Lecture 4- Techniques In Neuropharmacology Flashcards by Grace Marshall

What is in vitro approach

-Performed with tissue, cells, biological molecules
-Outside of their normal biological context

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What machines are used to slice brains

Cryostat or microtome

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What fluid is used on brain slices

-Paraformaldehyde
-Stops biological activity
-Preserves tissue

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What is used on a brain section in an autoradiography

-Radioligand
-Radioligand is a drug with a hydrogen group attached to it

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Dopamine transporters are heavily present in the

Striatum

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How is autoradiography used

-Used to find a molecule in brain sections
-Anything that is dark is where the molecule is present

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What is vivo approach

The effects of various biological entities are tested on whole living organism

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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

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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

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What is a “knock out” gene in vivo genetic manipulation

Specific gene inactivated

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What is a “knockin” gene in vivo genetic manipulation

Add foreign genetic material at specific gene locus

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What are the problems with genetic manipulation in vivo

-Developmental abnormalities
-Compensation and or functional redundancy
-Started at birth, last a lifetime

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Intracranial drug injections to manipulate

Receptor signalling locally in the brain

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When are direct drug brain injections used

-Experimental situations
-Can test whether a neurotransmitter plays a role in behaviour

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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

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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

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What do pyramidal cells contain that no other neuron has

VGLUT

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What does VGLUT stand for

Vesicular glutamate transporter

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What does VGAT stand for

Vesicular GABA transporter

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What is IHC used for

Protein detection

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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

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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