Neuroimaging

Question 1

What is the process of computer assisted tomography?

Answer 1

The head is placed between a source which emits a narrow beam of X-rays and an X-ray detector. A series of measurements is made of X-ray transmission. The source and detector are rotated as a pair through a small angle and a further series of measurements taken. This is repeated until the source and detector have rotated through 180∞. The radiodensity of each region of the head is computed from the transmission data for all of the beams that have traversed that region, and the results visually displayed.

Question 2

CAT scans provide a view through a single slice of brain lying at a known orientation. How is the whole brain able to be scanned?

Answer 2

By moving the head at right angles to the orientation plane for a short distance another section can be
imaged. This is repeated until the whole brain has been scanned.

Question 3

What is computerized tomography?

Answer 3

the algorithm—and the computer software to implement it— that calculates the radiodensity for each point in the brain slice.

Question 4

Computerized tomography is an example of an inverse problem, what does this mean?

Answer 4

starts with a data set from
which initial parameters, in this case source location, must be calculated. It contrasts with forward problems in which the source location is known and it is the data set which is calculated.

Question 5

What is the difficulty with inverse problems?

Answer 5

they do not have unique solutions. Hence they have to be constrained by assumptions and prior modeling based on earlier
results to find the most likely solution.

Question 6

What are the abilities of CAT scans?

Answer 6

CAT can distinguish tissues which differ in X-ray opacity by 1% (the lower the density the darker the image) with a spatial resolution of about 0.5 mm. Blood vessels can be seen by injection of radio-opaque dyes.

Question 7

What information does positron emission tomography provide?

Answer 7

insights into the function of the living brain as well as its anatomy.

Question 8

PET It uses the principles of computerized tomography in which g-ray detectors are located around the head and the source is a positron-emitting compound, either injected or inhaled, which enters the brain. What are these compounds?

Answer 8

Compounds used include neurotransmitters, receptor ligands, and glucose analogs which are used for studying brain activity. Typically they are radiolabeled with ** These isotopes have short half-lives, decaying to the element with atomic number one less

Question 9

In PET what happens to the positron produced when isotopes decay?

Answer 9

The positron (e+ , the antiparticle of the electron) travels a short distance before colliding with an electron (e-). The two particles annihilate with the production of two g-ray pho-
tons that shoot off in exactly opposite directions. These are detected simultaneously by a pair of detectors 180∞ apart. This coincidence detection permits localization of the site of the g-ray emission, which is between 2 and 8 mm from the positron source, depending on the isotope used.

Question 10

What is the spatial resolution of PET?

Answer 10

about 4–8 mm, not as good as CAT, but it can be used to
follow brain events over time.

Question 11

How the nonmetabolizable analog of glucose, 2-deoxyglucose (2-DG) used in PET functional studies?

Answer 11

This molecule crosses the blood–brain barrier, is transported into neurons and phosphorylated to 2-DG-6-phosphate, so it remains in the cell. However, it cannot be metabolized further. This means it acts as a marker for local glucose uptake and therefore of neuron activity. Imaging the distribution
of [18/9F]2-DG while subjects engage in sensory, motor, or cognitive tasks reveals how these functions are localized in the brain.

Question 12

What do PET studies show that implies that brief periods of brain activity can be supported by glycolysis?

Answer 12

these studies show that during transient increases
in neuronal activity, the rise in local cerebral oxygen consumption (as measured by 15OPET) does not match the increase in glucose utilization (as estimated from 2-DG PET).

Question 13

What gives rise to a net longitudinal magnetic field parallel to the scanner field in MRI?

Answer 13

Nuclei with odd mass number, for example, 1
1H, generate a magnetic field along their spin axis. In the powerful magnetic field of an MRI scanner, hydrogen nuclei can adopt one of two orientations; with their magnetic fields either parallel or antiparallel to the external field. The parallel state has a slightly lower energy and normally a small excess of nuclei will be in this state

Question 14

A cylindrical coil placed around the head broadcasts a radio frequency (rf) pulse to a slice of head at right angles to the main scanner field. What does this rf do to the nuclei in MRI?

