Animal models continue to be important tools for understanding disease mechanisms and for preclinical testing of potential therapeutics. Although the mouse is currently the most widely used species to model neurobiological disorders, we recognize that other model systems may also provide important insights.
Relative to the mouse, the rat has a larger brain and exhibits a more complex behavioural repertoire but still retains many tractable characteristics that make it amenable for laboratory research. Recent developments in genomic-editing technologies have facilitated the ability to manipulate the rat genome, thus spurring interest in the rat as a model for genetically linked disorders.
As such, our funder SFARI is working with the Medical College of Wisconsin (MCW) to generate and distribute CRISPR/Cas9 rat models of autism. Models will be maintained in the outbred Long-Evans background strain, as this is often the strain of choice for cognitive, behavioural and systems neuroscience studies. The intent is for these models to be available to any qualified researcher, with minimal cost and restrictions.
Syngap x 2
Fmr1 cON/y; Syngap1 cON/-
As an initial effort to characterize these lines, these models are being behaviorally phenotyped at SIDB and the Center for Development and Repair in Bangalore. Rat models are phenotyped according to a rigorous pipeline that assesses behaviours relevant to autism spectrum disorder, such as social and motor skills, and learning and sensory processing. Different cohorts of rats are run through different subsets of tasks to overcome potential order effects on behaviours and to limit the number of tasks each animal is put through.
Resulting data will be available pre-publication via downloadable behavioral datasheets (see links to available datasheets above).
The SIDB behavioral pipeline includes the following tasks:
In addition, prior to pipeline phenotyping, some rats are characterized using the following experimental set-ups/methods:
Eco-ethological housing system that permits rats to express a wide-range of innate behaviors that can be recorded and analyzed without disturbing animals.
The following schematic of the Habitat is an experimental set up devised by Peter Kind and colleagues at SIDB to study rat social, cognitive and motor behaviors in the lab. These complex behaviors include rat social hierarchies, fear and isolation, communication, play, memory, motor coordination, reaction to novelty and circadian rhythms.
Mimicking rat burrows
(Image courtesy of Peter Kind/Edinburgh University)
Between the SIDB Edinburgh and Bangalore sites, we have 15 - 20 different rat models, all monogenic forms of autism spectrum disorder, typically the high-confidence genes on the SFARI Gene list.
Earlier in 2021, we successfully generated our first conditional-on (c-on) using Crispr technology and the Flex system. To date, we have generated both the c-on models for Fmr1 and Syngap. We have also generated a tamoxifen inducible CAGG-Cre rat line to induce constitutive gene re-expression at selected ages.
These animals will allow us to discern whether particular monogenic forms of ASD have critical periods for gene re-expression.
Director of SIDB and professor at the University of Edinburgh
Professor at the University of California, San Francisco and a Howard Hughes Medical Institute Investigator
The Simons Foundation Autism Research Initiative (SFARI) recently announced the development of eight new genetic rat models for studies of autism spectrum disorder. The rat models, maintained in the outbred Long-Evans background strain, are available for use from the Medical College of Wisconsin. I recently spoke with SFARI Investigators Peter Kind and Loren Frank on their work with these rat models and their views on how these models will aid autism research.
Peter Kind is Director of the Simons Initiative for the Developing Brain (SIDB) and SFARI’s partner in the behavioral phenotyping of SFARI’s autism rat models. Kind is also a professor at the University of Edinburgh, where his laboratory focuses on understanding the roles that synaptic function and dysfunction play in the manifestation of neurodevelopmental conditions.
Loren Frank is a professor at the University of California, San Francisco, a Howard Hughes Medical Institute Investigator and a SFARI Investigator. Frank’s laboratory has an expertise in understanding how the hippocampus supports memory storage, memory retrieval and memory-guided decision-making. He was previously awarded a SFARI grant to examine learning and memory in Fmr1 rats, a model of fragile X syndrome.
The interview has been edited for clarity and brevity.
Peter: Between the SIDB Edinburgh site and the Bangalore site, we have somewhere between 15 and 20 different rat models, all to monogenic forms of autism spectrum disorder, typically the high-confidence genes on the SFARI Gene list.
We started with the fragile X and the SynGAP rats because Fmr1 and Syngap are two genes we were particularly interested in. We moved to the rat model primarily because of the complex social behaviors that rats have that mice do not. Rats evolved to live in large groups of up to 150 individuals, and they have numerous cooperative behaviors, such as hunting and playing. Mice are much more loner animals and have evolved far fewer social interactions.
But I would never say one model is better than another model. In fact, I’m a firm believer that we need more models, not fewer. We’ve learned a huge amount from flies, from fish[…]. It really comes down to the hypothesis you’re testing and the experiments you need to do to test that hypothesis. Different models are better than others to address particular questions. We thought the complex social repertoire of behaviors in the rat would be a particular advantage for studying autism.
