lecture 6 LOs Flashcards
what are microelectrodes used for
to record different aspects of electrical neural activity
intracellular/patch clamp technique
animal must be anesthetized or an in vitro brain slice penetration is used
permits recording of membrane potential, changes in electrical currents
provides detailed information of how drugs affect synaptic transmission or even individual ion channels that regulate neural activity
extracellular technique
electride is adjacent to neurons
only records action potentials
multiple cells can be recoded from at once
can be done in awake/behaving animal to assess changes in cell firing associated with beh
soup method of receptor locating and quantifying
tissue sample is isolated and ground into homogenate for analysis (often used to quantify amount of targets)
slice method for locating and quantifying receptors
uses intact pieces/slices of tissue (often used for localizing targets in the brain)
radioligands for labeling receptors in tissue
ligands embedded with radioactive atom
receptor binding can be indexed by amount of radioactivity in sample or through special photography
antibodies to label receptors in tissue
created by injecting an antigen (receptor) into a host animal and collecting blood samples
antibodies (proteins that bind to specifically targeted proteins) are tagged with coloured chemicals or fluorescent dyes for visualization
what is radioligand binding/how does it work
solution contained tissue homogenate or generic cells transfected with specific receptor is incubated with radioligand
bathe cells expressing receptor of interest in solution containing radioligand
after washout, unbound radioligand is removed and remaining amount that is bound to the tissue is measured, reflects the number of receptors in the tissue
how is radioligand used to indentify novel drugs
a standard radioligand is combined with a test one
displacement of the standard at different doses of the test compound indexes test compounds affinity/selectivity to the receptor
metabotropic receptor drug efficacy measure
measure second messenger enzyme activity in cells transfected with receptor
apply different concentrations of drug to cells to obtain dose/response function on enzyme activity
ionotropic receptor drug efficacy measure
typically use electrophysiological meaures to assess how drug activates ionic currents mediated by receptor
receptor autoradiography for receptor location and distribution in the brain
radioligand is incubated with tissue slices and then exposed to film sensitive to radiography
requires the drug to be highly selective
immunocytochemistry to visualize receptor location/distribution
brain slices are ficed and incubated with an antibody
antibodies incubated with brain slices, attaches to the antigen on cells that contain it
in situ hybridization for receptor measures
used to locate cells in tissue slices that manufacture a particular protein
detects specific mRNA molecules involved in synthesis of target proteins
in situ hybridization advantages and disadvantages
adv: highly selective, easier to analyze vs autoradiography or ICC
can be used to label cells that may express a protein even when no antibody is available
extremely sensitive - detects small amounts of cells that express a particular gene
disadv: just because cell contains mRNA doesn’t mean protein has been made or inserted where it need to go
quantifying receptors w enzyme linked immunoabsorbent assay (ELISA)
antibodies for target protein are linked another enzyme that acts on a substrate to form a coloured product
standard amounts of target protein placed in some wells, homogenized tissue from test samples place in others
amount of colour change is used to quantify amount of protein
positron emission tomograpy (PET) for brain activty measures
uses injected radioactive isotopes to identify areas of brain activity when subject is performing a task
low temporal resolution
what can PET be used to study when combined with radiolabeled drugs
locations/concentrations of those receptors in living human brains
NT release: by measuring displacement of radioligand, we can index changes in endogenous NT levels
PET measures of receptor binding
PET scan measures signal from radiotracer bound to receptor (used to index number of receptors or basal NT levels)
increased release of endogenous NT displaces radiotracer therefore a reduction in radiotracer binding refects and increase in NT release
fMRI brain activity measure
mesures BOLD (blood oxygenation level dependent) signal
better spatial and temporal resolution than PET
can be used to measure changes in brain activity when subject is performing certain tasks or patterns of activity across regions in resting state
pharmacological MRI
analyzes changes in brain function following drug administration, location, and time course of drug action
electroencephalography (EEG)
measure of synchronous firing in large groups of neurons
often used to measure event related potentials (ERPs)
better temporal resolution than fMRI (real time) but spatial resolution is poorer
knockout animals
gene for protein is deleter
knockin/transgene animals
original gene is removed, substituted for another
issues with genetically engineered animals
compensation by other genes for the altered one may mask the effect of the mutation
optogenetics
insert genes for light sensitive ion channels/pumps into neurons
genes are inserted via infusions of engineered viral vectors into brain region of interest
optogentic method channels
channels are activated when timulated with specific wavelength of light
channels can be excitatory (rhodopsin) or inhibitory (halorhodopsin)
this approach also permits greater temporal specificity (can turn on/off only a specific type of neuron for a few seconds)