Answer 14

The rf pulse makes the nuclei wobble around their magnetic axis—rather like a spinning top as it slows down—with the rate of wobbling in resonance with the pulse frequency. The wobble generates an electric field
that is received by the coil, producing a transverse magnetic field at right angles to the scanner field. When the rf pulse is turned off the nuclei return to their original state, and the longitudinal and transverse fields decay with relaxation times that are characteristic for the nucleus and its chemical environment (e.g., lipid or aqueous).

Question 15

How many coils is required to produce a MRI image?

Answer 15

actually requires a further three coils that produce magnetic field gradients in the x, y, and z directions.

Question 16

What is the resolution of an MRI image?

Answer 16

MRI has a resolution < 1 mm.

Question 17

An MRI method that records changes related to brain function in successive images is termed functional MRI (fMRI). Which is the most important one?

Answer 17

blood oxygen level
detection (BOLD) which provides a very sensitive measure of cerebral cortical activity with a voxel (volumetric pixel, the 3-D analog of a pixel in a 2-D image) size of 2 mm
on each side, following changes in activity with a time resolution of a few seconds.

Question 18

What does BOLD depend on?

Answer 18

on the ratio of oxygenated to deoxygenated hemoglobin and this varies with
blood flow and metabolism.

Question 19

What have BOLD studies shown about energy expenditure in the brain?

Answer 19

most of the energy expenditure of the brain is related to synaptic events
rather than the generation and propagation of action potentials. Indeed it seems that action potentials are produced using only 30% more energy than the calculated theoretical minimum.

Question 20

What is electroencephalography?

Answer 20

Recording the net electrical activity of the brain by means of surface electrodes attached to the scalp

Question 21

What are local field potentials?

Answer 21

Large numbers of cerebral cortical cells fire in synchrony and consequently their summed activity produces local field potentials (LFPs) big enough that they can be recorded with scalp electrodes. By using an
array of electrodes, activity of different brain areas can be examined simultaneously. The recording may be monopolar—each scalp electrode measures the potential with respect
to a distant indifferent electrode—or bipolar, in which the potential is measured between a pair of scalp electrodes

Question 22

What are the groupings of the frequencies of the LFPs?

Answer 22

alpha (8–13 Hz), beta (13–30 Hz), delta (1–4 Hz), theta (4–7 Hz). Activity in these frequency bands correlates with behavioral state, for example, sleep, arousal, or learning.

Question 23

What are evoked potentials (EPs) or event-related potentials (ERPs)?

Answer 23

They are brief fluctuations in the EEG generated by sensory, perceptual or cognitive stimuli. These potentials are used to
investigate the context, timing, and brain regions implicated in the process of interest.

Question 24

What is is measured in magnetoencephalography (MEG)?

Answer 24

the synchronized flow of currents along dendrites of about 50 000 cortical pyramidal cells all oriented in the same direction is sufficient to set up a measurable, if weak,
magnetic field

Question 25

How are these magnetic fields measured in MEG?

Answer 25

using an array of magnetometers called SQUIDS (superconducting quantum interference devices) which surround the head. The MEG signal is generated mostly by the flow of intracellular currents in dendrites generated by synaptic activity.

Question 26

Why must MEG be done in a magnetically shielded environment?

Answer 26

Because cortical activity generates a field of order 10-13 tesla (T) compared with the Earth’s magnetic field of 3.1 x 10-5T. SQUIDs are sensitive to magnetic fields as small as 10-18 T.

Question 27

What is the major advantage of MEG over EEG?

Answer 27

its temporal resolution of better than 1 ms, which is comparable to intracranial electrodes. Also, magnetic fields are less distorted by skull anatomy than electric fields, which gives MEG a better spatial resolution.

Question 28

A magnetic field changing with time (in strength or direction or both) induces an electrical field how is this exploited In transcranial magnetic stimulation (TMS)?

Answer 28

by using electromagnetic coils to induce currents in the brain which influence firing of neurons directly beneath the coil. The currents attenuate with distance from the coil.

Question 29

To what depths is TMS effective?

