Neuroscientists at IISc study monkey behaviour to answer fundamental questions about brain function
In 2021, Elon Musk took the internet by storm with his video game-playing monkeys. Using technology that transmitted neural data from the monkey’s brains directly to the computer screen, researchers from Musk’s Neuralink Corporation created systems in which the monkeys could play the game on the basis of their thoughts alone, without needing any physical movement. Hailed as a potential means to assist people with paralysis, this threw the spotlight on the significant applications of neuroscience research that involves our ancestral cousins – primates.
For a long time now, the human brain – with its thousands of neurons sending out signals that span our body – has enthralled and intimidated scientists in equal measure. Demystifying these neural connections is a monumental undertaking, and studying primates as model organisms has spearheaded our understanding of a vast array of neurological phenomena such as vision, memory formation and attention. What makes monkeys attractive candidates for such research is that since they share ancestry with human beings, they are highly intelligent, social animals whose body, brain and behaviour closely matches ours.
But this also means that conducting research on monkeys is more challenging than that on mice or fruit flies. For starters, there are understandably more ethical regulations for large animal experimentation. Investigators have to obtain ethics clearance from not only the Institutional Animal Ethics Committee, but also another Central Government body, the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).
The Indian Institute of Science has had a long history of primate research. Originally set up for biochemical research in 1965, the Primate Research Laboratory (PRL) housed hundreds of bonnet macaques (a species found in south India) at its peak. Ramesh V, who has been the Technical Officer-In-Charge of the Central Animal Facility for 36 years, recalls, “It was one of the largest facilities for bonnet monkeys in the world at the time, and was also used by other research institutes in the country.” In those days, monkeys were specifically bred for research in diverse areas such as reproductive biology, physiology and endocrinology. With the retirement of many of these researchers, the number of monkeys at the facility has dropped considerably to less than 50 animals. These days, the monkeys in the facility are used mainly for neuroscience research, which requires only a few animals per study.
Ramesh explains the efforts that go into ensuring that CPCSEA guidelines are followed to the letter, “We have dedicated staff for daily maintenance activities, such as cleaning the space, feeding animals and tending to any disruptions that break out. Even during the past three COVID waves, a smaller number of staff have come in daily so that the monkeys’ needs don’t go unattended.” Monkeys involved in research studies are kept separately from the others and are housed in large, spacious cages. Monkeys that are not used for research studies such as aged or pregnant animals, are kept in the larger, outdoor runs.
To enable brain recordings, the monkeys undergo a surgical procedure to implant a chip into their brain. This involves removing a small portion of the skull and placing a chip that has numerous electrodes contained in it. Each of these electrodes picks up electrical impulses from nearby neurons. The surgery is performed under anaesthesia with the guidance of a vet, while graduate students observe and assist with the process. Once the monkey recovers, the neurosensory chip allows scientists to record from hundreds of neurons. Interestingly, because data from each neuron is normally treated as a single unit, the scientists only need to study two to three monkeys to have enough samples to validate their hypotheses. As a result, very few monkeys are under experimentation at any given point of time. Usually, the monkeys chosen for experiments are young adults between 8-14 years of age, as cognitive functions can be different in juvenile and aged animals.
Laying the foundation
Owing to their higher intelligence and enhanced social capabilities, working with monkeys can be a challenge. “One of the most important things when working with monkeys is to establish a sense of trust between the student and the animal,” explains Supratim Ray, Associate Professor at CNS. Graduate students who choose to do primate research have to attend an orientation session where they are taught the basics of how to habituate the monkeys to their presence and the necessary precautions to take when working with monkeys. This includes wearing protective gear to maintain a sterile environment and prevent infections or injuries. Another key insight is to stay on guard and avoid eye contact, especially in the beginning, as monkeys often perceive it as a sign of aggression. New students initially approach the monkeys only with their seniors who are already acquainted with these animals.
Owing to their higher intelligence and enhanced social capabilities, working with monkeys can be a challenge
“Each monkey is unique,” says Surya Prakash, a graduate student in Supratim’s lab. “Some are very shy and retreat to the back of the cage when they see you coming, while others are more curious.” Usually, every student’s thesis starts with an outstretched arm holding out a treat for the assigned monkeys, hoping to entice the animals to come closer. Gradually, as the monkeys get used to the student’s presence, researchers spend longer hours in the facility petting the animals and learning which fruits they prefer so they can reward them with their favourite juice. To forge a deeper connection and establish a sense of intimacy, students play games such as making the monkey guess which of their fists contains food. Depending on the monkey’s inherent nature and the comfort level of the student, this can take anywhere from a few weeks to months.
