Neuroimaging Flashcards
What is the process of computer assisted tomography?
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.
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?
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.
What is computerized tomography?
the algorithm—and the computer software to implement it— that calculates the radiodensity for each point in the brain slice.
Computerized tomography is an example of an inverse problem, what does this mean?
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.
What is the difficulty with inverse problems?
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.
What are the abilities of CAT scans?
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.
What information does positron emission tomography provide?
insights into the function of the living brain as well as its anatomy.
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?
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
In PET what happens to the positron produced when isotopes decay?
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.
What is the spatial resolution of PET?
about 4–8 mm, not as good as CAT, but it can be used to
follow brain events over time.
How the nonmetabolizable analog of glucose, 2-deoxyglucose (2-DG) used in PET functional studies?
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.
What do PET studies show that implies that brief periods of brain activity can be supported by glycolysis?
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).
What gives rise to a net longitudinal magnetic field parallel to the scanner field in MRI?
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
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?
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).
How many coils is required to produce a MRI image?
actually requires a further three coils that produce magnetic field gradients in the x, y, and z directions.
What is the resolution of an MRI image?
MRI has a resolution < 1 mm.
An MRI method that records changes related to brain function in successive images is termed functional MRI (fMRI). Which is the most important one?
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.
What does BOLD depend on?
on the ratio of oxygenated to deoxygenated hemoglobin and this varies with
blood flow and metabolism.
What have BOLD studies shown about energy expenditure in the brain?
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.
What is electroencephalography?
Recording the net electrical activity of the brain by means of surface electrodes attached to the scalp
What are local field potentials?
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
What are the groupings of the frequencies of the LFPs?
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.
What are evoked potentials (EPs) or event-related potentials (ERPs)?
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.
What is is measured in magnetoencephalography (MEG)?
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
How are these magnetic fields measured in MEG?
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.
Why must MEG be done in a magnetically shielded environment?
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.
What is the major advantage of MEG over EEG?
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.
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)?
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.
To what depths is TMS effective?
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.
Why is the shape of the induced electrical field hard to model?
because of the nonuniform electrical properties of neural tissue
What is the resolution of TMS?
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.
There are two modes of TMS, single pulse and repetitive pulse. What is the advantage and disadvantage of single pulse?
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.
What is repetitive TMS?
This delivers exponentially rising and falling magnetic pulses with a frequency up to 50 Hz for several seconds.
What is repetitive TMS?
This delivers exponentially rising and falling magnetic pulses with a frequency up to 50 Hz for several seconds.
What does TMS have effects on?
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.
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?
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.
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?
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.
How does intracellular recording measure membrane potentials directly?
it is necessary to have two electrodes, one inside the cell, the other outside, both connected to a voltmeter of some description.
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?
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.
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?
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.
How do neurophysiologists stimulate neurons directly?
by injecting an electrical current into it via a stimulating microelectrode. The stimulator normally delivers a square wave
current pulse.
Which three variables can e altered at will on most neuron stimulators?
the duration of the pulse, the amplitude of the injected current, and the frequency of the pulses.
What determines the response of a neuron to a stimulator?
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.
How are the sizes and time courses of depolarizing and
hyperpolarizing potentials seen in nerve cell injected with small currents determined?
by the passive electrical properties of the neuron.
What does voltage clamping measure?
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.
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?
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.
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?
an early inward current followed by a late outward
current. These are the currents that normally flow during an action potential.
In voltage clamping experiments with a squid, how are the ions carrying the inward and outward currents determined?
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+.
Patch clamping is an in vitro technique that makes it possible to study the electrophysiology of single ion channels. How does it work?
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
What is the cell attached configuration of patch clamping used for?
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.
How does the whole cell configuration of patch clamping work and what does it measure?
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.
How does the outside-out configuration of patch clamping work and what is it used for?
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.
What is the inside-out configuration of patch clamping used for?
the detailed examination of second messengers because these can be applied directly to the inside face of the membrane via the bath solution.
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?
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.
How does calcium imaging make visible how Ca2+ signals spread in time and space through cells?
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.
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?
confocal microscopy.
Why can confocal microscopy image living tissue?
because it uses fluorescence to image a sample and fluorescent dye can be taken up by living cells,
In confocal microscopy, what happens to the fluorescent dye molecule (photoactivatable fluorophore) when exposed to light?
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.
In confocal microscopy, what exactly happens to the dyed sample?
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.
What is the resolution of confocal light microscopy limited by?
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.
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?
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.
How does STED work?
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.
In STED, how can offset laser light, stimulated emission, and fluorescence be separated?
