optogenetics and two photon microscopy

What is optogenetics?

The ability to use light to manipulate cell-type specific activity in the brain by turning cells on and off with light

What is two-photon imaging?

The ability to use light-based microscopy to monitor hundreds to thousands of neurons, useful for telling what population of cells are doing to explain things like rate codes

What was Francis Crick's challenge in neuroscience?

Need to control one type of cell in the brain while leaving others unaltered. Electrodes cannot precisely target defined cells and drugs act too slowly. He speculated light might have the properties to serve as control

Why are rhodopsins important for optogenetics?

Almost all major groups of organisms from algae to humans express rhodopsins - proteins that sense and respond to light. They contain retinal chloroform that changes when hit by light

How do rhodopsins work?

They are membrane proteins containing retinal chloroform that undergo conformational change when hit by light. This change can either directly move ions in or trigger a signaling cascade

What are the two broad classes of opsins?

Microbial opsins (found in bacteria, algae, fungi) and G-protein coupled opsins (involved in vision and circadian rhythms)

What are the key characteristics of microbial opsins?

Found in bacteria, algae, fungi. Directly gate ions across the membrane. Act as light driven ion channels or pumps. Don't need second messengers. Fast, single component proteins

What is phototaxis and how is it related to microbial opsins?

Phototaxis is moving toward or away from a light source. Algae use microbial opsins to control phototaxis

Where do channelrhodopsins come from?

Harvested from green algae ponds

Where do halorhodopsins come from?

Come from red fields (red archaean)

Why are microbial opsins optimal for optogenetics?

They are fast, reliable, express with a single gene, and directly control the current or voltage of the cell

What are the characteristics of G-protein coupled opsins?

Don't conduct ions directly. Activate G-protein signaling cascades, thus indirectly alter membrane potential. Involved in vision (rods and cones) and circadian rhythms. Much slower than microbial opsins

What does channelrhodopsin (ChR) do?

Light opens up a cation channel causing depolarization. Very fast depolarization occurs, which can trigger neuronal firing. This was the moment excitatory optogenetics was born

What was the first proof of concept for optogenetics?

Channelrhodopsin expressed in mammalian neurons showed that light alone could trigger neuronal firing

What does halorhodopsin do?

Light activated chloride pump that moves chloride into the cell, which hyperpolarizes the cell and inhibits neural firing

What is the functional relationship between channelrhodopsin and halorhodopsin?

Two tools that are functionally opposite: channelrhodopsin allows rapid and reversible induction of spiking by depolarization, while halorhodopsin suppresses spiking (inhibition) by hyperpolarization

What type of ion channel is channelrhodopsin?

Light-gated, non-selective cation channel that allows sodium, potassium, calcium, and any positively charged ions to flow through

What wavelength of light activates channelrhodopsin?

Blue light (sensitive to blue light)

What is the main ion driving current in channelrhodopsin and what does it cause?

Sodium is the main current driver, causing fast depolarization. The channel closes in 10-20ms

What is the temporal precision advantage of channelrhodopsin?

Not only able to turn cells on, but can ensure activation lasts for a very precise amount of time (10-20ms), which was almost impossible to do without optogenetics

What wavelength activates halorhodopsin (NpHR)?

Yellow light around 560 nm

How does halorhodopsin cause inhibition?

It's a light-driven chloride pump that actively moves chloride ions into the cell. Inward chloride movement drives hyperpolarization and suppresses spiking

What is the reversibility feature of halorhodopsin?

Inhibition stops immediately when you turn the light off, making it rapid and reversible inhibition

How can channelrhodopsin and halorhodopsin be used together?

Using two different colors of light (blue for ChR excitation, yellow for NpHR inhibition), you can potentially toggle cells to be on or off in the same animal

What is archaerhodopsin and what does it do?

An inhibitory opsin that is a light-driven outward proton pump. It pumps protons out of the cell, hyperpolarizes the neuron, and leads to inhibition/suppression of spiking

Why is archaerhodopsin considered better than halorhodopsin?

It prevents chloride from building up in the cell, has better stability and sustained hyperpolarization, and is a second generation inhibitory tool

What is the significance of having channelrhodopsin, halorhodopsin, and archaerhodopsin?

These three proteins have three different wavelengths and allow for almost complete optical control of a neural signal

What is the main limitation of electrical stimulation in neuroscience?

