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CAN > Circuits in the brain > Flashcards

Circuits in the brain Flashcards

1
Q

why is it important to map synaptic connections in the brain?

A

Understanding the structure of neuronal networks is the required step to then study the functional of neurons within the network
- allows us to study how neurons generate behaviour, memories, mood, etc.

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2
Q

For C. elegans, the identity and connections of all the neurons is unknown.

True or False?

A

FALSE

identity and connections are all known

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3
Q

Explain the Golgi method? and the scientists that pioneered it?

How were the neurons classified?

A

pioneered the science of connectomics

developed a method to stain neurons and fix tissue slices (Camillo Golgi)

Ramon y Cajal: credited with applying the Golgi method to systematically map, to a high degree of resolution, several types of neurons and classify them according to their shapes, their particular dendritic arborisations and axonal projections.

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4
Q

Explain the process of Biocytin staining?

A

Allows the targeted staining of neurons

Biocytin spreads throughout the entire neuron.

A sharp micropipette is pushed against the cell membrane of a neuron.

A chemical compound called biocytin is injected via the same pipette.

Biocytin can then be recognised by observing a specific antibody conjugated with an enzyme.

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5
Q

Does Biocytin give similar results to the Golgi method?

A

Yes, gives similar results to the one with the Golgi method, but with the added benefit that neurons can be targeted with high specificity

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6
Q

Once injected into the neuron, biocytin spreads throughout the entire cell

true or false?

A

TRUE

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7
Q

What is a limitation of this technique?

A

process requires that cells are still alive at the time of injection

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8
Q

How is the brain kept alive for this staining method?

A

brain has to be first sectioned, sections have to be kept alive in a culture medium, and subsequently patched and injected with biocytin

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9
Q

Explain lipophilic dyes?

and which is the most widely used lipophilic dye?

A

These are fluorescent molecules, highly soluble in the fatty lipid bilayer of the cell membrane.

There are several variances of lipophilic dyes, with DiI the most widely used.

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10
Q

How does DiI work?

A

diffuse passively along the cell membrane, eventually lighting up even very long axonal projections

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11
Q

explain Cholera toxin subunit B (CTB)?

  • the 2 subunits
  • can be conjugated with what
  • travels to what area
A

Subunit B of the cholera toxin is derived from Vibrio cholera.

Cholera toxin is formed from two components: A and B.

Component A is not used in this process: it causes the extrusion of water from the cells, leading to dehydration.

This technique uses only the subunit B, which binds to receptors on the cell surface and travels to the endoplasmic reticulum (ER).

It can be chemically conjugated with green fluorescent molecules.

At the ER, it is picked up by axons from the entorhinal cortex and transported back to the respective neuron somas.

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12
Q

Explain Anterograde tracers?

A

The tracer spreads in the same direction as the flow of information in a neuron within a network.

When an anterograde tracer is injected into area where neural cell bodies are present, it will spread towards the tip of the axon.

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13
Q

Explain retrograde tracers

A

The tracer works in the opposite way, moving against the direction of the information flow.

When a retrograde tracer is injected into area where axons are present, it will spread towards neuronal cell bodies.

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14
Q

Are Retrobeads and FluoroGold anterograde or retrograde tracers?

A

They are strictly retrogradely transported.

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15
Q

what is the process of retrobeads?

A

Retrobeads are injected into the hippocampus.

The beads are picked up by axon terminals of neurons that innervate the hippocampus.

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16
Q

FluoroGold is also known as?

A

hydroxystilbamidine

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17
Q

Explain Plant lectins?

A

Plant lectins are anterogradely transported molecules.

Lectins bind receptors on the cell membrane and are transported anterogradely to the tip of the axon.

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18
Q

They display a unique feature, which is?

A

they are released into the synaptic cleft and are picked up by the postsynaptic membrane.

In this way, they enter another neuron that is postsynaptic to the one initially targeted with lectin.

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19
Q

Part 2

Genetically-encoded tracers

A
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20
Q

One of the best-known vectors to deliver exogenous DNA sequences into neurons is the?

A

Adeno-associated virus (AAV)

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21
Q

explain the rAAV tracer in more detail?

A

Most content of the viral genome is removed and replaced with the gene of interest – for example, a fluorescent protein.

A suitable eukaryotic cell line is transfected with vectors that express all the missing viral genes and the recombinant genome.

