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• Abstract

SECTION I

## INTRODUCTION

The original reason for developing optical methods that monitor neuronal activity was to record the activities of many cells simultaneously during behavior [1], [2]. State-of-the-art optical techniques, like two-photon fluorescence (TPF) and second-harmonic generation (SHG) microscopy, allow studying detailed patterns of electrical activity from randomly located neuronal cells [3], [4], [5]. The spatial resolution of other techniques based on a linear approach like confocal microscopy allows monitoring time fluctuation of membrane potential in subcellular domains and can simultaneously interrogate diverse cellular compartments [6]. However, one-photon microscopy in intact tissue is limited by light scattering and out-of-focus fluorescence that blurs the image. On the other hand, using nonlinear laser techniques, like TPF and SHG microscopy, provides improved penetration depth in highly scattering samples [7], [8]. Nonlinear imaging allows visualizing populations of neurons with subcellular resolution in living animals up to 1000 $\mu\hbox{m}$ of depth [9]. Two-photon optical recordings have been successfully applied to the investigation of neural activity through membrane potential recording from subcellular structures like axons, dendrites, and spines in acute brain slices [10], [11], or simultaneous calcium recording from tens of neurons in vivo [5], [12], even in behaving animals [13], [14]. As neuronal circuits exhibit extremely complex connectivity, their functional dissection would crucially benefit from the activation of selected neurons. Optogenetic probes provide optical control over the activity of specific populations of neurons, not targetable through conventional electrophysiological techniques [15]. If combined with two-photon imaging, optogenetic tools can use infrared light to manipulate the electrical activity of neuronal cells singularly, with high spatial and temporal resolution [16], [17].

In this review we will briefly describe the state of the art in detecting and manipulating the activity of microcircuits of neurons. We will focus on two outstanding studies of 2012 on the optical investigation of neural activity, with voltage-sensitive dyes (VSDs) and optogenetic tools.

SECTION II

## NONLINEAR RECORDING AND MANIPULATION OF NEURAL ACTIVITY

Several optical techniques have been recently developed to measure the electrical activity of single neurons or neuronal populations. Two major classes of fluorescent indicators have been used, whose emission depends on calcium concentration or membrane potential. The most commonly used activity reporter is calcium, whose intracellular concentration fluctuates as a consequence of varying membrane potential and synaptic inputs. Calcium indicators are highly sensitive, allowing detecting a single action potential (AP) [12], [13], [14] and even a single calcium channel activity [18]. On the other hand calcium dynamics is slow compared to the variations in membrane potential, and this prevents resolving high-frequency spike in the neural activity. An alternative approach to probe membrane potential through light relies on specific VSDs [19]. These probes intercalate into one layer of the plasma membrane (i.e., the cell's outer membrane) and can directly report the intensity of the electric field across the membrane through several different mechanisms of voltage sensing that are common to both absorption and fluorescence [20]. VSDs have been employed to monitor, in real time, the electrical activity of large cortical areas with high spatial resolution (down to 20–50 $\mu\hbox{m}$) and high temporal resolution (< 1 ms) [21], [22], [23], [24], [25]. Light scattering and out-of-focus fluorescence again limit one-photon wide-field imaging of neuronal activity in brain slices and in vivo preparation. In combination with TPF or SHG microscopy, VSDs imaging in intact systems benefits from higher resolution in deep tissue [26], [27], [28], [29], [30], [31], [32]. However, most available VSDs lack spectral compatibility with two-photon excitation. Recently, Yan et al. synthesized and tested a palette of fluorinated hemicyanine dyes [33] that cover a broad spectral range, including a big section of the visible spectrum (440–670 nm) and a wide band in the infrared (900–1340 nm). Many applications of these new VSDs have been demonstrated, from intracellular AP recording in cardiomyocytes [34] to two-photon recording of back-propagating APs from single dendritic spines in cortical brain slices [33]. These dyes have been shown to allow APs recording from multiple neurons in acute slices (see Fig. 1). In detail, the authors took advantage of the dye di-4-AN(F)EPPTEA to investigate Purkinje cells (PCs) synchronization in the cerebellar cortex. The authors show, through simultaneous optical recording of the electrical activity, the lack of temporal correlation between the APs of the five monitored PCs (Fig. 1).

Fig. 1. Simultaneous AP recording from multiple cells by random access multiphoton microscopy. (a) TPF image (70-$\mu\hbox{m}$ depth) of an acute cerebellar slice stained with di-4-AN(F)EPPTEA. The two-photon excitation laser was rapidly scanned over five PCs indicated by the five red lines shown in A to measure the time-dependent fluorescence from these cells. An electrode was used to measure the electrical activity of PC1 in parallel. (b) Real-time multiplexed optical recording of spontaneous activities from the five PCs (black traces) with a temporal resolution $\sim\!400 \ \mu\hbox{s}$. PC1 electrical activity measured by the electrode (blue trace). The magnification of the simple spike in PC5 trace (red box) reveals the undershoot phase. Modified with permission from [33].

