Elsevier

Neuroscience Research

Volume 75, Issue 1, January 2013, Pages 76-81
Neuroscience Research

Technical Note
Probing the function of neuronal populations: Combining micromirror-based optogenetic photostimulation with voltage-sensitive dye imaging

https://doi.org/10.1016/j.neures.2012.11.006Get rights and content

Abstract

Recent advances in our understanding of brain function have come from using light to either control or image neuronal activity. Here we describe an approach that combines both techniques: a micromirror array is used to photostimulate populations of presynaptic neurons expressing channelrhodopsin-2, while a red-shifted voltage-sensitive dye allows optical detection of resulting postsynaptic activity. Such technology allowed us to control the activity of cerebellar interneurons while simultaneously recording inhibitory responses in multiple Purkinje neurons, their postsynaptic targets. This approach should substantially accelerate our understanding of information processing by populations of neurons within brain circuits.

Highlights

► An all-optical system for mapping circuits between neuron populations was developed. ► Inhibitory synaptic circuits in cerebellar cortex were detected and mapped. ► Circuits between molecular layer interneurons and Purkinje cells diverge broadly.

Introduction

Unraveling the function of brain circuitry is fundamental to understanding how the brain works. One of the challenges in such pursuits is to define how populations of neurons within brain circuits collectively communicate with each other to yield network function (Destexhe and Marder, 2004, Traub et al., 2004). Thus, there is a need for tools that enable interrogation of populations of neurons simultaneously. Traditional electrophysiology techniques allow stimulation of groups of presynaptic neurons, but it is hard to know precisely which neurons are being stimulated and which are not. Because electrodes are point detectors, electrophysiological detection of postsynaptic activity is typically restricted to one (or a very few) neurons.

Recent advances in optical techniques make it possible to stimulate and record from populations of neurons. For control of neuronal activity, optogenetic approaches based on light-sensitive channels and other photo-reactive molecules enable highly selective control of genetically defined populations of neurons (Mancuso et al., 2011, Yizhar et al., 2011). Such techniques offer high time resolution and high spatial precision, allowing stimulation of either individual neurons or large neuronal populations. In regard to detection of neuronal activity, a number of optical indicators enable monitoring of the activity of many neurons at once (Mancuso et al., 2011). Among these, voltage-sensitive dye (VSD) imaging is unique because it offers the most direct way to monitor the changes in neuronal membrane potential that are the foundation of neuronal activity (Cohen and Salzberg, 1978, Loew et al., 1979, Djurisic et al., 2003, Homma et al., 2009, Kee et al., 2008). Further, unlike extracellular electrophysiological recording of neuronal activity, VSD imaging enables observation of synaptic inhibition as well as excitation (Cohen and Yarom, 1998, Canepari et al., 2010). Combining these two optical approaches can provide a better way to examine neuronal circuitry. However, this all-optical approach is only beginning to be implemented (Wang et al., 2007a, Wyart et al., 2009, Zhang et al., 2010, Leao et al., 2012), with most work being done with either optical stimulation or optical detection (Homma et al., 2009, Mancuso et al., 2011).

Here, we describe a novel all-optical system that enables simultaneous optogenetic control of a population of neurons while using VSD imaging to detect responses of a population of postsynaptic neurons. Photostimulation via the light-sensitive cation channel, channelrhodopsin-2 (ChR2) (Nagel et al., 2003, Boyden et al., 2005), was done with a micromirror array system (digital micromirror device, or DMD) which allows arbitrary spatial patterns of illumination (Krause, 1996, Fukano and Miyawaki, 2003) and thereby permits photostimulation of populations of presynaptic neurons (Farah et al., 2007, Wang et al., 2007a, Wyart et al., 2009, Leifer et al., 2011, Sakai et al., 2012, Zhu et al., 2012). These experiments employed transgenic mice that express ChR2 in genetically defined populations of neurons (Wang et al., 2007b, Zhao et al., 2011a, Kim et al., submitted for publication). For detection of postsynaptic responses, we employed a recently developed VSD whose red fluorescence emission is compatible with optogenetic control of neuronal activity via ChR2 (Zhou et al., 2007, Kee et al., 2008).

By combining these two techniques – DMD-based optogenetic photostimulation and detection of postsynaptic responses via VSD imaging – we were able to detect the activity and spatial organization of a population of neurons within an inhibitory circuit in the cerebellar cortex. We chose this circuit as a proof-of-principle example because the inhibitory connections between GABAergic interneurons, located in the molecular layer (molecular layer interneurons, or MLIs), and Purkinje cells, the sole output neurons of the cerebellar cortex (Eccles et al., 1967), have been well-definited anatomically (Palay and Chan-Palay, 1974). Further, the small, subthreshold inhibitory postsynaptic potentials (IPSPs) generated by this circuit provide a good challenge for the sensitivity of VSD imaging. We demonstrate that this all-optical approach works and allows rapid and quantitative analysis of the spatial organization of neuronal circuits, paving the way for a deeper understanding of the functional dynamics of information processing by the brain.

Section snippets

Animals

Twenty one to twenty eight day old transgenic mice expressing channelrhodopsin-2 (hChR2 (H134R)) under the control of the nNOS promoter (nNOS-hChR2-YFP) were used (Kim et al., submitted for publication). All procedures were conducted under IACUC guidelines of Biopolis (Singapore) or the Marine Biological Laboratory (U.S.A.).

Brain slice preparation

Mice were anesthetized with isoflurane and sagittal cerebellar slices (200 μm thick) were prepared using a vibratome (Leica) in a cutting solution (in mM: 240 sucrose, 2.5

Photostimulation of neuronal populations via a digital micromirror device

The configuration of the optical system used in our experiments is illustrated in Fig. 1A. A 460 nm LED was used to activate channelrhodopsin-2 and thereby photostimulate the MLIs. Spatially patterned illumination was generated by the DMD and projected onto the slice via the microscope objective. The spatial pattern of illumination could be digitally controlled, with microsecond time resolution, by toggling each of the 777,600 (1080 × 720) micromirrors within the array (Krause, 1996). For VSD

Discussion

Here we describe a novel all-optical approach that enables dual control and detection of the activity of populations of neurons. Our technique is based on combining optogenetic activation of a variable number of presynaptic neurons through the DMD with detection of responses in populations of postsynaptic neurons by VSD imaging. This approach has some limitations, chiefly associated with VSD imaging of postsynaptic responses. First, because all cells are stained by extracellular application of

Conclusions

In conclusion, our approach provides a powerful tool to examine neuronal circuit function at both the cellular and population levels. This will pave the way for a deeper understanding of the functional dynamics of information processing by the cerebellum and other brain regions.

Acknowledgements

We thank L. Cohen and W. Ross for helpful discussion and technical advice, D. Lo, P. Namburi for technical support, and N. Chow for the histological image in Fig. 1B. This work was supported by a Grass Foundation fellowship, National Institutes of Health (NIH grant: R01 EB001963), Duke-NUS Signature Research Program (SRP) block grant, CRP grant from the National Research Foundation (Singapore) and by the World Class Institute (WCI) Program of the National Research Foundation of Korea (NRF)

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