This results in production of infective viral particles, released in the culture media.

The viral particles are purified and injected into the brain.

They are then picked up by cells and drive expression of the recombinant genes.

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22
Q

Recombinant AAVs are not transsynaptic and cannot infect other neurons other than those they entered at the first infection event.

true or false?

A

TRUE

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23
Q

AAVs are not thought to be pathogenic and are therefore a valid tool for gene therapy in humans.

true or false?

A

TRUE

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24
Q

EXPLAIN THE CreloxP system

A

Cre is a recombinase, normally found in simple viruses that infect bacteria.

The loxP sequence contains two left and right palindromic regions surrounding a unique region in between.

It recognises a specific DNA sequence known as the loxP site and cuts the double strand DNA molecule

Upon the double strand cut, the resulting DNA ends can be ligated back together with other compatible DNA ends.

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25
Q

Cre-mediated recombination between two loxP sites can lead to two very different outcomes, depending on the orientation of the loxP sites.

If the two IoxP sites are in opposite orientation what will occur?

A

then the result of Cre activity will be the inversion of the intervening DNA sequence.

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26
Q

If the two loxP sites are in identical orientation?

A

the result of Cre-mediated recombination will be the excision of the intervening sequence.

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27
Q

The organisation of a transgene that contains cDNAs encoding may contain what colour proteins?

A

green, yellow, red and blue fluorescent proteins

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28
Q

the ‘rainbow’ strategy: Without Cre activity, the transgene can only express what?

A

can only express GFP

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29
Q

Two incompatible lox sites can be used in combination to induce transgenic expression of a fluorescent protein only when what is there?

A

Cre recombinase is also present in the same cell

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30
Q

Is the pseudorabies virus vector a transsynaptic tool?

A

YES

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31
Q

EXPLAIN the pseudorabies virus vector in more detail?

A

Modified pseudorabies virus vectors (PRVs) retain the ability to replicate and to form infective viral particles- which are potentially toxic

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32
Q

They have been used successfully to detect, for instance, the entire neuronal network controlling pupil constriction in response to exposure to bright light

True or false?

A

True

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33
Q

The PRV can be modified to express a variety of fluorescent proteins which can then be coinfected in the same brain to study what?

A

parallel and or overlapping circuits.

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34
Q

explain how a classic, non-transsynaptic tracing system works?

A

Two sequential steps of tracer injection and retrograde tracing are required and false positives can be detected when unrelated neurons project to the site of the second injection

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35
Q

explain transsynaptic tools?

A

With a transsynaptic viral tracer, only one injection is needed, as the virus will then cross from post to pre synapse infecting the afferents neurons only.

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36
Q

Explain the Glycoprotein-deleted rabies virus (SADB19-DG)?

A

One gene – the G= or glyco-protein gene – is removed from the viral genome. It is typically replaced by the gene encoding for a fluorescent protein so that infected neurons can easily be detected.

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37
Q

How is the production of an infective viral particle achieved with this technique?

A

by providing all the missing viral sequences in cell lines in culture.

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38
Q

Define viral pseudo-typing?

A

Pseudotyping refers to the use of envelopes from different viruses. Viral pseudotyping changes the tropism of the virus.

eg. the rabies virus, which is a mammalian virus, changes tropism to avian cells because the envelope used is from an avian virus.

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39
Q

What is monosynaptic retrograde tracing?

A

the neurons of interest must be genetically engineered to express a receptor for EnvA

40
Q

They can then be infected with Cre-indicible AAVs containing the EnvA receptor TVA and the missing G protein to produce what?

A

infective particles that can cross synapses.

41
Q

The first injection (AAV helper viruses) induces selective expression of?

A

TVA and RG in Cre-expressing neurons (Cre+).

42
Q

The second injection (EnvA-coated rabies virus) results in?

A

rabies uptake by TVA-expressing neurons and transsynaptic retrograde transport of RG- coated rabies virus into upstream input neurons.

43
Q

explain mGRASP?

A

a simpler method to identify neurons that are connected by one synapse.

If the GFP protein is divided into two halves and these two halves are kept very close to each other, then they may behave as a intact GFP protein and emit fluorescent light.

44
Q

Must candidate pair be known beforehand? and why?

A

Yes

  • because each will be infected with a AAVs expressing one of the two halves of the GFP
45
Q

Part 3

Modifying GFP proteins

A
46
Q

Why do we modify tracer tools?

