In this presentation, Patrick Rothwell will cover why and when to use optogenetics in acute brain slices, also called ex vivo. Specifically, Rothwell will:

• Identify scientific questions that can be addressed using optogenetics in ex vivo preparations.
• Compare optogenetics to alternative ex vivo methodologies.

After watching this presentation, you should be able to better understand applications of optogenetic approaches in ex vivo preparations.

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

Patrick Rothwell, PhD

Patrick Rothwell is an assistant professor and MnDRIVE Neuromodulation Scholar in the department of neuroscience at the University of Minnesota Medical School. His lab studies neural circuit function in the striatum and basal ganglia, including modulation of these circuits by endogenous and exogenous opioids, and circuit dysfunction in brain conditions like addiction and autism spectrum disorders. Rothwell received his PhD in neuroscience from the University of Minnesota and completed postdoctoral training at Stanford University.

In this presentation, Stephanie Cragg and Yevgenia Kozorovitskiy will cover the basics of using optogenetics in acute brain slices, also called ex vivo. Specifically, they will:

• Identify key concepts in the design and interpretation of ex vivo optogenetic experiments.
• Describe critical parts and equipment for ex vivo optogenetics experiments.

After watching this presentation, you should be able to understand the basic setup and key concepts for ex vivo optogenetics experiments.

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

Speakers

Stephanie Cragg, DPhil

Stephanie Cragg is a professor of neuroscience in the department of physiology, anatomy, and genetics, at the University of Oxford and a co-founder of the Oxford Parkinson’s Disease Center. Her research focuses on understanding the control of dopamine neurotransmission, interactions with related circuits, and their dysfunction in disease. Craigg earned her PhD (DPhil) in neuropharmacology from the University of Oxford and undertook further training at Oxford and New York University.

Yevgenia Kozorovitsky, PhD

Yevgenia Kozorovitskiy is an assistant professor in the department of neurobiology at Northwestern University. Her laboratory focuses on neuromodulatory systems at the level of neural circuit development, function, and plasticity. Kozorovitskiy earned her PhD in neuroscience and psychology at Princeton University in the laboratory of Elizabeth Gould, and she carried out postdoctoral research at Harvard Medical School/HHMI with Bernardo Sabatini as a junior fellow of the Harvard Society of Fellows.

The resources below were selected by Patrick Rothwell, Stephanie Craigg, and Yevgenia Kozorovitsky, faculty from Module 3 of SfN’s Optogenetics Training Series. These resources supplement their presentations, Optogenetic Applications in Acute Brain Slices and Nuts and Bolts of Ex Vivo Optogenetics Experiments.

Use these resources to better understand appropriate applications and key concepts of designing and interpreting optogenetic experiments using ex vivo preparations.

The Optogenetics Wiki

This platform covers choice of opsins, opsin expression, and hardware with a supporting bibliography.

Gating of Dopamine Transmission by Calcium and Axonal N‐, Q‐, T‐ and L‐Type Voltage‐Gated Calcium Channels Differs Between Striatal Domains

In this 2015 report, Brimblecombe et al. use optogenetics for selective dopamine axon activation to help localize L-type calcium channels to dopamine axons.

Coupling Voltammetry With Optogenetics to Reveal Axonal Control of Dopamine Transmission by Striatal Acetylcholine

This book chapter by Kosillo et al. describes how to combine optogenetics with voltammetry in slices.

Neuromodulation of Excitatory Synaptogenesis in Striatal Development

Kozorovitskiy et al. use optogenetics with a transgenic cross to carry out in vivo induction of plasticity, and ex vivo readout of corticostriatal connectivity.

Subcellular Connectivity Underlies Pathway-Specific Signaling in the Nucleus Accumbens

This 2012 paper from MacAskill et al. uses optogenetics with physiology and two-photon imaging to examine different glutamatergic projections to the nucleus accumbens.

Input- and Output-Specific Regulation of Serial Order Performance by Corticostriatal Circuits

Rothwell et al. use optogenetics ex vivo to identify plasticity at specific corticostriatal synapses evoked by behavioral training.

Principles of Synaptic Organization of GABAergic Interneurons in the Striatum

In this 2016 report, Straub et al. use optogenetics ex vivo to identify and characterize synapses formed by rare types of striatal interneurons.

Glutamatergic Signaling by Mesolimbic Dopamine Neurons in the Nucleus Accumbens

Tecuapetla et al. use optogenetics ex vivo to characterize co-release of glutamate by mesolimbic dopamine neurons.

Striatal Dopamine Release Is Triggered by Synchronized Activity in Cholinergic Interneurons

This 2012 study by Threlfell et al. uses optogenetics with viral delivery to show that targeted activation of striatal cholinergic interneurons directly drives dopamine release.

Dopaminergic Neurons Inhibit Striatal Output through Non-Canonical Release of GABA

Tritsch, Ding, and Sabatini use optogenetics ex vivo to show that dopaminergic neurons also co-release GABA via an unconventional mechanism.

Biased Oxytocinergic Modulation of Midbrain Dopamine Systems

Xiao et al. use optogenetics with electrophysiology and anatomical assays to structurally and functionally define oxytocinergc projections to dopamine neurons, reporting opposing effects of oxytocin on tonic activity in the ventral tegmental area and substantia nigra pars compacta.

Equipment List

Explore these examples of components for optogenetics stimulation using LED illumination and a simple control circuit.

• LED Optical Path:
  • High power LEDs
  • Optical diffuser
  • Planoconvex lens: consider uncoated or coated for 350-700 nm light, and focal length 100-400 mm depending on space constraints around microscope
  • Dichromatic mirror: size depends on microscope constraints
• LED Control:
  • Resistors, trim pot
  • Linear current regulator
  • NPN bipolar transistor: for TTL pulse switch
  • Also required are a power supply, solderless breadboard, grounding, and connections wires. Single strand wire is used for the breadboard.

A more complex, flexible, and powerful open-source LED driver, called Cyclops, has been developed in Steve Potter’s laboratory at Georgia Tech University. This 2013 paper from Tchumatchenko, Newman, et al., Delivery of Continuously-Varying Stimuli Using Channelrhodopsin-2, describes Cyclops in more detail.

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

Complete this short survey to provide valuable feedback about Module 3 to SfN and the series faculty.