Answer 29

to depths of 1.5–3 cm so the cerebral and cerebellar cortices are the usual targets, but more powerful coils can be used to affect subcortical structures.

Question 30

Why is the shape of the induced electrical field hard to model?

Answer 30

because of the nonuniform electrical properties of neural tissue

Question 31

What is the resolution of TMS?

Answer 31

it has a high spatial resolution (< 1 cm) if a figure-of-eight coil is used which concentrates magnetic flux at the node of the coil, and a temporal resolution of tens of ms.

Question 32

There are two modes of TMS, single pulse and repetitive pulse. What is the advantage and disadvantage of single pulse?

Answer 32

it can be time-locked to the delivery of a stimulus so can allow precise timing of any effect. However, single pulses may not always be effective and hence repetitive TMS (rTMS) can be used.

Question 33

What is repetitive TMS?

Answer 33

This delivers exponentially rising and falling magnetic pulses with a frequency up to 50 Hz for several seconds.

Question 34

What is repetitive TMS?

Answer 34

This delivers exponentially rising and falling magnetic pulses with a frequency up to 50 Hz for several seconds.

Question 35

What does TMS have effects on?

Answer 35

on visual perception, movement control, attention, memory, language, and decision making. TMS can excite or inhibit depending on the stimulus characteristics. For example, 10 Hz rTMS to the motor cortex stimulates muscle contraction and improves performance in a motor learning task whereas I Hz rTMS impairs motor learning. When used to study cognition it is usually inhibitory so that it interferes with performance of cognitive tasks, either increasing reaction time or increasing the number of errors.

Question 36

The firing patterns of either single neurons or clusters of neurons in living animals in response to physiological stimuli are obtained by extracellular recording. What does this techniques use?

Answer 36

two fine electrodes usually of tungsten or stainless steel. One, the exploring (focal) electrode is placed as close as possible to a neuron. The second, indifferent electrode is placed at a convenient distance. Neuron activity will cause currents to flow between the two electrodes. These currents are amplified and fed to a computer. By convention, if the exploring electrode is positive with respect to the indifferent electrode an upward deflection is recorded. The polarity, shape, amplitude, and timing of the recorded waveform generated by neural activity will depend on the position of the electrodes. The closer the exploring electrode is to the neuron, the larger the measured signal. Changing the distance between the two electrodes or altering their relative positions will modify all the above parameters.

Question 37

The technique can be used in brain slices or other in vitro preparations or in vivo, for example, in anesthetized animals. What is a useful technique in animals?

Answer 37

inserts electrodes into the brain that are attached to a connector cemented into the skull. This is done under anesthetic. The animal is allowed to recover. As required, the recording circuitry (amplifier to computer) is plugged into the connector. This allows electrophysiology in conscious, behaving animals, using sophisticated experimental protocols over long periods.

Question 38

How does intracellular recording measure membrane potentials directly?

Answer 38

it is necessary to have two electrodes, one inside the cell, the other outside, both connected to a voltmeter of some description.

Question 39

Because neurons are small the tip of the intracellular electrode impaling the cell needs to be very fine when doing intracellular recording. How is this achieved?

Answer 39

glass micropipettes are manufactured to have a tip diameter of less than 1 μm. The micropipette is filled with an electrolyte (commonly KCl at a concentration of 0.15–3 M) to carry the current, so forming the microelectrode.

Question 40

Typically transmembrane potentials are less than 0.1 V and so must be amplified with an operational amplifier in intracellular recording. How does this work?

Answer 40

This has inputs from both the intracellular microelectrode that impales the cell and the reference (bath or indifferent) electrode, which is placed in the solution bathing the cell. If no potential difference exists between the microelectrode and the reference electrode the amplifier output will be zero. If a potential difference exists between the electrodes, however, the amplifier generates a signal, the magnitude of which is proportional to the potential. The output of the amplifier goes to the analog-to-digital port of a computer running software which allows display, storage, and analysis of the data.

Question 41

How do neurophysiologists stimulate neurons directly?

Answer 41

by injecting an electrical current into it via a stimulating microelectrode. The stimulator normally delivers a square wave current pulse.

Question 42

Which three variables can e altered at will on most neuron stimulators?

