Calibration of Neurotransmitter Release From Neural Cells for Therapeutic Implants

Abstract

In this work we quantified the in vitro calibration relationships between high frequency electrical stimulation and GABA and glutamate release in both mature retinoic acid differentiated P19 neurons and immortalized embryonic cortical cells engineered to express glutamic acid decarboxylase, GAD65. Extracellular glutamate and GABA was quantified by 2D gas chromatography and time of flight mass spectrometry after stimulation at varying amplitudes and frequencies. Amplitude sweeps resulted in a linear calibration for P19 neurons; the level of neurotransmitter varied over one order of magnitude from ~ 200 pg/neuron to ~ 1.2 ng/neuron for glutamate and ~ 1 ng/neuron to ~ 2 ng/neuron for GABA, depending on the stimulation amplitude. Frequency sweeps resulted in a peak release at 250 Hz for glutamate and 400 Hz for GABA in P19 cells. The GABA transporter inhibitor, nipecotic acid, increased extracellular GABA levels and decrease glutamate. In contrast the embryonic cortical cells had a strongly nonlinear dependency of release on stimulation amplitude, and a weak dependence on frequency. These cells had roughly equal extracellular glutamate and GABA levels after stimulation despite the expression of GAD65. In addition glutamate and GABA levels were insensitive to nipecotic acid. These results demonstrate an ability to calibrate and tune neurotransmitter release from neural cells using high frequency stimulation parameters.

A self-referencing glutamate biosensor for measuring real time neuronal glutamate flux

“…Under these conditions, approximately 16% of the cells are neurons and 7% are astrocytes (Jedlicka et al, 2007). Differentiated P19 cells respond to electrical stimulation by release of both Glu and GABA (Jedlicka et al, 2009). …”

“…Immunofluorescence staining was conducted using techniques in Jedlicka et al (2009). Differentiated P19 cells were fixed using 4% paraformaldehyde (10 min), washed in PBS, and cells were then permeabilized with 0.1% Triton X-100.…”

“…Using optical imaging, neurons, astrocytes, and neural processes were easily identified by their morphology (Jedlicka et al, 2009) . Fig.…”

“…These disadvantages limit the applicability of results regarding neurotransmitter kinetics. Analytical methods such as high performance liquid chromatography and gas chromatography/mass spectrometry (Jedlicka et al, 2009) and coupled techniques (e.g., microdialysis) are capable of quantifying Glu concentrations under physiological conditions (Watson et al, 2006), but cannot measure dynamic Glu flux, and are temporally constrained (due to bulk sampling) (Day et al, 2006). Thus, real-time autonomous techniques, which are capable of providing non-invasive quantification of Glu transport dynamics in neuronal cultures, are needed.…”

A novel method for simultaneous glutamate and extracellular activity measurement in brain slices with high temporal resolution

“…These disadvantages limit the applicability of any obtained Glu kinetic results. Analytical methods such as HPLC and MS [12] and coupled techniques (e.g., microdialysis) are capable of quantifying Glu concentrations under physiological conditions [13], but they cannot measure dynamic Glu flux and are temporally constrained (due to bulk sampling) [14].…”