Optogenetics
Optogenetics
Optogenetics
o A technique that revolves around expressing light receptors in cells of interest – such that you can
control the activity of those cells by shining light on them
Allows precise control (activation or inhibition) of cells of interest – in a temporally precise
manner
Tool for manipulating brain functions
o An approach which allows the control of neuronal activity with light
o A combination of optical and genetic techniques
Opto
Delivery of light to neurons expressing light-sensitive ions channels/pumps or GPCRs
Genetics
Targeting of specific neurons with genes encoding for light-sensitive ion
channels/pumps or GPCRs
Development of optogenetics
o In algae – channelrhodopsins function as visual proteins directing the algae towards or away from a
light source, or to find light conditions that are optimal for photosynthetic growth
Channelrhodopsins
A subfamily of retinylidene proteins that function as light-gated ion channels
Serve as sensory photo-receptors in unicellular green algae – controlling phototaxis =
movement in response to light
o ChR2 = channelrhodopsin 2
Binds to all-trans retinal causes conformational
change when retinal absorbs light forming 13-cis
retinal conformational change drives gating of ion
channels allowing entry of positively charged ions
Light absorption triggers subsequent
conformational change of the protein +
gating of the channel
o YFP-ChR2 fusion protein
If YFP is attached to ChR2 = label – any cell
expressing ChR2 glows = allows easy detection of
cells expressing channel rhodopsin
o ChR2 with reduced desensitisation characteristics
Responds to light in a more sustained fashion than ChR2
Optogenetic control of firing in vitro
o Boyden et al – investigated whether ChR2 could be used to control activity of neurons
Neurons grown in culture were transduced with ChR2-YFP fusion protein
Patched cultured neurons – held in voltage clamp
Applied blue light to neurons resulted in strong inward current =
strong depolarising current
Patch cultured neurons – in current clamp mode
Pulses of blue light 10ms or longer was sufficient to induce
action potentials
Optogenetic control of firing ex vivo
o Developed a viral construct which could be used to deliver ChR2 into mice
o Viral construct was infused into the hippocampi of adult mice
ChR2 was under the control of the EF-1α promotor – with YFP as a reporter
ChR2 was expressed in the adult mouse hippocampus
o Made slices from hippocampi
, Optogenetics
In voltage-clamp – blue light resulted in strong depolarising current
In current clamp – brief pulses of blue light could reliably drive spiking in the neurons
Optogenetic control of firing in vivo
o Developed transgenic mouse line which expressed ChR2 in cortical neurons
o Simultaneously made electrophysiological recordings from the cortex while they applied blue light
via a fibre optic cable
Showed that application of blue light could reliably drive spiking in recorded neurons in the
cortex
Spiking could be precisely timed with short pulses of blue light
Temporal and spatial precision
o Optogenetics allows ability to target it such that one can make temporally and spatially
manipulations of neuronal activity
Not possible with other stimulation techniques – electrical or pharmacological
o 1
Physical delivery of virus to a given anatomical location by syringe
Only cells within vicinity of injection site will take up optogenetic gene and express it
Deliver light to injection site causes selective activation in injection site
Uncovers circuit connectivity patterns by making use of axonal projections
o Increase selectivity by activating neurons expressing optogenetic construct
based on their axonal projections
By moving light to a different structure – only activates neurons
which project to this particular structure
Selects a subset of neurons within injection – activating a
specific pathway
o 2
Or genetically target neurons to be activated
Cell types can be addressed if the cell type of interest has a known genetic identity
o E.g. target to hippocampus use genetic techniques to make sure channel
rhodopsin is only expressed in a particular subset of cells or subtype of cells
o 3
Precision of illumination
Use lasers to individually target illumination to specific cells
Directing the illumination source to a given set of cells or even individual neurons –
useful when the targets of interest are separated in space
Delivering the genetic construct
o Optogenetic constructs are delivered to target cells via a virus
o Stages
Piece together genetic construct (transgene)
Promotor to drive gene expression + gene encoding opsin (light-sensitive ion
channel) + fluorescent reporter
Package construct into virus
Adeno-associated virus (AAV) is a small virus which can be used as a
vector to deliver target genes into mammals
o On its own, AAV is biologically inert = no chance of causing
infection or disease + non-replicating
