Exp tech in neuro - Intracellular staining methods

Question 1

How to label a cell intracellulary

Answer 1

Glass recording micro pipette containing a marker substance.

Marker injected either by ionophoresis or pressure injection

Question 2

Fluorescent markers

Answer 2

Lucifer yellow

Tetramethylrhodamine dextron

Question 3

Lucifer yellow

Answer 3

Good labelling of dendrites
Fluoresces over a large raange which is a disadvantage if using multiple markers
Not nescessary to perform further reactions to detect it

Question 4

Tetramethylrhodamine dextron

Answer 4

Fluoresces in red spectrum only
Not necessary to perform further chemical reactions in order to detect it
Fades if veiwed for too long with epifluorescence

Question 5

HRP

Answer 5

Provides physiological, morphological and ultrastructure info.
Non-toxic, does not kill neuron
Electron dense reaction products
Can be combined with other techniques
Limited use in thick slices /whole tissues
Difficult to label small cells due to HRP increasing the resistance of the micropippette
Incomplete axon labelling.
Requires chemical reaction with suspect carcinogen.

Question 6

Biocytin and Neurobiotin

Answer 6

Biotin has high affinity for avidin, this can be conjugated to a fluorescent marker which can be detected by LM, EM or fluoromicroscopy.
Stains axons more completely than HRP
Can be used in vivo, thick slices and whole mount preps.
Simple safe chemical reaction.
Can label small structures.
Cannott be combined with other ics metjods that use avidin.
Detergent must be used so that chemical Can penetrate tissue deeply, this is incompatable with good ultrastructural preservation.

Question 7

Prep of in vivo animals

Answer 7

Ics performed on deeply anaesthetised animal. Animal mounted in frame and brain/spinal cord is exposed to allow penetration with microelectrode
Physiological condition of animal monitored throughout

Question 8

Micro-electrodes

Answer 8

Made from glass tubing approx 1mm in diameter
Made in electrode puller
Filled with electrolyte solution containing marker
Modern electrodes have a fibre which allows solution to enter the tip via capillary action.
Some tips are too smal therefore resistance is too high, must be bevelled to prevent this

Question 9

Lebelling a cell

Answer 9

Possible to tell when a cell has been sucessfully penetrated by a micropipette by monitoring its resting membrane potential.
Once cell is penetrated it is important to map iwts receptor feild.
Once this is done the ionophoric current is applied and cell is filled with marker.

Question 10

Fization and sectioning

Answer 10

At the end of ecperiment animals are fixed by perfusion of a fixative into the CVS.
Type of fixative depends on what sort of study is to follow (LM, EM).
Once tissue is fixed, blocks containing labelled structures are made and then sectikns are cut with a vibratone and collefted in serial order.