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PMCOL 371 - Cellular Neuroscience > 16 - Optogenetics and Chemogenetics > Flashcards

16 - Optogenetics and Chemogenetics Flashcards

1
Q

6 methods to control neuronal activity:

A
  • Electrical Stimulation
  • Pharmacology
  • Loss of function
  • Genetic ablation
  • Optogenetics
  • Chemogenetics
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2
Q

Explain how the following is used to control neuronal activity: (precision/specificity?

  • Electrical Stimulation
  • Pharmacology
  • Loss of function
  • Genetic ablation
  • Optogenetics
  • Chemogenetics
A
  • Electrical Stimulation
    • Delivery of electrical current into the region of interest
    • High temporal precision
    • Low specificity (heterogeneous cell activation, fibres of passage)
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3
Q

Explain how the following is used to control neuronal activity: (precision/specificity?

  • Electrical Stimulation
  • Pharmacology
  • Loss of function
  • Genetic ablation
  • Optogenetics
  • Chemogenetics
A

Pharmacology (eg receptor agonists)

  • Local application of agonists that interact with specific receptors and open channels secondary to the messenger pathway
  • Good specificity (only receptor expressing cells will be activated)
  • Low temporal precision
  • Effect dependent on drug diffusion, metabolism, washout, degradation of drugs
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3
Q

Explain how the following is used to control neuronal activity: (precision/specificity?

  • Electrical Stimulation
  • Pharmacology
  • Loss of function
  • Genetic ablation
  • Optogenetics
  • Chemogenetics
A

Pharmacology (eg receptor agonists)

  • Local application of agonists that interact with specific receptors and open channels secondary to the messenger pathway
  • Good specificity (only receptor expressing cells will be activated)
  • Low temporal precision
  • Effect dependent on drug diffusion, metabolism, washout, degradation of drugs
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4
Q

Explain how the following is used to control neuronal activity: (precision/specificity?

  • Electrical Stimulation
  • Pharmacology
  • Loss of function
  • Genetic ablation
  • Optogenetics
  • Chemogenetics
A
  • Loss of function
    • via lesion of area of interest
      • Low specificity (in terms of size of lesion and cell phenotype)
      • Permanent damage
      • Repair mechanisms can affect neuronal activity
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5
Q

Explain how the following is used to control neuronal activity: (precision/specificity?

  • Electrical Stimulation
  • Pharmacology
  • Loss of function
  • Genetic ablation
  • Optogenetics
  • Chemogenetics
A
  • Genetic Ablation
    • of neurons with a specific phenotype
      • Highly specific
      • If conditional knock outs are used, developmental issues are eliminated
      • Low temporal precision, often irreversible
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6
Q

Explain how the following is used to control neuronal activity: (precision/specificity?

  • Electrical Stimulation
  • Pharmacology
  • Loss of function
  • Genetic ablation
  • Optogenetics
  • Chemogenetics
A
  • Optogenetics:
    • Control of neuronal activity with LIGHT (at specific wavelength) by inserting light sensitive proteins in cells of interest
    • High temporal precision (msec response)
    • High specificity (cell type targeting)
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7
Q

Explain how the following is used to control neuronal activity: (precision/specificity?

  • Electrical Stimulation
  • Pharmacology
  • Loss of function
  • Genetic ablation
  • Optogenetics
  • Chemogenetics
A

Chemogenetics:

  • Control of neuronal activity by inserting exogenous receptor that can be activated by exogenous ligands
  • DREADD receptors (Designer Receptor Exclusively Activated by Designer Drugs)
  • Low temporal precision
  • High specificity (cell type targeting)
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8
Q

Define optogenetics

A

Optogenetics is the integration of optics and genetics to achieve activation or inhibition of cellular function in living tissues

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9
Q

What does optogenetics involve?

A
  • Development of light-sensitive proteins
  • The strategies for delivering their genes to specific cells
  • The targeted illumination
  • Compatible readouts for reporting on changes in cell, tissue and animal behaviour
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10
Q

How do we control neuronal activity with light?

A
  • Opsins
    • - 7 transmembrane light sensitive proteins
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11
Q

What are opsins?

