PI: Hongkui Zeng, Allen Brain Atlases
Allen Institute for Brain Science
Title: “Establishing a Comprehensive and Standardized Cell Type Characterization Platform”
BRAIN Category: Census of Cell Types (RFA MH-14-215)
Dr. Zeng’s group will characterize cell types in brain circuits controlling sensations, such as vision and emotions, as a first step to better understand information processing across circuits. The data generated will be posted as a public online resource for the scientific community.
The brain circuit is an intricately interconnected network of a vast number of neurons with diverse molecular, anatomical and physiological properties. Neuronal cell types are fundamental building blocks of neural circuits. To understand the principles of information processing in the brain circuit, it is essential to have a systematic understanding of the common and unique properties for each of its components – the cell types, how they are connected to each other, and what are their functions in the circuit. From the study of numerous circuits, many types of mechanisms have been proposed regarding the roles of different cell types in signal processing. However, despite of the importance, we are far from a comprehensive understanding of the number and kinds of cell types in the brain or a given circuit. We do have a wealth of knowledge on the major cell types in each region, and many examples of specific types. But for the most part, due to the lack of systematic efforts, we don’t know the complete cell type composition of most circuits, and we have very little idea about the degree of variation and heterogeneity among single cells, both within a given type and between different types. To address this issue, we propose to establish a comprehensive and standardized cell type characterization platform that can be scaled up to systematically examine the properties and function of cell type components in any neural circuits throughout the brain. To implement this, we propose a model for collaboration between academic labs/centers and Allen Institute for characterizing cell types in specific brain circuits, with all the QC-passed data going into the Allen Institute Cell Types Database and becoming publicly available. We will test a range of experimental approaches, encompassing molecular, anatomical and physiological measurements and their integration at the single cell level. Our proof of principle studies are based on comparison of three major brain neural circuits in the mouse brain: two closely related cortical circuits – primary visual cortex (V1) and primary somatosensory cortex (S1), and a more distinct circuit – the hypothalamus/amygdala emotional pathway. These two axes of comparison should be very informative in assessing the reliability and generality of the cell type characterization approaches we will be testing. We thereby hope to determine the critical parameters and metrics necessary to classify neurons into discrete cell types, guided by their functions. Thus, we anticipate that this project and the resources it produces will have a broad impact and catalytic effect on the scientific community studying brain circuitry function and dysfunction.
NIH Spending Category
Mental Health; Neurosciences
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Public Health Relevance Statement
The brain circuit is an intricately interconnected network of a vast number of neurons with diverse molecular, anatomical and physiological properties, and many mental health disorders can be attributed to malfunction of brain circuitry. To understand the principles of information processing in the brain circuit, we propose to establish a comprehensive and standardized cell type characterization platform that can be scaled up to systematically examine the common and unique properties and function of cell type components in any neural circuit throughout the brain. Creating a publicly accessible cell types database will have broad impact on the study of brain circuitry as a foundation to further our understanding of brain function and brain diseases.
Allen Institute News 3/4/15
One of the most powerful ways to learn about the brain is to target specific types of cells and learn how their genes and behaviors contribute to the function of circuits. Scientists at the Allen Institute are working to catalog the thousands of different types of cells in the brain, often using techniques that manipulate the genes of a small subset of cells so that the cells glow under fluorescent microscopes.
“While there are thousands of types of cells, the tools we have are often not specific enough to identify just one cell type,” explains Hongkui Zeng, Senior Director of Research Science at the Allen Institute. “If you want to find a few needles in a haystack, you can hold a magnet over the pile and pull out just the metallic pieces. In the case of neurons, we need to first make them stand out enough to be picked up by our microscopes. We do this by manipulating unique gene markers for each cell type into fluorescent labels or probes, so that the structure and function of this cell type can be visualized and studied.”
As Zeng and her team report in the latest issue of Neuron, scientists at the Allen Institute have created an exciting and expansive new set of tools, including genetically altered mouse lines and viruses that can activate targeted genes, which make it much more feasible for scientists to study many new types of cells. They accomplished this in part by creating a system that relies on manipulating not just one, but two or more genes at a time, in order to increase the specificity of the types of cells studied.
