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NEUR3310 Neuroscience > OPTOGENETICS > Flashcards

OPTOGENETICS Flashcards

1
Q

How do neurons communicate and function? = 4

A
  1. Neurons are HIGHLY INTERCONNECTED cells
  2. that CONSTANTLY COMMUNICATE with each other.
  3. They GENERATE and PROPAGATE ELECTRICAL SIGNALS
  4. USES NEUROTRANSMITTERS FOR COMMUNICATION communication.
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2
Q

What is the typical resting membrane potential of neurons ? = 2

A
  1. Neurons at rest typically have a NEGATIVE RESTING MEMBRANE POTENTIAL
  2. OUTSIDE MORE POSITIVE, INSIDE MORE NEGATIVE
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3
Q

How is an action potential generated in neurons? = 3

A
  1. Voltage-gated SODIUM channels open in RESPONSE TO STIMULI,
  2. allowing SODIUM IONS TO DIFFUSE DOWN THEIR CONCENTRATION GRADIENT INTO THE NEURON
  3. This DEPOLARISES THE NEURON AND GENERATES AN ‘ACTION POTENTIAL’
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4
Q

What happens during the repolarization phase of an action potential? = 2

A
  1. During REPOLARISATION, POTASSIUM channels OPEN and SODIUM channels CLOSE,
  2. allowing POTASSIUM ions to EXIT THE NEURON and RESTORE THE NEGATIVE MEMBRANE POTENTIAL
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5
Q

What is non-invasive brain stimulation?

A

Non-invasive brain stimulation is a technique used to MANIPULATE BRAIN ACTIVITY with an EXTERNAL SOURCE, administered from OUTSIDE THE BODY.

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6
Q

Examples of Non-invasive brain stimulation = 2

A
  1. electrostimulation
  2. transcranial magnetic stimulation
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7
Q

How does ‘electrostimulation’ work in non-invasive brain stimulation?

= 3

A
  1. Electric current passed through electrodes on the
    scalp
  2. Various stimulation frequencies, positioning, brain targets
  3. Signal attenuated by skin, scalp, distance, etc
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8
Q

Transcranial magnetic stimulation = 3

A
  1. Magnetic fields delivered from a coil indue an electric
    field in brain tissue
  2. Typically delivered in repetitive trains
  3. Various stimulation frequencies, brain targets and intensities
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9
Q

examples of INVASIVE STIMULATION: 2

A
  1. BIONIC EYE
  2. DEEP BRAIN STIMULATION
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10
Q

Deep brain stimulation = 3

A
  1. An electrode is implanted into the brain and delivers
    electrical stimulation directly to neurons
  2. Powered by an external battery source
  3. Managing tremors in Parkinson’s disease
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11
Q

Bionic eye = 4

A
  1. Electrical prothesis is implanted into the eye, making contact with neurons in the retina.
  2. Paired with glasses or a camera that captures or
    filters light and sends this information to the implant,
    delivering electrical activity in neurons
  3. Varying levels of restoration of visual perception
    1. Electrode number
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12
Q

Spatial Resolution in Neuroscience

Question:
What challenge did Francis Crick identify in neuroscience in 1979, and what solution did he suggest?

A

Francis Crick identified the challenge of controlling a ‘POPULATION OF CELLS WHILE NOT STIMULATING OTHERS, ‘, which is a significant issue in neuroscience.

= Problem: Spatial resolution

SOLUTION:
He suggested using LIGHT TO CONTROL ACTIVITY AS A POTENTIAL SOLUTION

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13
Q

What is optogenetics?

A
  1. Using light to manipulate/control the activity of neurons

Named Nature Method of the Year in 2010

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14
Q

What is required for neurons to be manipulated by optogenetics?

A

Neurons need to be TARGETED TO EXPRESS LIGHT SENSITIVE PROTEINS

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15
Q

What are the advantages of optogenetics? = 2

A
  1. High spatial resolution
  2. High temporal resolution
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16
Q

How do we make neurons respond to light? = 3

A
  1. Through phototransduction
  2. Photoreceptors transduce light into electrochemical signals
  3. Rhodopsins are a type of photoreceptor involved in this process
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17
Q

What are opsins?

A

Opsins are G protein-coupled receptors (GPCRs) that respond to light.

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18
Q

What is the structure of opsins?

A

Opsin proteins are covalently bound to a chromophore.

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19
Q

What are the types of opsins found in vision?

A
  1. Rhodopsin is found in rods,
  2. OPN1SW, OPN1MW, and OPN1LW are found in cones.
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20
Q

What are the 2 types of Opsins?

A

TYPE 2 - ANIMALS

TYPE 1- MICROBIAL

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21
Q

What are Type 2 opsins and how do they function in animals?

