In this presentation, Christina Gremel will cover why and when to use optogenetics to investigate brain function. Specifically, Gremel will:

• Identify the advantages and disadvantages of optogenetic techniques compared to other techniques, such as lesions, pharmacological manipulation, and chemogenetics, to manipulate brain function.

After watching this presentation, you should be able to gain a general understanding on what optogenetics offers when exploring neural bases of behavior.

Visit the Community forum for all eight modules to share your insights and best practices, ask questions, and engage with other training series’ participants.

Speaker

Christina Gremel, PhD

Christina Gremel is an assistant professor in the department of psychology and a member of the neurosciences graduate program at the University of California, San Diego. Her research focuses on the behavioral and neural bases of decision-making and their perturbations in diseases states, such as addiction. She earned her PhD from Oregon Health & Science University and did her postdoctoral training at NIH.

In this presentation, Vikaas Sohal will provide an overview of the “basic operating parameters” for optogenetic tools. Specifically, Sohal will:

• Describe how to consider opsin kinetics when designing light trains for stimulation.
• Describe how to validate the expression of optogenetic tools.
• Identify important considerations when combining optogenetic tools with each other or other optical methods.

After watching this presentation, you should be able to understand the critical considerations when selecting optogenetic tools.

Visit the Community forum for all eight modules to share your insights and best practices, ask questions, and engage with other training series’ participants.

Speaker

Vikaas S. Sohal, MD, PhD

Vikaas Sohal, MD, PhD, is a professor of psychiatry and behavioral sciences at the University of California, San Francisco. He directs a laboratory which studies emergent patterns of activity in limbic circuits and how they contribute to normal and pathological aspects of cognition and emotion. He received his bachelor’s and master’s degrees in applied mathematics from Harvard, a master’s degree in mathematics from the University of Cambridge, and an MD and PhD from Stanford. He also completed postdoctoral training during his psychiatry residency at Stanford, during which time he carried out some of the first experiments using optogenetics in the laboratory of Karl Deisseroth.

In this presentation, Mario Penzo will describe the various strategies commonly employed for the delivery of bacterial opsins to the nervous system. Specifically, Penzo will:

• Describe different opsin delivery methods, such as virus injection, electroporation, and use of transgenic mice.
• Identify, for a particular delivery strategy (adeno-associated virus vectors), the importance of the various serotypes available.

After watching this presentation, you should be able to understand the pros and cons of the different opsin delivery methods.

Visit the Community forum for all eight modules to share your insights and best practices, ask questions, and engage with other training series’ participants.

Speaker

Mario A. Penzo, PhD

Mario A. Penzo is the chief of the unit on the neurobiology of affective memory of NIH National Institute of Mental Health (NIMH). His group’s research aims to understand the neuronal mechanisms underlying the formation and regulation of affective memories. To do so, they combine various technical approaches, which include behavioral assays, electrophysiology, neuroanatomy, and optogenetics. Penzo obtained his PhD. at the Albert Einstein College of Medicine and completed his postdoctoral fellowship at Cold Spring Harbor Laboratory.

The articles below were selected by Christina Gremel, Vikaas Sohal, and Mario Penzo, faculty from Module 2 of SfN’s Optogenetics Training Series. These resources supplement their presentations, Implementing Optogenetics in the Lab: Why Use Optogenetic Methods, Selection of Opsins, and Delivery of Opsins.

Use these resources to understand when to choose optogenetics versus other approaches, pros and cons of different opsin delivery methods, and available sources of optogenetic tools.

Resources Related to Christina Gremel’s Presentation, Implementing Optogenetics in the Lab: Getting Started

Differential Corticostriatal Plasticity During Fast and Slow Motor Skill Learning in Mice

In this 2011 article, Costa, Cohen, and Nicolelis demonstrate putative single neuron recordings in mice during behavior.

Causal Role for the Subthalamic Nucleus in Interrupting Behavior

Fife et al. demonstrate the use of an inhibitory opsin to test the necessity of a population of cells for a given behavior.

Visualizing Hypothalamic Network Dynamics for Appetitive and Consummatory Behaviors

This 2015 report from Jennings et al. demonstrates the use of multiple methods, including chemogenetic and optogenetic activity tools, to examine the contribution of a specific cell-type to a given behavior.

Distinct Behavioral and Network Correlates of Two Interneuron Types in Prefrontal Cortex

Kvitsiani et al. reveal how optogenetic tagging can be used to examine the neural activity of an isolated cell type during a given behavior.

PINP: A New Method of Tagging Neuronal Populations for Identification During In Vivo Electrophysiological Recording

Lima et al. show how optogenetics can be used to tag neural activity in an identified neuronal population.

