Leaky Optical Neural Probe for Optical Stimulation and Real Time Electrochemical Detection of Dopamine Exocytosis from Optogenetically Modified Human Neural Stem Cells

Abstract

Parkinson’s disease (PD) is characterized by the degeneration of dopaminergic neurons in the midbrain. The most effective therapy for the treatment of PD is administration of levodopa. However, it leads to the development of motor complications. Continuous delivery of dopamine has been shown to reduce the risks associated with chronic motor complications [1]. This work presents the fabrication and characterization of a microfabricated implantable leaky optical neural probe to fulfil three functions: i) it acts as a cell culture substrate for application in cell replacement therapy (CRT). ii) the leaky waveguide on the probe enables optical stimulation of neural stem cell derived optogenetic neurons differentiated on the probe. iii) pyrolytic carbon electrodes allow electrochemical detection of dopamine exocytosis and modulation of its continuous supply in the striatum for application in Parkinson’s disease (PD) therapy. The neural probes are optimized for a mouse brain (4 mm long, 50 µm thick and 200 µm wide). The width is necessary for differentiating a large number of stem cells for application in CRT. Electrodes with micropillars are patterned in SU-8 and pyrolysed to obtain the working (WE) and counter electrodes (CE) while gold is used as a pseudo reference electrode (RE) for electrochemical measurements. Pyrolytic carbon is used as cell substrate as it supports differentiation of neural stem cells into dopaminergic neurons [2]. ASU-8 waveguide was designed along the probe edge to encapsulate the cells during implantation and enable optogenetic stimulation of a large population of neurons in the immediate vicinity of the waveguide without the need to increase input optical power. The probes are fabricated using a combination of front and backside silicon etching. The backside etch defines the probe thickness while the front side etch releases the probe and defines the groove for the placement of an optical fiber for coupling light into the waveguide. Figure 1a shows the probe shank containing the pyrolytic carbon electrodes surrounded by the leaky waveguide. The micro-pillars act as anchor points for the adhesion of stem cells. The design of a leaky waveguide was optimized using COMSOL. Figure 1b shows light leak pattern from the fabricated waveguide. The intensity of light leaking from the waveguide is measured to be 10 mWmm-2 (laser pulsed at 2 ms) which is sufficient for the stimulation of the optogenetically modified cells. The ability of the electrodes to detect different concentrations of dopamine (Figure 1c) demonstrate the suitability of the probes for optogenetic stimulation and electrochemical detection of dopamine. Furthermore, optogenetic human neural stem cells differentiated on the probes show excellent viability (Figure 1d). Experiments are currently in progress to evaluate dopamine exocytosis from optical stimulation of neurons.