Gathering Self-Initiated Rat Behavioral Data to Characterize Post-Stroke Deficits

Summary

A system for acquiring data from self-initiated individual behavior sessions within a social colony cage setting is presented. The efficacy of this system is demonstrated using an automated skilled reach assessment, enabling the characterization of post-stroke motor impairments, potential behavioral alterations related to motivation, circadian variations, and other innovative dependent variables.

Abstract

Behavioral testing in rat models is frequently utilized for diverse purposes, including psychological, biomedical, and behavioral research. Many traditional approaches involve individual, one-on-one testing sessions between a single researcher and each animal in an experiment. This setup can be very time consuming for the researcher, and their presence may impact the behavioral data in unwanted ways. Additionally, traditional caging for rat research imposes a lack of enrichment, exercise, and socialization that would normally be typical for the species, and this context may also skew the results of behavioral data. Overcoming these limitations may be worthwhile for several research applications, including the study of acquired brain injury. Here, an example method is presented for automatically training and testing individual rat behavior in a colony cage without the presence of humans. Radio frequency identification can be utilized to tailor sessions to the individual rat. The validation of this system occurred in the example context of measuring skilled forelimb motor performance before and after stroke. Traditional characteristics of post-stroke behavioral impairments and novel measures enabled by the system are measured, including success rate, various aspects of pull force, bout analysis, initiation rate and patterns, session duration, and circadian patterns. These variables can be collected automatically with few limitations; though the apparatus removes experimental control of exposure, timing and practice, the validation produced reasonable consistency in these variables from animal to animal.

Introduction

Behavioral training and testing with rat models are important in countless research areas, from the exploration of cognitive processes to disease states and more¹. Typically, this training and testing is conducted with single animals in one-on-one sessions, with a researcher manually removing the animal from their home caging and temporarily placing them in some kind of apparatus. Unfortunately, there are several difficulties and limitations with this approach. First, behavioral testing can take a great deal of time for researchers, and when training is necessary, that time requirement becomes even greater. Second, this approach automatically affects-or even potentially confounds-the acquired data, as has been established elsewhere². These confounds are especially salient when considering enrichment-related variables. Specifically, laboratory rats are traditionally housed in small cages that are just big enough for one or two rats³, and if running wheels are not provided, they may go a lifetime without meaningful opportunities to exercise. Additionally, isolated housing can be a major source of stress in a social species such as the rat⁴. Some of these welfare-related drawbacks likely impact rat physiology^5,6, which may preempt the development of species-typical behavioral expression⁴ and impact the quality of rodent models as applied to human contexts.

Researchers have pursued several types of solutions to these problems in recent years. The simplest type of solution has been to automate behavioral testing and training^7,8,9,10, thus removing the requirement for a single researcher to attend to a single animal. An additional solution has been to automate animal transfer to experimental chambers^11,12, further removing the need for human involvement. Last, several setups have been explored which allow animals to be housed in colony caging with other animals and with more room for exploration and enrichment¹³. Despite these advantages, such colony setups can limit or complicate the efforts to gather individually differentiated behavioral data (though see efforts to use computer vision)^14,15. If individual behavioral data is required, it can be more difficult or complex to identify and retrieve animals from colony caging for behavioral sessions as well. At present, few systems exist for collecting individual behavioral data from (enriched) colony housing^16,17,18.

These drawbacks may specifically impact research on the behavioral effects of acquired brain injury. First, it is clear that the presence and/or sex of humans as well as handling practices affect rodent behavior^2,19, and these variables may differentially impact the behavior of rats before vs. after stroke. Second, human behavioral outcomes after stroke can be worsened by voluntarily decreased engagement with the recommended dosage of rehabilitation exercises²⁰. Currently, rodent experiments tend to not model this sort of context, because rats are not free to choose to engage or abstain from behavioral sessions.

