Optical Recording of Neuronal Activity with a Genetically-Encoded Calcium Indicator in Anesthetized and Freely Moving Mice

Fluorescent calcium (Ca2+) indicator proteins (FCIPs) are promising tools for functional imaging of cellular activity in living animals. However, they have still not reached their full potential for in vivo imaging of neuronal activity due to limitations in expression levels, dynamic range, and sensitivity for reporting action potentials. Here, we report that viral expression of the ratiometric Ca2+ sensor yellow cameleon 3.60 (YC3.60) in pyramidal neurons of mouse barrel cortex enables in vivo measurement of neuronal activity with high dynamic range and sensitivity across multiple spatial scales. By combining juxtacellular recordings and two-photon imaging in vitro and in vivo, we demonstrate that YC3.60 can resolve single action potential (AP)-evoked Ca2+ transients and reliably reports bursts of APs with negligible saturation. Spontaneous and whisker-evoked Ca2+ transients were detected in individual apical dendrites and somata as well as in local neuronal populations. Moreover, bulk measurements using wide-field imaging or fiber-optics revealed sensory-evoked YC3.60 signals in large areas of the barrel field. Fiber-optic recordings in particular enabled measurements in awake, freely moving mice and revealed complex Ca2+ dynamics, possibly reflecting different behavior-related brain states. Viral expression of YC3.60 – in combination with various optical techniques – thus opens a multitude of opportunities for functional studies of the neural basis of animal behavior, from dendrites to the levels of local and large-scale neuronal populations.


INTRODUCTION
Neuronal circuits are organized at diverse spatial scales, from subcellular compartments such as dendrites to local neuronal populations to whole brain areas. For understanding how information is encoded in neuronal circuits, it is essential to record activity from a large number of neurons in living and, preferably, in freely moving animals. Advanced in vivo fl uorescence staining and imaging techniques, in particular using fl uorescent Ca 2+ indicators, permit functional studies of neuronal activity in the living brain across all spatial scales (Helmchen and Denk, 2005;Kerr and Denk, 2008;Grewe and Helmchen, 2009;Wilt et al., 2009). Because action potentials induce Ca 2+ transients in soma and dendrites via opening of voltage-gated Ca 2+ channels (Markram and Sakmann, 1994;Helmchen et al., 1996), neuronal spiking activity can be inferred from the fl uorescence measurements. Loading of synthetic Ca 2+ Larkum, 2009a;Murayama et al., 2009). In spite of their strengths, synthetic Ca 2+ indicators have disadvantages and limitations. In particular, they cannot label specifi c subpopulations of cells and subcellular compartments. Moreover, synthetic dye loading is not stable over time and potentially damaging to the brain tissue, limiting the duration of imaging to a few hours.
Genetically encoded fl uorescent Ca 2+ indicator proteins (FCIPs, Miyawaki et al., 2005;Hires et al., 2008; allow for long-term, cell-type specifi c imaging of neural activity in living animals. Following the introduction of the fi rst Ca 2+ -sensitive protein indicator 'cameleon' over a decade ago (Miyawaki et al., 1997), great progress has been made in developing improved FCIPs for functional expression in the mammalian brain (Hasan et al., 2004;Nagai et al., 2004;Heim et al., 2007;Wallace et al., 2008;Tian et al., 2009). First generations of 'yellow cameleons' (YC) showed functional responses in cultured mammalian cells and in invertebrates but their performance in mammalian neurons in an intact tissue was disappointing (Hasan et al., 2004), possibly due to (a) poor fl uorescence-resonance-energy-transfer (FRET) effi ciency in response to Ca 2+ increases and (b) potential interaction of Ca 2+ sensing domains in YCs with cellular targets. Subsequently, the use of a circular permuted Venus 173 variant (cpVenus 173) as a yellow fl uorescent protein (YFP) FRET partner of cyano fl uorescent protein (CFP) produced a YC sensor, yellow cameleon 3.60 (YC3.60), that showed effi cient FRET responses with a large dynamic range in cuvette and also in cultured HeLa cells . Moreover, to reduce interaction of Ca 2+ sensing modules with cellular targets, mutant CaM/M13 pairs and the skeletal muscle specifi c Ca 2+ sensing protein troponin were used to engineer novel FCIPs, D3cpv  and TN-XXL , respectively. In D3cpv, cpVenus 173 was used with CFP while in TN-XXL a circular permuted Citrine 174 was used with CFP. All three FCIPs, YC3.60 Kuchibhotla et al., 2008), D3cpv Wallace et al., 2008) and TN-XXL  have been shown to be functional in vivo. Unlike for D3cpv and TN-XXL, however, functional characterization of YC3.60 has remained incomplete. Since these three FCIPs have the circular permuted YFP variants, cpVe-nus173 (YC3.60 and D3cpv) and cpCitrine 174 (TN-XXL), as a key feature in common, it appeared promising to further examine the suitability of YC3.60 for in vivo imaging of neural activity and deploy recombinant adeno-associated viruses (rAAVs) as a method of gene delivery that has been successfully applied in previous studies Tian et al., 2009).
