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Neurons in the mammalian brain receive thousands of synaptic inputs on their dendrites. In many types of neurons, such as cortical pyramidal neurons, excitatory synapses are formed on fine dendritic protrusions called spines. Usually, an individual spine forms a single synaptic contact with an afferent axon. In this protocol, we describe a recently established experimental procedure for measuring intracellular calcium signals from dendritic spines in cortical neurons in vivo by using a combination of two-photon microscopy and whole-cell patch-clamp recordings. We have used mice as an experimental model system, but the protocol may be readily adapted to other species. This method involves data acquisition at high frame rates and low-excitation laser power, and is termed low-power temporal oversampling (LOTOS). Because of its high sensitivity of fluorescence detection and reduced phototoxicity, LOTOS allows for prolonged and stable calcium imaging in vivo. Key aspects of the protocol, which can be completed in 5-6 h, include the use of a variant of high-speed two-photon imaging, refined surgery procedures and optimized tissue stabilization.

Neurons in cortical sensory regions receive modality-specific information through synapses that are located on their dendrites. Recently, the use of two-photon microscopy combined with whole-cell recordings has helped to identify visually evoked dendritic calcium signals in mouse visual cortical neurons in vivo. The calcium signals are restricted to small dendritic domains ('hotspots') and they represent visual synaptic inputs that are highly tuned for orientation and direction. This protocol describes the experimental procedures for the recording and the analysis of these visually evoked dendritic calcium signals. The key points of this method include delivery of fluorescent calcium indicators through the recording patch pipette, selection of an appropriate optical plane with many dendrites, hyperpolarization of the membrane potential and two-photon imaging. The whole protocol can be completed in 5-6 h, including 1-2 h of two-photon calcium imaging in combination with stable whole-cell recordings.