Answer 42

the duration of the pulse, the amplitude of the injected current, and the frequency of the pulses.

Question 43

What determines the response of a neuron to a stimulator?

Answer 43

The direction of the current (which is defined as the flow of positive charge). If a small inward current is injected into a cell it will become a little more inside positive. This is a decrease in the membrane potential because Vm gets closer to zero and is called a depolarization. If, on the other hand, an outward current is injected (that is, if current is withdrawn from the cell) then the membrane potential increases; this is called hyperpolarization.

Question 44

How are the sizes and time courses of depolarizing and hyperpolarizing potentials seen in nerve cell injected with small currents determined?

Answer 44

by the passive electrical properties of the neuron.

Question 45

What does voltage clamping measure?

Answer 45

the currents that flow across an excitable cell membrane at a fixed potential. Measuring currents is important because it provides information on which ions might be responsible for changes in membrane potential.

Question 46

Only if both V and R are known can I be calculated from Ohm’s law, V = IR. How does voltage clamping circumvent this problem?

Answer 46

by measuring the transmembrane potential and having a feedback amplifier in the circuit which injects into the cell the current that is needed to keep the membrane potential constant; that is, the voltage is clamped. The current that must be injected by the circuit to keep the voltage fixed has to be the same size as the current flowing through the ion channels that would normally cause the potential to change. The voltage at which the membrane is clamped is called the command voltage. By examining the currents that flow across a membrane over a range of command voltages it is possible to determine which ions carry the currents.

Question 47

During voltage clamping, when the command voltage is switched to 0; it gives rise to a capacitance current, as the change in voltage alters the amount of charge stored on the nerve cell membrane, what happens after this?

Answer 47

an early inward current followed by a late outward current. These are the currents that normally flow during an action potential.

Question 48

In voltage clamping experiments with a squid, how are the ions carrying the inward and outward currents determined?

Answer 48

When the squid axon is bathed in a solution that contains no sodium the early inward current is abolished. This shows that the early inward current is carried by Na+. The same result is seen if an axon immersed in normal seawater is poisoned with the neurotoxin tetrodotoxin (TTX). By binding to the external mouth of the Nav, TTX prevents Na+ from permeating through the channel. TTX added to any nerve preparation abolishes action. potentials. Similarly, tetraethylammonium (TEA) which blocks Kv s, when added to the bathing medium abolishes the late outward current, showing that it is carried by K+.

Question 49

Patch clamping is an in vitro technique that makes it possible to study the electrophysiology of single ion channels. How does it work?

Answer 49

by forming a very high electrical resistance seal between a glass micropipette and the surface of a cell. Only currents flowing through the patch under the electrode will be recorded. This permits the extremely small currents that flow through single ion channels (about 1 pA) to be measured. The electronics allows the voltage of the patch to be clamped so voltage-clamping experiments can be performed

Question 50

What is the cell attached configuration of patch clamping used for?

Answer 50

used for measuring single channel currents in intact cells. Second-messenger-induced modifications of the patched channels can be investigated in response to bathing the cell with specific agents, for example neurotransmitters.

Question 51

How does the whole cell configuration of patch clamping work and what does it measure?

Answer 51

the patch under the microelectrode is ruptured. The current flowing through the electrode represents the sum of all the currents flowing through individual ion channels on the cell surface. Hence whole cell patching measures macroscopic currents.

Question 52

How does the outside-out configuration of patch clamping work and what is it used for?

Answer 52

used to study single-channel currents in which the patch is removed from the cell but remains sealed to the pipette tip. This configuration is used to study the effects of ligands such as neurotransmitters, hormones, or externally acting drugs on channels. These ligands are added to the bath because bath solutions can be changed much more easily and rapidly than the pipette solution. This has obvious advantages for performing complicated experiments such as investigating dose–response relationships.

Question 53

What is the inside-out configuration of patch clamping used for?

Answer 53

the detailed examination of second messengers because these can be applied directly to the inside face of the membrane via the bath solution.

Question 54

Each of the square wave events is the opening of a single channel in a patch clamp recording, what do the parameters of the waves measure?

