APMDA boutelle L3&L4

• 
• 1. Solution pumped into the probe is sterile water called perfusate
• 2. It washes over the inside of the semi-permeable membrane tip of the microdialysis probe
• 3. Chemical speceies in the extracellular fluid that are not in the perfusate  in the same concentration, DIFFUSE down the concentration gradient, cross the semi-permeable membrane and are then carried away form the membrane area to give the dialysate.
• 4. Dialysate collected for ex-vivo analysis

If solution is well stirred, concentration gradient appears across the membrane

Dialysate concentration limited by diffusive flux and internal flow rate.

In vitro, you need a vortex created by high speed stirring. The conc grad is across the memb only.

• Passive effects:
• Excluded volume and totuous difffusion path
• Tissue effects are rate limiting
• Prove recovery<invitro

• Active effects:
• Release rate to local ECF conc
• Buffering of extracellular environment
• In vitro>probe recovery> in vivo passive

• Results in Probe recovery < in vitro case
• 
• Excluded volume and totuous difffusion path
• Lack of stirring
• Tissue effects are rate limiting

These molecules therefore diffuse to the probe much less efficiently. Hence the concentration is lower and probe recovery is lower than in vitro

• In vitro> probe recovery in vivo active> in vivo passive
• 
• This is due to the tissue actively responds to a drop in local concentration by releasing more and uptaking less. Summarised as

- Buffering of extracellular environment


This causes the diffusion projle to move towards the in vitro value

• Zero nett flux
• Extrapolation to zero flow.

• Graphically find the point at which the microdialysis probe neither removes or adds molecules to the ECF. 
• The slope of the graph is the probe recovery E
• The y intercept is the normal mode microdialysis conc.
• The x-intercept is the ECF conc
• Vary C_in and measure C_out

• The flow rate through probe is reduced and dialysate conc measured
• Process continued until dialysate conc = true ECF value

(so youre like plotting this decaying exponential until you get to 100% which takes ages)

It can have errors if there is significant damaged tissue

• Insert probes slowly (0.3-4uL/s): to minimise tissue damage
• Allow 12 hrs for recovery: 
• Perfuse with correct ionic solution: tissue is sensitive to ionic composition of perfusate. If it is incorrect, the tissue might be more/less electrically active. 
• Check signal integrity

Use TTX to lock voltage gated sodium channels.

• 1. Monitor stimulated response and ensure it is reproduceable
• 2. Challenge with local drug through probe
• 3. Check that response returns on drug washout. 

This lets us use a pairwise statistics approach: Lets us compare control and drug response in the same animal.

• 1. Collect a sample: Takes 10-20 mins. Stored in an autosampler.
• 2. Use chromatography to separate the different components
• 3. analyse the peaks as they elute from the column
• 4. If the levels are too low inject more sample- hence its mass sensitive.

• The probe is much larger than cells
• The number of intact neurons can be reduced which is important if you want to measure substances with short lifetimes and hence short diffusional lengths.

• Second example
• The probe caused a proliferation of glial cells to occur close to the membrane.
• The consequence of this is that you get a lower proportion of neurons

• Idea is to make capture of released molecules more effective by trapping them on antibody-coated nano-particles
• They bind the molecule targeted by the antibody very tightly.
• This leaves a very low 'free' concentration left in the dialysate
• Additionaly, it stops 'sticky' molecules such as peptides from sticking to the connecting tubing in non-specific binding.

As temporal resolutio increases, moles available to detect decrease, but the number of samples dramatically increases.

You have to use an automatic sampling to use high temporal resolution

• Very fast response ( much less dead volume to allow faster changes to be seen)
• Very sensitive
• Tissue damage is small

• Fast voltage ramp applied to electrode over 10ms 
• Dopamine oxided to dopamine-o-puinone to give a current peak
• On the reverse scan, the dopamine-o-quinone is reduced back to dopamine to give a reverse peak.
• Perform background scan which is subsequently removed from subsequent scans
• Position of oxidation reduction peak is diagnostic for the chemical species
• The positions of the peaks is diagnostic for the anlyste.

• Can only detect species that can be oxidised or reduced by the electrode
• To get a signal, you must subtract the baseline signal, so you cannot measure basal concentrations.

• 1. Ability to handle small volumes
• 2. Ability to handle large number of samples
• 3. Analytical sensitivity of the detection system.