Carbon Nanotube Sensors Enable Visualization of Dopamine Neuromodulation at the Resolution of a Single Chemical Synapse

Abstract

Among the organs in our body, the brain easily remains the most intriguing in terms of its complexity and function. Nerve cells, which are the functional building blocks of the brain, operate in complex networks that underpin most of the brain’s capabilities, including ability to learn, remember, initiate and orchestrate complex movement. In systems neuroscience, behavioral assays and large-scale neuronal activity recording have broadened our understanding of the role that specific brain regions and neuronal circuits play in a behaving animal. An equally exciting aspect of neuroscience concerns itself with the cellular and molecular underpinnings of nerve cell function. Unique among all the organs, the brain’s constituent cells, neurons, need to communicate with another in intricate networks that are responsible for circuit function, and most of this communication is enabled by chemical cues that are released between neurons. Better understanding of neuronal communication via chemical release from synapses requires technologies that can help us visualize and measure the spatial and temporal dynamics of these chemical signals. Our lab seeks to address this challenge by developing optical biosensors with ultra-low detection capabilities, high spatial resolution and signal-to-noise ratio.To study chemical synapses at high spatial resolution, we use primary dopamine neuron cultures derived from rats and mice as a model system. Dopamine neuron signaling is critical for learning and motor control, and its aberration is implicated in a wide range of neurological and psychiatric disorders. Furthermore, dopamine is a neuromodulator, and it is a neurochemical that is predicted to be highly diffusive in its spatial signaling. Our lab and several others have developed ssDNA functionalized SWCNT optical sensors to study catecholaminergic neurotransmitters, including dopamine. In a recent publication we reported an assay to study dopamine effluxes from primary dopaminergic neurons using a chemi-sensitive 2D substrate (DopaFilm). DopaFilm is fabricated from solution phase SWCNT-sensors in two simple steps: (1) surface functionalization of glass coverslips using silane chemistry to anchor nanosensors (2) drop casting of nanotube sensor solution to create the 2D nanofilm followed by passivation with a thin layer of poly(D-lysine). Primary dopamine neurons plated on DopaFilm are allowed to grow and mature (2 – 6 weeks depending on the experiment). Dopamine release activity can be imaged by recording the NIR emission (900-1400 nm) and at excitation with a 785nm laser. With this technology we were able to faithfully measure dopamine concentrations as low as 1nM with DopaFilm, which has an apparent dissociation constant of 268nM. In 2D cultures, we routinely observed spontaneous and evoked dendritic and axonal DA release events with bouton level spatial resolutions and sub-second temporal resolutions. With signal-to-noise ratios above 50 and comparable on and off-kinetics to latest genetically encoded dopamine sensors (dLight1 and GRABDA), DopaFilm is by far the most sensitive technology to study dopamine chemical signaling with the resolution of a single synapse. The technology allows the study of an entire neuron at the resolution of a single synapse. We combine functional imaging data with pharmacological and genetic perturbations to dissect the role that circuit effects and specific genes (proteins) play in dopamine synthesis, packaging, and release. Additionally, by taking advantage of the NIR emission of SWCNTs, we are able to multiplex dopamine release imaging with Ca2+ activity in orthogonal channels, a feat that is yet to be demonstrated with any competing technology.