Brainscape's Knowledge GenomeTM


Top Flashcards (Certified)
All Flashcards

PMCOL 371 > 17 - Optogenetics and Chemogenetics > Flashcards

17 - Optogenetics and Chemogenetics Flashcards

(71 cards)

Start Studying
1
Q

6 methods to control neuronal activity:

A
  • Electrical Stimulation
  • Pharmacology
  • Loss of function
  • Genetic ablation
  • Optogenetics
  • Chemogenetics
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

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

  • Electrical Stimulation
A

Electrical Stimulation
- Delivery of electrical current into the region of interests
- High temporal precision
- Low specificity (heterogeneous cell activation, fibres of passage)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

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

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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

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

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

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

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

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

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)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Explain how the following is used to control neuronal activity: (precision/specificity?
-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)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Define optogenetics

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

How do we control neuronal activity with light?

A

Opsins
- 7 transmembrane light sensitive proteins

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What are opsins?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

____ opsins are present in prokaryotes, algae and fungi

A

Type 1 opsins are present in prokaryotes, algae and fungi

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

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

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Type 1 opsins require ____ as the photon sensing cofactor

A

Type 1 opsins require retinal as the photon sensing cofactor

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

All-trans-retinal ←→ ____

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

What are the two light sensitive proteins in C. Reinhardtii

A

ChR1 and ChR2
- slow activation and inactivation kinetics
- Low current

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
What drives an AP in cultured neurons transfected with ChR2?
Brief pulses of blue light
26
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)
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)
27
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).
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).
28
In mammalian systems, do opsins need Retinal to work?
Retinal in mammalian cells is sufficient for the opsins to work (no need for additional retinal)
29
How to express opsins in neurons?
- In culture, transfections - In vivo, viral targeting/ transgenic animal
30
How to use opsins to selectively activate or inhibit a subpopulation of neurons
Driving the expression via specific promoters
31
What are two in vivo delivery and targeting strategies of optogenetic tools into mammalian neuronal systems
Viral targeting Transgenic animal targeting
32
Viral systems: - uses: - expression? - detection of expressing cells - Cellular specificity?
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
Transgenic animals: Involve expression of opsins ____
Transgenic animals: Involve expression of opsins *directly in cells of interest (VGluT2-ChR2-EYFP mice)*
34
Expression of opsins in Cre-driver using:
Expression of opsins in Cre-driver using: *viral infection of Cre recombinase-dependent viruses (DIO, double floxed inverted open-reading-frame)*
35
What are the first 3 steps to optogenetics?
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
36
What are the last 3 steps to optogenetics:
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
37
Optogenetics: Which cells express light-sensitive channels depends on:
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
Optogenetics: Once the genes have been inserted, it can take how long for them to be fully expressed?
1-4 weeks
39
What is the light source for optogenetics?
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
What are the two main challenges of all optical investigation of neuronal circuites?
- Reliable delivery and expression of the sensors and actuators in the same neurons - Elimination of cross talk between the imaging and manipulation channels
41
What are two uses of optogenetics?
- Understand how a complex biological system works - Fix a “defective” system by enabling its behaviour to be steered
42
Targeting optogenetic tools in vivo: How is direct stimulation of neuronal cell bodies achieved? | Targeting Optongentic Tool In Vivo- Local Somata
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
Targeting Optogenetic Tools in vivo: How is additional cell-type specificity attained | Targeting Optongentic Tool In Vivo- Recominant or promoter- dependent
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
Targeting Optogenetic tools in vivo: How is projection targeting achieved?
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
Targeting optogenetic tools in vivo: What is projection termination
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
Tools Targeting optogenetic in vivo What is Combinatorial local somata?
- 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
____ is a key structure in motivated behaviour, including feeding
*Lateral hypothalamus (LH)* is a key structure in motivated behaviour, including feeding
48
Bed nucleus of the ____ is a key integrator of motivational behaviours, including feeding
Bed nucleus of the *stria terminalis (BNST, in the amygdala) *is a key integrator of motivational behaviours, including feeding
49
Which area of the amygdala is activated during food consumption?
BNST Bed nucleus of the stria terminalis
50
BNST (Bed neurons of stria terminalis) contains ____ neurons and projects to both LH and VTA LH = lateral hypothalamus VTA = ventral tegmental area
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
What is Vgat in VGAT-Cre Mice? Where is it found?
Vgat = transporter expressed in GABAergic neurons
52
What is the optogenetic virus in BNST (bed nucleus of stria terminalis)
Double floxed inverted open-reading-frame (DIO) strategy
53
____ circuit activation induces feeding in well fed mice - when laser off = immediate cessation of feeding response
*Vgat BNST→LH* circuit activation induces feeding in well fed mice - when laser off = immediate cessation of feeding response
54
What is preBotzinger complex?
Respiratory rhythmogenic center Neuronal control of breathing
55
Optogenetic excitation of ____ potently drives inspiratory activity in vivo
Optogenetic excitation of *preBotzinger complex neurons *potently drives inspiratory activity in vivo
56
What are some limitations of optogenetics in human studies and therapies?
- 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
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 ____
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
Potential therapies from optogenetics? (8)
- In disease where neuronal population is hyper/hypoactive (epilepsy, depression, alzheimers) - Vision disorders - anxiety - addiction - chronic pain - sleep disorders - ADD - Migraine
59
What was the first clinical-safety trial of an optogenetic therapy meant to treat?
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
How is optogenetics being studied to treat Chronic Pain?
By expressing inhibitory opsin in nerves that are responsible for pain transmission in order to prevent neurons from firing and reduce pain sensation
61
What is chemogenetics
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
Chemogenetics uses ____ that are not activated by endogenous ligands but that can be activated specifically by pharmacologically inert ligands (eg ____)
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
DREADD?
Designer Receptors exclusively activated by designer drugs
64
Mutant M3 and M4 muscarinic receptors are activated by ____ and are insensitive to ____
Mutant M3 and M4 muscarinic receptors are activated by*nM concentration of CNO*and are insensitive to **Ach**
65
What type of receptor are mutant M3 and M4 muscarinic receptors?
GPCR
66
3 types of G-proteins associated with mutant M3 and M4 muscarinic receptors
Gq, Gs, Gi
67
CNO acts on ____ to cause ____ via ____ g protein
CNO acts on *hM3Dq* to cause *Increased intracellular calcium (depolarization) and increase cell excitability* via **Gq** g protein
68
Perlapin acts on ____ to cause ____ via ____ g-protein
Perlapin acts on *GsD* to *activate adenylyl cyclase and increase cAMP* via **Gs** g-protein
69
Compound 21 acts on ____ to cause ____ via ____ g-protein
Compound 21 acts on *hM4Di* to *activate K+ channels (GIRKs) (hyperpolarization) and inhibit neurotransmitter release (neuronal silencing)* via **Gi** g-protein
70
Salvinorin B acts on ____ to cause ____ via ____ g-protein
Salvinorin B acts on *KORD* to *Activate K+ channels (GIRKs) (hyperpolarization) and inhibit neurotransmitter release (neuronal silencing)* via **Gi** g-protein
71
In vivo delivery and targeting strategies of chemogenetic tools into mammalian neuronal systems:
- 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

PMCOL 371 (18 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2025 Bold Learning Solutions. Terms and Conditions