A

7 transmembrane light-sensitive proteins that control neuronal activity with light

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12
Q

_______ opsins are present in prokaryotes, algae and fungi

A

Type 1 opsins are present in prokaryotes, algae and fungi

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13
Q

________ opsins are present in higher eukaryotes (mostly involved in vision - GPCR)

A

Type II opsins are present in higher eukaryotes (mostly involved in vision - GPCR)

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14
Q

________ single protein that combine light sensation and ion flux, encoded by a single gene

A

Type I Opsins single protein that combine light sensation and ion flux, encoded by a single gene

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15
Q

Microbial (type 1) opsins are present in ____, ____, _____

A

Microbial (type 1) opsins are present in algae, bacteria, and fungi

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16
Q
  • Type 1 opsins require _____ as the photon sensing cofactor
A

Type 1 opsins require retinal as the photon sensing cofactor

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17
Q

All-trans-retinal ←→ _______

A

All-trans-retinal ←→ 13-cis retinal configuration

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18
Q

Retinal acts as an antenna for _____ and when retinal is bound, the functional opsin is termed _______

A

Retinal acts as an antenna for photons and when retinal is bound, the functional opsin is termed Rhodopsin

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19
Q

___________ is light sensitive - detects light and move towards it to feed itself through photosynthesis

A

Chlamydomonas Reinhardtii is light sensitive - detects light and move towards it to feed itself through photosynthesis

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20
Q

__________ is light-sensitive protein which is located on the boundary of the algae’s eye-like structure (primitive visual system), called an ______

A

Channelrhodopsin is light-sensitive protein which is located on the boundary of the algae’s eye-like structure (primitive visual system), called an eyespot

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21
Q

When hit by light, Channelrhodopsin _________

A

When hit by light, Channelrhodopsin converts light into an electric current → change of shape and open channel through the boundary of the eyespot → positively charged ions enter the eyespot → the flow of charged particles generates an electric current that, through a cascade of events, forces the algae’s two flagella to steer the organism towards the light

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22
Q

What are the two light sensitive proteins in C. Reinhardtii

A
  • ChR1 and ChR2
    • slow activation and inactivation kinetics
    • Low current
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23
Q

Mutagenesis of sequence of light sensitive proteins in order to ________________ in order to better control changes in voltage potentials of excitable cells

A

Mutagenesis of sequence of light sensitive proteins in order to design opsins with improved expression, photocurrent, deactivation time, alternative wavelength sensitivity in order to better control changes in voltage potentials of excitable cells

Light can be used as an on/off switch to control neuronal activity

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24
Q

What are the three key properties of different classes of opsins?

A
  • Inhibition vs excitation
  • Wavelength of excitation
  • Kinetics of activation and inactivation
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25
Q

What drives an AP in cultured neurons transfected with ChR2?

A

Brief pulses of blue light

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26
Q

Swimming of a transgenic Caernorhabditis elegans expressing inhibitory opsin (NpHR) in muscles is instantaneously, and repeatedly, inhibited by ________ ___\_ (duration of illumination is indicated by appearance of a yellow dot)

A

Swimming of a transgenic Caernorhabditis elegans expressing inhibitory opsin (NpHR) in muscles is instantaneously, and repeatedly, inhibited by photoactivation of HR (duration of illumination is indicated by appearance of a yellow dot)

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27
Q

Halorhodopsin expression in motoneurons

Swimming of a transgenic Caernorhabditis elegans expressing inhibitory opsin (NpHR) in cholinergic motoneurons is instantaneously inhibited by ________ (duration of illumination is indicated by appearance of a yellow dot).

A

Swimming of a transgenic Caernorhabditis elegans expressing inhibitory opsin (NpHR) in cholinergic motoneurons is instantaneously inhibited by photoactivation of NpHR (duration of illumination is indicated by appearance of a yellow dot).

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28
Q

In mammalian systems, do opsins need Retinal to work?

A
  • Retinal in mammalian cells is sufficient for the opsins to work (no need for additional retinal)
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29
Q

How to express opsins in neurons?

A
  • In culture, transfections
  • In vivo, viral targeting/ transgenic animal
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30
Q

How to use opsins to selectively activate or inhibit a subpopulation of neurons

A

Driving the expression via specific promoters

31
Q

What are two in vivo delivery and targeting strategies of optogenetic tools into mammalian neuronal systems

A

Viral targeting

Transgenic animal targeting

32
Q

Viral systems:

  • uses:
  • expression?
  • detection of expressing cells
  • Cellular specificity?
A

Viral systems:

  • uses:
    • Adeno-Associated virus (AAV) or Lentivirus (LV)
  • fast expression
  • Sequence of fluorescent proteins to detect expressing cells (EYFP, mCherry)
  • Cellular specificity
    • Use of specific promoters
    • Injection of virus in targeted site
    • Targeted light delivery
33
Q

Transgenic animals:

Involve expression of opsins __________

A

Transgenic animals:

Involve expression of opsins directly in cells of interest (VGluT2-ChR2-EYFP mice)

34
Q

Expression of opsins in Cre-driver using:

A

Expression of opsins in Cre-driver using: viral infection of Cre recombinase-dependent viruses (DIO, double floxed inverted open-reading-frame)

35
Q

What are the first 3 steps to optogenetics?

