Evaluation of metaquant microdialysis for measurement of absolute concentrations of amphetamine and dopamine in brain: A viable method for assessing pharmacokinetic profile of drugs in the brain

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Abstract

Direct measurement of absolute brain concentration of amphetamine and dopamine were obtained using metaquant (MQ) microdialysis, which achieves near 100% recovery, in the caudate nucleus. Conventional microdialysis monoprobes were also implanted in the caudate nucleus in the contralateral side of the same animals to compare the brain concentrations obtained from these two probe types. In addition plasma concentrations of amphetamine were obtained simultaneously from the same animals. The distribution of amphetamine in the plasma and of amphetamine and dopamine in both probe types followed same profile at each time interval. The basal dialysate concentration of dopamine in the caudate nucleus measured by MQ, was 9.40 ± 0.60 nM, while measured by conventional microdialysis it was 6.35 ± 0.36 nM. This study demonstrates that MQ microdialysis is an appropriate method for determination of true extracellular levels of drugs and neurotransmitters in the brain, under dynamic conditions. Since these measurements, together with measurements of plasma concentrations of the drug, can be made in a single animal, the method can be used to study pharmacokinetic–pharmacodyamics profile of psychoactive agents.

Introduction

Intracerebral microdialysis has many advantages for measuring endogenous and exogenous compounds, in localised brain areas in anaesthetised or freely moving animals, and enables local administration of drugs to the tissue being sampled, through the probe. It is a widely used technique in neuroscience, which enables measurement of chemical constituents in the brain extracellular fluid (ecf) in vivo. Although the majority of studies have used microdialysis to measure the levels of neurotransmitters and their response to pharmacological and behavioural challenge (Zetterstrom et al., 1983, Ungerstedt, 1984, Young, 1993, Westerink, 1995), free (unbound) drug concentrations in the brain can also be measured (Morrison et al., 1991, de Lange et al., 1995a, de Lange et al., 1995b). However, one of the drawbacks of microdialysis has been the inability to measure absolute ecf concentrations. The movement of solutes between the ecf and dialysate across the semi-permeable membrane down their concentration gradients occurs under non equilibrium conditions due to continuous perfusion of the probe with artificial cerebrospinal fluid (aCSF). Therefore the concentration of the solutes found in the microdialysate samples represents only a fraction of their concentration in the ecf. The ratio of the two concentrations is termed the recovery; this recovery is easily obtained in vitro as the concentration surrounding the probe in vitro is known. However, recovery in vivo is very different, principally due to the diffusion resistance of the tissue, and active processes occurring in the living tissue (e.g. Bungay et al., 1990, Morrison et al., 1991). Assessing recovery in vivo is problematic as the actual brain concentration of the substance surrounding the probe is unknown.

This problem is generally circumvented in microdialysis studies, where changes in endogenous substances (e.g. neurotransmitters) are expressed as dialysate concentrations rather than actual brain concentrations. This approach overcomes the necessity of knowing the in vivo recovery of the substance and is appropriate for many studies where the changes in concentration to drug challenge (as opposed to absolute brain concentrations) are important. However, it is severely limited for studies, such as pharmacokinetic studies, where knowledge of the absolute brain concentrations of drugs and neurotransmitters becomes important (see reviews by de Lange et al., 1995a, de Lange et al., 1995b, de Lange et al., 2000).

Several approaches have been used to attempt to address this problem, and make reliable estimates of the ecf concentrations of solutes. One method is simply to assume that recovery in vitro provides a reliable estimate of recovery in vivo. This method is severely limited as physiological factors in vivo interact with the dialysis membrane performance (e.g. extracellular tissue tortuosity, active processes in the tissue) are not accounted for in vitro recovery.

A second approach is to extrapolate to zero flow (Jacobson et al., 1985). This method relies on the concept that as the flow rate through the probe approaches zero, the recovery approaches 100% (i.e. equilibrium). Thus by measuring dialysate concentrations at a number of flow rates, one can extrapolate to zero flow, and thus make an estimate of the concentration.

