We use patterned light to initiate scattered photons that are obliquely directed to excite voltage indicators, which report changes in the membrane potential. Our proposed scheme reduces the excitation of voltage indicators elsewhere in the cell that constitutes the background signal and therefore enables us to amplify the detected fluorescence carrying the voltage information.
We aim to understand how brain circuits, learn, memorize or process information. To achieve this aim, we follow a bottomup approach by focusing on single neurons from rat brains and study how different synaptic inputs of a single neuron translate to an output or an action potential. We have custom-built a unique two-photon laser microscope that incorporates a holographic projector, which transforms the incident laser into multiple foci at the sample volume. The hologram is programmable so we can position the different foci anywhere around the neuron in 3D. Each focus can be used to trigger a synaptic input or used as an optical probe to record the activity of the neuron. We can therefore stimulate and probe the activity from multiple locations within the neuron’s dendritic tree using light. For triggering inputs, a focal stimulation represents a synaptic input via two-photon photolysis of caged neurotransmitters. For recording, a laser focus excites a calcium indicator that changes in fluorescence whenever the neuron is active. Using these techniques, we have now identified a novel function of a specific set of dendrites that can have a significant role in learning and memory. The set of dendrites we are probing are currently unexplored due to their very thin morphology. We were able to observe unique properties that allow these dendrites to be more receptive to inputs whenever the neuron fires a series of action potentials. Hence, they have a functional role in the brain's capacity to learn and memorize.
We use complex light patterns to simultaneously record the neuronal activity along the dendrites of a single neuron. We use holographic projection to produce multiple foci directed onto different dendritic regions of the neuron. Each focus excites neuronal activity reporters via either two-photon (2P) or single-photon (1P) excitation. The fluorescence emanating from all foci are simultaneously recorded using an electron-multiplying charge-coupled device (EMCCD) camera thereby enabling simultaneous multi-channel recording of the neuronal activity from multiple sites at high frame rates (up to 400Hz). We report recording of neuronal activity from two types of reporters: (1) Ca2+ indicator, Cal-520; and (2) voltage indicator, JPW-1114. We optically recorded the activity evoked by the neuron following injection of current onto the soma. Holographic multi-site Ca2+ imaging resulted in high signal-to-noise ratio but with poor temporal resolution. On the other hand, multi-site voltage imaging produced noisy and low SNR signals but with high temporal resolution that is able to resolve action potentials.
The spectacular facets of light have made light ubiquitous in all fields of science. Light’s interaction with matter allows for accurate manipulation of atomic and molecular structures that enabled fundamental breakthroughs in physics, chemistry, and biomedical research. The transfer of light’s energy on molecules and genetically expressed proteins can be used to stimulate cells and emulate cellular processes such as synaptic inputs spatially distributed along the neuron’s dendritic tree. Here, we show basic neuronal functions derived via numerical modelling and describe how we can use light to emulate these functions in order to provide a systematic study of the neuron’s response. We focus on cortical pyramidal neurons and use the NEURON simulation environment to analyze how spatio-temporal stimulation patterns along various dendritic locations sets the neuron to fire an output. We then show an equivalent response from experiments via complex spatial light patterns for stimulating across different regions along the dendritic tree. Furthermore, we use the same spatial light patterns to simultaneously visualize neuronal responses via functional calcium imaging predicted via the same neuron model. Visualizing dendritic responses from back-propagating action potentials can provide new insights to some important features of dendritic computation.