methods in modern neuroscience

Question 1

what are the pros and cons to using certain model organisms?

humans, primates, rodents, zebrafish, drosophila

Answer 1

humans - verbal feedback, restrictions due to ethics

Primates - similar to humans but can be used in ways humans cannot. Still ethical issues present

Rodents/zebrafish/drosophila - short life cycle - can make transgenic animal to observe mutations
Zebrafish - transparent and therefore can image the entire brain
Drosophila - invertebrates so very different to humans

Question 2

once you’ve chosen your model organism, what three questions do you need to answer when experimenting?

Answer 2

1. Describe the phenomenon or behaviour (behavioural and psychophysical techniques) e.g. object recognition when concerning the visual system
2. Which part of the brain is involved in a particular processing?
3. What is the circuit mechanism? How the neurons are organised, how the circuits develop/change with age

Question 3

how can we identify what area of the brain controls a certain process?

freebie - fMRI, what are pros and cons?

Answer 3

Lesions - directed in animal models, or opportunities in humans after strokes/neurodegenerative diseases

Whole brain imaging techniques - see what areas are active during certain behaviours

Multielectrode recordings in different brain errors

fMRI
Identifies metabolically active areas of the brain - telling us which part of the brain is involved in certain behaviours, can be used in humans. Con = low resolution, not for individual neurons/synapses etc…

Question 4

what two main kinds of staining are used and what are they good for?

what is used more often now?

Answer 4

Golgi staining (silver chromate) - sparsely labels neurons, by not staining all neurons individual ones can be viewed and morphology described
As seen in retinal sections - you can characterise different neurons

Nissl staining - to distinguish glia and neurons. The +ve dye binds to negative RNA, staining the nucleolus in both kinds of cells, but additionally staining Nissl bodies in neurons

Now we use fluorescent microscopy more often, e.g. GFP

Question 5

what are the limitations of staining?

Answer 5

Trying to stain individual neurons - can’t really do it
You cannot do both morphology experiments (like with the dyes) and electrophysiological experiments in the same cell

Question 6

how do sharp electrode recordings work?

Answer 6

Inserting a sharp electrode into a neuron to image the membrane potential of a neuron to view action potentials etc…
Typical neuron cell body = 50 microns in diameter
Tip of electrode = around 1 micron

Question 7

what are the disadvantages of sharp electrode recording? X3

Answer 7

Can’t change the solution inside and outside like you can with patch clamp

Can’t control membrane potential very well as the electrode does not form a tight seal with the membrane, there’s a big gap

Cannot look at single channels, just the neuron as a whole

Question 8

generally, what is the set up for a patch clamp experiment. ‘cell-attached’ configuration?

Answer 8

A glass micropipette with a fine tip (tip of electrode 1 x 10^-6 m or 1 micron) is filled with an electrolyte solution and brought into contact with the cell membrane

Seal Formation: Gentle suction applied to micropipette, forms tight seal (gigga seal) between the pipette tip and a small patch of the cell membrane, isolating a tiny area of the membrane, allowing precise measurements of ion channel activity

Question 9

how is data recorded from the patch-clamp technique? what is it useful for?

Answer 9

current generated by ion channel activity in the patch of membrane (one or two channels) are recorded using sensitive amplifiers, in picoAmps (1x10-12). These signals can provide information about the type and behaviour of ion channels present in the membrane, including their opening and closing (activation and inactivation) kinetics, conductance, and selectivity

Manipulation: Various drugs, ions, or other molecules can be applied to the extracellular solution to modulate ion channel activity, allowing researchers to investigate the effects of different compounds on channel function
Can look at single channels

Question 10

explain the ‘whole cell’ configuration of the patch clamp technique

what are you measuring/recording?

Answer 10

Stronger suction is applied to rupture the membrane patch and create a direct electrical connection between the pipette interior and the cell’s cytoplasm.

Recording: current from the entire cell interior, rather than just a small patch of membrane like in cell attached, is recorded using the micropipette.

This allows for more comprehensive analysis of cellular electrical activity,
measures the current from whole populations of ion channels/current value (sum of current created by all populations of ion channels)

Question 11

Explain the ‘inside out’ configuration of the patch clamp technique

what are you measuring/recording?

Answer 11

Going from cell attached, - you’ve formed the seal between micropipette and cell as normal, but now you pull away the pipette, and in doing so pull away a section of the membrane

Forget the rest of the cell left behind (unlike whole cell), you can change the ‘intracellular solution and measure the resultant current through the patch of membrane you pulled away

Question 12

explain the ‘outside out’ configuration of the patch clamp technique

Answer 12

Preparation of the cell: imagine you’ve got as far as the whole cell configuration - so you’ve formed a seal with the micropipette, applied suction to remove a section of the membrane and your pipette is continuous with the interior of the cell.
Now you retract/pull away the micropipette, resulting in an isolated patch of membrane with the interior exposed to the micropipette’s salt solution, and the exterior facing away from the pipette - i.e. you can change the extracellular medium (opposite of inside out)

Recording the electrical activity: A voltage-clamp amplifier is used to measure the flow of ions through the ion channels in the patch of membrane.
By controlling the voltage across the membrane and monitoring the resulting currents, researchers can characterise the properties of the ion channels, such as their conductance, gating kinetics, and pharmacological sensitivity

Question 13

how can we kind of do morphological and electrophysiological experiments in one?