A
  1. Piece together genetic construct:
    • promoter to drive expression
    • Gene encoding opsin (light-sensitive ion channel)
  2. Insert construct into virus
  3. Inject virus into animal brain; opsin is expressed in targeted neurons
  4. Insert ‘optode’, fibre-optic cable plus electrode
  5. Laser light of specific wavelength opens ion channel in neurons
  6. Record electrophysiological and behavioural results
36
Q

What are the last 3 steps to optogenetics:

A
  • Insert ‘optode’, fibre-optic cable plus electrode
  • Laser light of specific wavelength opens ion channel in neurons
  • Record electrophysiological and behavioural results
37
Q

Optogenetics:

Which cells express light-sensitive channels depends on:

A

the promoter region of the inserted DNA sequence

Cells which contain transcription factors that recognize the promoter sequence will express these channels while cells that lack specific transcription factors for the sequence will not

38
Q

Optogenetics:

Once the genes have been inserted, it can take how long for them to be fully expressed?

A

1-4 weeks

39
Q

What is the light source for optogenetics?

A

LED light/ laser system

  • specific wavelength of excitation
  • Ability of delivering light in fashion similar to neuronal activity (frequency and intensity)
  • Localized optostimulation via optic fibres (100-200 micrometers in diameter)
40
Q

What are the two main challenges of all optical investigation of neuronal circuites?

A
  • Reliable delivery and expression of the sensors and actuators in the same neurons
  • Elimination of cross talk between the imaging and manipulation channels
41
Q

What are two uses of optogenetics?

A
  • Understand how a complex biological system works
  • Fix a “defective” system by enabling its behaviour to be steered
42
Q

Targeting optogenetic tools in vivo:

How is direct stimulation of neuronal cell bodies achieved?

A

A) By injecting virus at the target region and then implanting a light delivery device above the injected region. Even this simple experiment can provide specificity with viruses that will not transduce afferent axons and fibers of passage

43
Q

Targeting Optogenetic Tools in vivo:

How is additional cell-type specificity attained

A

B) Additional cell-type specificity is attained either by cell-type-specific promoters in the viral vector or via a recombinase-dependent virus, injected in a transgenic animal expressing a recombinase such as Cre in specific cells, leading to specific expression of the transgene only in defined cell types

44
Q

Targeting Optogenetic tools in vivo:

How is projection targeting achieved?

A

C) Projection (axonal) targeting is achieved by viral injection at the region harboring cell bodies, followed by implantation of a light-delivery device above the target region containing neuronal processes from the virally transduced region; in this way cell types are targeted by virtue of their projections

45
Q

Targeting optogenetic tools in vivo:

What is projection termination

A

D) Projection termination labeling is a more refined version of projection targeting in which cells are targeted by virtue of synaptic connectivity to the target region and likely excluding cells with axons simply passing through the region

Transcellular labeling using a recombinase-dependent system is shown

Viruses expressing Cre fused to a transneuronal tracer (lectin) are delivered at the synaptic target site and a Cre-dependent virus is injected into the region with cell bodies

Cells that project to the cre-injected area express the Cre-dependent virus and become light sensitive

This can also be achieved with axon terminal transducing viruses although without control over the postsynaptic cell types

46
Q

Tools Targeting optogenetic in vivo

What is Combinatorial local somata?

A
  • Expression of two opsins with different characteristics in one brain region using a combination of the promoter or Cre-based approaches.
  • Light delivery to the somata is performed using two different wavelengths designed to minimize cross-activation
47
Q

_________ is a key structure in motivated behaviour, including feeding

A

Lateral hypothalamus (LH) is a key structure in motivated behaviour, including feeding

48
Q

Bed nucleus of the ________ is a key integrator of motivational behaviours, including feeding

A

Bed nucleus of the stria terminalis (BNST, in the amygdala) is a key integrator of motivational behaviours, including feeding

48
Q

Bed nucleus of the ________ is a key integrator of motivational behaviours, including feeding

A

Bed nucleus of the stria terminalis (BNST, in the amygdala) is a key integrator of motivational behaviours, including feeding

49
Q

Which area of the amygdala is activated during food consumption?

A

BNST

Bed nucleus of the stria terminalis

50
Q

BNST (Bed neurons of stria terminalis) contains _______ neurons and projects to both LH and VTA

LH = lateral hypothalamus

VTA = ventral tegmental area

A

BNST (Bed neurons of stria terminalis) contains inhibitory GABAergic neurons and projects to both LH and VTA

LH = lateral hypothalamus

VTA = ventral tegmental area

51
Q

What is Vgat in VGAT-Cre Mice? Where is it found?

A

Vgat = transporter expressed in GABAergic neurons

52
Q

What is the optogenetic virus in BNST (bed nucleus of stria terminalis)

A

Double floxed inverted open-reading-frame (DIO) strategy

53
Q

________ circuit activation induces feeding in well fed mice - when laser off = immediate cessation of feeding response

A

Vgat BNST→LH circuit activation induces feeding in well fed mice - when laser off = immediate cessation of feeding response

53
Q

________ circuit activation induces feeding in well fed mice - when laser off = immediate cessation of feeding response

A

Vgat BNST→LH circuit activation induces feeding in well fed mice - when laser off = immediate cessation of feeding response

54
Q

What is preBotzinger complex?

