Microdialysis experiment

SSR-504734 is a potent, selective, and reversible inhibitor (IC50 = 18 nM) that is competitive with glycine [47,51]. The inhibitor rapidly and reversibly blocked the uptake of [14C]glycine in mouse cortical homogenates, which was sustained for up to 7 h. Complete cessation of blockade and return to glycine basal levels occurred prior to 24 h, which is in stark contrast to NFPS (>24 h). SSR-504734 potentiated a nearly twofold increase of NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) in rat hippocampal slices and produced an increase in contralateral rotations in mice when microinjected into the striatum. Microdialysis experiments indicated that the inhibitor induced a rapid and sustained increase in extracellular glycine levels in the PFC of freely moving rats [51]. The compound also demonstrated efficacy in a variety of psychosis models [51-53]. SSR-504734 was reportedly in clinical trials for schizophrenia but discontinued after Phase I (data not disclosed) [54]. 

After the completion of the in vivo microdialysis experiment, rats are euthanized with an overdose of sodium pentobarbital (100 mg/kg) and perfused intracardially with 0.9% saline followed by 10% formalin. The brains are extracted and stored in a 10% formalin solution until they are sectioned into slices (40-60 mm) consecutively through the guide cannula tract. The sections are mounted onto gelatin-coated glass slides and stained with thionin. An observer unaware of the rat s treatment or results verifies the cannula location for each rat. 

Camp DM, Robinson TE. 1992. On the use of multiple probe insertions at the same site for repeated intracerebral microdialysis experiments in the nigrostriatal dopamine system of rats. J Neurochem 58(5) 1706-1715. 

Wellman PJ. 1990. An inexpensive guide cannula and collar for microdialysis experiments. Brain Res Bull 25(2) 345-346. 

Potschka, H., M. Fedrowitz, and W. Loscher. 2002. P-Glycoprotein-mediated efflux of phenobar-bital, lamotrigine, and felbamate at the blood-brain barrier Evidence from microdialysis experiments in rats. Neurosci Lett 327 173. 

Microdialysis experiments show that BEO (0.5 ml/kg) did not affect basal amino acid levels, whereas it significandy reduced the efflux of excitatory amino acid, namely aspartate and glutamate, in the frontoparietal cortex typically observed following MCAo (Fig. 2). Extracellular levels of glycine, GABA,... 

Fig. 2. Transient retinal ischemia increases intravitreal glutamate in rat. Neurochemical data from microdialysis experiments carried out in anesthetized rats to demonstrate that ischemia/reperfusion insult increases intravitreal glutamate. The extracellular level of glutamate shows a moderate increase during the first 10 min of ischemia, more evident toward the end of the ischemic period, to reach statistical significance at 10 and 150 min of reperfusion. Baseline glutamate concentrations (basal values) are the mean concentrations obtained by averaging six samples collected consecutively at 10 min intervals immediately before the onset of ischemia (n — 6 rats). Glutamate values (pM) are expressed as mean S.E.M. Statistical significance was assessed by ANOVA followed by Dunnett s test for multiple comparisons. P < 0.05 and P < 0.001 versus basal values.

Perfusion solution used in microdialysis experiments vary widely in composition and pH. Ideally the composition, ion strength, osmotic value and pH of the perfusion solution should be as close as possible to those of the extracellular fluid of the dialyzed tissue. Perfusion fluids should be at body temperature. 

The microdialysis probe is the heart of the method, as a chromatographic column is the heart of the HPLC instrument. Rigid CMA probes, Models 10, 11, and 12, are used for stereotaxic implantations into the brain, where the probe can be fixed (cemented) to the skull. A flexible probe design (CMA 20) allows the placement of such a catheter into the moving tissues (muscle) or peripheral organs for studies in freely moving animals. The technical difficulties of microdialysis experiments impose requirements for precise liquid delivery, minimized dead volumes, and the capability of handling small sample volumes. 

Figure 6.3 Basic setup for microdialysis experiments on small laboratory animals. The system can be used for simultaneous collection from up to three microdialysis probes. The main components are (A) CMA/100 Microinjection Pump, (B) CMA/140 Microfraction Collector, (C) temperature controller, ( >) liquid switch for switching between different perfusion fluids, and ( ) in vitro stand for storage of microdialysis probes.

Tang A, Bungay PM, Gonzales RA. Characterization of probe and tissue factors that influence interpretation of quantitative microdialysis experiments for dopamine. J. Neurosci. Methods 2003 126 1-11. 

Using microdialysis experiments the relative oral bioavailabilities of the compounds 34, 35 and 5-OH-DPAT could be calculated.215 The relative oral bioavailabilities were calculated by comparing the Areas Under the Curve (AUCs) after oral and subcutaneous administration. When there was no significant difference between the AUCs the subcutaneous dose was divided by the oral dose and multiplied by 100 to give the relative oral bioavailability. These data show that, although the affinities of the benzo[ ]thiophenes (34 and 35) for the dopamine receptors are lower as compared to 5-OH-DPAT, the relative oral bioavailability is higher. Therefore, the benzo[/i]thiophenes are interesting compounds for further research. 

Data of the microdialysis experiments were converted into percentage of the basal levels. The basal levels were determined from four consecutive samples (less than 20% variation), and set at 100%. During a period of 165 min after administration of the test compound the dopamine... 

The results of the microdialysis experiments of the compounds 34, 35, 5-OH-DPAT, 38 and 39 are shown in Figure 4.1-4.4. S.c. administration of all compounds, except compound 38, induced a dose-dependent and significant decrease in the release of dopamine in the striatum. Furthermore, compounds 34, 35 and 5-OH-DPAT also induced a significant decrease in the release of dopamine in the striatum after p.o. administration. Effects of compounds 38 and 39 were not studied upon p.o. administration. 

The relative oral bioavailabilities, as determined by comparing the AUC after s.c. and p.o. administration, of 6-(N,N-di- -propylamino)tetrahydrobenzo[/>]thiophene (34), 5-(N,N-di- -propylamino)tetrahydrobenzo[/>]thiophene (35) and 5-OH-DPAT (9) were calculated from Figures 4.1-4.3, and are shown in Table 4.2. For compounds 34 and 35 the relative oral bioavailabilities were > 10 %, while for the reference compound 5-OH-DPAT it was 1 %. In order to verify the fact that the decrease induced by a dose of 10 pmol/kg p.o. was not already induced by a lower dose, we have found that a dose of 1 pmol/kg p.o. of 5-OH-DPAT induced a decrease in the release of dopamine in the striatum of only 50-55 %. Furthermore, microdialysis experiments in our laboratory with the (-)-enantiomer of 5-OH-DPAT also showed that the relative oral bioavailability was about 1-3 % (Chapter 7). 

Table 4.2 AUCs of the microdialysis experiments of 6-(N,N-di- -propylamino)tctrahydrobenzo thiophene (34), 5-(N.N-di- -propylamino)tctrahydrobenzo A thiophcne (35) and 5-OH-DPAT (9) after s.c. and p.o. administration.

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