Here, we report that viral expression of YC3.60 in the mouse barrel cortex allows in vivo measurements of spontaneous and whiskerevoked neuronal activity with high sensitivity and dynamic range. In combination with two-photon microscopy, YC3.60 permits Ca 2+ measurements from individual apical dendrites of cortical neurons as well as from small populations of neurons. Moreover, we demonstrate that YC3.60 Ca 2+ signals in barrel cortex can be read out in a bulk fashion, in particular through an optical fi ber, which enables optical recording of neocortical activity during behavior in freely moving mice. Based on the excellent in vivo performance of virally-expressed YC3.60, we propose it as a sensitive and versatile tool for optical studies of brain function.

MATERIALS AND METHODS
All experiments were performed in accordance with the animal welfare guidelines of the Max Planck Society and the guidelines of the Federal Veterinary Offi ce of Switzerland, respectively. All experimental procedures were approved by the local authorities (Regierungspräsidium Karlsruhe and Cantonal Veterinary Offi ces in Zurich and Bern, respectively).

AAV-MEDIATED GENE TRANSFER INTO MOUSE NEOCORTEX
rAAV equipped with YC3.60 under control of a human synapsin promoter ( Figure 1A) was co-transfected with pDp1, pDp2 (ratio: 3:1) helper plasmids in HEK293 cells (Hasan et al., 2004;Wallace et al., 2008). Seventy-two hours after transfection, HEK293 cells were collected and packaged viruses were released by repeated freeze-and-thaw on dry-ice-ethanol bath. Viruses were purifi ed fi rst on the iodixanol gradient and later by pre-casted 1 ml Heparin columns (Amersham) using FPLC . Infectious virus titers were determined in primary neuron cultures and was 3 × 10 8 transducing units per microliter. Before virus injection, 6-8 weeks old BL/C57 mice were anesthetized with ketamine plus xylazine by intraperitoneal injection (ketamine, 80 mg per kilogram body weight; xylazine, 10-16 mg/kg). Viruses (200-300 nl) were delivered through thin glass pipettes (tip size 8-12 µm) at a depth of about 250 µm to the whisker-related somatosensory cortex (L2/3) by stereotaxic injection (Hasan et al., 2004;Wallace et al., 2008). To facilitate intraparenchymal administration we included 20% hypertonic D-mannitol in the solution (Mastakov et al., 2001). Infected animals were kept for at least 21 days before imaging or analysis of brain tissues.

TISSUE FIXATION AND IMMUNOHISTOCHEMISTRY
Mouse brains were fi xed for a few hours in 4% paraformaldehyde and YFP fl uorescence was visualized as a bright spot at the virusinjection site using a stereomicroscope (SV11; Zeiss) ( Figure 1B). Brains were coronally sliced to a thickness of 75-100 µm using a vibratome (VT 1000S; Leica Instruments). Slices were counterstained for neuron-specifi c marker, NeuN, using a mouse antineuronal nuclei (NeuN) monoclonal antibody (1:1,000 dilution) (Millipore) and a Cy3-conjugated goat anti-mouse IgG (1:200 dilution; Jackson Immuno Research laboratories). Green (GFP) and red (Cy3) fl uorescence were visualized with the Zeiss LSM 5 Pascal laser scanning confocal imaging system equipped with GFP fi lters.

