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Neurotransmitter Recovery  

Recovery is affected by many different variables.  It is therefore important to optimize each step of the process. As a rough idea, the sampling time and probe membrane length will decide the absolute content level of analyte in the microdialysis samples.

1. Flow rate: To increse concentration of analyte, decrease the pump speed. To increase absolute content of analyte in the samples, simply allow for a longer collection time. Recovery rate is directly proportional to the flow rate of the perfusion liquid via the microdialysis probe.  A slower flow rate allows the analytes more time to diffuse across the probe membrane. Additionally, a low flow rate will generate less pressure inside the microdialysis probe, which positively effects the diffusion of molecules across the membrane. Typically 0.5 - 2 ul/min flow rates are employed to ensure optimal recovery of neurotransmitters.(see data). No net flux, near equilibrium, and ultra slow flow microdialysis are methods used to determine/calculate absolute content of the target substance around the probe surface. Perfusion liquid:  The perfusion liquid should be as close as possible to the extracellular fluid.  Many use a Ringer’s solution (148 mM NaCl, 4 mM KCl, 3 mM CaCl2) which has higher calcium and potassium levels than aCSF (148 mM NaCl, 4 mM KCl, 1.2 mM CaCl2 ,0.85 mM MgCl2). The aCSF solution more closely mimics physiological salt conditions in-vivo which, in turn, yields more accurate readings from the animal.  
2. Microdialysis Probe: Different probe membrane types are available depending on the application (peptide vs. monoamine).  Increasing the membrane length increases the recovery rate.   
3. Tubing: Minimizing the tubing length from the microdialysis probe to the collector can increase sample recovery as well.  The shorter the tubing the less chance for back pressure to accumulate which can affect the neurotransmitter flux at the membrane.  Additionally, the faster the sample is chilled, injected into the HPLC, or combined with an antioxidizing agent the better recovery you will have. 

From Sample Collection to Analysis (Microdialysis-HPLC On-line/Off-line)

Once the microdialysis sample is collected via the probe it can either be injected directly (on-line) into a high performance liquid chromatography (HPLC), or sampled into a 96-well plate via a fraction collector for later analysis (off-line).  Depending on the analytes of interest, different analytical techniques can be used.  Most commonly used are HPLC coupled to an electrochemical detector (ECD) for detection of serotonin, dopamine, norepinephrine, acetylcholine, GABA, and glutamate or fluorescence for the detection of histamine.

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