Anything in vicinity of electrode gets activated - can't selectively activate specific cell types (e.g., only excitatory vs inhibitory). Lacks cell-type specificity

What is the main deep value of optogenetics?

Cell type specification - the ability to achieve both excitation and inhibition of specific cell types

How does optogenetic excitation overcome electrical stimulation limitations?

Can molecularly identify specific neurons (e.g., interneurons) and introduce channelrhodopsin only to that neuron type, allowing selective activation with blue light

Describe the hypothalamus and aggression optogenetics experiment

Scientists found that the ventral medial hypothalamus (ventral lateral division) drives aggression. Using channelrhodopsin in VMHLD neurons (which project to PAG), optical fiber was implanted deep in brain. Light activation led mouse to attack another mouse or inflated glove

What motivated behavior was shown in the hypothalamus aggression study?

When activating the particular class of neurons with light, the mouse would push a lever to bring out a submissive mouse, showing motivation to attack circuit

What happens with temporary inactivation of a brain region?

If you need that region, performance goes down showing it contributes acutely to behavior. However, this doesn't definitively tell you if it's permanently required

What can happen with permanent lesion of a non-critical region?

Initial decline in behavior, but over time performance can recover as other regions compensate and the system learns to reorganize

What happens with permanent lesion of a critical region?

No matter how long you wait, performance will always be poor because the region is so important

What is the key lesson about determining brain region function?

Can't just get rid of a region to determine what it does - different time courses and plasticity affect causality. Use transient inactivation to understand acute usage and permanent lesions to understand true necessity

How is optogenetics implemented using gene therapy?

Package channelrhodopsins and halorhodopsins into a viral vector (tool to transport genome sequences into living cells), wait two weeks until opsin is expressing, then implant optical fibers or wireless stimulation to shine light with high precision

What is an alternative to viral vectors for optogenetics?

Transgenic lines, which cuts out the viral injection step

What was the question in the auditory cortex optogenetics case study?

What/when is the role of auditory cortex in frequency discrimination task, especially given that plasticity leads to expansion of representation for important tones

What did lesion studies show about auditory cortex and complex frequency modulated sweeps?

When auditory cortex (AC) was bilaterally removed, gerbils learned a little bit but never got really good at complex sweeps, showing AC is needed for this task

What did lesion studies show about auditory cortex and pure tone discrimination?

Animals were good at pure-tone frequency discrimination even with AC lesions, showing they don't need AC for this plasticity

What is the limitation of lesion studies in the auditory cortex?

Lesion studies address whether a behavior can be learned in the absence of sensory cortex, NOT whether the sensory cortex is actually used or optimal for learning

Describe the auditory GO-NO-GO task used in AC optogenetics study

Head-fixed mouse with optical beam. If mouse licks correctly to the correct tone, it breaks the beam and gets water droplet. Mouse tries not to lick to wrong tone

How was the auditory cortex silenced in the optogenetics study?

Placed channelrhodopsin into inhibitory neurons. Shining light into interneurons which transmit GABA causes local network to not respond. This is more efficient than halorhodopsins because activating inhibitory interneurons shuts down excitatory neurons

What did silencing AC during learning show?

Mouse normally goes from chance to very accurate within first few days. When AC was inactivated, they learned much more slowly, showing AC is absolutely being used during learning

What is the default pathway for learning task contingencies?

The auditory cortex (AC) is part of the default pathway for learning task contingencies

What did temporal precision optogenetics reveal about AC's role over time?

If AC is inhibited early in learning, performance is severely impaired. But if inhibited later on, mouse gets better at task even without AC. AC is used during learning but at end of learning it is no longer needed

Where does learning consolidate after initial AC involvement?

After learning, information consolidates into some subcortical area. Cortex is expensive real estate, so after learning consolidates it somewhere else - AC tutors into subcortical areas

What are the advantages of extracellular recording?

Electrode sits outside neurons and can record from dozens of neurons at the same time

What are the advantages of intracellular recording?

Electrode/patch pipette goes into neuron to directly record electrical signals with high temporal resolution. Can record dozens of neurons extracellularly with precise targeting in specific regions

What are the limitations of electrophysiology?

Little to no microscopic spatial resolution (hard to figure out precise spatial arrangement). Place codes are hard to determine. Challenging to dissociate different cell types (can only use waveform to classify excitatory vs inhibitory). Challenging to record more than dozens simultaneously. Difficult to monitor same cell over days to weeks

What three components enable optical measurement of neural activity?

