A novel genetically encoded voltage indicator for studying motor cortical circuitry
Scholtz, David Johannes
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Scholtz, D. J. (2014). A novel genetically encoded voltage indicator for studying motor cortical circuitry (Thesis, Bachelor of Biomedical Sciences with Honours). University of Otago. Retrieved from http://hdl.handle.net/10523/5210
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Abstract:
The primary motor cortex (M1) consists of layers that are occupied by distinctive excitatory pyramidal neuron and inhibitory interneuron populations. Neurons within each layer receive inputs from numerous cortical and subcortical structures that relay proprioceptive and sensory feedback to modulate motor outputs and facilitate motor learning. The neurons within the upper layers (layer 2/3) are linked with processing and integrating these inputs and activating the circuitry that generates motor output commands that drive voluntary movement. To date our understanding of how these circuits achieve this remains elusive. Our poor understanding arises from technical challenges associated with studying the simultaneous behaviour of the electrical activity of the vast diversity and complex connections of the neurons within these circuits. To overcome this limitation we aim to use a Genetically Encoded Voltage Indicator (GEVI) called VSFP-Butterfly 1.2 that is endogenously expressed in layer 2/3 pyramidal neurons M1 in a transgenic mouse. We aim to determine the fidelity of VSFP-Butterfly 1.2 expression in the transgenic mouse and its ability to report subthreshold synaptic fluctuations in electrical membrane potential as changes in fluorescence.
VSFP-Butterfly 1.2 is engineered to be expressed in layer 2/3 pyramidal neurons downstream of the Ca2+ Calmodulin-dependent protein kinase 2 (CAMKII). Immunohistochemistry for CAMKII in layer 2/3 of M1 slices found that the majority of neurons that express VSFP-Butterfly 1.2 also clearly express CAMKII (99.24 ± 0.567 %, n = 9 slices from 6 mice). Simultaneous recording of local field potential (LFP) and VSFP-Butterfly 1.2 fluorescent optical signals from layer 2/3 of slices from the M1 in response to extracellular electrical stimulation revealed a clear voltage-response relationship for VSFP-Butterfly 1.2 (n = 8 slices from 4 mice). Pharmacological excitatory synaptic antagonists dampened both the optical VSFP-Butterfly 1.2 (P < 0.0001, One-way ANOVA multiple comparisons, n = 4 slices from 2 mice) signals and LFP responses (P < 0.0001, One-way ANOVA multiple comparisons, n = 4 slices from 2 mice); and all responses were eliminated by tetrodotoxin which is known to block all voltage dependent electrical activity in neurons (P < 0.05, One-way ANOVA multiple comparisons, n = 2 slices from 1 mouse). In addition, we provide evidence that VSFP-Butterfly 1.2 can report membrane potential fluctuations at distances as far as 793.6 μm from the recording column (P < 0.0001).
Our results show that VSFP-Butterfly 1.2 is reliably expressed exclusively in layer 2/3 neurons of the M1 in the transgenic mouse where it accurately reports physiologically relevant electrical synaptic responses. Our validation supports the future use and exciting benefit of this mouse to begin to understand the basis of network and circuit connectivity during motor output and motor learning.
Date:
2014
Advisor:
Empson, Ruth M
Degree Name:
Bachelor of Biomedical Sciences with Honours
Degree Discipline:
Physiology
Publisher:
University of Otago
Keywords:
VSFP; genetically encoded voltage indicator; voltage sensitive fluorescent protein; primary motor cortex; Butterfly; FRET
Research Type:
Thesis
Languages:
English
Collections
- Physiology [152]
- Thesis - Honours [339]