Besides monitoring cortical connectivity and synchronization through VSDs, an elegant alternative to dissect neuronal circuitry implies controlling network firing. Optogenetics offers the unique opportunity of manipulating the activity of a specific population of genetically targeted neurons with light [35], [36], [37]. The introduction of genes (so-called opsin genes) encoding for light-sensitive protein in neurons has been applied to a number of studies on, for example, circuit mapping [38], [39], memory storage [40], [41], anxiety [35], and Parkinson's disease [42], [43]. If combined with nonlinear excitation, optogenetic tools can benefit from increased control of neuronal activation within the intact tissue, achieving single-cell precision. Up to now, few groundbreaking studies have coupled optogenetics with two-photon excitation [44], [45], [46], [47]. However, the commonly used channelrhodopsin-2 has low single-channel conductance and displays fast kinetics [48]; therefore two-photon excitation of a neuron required either complex stimulation strategies or high expression levels of the opsin. Two parallel studies [16], [49] have recently reported the optimization of a new red-shifted chimeric opsin, ${\rm C}1{\rm V}1_{\rm T}$, that displays slower off-kinetics and larger photocurrents than other channelrhodopsins [50]. After optimizing the illumination parameters on cell cultures, the authors showed the application of this red-shifted opsin in cortical or hippocampal slices and, finally, in a mammalian in vivo preparation.

In this case, the concomitant expression of EYFP and ${\rm C}1{\rm V}1_{\rm T}$ was induced in layer 2/3 neurons of the somatosensory cortex of adult mice by injection of a specifically engineered adeno-associated virus [Fig. 2(a) and (b)]. The targeted neurons can be thus visualized [to the smaller structures, see the YFP-labeled spines in Fig. 2(c)] and singularly activated [Fig. 2(d)] in vivo. The high temporal and spatial resolution of the two-photon stimulation of single neurons is highlighted in Fig. 2(d), where the authors show the precise spike-train control with the 5-Hz 1040-nm raster-scanning illumination (lower left) and the failed activation of a pyramidal neuron by lateral or axial displacement of the scanning region of interest.

Fig. 2. In vivo two-photon optogenetic control of spike firing. (a) Experimental scheme for in vivo two-photon manipulation of superficial somatosensory neurons transduced with ${\rm C}1{\rm V}1_{\rm T}$-p2A-EYFP. (b) Left, two-photon image of layer 2/3 pyramidal neurons transduced with ${\rm C}1{\rm V}1_{\rm T}$-p2A-EYFP in somatosensory cortex (150–250 $\mu\hbox{m}$ deep). Right, two-photon image of layer 1 pyramidal neurons transduced with ${\rm C}1{\rm V}1_{\rm T}$-p2A-EYFP in somatosensory cortex (50–150 $\mu\hbox{m}$ deep). (c) Two-photon image of dendritic spines on pyramidal cells transduced with ${\rm C}1{\rm V}1_{\rm T}$-p2A-EYFP in layer 2/3 of somatosensory cortex. (d) Upper left, in vivo two-photon image of layer 2/3 pyramidal cells transduced with ${\rm C}1{\rm V}1_{\rm T}$-p2A-EYFP (imaged during loose patch). Lower left, trace showing precise spike-train control with 5-Hz 1040-nm raster-scanning illumination; the amplitude and waveform of these evoked spikes recorded in cell-attached mode matched the spontaneous spikes in each cell. Upper right, axial resolution of two-photon optogenetic control of spiking in vivo. Spiking of layer 2/3 pyramidal neurons (blue triangles) as a function of the position of the scanned region of interest (red boxes) is shown. Lower right, lateral resolution of two-photon optogenetic control of spiking in vivo. Modified with permission from [16].
SECTION III

## CONCLUSION

The investigation of neuronal signaling has been recently targeted by optical tools that can both interrogate and manipulate neural electrical activity. If combined with VSDs or optogenetic tools, nonlinear microscopy provides nice spatial control over the triggering and the recording of single cell activity in intact tissue. The studies reported here demonstrate the possibility of performing precise monitoring and activation of individual neurons through two-photon excitation.

We believe that these studies paved the way to study neural function, from synaptic weights to entire dynamics of optogenetically targeted populations of neurons. This, in conjugation with multiple laser beams, will allow dissecting the function of microcircuits with single-cell precision by simultaneous 3-D stimulation and recording from multiple neurons.

The future goal for voltage imaging would be to measure individual APs and subthreshold events without averaging both from neuronal somas and individual spines of genetically targeted cell types. This attainable achievement, together with improvements in voltage sensors, could open a new epoch for in vivo study of neuronal activity through genetically selected mammalian circuits.

## Footnotes

This work was supported in part by LASERLAB-EUROPEunder Grant Agreements 228334 and 284464, EC's Seventh Framework Programme; by the Italian Ministry of Education, University and Research in the framework of the Flagship Project NANOMAXunder Research Grant RGP0027/2009; by the “Ente Cassa di Risparmio di Firenze.” and by the Italian Ministry of Health in the framework of the “Stem Cells Call for proposals.” This work has been carried out in the framework of the research activities of ICON Foundation supported by“Ente Cassa di Risparmio di Firenze.” Corresponding author: A. L. Allegra Mascaro (e-mail: allegra@lens.unifi.it).

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