Answer 54

The height of the wave is the unitary channel current, the duration of the wave is the time for which it is open. Statistical analysis of large numbers of opening events provides estimates for parameters such as mean channel open time, and allows models of channel kinetics to be tested. Such studies are very useful in fathoming out how neuroactive drugs act at the molecular level.

Question 55

How does calcium imaging make visible how Ca2+ signals spread in time and space through cells?

Answer 55

Fluorescent dyes are used that on binding Ca2+ absorb UV light at a different wavelength than they do in the unbound state. Neurons are preloaded with the dye and the emission of UV from the dye is observed in response to its excitation by the two distinct absorption wave- lengths. This gives a quantitative measure of how the concentration of Ca2+ changes in the neuron in real time.

Question 56

Conventional optical microscopy has a resolution limited approximately by half the wavelength of light. What method of light microscopy gets closest to the limits imposed by the physics of light diffraction?

Answer 56

confocal microscopy.

Question 57

Why can confocal microscopy image living tissue?

Answer 57

because it uses fluorescence to image a sample and fluorescent dye can be taken up by living cells,

Question 58

In confocal microscopy, what happens to the fluorescent dye molecule (photoactivatable fluorophore) when exposed to light?

Answer 58

is excited by light of a specific wavelength so that electrons are boosted from the ground state to high quantum energy states. After a few nanoseconds the molecule de-excites back to the ground state, emitting lower energy photons with longer wavelength because some of the energy of the exciting photon is dissipated internally within the molecule.

Question 59

In confocal microscopy, what exactly happens to the dyed sample?

Answer 59

sample is illuminated by laser light tuned to excite the fluorescent dyes in the sample. Light is brought to focus on a small region of the sample. Scanning mirrors move the laser across the specimen so that each small region of it can be excited in turn. The fluorescent light emitted by the specimen passes through a pinhole before entering a digital camera. This eliminates all out of focus light; that is, only light from the focal plane reaches the camera.

Question 60

What is the resolution of confocal light microscopy limited by?

Answer 60

by the size to which the excited region can be focused and means that structures closer than 200 nm in the focal plane cannot be resolved. The depth resolution (along the optical axis) is worse, around 500 nm.

Question 61

Several super-resolution imaging techniques have been developed which allow light microscopy to obtain information far below the l/2 diffraction limit of visible light. One is stimulated-emission depletion microscopy (STED), how does it circumvent the diffraction limit?

Answer 61

limit by targeted de-excitation of dye molecules without making them fluoresce. Essentially this switches the dye molecules off, reducing the size of the excited region.

Question 62

How does STED work?

Answer 62

by using two lasers, an excitation laser and an offset laser. Fluorescent dyes which label specific sites of a sample are excited by light of a particular wavelength produced by the excitation laser. After a few nanoseconds the dye molecule spontaneously relaxes to the ground state by emitting a fluorescence photon of longer wavelength; that is, a redder light. However, an excited molecule can also return to its ground state by stimulated emission instead of spontaneous relaxation and fluorescence. This can be triggered by irradiating the excited dye molecule with light of similar wavelength to the fluorescence light. This is done with the offset laser. In response, the dye molecule returns to the ground state, emitting a photon of exactly the same wavelength but without emitting a fluorescence photon.

Question 63

In STED, how can offset laser light, stimulated emission, and fluorescence be separated?

Answer 63

by appropriate optics. The offset laser is focused so as to de-excite (by stimulated emission) almost all of the dye molecules in a circular region activated by the excitation laser. The de-excitation zone is a broad annulus, leaving just a few fluorophores in the center of the region excited and hence able to fluoresce. This allows nanoscale resolution (< 10 nm) permitting extremely fine detail to be visualized in neuron morphology

Question 64

Before STED, how was fine detail visualized in neuron morphology?

Answer 64

approximated by three-dimensional reconstruction from ultra- thin (~50 nm) serial sections viewed under electron microscopy, a far more laborious technique.

Question 65

Other than labour required, what is the other advantage of STED over electron microscopy?

Answer 65

can be done with live neural tissue, unlike electron microscopy. This means that nanoscale changes in neuroanatomy can be followed over time.

