The circuits of the brain linking together the regions known as the midbrain, the striatum and the cortex are critical to understanding disorders such as Parkinson’s disease, addiction and autism. Understanding and modelling how the circuit works in human neurons has been difficult, mainly due to the inaccessibility of the human brain and the limitations of current technologies to reliably grow circuits of human neurons in the laboratory.
To tackle this challenge, a new Oxford collaborative team of biologists and engineers led by Professor Richard Wade-Martins (DPAG/Kavli), Professor Ed Walsh (Dept. Engineering Sciences) and Dr Ricardo Marquez Gomez (DPAG/Kavli) has been awarded a grant of £2 million from the BBSRC to bring together the technologies of human stem cells and oil-wall microfluidics. The human stem cells will be used to generate each specific neuronal subtype of the cortical-striatal-midbrain circuit held within oil-walled chambers. Such chambers can reliably construct microenvironments and circuits with long-term accessibility, contrasting with current microfluidic technologies based on rigid and single use plastics.
The project will study the physiology, regulation and cellular architecture of neurons in the circuit. Taking advantage of the adaptability properties of the fluid-walls technology, the team will also explore the regulation of gene expression in the cell body and along the neuronal axons which form the circuits to understand how genes control neuronal circuit function.
Early work from the team published in 2024 illustrated how the three neuron types could be grown together to help study Parkinson’s disease (Do et al NPJ Parkinson’s 2024), and that the oil-walls system supports neuronal growth (Nebuloni, Do et al Lab on a Chip 2024). The new BBSRC project kicked off in January 2025 for five years.
The research
The striatum is a brain region responsible for the correct integration of sensorial, motor, and cognitive information. This information arrives from the cortex and the striatum transforms it in a go or no-go instruction to the rest of the body. Given the vast amount of information arriving from the cortex, the striatum needs help modulating the cortex-striatal communication. This help comes in the form of small molecules such as dopamine, which modulates the conversation between the cortex and the striatum so the sensorial, motor, and cognitive information gets integrated properly and a given task can be performed successfully. These three brain regions (cortex, striatum and dopamine) form an important neuronal circuit that plays a major in movement processes for example, and when it fails it gives rise to disorders such as Parkinson’s disease. As important as it is, most of what we know about this circuit comes from studies performed in animals, and its study in humans has been limited to post-mortem or scan-based studies, without going in detail inside this human circuits. These limitations, point to the necessity of developing new tools to study this circuit in a human-like manner outside of the brain.
A major constrain of studying neuronal circuits outside of the brain, is their compartmentalization. The cortex, striatum and dopamine have their main centres in different parts of the brain and communicate through projections that come together in the striatum. Therefore, to study this neuronal triad we need to develop a system that allow us to segregate the neurons in different regions, while allowing them to communicate through projections send between them. To do this, we have developed a novel method using oil microfluidics. Oil-based microfluidics is a technique that allows to create microenvironments where neurons of human origin can be grown, generating a microcircuit on-a-dish. Moreover, these neurons can send projections through design conduits allowing us to control growth directionality by using microfluidic principles, ensuring that the communication between neuronal regions is as much alike as in a human brain.
In this project, we aim to use our oil-based microfluidic system to generate a circuit that contains the three neurons involved in the circuitry: cortical, striatal and dopamine neurons. These neurons will be differentiated from human pluripotent stem cells (hiPSCs) obtained from healthy individuals. First, we aim to build the microfluidic microenvironments in a miniaturized platform that allow us to study the circuitry with different experimental approaches. We will focus our study on the striatal cells, as centre of cortical inputs and dopamine modulation. Second, we will study the cellular structure of the striatum, which in the human brain, has two main neuronal populations that can be identified by expression of dopamine receptors named D1 and D2 receptors. We will generate hiPSCs neurons containing D1 or D2 receptors bound to fluorescent markers to study their molecular fingerprint within their projections using live high-resolution microscopy. Third, we will study the communication of the cortex and dopamine neurons with the striatum by using light-activatable proteins (opsins), allowing for unidirectional control of the communication between them and patch clamping, which allow to record neuronal activity of a single neuron. We will also study calcium dynamics within the neuronal projections and how this gets affected by neuronal activity controlled by opsins. Fourth, we will deepen in the neuron biology to study which genes and proteins are being expressed in neurons and in their neuronal projections. This will be achieved by a novel cutting and collecting technique that is feasible due to the flexibility of the oil-microfluidics and out cutting-edge robotics technology. Finally, we will combine Multielectrode array technology with our oil-microfluidics to generate the first-of-a-kind microcircuit recordings of neuronal activity.
This project represents an effort to apply oil-microfluidics technology to the study of human neuronal circuits, with unprecedented detail.