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
Before STED, how was fine detail visualized in neuron morphology?
approximated by three-dimensional reconstruction from ultra- thin (~50 nm) serial sections viewed under electron microscopy, a far more laborious technique.
Other than labour required, what is the other advantage of STED over electron microscopy?
can be done with live neural tissue, unlike electron microscopy. This means that nanoscale changes in neuroanatomy can be followed over time.
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?
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.
What is the spatial resolution of PLM limited by?
not limited by diffraction, but only by the precision with which each fluorophore can be localized.
In PLM how exactly is each protein observed individually?
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.
What is the precision of the position measurement of PLM dependant on?
on the contrast between the
brightness of the fluorophore compared with the background; the greater the contrast, the higher the precision.
What is an advantage of PLM?
that each dye molecule only undergoes a few photoexcitation cycles and this avoids a problem
called photobleaching.
What is a disadvantage of PLM?
the time it takes to acquire an image. The greater the fluorophore density in the sample, the longer the imaging time.
How can acquisition time in PLM be reduced?
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.
What is the brainbrow technique?
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.
What is the importance of the brainbrow technique?
it allows detailed studies of neural connectivity and circuitry.
What is the importance of the brainbrow technique?
it allows detailed studies of neural connectivity and circuitry.
Differential expression of which five protein fluorophores allows upwards of 90 distinct
hues to color individual cells?
yellow, orange, red, green, and cyan derivatives of the original green fluorescent protein (GFP)
When was the brainbrow technique invented?
in 2007 and originally allowed mapping of only a few neurons, but currently over 100 neurons can be mapped simultaneously.
The brainbrow technique relies on which genetic engineering system?
cre/loxP genetic engineering system which has been used extensively in brain research since 1998 to control gene expression in particular cell types.
What does the cre/loxp system allow?
selective gene deletion or activation to be generated by
recombination in specific cell types, and at particular times (e.g., stages in development).
What does the cre/loxp system require?
two types of transgenic animal (usually mice) to be engineered which are sub-
sequently crossed
How are Cre animals created?
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).
How are floxed animals created?
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.)
In both cases of floxed and cre animals, what is common in how are they genetically engineered?
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.
What is the principle of the cre/loxp system?
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.
The cre/loxP system can also be used to study the effect of activating a transgene. How is this achieved?
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.
In the brainbow technique constructs are created with several loxP sites flanking fluorescent protein genes. What does this allow?
a variety of excisions to take place resulting in a range of
differently color-coded neurons
What does optogenetics provide?
a set of techniques which allow millisecond control and sensing of brain processes in targeted populations of neurons using light.
What are the two types of optogenetic devices?
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.
How are the sensor and actuator proteins manufactured in optogenetics?
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.
What is the capacity for genetic engineering techniques in terms of optogenetics limited to?
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.
What do some sensors and actuators require, in addition to genetically
encoded proteins?
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.
What is the disadvantage of introducing DNA in the form of viruses/ plasmids in optogentics?
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.
What have protein based optical sensors been developed for?
membrane voltage, intracellular calcium concentration, neurotransmitter release, and for a number of second messengers, for example cAMP.
What are sensors based on?
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.
What modulates the light emission from GFP derivatives?
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
What is necessary to detect rapid events with optogenetics?
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.
Why increasing the expression of the sensor often not the solution to sampling at a higher rate in optogenetics?
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.
What are most actuators?
at present they are ion channels, proteins which regulate ion channels, or ion
pumps.
What makes actuators light responsive?
Many are based on visual rhodopsin which is coupled to G protein or microbial rhodopsins that are proton or chloride pumps.
What do actuators allow control over?
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.
What can actuators be stimulated by?
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.
An extremely promising optogenetic device is channelrhodopsin-2 (ChR2), an algal protein. How does it respond to blue light?
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.
Other than its use as an actuator, what is ChR2 used for?
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.
What does the gene archaerhodopsin-3 (Arch) code for?
an outward proton pump
When Arch is virally expressed in the mouse cortex and illuminated with yellow light, what happens?
it almost completely silences
those neurons that express the protein.
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?
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.
What is Mac (another actuator protein)?
a light-driven proton pump derived from a fungus, silences neurons when
illuminated by blue light.
What does using Arch and MAc together in an optogenetic protocol allow?
silencing of two neural populations independently with two different wavelengths of light.
What was the first optogenetics experiment and when was it done?
triggering neuronal action potentials using light—was done in 2002.
Which animal models have optogenetic experiments been done in?
nematode Caenorhabditis elegans, the fruit fly Drosophila, zebra fish, mice, and primates
Which three broad classes of investigation has optogentics been used for?
● 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.
Optogenetics is being used in combination with other techniques, such as…?
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).