CA 2+ IMAGING AND ELECTROPHYSIOLOGY IN SLICE CULTURES
Organotypic hippocampal slices were prepared as previously described (Stoppini et al., 1991) and superfused during recording at room temperature with artifi cial CSF (ACSF; Biometra) containing (in mM): 125 NaCl, 25 NaHCO 3 , 2.5 KCl, 1.25 NaH 2 PO 4 , 1 MgCl 2 , 2 CaCl 2 , 25 Glucose, saturated with 95% O 2 /5% CO 2 . Loose patch-clamp recordings were performed from CA3 pyramidal cells with ACSF-fi lled pipettes (50-100 MΩ). Cell spiking was elicited by monopolar electrical stimulation delivered at a rate of <0.1 Hz with an ACSF-fi lled glass pipette placed in the region of the apical dendrites (stratum lucidum or stratum radiatum). Multiple spikes were generated by varying the number of electrical stimuli delivered at 100 Hz. Electrophysiological signals were acquired with a software-controlled patch-clamp amplifi er (EPC-9, Pulse 8.11; Heka Elektronik). For the in vitro characterization of YC3.60 we used hippocampal neurons because hippocampal slice cultures are routinely available in the lab. Neocortical and hippocampal neurons are known to exhibit very similar AP-evoked Ca 2+ signaling (Helmchen et al., 1996). Two-photon imaging of slice cultures was performed with a mode-locked femtosecond Ti-sapphire laser (Cameleon XR; Coherent) set at an excitation wavelength of 840 nm. Fluorescence signals were acquired with an upright laser scanning microscope (Zeiss LSM 510 NLO) equipped with a 63× water immersion objective (NA 1.0). Frame-scan acquisition was performed from somatic regions of interest (ROIs) at a rate of 24.1 Hz. Frames typically were 30 × 27 pixels (pixel size 140 nm × 140 nm). While ROI width could vary the number of lines was kept constant to ensure the same sampling rate for all recordings.

IN VIVO CA 2+ IMAGING AND ELECTROPHYSIOLOGY
Animals were surgically prepared for in vivo imaging as described previously (Waters et al., 2003;Nimmerjahn et al., 2004;Wallace et al., 2008). Briefl y, mice were anesthetized with urethane (∼1.5 g/kg) and a stainless steel plate was attached to the exposed skull. For experiments combining electrophysiology and imaging the dura was carefully removed. The exposed tissue was superfused with normal rat Ringer solution (in mM: 135 NaCl, 5.4 KCl, 5 HEPES, 1.8 CaCl 2 , pH 7.2 with NaOH). To dampen heartbeat and breathing-induced motion, we fi lled the cranial window with agarose (type III-A, Sigma; 1% in NRR) and covered it with an immobilized glass coverslip. Body temperature was maintained at 37°C with the help of a heating blanket. For in vivo imaging (Figure 2), we used a custom-built two-photon microscope with ∼100-fs laser pulses at 870 nm wavelength provided by a Ti:sapphire laser (Spectra-Physics) and a 40× water-immersion objective (NA 0.8; Olympus). CFP and YFP fl uorescence were collected with blue (450-475 nm) and green (535-550 nm) emission fi lters (AHF AG). Frame scans were acquired at 7.81 Hz with 128 × 128 pixel resolution. Juxtacellular recordings were obtained from YC3.60-expressing L2/3 neurons with glass pipettes (5-7 MΩ) containing NRR solution and 0.025 mM Alexa-594 for pipette visualization. Neurons were visually targeted using the two-photon microscope. Action potentials were recorded in current-clamp using an Axoclamp 2-B amplifi er (Axon Instruments, Molecular Devices) and digitized using software custom-written in LabView (National Instruments). A commercial camera system (Optical Imaging) and custom made optics were used for wide-fi eld recordings of single-photon excited YFP-fl uorescence (excitation fi lter 440 nm, dichroic mirror 460 nm, emission fi lter 535 nm). Movies were acquired at 10 Hz from a 4.2 mm × 4.2 mm fi eld of view (FOV) and the mean YFP-fl uorescence signals from the entire FOV (expressed as relative fl uorescence changes ΔF/F YFP ) were analyzed during spontaneous activity and upon air-puff whisker stimulation. Control measurements using an emission fi lter for detecting CFP-fl uorescence revealed signal decreases as expected (data not shown). Simultaneous local fi eld potential (LFP) recordings were performed with a 16-site single shank probe (Neuronexustech) on a multichannel recording system (Cheetah, Neuralynx). The probe was inserted under visual control in the middle of the fl uorescent spot and LFP signals from L2/3 were recorded.