“We want to understand the neural code, that is, how the brain encodes information about the external world,” explains Aleena Garner, scientist at the Allen Institute, “so having methods that let us monitor the activities of many different types of cells in a consistent manner is key to studying the roles of these cells in processing information and driving the animal’s behavior.”
Another exciting facet of these new tools, beyond making certain cell types fluorescently glow, is the ability to use light to make the cells actually fire a signal, using a technique called optogenetics.
“The combination of fluorescent imaging and optogenetic stimulation is a powerful way to learn both where cells are in space, when they are active or silent, and how they interact with other cells in the circuits they form,” says Zeng.” The new tools we have created open doors to identifying and learning more about the many different types of cells in our brains: the first crucial step to understanding how information is encoded by neural circuits.”
The work reported in the Neuron article is a result of collaborations between Allen Institute scientists and researchers at University College London, MIT, Imperial College London, University of Otago, University of Zurich, RIKEN Brain Science Institute, and Mount Sinai Hospital.
Transgenic Mice for Intersectional Targeting of Neural Sensors and Effectors with High Specificity and Performance
Article in Neuron 3/4/15
by Linda Madisen1, Aleena R. Garner1, Daisuke Shimaoka2, Amy S. Chuong3, Nathan C. Klapoetke3, Lu Li1, Alexander van der Bourg4, Yusuke Niino5, Ladan Egolf4, Claudio Monetti6, Hong Gu1, Maya Mills1, Adrian Cheng1, Bosiljka Tasic1, Thuc Nghi Nguyen1, Susan M. Sunkin1, Andrea Benucci2, 5, Andras Nagy6, Atsushi Miyawaki5, Fritjof Helmchen4, Ruth M. Empson7, Thomas Knöpfel8, Edward S. Boyden3, R. Clay Reid1, Matteo Carandini2, Hongkui Zeng1
- Evaluated multiple mouse transgenic intersectional strategies
- Established TIGRE locus as a novel permissive docking site for transgene expression
- Developed 21 new intersectional driver and reporter lines expressing novel tools
- Demonstrated functionality of new voltage or calcium sensing or optogenetic lines
An increasingly powerful approach for studying brain circuits relies on targeting genetically encoded sensors and effectors to specific cell types. However, current approaches for this are still limited in functionality and specificity. Here we utilize several intersectional strategies to generate multiple transgenic mouse lines expressing high levels of novel genetic tools with high specificity. We developed driver and double reporter mouse lines and viral vectors using the Cre/Flp and Cre/Dre double recombinase systems and established a new, retargetable genomic locus, TIGRE, which allowed the generation of a large set of Cre/tTA-dependent reporter lines expressing fluorescent proteins, genetically encoded calcium, voltage, or glutamate indicators, and optogenetic effectors, all at substantially higher levels than before. High functionality was shown in example mouse lines for GCaMP6, YCX2.60, VSFP Butterfly 1.2, and Jaws. These novel transgenic lines greatly expand the ability to monitor and manipulate neuronal activities with increased specificity.
The brain comprises a large number of neuronal and non-neuronal cell types, whose connections and interactions are fundamental to its function. To observe and manipulate their activities selectively, the best available approach is genetic targeting of protein-based sensors and effectors to specific cell types (Huang and Zeng, 2013). In mice, the Cre/lox recombination system is the most widely used approach to access specific cell types, utilizing gene promoters or loci with specific expression patterns (Gerfen et al., 2013,Gong et al., 2007, Madisen et al., 2010 and Taniguchi et al., 2011). However, cell populations defined by Cre driver lines are often heterogeneous, encompassing multiple brain regions and/or multiple cell types (Harris et al., 2014). Fundamentally, cell types are rarely defined by single genes, but rather by intersectional expression of multiple genes. Thus, it is imperative to develop intersectional genetic targeting approaches, combining regulatory elements from two or more genes to increase specificity of transgene expression. Important efforts have been made to develop transgenic intersectional approaches, most successfully with the combination of Cre and Flp site-specific recombinases (SSRs) (Dymecki and Kim, 2007, Dymecki et al., 2010, Kranz et al., 2010, Ray et al., 2011 and Robertson et al., 2013). However, thus far, intersectional approaches have not been widely used in functional studies, due to the limited number of validated transgenic tools available.