This article introduces a protocol designed to facilitate individual behavioral testing within the framework of enriched colony caging. This approach not only addresses the constraints of current practices but also opens avenues for the exploration of innovative measures. A one-rat turnstile (ORT) has been developed and can be affixed to a colony cage, enabling animals to enter behavioral chambers independently and initiate their own training and testing sessions. The system is affordable; each ORT can be assembled at low cost (given access to a 3D printer). In the past, validation of this system was carried out using a basic operant chamber, showing that animals could be consistently trained to perform a simple operant lever press without the presence of an experimenter¹⁶. Nevertheless, the question of whether this configuration is applicable to other scenarios remains unresolved. The aim is to validate the effectiveness of the ORT-colony caging setup, which was previously established, for training and quantifying skilled reach behavior relevant to motor impairment following a stroke. The configuration was utilized to generate novel variables that are typically not explored in stroke research. These variables include performance metrics for the skilled reach task and measurements of self-initiation, which could be pertinent to motivation and decision-making. Furthermore, stroke-induced changes in the circadian patterns of daily self-initiation across the entire 24 h period were effectively detected.

Protocol

All procedures and animal care were approved by the University of North Texas institutional animal care and use committee (IACUC) and adhered to National Institutes of Health guide for the care and use of Laboratory animals. Adult male and female Long-Evans rats (400-800 g, 1.5 years old), used in the present study, were housed in colony caging.

1. Equipment preparation

1. Obtain or assemble the one-rat turnstile (ORT) according to the design files and instructions for construction (see Supplementary File 1 and Supplementary Coding File 1). Refer to Butcher et al.¹⁶ for further details.
   NOTE: ORTs are specific to rat size, so a colony cage should include animals that are approximately the same size. If one doesn't wish to self-assemble ORTs, they may be purchased pre-assembled (see Table of Materials).
2. Obtain and attach a radio-frequency identification (RFID, see Table of Materials) reader and obtain and inject animals with RFID tags.
   NOTE: When injecting RFID full duplex (FDX) tags, the orientation must be perpendicular to the RFID antenna as the rat walks through the ORT. In this validation, tags were implanted subcutaneously between the shoulder blade on a plane parallel with the spine.
3. Affix the RFID antenna to the tube of the ORT.
4. Construct and/or obtain the behavioral apparatus(es) and colony caging appropriate for the experimental question. In this example, custom-built colony caging^21,22 was used in conjunction with commercially available operant chambers (see Table of Materials), though any equipment could theoretically be used.
   NOTE: Competition of colony-housed animals for access to the behavioral apparatus(es) via the ORT should be considered. Anticipate needing one ORT + behavioral apparatus for every 4 to 6 animals.
5. Attach the ORT(s) between the behavioral apparatus and colony caging.
6. Cut a portal hole in the behavioral apparatus and colony caging using a Dremel rotary tool (see Table of Materials) or similar instrument. The internal diameter should be equal to the outer diameter of the constructed ORT tunnel.
   ​NOTE: The ORT must be elevated a few inches to operate, so a small platform or stand will be needed to align the colony caging and apparatus heights.
7. Install RFID system to read animals as they pass through the ORT and, if desired, integrate it with the behavioral apparatus.

2. Presurgical behavioral training

1. Obtain same-sized cohort of rats and introduce them into the colony caging.
   NOTE: Animals that have been reared or housed extensively in isolation or with few conspecifics may have more trouble exploring the chamber, especially when it involves traversing social areas of the colony caging. Animals should be exposed to group caging early in life to avoid this pitfall.
2. Remove access to any manipulanda within the behavioral apparatus and set the chamber to auto-deliver rewards every 60 s, on average, when occupied.
   NOTE: This study used sucrose water (30% to 40%) as the reward, but sweetened condensed milk is also effective.
3. Train all rats to regularly enter the behavioral apparatus(es) via the ORT.
4. At least once per day, check the data to ensure all animals are entering the ORT. If animals are not entering, insert a pen-sized object into the locking mechanism to prevent it from locking temporarily allow animals to explore more freely. If animals are still not entering, remove the turnstile and attach a temporary sidewall to allow free tunnel access to the chamber.
5. Once all animals are regularly entering the chamber, return the lock (and turnstile) and reassess.
   NOTE: Animals may also occupy the ORT and chamber as a temporary reprieve from other rats. One way to preempt this sort of monopolization of the chamber is to attach an additional ORT that bridges to a simple isolation chamber.
6. Introduce the manipulandum-the pull handle, in this example case-and set to the highest sensitivity. Insert the handle just inside the box (up to 2 cm) or just outside the box.
   NOTE: Painter's tape can evoke reach attempts if affixed to the back of the handle, just out of reach.
7. Reduce the frequency that the reward (i.e., 30% sucrose water) is auto-delivered (e.g., every 90-120 s). Remember that any reward can be utilized that fits the needs of the experimenter and the animals' preferences.
8. Check the data daily to ensure that all animals have learned to activate the lever. Bait the lever and/or change insertion level until all animals are pulling.
9. Discontinue auto-delivery of rewards so that they are only available via activation of the pull handle.
10. If previously inserted, retract the lever each day (provided that all rats continue to pull at that retraction level) by 0.25 mm to 0.5 mm until the lever is in its final position, 1 cm to 1.25 cm outside the chamber.
    NOTE: The exact position of the lever depends on the size of the rats. Ensure to choose a position which results in the desired reaching topography.
11. Initiate a percentile or other training program to progressively increase required pull forces to activate the handle.
    NOTE: This study used a percentile schedule that sets the criterion for reinforcement at the upper quartile of the previous 15 responses. Alternatively, stepwise increases of the pull criterion can be used⁷.
12. Once animals reliably reach the final criterion range of 120 g pulls, remove the percentile training program and fix the criterion for handle activation at a constant of 120 g.
13. Collect baseline data at this force requirement until success rates have been steady (non-trending) for about a week.