A

Respiratory rhythmogenic center

Neuronal control of breathing

55
Q

Optogenetic excitation of __________ potently drives inspiratory activity in vivo

A

Optogenetic excitation of preBotzinger complex neurons potently drives inspiratory activity in vivo

56
Q

What are some limitations of optogenetics in human studies and therapies?

A
  • Limitations include the introduction of a foreign gene into the human brain
  • Fiber optics could pose the threat of infection and being uncomfortable and having to carry heavy batteries
  • Cost of procedure and equipment
57
Q

Is optogenetics applicable to human studies:

  • Viruses appear to be safe in primates (AAVs have ________, overexpression of ______?)
  • Bioengineers are continuously developing laser stimulation devices that are ____, ______, ______
  • Molecular biologists are continuously developing opsins that are more sensitive to ______ and _______
A

Is optogenetics applicable to human studies:

  • Viruses appear to be safe in primates (AAVs have low immuno response, overexpression of opsins?)
  • Bioengineers are continuously developing laser stimulation devices that are smaller, more effecient, more precise and wearable
  • Molecular biologists are continuously developing opsins that are more sensitive to light and different wavelengths (red shift)
58
Q

Potential therapies from optogenetics? (8)

A
  • In disease where neuronal population is hyper/hypoactive (epilepsy, depression, alzheimers)
  • Vision disorders
  • anxiety
  • addiction
  • chronic pain
  • sleep disorders
  • ADD
  • Migraine
59
Q

What was the first clinical-safety trial of an optogenetic therapy meant to treat?

A

Retinitis Pigmentosa - disorder that destroys photoreceptors in the eye

Treatment seeks to compensate for photoreceptors loss by conferring light sensitivity to retinal ganglion cells (which usually transfer visual signals from photoreceptors to the brain)

60
Q

How is optogenetics being studied to treat Chronic Pain?

A
  • By expressing inhibitory opsin in nerves that are responsible for pain transmission in order to prevent neurons from firing and reduce pain sensation
61
Q

What is chemogenetics

A

Chemogenetics is the integration of genetically engineered receptors that selectively interact with small exogenous molecules and genetics to achieve activation or inhibition of cellular function in living tissues

62
Q

Chemogenetics uses ______ that are not activated by endogenous ligands but that can be activated specifically by pharmacologically inert ligands (eg ______)

A

Chemogenetics uses mutated receptors that are not activated by endogenous ligands but that can be activated specifically by pharmacologically inert ligands (eg Clozapin N-Oxide, CNO*-clozapine))

63
Q

DREADD?

A

Designer Receptors exclusively activated by designer drugs

64
Q

Mutant M3 and M4 muscarinic receptors are activated by ______ and are insensitive to _____

A

Mutant M3 and M4 muscarinic receptors are activated by nM concentration of CNO* and are insensitive to Ach

65
Q

What type of receptor are mutant M3 and M4 muscarinic receptors?

A

GPCR

66
Q

3 types of G-proteins associated with mutant M3 and M4 muscarinic receptors

A

Gq, Gs, Gi

67
Q

CNO acts on ______ to cause _____ via ___ g protein

A

CNO acts on hM3Dq to cause Increased intracellular calcium (depolarization) and increase cell excitability via Gq g protein

68
Q

Perlapin acts on ______ to cause ______ via _____ g-protein

A

Perlapin acts on GsD to activate adenylyl cyclase and increase cAMP via Gs g-protein

69
Q

Compound 21 acts on ______ to cause ______ via _____ g-protein

A

Compound 21 acts on hM4Di to activate K+ channels (GIRKs) (hyperpolarization) and inhibit neurotransmitter release (neuronal silencing) via Gi g-protein

70
Q

Salvinorin B acts on ______ to cause ______ via _____ g-protein

A

Salvinorin B acts on KORD to Activate K+ channels (GIRKs) (hyperpolarization) and inhibit neurotransmitter release (neuronal silencing) via Gi g-protein

71
Q

In vivo delivery and targeting strategies of chemogenetic tools into mammalian neuronal systems:

A
  • Viral targeting
  • Transgenic animal targeting

G protein will be activated in the presence of CNO* (usually delivered systemically) and the pharmacological effects persist for up to 10 hours

72
Q

Explain the image

A
  • Use DREADD virus in the preBotC to hyperpolarize cells and apneas (pauses in breathing) occurred mainly in REM sleep, when excitatory drive to preBotC is mostly reduced

PMCOL 371 - Cellular Neuroscience (19 decks)

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