Chapter 10 - Mouse transgenic approaches in optogenetics

https://doi.org/10.1016/B978-0-444-59426-6.00010-0Get rights and content

Abstract

A major challenge in neuroscience is to understand how universal behaviors, such as sensation, movement, cognition, and emotion, arise from the interactions of specific cells that are present within intricate neural networks in the brain. Dissection of such complex networks has typically relied on disturbing the activity of individual gene products, perturbing neuronal activities pharmacologically, or lesioning specific brain regions, to investigate the network's response in a behavioral output. Though informative for many kinds of studies, these approaches are not sufficiently fine-tuned for examining the functionality of specific cells or cell classes in a spatially or temporally restricted context. Recent advances in the field of optogenetics now enable researchers to monitor and manipulate the activity of genetically defined cell populations with the speed and precision uniquely afforded by light. Transgenic mice engineered to express optogenetic tools in a cell type-specific manner offer a powerful approach for examining the role of particular cells in discrete circuits in a defined and reproducible way. Not surprisingly then, recent years have seen substantial efforts directed toward generating transgenic mouse lines that express functionally relevant levels of optogenetic tools. In this chapter, we review the state of these efforts and consider aspects of the current technology that would benefit from additional improvement.

Introduction

Transgenic mice have been widely used in neuroscience research to facilitate the deciphering of gene and cellular functions. Perhaps the greatest advantage of using a transgenic approach in such studies is that cell population-restricted transgene expression can be achieved using specific promoters, and this restricted pattern of expression can be passed on to subsequent generations fairly reproducibly. In functional studies of the mouse brain, a variety of transgenic strategies have been used to inactivate or overexpress particular genes, label specific cell populations or their subcellular compartments, and manipulate the activity or function of specific cell populations (Luo et al., 2008). For example, the strong, neuronally restricted expression of fluorescent reporter in Thy1-EYFP mice has made possible studies of morphology, connectivity, electrophysiology, and mRNA content of a single neuron and has permitted long-term in vivo imaging of neurons (Feng et al., 2000, Micheva et al., 2010, Sugino et al., 2006). Further, by incorporating a strategy for combinatorial expression of fluorescent proteins, BrainBow mice have enabled the simultaneous mapping of projections and connectivity among multiple neurons (Livet et al., 2007). Given the wealth of information transgenic mice have yielded in past studies of neural circuits, it is not surprising that considerable efforts have been expended to establish lines in which the activity of populations of neurons can be both easily observed and reliably and reversibly manipulated.

One of the most exciting recent advances in experimental neuroscience has been the development of genetically encoded light-sensitive proteins, giving rise to the burgeoning field of optogenetics. In its broadest sense, optogenetic tools include both optical indicators of neuronal activity, such as genetically encoded calcium or voltage sensors, and optical actuators of neuronal activity, such as light-activated membrane channels and pumps. Although both types of tools are of intense interest to the neuroscience community, the latter group of molecules has been especially pursued given the opportunity they offer for being able to activate and inactivate particular neurons in live, behaving animals. Recent work incorporating three of these molecules, the neural-activating cation channel, channelrhodopsin-2 (ChR2) (Boyden et al., 2005, Nagel et al., 2003), the neural-silencing chloride transporter, halorhodopsin (NpHR) (Han and Boyden, 2007, Zhang et al., 2007), and the neural-silencing proton pump, archaerhodopsin (Arch) (Chow et al., 2010), has demonstrated the power of these tools to activate or silence neurons with unparalleled specificity and temporal precision on a millisecond scale. In addition, ChR2 has already been widely used in rodents to map circuits between defined neuronal populations.

Optogenetic actuators, such as ChR2, function by regulating the membrane potential of excitable cells. To generate sufficient membrane depolarization for light-induced action potentials, functional ChR2 protein must be expressed on the cell membrane at very high level or density due to the low single-channel conductance. In the past, such high-level expression has routinely been achieved using strategies that rely on delivering high copy numbers of transgene to cells, such as by in utero electroporation or viral infection. With a few notable exceptions, it has proven more difficult to obtain transgenic mice that express these genetic tools both robustly and widely enough to allow for probing the functionality of a wide range of cell types. For example, although the Thy1 promoter directed sufficient ChR2 expression to investigate the cortical and olfactory circuits (Arenkiel et al., 2007, Wang et al., 2007), this promoter is sensitive to inhibitory positional effects when randomly integrated into the genome, and it lacks ubiquitous neuronal expression. Clearly, to exploit the full potential of current and future optogenetic tools for elucidating neural circuitry, transgenic lines need to be developed that will allow for high-level transgene expression in any specific cell type of interest. Recently developed Cre-dependent reporter mouse lines with the ability to robustly express a variety of opsins proffer great promise to fulfill this need.