FIBER-OPTIC CA 2+ RECORDINGS
Fiber-optic recordings were performed as described previously Murayama and Larkum, 2009b). Briefl y, eight mice (∼10 weeks old) were used in these experiments and were deeply anesthetized by isofl urane (1.5-3%, Baxter). Following surgery, an analgesic was administered (buprenorphine, twice per day; Essex Chemie) and local anesthetic (lidocaine; Sigma-Aldrich) applied to the scalp. On the day of the experiment, the head was fi xed in a stereotaxic instrument (Narishige) and body temperature maintained at 36-37°C. A craniotomy was performed above the virus injected area (somatosensory barrel cortex, 1 mm diameter). In one experiment, the skull was thinned above the injected area. The dura mater remained intact. After anesthesia experiments, animals recovered for 1-2 h and were then transferred to the arena for behavioral observation and freely moving fl uorescence imaging. Fixation of the fi ber-optic mount to the animal's head was performed as described previously (Murayama and Larkum, 2009b). A blue LED (IBF + LS30ROB-3W-Slim-RX or IBF + LS30W-3W-Slim-RX, Imac Co.) was used as a light source. An excitation fi lter (FF01-438/24-25, Semrock or D480/30x, Chroma Technology), a dichroic mirror (FF520-Di01-25x36, Semrock), and an emission fi lter (FF01-542/27-25, Semrock) were used for epifl uorescence Ca 2+ recordings. A 10x objective (Edmund Optics) was used for illuminating and imaging an optical fi ber (NT57-069, NA, 0.22, core diameter of 440 µm; total diameter of 470 µm, Edmund Optics). A CCD camera (MicroMax, Roper Scientifi c) was used for collecting fl uorescence. Sensory responses were evoked by a brief air puff (50 ms-duration) delivered to the contralateral whiskers. Fluorescence changes were sampled at 100 Hz. Data was acquired on a PC using WinView software (Roper Scientifi c). ROIs were chosen offl ine for measuring fl uorescence changes (see below). Animal behavior was observed by using a CCD camera (Logitech, Japan). The video recording was acquired and stored to disk using QuickCam software (Logitech). Cadmium chloride was from Fluka.

DATA ANALYSIS
For all experiments in slices cultures as well as for in vivo two-photon imaging, Ca 2+ signals were expressed as relative YFP/CFP ratio changes ΔR/R after background subtraction and further analyzed using Igor (Wavemetrics Inc.) and Matlab (Mathworks). Peak amplitudes of Ca 2+ transients were determined as the mean of three sampling points around the peak location. Decay time constants were obtained from single-exponential fi ts. To quantify AP detection, we calculated the signal-to-noise ratio (SNR) of AP-evoked transients as the ratio of peak amplitude to SD of the unfi ltered trace 750 ms prior to the fi rst AP. Similarly, the SNR of baseline traces was computed as the ratio of peak to SD of the respective trace. The distribution of baseline SNR values was estimated by fi tting a Gaussian and the detection threshold for AP-evoked transients was determined as the SNR value above which less than 5% of baseline traces would be classifi ed as false positives (see Figure 4E). For fi ber-optic recordings, fl uorescence signals were quantifi ed by measuring the mean pixel value of a manually selected ROI for each frame of the image stack using Igor Pro (Wavemetrics) software. Ca 2+ changes were expressed as ΔF/F = F t /F 0 , where F t was the average fl uorescence intensity within the ROI at time t during the imaging experiment and F 0 was the mean value of fl uorescence intensity before stimulation.