Answer 13

Using whole-cell configuration, with a fluorescent dye in the micropipette, you can label the individual cell AND current from the neuron at the same time (gap junctions may result in staining of connected neurons)

So neurons with a certain morphology can be linked to a certain function/response type

Question 14

what are the limitations of using a fluorescent dye in whole cell patch clamp to see morphology and electrophysiology?

Answer 14

Cannot label many cells
Limited ability to label specific cell type
Limited ability to label cellular compartments
Limited ability for live labelling (cytotoxic dyes)

Question 15

what is GFP?

Answer 15

fusion protein
You can add it into the gene sequence of a protein of interest in order to follow that protein, or label a specific kind of cell/neuron (by choosing a protein it uniquely expresses, or a promotor?)

Question 16

what does GFP do and what are today’s developments in the area?

Answer 16

Stimulated by blue light to…
Emits a green light
excitation peak at 498 nm
emission peak at 508 nm

Now a load more colours have been developed, excitated by different wavelengths of light

You can label different cells with different colours, viewing them separately under different light, overlap to see all the different cells etc…

Question 17

explain the function of the components of a simple fluorescence (epifluorescence) microscope

Answer 17

two lenses, the objective lens which is focused onto the sample, and the tube lens which focuses the image onto your eye/camera

excitation filter - lets in only the wavelength of light that excites your protein (e.g. for GFP, blue light)

dichroic mirror - this reflects blue light (that’s passed through the excitation filter) onto the sample to stimulate your fluorescent protein
it then lets the emitted green light through to reach the tube lens (to be seen)

emission filter - just before the tube lens, only lets through light emitted by your fluorescent protein

Question 18

what are some features/abilities of more advanced fluorescent microscopes?

Answer 18

most fluorescent microscopes will have multiple filters you can change between

There are even microscopes with two different objective lenses allowing the sample to be viewed in a much higher resolution

Question 19

what version of GFP can we use to look at neuron activity?

Answer 19

GCaMP

this is a GFP based calcium indicator (active neurons have higher calcium levels so fluoresce more)

It visualises and measures calcium ion levels in living cells (corresponding to level of activity)

Question 20

what are the components of GCaMP and how do they work?

Answer 20

Circularly permutated/altered green fluorescent protein (GFP)

Calmodulin (CaM) domain

Calcium-binding M13 peptide

there is a conformational change in GFP upon calcium binding to M13 and calmodulin domains,
increasing fluorescence intensity as a result

Question 21

give three applications of GCaMP?

Answer 21

Neuroscience research
Monitoring neuronal activity and synaptic transmission
Studying physiological processes in living organisms (e.g., mice, fruit flies, zebrafish - entire brain can be imaged for example)

Question 22

what is a confocal microscope, and how does it work?

Answer 22

A way of improving image quality, using a pinhole between the objective and tube lenses

using a pinhole eliminates out-of-focus light, allowing only focused light from the focal plane to reach the detector

Question 23

how exactly is the image improved by using a confocal microscope?

Answer 23

Illumination of the sample point-by-point or line-by-line, reduces photobleaching and phototoxicity

Reduced background noise and improved contrast due to elimination of out-of-focus light

Improved axial (depth) resolution, as only the focused light contributes to the image

Question 24

what are three problems with using model organisms when investigating neuronal activity?

what are some solutions to this?

Answer 24

Animal is sedated using Na+ channel blockers

Animal is stressed due to being handled

Animal does not perform behaviour that it usually does in nature

solutions = freely moving animal (so less handling and no sedation), virtual reality to create scenario you want

Question 25

how can epifluorescence microscopy be used on moving model organisms?

Answer 25

In mice, embedding a tiny epifluorescence microscope in their skull to view a specific group of neurons/area of the brain during certain activities/in response to certain stimuli, while the mouse is free to move

Someone also invented a microscope to track movements of zebra fish while viewing their neurons or something idk

Question 26

what is channelrhodopsin?

Answer 26

An ion channel, modified so that stimulation by light (blue light, 480 nm) the channel opens

The channel is non-selective cation one, and therefore depolarises the membrane when open

So you can stimulate a neuron/increase its activity (if it expresses channelrhodopsin) by exposing it to blue light

Question 27

what is halorhodopsin?

Answer 27

An ion channel modified so that it is stimulated by yellow light - 570 nm

It is selective for Cl- and therefore hyperpolarises a neuron, reducing it’s activity/inhibiting it

Question 28

what’s the problem with large scale studies/whole organisms?

Answer 28

lots of neurons you’re looking at, you will have different morphologies, lots of different connections, different functions you need to align with morphology, different gene expression that needs to be investigated

Question 29

how well has the neuronal structure/connections of C. elegans been investigated? how was this done?

Answer 29

total of 302 neurons, all of which has been mapped in the C.Elegans connectome
Each neuron is individual and specific, with it’s own connections and function

By pairing fluorescent dye in different neurons you can map the connections between neurons

Question 30

what technique can be used to map the brain of larger animals?

where has this been done?

Answer 30

Take thin slices of the brain, use electron microscopy to image it

Repeat with loads and loads of slices and compile it together

This allows you to see individual synapses and the borders of neurons
You can look at neuron morphology and neuron connections

Drosophila - entire central brain has been mapped (125000 neurons, 20 million connections)

Question 31

you can map an organism’s entire central brain, but for this to be useful you need…?

Answer 31

a specific question to investigate

Question 32

how do you profile gene expression? (R_ _ To______)

Answer 32

RNA tomography

Only applicable to certain animals, but you section e.g. zebrafish embryos into 50-100 slices and sequence the RNA present in individual sections

Mathematical image reconstruction is used to determine 3D genome-wide expression patterns