Neurobiological Techniques

Question 1

High Performance Liquid Chromatography (HPLC):

Answer 1

• Measures neurotransmitter levels
• Separates brain sample into individual substances
• High pressure forces air into the tube, mixes with sample (mobile phase) and into the stationary phase
• Substances are separated in the stationary phases, leaves stationary phase (effluent) at different times
• Detector detects when each effluent comes out
Chromatograph provides peaks of each substance (peak of Ach, dopamine…etc)

Diagram

Question 2

In-vivo Microdialysis:

Answer 2

• Can take brain sample while animal is awake and performing a task (can see what neurotransmitters are released while the animal is performing that specific task)
• Artificial cerebral spinal fluid (CSF) is pumped into the out of the brain of an animal, output is collected and submitted to HPLC for analysis
• CSF is pumped in the brain through a surgically implanted microdialysis probe
• Tip of probe is semipermeable, pumping CSF through it creates a concentration gradient
Released neurotransmitters move down the concentration gradient, into the CSF and then out of the brain

Diagram

Question 3

Stereotaxic Surgery:

Answer 3

• Paxinos & Watson developed a coordinate system (brain atlas) to determine where brain regions are
• Coordinates are based on specific reference points of the skull
• Lambda, Bregma are common reference points
○ Lambda- Rear suture creates a lambda like structure
Bregma- coronal suture intersects perpendicularly with the sagittal suture

	• 3 sets of coordinates
		○ Anterior- Posterior
			§ Front to back
		○ Medial-Lateral
			§ Away from longitudinal fissure
		○ Dorsal-Ventral
Depth in brain

Question 4

Electrolytic Lesion:

Answer 4

• Passing a strong current through an electrode inserted into the brain
• Creates a hole in the brain
• Not very selective, damages neurons in brain region as well as connecting nerve fibers
○ Nerves connecting different brain regions
i.e. Want to damage nucleus accumbens, but also kills neurons connecting the amygdala to the hypothalamus

Question 5

Excitotoxic Lesions:

Answer 5

• Selective disruption of cells
• Kills neurons in that brain region, but spares connective nerve fibers
• Causes cell death by overstimulating glutamate receptors (too much glutamate causes cells to kill themselves)
i..e NMDA, Kainic acid, ibotenic acid

Question 6

Specific Toxin Lesions:

Answer 6

• Selective destruction of specific cell types
• i.e. Want to disrupt dopaminergic input to the hippocampus, hippocampus still intact
• Toxins are taken into cells via reuptake transporters, kills cells
• i.e. 6-hydroxydopamine affects norepinephrine and dopamine, 192 IgG-saporin affects Ach
• 192 IgG-saporin is an immunotoxin (has an antibody that recognizes receptors found only on cholinergic neurons in the basal forebrain)
Injecting saporin into brain, will only attach to cholinergic neurons, only kills those neurons

Question 7

Temporary Inactivation of Cells:

Answer 7

• Tetrodotoxin (TTX), and lidocaine are sodium channel blockers- prevents action potentials
• GABA agonists cause overinhibition
• No neurochemical specificity, but provides temporal specificity (turning off all neurons in one specific region)
TTX, lidocaine affects fibers of passage (stop action potentials by blocking sodium channels), sodium channels are found all along the axon, including on fibres of passage as well

Question 8

Immunohistochemistry (IHC):

Answer 8

• Using an labelled antibody to identify proteins
• Antibodies are highly specific for a foreign substance
• Take a cholenergic neurons out of a rat brain, put it into a mouse brain
• Mouse will develop antibodies to that antigen, collect antibodies from blood of mouse, attach visible label
Inject labelled antibody into rat, can tag cells that are expressing cholinergic neurons

Question 9

ChAt and Parvalbumin IHC in Rat Basal Forebrains:

Answer 9

• Rat tissue synthesized antibodies of ChAT (Choline acetyltransferase- synthesizing enzyme of Ach- only found in Ach producing neurons)
192 IgG-saporin injected into basal forebrain, use antibodies to measure amount of ChAt

Diagram

Question 10

In-situ Hybridization (ISU):

Answer 10

• Synthesizing labelled, complementary sequence to mRNA sequence
Binds to mRNA sequence, causing hybridization, can be used to detect mRNA

Diagram

Question 11

Zif268:

Answer 11

Diagram