Self-Stimulation of the Brain

In this seminal paper from 1958, Olds reports using intra-cranial self-stimulation to identify a brain area that supports food seeking.

Fast-Spiking Interneurons Supply Feedforward Control of Bursting, Calcium, and Plasticity for Efficient Learning

Owen, Berke, and Kreitzer provide a demonstration of combining methods, including cell-type-specific ablation, optogenetics, electrophysiology, imaging, and behavior, to modulate one cell population while recording the activity of a connected cell population.

Whole-Cell Recording of Neuronal Membrane Potential During Behavior

In this 2017 review article, Peterson gives a comprehensive overview of in vivo whole cell recording methods.

Lateral Hypothalamic Circuits for Feeding and Reward

Stuber and Wise review what optogenetics offers in the probing of behavior compared to other techniques.

Resources Related to Vikaas Sohal’s Presentation, Selection of Opsins

Multimodal Fast Optical Interrogation of Neural Circuitry

This seminal 2007 paper from Zhang et al. presents halorhodopsin (NpHR), a complement to channelrhodopsin (ChR2), as a tool capable of generating a chloride flux when activated by light — thereby inducing rapid and reversible hyperpolarization.

Multiple-Color Optical Activation, Silencing, and Desynchronization of Neural Activity With Single-Spike Temporal Resolution

Han and Boyden further characterize the halorhodopsin/channelrhodopsin system for multichannel photoinhibition and photostimulation of virally or transgenically targeted neural circuits.

Parvalbumin Neurons and Gamma Rhythms Enhance Cortical Circuit Performance

In this 2009 article, Sohahl, Zhang, et al. use optogenetics to report evidence for the involvement of the specific activation of parvalbumin positive, fast-spiking interneurons in the production of cortical gamma oscillations.

High-Performance Genetically Targetable Optical Neural Silencing by Light-Driven Proton Pumps

Chow et al. describe archaerhodopsin-3 (Arch), a light-driven proton pump that mediates functional neuronal silencing.

Lateral Competition for Cortical Space by Layer-Specific Horizontal Circuits

Adesnick and Scanziani use a combination of optogenetics and electrophysiology to characterize the activity of horizontal projections between vertical domains of the mouse somatosensory cortex.

Ultrafast Optogenetic Control

In this 2010 technical report, Gunaydin et al. describe a novel engineered opsin gene, ChETA, that overcomes many of the technical limitations of channelrhodopsin and allows for sustained spike trains up to at least 200 Hz.

Neocortical Excitation/Inhibition Balance in Information Processing and Social Dysfunction

Yizhar and Fenno, et al. design and use several novel optogenetic tools, including stabilized step function opsins (SFOs), to test the hypothesis that abnormal circuit level physiology (via a cellular excitation/inhibition balance) underlies neuropsychiatric disease-related symptoms.

Principles for Applying Optogenetic Tools Derived From Direct Comparative Analysis of Microbial Opsins

Mattis et al. present a systematic comparison of microbial opsins — including depolarizing, ultrafast depolarizing, and hyperpolarizing tools — under matched, ex vivo experimental conditions. The authors identify key parameters for the conduct, design, and interpretation of experiments involving optogenetic techniques.

Independent Optical Excitation of Distinct Neural Populations

In this 2014 article, Klapoetke et al. describe two channelrhodopsins, Chronos and Chrimson, that enable two-color activation of independent neural populations without discernable cross-talk.

Projections From Neocortex Mediate Top-Down Control of Memory Retrieval

Rajasethupathy et al. employ a variety of optogenetic approaches to identify a projection from the prefrontal cortex to the hippocampus which, when selectively activated, elicits memory retrieval.

Structural Foundations of Optogenetics: Determinants of Channelrhodopsin Ion Selectivity

Berndt and colleagues identify structural determinants of channelrhodopsin ion selectivity and use their parameters to engineer chloride-conducting channels with higher chloride conductivity and selectivity and test their efficacy in vivo.

Resources Related to Mario Penzo’s Presentation, Delivery of Opsins

Sources of Optogenetic Tools: Viral Vectors

You can purchase a stock viral vector from academic or non-profit viral vector cores. Alternatively, these cores will also develop custom viral vectors from a plasmid that you provide.

• UNC Vector Core at the University of North Carolina at Chapel Hill
• Penn Vector Core at the University of Pennsylvania
• Addgene, the nonprofit plasmid repository
• PVM at IGMM (Platforme de Vectorologie de Montpellier at Institut Génétique Moléculaire Montpellier)
• Private companies can also custom-make viruses from a plasmid that you provide.

