Membrane microdialysis

Figure 4.6 The tip of a microdialysis probe, expanded to show dialysis tubing around a steel cannula through the base of which fluid can flow out and then up and over the membrane. The length of membrane below the probe support can be altered (1-10 mm) to suit the size of the animal and the brain area being studied. Flow rates are normally below 2 pl/min...

ATP certainly fulfils the criteria for a NT. It is mostly synthesised by mitochondrial oxidative phosphorylation using glucose taken up by the nerve terminal. Much of that ATP is, of course, required to help maintain Na+/K+ ATPase activity and the resting membrane potential as well as a Ca +ATPase, protein kinases and the vesicular binding and release of various NTs. But that leaves some for release as a NT. This has been shown in many peripheral tissues and organs with sympathetic and parasympathetic innervation as well as in brain slices, synaptosomes and from in vivo studies with microdialysis and the cortical cup. There is also evidence that in sympathetically innervated tissue some extracellular ATP originates from the activated postsynaptic cell. While most of the released ATP comes from vesicles containing other NTs, some... 

As described above, because MAO is bound to mitochondrial outer membranes, MAOIs first increase the concentration of monoamines in the neuronal cytosol, followed by a secondary increase in the vesicle-bound transmitter. The enlarged vesicular pool will increase exocytotic release of transmitter, while an increase in cytoplasmic monoamines will both reduce carrier-mediated removal of transmitter from the synapse (because the favourable concentration gradient is reduced) and could even lead to net export of transmitter by the membrane transporter. That MAOIs increase the concentration of extracellular monoamines has been confirmed using intracranial microdialysis (Ferrer and Artigas 1994). 

IL-ip is a well documented sleep factor (reviewed by Obal Krueger, 2003). Its administration increases sleep, its blockade decreases sleep and sleep rebound, and its transcription increases during waking. IL-1 receptor knock-out mice sleep less. Local application of IL-1 p in POA also stimulates NREM sleep. We examined the effects of local administration of IL-1 p and an antagonist through microdialytic application adjacent to lateral POA neurons (Alam et at, 2004). Neuronal activity is recorded within 0.5-1.0 mm of a microdialysis membrane in unrestrained rats. IL-ip potently inhibited the activity of 79% of wake-active neurons. The inhibitory response to IL-ip of wake-active neurons could be blocked by pre-treatment with IL-lra, an IL-ip antagonist. IL-ip application also excited some sleep-active neurons, but this response was inconsistent. 

The technique consists of a microdialysis probe, a thin hollow tube made of a semi-permeable membrane usually around 200-500 /xm in diameter, which is implanted into the skin and perfused with a receiver solution that recovers the unbound permeant from the local area. In principle, the driving force of dialysis is the concentration gradient existing between two compartments separated by a semi-permeable membrane. For skin under in vivo conditions, these compartments represent the dermal or subcutaneous extracellular fluid (depending on the probe position) and an artificial physiological solution inside the probe [36-38],... 

The relative recovery (RR) of the probe, essential for data interpretation, is calculated using the retrodialysis method, which assumes that the net transport through the microdialysis membrane from the perfusate to the surrounding tissues equals the net transport from the tissues into the perfusate. The equation for calculation is represented as follows [39] ... 

In vivo microdialysis is based on the principle of dialysis, the process whereby concentration gradients drive the movement of small molecules and water through a semipermeable membrane. In vivo microdialysis involves the insertion of a small semipermeable membrane into a specific region of a living animal, such as the brain. The assembly that contains this semipermeable membrane is called a probe, which is composed of an inlet and an outlet compartment surrounded by a semipermeable membrane (see O Figure 9-1). Using a microinfusion pump set at a low flow rate (0.2-3 /rL/min), an aqueous solution known as the perfusate is pumped into the inlet compartment of the microdialysis probe. Ideally, the... 

Neurotransmitters, peptides, and other endogenous substances in the extracellular space can be sampled using in vivo microdialysis. The dialysis membrane typically excludes the transport of larger molecules and enzymes that could otherwise interfere with analysis of the substances of interest. In the case of neurotransmitters, the levels in dialysates are the net result of the interaction between processes affecting release into and removal from the extracellular space. Consequently, in vivo microdialysis can only sample a neurotransmitter that has not yet been removed by clearing mechanisms and this method does not provide information regarding intracellular levels of substances. 

Graph used to calculate the point of no net flux for dopamine (DA). Using regression analysis, the extracellular concentration of DA is estimated via the difference method [the DA concentration in the perfusate minus the concentration of DA in the dialysate] plotted against the DA concentration in the perfusate. Values above the zero on the y-axis indicate diffusion to the brain, whereas values below the zero indicate diffusion from the brain. The zero point on the y-axis represents a steady state, at which no net flux of DA occurs across the dialysis membrane and represents the extracellular concentration of DA on the x-axis. Figure from Parsons, L.H., Justice, J.B., Jr. (1994). Quantitative approaches to in vivo brain microdialysis. Crit Rev Neurobiol. 8(3) 189-220... 