Optical tools (microscopes) + neural activity-dependent fluorescence (use changes of light to understand what neurons are doing) + techniques to get sensors into brain

What is fluorescence?

Fluorescent molecules absorb light and then emit it at a longer wavelength

How does one-photon fluorescence work?

Single photon of high energy excites molecule from ground to excited state, then emits lower energy, longer wavelength fluorescent photon

What is the main challenge of one-photon fluorescence?

A lot of light scattering

How does two-photon fluorescence work?

Take two lower energy photons that arrive at the same time (femtosecond timing). Together they provide same energy as blue light. Two infrared photons do what one blue photon could do, but only in one tiny spot

What are the advantages of two-photon fluorescence?

Get deeper image, less photo damage, less out of focus fluorescence. Also called intrinsic optical sectioning. Remedies the challenge of one-photon fluorescence

Who discovered the two-photon effect and when?

Maria Goppert Meyer discovered the two-photon effect in 1931

What are two major purposes of two-photon imaging?

1) Get high resolution structural image of tissue and blood flow with fluorescent tracers, 2) Image activity of neurons in vivo through calcium imaging

Why is calcium a good target for imaging neural activity?

External [Ca] ~2mM, internal [Ca] ~50nM - a 40,000 fold difference with >100-fold change during activity. Ca signals are comparatively easy to detect because relative changes are huge

Compare calcium, sodium, and potassium concentration gradients

Ca: 40,000-fold difference (easiest to detect). Na: 9-fold difference (external>internal, needs sensitive indicator). K: 20-fold difference (internal>external, needs sensitive indicator)

How do calcium indicators work?

Excited by specific wavelength of light. Can be genetically encoded in viruses/transgenic animals or as dye. Work by emitting more light when bound to calcium

Who developed the first really useful calcium indicator and when?

Roger Tsien in 1980 (Nobel Prize winner)

What are the two main components of synthetic calcium sensors (Fura-2, OGB)?

1) Ca2+ chelating site, 2) Fluorophore that emits light (dependent on whether bound to calcium)

How does fluorescence report calcium concentration in synthetic sensors?

Fluorescence intensity or wavelength can report calcium concentration. When calcium binds, the molecule fluoresces differently - like a molecular switch. Excite 350-380 nm, measure 505-520 nm, ratio of two signals reflects intracellular Ca2+

How were synthetic calcium indicators originally delivered to neurons?

Take patch pipette, find neuron, suck off membrane and pass indicator in

How are synthetic calcium indicators delivered to neurons now?

Attach acetyl methyl ester on it, which allows it to pass over and through membranes

How do genetically encoded calcium indicators (GECI) work?

Introduce fluorophore that is genetically encoded. Take GFP molecule with two domains (M13 and calmodulin that's sensitive to calcium). When calcium binds, conformational change changes brightness of molecule

How well do calcium indicators report action potentials?

Brightness of neuron is proportional to how many action potentials occur - you get a response to calcium every time there is a spike

What are the pros of 2-photon calcium imaging vs electrophysiology?

Image many single cells simultaneously (see spatial layout). Identify cells by projections, genetic identity, protein expression. Easier to record from dendritic spines, axons, dendrites (get lots of data from one animal)

What are the cons of 2-photon calcium imaging vs electrophysiology?

It's slow (calcium signaling slower than voltage changes, takes time for calcium to bind and indicator to change fluorescence). It's indirect - although highly correlated with cell activity, not the same exact thing

What was being studied in the two-photon calcium imaging auditory system case study?

Functional mapping of cholinergic and cortical activity with two-color, two-photon imaging. Role of nucleus basalis (NB) as it sends projections to auditory cortex (AC)

What are the two auditory pathways to AC?

Primary: ventral division of MGB sends input to AC. Secondary (for behaviorally salient sounds): medial division of MGN to amygdala to nucleus basalis back to AC

What was the research question about nucleus basalis projections?

Don't know whether NB projection axons themselves have native auditory response or only respond to behaviorally relevant stimuli

How was two-color, two-photon imaging used in the NB/AC study?

Put red fluorescent (R-gecko) into AC neurons. Green fluorescence in NB axons. Little flash every time sound stimulus occurred

What did the NB/AC two-photon imaging study reveal?

NB axons respond to sound. Heterogeneity in responses. Axons are not tonotopically organized, but neurons in AC are organized via characteristic frequency. Auditory frequencies in NB being consistent in neurons allows them to be recruited by AC to learn from emotionally salient stimuli