AAV-INDUCED EXPRESSION OF THE CA 2+ SENSOR YC3.60 IN MOUSE BARREL CORTEX
YC3.60 is a genetic Ca 2+ sensor based on a CFP, a Ca 2+ -sensitive linker, and a bright circularly permuted YFP .
Here, we characterized YC3.60 as a promising candidate protein for reporting neuronal activity in the mammalian cortex in vivo. We expressed YC3.60 in mouse barrel cortex under control of a human synapsin promoter using a rAAV vector ( Figure 1A). Three weeks or longer after stereotactic virus injection, YC3.60 expression appeared as a bright fl uorescent spot with a diameter of 0.5-1 mm ( Figure 1B). Analyses of fi xed brain slices showed that near the injection site a majority of cortical neurons (>80%) in layer 2/3 (L2/3) and L5 were fl uorescently labeled (Figures 1C,D). Neurospecifi city of YC3.60 expression was confi rmed by overlap with NeuN-staining. Cells showed bright staining in soma excluding the nucleus (Figures 1E,F) as reported for other FCIPs (Hasan et al., 2004;Wallace et al., 2008;Tian et al., 2009). Due to the dense expression, the neuropil (including dendrites and axons) was also brightly stained near the injection site. YC3.60-expressing cells could be imaged by two-photon microscopy in the living mouse brain down to about 400 µm covering the supergranular layers (Figure 2A). Vertically running apical dendrites of pyramidal neurons were clearly visible in side projections of image stacks ( Figure 2B). In vivo counterstaining with the astrocyte-marker sulforhodamine 101 (SR101) (Nimmerjahn et al., 2004) revealed a complete lack of overlap with YC3.60 staining confi rming neurospecifi city (Figure 2C).
YC3.60 reports increases in intracellular free Ca 2+ concentration as a FRET change caused by a conformational change that brings CFP and YFP closer together . The acceptor (YFP) fl uorescence intensity is expected to increase while the donor (CFP) fl uorescence should decrease. Indeed, YFP-increases and concomitant CFP-decreases were observed in initial experiments on rat organotypic hippocampal slices, in which increasing number of synaptic shocks were delivered to the mossy fi bers and fl uorescence changes were recorded in the neuropil of the CA3 region ( Figure 3A). Expressed as relative percentage change of the YFP/CFP ratio (ΔR/R), the evoked signals showed a large Imaging neuronal activity with YC3.60 dynamic range, with peak signals up to 50% and little indication of saturation. AP-evoked calcium transients of smaller amplitude were readily observed in single cells, both in vitro ( Figure 3B) and in vivo ( Figure 3C). Even though a ratiometric measurement with simultaneous acquisition of fl uorescence in the YFP-and CFPchannel is advantageous as it normalizes for common baseline drifts, the relative fl uorescence changes in the YFP-channel alone (ΔF/F YFP ) also clearly indicated Ca 2+ transients (Figures 3B,C).
In the following sections, we will present Ca 2+ transients either as relative percentage change of the ratio YFP/CFP (ΔR/R) or as ΔF/F YFP .

YC3.60 REPORTS AP FIRING IN VITRO AND IN VIVO
We next examined how sensitively YC3.60 reports AP fi ring in vitro and in vivo by combining targeted electrical recordings with twophoton imaging (see Materials and Methods). Synaptic stimulation of pyramidal neurons in slice cultures elicited single APs or bursts of AP trains as revealed by juxtacellular recordings (Figure 4A). Single APs evoked Ca 2+ transients with an average ΔR/R amplitude of 5.9 ± 0.3% (mean ± SEM, n = 32), while high-frequency bursts of 2, 3, and 5 APs elicited successively larger transients (10.1 ± 0.3% (40)  mated by a linear fi t (Figure 4C; slope = 3.15%/AP; r 2 = 0.98). The decay of Ca 2+ transients was relatively fast and did not signifi cantly depend on AP number (Figure 4D; time constants of exponential fi ts: 0.78 ± 0.05, 0.82 ± 0.03, 0.91 ± 0.04, and 0.80 ± 0.06 s for 1, 2, 3 and 5 APs, respectively; slope of linear regression 4 ms/AP).
