Lecture 4- Techniques In Neuropharmacology Flashcards
What is in vitro approach
-Performed with tissue, cells, biological molecules
-Outside of their normal biological context
What machines are used to slice brains
Cryostat or microtome
What fluid is used on brain slices
-Paraformaldehyde
-Stops biological activity
-Preserves tissue
What is used on a brain section in an autoradiography
-Radioligand
-Radioligand is a drug with a hydrogen group attached to it
Dopamine transporters are heavily present in the
Striatum
How is autoradiography used
-Used to find a molecule in brain sections
-Anything that is dark is where the molecule is present
What is vivo approach
The effects of various biological entities are tested on whole living organism
How does a PET scan work
-Used to visualise things like receptors in a live human brain
-Injected with radioactive molecule to find where the molecule travels
How does in situ hybridisation work
-RNA probe is created to mimic RNA
-Often radioactive or fluorescent
-RNA probe used to find mRNA in chosen protein
What is a “knock out” gene in vivo genetic manipulation
Specific gene inactivated
What is a “knockin” gene in vivo genetic manipulation
Add foreign genetic material at specific gene locus
What are the problems with genetic manipulation in vivo
-Developmental abnormalities
-Compensation and or functional redundancy
-Started at birth, last a lifetime
Intracranial drug injections to manipulate
Receptor signalling locally in the brain
When are direct drug brain injections used
-Experimental situations
-Can test whether a neurotransmitter plays a role in behaviour
What is the process of stereotaxic surgery
-Used in intracranial drug injections
-Removal of a piece of skull to inject
-Accurate targeting of specific brain structures
-Implant electrodes, inject drugs/ viruses
How can ISH be used to examine neuronal activity on excitatory pyramidal cells and inhibitory inter neurons in the cortex
-Pyramidal cells express Vglut
-Inhibitory interneurones express VGAT
-Activated neurons express IEG cfos or fos
-fos+VGAT = activated interneuron
What do pyramidal cells contain that no other neuron has
VGLUT
What does VGLUT stand for
Vesicular glutamate transporter
What does VGAT stand for
Vesicular GABA transporter
What is IHC used for
Protein detection
What is the process of IHC
-Inject antigen to produce antibodies
-Tale slice of brain
-Purify antibodies
-This primary antibody detects protein of interest
-Secondary antibody detects primary
What methods are used for IHC
-Colorimetric reaction
-Fluorescence
What are the advantages of IHC
-High specificity and spatial resolution
-Can show a single neuron activated
What are the disadvantages of IHC
-Provides a static snapshot of the brain
-Doesn’t show development over time
Neuronal activity increasing causes
Increased oxygen demand
What does fMRI not tell you
Specific neurons which are activated
With fMRI, haemoglobin is
Diamagnetic
When haemoglobin is oxygenated it becomes
Paramagnetic
In fMRIs, difference in magnetic attraction leads to
Small differences in the MR signal of blood depending on the degree of oxygenation
fMRI scans show what effect of cocaine
Increased activity in the nucleus accumbens when compared to saline
What are the advantages of EEG scans
-Non invasive
-Good milli-second time resolution compared to fMRI
Disadvantages of EEGs
Poor spatial resolution
Process of stereotaxic surgery
-Implant electrodes, inject drugs/viruses, produce leisons
Advantages of intracranial electrophysiology
-Record fast real time changes in activity
-Measure neuronal populations, single neurons and specific brain areas
Disadvantages of intracranial electrophysiology
-Simply detects activity
-Doesn’t say anything about the physical identity of the activated neuron
Disadvantages of local brain lesions
-No neuronal specificity
-Damage is permanent
Induce local brain damage via
Electrical current, toxins
Intracranial drug injections are used to
Manipulate brain activity locally
Direct brain injections are either
-Inhibitors of activity
-GABA receptor agonists
How do intracranial drug injections differ to lesions
Reversible
What is optogenetics
Light induced neuronal activity manipulations using viruses
Stages of optogenetics
-Transfected neurons with opsins
-Fibre optic cable shines a light that can affect opsin
-Measures changes in the behaviour once activated or inhibited
What are the two lights for optogenetics
-Blue (channelrhodopsin)
-Yellow (halorhodopsin)
What does channelrhodopsin do
-Optogenetics
-When a blue light is shone, this opsin is activated in the neuron
-Allows you to find out sufficiency needed for activation of desired behaviour
What does halorhdopsin do
-Optogenetics
-When yellow light is shone, this opsin is activated in the neuron
-Allows you to shut down neurons
Advantages of optogenetics
-Fast
-Tailored to specific neuronal populations
-Light manipulation of terminals affects only neurons from a specific source
Disadvantages of optogenetics
-Gene inactivation affects all neurons in an area
-Drugs turn on all neurons everywhere, uncontrolled
Method of microdialysis
-Pump aCSF into sample brain
-Neurotransmitters collected through dialysate
-Measures changes in neurotransmitters over minutes
Disadvantages of microdialysis
-Doesn’t tell you specific neurons that are releasing the neurotransmitters
Addictive drugs cause dopamine release in the
Striatum
Method of voltammetry
-Stick electrode into extra cellular fluid
-Rapid sampling detecting changes
Method of whole cell electrophysiology in brain slices
(ex vivo electrophysiology)
-Record from single neuron
-AMPAR- mediated EPSC
-Kept alive in aCSF
-Stimulating electrode that activates the synapse
-Can record human neurons
Disadvantages of whole cell electrophysiology in brain slices
(ex vivo electrophysiology)
-Only measures synapse
-Does not measure whether a behaviour is due to synaptic pathway
Terminals are still alive when
Cell body is dead