A
  1. Type 2 opsins are found in animals.
  2. When the CHROMOPHORE ABSORBS LIGHT, it CAUSES A CONFORMATIONAL CHANGE that ACTIVATES THE G-PROTEIN, leading to the INDIRECT OPENING OF CHANNELS
22
Q

What are Type 1 opsins and where are they found?

A

Type 1 opsins are found in microbial organisms.

They DIRECTLY ACT AS ION CHANNELS THAT OPEN IN RESPONSE TO LIGHT

23
Q

What is Channelrhodopsin (ChR) and where is it found?

A

Channelrhodopsin (ChR) is found in the microorganism Chlamydomonas Reinhardtii,

= a single-celled green algae.

24
Q

What is the function of Channelrhodopsin?

A

Channelrhodopsin is a light-gated ion channel.

25
Q

How does Channelrhodopsin respond to light?

A

It opens in the presence of 473nm light.

26
Q

What ions does Channelrhodopsin allow to pass through?

A

Channelrhodopsin is a non-specific cation channel.

27
Q

How has Channelrhodopsin (ChR) been utilized in mammalian cells?

A

Channelrhodopsin (ChR) has been isolated and expressed on mammalian cells.

28
Q

What technique is commonly used to study Channelrhodopsin function in mammalian cells?

A

Whole-cell patch clamp recordings are often employed to study Channelrhodopsin function.

29
Q

What types of mammalian cells are commonly used in Channelrhodopsin experiments?

A

Xenopus oocytes and HEK293 cells are frequently utilized for Channelrhodopsin studies.

30
Q

What effect does Channelrhodopsin activation have on these cells?

A

Activation of Channelrhodopsin leads to depolarization of the mammalian cells.

31
Q

What is the objective of optogenetics experiments involving ChR2 expression in hippocampal neurons?

A

The objective is to express Channelrhodopsin-2 (ChR2) in hippocampal neurons and stimulate them with light in vitro.

32
Q

What can be achieved by making neurons express ChR2?

A

Neurons can be made to express ChR2, a light-gated ion channel.

33
Q

What effect does light stimulation have on these neurons?

A

Light stimulation can elicit action potentials in the hippocampal neurons expressing ChR2.

34
Q

What are some opsins used for neuronal silencing?

= 2

A
  1. Archaerhodopsin, found in Archaea,
  2. Halorhodopsin, found in Halobacterium,

are commonly used for this purpose

35
Q

Opsin variants- Neuronal silencing…UNDERSTANDING

Archaerhodopsin- Found in Archaea = 4

A
  1. -Activation (~540nm) causes pumping of protons OUT the cell
  2. -Lowers resting membrane threshold preventing APs
  3. -Slightly longer response time than ChR2
  4. -pH changes
36
Q

Opsin variants- Neuronal silencing…UNDERSTANDING

Halorhodopsin-Found in Halobacterium = 3

A
  1. -Activation (~570nm) causes pumping of chloride INTO the cell
  2. -Longer lasting inactivation after light stimulation ends
  3. -Longer response time than Arch
37
Q

Why is it important to choose the right opsin for an experiment?

A

It’s crucial to consider SPATIAL or TEMPORAL FACTORS and the desired function of the opsin.

38
Q

What is the function of Step Function Opsins (SFOs)?

A
  1. Step Function Opsins, like certain ChR2 mutants, stay open for a prolonged period of time once activated.
  2. They can also be inactivated by light of a different wavelength (e.g., 560nm).
39
Q

What is the purpose of Bidirectional Pair of Opsins for Light-induced Excitation and Silencing (BIPOLES)? = 2

A
  1. BIPOLES consist of
    • a red-shifted ChR2 for activation
    • blue-shifted activated GtACR2, which is a light-gated anion channel.
  2. This pair allows both EXCITATION and SILENCING of neurons using DIFFERENT WAVELENGTHS OF LIGHT
40
Q

How can neurons be made to express ChR2 for in vivo optogenetics experiments?

A

Neurons can be genetically modified through techniques such as transgenic animals or viral delivery.

41
Q

What are the advantages of using transgenic animals for ChR2 expression?

A

Transgenic animals offer good expression throughout the entire neuronal population and can utilize methods like the Cre-lox system for temporal control of expression.

42
Q

What are the disadvantages of using transgenic animals?

A

Transgenic animals require high maintenance and are costly to maintain.

43
Q

How does viral delivery compare to transgenic animals for ChR2 expression?

A

Viral delivery allows for specific targeting of certain brain regions while leaving others intact, providing great control over ChR2 expression.

It is relatively cheap compared to transgenic methods.

44
Q

What is a drawback of using viral delivery for in vivo optogenetics?