To determine YC3.60 sensitivity in vivo, we performed two-photon targeted juxtacellular recordings from YC3.60-expressing L2/3 neurons in barrel cortex of anesthetized mice (Figure 4B). In many cases, single APs were associated with clear ΔR/R transients. On average, single APs elicited transients of 2.00 ± 0.09% peak amplitude (n = 138 transients from 11 cells). Bursts of up to 7 APs elicited transients with successively larger amplitude ( Figure 4C). The relationship between Ca 2+ transient amplitude and AP number again was well approximated by a linear fi t (slope = 1.39%/AP; r 2 = 0.98), indicating that YC3.60 fl uorescence changes for bursts of APs were well below saturation. Indeed, much larger events (20-30% ΔR/R) were occasionally observed in response to trains of 10 or more APs (see Figure 5B). We estimated AP-detection effi ciency by comparing the SNR of AP-evoked transients to the baseline noise level (see Materials and Methods). Detection rates were 71% for single APs and  fi eld. On the fi nest scale, we simultaneously measured spontaneous dendritic and somatic Ca 2+ transients in L2/3 pyramidal neurons using 'arbitrary plane' imaging (Göbel and Helmchen, 2007b) ( Figure 5A). As expected (Schiller et al., 1995), Ca 2+ transients showed faster decay times in apical dendrites compared to somata (0.35 ± 0.04 s vs. 0.47 ± 0.04 ms; n = 23; p < 0.01; paired two-tailed t-test), confi rming relatively rapid kinetics of YC3.60. A large dynamic range of YC3.60 is demonstrated in Figure 5B, where Ca 2+ transients evoked by complex AP patterns (ranging from individual APs to trains of 10 or more APs) were revealed with high fi delity. The peak ΔR/R amplitudes of around 30% in these recordings presumably still are far from indicator saturation (see Figure 3A).
On the level of local neuronal populations, air-puff whisker stimulation (5 Hz) elicited clear Ca 2+ transients in neuronal somata but also in the neuropil (Figure 5C). Bursts of AP fi ring could be distinguished even at relatively slow frame rates (7.81 Hz), highlighting the rapid kinetics of the Ca 2+ sensor. Combined electrical recordings 80 and 93% for bursts of 2 and 3 APs, respectively ( Figure 4E). Similar to the in vitro results, Ca 2+ transients showed fast decays, which did not depend on AP number (Figure 4D; for single APs 0.60 ± 0.05 s; n = 78 transients from nine cells; slope of linear regression −16 ms/ AP). The differences in absolute values for peak amplitudes and decay time constants between in vitro and in vivo experiments most likely can be attributed to the differences in experimental conditions. In particular, a similar reduction of AP-evoked Ca 2+ transient amplitude at physiological compared to room temperature has also been reported for other FCIPs (Mao et al., 2008;Tian et al., 2009). We conclude that YC3.60 sensitively reports AP fi ring both in vitro and in vivo and that it can even resolve single APs.