Hsiao JK, Ball BA, Morrison PE, Mefford IN, Bungay PM. 1990. Effects of different semipermeable membranes on in vitro and in vivo performance of microdialysis probes. J Neurochem 54(4) 1449-1452. 

West AR, Moore H, Grace AA. 2002. Direct examination of local regulation of membrane activity in striatal and prefrontal cortical neurons in vivo using simultaneous intracellular recording and microdialysis. J Pharmacol Exp Ther 301(3) 867-877. 

Aliquots of membranes (0.35 ml) are introduced into one compartment of the microdialysis cell and aliquots of [ H]acetylcholine are introduced into the other compartment. 

Microcalorimetric combustion, dioxetane enthalpy of formation, 163 Microdialysis membrane, hydrogen peroxide determination, 651... 

Microdialysis probe. Enlarged view of the lower end shows a semipermeable membrane that allows small molecules to pass in both directions, but excludes large molecules. 

Dialysis is the process in which small molecules diffuse across a semipermeable membrane that has pore sizes large enough to pass small molecules but not large ones. A microdialysis probe has a semipermeable membrane attached to the shaft of a hypodermic needle, which can be inserted into an animal. Fluid is pumped through the probe from the inlet to the outlet. Small molecules from the animal diffuse into the probe and are rapidly transported to the outlet. Fluid exiting the probe (dialysate) can be analyzed by liquid chromatography. 

You can demonstrate the size of colloidal particles with a dialysis experiment in which two solutions are separated by a semipermeable membrane that has pores with diameters of 1—5 nm.3 Small molecules diffuse through these pores, but large molecules (such as proteins or colloids) cannot. (Collecting biological samples by microdialysis was discussed at the opening of Chapter 25.)... 

Using microdialysis to simultaneously administer histamine and monitor changes in ACh release, we found that perfusion of the cortex with histamine does not alter spontaneous release of ACh, but results in a concentration-dependent inhibition of 100 mM potassium-evoked release of ACh [55], up to more than 60% (figure 1). This concentration of potassium is only apparently high, for the low recovery of potassium through the microdialysis membrane [59], and the rapid dilution of potassium in the extracellular space necessitate... 

It should also be pointed out that in the case of an in vivo measurement, the microdialysis probe will be able to recover not only glucose but also many other biological compounds with low molecular weight from the subcutaneous tissue. The electrochemical interferents are greatly reduced by the use of PB at a low applied potential. However, other biological compounds could negatively affect the stability of the enzymatic membrane. Also, it is possible to have a sort of passivation or fouling of the electrode surface due to the absorption of... 

A system underpinned by commercially made screen-printed electrochemical cells was described by Palmisano et al. [19]. The cells were converted into biosensors for lactate in milk and yoghurt by addition of an electrochemically polymerised barrier to interference and a layer composed of lactate oxidase, glutaraldehyde and BSA. These steps appeared to have been carried out by hand. As there was no outer diffusion-limiting membrane, the linear range of the sensors was quite small (0-0.7 mM). They were incorporated into a FIA with a microdialysis unit based on a planar membrane and a buffer reservoir (earlier work used a microdialysis fibre with a platinum electrode [29]. The concentration of lactate was determined in various milks (0.27-1.64 mM), and in raw milk (c. 0.5-0.9 mM) left to degrade on the laboratory bench. The recovery of the microdialysis unit, 2.6%, implied that the sensor had an ability to return measurable currents for very low concentrations of lactate. A further implication is that the electro-polymerised layer was very effective at preventing interference. 

F. Palmisano, D. Centonze, M. Qunito and P.G. Zambonin, A microdialysis fibre based sampler for flow injection analysis determination of L-lactate in biofluids by an electrochemically synthesised bilayer membrane based biosensor, Biosens. Bioelectron., 11 (1996) 419-425. 

Figure 6.2. Schematic drawing of the microdialysis probe used to sample the concentration of dopamine, DOPAC, 5HIAA and HVA in the extracellular space in rat brains. The fluid is delivered by a pump and passes via the vertical inlet and the inner cannula down to a 3.0 0.5 mm part of the probe, where a semipermeable membrane surrounds the inner cannula (stippled lines). The dialysis process occurs when the fluid passes between the inner cannula and the dialysis membrane. The perfusion medium flows inside the outer cannula to the horizontal outlet and is collected in 20 min (40 pi)...

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