Optogenetics
Optogenetics
o A technique that revolves around expressing light receptors in cells of interest – such that you can
control the activity of those cells by shining light on them
Allows precise control (activation or inhibition) of cells of interest – in a temporally precise
manner
Tool for manipulating brain functions
o An approach which allows the control of neuronal activity with light
o A combination of optical and genetic techniques
Opto
Delivery of light to neurons expressing light-sensitive ions channels/pumps or GPCRs
Genetics
Targeting of specific neurons with genes encoding for light-sensitive ion
channels/pumps or GPCRs
Development of optogenetics
o In algae – channelrhodopsins function as visual proteins directing the algae towards or away from a
light source, or to find light conditions that are optimal for photosynthetic growth
Channelrhodopsins
A subfamily of retinylidene proteins that function as light-gated ion channels
Serve as sensory photo-receptors in unicellular green algae – controlling phototaxis =
movement in response to light
o ChR2 = channelrhodopsin 2
Binds to all-trans retinal causes conformational
change when retinal absorbs light forming 13-cis
retinal conformational change drives gating of ion
channels allowing entry of positively charged ions
Light absorption triggers subsequent
conformational change of the protein +
gating of the channel
o YFP-ChR2 fusion protein
If YFP is attached to ChR2 = label – any cell
expressing ChR2 glows = allows easy detection of
cells expressing channel rhodopsin
o ChR2 with reduced desensitisation characteristics
Responds to light in a more sustained fashion than ChR2
Optogenetic control of firing in vitro
o Boyden et al – investigated whether ChR2 could be used to control activity of neurons
Neurons grown in culture were transduced with ChR2-YFP fusion protein
Patched cultured neurons – held in voltage clamp
Applied blue light to neurons resulted in strong inward current =
strong depolarising current
Patch cultured neurons – in current clamp mode
Pulses of blue light 10ms or longer was sufficient to induce
action potentials
Optogenetic control of firing ex vivo
o Developed a viral construct which could be used to deliver ChR2 into mice
o Viral construct was infused into the hippocampi of adult mice
ChR2 was under the control of the EF-1α promotor – with YFP as a reporter
ChR2 was expressed in the adult mouse hippocampus
o Made slices from hippocampi
, Optogenetics
In voltage-clamp – blue light resulted in strong depolarising current
In current clamp – brief pulses of blue light could reliably drive spiking in the neurons
Optogenetic control of firing in vivo
o Developed transgenic mouse line which expressed ChR2 in cortical neurons
o Simultaneously made electrophysiological recordings from the cortex while they applied blue light
via a fibre optic cable
Showed that application of blue light could reliably drive spiking in recorded neurons in the
cortex
Spiking could be precisely timed with short pulses of blue light
Temporal and spatial precision
o Optogenetics allows ability to target it such that one can make temporally and spatially
manipulations of neuronal activity
Not possible with other stimulation techniques – electrical or pharmacological
o 1
Physical delivery of virus to a given anatomical location by syringe
Only cells within vicinity of injection site will take up optogenetic gene and express it
Deliver light to injection site causes selective activation in injection site
Uncovers circuit connectivity patterns by making use of axonal projections
o Increase selectivity by activating neurons expressing optogenetic construct
based on their axonal projections
By moving light to a different structure – only activates neurons
which project to this particular structure
Selects a subset of neurons within injection – activating a
specific pathway
o 2
Or genetically target neurons to be activated
Cell types can be addressed if the cell type of interest has a known genetic identity
o E.g. target to hippocampus use genetic techniques to make sure channel
rhodopsin is only expressed in a particular subset of cells or subtype of cells
o 3
Precision of illumination
Use lasers to individually target illumination to specific cells
Directing the illumination source to a given set of cells or even individual neurons –
useful when the targets of interest are separated in space
Delivering the genetic construct
o Optogenetic constructs are delivered to target cells via a virus
o Stages
Piece together genetic construct (transgene)
Promotor to drive gene expression + gene encoding opsin (light-sensitive ion
channel) + fluorescent reporter
Package construct into virus
Adeno-associated virus (AAV) is a small virus which can be used as a
vector to deliver target genes into mammals
o On its own, AAV is biologically inert = no chance of causing
infection or disease + non-replicating
