LOTOS-based two-photon calcium imaging of dendritic spines in vivo

Abstract

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.

Functional imaging of neuronal activity of auditory cortex by using Cal-520 in anesthetized and awake mice

“…The speaker was placed ~6 cm away from the contralateral ear of the mouse. Background noise (~60 dB SPL) was dominated by low-frequency components, comparable to the setup used previously [11, 42,43]. For more details of the background noise, in the low frequency range (0 to 10 kHz), the main peak of power is below 1 kHz with spectral density of 33 dB/sqrt(Hz).…”

“…We exposed the right Au1 of each mouse for two-photon imaging under anesthesia, as described in previous studies [11,15,42,43]. Briefly, each mouse was anesthetized with 1.5% isoflurane in pure oxygen and kept on a warm plate (37.5-38°C).…”

Intravital imaging of dendritic spine plasticity

“…67,68 Although most studies on dendritic spines have been focusing on structural plasticity due to various technical challenges, recent advances in calcium imaging techniques enable the observation of single spine activity in vivo. 20,22,69 The first report of this approach combined whole-cell patch clamp recording and synthetic calcium sensor (OGB-1) to investigate the soundevoked responses on individual spines in the auditory cortex. 20 Except for a few instances of local clustering of similar frequency tuned spines, most similarly tuned spines were found to be widely spread throughout both apical and basal dendrites in layer 2/3 pyramidal neurons.…”

Optimizing and extending light-sculpting microscopy for fast functional imaging in neuroscience

“…Strategies to overcome this speed limit include random access scanning using acousto-optical deflectors (AODs) [4][5][6], a method designed for imaging with rates of up to ~50kHz/N, where N is the number of points. As a second possibility, fast plane scanning using AODs [11] or resonant scanners [12][13][14] can increase the frame rate considerably. For example, a typical 8 kHz resonant scanner would achieve video rate (30 Hz) in bidirectional scanning mode for a 512x512 pixel image.…”