There is also a lot of autism data coming out of mice1, and we thought that cross-species validation would be very important to understand any translatability across mammalian species2. I know people look at rats and just see big mice, but there’s actually about 12 million years of evolution between them. To put that into perspective, there’s six million years of evolution between a chimpanzee and a human. So rats and mice are very distinct species, but offer many of the same advantages in terms of large litter size and shortened developmental periods. Rat neurons are also much more robust in culture than mouse neurons, and that’s turned into a huge advantage for many of our colleagues. Also, from a lab-specific point of view, we are very interested in using longitudinal functional magnetic resonance imaging, and because of the rat’s bigger brain, we can get much more resolution using rats.
Loren: We have always been a rat lab for some of the reasons that Peter mentioned, including how social rats are. Also, just cognitive capacity-wise, we can get our rats to do more complicated behaviors that we cannot get a mouse to do.
When we started to look at autism rat models, we started with an inbred Sprague Dawley strain. From a genetic homogeneity perspective, inbred is the way to go. But it’s not obvious from a ‘modeling human conditions’ perspective. In fact, we have found that the wild-type inbred Sprague Dawley strain performs at the same cognitive level as hippocampally lesioned Long-Evans animals. Sprague Dawley rats are also closer to blind, so you need really bright lights for behavioral tasks with them. Since we are interested in hippocampal-dependent cognition and behavior in our lab, we always work with an outbred Long-Evans strain.
Peter: I completely agree with what Loren said. Both the Sprague Dawley and Long-Evans models we use are all outbred strains. And the cognitive capacity of the rats is a big plus. We wanted to use some of the complex behavioral tasks developed in Richard Morris’ and Emma Wood’s laboratories, here at the University of Edinburgh, with our autism models, and we can’t get mice to do those tasks.
The accompanying figure shows that spatial learning and memory in the water maze are not affected by the loss of the fragile X mental retardation protein (FMRP) in rats. Fmr1 KO rats exhibited learning that was similar to wild-type rats, as measured as decreased path length to reach a submerged escape platform (A) and increased crossing of the platform (B) over trials. In contrast, prior work has shown that Fmr1 KO mice exhibit learning deficits in this same spatial task when compared to wild-type mice (data not shown; e.g., Baker K.B. et al.1).
Peter: When we set up the pipeline, we wanted our tasks to be as unbiased and as comprehensive as possible. In that context, it’s important to remember that the behavioral repertoire of a rat is very distinct from the behavioral repertoire of a human. So, we don’t ask if our rats look like a human with the corresponding condition, something often referred to as ‘face validity.’ Instead, we ask how a given mutation affects the natural behaviors of a rat, including a lot of spontaneous behaviors that rely on a wide range of brain areas.
To keep the pipeline standardized and to make sure that we can reproduce behaviors across sites, we also set it up at two different locations, running many of the tasks in both Bangalore, India, and here in Edinburgh, Scotland. All of the people across those sites are trained to do the behavioral assays using identical, and very detailed, protocols that take into account not just what the tasks are but how the tasks are run and the context in which they are run. For example, tasks are run for every rat model in the exact same order and at the same point in the animal’s light-dark cycle. Everybody running the tasks and the Principal Investigators involved also meet once a week to make sure things remain standardized. And at those sessions, the discussions can get very lively and everyone contributes to how the data should be analyzed and interpreted. With all of this, we’re trying to build what we loosely call an ‘ethogram’ for each animal model.
Loren: It is deeply heartening, Peter, to hear what you are doing. It’s really important to understand the complex behaviors of these animals as a starting point. The brain has changed in a whole bunch of complicated ways as a result of a genetic mutation. We have to really understand the whole process of what makes these animals different, behaviorally, so that we know how to relate these differences at the neural level. It’s an incredibly important and an incredibly hard problem.
Phenotyping rats. Rat models are phenotyped according to a rigorous pipeline that assesses behaviors relevant to autism spectrum disorder, such as social and motor skills, learning and sensory processing. Different cohorts of rats are run through pipeline 1 and pipeline 2 to overcome potential order effects on behaviors and to limit the number of tasks each animal is put through. For both pipeline 1 and 2, some groups live in the Habitat prior to and during participation in the pipelines. (*MoSeq was originally developed by SFARI Investigator Sandeep Robert Datta to study mouse behaviors3.
Loren: Our lab has always been interested in fundamental cognitive abilities, like creating memories through the events of daily life, and using those to make decisions. In early conversations with SFARI, there was a recognition that we might understand enough about these processes to be able to apply what we know to disease models. So we picked Fmr1 because it was well studied on the mouse model side, so we knew we would have something to compare to.