A

Viral delivery is invasive,

  • as it involves the injection of viruses into the brain tissue.
45
Q

Cell-Specific Promoters + Opsins

Study: Haery et al., 2019, Frontiers in Neuroanatomy

A
  1. AAV9-CaMKII-ChR2-eGFP: Expresses ChR2 in excitatory neurons and tags them with GFP.
  2. AAV5-hSyn1-ArchT-TdTomato: Expresses ArchT in both excitatory and inhibitory neurons and tags them with TdTomato.
  3. AAV1-Aldh1/1-ChR2(C128/D156)-YFP: Expresses ChR2 SFFO in astrocytes and tags them with YFP.
46
Q

In Vivo Optogenetics with Fibre Implants (Optrodes) - METHOD AND COMPONENTS = 3

A
  1. Fibre implants, also known as optrodes, involve the implantation of optic cannulas directly to the target site in the brain.
  2. Components:
    A. Outer Casing: Protrudes above the skull and connects implants to external optic fibre and light source. Approximately 10mm in length.
  3. B. Cannula Outer Casing: Encases the optic cannula, secured with dental cement onto the skull.
47
Q

Importance of Numerical Aperture in Optrodes

= CONCEPT? EFFECT ON LIGHT DISPERSION? OPSIN ACTIVATION REQUIREMENTS? = 5

A
  1. Concept:
    The numerical aperture (NA) of optrodes determines the angle of the cone of light emitted from the device.

Effect on Light Dispersion:
2. Low NA results in a narrower cone of light, while high NA produces a wider cone of light.

  1. Higher NA concentrates light intensity over a smaller area, whereas lower NA spreads intensity over a wider area.
    Opsin Activation Requirements:

Opsins, like
4. Channelrhodopsin, require a minimum light intensity for channel activation.
5. Channelrhodopsin typically needs about 5-12mW/mm^2 of light intensity for activation.

48
Q

Light Intensity and Depth in Optogenetic Fibre Implants = 3

A

Concept:

  1. In vivo optogenetic fibre implants (optrodes) face light intensity challenges when targeting deeper brain structures.

Impact of Numerical Aperture (NA):

  1. Higher NA results in narrower cones of light, leading to decreased light intensity with depth.
  2. Deeper brain structures receive less light intensity with higher NA optrodes.
48
Q

Fibre Implants in In Vivo Optogenetics

METHOD, PURPOSE, IMPLEMENTATION, CONTROL

A
  1. Method:
    In vivo optogenetics involves the use of fibre implants, which connect optic fibres to an external light source.

2.Purpose:
Fibre implants enable the delivery of light to specific brain regions for precise manipulation of neuronal activity.

  1. Implementation:
    Optic fibres are surgically implanted into the brain, allowing for the transmission of light signals to targeted neurons.
  2. Control:
    The external light source provides control over the timing, duration, and intensity of light stimulation, facilitating precise experimental conditions.
49
Q

In Vivo Optogenetics for Behavior = 5

A
  1. Application:
    In vivo optogenetics extends beyond neuronal manipulation to study behavior in live animals.
  2. Method:
    Optogenetic techniques are employed to selectively activate or inhibit specific neuronal populations in the brain.
  3. Behavioral Observations:
    - Researchers observe and analyze the behavioral responses elicited by optogenetic stimulation.
    - Changes in behavior provide insights into the functional roles of targeted brain circuits.
  4. Experimental Design:
    - Optogenetic stimulation is applied during behavioral tasks or in specific contexts to assess its impact on behavior.
  • Control experiments, including sham stimulation or stimulation of non-targeted brain regions, help elucidate the effects of optogenetic manipulation.
  1. Insights:
    In vivo optogenetics for behavior allows researchers to link neuronal activity to behavioral outcomes, shedding light on the neural basis of complex behaviors.
50
Q

Neuronal Manipulation with Optogenetics and DREADDs

A
  1. Method:
    Optogenetics and Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) enable precise manipulation of neuronal activity with high spatiotemporal resolution.
  2. Capabilities:
    - These techniques allow for both excitation and inhibition of specific neuronal populations in the brain.
  3. Applications:
    - Utilized in various research domains, including behavioral testing, studying plasticity, analyzing functional outputs of neurons, and exploring therapeutic interventions.
    - Offers versatility in experimental design, facilitating investigations into the neural mechanisms underlying complex behaviors and diseases.
  4. Benefits:
    - Optogenetics and DREADDs provide unprecedented control over neuronal activity, offering insights into the causal relationships between neural circuitry and behavioral outcomes.
  • Enables the development of targeted interventions for neurological disorders and the exploration of novel therapeutic strategies.

NEUR3310 Neuroscience (18 decks)

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