Two-photon photostimulation and imaging of neural circuits Volodymyr Nikolenko, Kira E Poskanzer &Rafael Yuste We introduce an optical method to stimulate individual neurons in brain slices in any arbitrary spatiotemporal pattern, using two-photon uncaging of MNI-glutamate with beam multiplexing. This method has single-cell and three-dimensional precision. By sequentially stimulating up to a thousand potential presynaptic neurons, we generated detailed functional maps of inputs to a cell. We combined this approach with two-photon calcium imaging in an all-optical method to image and manipulate circuit activity. Neuronal circuits are composed of many cell types, and it is likely that each cell type carries out a specialized function 1 . Therefore, as a prerequisite to understanding the function of a circuit, it appears necessary to map synaptic connections among different types of neurons or to map all connections made onto a given cell 2 . After early work using fluorescent membrane probes 3 , photo- stimulation of neurons using caged glutamate 4 has greatly advanced this research program, generating high-resolution input maps of neurons in brain slices 4–10 . In this method, glutamate is uncaged by focusing ultraviolet light at a particular position in the brain slice and simultaneously recording intracellular responses from a neuron at a different location. By moving the uncaging beam systematically across the brain slice, one can map the territories that generate excitatory or inhibitory responses in the recorded cell. Although very useful, this method suffers from the problem that, because of the inherent scattering of light in living tissue and the large uncaging area generated by one-photon excitation, the stimulated area contains more than one neuron. Thus, one-photon photostimulation has not revealed synaptic connections between cells, but instead connections between a particular territory and a recorded neuron. To stimulate individual cells, we took advantage of the exqui- site spatial resolution of two-photon excitation 11 . We developed a two-photon photostimulation method, wherein a beam- multiplexed two-photon laser is moved from neuron to neuron to uncage glutamate and sequentially make each neuron fire, while the resulting synaptic potential in a particular cell is simultaneously recorded. This allows for the detection of mono- synaptically connected cells and for input maps to be built with single-cell resolution. We combined this method with two- photon calcium imaging to manipulate and simultaneously record circuit activity. RESULTS Optical design and labeling of neurons We developed a method to photostimulate and image the activity of large neuronal populations using a single laser (Fig. 1 and Supple- mentary Fig. 1 online). Custom software allowed us to position of the laser beam in any arbitrary point in the field of view and to quickly switch between two-photon calcium imaging of indo-1 acetoxymethyl (AM) ester (indo-1 AM) and two-photon uncaging of 4-methoxy-7-nitroindolinyl (MNI)-caged L-glutamate 12,13 on individual neurons, causing them to fire action potentials. We used an electro-optical modulator for switching between two laser light intensities: a lower intensity for imaging and a higher intensity for uncaging. Thus, we used the same laser beam (at 725 nm) to trigger and monitor circuit activity. To visualize cell bodies of neurons and detect their coordinates, we loaded acute neocortical slices from mouse somatosensory and visual cortex with membrane-permeant AM-ester calcium indica- tors. Because 700–735 nm light is required for two-photon unca- ging of MNI-caged L-glutamate, we chose indo-1 AM as the calcium indicator. Indo-1 AM produced good loading of neurons 14 and excellent calcium sensitivity in response to action potentials (Fig. 1). In addition, the low-affinity calcium indicator mag-indo-1 AM loaded neocortical slices more efficiently than indo-1 AM. Even though mag-indo-1 AM is not suitable for optical monitoring of action potentials (due to its low affinity for calcium), it proved very useful for fluorescent labeling of neuronal somata (Supple- mentary Fig. 2 online) for experiments that did not require calcium imaging (for example, in input mapping experiments). For combined imaging/uncaging experiments, it was possible to jointly label with both indicators to obtain robust labeling of the neurons by mag-indo-1 AM, while still benefiting from the func- tional calcium sensitivity of indo-1 AM. Indo-1 AM and mag-indo-1 AM loaded most neurons but not glia, as determined by dual labeling with sulforhodamine 101 (ref. 15; Supplementary Fig. 3 online). Only 5.7 ± 1.56% of mag- indo-1 AM–loaded cells were also loaded with sulforhodamine 101, p u o r G g n i h s i l b u P e r u t a N 7 0 0 2 © e r u t a n / m o c . e r u t a n . w w w / / : p t t h s d o h t e m RECEIVED 3 MAY; ACCEPTED 23 SEPTEMBER; PUBLISHED ONLINE 28 OCTOBER 2007; DOI:10.1038/NMETH1105 Howard Hughes Medical Institute, Department of Biological Sciences, Columbia University, 1212 Amsterdam Avenue, Box 2435, New York, New York 10027, USA. Correspondence should be addressed to V.N. (vn59@columbia.edu). NATURE METHODS | VOL.4 NO.11 | NOVEMBER 2007 | 943 ARTICLES