Development of optical sensors for the study of neurotransmission
Creators: name: Zhang, William Hongyu
Publisher: name: The Australian National University
Year: 2016
Subjects: optical sensor
protein engineering
FRET
glycine
GABA
neurotransmitter
long term potentiation
long term depression
fluorescent protein
synthetic dye
hippocampus
learning

description: The study of neurotransmission allows for greater understanding the central nervous system and can also improve our ability to understand and treat neurological disorders. The use of optical sensors to study neurotransmission can give insights that cannot be obtained through other techniques, as optical sensors are capable of providing spatial and temporal information about neurotransmission on a sub-cellular scale. Of particular interest is glycine, as while it is known as a primary inhibitory neurotransmitter in the central nervous system, it has also been shown to be important for the synaptic plasticity of glutamatergic neurons in the hippocampus. While this suggests that glycine plays a role in long term memory formation and learning, the exact contribution and regulation of this neurotransmitter is currently debated. An optical sensor for this neurotransmitter can provide new insight into the usage, release and distribution of glycine, which would improve our understanding of how learning and memory is moderated in the central nervous system. The main obstacle facing the use of optical sensors in biological imaging is that a specific sensor must be developed for a specific ligand, which is often a non-trivial process. As there is currently no optical sensor for glycine, one must be developed in order to allow for the study of this neurotransmitter. In this work we describe the engineering of a genetically encodable glycine specific optical sensor, GRIP (Glycine Ratiometric Indicator Protein) as well as the development of the semi-synthetic sensors GRIPPED (Glycine Ratiometric Indicator Protein Potency Enhanced by a Dye) and GASP (GABA Sensing Protein), which are a more sensitive optical sensor for glycine and a GABA sensor, respectively. The methodology and sensor designs employed in the creation of these sensors (GRIP, GRIPPED and GASP) could be useful for the development of optical sensors for other ligands, making optical sensor development for other neurotransmitters of interest more accessible in general. The genetically encodable sensor GRIP was also applied in situ within acute hippocampal brain slices from rats, in order to both demonstrate its functionality as well as to study glycine neurotransmission in the context of neuronal synaptic plasticity. Of the insights gained from the application of this sensor, two particularly noteworthy findings include differences in glycine availability between different neuron substructures with micron scale resolution and the time-correlated release of glycine in response to long term potentiation inducing stimulus (high frequency stimulation). The physiological results are a direct confirmation of differential glycine regulation as a component of neuronal synaptic plasticity and the results also demonstrate that the GRIP sensor is able to report spatial and temporal information, as initially desired.