Question 66

To spatially resolve closely spaced fluorescing dye molecules (fluorophores), a variety of techniques such as photoactivated localization microscopy (PLM), work by separating the fluorescence of each emitter in time. What is the fundamental principle of these techniques?

Answer 66

Instead of imaging all the fluorophores simul- taneously, these techniques image each individual fluorophore one at a time, making it possible to find the position of each molecule with high precision. Once all of the positions have been found, they are plotted as points in space to construct an image.

Question 67

What is the spatial resolution of PLM limited by?

Answer 67

not limited by diffraction, but only by the precision with which each fluorophore can be localized.

Question 68

In PLM how exactly is each protein observed individually?

Answer 68

the excitation light illuminates the entire sample but at low intensity so that only a few fluorophores are excited at a time, and this fluorophore excitation is stochastic; that is, whether a given dye molecule is excited at a given time is random. This enables different fluorophores to be excited at different times, so they can be imaged individually. Computer algorithms are used to construct an image of the sample from the locations of each fluorophore.

Question 69

What is the precision of the position measurement of PLM dependant on?

Answer 69

on the contrast between the brightness of the fluorophore compared with the background; the greater the contrast, the higher the precision.

Question 70

What is an advantage of PLM?

Answer 70

that each dye molecule only undergoes a few photoexcitation cycles and this avoids a problem called photobleaching.

Question 71

What is a disadvantage of PLM?

Answer 71

the time it takes to acquire an image. The greater the fluorophore density in the sample, the longer the imaging time.

Question 72

How can acquisition time in PLM be reduced?

Answer 72

by using higher excitation intensity (the fluorophore turns off within nanoseconds of it being excited), because imaging time is determined by how rapidly each fluorophore turns on and off. but this can limit resolution.

Question 73

What is the brainbrow technique?

Answer 73

Individual neurons and glia in the living brain can be made to express specific fluorescent proteins using genetic engineering techniques. The cells can be imaged in brain sections with confocal microscopy, making it possible to map each one because it has a distinctive color.

Question 74

What is the importance of the brainbrow technique?

Answer 74

it allows detailed studies of neural connectivity and circuitry.

Question 75

What is the importance of the brainbrow technique?

Answer 75

it allows detailed studies of neural connectivity and circuitry.

Question 76

Differential expression of which five protein fluorophores allows upwards of 90 distinct hues to color individual cells?

Answer 76

yellow, orange, red, green, and cyan derivatives of the original green fluorescent protein (GFP)

Question 77

When was the brainbrow technique invented?

Answer 77

in 2007 and originally allowed mapping of only a few neurons, but currently over 100 neurons can be mapped simultaneously.

Question 78

The brainbrow technique relies on which genetic engineering system?

Answer 78

cre/loxP genetic engineering system which has been used extensively in brain research since 1998 to control gene expression in particular cell types.

Question 79

What does the cre/loxp system allow?

Answer 79

selective gene deletion or activation to be generated by recombination in specific cell types, and at particular times (e.g., stages in development).

Question 80

What does the cre/loxp system require?

Answer 80

two types of transgenic animal (usually mice) to be engineered which are sub- sequently crossed

Question 81

How are Cre animals created?

Answer 81

with a construct that has a gene for a viral recombinase enzyme, cre (causes recombination) downstream of a cell specific promoter. These can be specific for glia (glial fibrillary acidic protein promoter), neurons (e.g., synapsin 1 or neuron-specific enolase promoters), or even for specific neuron types (e.g., CaMKII promoter is selective for CA1 cells of the hippocampus).

Question 82

How are floxed animals created?

Answer 82

are created in which all nucleated cells contain the target gene flanked on both sides by a 34 base pair loxP sequence, derived from bacteriophage P1. (Bacteriophages are viruses that infect bacteria.)

Question 83

In both cases of floxed and cre animals, what is common in how are they genetically engineered?

