New Optical Nanosensors Improving Brain Mapping Technology

By Benedette Cuffari, M.Sc.Mar 9 2017

                                                                                                                      Jolygon/Shutterstock

Nerve cells, otherwise known as neurons, are the basic structural and functional units of the nervous system. Comprised of a cell body, axon and dendrites, neurons relay the information from one another through an essential process of the propagation of action potentials (AP)¹. When an appropriate signal or stimulus is received by the dendrites, the cell membrane of the neuron depolarizes with the influx of sodium (Na⁺) ions by the opening of sodium channels. Upon depolarization, repolarization occurs where an outflow of potassium (K⁺) ions will follow the opening of the potassium channels¹. However, a brief phase of hyperpolarization will occur in between these two processes by the increasing influx of K⁺ ions. This hyperpolarization phase ensures that the signal is transmitted in one direction by making the neurons incapable of receiving a new action potential until the neurons are repolarized. The neuron will only be able to propagate a new AP after the Na⁺ and K⁺ channels close, and the cell attain a resting membrane potential (RMP).

The release of K⁺ ions into the extracellular spaces (ECS) during repolarization causes an increase in the extracellular K⁺ concentration. Due to the importance of these K⁺ ions in the determination of the membrane potential, the concentration of K⁺ ions is strictly regulated by mechanisms such as passive diffusion through ECS and the reuptake of K⁺ by various channels and transporters of neurons and glia².

Pathological states such as epilepsy, cortical spreading depression and ischemic stroke could result with imbalances in the K⁺ homeostasis². Therefore, the ability to monitor the spatial and temporal K⁺ ions would serve a great purpose in the diagnosis and management of such pathological conditions.

Potassium-sensitive microelectrodes are one of the most widely used tools for measuring K⁺ ions for single-point measurements of K⁺ ions, however, their use is limited by their inability to monitor spatiotemporal flux necessary to understand the kinetics and dynamics of these ions. Other available methods employ ion sensitive dyes to monitor the K⁺ ions². While these dyes could be used to study the dynamics of the K⁺ ions, a lack of dyes with proper affinity to the K⁺ ions and the immediate diffusivity of dyes into the ECS, due to the small size of dyes, limits their use for this purpose.

In an attempt to solve these problems, researchers from the University of Laussane developed a potassium-sensitive nanosensor to study K⁺ dynamics. Jean-Yves Chatton’s team developed a dendrimer-based potassium nanosensor by encapsulating a potassium sensitive indicator, Asante Potassium Green 4 (APG4) in a branched structure called a dendrimer². The generation 5 (poly)amidoamine (PAMAM) dendrimer has a 1,4 diamino butane core and is covalently functionalized with polyethyleneglycol (PEG) dodecamers.

The surface functionalization with PEG assists in solubility and eliminates the possibility of toxicity associated with the naked PAMAMs, which are known to cause transient holes in the cells due to their cationic amine terminals². The dendrimer-based nanosensors (APG4 PAMAM-PEG) used in this study measured at 6.6 nm in diameter with a molecular mass of 100 kDa, making them difficult to diffuse into the ECS, unlike the considerably smaller traditional dyes, which typically have molecular weights measuring less than 1500 Da.

Upon comparison with two other K⁺ sensitive dyes, APG2 and PBFI enclosed in a similar dendrimer-based structure, the APG4 PAMAM-PEG nanosensors showed the highest sensitivity². In vitro studies revealed that the APG4 PAMAM-PEG nanosensors exhibited a concentration-dependent increase in fluorescence, with great selectivity to K⁺ ions by remaining unaffected by the increase in the concentrations of the monovalent Na⁺ cations.

Although the fluorescence of these nanosensors also stayed unaffected by increasing the concentration of divalent cations, calcium (Ca²⁺) and magnesium (Mg²⁺) ions, the fluorescence is affected by an increase in the concentration of barium (Ba²⁺) ions².

Fluorescence recording by the single-photon fluorescence imaging of APG4 PAMAM-PEG immobilized in a silica sol-gel confirmed the responsiveness and reversibility of the sensor response². Two-photon imaging of the same nanosensors immobilized in a silica sol-gel showed a radial increase in the local fluorescence from the focal point when subjected to an iontophoretic pulse. Furthermore, when injected into the ECS of the acute brain slice of mouse, the two photon microscopy images of the APG4 PAMAM-PEG nanosenors showed much longer APG4 fluorescence retention as compared to the free APG4 alone².

This group of researchers also designed dual dye nanosensors with a similar structure to the APG4 PAMAM-PEG nanosenor. The difference between this nanosensors involved the encapsulation of two dyes, APG4 and a K⁺-insensitive dye, Alexa Fluor 568 (AF568) to counteract the sensor’s sensitivity to small movements or drifts, which also showed a concentration-dependent fluorescence increase².

Chatton’s team successfully used the dendrimer-based nanosensors for imaging the K⁺ dynamics in the ECS, which highlights the range of possible extensions of this nanosensor strategy³. The researchers believe that this nanosensor design could be used in combination with several optical readouts, therefore laying the path for the design of multimodal optical nanosensors that can be applied in a broader range of possible applications².

References:

1. "Action Potentials." Hyper Physics. Web. http://hyperphysics.phy-astr.gsu.edu/hbase/Biology/actpot.html.
2. Wellbourne-Wood J, Rimmele TS, Chatton J; Imaging extracellular potassium dynamics in brain tissue using a potassium-sensitive nanosensor. Neurophoton. 0001;4(1):015002.
3. "New Optical Nanosensor Improves Brain Mapping Accuracy, Opens Way for More Applications." Phys.org. 2 March 2017. Web. https://phys.org/news/2017-03-optical-nanosensor-brain-accuracy-applications.html.

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