Shedding light on brain function:

Optogenetics and beyond

Electrical and chemical signals in neuroscience

Action potential

Input
Chemical synaptic
Retrograde transmission
transmission

Dendritic Synaptic
backpropagation
potential

Action potential

Output
 Electrical and chemical reporters in neuroscience

Action potential

Input
Voltage sensors
Chemical synaptic
Retrograde transmission
transmission

Chemical sensors
Dendritic Synaptic
backpropagation
potential

Synthetic reporters Action potential

Genetically-encoded reporters
DNA –> RNA –> protein Output
 actuators and reporters/sensors

Electrical and chemical actuators in neuroscience

Action potential

Input
Voltage actuators
Chemical synaptic
Retrograde transmission
transmission
Stimulating
Silencing
Chemical actuators Dendritic Synaptic
backpropagation
potential
Stimulating
Silencing Action potential

Genetically-encoded actuators
DNA –> RNA –> protein Output
 Optogenetics

• Definition:
Experimental use of light-responsive proteins
(“opto-”) encoded in DNA (“-genetic”).
(Miesenböck, 2009).
 Optogenetic tools

• Reporters (sensors)
– Membrane potential
– Ca2+
– Synaptic activity
• Effectors (actuators)
– Excitation (activators)
– Inhibition (silencers)
– Second messenger cascades
The growth of optogenetics

upshoot in 2005 when

channelrhodopsin was first
expressed in neurons.

Deisseroth, 2011
 causal relations between neuronal and behavioural science.

Causal neuroscience

• Recording
– E-phys
imaging more replacing
– Imaging elecrtophysiology

• Stimulation
– Electrical
– Optical
• Silencing Knife cut

– Lesions
– Optical
more precise activation/silencing
Rersible activation and silencing.
 Optogenetic tools

• Reporters (sensors)
– Membrane potential
– Ca2+
– Synaptic activity
• Effectors (actuators)
– Excitation (activators)
– Inhibition (silencers)
– Second messenger cascades
 Sensors: GFP-based reporters
fluorescent intensity changes with exact conditions. Conformation of molecule influences how well it fluoresces

Aequorea victoria

The Nobel Prize in Chemistry 2008

was awarded jointly to Osamu Shimomura,
Martin Chalfie and Roger Y. Tsien "for the
discovery and development of the green
fluorescent protein, GFP".
Ormö et al. 1996
GEVI: Membrane potential
FlaSh

graded change in
fluorescence as the
voltage changed

Note the very slow change in

indicator fluorescence.
Can't indicate action potentials.

Knöpfel et al., 2006

Siegel & Isacoff, 1997
Accelerated Sensor of Action Potentials 1 (ASAP1)
this sensor quite good at picking up the signal as seen with
electrophysiology.
Now you can see voltage at all parts of cell; with recording electrode
can only see where you record from (e.g. just from cell body or
dendrites).

St Pierre et al., 2014

 Voltage-imaging of multiple identified neurons
today there are voltage sensors that work with increasing fluorescence with either depolarisaiton or hyperpolarisation. Different reporters > see two
cell populations imaged simultaneously in brain of behaving mice.

But voltage sensors can change cell properties and can be toxic...

Kannan et al., Science 2022

 ...Ca2+ concentration often used as a proxy. Done most often using GCaMP6. GFP coupling to Calmodulin with CaM
interacting peptide. We are now on generation 8, although 6 is most used.

GECI: [Ca2+] as proxy for AP activity

GCaMP6
firing rate too
high - can't
resolve

slow response

but investigators mostly interested in whether neurons

are active or not

Faster responses

cpFP circularly permuted GFP

CaM calmodulin
M13 CaM-interacting peptide

Knöpfel et al., 2006 Chen et al., 2013

 Synaptic transmission

Dreosti et al., 2009

calcium transient detected with GCaMP
Target to synaptophysin - detect Ca2+ at vesicles and not rest of the terminal

vesicle fusion measured with synaptophluorin - pH sensitive form of GFP.

Since there's a difference between intravesicular pH and exrtacellular
medium, at time of exocytosis pH change can be detected with pH sensitive
version of GFP

Dreosti & Lagnado, 2011

 Synaptic transmission

Ca2+

SynaptopHluorin

Dreosti & Lagnado, 2011 Royle et al., 2008

 Synaptic transmission
transitter release
reporter that binds glutamate and change
fluorescence - glutamate sensitive fluorescence
reporter

Ca2+

SuperGluSnFR

GluSnFR = Glutamate-sensitive fluorescent reporter

Dreosti & Lagnado, 2011 Hires et al., 2008

 Synaptic transmission

Ca2+

Exocytosis Release

Dreosti & Lagnado, 2011

 much faster version used today. Can detect e.g. in barell cortex, can express in
pre or postsynaptic neuron, then record using camera the change in fluorescence
when you for example wiggle a whikster of mouse

iGluSnFR3

Record the transients with each glutamate stimulus.

Now there are sensors for any imaginable protein to be assayed... Aggarwal et al., 2023
 G-protein-coupled receptor-activation-based
(GRAB) sensors
link GFP with molecule
that's responsive to transmitter/ molecule of interest. Use naturally occuring receptor for that ligand, just couple that
receptor with GFP.