A

to provide information about neuronal activity and activation status

to allow us to control when they are active or silent

47
Q

explain the process of GCaMP?

A

The process involves generating a chimeric GFP fused with two calcium binding domains from other proteins

When the concentration of calcium in the cytoplasm is low, GFP fluorescence emission becomes very inefficient.

When calcium is available in the cytoplasm, the fluorescence is emitted with high efficiency.

48
Q

Define Optogenetics?

A

Optogenetics is the process of controlling neurons using light.

A combination of genetic engineering and use of light of defined wavelengths

49
Q

Channelrhodopsin (ChR2): light gated non specific ion channel does what?

A

An ion channel that activates on exposure to blue light. It allows the entry of positive ions into the cell and firing of action potentials.

50
Q

Halorhodopsin (NpHR): light gated ion pump does what?

A

Activates on exposure to yellow light and allows the entry of negative ions – it therefore suppresses neuronal firing.

51
Q

how can the system be implemented so that neurons can be controlled in a living mouse and the behaviours can be studied as a function of activation or inhibition of a specific neuron?

A

light has to be delivered where the neurons are.

light is generated using typically using LEDs or lasers

then requires an optic fibre that goes all the way into the brain to target those neurons that are supressed in the channelrhodopsin or the halorhodopsin.

52
Q

Designer receptors exclusively activated by designer drugs (DREADDs)

explain what these are?

A
  • a method for switching neurons on and off
  • uses compounds that are only recognised by one receptor type to control individual receptor types

Activation of DREADD receptors can lead to either neuronal firing or neuronal silencing

53
Q

What are the advantages of genetically-encoded tracers?

A
  • can be targeted to genetically defined neurons, via Cre activity
  • allow for combinatorial use of fluorophores

• engineered version of GFP can be useful to monitor
connectivity and activity

  • useful tools to modulate circuit activity
  • some recombinant viruses are good transsynaptic tracers
  • mapping of monosynaptic-restricted afferents
  • AAV potential for gene therapy in humans
54
Q

What are the disadvantages of genetically-encoded tracers?

A
  • the Cre-lox system can only use in genetically amenable model systems (eg drosophila, zebrafish, mouse)
  • their mammalian use is largely limited to the mouse
  • some recombinant viral vectors are toxic at cellular and/or systemic level
55
Q

Connectivity can be studied at differing resolutions.

The techniques described previously are on the what scale?

A

mesoscale

56
Q

The area of the thalamus that receives retinal innervation is known as?

A

dorsal lateral geniculate or dLGN.

57
Q

What are two ways in which the thalamus coordinates activity in the cortex?

A

a) transferring sensory information from the periphery of the body (such as the eyes) to higher cortical regions
b) By coordinating of the interaction between different areas of the cortex to support the elaboration of sensory information into cognition and conscious awareness

58
Q

From the thalamus, thalamocortical neurons of the dLGN extend long axons to relay visual information to the what?

A

primary visual cortex V1

59
Q

as from V1, information is then integrated with other senses and conscious experience in different parts of the brain cortex.

true or false?

A

TRUE

60
Q

The visual pathway is an ensemble of what?

A

parallel pathways, each one encoding a particular feature of the visual field.

61
Q

Visual information is detected by the?

A

separate components of the retinal circuitry

62
Q

What is feedforward inhibition?

A

information from the retina is transferred forward along the visual pathway but in a way that generates inhibition of downstream neurons, not excitation

63
Q

What is the origin of the visual pathway?

A

The retina

64
Q

What are the six main types of neurons that make up the layers of the retina?

A

1: rods
2: cones
3: horizontal cells
4: bipolar cells
5: amacrine cells
6: retinal ganglion cells (RGCs)

65
Q

Light will hit first the bottom layer (6) as this is the layer that faces the pupil. However, the vast majority of photoreceptors are organised on the opposite side of the retina. Photoreceptors are what?

A

Rods and cones

66
Q

What do Bipolar cells establish?

A

contacts with the ganglion cells that form the ganglion cell layer (GCL)

67
Q

These ganglion cells eventually extend long axons that leave the retina and form the what?

A

Optic nerve

68
Q

In-between bipolar cells and the retinal ganglion cells there is a layer of what cells?

A

amacrine cells

69
Q

what do the amacrine cells contribute to ?

A

contribute to the inner plexiform

layer (IPL).

