Deuterated Glutamate-Mediated Neuronal Activity on Micro-Electrode Arrays

Abstract

The excitatory synaptic transmission is mediated by glutamate in neuronal networks of the mammalian brain. In addition to the synaptic glutamate, extra-synaptic glutamate is known to modulate the neuronal activity. In neuronal networks, glutamate uptake is an important role of neurons and glial cells for lowering the concentration of extracellular glutamate and to avoid the excitotoxicity by glutamate. Monitoring the spatial distribution of intracellular glutamate is important to study the uptake of glutamate, but the approach has been hampered by the absence of appropriate glutamate analogs that report the localization of glutamate. Deuterium-labeled glutamate (GLU-D) is a promising tracer for monitoring the intracellular concentration of glutamate, but physiological properties of GLU-D have not been studied. Here we study the effects of extracellular GLU-D for the neuronal activity by using primary cultured rat hippocampal neurons that form neuronal networks on microelectrodes array. The frequency of firing in the spontaneous activity of neurons increased with the increasing concentration of extracellular GLU-D. The frequency of synchronized burst activity in neurons increased similarly as we observed in the spontaneous activity. These changes of the neuronal activity with extracellular GLU-D were suppressed by antagonists of glutamate receptors. These results suggest that GLU-D can be used as an analog of glutamate with equivalent effects for facilitating the neuronal activity. We anticipate GLU-D developing as a promising analog of glutamate for studying the dynamics of glutamate during neuronal activity.

Keywords: cultured neuronal network; deuterium-labeled glutamate; glutamate receptor; microelectrode array; neurotransmitter; spontaneous activity.

Figure 1

Culture of rat hippocampal neuronal network. (a) A photograph of the MED probe. Scale bar, 1 cm. (b) A phase-contrast micrograph of cultured rat hippocampal cells on the MED probe at 21 DIV. Black squares are electrodes. Scale bar, 50 μm.

Figure 2

Chemical structure of L-glutamate-d5 (GLU-D).

Figure 3

Raman spectra of GLU and GLU-D solutions. Blue and red lines show spectra of GLU and GLU-D, respectively. The peaks of oxygen and nitrogen appeared at around 1555 and 2332 cm⁻¹, respectively. Red and blue squares correspond to wavenumber range of appearing CD₂ stretching vibration of GLU-D and CH₂ stretching vibration of GLU, respectively.

Figure 4

The effects of extracellular GLU/GLU-D on the spontaneous activity of primary cultured neurons at 30 DIV (for GLU) and 31 DIV (for GLU-D). (a) Spontaneous activity of neurons in the media with different concentrations of GLU/GLU-D. Upper panels are raster plots of 64 channels and lower panels are the typical waveforms. (b) Normalized mean firing rates at each GLU (blue) and GLU-D (red) concentration.

Figure 5

The effects of extracellular concentrations of GLU/GLU-D on the synchronous firing activity of the neuronal networks at 30 DIV. (a) Typical waveform of normalized spike frequency in each 100 ms time bin. (b) Normalized mean spike frequency in SBFs at each concentration of GLU (blue) and GLU-D (red).

Figure 5

The effects of extracellular concentrations of GLU/GLU-D on the synchronous firing activity of the neuronal networks at 30 DIV. (a) Typical waveform of normalized spike frequency in each 100 ms time bin. (b) Normalized mean spike frequency in SBFs at each concentration of GLU (blue) and GLU-D (red).

Figure 6

The effects of glutamate receptors blockers on extracellular glutamate-induced neuronal activity. (a) Mean firing rates at different concentrations of GLU (blue) and GLU-D (red) in the media with 10 µM of CNQX. (b) Mean firing rates at different concentrations of GLU (blue) and GLU-D (red) under 50 µM of D-APV.