GRABACh3.0

selective, almost as much as natural cholinergic signalling, but this changes

fluorescence.

but you take away some of the acetylcholine used for signalling.
Tio = Tiotropium

M3R = type 3 muscarinic ACh receptor

cpEGFP = circularly permutated EGFP Jing et al., 2020
 Optogenetic tools

• Reporters (sensors)
– Membrane potential
– Ca2+
– Synaptic activity
• Effectors (actuators)
– Excitation (activators)
– Inhibition (silencers)
– Second messenger cascades
 Channelrhodopsin-2
first paper where they tried to express ChR in neurons. Light sensitive current when
blue light shone, and light sensitive current can drive action potentials. Retinal used as
light sensitive molecule, and neurons have natural retinal.

Nagel et al., 2003 Boyden et al., 2005

 Crystal structure of ChR2
can do targeted mutations to change properties.

Kato et al., 2012

 How ChR2 works

need to go from light adapted to dark adapted state before can

be open again.

instead of isomerising around AA11,

its around AA13. Changes from all trans
to 13 cis form. ChR exists in several forms. Two different
open states, and dark and light adapted state.

Hegemann & Möglich, 2011

 Improvements

• Speed
• Optical
switches
• Multiple
wavelengths
• Inhibition
inhibitory opsins

Pastrana, 2011
 Speed

WT

E123T
can drive neurons at higher rate and
for longer.

Gunaydin et al., 2010

can also make ChR really slow so it acts as switch - can turn it on, and stays on until turned off.

Optical switches
turn on and off with different colour
light.

Berndt et al., 2009

Multiple wavelengths
can express different ChRs in different cell tyeps, and using
blue/green light to turn on one cell type vs another.

Zhang et al., 2008

 first inhibitory opsin tool

Optogenetic inhibition: Halorhodopsin

NpHR = halorhodopsin.

both rhodopsins expressed.

Zhang et al., 2007

 today, archaerhodopsin used more commonly. Hyperpolarises the cell, and also
changes the pH in the cell, which can prevent transmitter release at neuronal

Optogenetic inhibition: Archaerhodopsin

terminals.

Action spectrum of Arch

Silencing of neocortical neurons in awake mice

Chow et al., 2010

 both halorhodopsin and archaerhodopsin are pumps and therefore relatively slow. So change ChR so it becomes chloride permeable.

Optogenetic inhibition: A Chloride-conducting

Channelrhodopsin (ChloC)

Wietek et al., 2014; Berndt et al., 2016

Optogenetic modulation of intracellular signalling
can just take rhodopsin and instead of coupling to transducin, change ICLs to something that
resembles either beta or alpha adrenergic receptor (or whichever type of receptor that
operates a specific type of G protein) to change intracellular signalling cascade based on
using light rather than using natural ligand.

Airan et al., 2009

Optogenetic control of gene expression
can use light to turn on and off the gene of interest.

Wang et al., 2012

 Specificity

• Cell type use promoters to drive protein in specific cell types.

Genetic targeting

• Spatial
spatial specificity with both genetic targeting and light source.

Light source and delivery

• Temporal
 Genetic targeting
need high level of expression, strong promoter therefore also needed. But how can you get cell type specificity
with these general promoters like CMV.

• Photostimulation efficiency depends on level of expression,

and usually requires a strong promoter (e.g. CMV, EF-1α).
• Cell-type-specific expression can be achieved CaMKIIa promoter expressed in
pyramidal cells, is strong enough to
– Developmental delivery (in utero electroporation) drive channelrhodopsin expression
specifically
– Viral vector with strong cell-type-specific promoter (e.g. CaMKIIα)
– Cre-lox system: Transgenic mouse line expressing Cre recombinase in desired
cell type, combined with either floxed-stop transgenic line or viral vector with
Cre recombinase dependent expression

e.g. use parvalbumin promoter to drive Cre expression

Then use flox stop or double inverted reading frame
flox sites to control expression.

flex system?
 Light sources and delivery

• Light sources
– Hg lamp with shutter
– LED
– Laser
• Delivery
– Light flashes
– Objective
– Optical fibre most common if you work in behaving animal
Optical stimulation in vivo

can calculate depth

of penetration

Aravanis et al., 2007

 Summary

• Optogenetic tools
GCaMP6

– Sensors
– Effectors

• Specificity
– Cell type
– Spatial
– Temporal

• Experimental use
– Causal relations
to what extent can this be demonstrated?
Critical assessment of recent papers using optogenetic tools
1. A Unified Framework for Dopamine Signals across Timescales.
Kim HR, Malik AN, Mikhael JG, et al.
Cell. 2020 Dec 10;183(6):1600-1616.e25. doi: 10.1016/j.cell.2020.11.013.

2. Encoding of Discriminative Fear Memory by Input-Specific LTP in the Amygdala.

Kim WB, Cho JH.
Neuron. 2017 Aug 30;95(5):1129-1146.e5. doi: 10.1016/j.neuron.2017.08.004.

3. Associative and predictive hippocampal codes support memory-guided behaviors.

Liu C, Todorova R, Tang W, Oliva A, Fernandez-Ruiz A.
Science. 2023 Oct 20;382(6668):eadi8237. doi: 10.1126/science.adi8237.

4. Gamma frequency entrainment attenuates amyloid load and modifies microglia.

Iaccarino HF, Singer AC, Martorell AJ, et al.
Nature. 2016 Dec 7;540(7632):230-235. doi: 10.1038/nature20587.

5. Volume-transmitted GABA waves pace epileptiform rhythms in the hippocampal network.

Magloire V, Savtchenko LP, Jensen TP, et al.
Curr Biol. 2023 Apr 10;33(7):1249-1264.e7. doi: 10.1016/j.cub.2023.02.051.