Single-spike detection in vitro and in vivo with a genetic Ca2+ sensor

Measurement of in vivo neuronal activity with single neuron and single action potential resolution is important for studying neuronal function. Delivery of a FRET-based fluorescent Ca 2+ indicator protein using adeno-associated virus results in high expression levels allowing in vivo detection of single action potentials at low firing rates. Griesbeck et al ., also in this issue, describe the use of a similar sensor for recording neuronal activity in vivo . Measurement of population activity with single-action-potential, single-neuron resolution is pivotal for understanding information representation and processing in the brain and how the brain's responses are altered by experience. Genetically encoded indicators of neuronal activity allow long-term, cell type–specific expression. Fluorescent Ca 2+ indicator proteins (FCIPs), a main class of reporters of neural activity, initially suffered, in particular, from an inability to report single action potentials in vivo . Although suboptimal Ca 2+ -binding dynamics and Ca 2+ -induced fluorescence changes in FCIPs are important factors, low levels of expression also seem to play a role. Here we report that delivering D3cpv, an improved fluorescent resonance energy transfer–based FCIP, using a recombinant adeno-associated virus results in expression sufficient to detect the Ca 2+ transients that accompany single action potentials. In upper-layer cortical neurons, we were able to detect transients associated with single action potentials firing at rates of