The ongoing development of increasingly effective sensors and effectors offers extraordinary opportunities for studies of neuronal interactions and functions (Fenno et al., 2011, Huang and Zeng, 2013 and Knöpfel, 2012). One issue with the practical utility of these tools is that they require high-level expression in cell populations of interest. Such high levels of expression can be obtained with techniques that result in high transgene copy numbers in individual cells, such as in utero electroporation and adeno-associated virus (AAV) infection. However, these approaches have limitations, including invasive surgical delivery, incomplete coverage of the desired cell population, variable levels of expression in different cells, and, in the case of viruses, potential cytotoxicity associated with long-term viral infection and/or uncontrolled gene expression.
Transgenic mouse lines that express high and heritable patterns of genetic tools in specific cell populations provide an alternative that can overcome at least some of these limitations (Zeng and Madisen, 2012 and Zhao et al., 2011). We previously establish a standardized Cre-reporter system in which transgene expression was driven by a strong, ubiquitous CAG promoter targeted to the Rosa26 locus (Madisen et al., 2010 and Muzumdar et al., 2007), expressing fluorescent proteins, calcium sensor GCaMP3, and optogenetic activator ChR2(H134R) and silencers Arch and eNpHR3.0 (Madisen et al., 2010, Madisen et al., 2012 and Zariwala et al., 2012). Although proved useful in many applications (Ackman et al., 2012, Haddad et al., 2013, Issa et al., 2014, Jackman et al., 2014, Kheirbek et al., 2013, Lee et al., 2014, Nguyen-Vu et al., 2013 and Pi et al., 2013), we and others have also identified limitations in the sensitivity or functionality of these reporters in other situations.
Currently the only transgenic mouse approach demonstrated to reliably achieve AAV-like high-level expression is the use of the Thy1.2 promoter in randomly integrated transgenes (Arenkiel et al., 2007,Dana et al., 2014, Feng et al., 2000 and Zhao et al., 2008), presumably with multiple copies at the insertion site. Although powerful in driving tool gene expression, this approach also has drawbacks. Expression of transgenes driven by the Thy1.2 promoter is strongly position dependent, necessitating a screen of multiple founder lines to find potentially useful ones. Furthermore, adding Cre-dependent control, e.g., a floxed-stop cassette, to a multi-copy transgene is problematic because Cre could then induce recombination both within and between the different copies, resulting in reduced transgene copy numbers and variability of transgene expression among different cells.
We therefore undertook a systematic evaluation of multiple approaches aiming at more specific and more robust transgene expression. To complement existing Cre driver lines, we focused on the intersection of Cre with another recombinase or with a transcriptional activator. In addition, we built and validated a new docking site in a permissive genomic locus, the TIGRE locus (Zeng et al., 2008), which supports repeated targeting. By introducing a tTA-based transcriptional amplification approach to the TIGRE locus, all reporter lines doubly regulated by Cre and tTA drove robust expression of sensors and effectors at levels substantially higher than those in comparable Rosa-CAG-based reporters. Functional characterization of lines carrying representative optical tools under Cre and tTA control demonstrates their enhanced efficiency for studies of neuronal activity, both in vitro and in vivo.
In our effort to improve upon current strategies for cell-type-specific transgene expression, we explored three strategies for intersectional control: (1) reporter expression that depends on two independent SSRs from the ubiquitous Rosa26 locus, (2) Cre-dependent reporter expression from an endogenous locus targeted because of its cell-type-specific expression pattern, (3) reporter expression dependent on Cre and the transcriptional transactivator tTA from another ubiquitous genomic locus, TIGRE. Since the third strategy resulted in the most strongly enhanced transgene expression, we created a series of TIGRE reporter lines that show high-level expression of novel calcium, voltage, and glutamate sensors and optogenetic effectors. The complete list of new intersectional transgenic mouse lines and AAVs introduced in this paper (17 reporter lines, 4 driver lines, and 10 AAVs) is shown in Table 1.