David Kastner, a postdoc in my lab who’s also an M.D. psychiatrist, asked what behavioral assays we could use. One assay used a lot in studies of psychiatric disease in humans, as well as mice and rat models, is something called ‘prepulse inhibition.’ It’s a really simple paradigm. You basically give a first sound and then a loud second sound follows, and then you ask how much the animal jumps. This seems sort of silly in a way, but this behavior is altered in schizophrenia, and it can be different between males and females. At the time, there were various reports in literature that Fmr1 mice were either the same as humans, the opposite, or showed no effect with prepulse inhibition.
And what we found was that we had to reengineer the whole paradigm. The way it had been done before was with an arbitrarily loud first sound, an arbitrary delay and an arbitrarily loud second sound, and then a small number of trials to measure how much that affected the animal. So we came up with a new, consistent set of paradigms and a new mathematical model for understanding prepulse inhibition, and we found no consistent differences in prepulse inhibition across Fmr1 mutant and non-mutant rats. But we did find consistent differences between males and females, so we know that it’s not that we’re just ignoring things4.
It was a real lesson for us. Behavior has to be done really carefully and thoughtfully. Behavior is the output of an incredibly complicated neural process. So all the things Peter was talking about needing, including replicating things across cohorts, are important to really be confident about behavioral results.
Peter: I’ll just add something to that, following on beautifully from what Loren just said. We’ve just characterized another rat autism model that lacks Nlgn3 and found that the animals freeze less in a standard, cued fear-conditioning task5. This would have been typically interpreted as the rats having a problem with fear learning. But by running numerous different tasks, we found that the animals were actually learning, but using a different expression of fear. Whenever you’re faced with fear, you either do fight, flight or freeze. Normally, rats freeze, but our animals were responding with flight. As Loren said, behavior is incredibly complicated, and you need really good behaviorists, and you need to be very careful with your interpretations on what are believed to be standard tests. The tests are standard, but the interpretations can be often very difficult.
Peter: When people think of treatments, they typically think of therapeutic testing in animal models. Pharmaceutical companies have used rats for decades because they are particularly well suited, as the metabolism of the rat is closer than mice’s to that of a human. However, there is also still much to be learned from fundamental science that has important implications for neurodevelopmental conditions. For example, Adrian Bird’s work on Rett Syndrome showed that if you re-express the gene even late in an animal’s life, the animal recovers amazing function6. That challenged the whole idea that there are critical periods during development when treatments would be more effective. But we have no idea how generalizable this is. So a focal point for SIDB is to determine whether the monogenic forms of autism are issues of neural development or neural maintenance. I’d like to see the field start classifying, gene by gene, which conditions have a critical period for intervention — those that are truly neural developmental — and which are associated with neural maintenance and able to be corrected even later in life. That stratification has huge implications for treatment strategies in humans.
In terms of testing pharmaceuticals, we also need to remember that it’s not just about bringing things into clinical trials. Every time you give a drug, that’s a valid fundamental experiment on cellular biochemistry and physiology as well as the circuits involved.
Loren: I can just add one other perspective on this. People are starting to think about therapies that might live at a circuit level, as opposed to a pharmacological level. These animal models have real power to help us figure out the systems and to know how they respond.
Systematic examination of prepulse inhibition. When an animal or person responds less to a startlingly loud noise that has been preceded by a quieter sound, this is referred to as prepulse inhibition (PPI) of the startle reflex. Changes in PPI have been linked to a variety of neuropsychiatric conditions. Using a new method for assessing PPI, Frank’s laboratory recently showed that Fmr1 KO rats exhibited the same levels of PPI response as wild-type rats, but that there were differences between wild-type males and females. Image from Miller E.A. et al.4
Loren: The question for me is: What are the things that SFARI can do that nobody else is going to do? Their amazing human genetic work, in terms of putting together the Simons Simplex Collection, Simons Searchlight and SPARK cohorts, has really been fairly unique. And the addition of all of these animal models, doing all the work of creating them and making them easily available, is a huge service to the field. It allows you to ask questions about whether there are common phenotypes and common underlying mechanisms.
I’d also like to see them continue the support for fundamental biology that helps everybody in the community. And I’d like them to think hard about other long-term initiatives, like they did with the Circuit Dynamics request for applications. Things that we could really push and that allow people to try out some slightly crazy things that could move the needle and really change things. For example, our lab is thinking about whether we can teach animals to amplify patterns of brain activity — you know, neurofeedback. And if so, can we then translate that to disease models where we’re using in-built learning mechanisms to push the circuits to a state that’s back toward the standard behavior.
Peter: SFARI has always appreciated the importance of fundamental biology and exploratory science and that you often don’t know where the next big discovery is going to come from. We can learn amazing things about fundamental and autism biology. I think SFARI has done an incredible job at casting that wide net. The SFARI meetings are definitely amazing, bringing everyone together and fostering collaborative work.
SFARI could also play a big part in guiding clinical trials. If a scientific standard was set by SFARI, the premier foundation for autism research, it would change how we think about translating something through to a clinical trial.