SENSORY-EVOKED AND SPONTANEOUS SUBCELLULAR, CELLULAR AND WIDE-FIELD CA 2+ SIGNALS
We next investigated in how far YC3.60 can be used to study sensory-evoked cortical activity on various spatial scales, ranging from dendrites of individual neurons to large areas of the barrel April 2010 | Volume 4 | Article 9 | 9 Lütcke et al. Imaging neuronal activity with YC3.60 allowed us to directly assess the degree of potential contamination of somatic signals by Ca 2+ signals in the surrounding neuropil (Kerr et al., 2005;Göbel and Helmchen, 2007a). Whisker-evoked events were associated with signifi cantly larger Ca 2+ transients in somata compared to the neighboring neuropil (4.0 ± 0.5 vs. 1.6 ± 0.2% ΔR/R, n = 32, p < 0.01, paired two-tailed t-test). In contrast, spontaneously occurring APs elicited Ca 2+ transients in somata but not in the neuropil (2.3 ± 0.4 vs. 0.4 ± 0.1%, n = 37, p < 0.01) ( Figure 5D). From these observations we conclude that sensory stimulation can evoke Ca 2+ transients in the neuropil in addition to somatic signals, presumably refl ecting excitation of afferent axonal pathways (Kerr et al., 2005), but that high-resolution two-photon imaging clearly distinguishes the cellular AP-evoked signals. Activation of a substantial fraction of neurons in the local population as well as of the surrounding neuropil might cause suffi cient YC3.60 signals for large-scale bulk recording of barrel cortex excita-tion. Indeed, in addition to sensory-evoked responses spontaneous fl uctuating calcium signals were apparent in the neuropil when entire two-photon imaging frames were used as ROI ( Figure 6A). Similar spontaneous YC3.60 signals were observed with wide-fi eld single-photon-excited fl uorescence with a CCD-camera positioned above barrel cortex ( Figure 6B). These slow oscillations closely corresponded to the simultaneously measured LFP and most likely are due to synchronous activity during anesthesia. In addition, air puff stimulation of whiskers evoked stimulus-locked LFP signals and bulk fl uorescence changes with an average ΔF/F YFP amplitude of 0.47 ± 0.03% and an average decay time of 0.48 ± 0.04 s (n = 3 animals). Bulk-activity in barrel cortex could also be read out using single-photon excitation and fl uorescence collection through a single-core optical fi ber ( barrel cortex, we observed both spontaneous and air puff-evoked ΔF/F YFP signals ( Figure 6C) that were similar to the signals observed by wide-fi eld camera imaging.

FIBER-OPTIC RECORDING OF BARREL CORTEX ACTIVITY IN FREELY MOVING MICE
Fiber-optic recordings enable measurements of neuronal activity in awake, freely behaving animals (Adelsberger et al., 2005;Murayama et al., 2007;Murayama and Larkum, 2009b; for review see Grewe and Helmchen, 2009;Wilt et al., 2009). We tested if YC3.60 expression is suitable for such an application. Using fi rm attachment of the single optical fi ber to the animal's head (Murayama and Larkum, 2009b) we measured bulk fl uorescence from the barrel fi eld in awake, freely moving mice (Figure 7). While the mice were either passively sitting or actively exploring, we recorded complex ΔF/F YFP signals (Figures 7A-C). We could block the Ca 2+ transients by application of Cd 2+ , a Ca 2+ channel blocker, to the cortical surface (Figure 7D), indicating that the YC3.60 sensor was reporting Ca 2+ transients. Mechanically jolting the fi ber did not result in obvious fl uorescence changes in YC3.60expressing mice as in previous fi ber-optic studies Murayama and Larkum, 2009b). Furthermore, fl uorescence traces in a wild-type mouse (without YC3.60) were fl at without any movement-related changes ( Figure 7E). Together these fi ndings exclude opto-mechanical artifacts as source of the observed fl uorescence changes and instead indicate that YC3.60 signals are caused by Ca 2+ channel activation and thus provide a readout of the complex activation pattern in the barrel cortex. Fiber-optic recordings using YC3.60 thus should permit closer investigation of behavioral-related activity in specifi c neocortical areas.

DISCUSSION
In vivo Ca 2+ imaging in the mammalian neocortex so far mainly relied on synthetic indicator dyes (Garaschuk et al., 2006;Göbel and Helmchen, 2007a;Kerr and Denk, 2008;Grewe and Helmchen, 2009). Recently, different FCIPs (also called genetically encoded Ca 2+ indicators or GECIs) have been developed that will allow for a more specifi c interrogation of various aspects of neural activity, compared to synthetic sensors. Here, we have shown that upon neuron-specifi c expression in the mouse neocortex, the FCIP YC3.60 can be used to read out activation of neocortical areas at different spatial scales both in anesthetized and awake, freely moving animals.