Answer 83

genetically engineered by having the construct inserted into a vector (often based on the double stranded circular DNA molecules, plasmids, that make up the genome of bacteria). The vector can be engineered with a variety of reporter genes so that its presence can be detected. It can then be injected into the pronucleus of fertilized oocytes or incorporated in embryonic stem cells that are then introduced into blastocysts (early embryos with 8–16 cells). The oocyte or blastocyst is subsequently implanted into a foster mother and the resulting offspring screened to ensure they have incorporated the constructs.

Question 84

What is the principle of the cre/loxp system?

Answer 84

the cre enzyme recognizes two loxP sequences if they are in the same 5¢–3¢ orientation, bringing them together so that recombination effectively excises the target gene between them, leaving behind one loxP site. This happens in the offspring (F1 hybrids) of cre and floxed animal crosses. The floxed constructs are expressed in all nucleated cells in the resulting offspring, but the cre construct is expressed only in the cell type targeted by the promoter. Hence the cre/loxP recombination is cell specific.

Question 85

The cre/loxP system can also be used to study the effect of activating a transgene. How is this achieved?

Answer 85

a floxed construct is produced in which loxP sequences flank a stop codon upstream of the target gene. Now recombination cuts out the stop codon in the cre- expressing cells and the transgene will be transcribed by DNA polymerase. The transgene will remain inactive in all other cells because these do not express cre. Hence the target gene is expressed in a cell-specific way.

Question 86

In the brainbow technique constructs are created with several loxP sites flanking fluorescent protein genes. What does this allow?

Answer 86

a variety of excisions to take place resulting in a range of differently color-coded neurons

Question 87

What does optogenetics provide?

Answer 87

a set of techniques which allow millisecond control and sensing of brain processes in targeted populations of neurons using light.

Question 88

What are the two types of optogenetic devices?

Answer 88

Sensors are proteins which transduce physiological signals in cells to light emissions so as to make specific cell functions visible. Actuators are proteins that respond to light signals by altering a physiological process.

Question 89

How are the sensor and actuator proteins manufactured in optogenetics?

Answer 89

produced by animals genetically engineered to manufacture them by introducing the encoding DNA into their genome. Genetic engineering techniques can in some instances ensure that the DNA is restricted to particular cell types.

Question 90

What is the capacity for genetic engineering techniques in terms of optogenetics limited to?

Answer 90

limited to date by our knowledge of gene expression in specific cell types. For example, whilst a requirement of dopaminergic cells is that the gene for the enzyme tyrosine hydroxylase (TH) must be switched on, this is also true of other catecholaminergic neurons, so animals engineered so that TH can be light activated or will emit light so as to indicate the enzyme’s activity, cannot be interpreted as just reflecting dopaminergic neuron function.

Question 91

What do some sensors and actuators require, in addition to genetically encoded proteins?

Answer 91

small molecules that must be injected or ingested. Alternatively, the DNA is introduced in the form of plasmids or viruses. These are subsequently taken up by the cells for which these vectors are the targets.

Question 92

What is the disadvantage of introducing DNA in the form of viruses/ plasmids in optogentics?

Answer 92

each animal has to be treated individually, making experiments time-consuming and laborious. However, it is sometimes needed to gain sufficiently high levels of expression of a protein.

Question 93

What have protein based optical sensors been developed for?

Answer 93

membrane voltage, intracellular calcium concentration, neurotransmitter release, and for a number of second messengers, for example cAMP.

Question 94

What are sensors based on?

Answer 94

a green fluorescent protein (GFP) chromophore. In one guise fusion proteins are created between GFP and another protein which is able to report some biochemical change. For example, a GFP–Shaker potassium channel fusion protein is able to signal voltage-dependent conformational changes in the channel as alterations in GFP fluorescence.

Question 95

What modulates the light emission from GFP derivatives?

Answer 95

Either: ● Protonation/deprotonation which is in turn affected by the conformational state of the protein that controls access of the chromophore to the solvent; or ● Variations in the electrical properties of neighboring chromophores brought about by changes in their proximity or orientation to each other

Question 96

What is necessary to detect rapid events with optogenetics?

Answer 96

to sample at a sufficiently high rate. Thus, to detect individual action potentials lasting one millisecond it is necessary to sample with a frequency greater than 1 kHz if all are to be captured. Short events produce fewer photons and so are harder to see.

