Amperometric Microsensors Monitoring Glutamate-Evoked In Situ Responses of Nitric Oxide and Carbon Monoxide from Live Human Neuroblastoma Cells

Abstract

In the brain, nitric oxide (NO) and carbon monoxide (CO) are important signaling gases which have multifaceted roles, such as neurotransmitters, neuromodulators, and vasodilators. Even though it is difficult to measure NO and CO in a living system due to their high diffusibility and extremely low release levels, electrochemical sensors are promising tools to measure in vivo and in vitro NO and CO gases. In this paper, using amperometric dual and septuple NO/CO microsensors, real-time NO and CO changes evoked by glutamate were monitored simultaneously for human neuroblastoma (SH-SY5Y) cells. In cultures, the cells were differentiated and matured into functional neurons by retinoic acid and brain-derived neurotrophic factor. When glutamate was administrated to the cells, both NO and CO increases and subsequent decreases returning to the basal levels were observed with a dual NO/CO microsensor. In order to facilitate sensor's measurement, a flower-type septuple NO/CO microsensor was newly developed and confirmed in terms of the sensitivity and selectivity. The septuple microsensor was employed for the measurements of NO and CO changes as a function of distances from the position of glutamate injection. Our sensor measurements revealed that only functionally differentiated cells responded to glutamate and released NO and CO.

Keywords: amperometric sensor; carbon monoxide; glutamate stimulation; neuroblastoma cells; nitric oxide.

Figure 1

The experimental setup for real-time NO and CO measurements for functional human neuroblastoma cells with a glutamate injection. A vertical distance between the end plane of a sensor and cell monolayer surface is approximately 10 μm. NO/CO sensor in this figure represent either dual or septuple sensors.

Figure 2

Phase contrast images of cultured cells (A) with and (B) without cell differentiation factors, RA and BDNF. Western blot analysis (C) with differentiated, D+, and non-differentiated, D−, cells and the graph (D) showed the quantified protein levels. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), retinoic acid (RA), and brain derived neurotrophic factor (BDNF).

Figure 3

NO and CO concentration changes responding to sequential glutamate injections (time points are marked with green arrows), monitored for (A) differentiated cells; (B) non-differentiated cells; and (C) only serum-free medium with a dual NO/CO microsensor.

Figure 4

The concentration changes of NO (blue, locations 2, 4, 6) and CO (red, locations 1, 3, 5, 7) responding to 50 nM glutamate administration (marked with green arrows), measured with a septuple NO/CO sensor for differentiated cells (solid lines) and in only medium without cells (dashed lines). The glutamate injection site is marked with a pink triangle.