Optogenetic indicators, such as genetically encoded calcium indicators (GECIs) (also called fluorescent calcium indicator proteins) and voltage sensitive fluorescent proteins (VSFPs), have had a longer history of development than the optogenetic actuators. In particular, a variety of GECIs have been engineered, based on combinations of different types of calcium-binding proteins and fluorescent proteins (Mank and Griesbeck, 2008). Major advantages of using genetically encoded optical sensors over synthetic indicators to monitor cell activity include their suitability for long-term tracking of particular cells over time, as well as their ability to target specific cell types or populations. Although recent work has improved upon the sensitivity and stability of early generation optical indicators, current versions of both GECIs and VSFPs still require very high-level expression to present changes in relative fluorescence at a sufficiently high signal-to-noise ratio (SNR). Thus, there is a continued need to improve both the SNR and the sensitivity to subthreshold and single spike-induced changes in calcium or voltage. Similar to optogenetic actuators, genetically encoded optical indicators are most commonly delivered through viral or DNA plasmid transduction in functional studies. The most recent versions of these indicators, such as GCaMP3 (Tian et al., 2009), possess greatly improved properties over earlier iterations, making the transgenic approach feasible for their application. Indeed, promising mouse lines that express these molecules have been developed and are currently being characterized.

In the following sections, we will review common strategies for generating transgenic mice, discuss the significant progress that has been made over the past few years in developing transgenic lines that express optogenetic molecules to functional levels, and consider what improvements to current technologies are needed to allow transgenic lines to capitalize on the exceedingly powerful tools offered by optogenetics.

Section snippets

General transgenic approaches

There are two general strategies for expressing a transgene in a cell population-specific manner. The first is to express the transgene directly under a promoter that is active in only particular cell types. The second is to use a binary system, in which expression of the transgene is regulated by another “driver” gene, whose own expression is controlled by a specific promoter.

Transgenic expression of optogenetic tools

Successful application of optogenetic tools to in vivo studies requires very high-level expression of these genes in the cells whose activity is to be manipulated. For this reason, investigators have typically expressed optogenetic proteins by methods that deliver high copy numbers of transgenes to target cells, such as with recombinant viral vectors, or by in utero electroporation (Zhang et al., 2010). Although these strategies can effectively achieve sufficient transgene expression for

Future directions

Tremendous progress has been made toward the goal of creating functionally relevant transgenic mice for optogenetic studies. The characterization of early generation transgenic lines carrying light-activatable molecules has provided insight as to the prerequisites for an effective transgenic approach to applying these tools. Key among these is the requirement for targeted cells to be more light sensitive and to exhibit more rapid on/off kinetics. Increased light sensitivity of targeted cells

Acknowledgments

This work was funded by the Allen Institute for Brain Science and NIH grants (MH085500, DA028298) to H. Z. The authors wish to thank the Allen Institute founders, Paul G. Allen and Jody Allen, for their vision, encouragement, and support.

References (68)

  • C. Monetti et al.

    PhiC31 integrase facilitates genetic approaches combining multiple recombinases

    Methods (San Diego, Calif)

    (2011)
  • G. Nagel et al.

    Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses

    Current Biology

    (2005)
  • J. Ren et al.

    Habenula “cholinergic” neurons co-release glutamate and acetylcholine and activate postsynaptic neurons via distinct transmission modes

    Neuron

    (2011)
  • C.R. Yu et al.

    Spontaneous neural activity is required for the establishment and maintenance of the olfactory sensory map

    Neuron

    (2004)
  • H. Zong et al.

    Mosaic analysis with double markers in mice

    Cell

    (2005)
  • B.K. Andrasfalvy et al.

    Two-photon single-cell optogenetic control of neuronal activity by sculpted light

    Proceedings of the National Academy of Sciences of the United States of America

    (2010)
  • A. Berndt et al.

    High-efficiency channelrhodopsins for fast neuronal stimulation at low light levels

    Proceedings of the National Academy of Sciences of the United States of America

    (2011)
  • E.S. Boyden et al.

    Millisecond-timescale, genetically targeted optical control of neural activity

    Nature Neuroscience

    (2005)
  • E. Chaigneau et al.

