMING ~1 d

Fig. 6 |. Hardware and software updates for optogenetic manipulation.

Fig. 7 |. Commutator setup for optogenetic manipulation.

Day 2+: conduct training and testing using optogenetic manipulation

● TIMING ≥1 d

Day 3+: optogenetic data analysis

Procedure 2: combining touchscreen testing with FP

Fig. 8 |. Equipping touchscreen chambers for FP.

Day 1: set up FP hardware and software

● TIMING 1 d

Day 2: collect FP data during touchscreen testing

● TIMING ≥1 d

FP data analysis

Procedure 3: combining touchscreen testing with microendoscopic imaging

Fig. 9 |. Equipping touchscreen chambers for microendoscopic imaging.

Day 1: set up microendoscope and DAQ

● TIMING 1 d

Setting up the ‘dummy’ microendoscope to habituate mice to the tether

Setting up the functional microendoscope

Fig. 10 |. Commutator and software setup for microendoscopic imaging.

Day 2+: conduct microendoscopic imaging during touchscreen behavior

● TIMING ≥1 d

Day 3+: analyze microendoscopic imaging data

Troubleshooting

Table 1 |.

Timing