Multi-area two-photon microscopy

Summary

Principal Investigator: Fritjof Helmchen
Zurich Brain Research Institute
Title: "Multi-area two-photon microscopy for revealing long-distance communication between multiple local brain circuits"
BRAIN Category: Large-Scale Recording-Modulation – Optimization (RFA NS-14-008)

Dr. Helmchen and his colleagues propose a system to simultaneously record neuronal activity in four different areas of the neocortex and discover how brain cells in different regions interact during specific behaviors.

Principal Investigator: Fritjof Helmchen
Zurich Brain Research Institute
Title:
“Multi-area two-photon microscopy for revealing long-distance communication between multiple local brain circuits”
BRAIN Category: 
Large-Scale Recording-Modulation – Optimization (RFA NS-14-008)

Dr. Helmchen and his colleagues propose a system to simultaneously record neuronal activity in four different areas of the neocortex and discover how brain cells in different regions interact during specific behaviors.

NIH Webpages

Two-photon laser-scanning microscopy has become the method of choice for imaging nerual dynamics on the cellular level in the intact brain. The particular advantage of 2-photon microscopy is its reduced sensitivity to light scattering so that individual cells with their dendrites can be resolved several hundreds of micrometers deep in neural tissue in living animals

Two-photon laser-scanning microscopy has become the method of choice for imaging nerual dynamics on the cellular level in the intact brain. The particular advantage of 2-photon microscopy is its reduced sensitivity to light scattering so that individual cells with their dendrites can be resolved several hundreds of micrometers deep in neural tissue in living animals

Project Description

Two-photon microscopy is a widely used, key method for functional imaging of cellular activity in living animals. Most recently, in vivo calciu imaging experiments have started to reveal the spatiotemporal activity patterns that occur in various areas of the neocortex during head-fixed mouse behavior. Typically, however, the field-of- view for imaging cellular activity is fairly small, on the order of a few hundred micrometers. This restriction limits the size of neuronal networks that can be studied and thus leaves open the question how local neuronal networks communicate with distant, synaptically connected regions. What is therefore needed is a new imaging instrument that allows high-resolution functional imaging from two regions simultaneously, in the best case identifying the mutual projection neurons. We have developed a novel multi-area 2-photon microscope (MA2PM) that fulfills this need by enabling simultaneous imaging of two sub-areas within a large global scan- field (1.8 mm diameter, a distance equivalent to lining 100,000 neuronal cell bodies side-by-side assuming 20 um per neuron). Such two areas can be independently and flexibly positioned, making it for example possible to simultaneously measure neurons in the primary and secondary somatosensory areas of mouse neocortex (> 1 mm apart) while the mouse is performing a tactile texture discrimination task with its whiskers. The Using the MA2PM prototype we have conducted first proof-of-principle experiments in somatosensory cortex of awake, behaving mice using a genetically-encoded calcium indicator. We apply viral retrograde labeling strategies to identify the subsets of neurons that give rise to the inter-areal connection. Our goal in the proposed project is to further optimize and extend this innovative, transforming microscopy technology in several ways. Aim 1: We will work on finalizing a full microscope design for 2-area imaging and demonstrate its usefulness for research on corticocortical processing. We are fully dedicated to build a modular, carefully designed instrument, perform a quantitative system characterization, and disseminate this new instrument to the broader neuroscience community, especially to the growing number of research groups applying two-photon imaging for the analysis of cortical processing. Aim 2: We will furthermore expand the system to an even larger field-of-view and extend it for simultaneous imaging of four sub-areas. To this end we will apply newest laser technology and employ state-of-the-art red-shifted genetically encoded calcium indicator for deep imaging. We particularly aim at instantiating two-layer imaging (e.g. in layers L2/3 and L5) in two connected cortical areas. Overall, we expect that the new multi-area imaging technology will be an enabling technology to bridge the level of local microcircuits to the ‘macrocircuit’ level of communicating brain areas and thus will be of immediate and broad interest and highest significance to the neuroscience community.

Public Health Relevance Statement

Information processing in the brain is implemented by complex neuronal networks that are organized across multiple spatial scales, from synapses to local neural circuits to communicating brain regions. An overarching understanding of neural circuit function across these scales is still missing but novel optical imaging methods hold great promises to build a bridge. We have devised a novel 2-photon fluorescence microscope, which on the one hand resolves individual neurons ‘at work’, measuring their activity in mouse neocortex during behavior, on the other hand allows to perform high-resolution imaging in distant, connected regions, up to almost 2 mm apart. In the proposed research we will optimize this new microscope type and extend it to even larger areas and to simultaneous imaging from different cortical layers. These advances are aimed at enabling new biological experiments that may reveal the cellular basis and the significance of corticocortical communication.

NIH Spending Category

Bioengineering; Neurosciences

Project Terms

Address; Animals; Area; awake; base; Behavior; Behavioral; Biological; Brain; Brain region; Calcium; calcium indicator; Caliber; cell body (neuron); cell type; Cells; cellular imaging; Communication; Communities; Complex; Computer software; Custom; Dendrites; design; Detection; Discrimination (Psychology); Distant; Ensure; experience; Fiber; Fluorescence; fluorescence microscope; Functional Imaging; Generations; Goals; Head; Image; Image Analysis; imaging modality; Imaging technology; improved; in vivo; in vivo imaging; Individual; information processing; innovation; instrument; interest; Label; Lasers; Life; Measurement; Measures; Methods; Microscope; Microscopy; Monitor; Mus; Neocortex; neocortical; neural circuit; Neurons; Neurosciences; novel; Operating System; optic imaging; Optics; Pattern; Performance; Population; Population Dynamics; Positioning Attribute; Process; Property; prototype; public health relevance; relating to nervous system; Research; research study; Resolution; Rodent; Scanning; Sensory; Side; Signal Transduction; somatosensory; Somatosensory Cortex; spatiotemporal; Speed (motion); Support System; Synapses; System; Tactile; Technology; Testing; Texture; Tissues; two-photon; Vibrissae; Viral; Work

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