    The relationship between blood flow and neuronal activity in the rodent olfactory bulb

    Journal of Neuroscience

    (2007)
  • J. Chen et al.

    Transgenic animals with inducible, targeted gene expression in brain

    Molecular Pharmacology

    (1998)
  • B.Y. Chow et al.

    High-performance genetically targetable optical neural silencing by light-driven proton pumps

    Nature

    (2010)
  • N. Chuhma et al.

    Functional connectome of the striatal medium spiny neuron

    Journal of Neuroscience

    (2011)
  • D. De Saint Jan et al.

    External tufted cells drive the output of olfactory bulb glomeruli

    Journal of Neuroscience

    (2009)
  • A.K. Dhawale et al.

    Non-redundant odor coding by sister mitral cells revealed by light addressable glomeruli in the mouse

    Nature Neuroscience

    (2010)
  • J. Diez-Garcia et al.

    Activation of cerebellar parallel fibers monitored in transgenic mice expressing a fluorescent Ca2 + indicator protein

    European Journal of Neuroscience

    (2005)
  • M.L. Fletcher et al.

    Optical imaging of postsynaptic odor representation in the glomerular layer of the mouse olfactory bulb

    Journal of Neurophysiology

    (2009)
  • S. Gong et al.

    Targeting Cre recombinase to specific neuron populations with bacterial artificial chromosome constructs

    Journal of Neuroscience

    (2007)
  • S. Gong et al.

    A gene expression atlas of the central nervous system based on bacterial artificial chromosomes

    Nature

    (2003)
  • V. Gradinaru et al.

    eNpHR: A Natronomonas halorhodopsin enhanced for optogenetic applications

    Brain Cell Biology

    (2008)
  • M. Hagglund et al.

    Activation of groups of excitatory neurons in the mammalian spinal cord or hindbrain evokes locomotion

    Nature Neuroscience

    (2010)
  • X. Han et al.

    Multiple-color optical activation, silencing, and desynchronization of neural activity, with single-spike temporal resolution

    PLoS One

    (2007)
  • M. Hara et al.

    Imaging endoplasmic reticulum calcium with a fluorescent biosensor in transgenic mice

    American Journal of Physiology

    (2004)
  • M.T. Hasan et al.

    Functional fluorescent Ca2 + indicator proteins in transgenic mice under TET control

    PLoS Biology

    (2004)
  • J. He et al.

    Encoding gender and individual information in the mouse vomeronasal organ

    Science (New York, NY)

    (2008)
  • Cited by (77)

    • Optimised induction of on-demand focal hippocampal and neocortical seizures by electrical stimulation

      2020, Journal of Neuroscience Methods
      Citation Excerpt :

      Other studies have also combined kindling with optogenetics in this manner to give rise to a gradual intensification of stimulus-induced seizures in mice over time (Wang et al., 2017; Berglind et al., 2018). Although such approaches can circumvent the limitation of electrical stimulation in identifying the specific neuronal networks responsible for focal epileptogenicity, establishing transgenic lines that express functionally relevant levels of optical actuators of neuronal activity through genetic manipulation can be laborious, costly and time-consuming (Zeng & Madisen, 2012). Moreover, rats offer several advantages over mice as a disease model for use in EIT experiments and other imaging studies aimed at investigating neural network dynamics in epilepsy due, for example, to their larger body size and thus ability to accommodate a greater number of electrodes (Anikeeva et al., 2011).

    • Functional interrogation of neural circuits with virally transmitted optogenetic tools

      2020, Journal of Neuroscience Methods
      Citation Excerpt :

      While opsin-expressing mouse lines have been extensively used, the cost and effort to maintain the lines led to the development of the more flexible conditional cre-loxP lines (Fig. 1). Those lines can be used for many applications, e.g. optogenetic or chemogenetic manipulations, tracing studies, or calcium imaging (Zeng and Madisen, 2012). In the last decade, the development of bacterial artificial chromosome (BAC) transgenic lines facilitated and sped up the development of cre-mice.

    • Light-Based Neuronal Activation: The Future of Cranial Nerve Stimulation

      2020, Otolaryngologic Clinics of North America
      Citation Excerpt :

      gene transfer technology to deliver opsin genes to target cells. Opsin-expressing transgenic murine lines are commercially available and readily generated.38 Although the transgenic approach is commonly used in the laboratory, it is not directly translatable to human studies.

    View all citing articles on Scopus
    View full text