European Observatory for Nanomaterials

The human brain, the 1.3 kg organ that is safeguarded inside the skull, is one of the most mysterious biological objects in the known universe. While mostly composed of water by mass, it is home to roughly 85 billion neurons (building blocks of the brain). These neurons make approximately one quadrillion connections (synapses) (Figure 1a) to furnish movement, learning, emotions and ideation that makes humans the most intelligent species on earth. While manmade integrated circuits use electrical signals to communicate and enable downstream applications, neurons employ both electrical and chemical signals to pass information between each other. 

In general, neurons signal to one another through electrical impulses called action potentials. Following the initiation of an action potential, electrical signals propagate from the cell body to the axon and release neurochemicals to target neurons from axonal terminals where synaptic connections are located (Figure 1a). To peer into brain activity, one could follow electrical activity or neurochemical activity. Interestingly, electrical activity that enables action potential propagation in neurons results from the flux of cations from the extracellular space (outside) into the cytoplasm (inside), including calcium (Ca2+ ). This results in Ca2+ concentration dynamics, which can be coupled with fluorescence microscopy and used as a proxy for measuring electrical activity in neurons. 

While Ca2+ is a good surrogate for monitoring neuronal electrical activity, chemical signaling is what ultimately dictates flow of information between neurons (see synapse in Figure 1a). Generally, chemical signaling is generated by the biochemical reactions that are triggered by the electrical activity of action potentials. However, neurochemicals can also be released independent of action potentials. In such a scenario, action potential-independent release of neurochemicals is decoupled from electrical, and hence Ca2+ activity. As we broaden our knowledge on neural circuits with Ca2+ imaging, it is imperative to supplement Ca2+ activity data with measurements of neurotransmitter release events. 

Figure1.  (a) A rendering of a neuron with cell body, dendrites, an axon and a synapse at the end of an axon terminal. Chemical release from a synapse signals to the post synaptic neuron (b) Primary dopamine neurons grown on a dopamine-sensitive, fluorescent composite nanofilm.

Adapted from Bulumulla et al. 2022 (PMID: 35786443, DOI: 10.7554/eLife.78773)

Direct measurement of neurotransmitters is no easy task and is in fact far more challenging than measuring Ca2+ dynamics. During neuronal firing events, intracellular Ca2+ concentrations could rise from nanomolar to micromolar levels while changes in neurotransmitters concentrations are modest. Also, it is challenging to distinguish neurotransmitters from structurally similar compounds and metabolites. While there are few ways of measuring neurotransmitters, fluorescence imaging using biosensors is the preferred technique that provides adequate spatial and temporal resolutions. This effort is mostly enabled by genetically encoded protein-based sensors.

We, along with a handful of other labs, are developing a new class of biosensors constructed from single walled carbon nanotubes (SWCNT). SWCNTs represent a class of nanostructures with unique and advantageous photophysical properties including photoemission in the near infrared and short-wave infrared (NIR/SWIR) region of the spectrum (900 - 1400 nm) and high photostability. There are several ways of engineering biosensors out of SWCNTs, and one method employs non-covalent functionalization with single strand DNA (ssDNA) oligonucleotides. The selectivity towards a specific molecule of interest is imparted by the ssDNA sequence and the modular nature of DNA affords additional tunability. Out of many analytes, our lab focusses on an important neuromodulator called dopamine and seeks to elucidate the molecular machinery behind dopamine release at a cellular level.

In a recent report we disclosed a method that can help visualize dopamine effluxes from individual neurons with the spatial resolution of a single synapse and sub second temporal resolution. In addition to dopamine release events, we were able to visualize diffusion of dopamine outside of neurons following release. To visualize dopamine efflux and diffusion events, we first immobilized nanosensors on a glass substrate, passivated the nanosensor layer with polylysine, and cultured rat primary dopamine neurons on the composite film (Figure 1b). After maturation of dopamine neurons, we optogenetically stimulated the neurons to induce action potential firing and observed how and where dopamine is released from these neurons. A custom-made microscope enabled imaging in the NIR/SWIR window of the spectrum, where most commercial microscopes are not optimized for imaging.

With this new technology we were able to image dopamine release events in axonal terminals (Figure 2a) and more importantly shed light on poorly understood dendritic dopamine release events (Figure 2b and 2c). Typically, neurons release neurochemicals from their axonal terminals. While this mechanism holds true for many types of neurons in the brain, neurons that release neuromodulators such as dopamine are known to deviate from the norm in being release capable from their dendrites (Figure 1a). Our method made it possible to image dopamine release events in dendritic segments in ways conventional methods of inquiry have not able to do. 

Figure 2. Bright hotspots depict dopamine release from (a) axonal terminals, (b) dendritic segments and (c) dendritic processes close to the cell body.

Adapted from Bulumulla et al. 2022 (PMID: 35786443, DOI: 10.7554/eLife.78773)

Compared to other fluorescent sensors used in neuroscience, nanosensors offer unique advantages. Imaging in the NIR/SWIR window allows deeper tissue imaging with less scattering and minimal tissue autofluorescence. These are especially critical criteria when it comes to imaging brain tissue. For example, we can study quantal (that is, unitary) release events at synaptic resolutions, which has never been demonstrated before. Another key feature of nanosensors is the pharmacological compatibility to faithfully interpret effects of drugs, with the added possibility of screening drugs for various applications. Compared to genetically encoded sensors, nanosensors are ready to use immediately after application to the biological specimen. The construction of sensors is relatively straightforward and can be done in under two hours without advanced synthetic expertise. Importantly, this tool could enable study of dopamine dynamics in species where deployment of genetically encoded probes could be challenging.

While there are many benefits from SWCNT-based class of biosensors, there are also opportunities for improvement. First, genetically encoded sensors are targetable to a subpopulation of neurons via genetic strategies. However, nanosensors cannot be specifically guided/targeted to cells of choice. Second, it is difficult to traffic nanosensors beyond cell membranes into the cytoplasm to measure intracellular analytes. Structurally similar and sometimes off target analytes respond to SWCNT-DNA sensors and tuning the selectivity of these class of sensors is still an active area of research. This will be particularly important for in vivo applications. Moreover, the sensing mechanism for SWCNT-based nanosensors is not completely understood and this impairs our ability to rationally design sensors for a broad class of analytes. Finally, the unique photophysical properties of SWCNTs necessitates custom microscopy solutions that can be a hinderance to their widespread use.

In summary, the nanoscale size and advantageous photophysical properties of SWCNT-based sensors offers an opportunity to carry our biological measurements at length and time scales previously not possible and addressing the aforementioned drawbacks will help facilitate large scale adoption of the technology by the scientific community. The dopamine nanosensors discussed herein could find utility as a scientific tool in laboratories that study the mechanism of neurochemical synthesis and release and could help explore how such mechanisms go awry during disease states, such as the neurodegenerative loss of dopamine neurons that lead to Parkinson`s disease.

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