Question 97

Why increasing the expression of the sensor often not the solution to sampling at a higher rate in optogenetics?

Answer 97

because the presence of sensor disturbs the variable under study. For example, the voltage sensors needed to detect action potentials change the capacitance of the cell membrane in which they are expressed, and this suppresses synaptic potentials thereby altering the behavior of the neurons under study.

Question 98

What are most actuators?

Answer 98

at present they are ion channels, proteins which regulate ion channels, or ion pumps.

Question 99

What makes actuators light responsive?

Answer 99

Many are based on visual rhodopsin which is coupled to G protein or microbial rhodopsins that are proton or chloride pumps.

Question 100

What do actuators allow control over?

Answer 100

the same variables that the sensors respond to: membrane voltage, intracellular calcium concentration, and secretion of signaling molecules. There are also light-activated G-protein-coupled receptors, second messengers, kinases, and transcription factors. A light-activated glutamate receptor (LiGluR) is a calcium channel and hence allows control over calcium dependent processes such as secretion.

Question 101

What can actuators be stimulated by?

Answer 101

by LED or laser light delivered via optical fibers. These can be used in vitro (e.g., in brain slices) or in vivo, implanted into the brain ahead of time: this allows optogenetic experiments to be done in awake, behaving animals. Actuators available now allow control over processes with a timescale of a few milliseconds to seconds.

Question 102

An extremely promising optogenetic device is channelrhodopsin-2 (ChR2), an algal protein. How does it respond to blue light?

Answer 102

It responds to blue light by opening a nonspecific cation conductance. The resulting inward Na+ current excites any neuron that has incorporated ChR2 into its membrane.

Question 103

Other than its use as an actuator, what is ChR2 used for?

Answer 103

fluorescently labeled ChR2 reveals light-stimulated axons and synapses in intact brain tissue. This has been used to study the molecular events in spike timing-dependent plasticity. In addition ChR2 has been used to map long-range cortico-cortical connections in the brain, and to map the spatial location of specific inputs on the dendritic tree of individual cortical pyramidal neurons.

Question 104

What does the gene archaerhodopsin-3 (Arch) code for?

Answer 104

an outward proton pump

Question 105

When Arch is virally expressed in the mouse cortex and illuminated with yellow light, what happens?

Answer 105

it almost completely silences those neurons that express the protein.

Question 106

Arch spontaneously recovers from light-dependent inactivation, unlike light-driven chloride pumps that enter long-lasting inactive states in response to light. What does this mean in terms of optogenetics?

Answer 106

that Arch could mediate several cycles of optical silencing during an experiment. Expressed in specific cells this could be used to investigate their role in any particular neuron function by temporarily inhibiting the cells. Unlike gene knockout experiments, optogenetic silencing can be transient and can be repeated any number of times, allowing animals to be used as their own controls.

Question 107

What is Mac (another actuator protein)?

Answer 107

a light-driven proton pump derived from a fungus, silences neurons when illuminated by blue light.

Question 108

What does using Arch and MAc together in an optogenetic protocol allow?

Answer 108

silencing of two neural populations independently with two different wavelengths of light.

Question 109

What was the first optogenetics experiment and when was it done?

Answer 109

triggering neuronal action potentials using light—was done in 2002.

Question 110

Which animal models have optogenetic experiments been done in?

Answer 110

nematode Caenorhabditis elegans, the fruit fly Drosophila, zebra fish, mice, and primates

Question 111

Which three broad classes of investigation has optogentics been used for?

Answer 111

● Tracing of neuronal connections. This has shown, for example, that neocortical pyramidal cell dendritic trees are segregated into functional domains according to input with local, ascending, and descending axons going to specific domains. ● Neural network activity. This has revealed that brain circuits exist for behaviors an animal would not normally exhibit, such as male courtship display in a female. ● Neural mechanisms of sensation, reward, and cognition.

Question 112

Optogenetics is being used in combination with other techniques, such as...?

Answer 112

extracellular recording from awake behaving animals and with fMRI (in this case to establish precisely what cellular events were required to explain the fMRI signal).