70
Q

visual information is detected by the retinal circuitry in its separate components, so that some cells will only respond and encode contours, other specific colours, others movement etc

true or false

A

TRUE

71
Q

the visual pathway is an ensemble of what type of pathways

A

parallel pathways

72
Q

each parallel pathway encoding a particular feature of the visual field. We know that it is only when this information has reached the
(WHAT PART) of the brain that it gets combined again to re-create an image that closely resembles the outside world?

A

neocortex

73
Q

What protein is a marker of dLGN interneurons?

A

Sox14

74
Q

Part 2

Sox14 expressing neurons

A
75
Q

What are ES cells?

A

totipotent stem cells that retain the potential to divide and differentiate into all cell types of the body.

76
Q

the lateral geniculate is divided in three regions. What are the 3 regions?

A
  • ventral LGN (vLGN)
  • intergeniculate leaflet (IGL)
  • dorsal LGN (dLGN)
77
Q

Are they retino-recipient areas?

A

Yes

78
Q

all the LGN divisions are retinorecipient, only the what area receives the information destined for conscious vision?

A

dLGN

79
Q

The other regions do not transfer visual information to the cortex and therefore are thought to be involved in non-visual functions such as

A

the regulation of the circadian body clock.

80
Q

Sox14 neurons in the dLGN are therefore GABAergic or Glutamatergic local interneurons?

A

GABAergic

81
Q

Explain the strategy that is followed based on the modified rabies system?

A

Step 1: modify the rabies in one of two ways:

(a) stop it from spreading across synapses and infect new neurons by
removing the gene encoding for the viral glycoprotein G

(b) pseudotyping the rabies with an envelope protein from an avian virus so that it can only infect either bird or mammalian neurons
that are made to express the avian receptor called TVA

Step 2: Target neurons with Cre-dependent AAV viruses

use the modified rabies to study the direct synaptic afferents to the neurons of interest by:

Targeting these neurons first with Cre-dependent AAV viruses, making them express the EnvA receptor TVA and the rabies G.

Step 3: perform a precise brain injection in the dLGN to deliver the modified rabies virus (made to express a red fluorescent protein):

The only cells that can be infected by the modified rabies are those GABA dLGN interneurons that were previously targeted during embryonic development.

82
Q

What is retino-geniculate convergence?

A

The phenomenon where we have three RGCs (retinal ganglion cells) of the same type projecting onto one inter neuron

83
Q

TOPIC 3: MINDFULNESS

A
84
Q

What is mindfulness?

A

a term derived from the religious teachings of Buddhism

active watchful mind, the activity of mind and constant presence of mind, which is one of the duties most frequently inculcated on the good Buddhist.

‘the awareness that arises from paying attention on purpose, in the present moment and non-judgmentally’

85
Q

we know that the practice of sati is important in enabling us to develop what?

A

insight into the true nature of reality.

86
Q

mindfulness training can provide a way of changing what?

A

the way we relate to our symptoms rather than changing the symptoms themselves, and the emphasis is on an integrated awareness of the body and the mind as a whole rather than working with specific aspects of experience.

87
Q

MBSR (mindfulness based stress reduction) is the overarching framework with components of CBT integrated. MBCT was developed by John Teasdale, Mark Williams and Zindel Segal with a specific aim of preventing what?

A

relapse of depression

88
Q

In MBCT, the core skill to be developed is the ability to do what?

A

step away from our own thinking processes and to see them more clearly.

89
Q

MBSR has been found to be effective in what type of clinical practices?

A

chronic pain, cardiovascular disease and as an adjunct treatment for many different types of cancer

90
Q

What is MBCT?

A

mindfulness-based cognitive therapy

91
Q

The Anterior cingulate cortex (ACC) is part of?

A
  • limbic system
  • ‘default mode network’
  • frontoparietal attention networks
92
Q

What is the anterior cingulate cortex associated with?

A
  • emotional regulation
  • pain: response to nociceptive stimuli
  • cognition
93
Q

Regions that mediate anxiety and fear-related responses?

A

Amygdala

Periaqueductal grey matter

94
Q

Short-term practice of a form of mindfulness meditation was shown to?

A

reduce levels of anxiety and cortisol produce

changes in the ACC

increase activity in ventral ACC

95
Q

What is the ventral AC involved in?

A

Involved in the regulation of the amygdala

CAN (5 decks)

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