p u o r G g n i h s i l b u P e r u t a N 0 1 0 2 © natureprotocols / m o c . e r u t a n . w w w / / : p t t h PROTOCOL NATURE PROTOCOLS | VOL.5 NO.5 | 2010 | 849 INTRODUCTION During early development, brain neurons undergo extensive activity-dependent morphological 1–5 and synaptic 6–9 refinement. Experience-driven refinement results in progressive and persist- ent changes in the functional output of both single neurons and ensemble populations to influence normal or abnormal brain function later in life. Examples of such refinement in higher verte- brates include the activity-dependent structural growth of cortical neurons and the formation and maintenance of ocular dominance columns 10–12 . In the developing embryonic retinotectal system of Xenopus tadpoles, tectal neurons receiving direct innervation from the optic nerve show functional changes reflected by both an age- dependent reduction in receptive field (RF) size 13–15 and rapid potentiation or depression of firing rates induced by brief, RF- specific, visual experience 9,16,17 . A fundamental question of develop- mental neuroscience is how sensory experience modifies long-term firing behavior and RF response properties of both individual cells and ensemble neuronal populations in vivo. Neural network plasticity Historically, research into neuronal function and plasticity has been limited to the study of either individual neurons or bulk popu- lation responses. As such, our present understanding of neural network plasticity induced by sensory stimuli has relied on com- piling statistics of single-neuron recordings across many animals or investigating averaged neuronal responses through bulk record- ings. Both of these approaches, however, have significant limitations when investigating ensemble plasticity. Single-neuron recordings do not allow investigation of variable plasticity effects within a single animal. They are also technically arduous, making it difficult to compile large statistics. Bulk recordings, in contrast, can record the average responses of many neurons simultaneously within a single animal. However, the collective function of individual neuronal out- put is not linear—simply averaging response properties overlooks the complex computational relationships of neural population cod- ing, where firing rates of individual neurons in context with the full neuronal ensemble convey information about the input stimulus. For example, a neuronal population may potentiate on average, but a subpopulation of neurons may depress at the same time 17,18 . Bulk recordings would miss this depressing subpopulation, which may be extremely significant for coding information about the training effects. To accurately decode neural network function and to under- stand how neural circuits dynamically change to mediate plasticity requires simultaneous recording of large populations of neurons, with single-neuron resolution. Currently, few techniques allow researchers to capture network dynamics at the single-cell level within intact neural systems. Single- unit electrophysiological recording using single or multielectrodes provides simultaneous recording from up to dozens of neurons 19 . Spike-sorting algorithms are then used to classify action potentials (APs) from individual neurons. Although single-unit recordings provide high temporal resolution of neuronal firing, they require invasive insertion of electrodes, each of which can only sample activ- ity from a small group of neurons. Moreover, electrode data do not provide information on anatomical localization of recorded neurons. Functional multineuron calcium imaging Functional multineuron calcium imaging allows us to bridge the gap between systems and cells 20 . This technique provides simulta- neous imaging of large populations of neurons (~200 neurons) within intact tissues with accurate identification of anatomical relationships between all cells. Importantly, after minimally inva- sive infusion with calcium-sensitive dyes, imaging can be carried out noninvasively within the intact and awake organism, providing the most powerful means to record native activity of large popula- tions of brain circuit neurons for the study of neural encoding and plasticity. In this protocol we describe application of functional multineuron calcium imaging for the study of network ensemble functional activity and plasticity within the optic tectum of the intact and awake developing brain of the transparent Xenopus laevis tadpole. We used a specialized technique called single-cell excitability In vivo single-cell excitability probing of neuronal ensembles in the intact and awake developing Xenopus brain Derek Dunfield & Kurt Haas Department of Cellular and Physiological Sciences and the Brain Research Centre, University of British Columbia, Vancouver, British Columbia, Canada. Correspondence should be addressed to K.H. (kurt.haas@ubc.ca). Published online 8 April 2010; doi:10.1038/nprot.2010.10 Sensory experience can elicit long-lasting plasticity of both single neurons and ensemble neural circuit response properties during embryonic development. To investigate their relationship, one must image functional responses of large neuronal populations simultaneously with single-cell resolution. In this protocol, we describe a noninvasive approach to assay functional plasticity of individual neurons and neuronal populations in vivo using targeted infusion of calcium-sensitive dyes, two-photon microscopy and synchronized visual stimuli presentations. This technique allows visualization of ~200 neurons while probing visual responses in the optic tectum of awake, immobilized Xenopus laevis tadpoles. The protocol includes visual training paradigms that elicit long-lasting potentiation or depression of functional responses, allowing investigations of population and single-neuron plasticity induced by natural sensory stimuli in the awake, intact, developing brain. Setup time for this protocol, including dye injection and chamber preparation, is ~2 h. Excitability probing experiments can then be performed for at least 3 h.