Imaging Sodium Flux during Action Potentials in Neurons with Fluorescent Nanosensors and Transparent Microelectrodes

Abstract

Sodium flux plays a pivotal role in neurobiological processes including initiation of action potentials and regulation of neuronal cell excitability. However, unlike the wide range of fluorescent calcium indicators used extensively for cellular studies, the choice of sodium probes remains limited. We have previously demonstrated optode-based nanosensors (OBNs) for detecting sodium ions with advantageous modular properties such as tunable physiological sensing range, full reversibility, and superb selectivity against key physiological interfering ion potassium. (1) Motivated by bridging the gap between the great interest in sodium imaging of neuronal cell activity as an alternative to patch clamp and limited choices of optical sodium indicators, in this Letter we report the application of nanosensors capable of detecting intracellular sodium flux in isolated rat dorsal root ganglion neurons during electrical stimulation using transparent microelectrodes. Taking advantage of the ratiometric detection scheme offered by this fluorescent modular sensing platform, we performed dual color imaging of the sensor to monitor the intracellular sodium currents underlying trains of action potentials in real time. The combination of nanosensors and microelectrodes for monitoring neuronal sodium dynamics is a novel tool for investigating the regulatory role of sodium ions involved during neural activities.

Keywords: dorsal root ganglion; ion sensing; nanosensor; optode; ratiometric imaging; sodium; transparent microelectrode.

Figure 1.

Sensor mechanism and optical setup: (a) Chromoionophore (C), Sodium Ionophore (I), Rhodamine (RhD), and Ion Exchanger (R) are all encapsulated in the PEG-lipid coated polymer matrix. The selective recruitment of sodium ion to the sensor results in deprotonations of two fluorophores, C and RhD, to maintain the charge neutrality. This leads to ratiometric fluorescence intensity change in signal readout. (b) OBNs (red) are first microinjected to the DRG on one of the 2 × 4 stimulation TMEs (expanded image, 80 × 80 μm², superimposed with schematic stimulation current pulse sequences). The dimensions of the TME schematic drawing are not to scale. The emission light collected from the OBN during stimulation is chromatically separated (585 BP40 and 685 BP40) and recaptured on two translated areas of the camera. Scale bar: 20 μm.

Figure 2.

In solution, physiological buffer pH 7.2 (blue) and in situ cell (red) calibrations of the Na⁺ OBNs. For intracellular applications, the sensor response (EC₅₀) derived from the Hill fit is tuned to the physiological Na⁺ concentration between 5 and 15 mM. EC₅₀ of the in solution sensor response is 7.6 ± 0.4 mM. In situ cell calibration (EC₅₀: 18.5 ± 2.4 mM) was performed by superfusing different concentrations of Na⁺ in the presence of gramicidin and monensin to equilibrate Na⁺ concentration across cell membrane. Ratiometric sensor responses are normalized to deprotonation degree (α) on the calibration curve.

Figure 3.

Transparent stimulation microelectrodes. (a) Optical image of transparent microelectrodes (left). Scale bar: 300 μm. Inset: microscope image of nanomesh microelectrode. Scale bar: 20 μm. SEM image of nanomesh structure (right). Scale bar: 500 nm. (b) Voltage transient of Au/ PEDOT:PSS nanomesh microelectrode and ITO microelectrode under 0.4 mC/cm² current pulse stimulus.

Figure 4.

Time-lapse measurements (exposure time: 50 ms, frame rate: 1 Hz) of sensor response to intracellular Na⁺ dynamics induced by electrical stimulation. (a) Single-trial sensor response to Na⁺ transients at the DRG injection site to stimulation pulse trains (2 μA, 2 ms duration, 50 Hz, repetition rate 1 Hz, stimulation time 5 s, 100 s between each stimulus). (b) Sensor response to increasing stimulation currents. Numbers above arrowheads indicate magnitude of stimulation current (μA). (c) Average sensor intensity ratio change ΔR plotted against stimulus current indicate sensor response is proportional to the stimulus intensity (Redline: linear fit, R² = 0.95, n = 3). (d) Fluorescence transients of CoroNa response to the stimulus under the same experiment protocol in (a). (e) pH sensor response to the stimulus under the same experiment protocol in (a). Arrowheads above traces indicate starting points in the stimulus sequences.