Original characterization of YC3.60  defi ned it as an indicator with high sensitivity and dynamic range in vitro. However, reliable Ca 2+ imaging in vivo was not demonstrated in transgenic animals , possibly due to the use of a plasma-membrane bound version of the sensor which may have resulted in a reduced AP detection effi ciency (Mao et al., 2008). Subsequently, YC3.60 was employed to obtain quantitative Ca 2+ concentration measurements in brain slices (Liu et al., 2008) and resting Ca 2+ concentrations in vivo (Kuchibhotla et al., 2008). The current study extends these fi ndings by demonstrating that viral delivery of YC3.60 permits in vivo measurements of sensory-evoked Ca 2+ signals that closely correspond to cellular AP fi ring patterns. Furthermore, we have shown that YC3.60 can be applied to monitor neural activity at diverse spatial scales, covering dendritic Ca 2+ signals, AP fi ring in local neuronal populations as well as large scale brain areas.
Gene delivery by AAV-vectors  provides a highly fl exible, straightforward and safe approach for dense and wide-spread expression of FCIPs in neurons (Kootstra and Verma, 2003). For these reasons, viral delivery has been the method of choice in recent characterizations of FCIPs in the mammalian brain Wallace et al., 2008;Tian et al., 2009). An alternative approach for gene delivery of FCIPs into the brain is in utero electroporation, which provides the potential benefi t of celltype specifi city, depending on the precise electroporation protocol (Borrell et al., 2005) but which results in relatively sparse labeling of neurons compared to viral delivery . Finally, the production of transgenic mice expressing a number of different FCIPs has been reported (Hasan et al., 2004;Heim et al., 2007) including membrane-bound YC3.60 . In one of these studies, sensory-evoked Ca 2+ transients were demonstrated using wide-fi eld imaging of the olfactory bulb with two FCIPs expressed under a tetracycline-inducible promoter (Hasan et al., 2004). The widespread use of these mouse lines has been limited, however, by the failure to clearly monitor AP-evoked cellular Ca 2+ signals in vivo, possibly due to low protein expression levels.
Comprehensive analysis of neural circuits will require reliable detection of single APs as well as estimation of the fi ring frequency during bursts of APs. Here we demonstrate that YC3.60 can detect the occurrence of single APs in pyramidal cells of mouse barrel cortex in vivo with a sensitivity comparable to recent reports of another ratiometric FCIP, D3cpV . Unlike D3cpV, however, YC3.60 shows faster kinetics and minimal saturation for bursts of up to at least 10 APs, thus making it a suitable tool for quantitative investigation of local neural network dynamics in vivo. Overall our analysis revealed that YC3.60 shows comparable signals in terms of sensitivity and decay times to the commonly used synthetic indicator Oregon Green BAPTA-1 (Kerr et al., 2005), which has been used extensively for optical monitoring of AP fi ring in populations of neurons. Recently, in addition to D3cpV, two other novel FCIPs have been proposed for in vivo two-photon Ca 2+ imaging in the mammalian brain. First, in utero electroporation of TN-XXL, a troponin-based ratiometric FCIP, allowed repeated Ca 2+ imaging from the same neurons over days; however its sensitivity to AP fi ring was relatively low with single AP detection only achievable in brain slices . Second, following AAV-delivery the single-fl uorescent protein sensor GCaMP-3 has recently been shown to exhibit large fl uorescence changes and to detect APs with fast kinetics and little saturation in mouse somatosensory cortex in vivo (Tian et al., 2009). This Ca 2+ sensor does not, however, permit ratiometric imaging and thus will be more susceptible to motion artifacts compared to YC3.60. It is likely that in the near future improved versions of either cameleons or GCaMPs will provide even better optical readout of neuronal spiking. Furthermore, single-fl uorophore FCIPs may be co-expressed with a second indicator that is not Ca 2+ -sensitive in order to reduce their susceptibility to motion artifacts.
In addition to two-photon Ca 2+ imaging at the level of single cells and small populations of neurons, we demonstrated the use of FCIPs to record large-scale neuronal activity in awake, freely human primates (Stettler et al., 2006) and is therefore likely to play an important role in the dissection of the neural underpinnings of complex behaviors.