But, as always, the proof is in the pudding. Once the students are reasonably certain of the bond they share with the monkeys, they start training them to engage in tasks that would lead them towards answering a research problem. “It is completely the choice of the monkey if he wants to perform the task.” explains Sini Simon, a graduate student in SP Arun’s lab at CNS, underlining the main hurdle in primate research. Both Arun’s and Supratim’s labs rely on the monkeys performing specific behavioural tasks to glean insights into different aspects of brain function. To induce monkeys to do their tasks, the researchers offer solid or fluid rewards to the animal for each step of training. They start small, initially encouraging the monkeys to open the researcher’s hand to get treats. With time, the monkeys begin to sit on a specially designed chair, and learn to perform the task either by moving their eyes or touching a screen. Each of these steps is reinforced by rewarding the monkey with juice or fruit, and never through punitive measures.
Getting down to monkey business
Once the monkey is wheeled into the experiment room, students grapple with the herculean challenge of training the monkeys to perform the experiment. Supratim’s lab studies neural mechanisms underlying the cognitive abilities of these primates by measuring a specific class of waves called “gamma”. Using an elaborate setup involving an eye tracker, a computerised reward system and a dot on the computer screen, these scientists slowly piece together the neuronal activity that regulates their attention. In this process, the monkey is required to stare at a small dot on the screen without blinking, while the eye tracker (essentially, an infrared camera) monitors its movements and a reading of gamma oscillations is taken through the chip implanted earlier. Every time the monkey looks at the screen for the required length of time, the automated system releases a few drops of juice via a pipe located close to the monkey’s mouth. While this process sounds pretty straightforward, training the monkeys to look at the screen for long durations can be cumbersome. “We usually increase the duration of time gradually, giving out the juice at longer intervals once the monkey starts adapting to the experiment.” explains Supratim.
At Arun’s lab, which studies the neural basis of vision and object recognition, the monkeys are trained to find differences between two objects. Known as the ‘same-different’ task, the experiments involve showing monkeys two images in rapid succession and training them to press a button if the two images are the same, and another button if the two images are different. Another task consists of displaying a series of photos on the screen and requires the monkeys to press the button if one of the images in the array is unrelated to others. Needless to say, because of the complexity that these processes demand, it takes a long time and a significant amount of effort (and, of course, juice) before the monkeys become proficient at these tasks and the researchers are able to record neuronal activity. Often, the monkeys understand the task, but have trouble touching the button with just one of their fingers, thereby generating faulty data by registering multiple touches at the wrong time. To circumvent such issues, scientists ease them into the process by starting with larger buttons and gradually scaling the button down to the desired size.
Recently, a new initiative by Arun’s lab has been to create a naturalistic environment in which to record brain activity. Unlike the traditional approach wherein monkeys enter the artificial environment of a lab to be studied, this method emulates the wild by including trees and open areas, increased natural light and foraging opportunities. Bringing in a touchscreen workstation, a juice pipe and a chinrest that holds monkeys’ heads immobile to enable gaze tracking, scientists at Arun’s lab achieved remarkable success as the monkeys performed the same-different task of their own volition, coming to the setup as and when they desired, while obtaining highly accurate gaze signals. Incredibly, naive monkeys that were allowed to mingle with trained monkeys in this space also picked up on the experiment within a few weeks – a novel finding that enables the easy training of more animals.
The road ahead
While results from research with primates have led to path-breaking discoveries across the world, training monkeys to perform any of these behavioural tasks is lined with difficulties. “Every monkey has its own challenges,” reveals Surya. “Some might be easy to approach but won’t get into the chair. Others will enter the lab in record time only to take a long time to figure out how the experiment works.” Armed with juice and incredible amounts of perseverance, it is the researcher’s job to patiently untangle these knots until the experiment is smoothly performed. Sini, from Arun’s lab, reiterates this sentiment, “You can’t just sit down and tell the monkey what the experiment is. We are at the mercy of the animals we record, so if the animal decides it is not going to come in for the recording, that’s it. You’re done.”
Despite the unpredictability of the animals’ behaviours, Surya and Sini agree that the experience has been extremely rewarding. After five years of working in this area, Surya admits that he is enthusiastic about working with these animals for his long-term research goals. “The one advantage you have of working with monkeys is that you get access to the kind of recordings that you won’t be able to get with humans. You can actually record at the single cell level. And you can also record from a population of neurons and examine how they communicate with another part of the brain,” he explains.
The future is brimming with possibilities. Arun has high hopes for experiments in the naturalistic environment. He believes that this could be a gateway to identifying the mechanisms that link vision to natural movements, such as seeing a fruit and jumping to another tree to retrieve it. “We want to ultimately understand how the visual areas in the brain talk to other regions of the brain while animals engage in natural behaviours,” he says. In tandem, Supratim’s lab has discovered that the gamma waves generated in the monkey brain are colour sensitive. He plans to eventually expand to other behavioural tasks for the monkeys to understand which colour-identifying neural circuits induce gamma wave production. Since gamma waves are associated with higher-level cognitive processes such as memory and attention, this research could also help us understand the neural circuits in both health and disease.
Anusha Rastogi is an Integrated PhD student in the Division of Biological Sciences and a former science writing intern at the Office of Communications, IISc