3. Inducing stroke

1. Surgically induce stroke in all colony caged animals at the same time.
   NOTE: To induce the stroke an endothelian-1 model of stroke was used, which has been described elsewhere²³.
2. Allow animals to recover in traditional caging, isolated individually, for 3-7 days.

4. Postsurgical behavioral testing

1. After recovery, return animals to the colony caging with the ORT-attached skilled reach apparatus.
2. Perform the behavioral testing, keeping the pull requirements at the final of 120 g (follow step 2) until sufficient data is collected to evaluate post-stroke deficits (from one to several days).
3. Implement any post-stroke or recovery-related independent variables during subsequent days while animals are accessing the chamber.

Results

The animals were trained and tested with four female rats in one colony cage and four male rats in a separate colony cage. All rats learned to pass through the ORTs in four days or less. The four female rats reached >85% successful bouts at the 120 g force requirement in approximately 6 weeks of training and the male rats reached the same criterion in 10 weeks (compared to roughly 3 weeks with standard training with deprived rats)⁷. This training duration was greatly lengthened due to several ...

Discussion

This protocol has multiple uses. First, and most broadly, the ORT was developed for the purpose of enabling automated single-subject behavioral training and data collection in the context of social, enriched housing. While this study tested the idea of collecting typical behavioral measures and elaborating upon them in the context of stroke, the same can be done for other applications and behavioral tasks. Even the measures gathered in this validation can also be adjusted as needed to include alternative reinforcement sc...

Materials

Name	Company	Catalog Number	Comments
3D printer			Consult with local makerspace
bolt	Boltdepot	1346	6-32 or 8-32 by  0.5"
bolt	Boltdepot	1348	6-32 or 8-32 by  0.75"
door hinge	XJS (Amazon)	43398-16234	1" cabinet stainless steel door hinge set; Optional (if "perfect hinge" is not printed)
drill			Any electric drill works
extension spring	Nieko (Amazon)	50456A	Choose and adjust spring based on ORT sized and desired tension
granulated sugar
lock nuts	Boltdepot	2551	6-32 or 8-32
measuring tape
microcontroller	Arduino	A000066	Arduino Uno
microswitch	Sparkfun	KW4-Z5F	mini microswitch (SPDT-roller lever)
One Rat Turnstile (ORT)	Vulintus		Contact company to request quote if not self-assembling
Operant Chambers as desired for behavioral assessment: For this experiment we used automated isometric pull chambers from Vulintus	Vulintus	No cat #: contact Vulintus	Contact Vulintus for quote
PLA filament	OVERTURE (Amazon)	UK-MATTEPLA17511
plexiglass	Lesnlok (Amazon)	B09P74K7BR	clear, 1/8" thickness, Cut to size
plexiglass cutter
python program	Python Software Foundation		software available on request
RFID reader	Priority 1 Design	RFIDRW-E-USB	With antenna
RFID tag	Unified Information Devices	UC-1485-10
rod	Boltdepot	23632	cut to > 3.5"
Rotary tool			Used to bore hole in apparatus and colony caging for ORT; any hardware usable
sand paper	HSYMQ (Amazon)	TOMPOL-1118-1915-11
socket wrench set			Any socket wrench set works
soldering iron
super glue		234790
wire	Plusivo (Amazon)	EAN0721248989789