Viral Vector Types:

Viral vectors provide a powerful approach for transducing DNA into cells. Many factors must be considered when selecting the appropriate viral vector, including pathogenesis, target cell type, target expression levels, and the size of the genetic cargo.

The table below summarizes the primary factors to consider when making this selection for gene delivery. Table 1 is adapted with permission from AAV, Adenovirus and Lentivirus-Based Gene Delivery Handbook of Vigene Biosciences.

Adeno-Associated Virus Serotypes:

Adeno-associated viruses, or AAVs, are small, single-stranded DNA viruses that are non-pathogenic and can mediate long-term gene expression in vivo. Selecting the right serotype is critical for the success of gene delivery, as one must consider different tissue tropism, anterograde vs. retrograde expression, and other factors.

Table 2 below, adapted with permission from the AAV, Adenovirus and Lentivirus-Based Gene Delivery Handbook of Vigene Biosciences, lists different AAV serotypes often used in neuroscience experiments; commonly used AAVs are in red.

For more information on viral vectors, please consult:

• AAV guides, such as these from Addgene and Vigen Biosciences
• Retrograde AAV guides, such as from Addgene
• Canine adenovirus type 2( CAV2), an alternative to retrograde AAV, with more information available here

Commonly Used Promoters for Viral Vectors:

Promoters allow for selective expression in viral vectors in different tissues or different brain cells (neurons versus astrocytes) based on gene expression.

The table below, adapted with permission from the AAV, Adenovirus and Lentivirus-Based Gene Delivery Handbook of Vigene Biosciences, lists different promoters that are compatible AAVs and often used in neuroscience experiments.

Supporting References:

Engineered AAVs for Efficient Noninvasive Gene Delivery to the Central and Peripheral Nervous Systems

Chan et al., describe a cell type-specific capsid selection method, known as CREATE, was used to produce AAVs that allow gene delivery to the entire central and peripheral nervous systems, with multicolor labeling of single cells.

Non-Invasive, Focused Ultrasound-Facilitated Gene Delivery for Optogenetics

In this 2017 paper, Wang et al. present a noninvasive, ultrasound-based technique to introduce optogenetic channels into the brain by temporarily opening the blood-brain barrier.

Analysis of Transduction Efficiency, Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8, and 9 in the Mouse Brain

Here, Aschauer, Kreuz, and Rumpel provide a detailed and quantitative analysis of the transduction profiles of rAAV vectors based on six of the most commonly used serotypes (AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9) that allows systematic comparison and selection of the optimal vector for a specific application.

A Designer AAV Variant Permits Efficient Retrograde Access to Projection Neurons

Ternvo et al. describe the engineering of a novel AAV vector with potent retrograde functionality (rAAV2-retro) and the resulting retrograde access to projection neurons.

AAV-Mediated Anterograde Transsynaptic Tagging: Mapping Input-Defined Functional Neural Pathways for Defense Behavior

In this 2017 article, Zingg et al. report that adeno-associated virus (AAV1 and AAV9) exhibit anterograde transsynaptic spread properties and allow the tracing and functional manipulation of axonal projections from input-defined neuronal populations.

Nucleus Accumbens D2R Cells Signal Prior Outcomes and Control Risky Decision-Making

Zalocusky et al. report how using a D2 specific promoter enables genetic access to D2R-expressing cells in the rat striatum to exert optogenetic control during decision-making and reveal a causal effect of these cells in driving risk-preference.

Optogenetic Astrocyte Activation Modulates Response Selectivity of Visual Cortex Neurons In Vivo

Perea et al. provide an illustrative example of using astrocyte-specific promoter to drive channelrhodopsin-2 expression. The authors report selective photostimulation of astrocytes with channelrhodopsin-2 in primary visual cortex enhances excitatory and inhibitory synaptic transmission, and influences neuronal integrative features critical for sensory information processing.

A Direct GABAergic Output From the Basal Ganglia to Frontal Cortex

This 2015 paper from Saunders et al. provides an example of using a transgenic mouse line to drive channelrhodopsin-2 expression. The authors use this approach to identify a direct, GABAergic projection from the basal ganglia to frontal regions of the cerebral cortex. 

Experience-Dependent Modification of a Central Amygdala Fear Circuit

In this 2013 article, Li, Penzo et al. use a combination of viral vector transduction with a transgenic, Cre-expressing mouse line to express channelrhodopsin-2 or archaerhodopsin and demonstrate robust fear conditioning-induced synaptic plasticity in the central amygdala.

Visit the Community forum for all eight modules to share your insights and best practices, ask questions, and engage with other training series’ participants.

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