A third approach (Lonnroth et al., 1987) derived from the concept that when the concentration of a solute inside the probe (dialysate concentration) is the same as that outside (ecf concentration), there will be no concentration gradient, and there will be no net flux of the solute between the two compartments. By applying different concentrations of the solute of interest in the perfusate, and measuring the flux in or out of the dialysis stream, it is possible to calculate the theoretical point of no net flux, which represents the external concentration of the solute (Parsons and Justice, 1992, Justice, 1993, Olson and Justice, 1993).

A fourth approach is to perform in vivo calibration of the microdialysis probe using retrodialysis of a reference compound (calibrator). Recovery is determined as the relative loss of the calibrator into extracellular fluid and assumed that relative loss is equal to the relative gain (de Lange et al., 2000). In vivo calibration using the actual compound of interest can be performed prior and/or subsequent to in vivo experiment.

All these methods have severe limitations in application to studies of dynamic process in vivo, such as changes in drug and neurotransmitter concentration after drug application. Both extrapolation to zero flow and no net flux require multiple measurements over a relatively long period of time, and therefore require the solute of interest to be in steady state for a period of hours, while retrodialysis calibration requires the addition of the calibration compound prior to the experiment which may interfere with its distribution and its effect for the actual study (de Lange et al., 2000) and assumes that recovery into the probe is the same as retrodialysis from the probe.

Given that when there is no flow through the tip, equilibrium is achieved, and the recovery is 100%, it follows that at very low flow rates, the recovery is close to 100%. However, at such low flow rates the sample handling and analysis of the amount of dialysate becomes problematic. To circumvent this problem, a novel metaquant (MQ) method has been developed (Cremers et al., 2009). The MQ probe differs from a conventional probe in that it has two inflow lines: one, the ultra-slow flow, perfuses the dialysis tip in much the same way as in conventional microdialysis probes; the other, the carrier flow, goes only as far as the probe body (Fig. 1). Thus the flow through the dialysis tip is at ultra-slow flow rate (in this case 0.15 μl/min), and achieves near 100% recovery. This flow then mixes with the carrier flow in the body of the probe, diluting the outflow from the dialysis tip to give manageable flow in the output line, and sufficient sample volumes. Since the dilution factor within the probe body is defined by the relative flow rates in the ultra-slow flow and the carrier flow, it can be easily determined and used to calculate the concentration in the dialysis tip from the concentration measured in the sample. This allows accurate quantitative microdialysis with convenient sample volumes for fluid handling and short lag times inside the outlet tubing (Cremers and Ebert, 2007, Cremers et al., 2009).

The main objective of our study was to investigate the use of MQ dialysis probes for measuring absolute brain concentrations of amphetamine and dopamine. Amphetamine was chosen for these studies, since its central actions, mainly by acting on dopamine transporters are well characterised (e.g. Rothman and Baumann, 2003, Sulzer et al., 2005). As an additional comparison, conventional microdialysis measurements were made in the contralateral caudate nucleus, and plasma amphetamine measurements were made, in the same animals.

Section snippets

Materials and method

MQ microdialysis probes, comprising a cellulose dialysis tip (length 4 mm; diameter 216 μm: cut off of 18 kDa (Cremers et al., 2009) were supplied by BrainsOnLine (Netherlands). Conventional monoprobes comprising a Cuprophan dialysis tip (length 2 mm; diameter 320 μm; cut off 60 kDa) were constructed in our laboratory as previously described (Young et al., 1992).

Results

In vitro probe recovery for MQ probes for amphetamine was 92 ± 5.0% and for dopamine was 95 ± 1.8%. In vitro recovery for monoprobes for amphetamine was 12 ± 0.7% and for dopamine was 15 ± 0.9%.

Amphetamine concentrations in the caudate nucleus showed a time response relationship (main effect of sample: F (5, 24) = 7.82, p = 0.002: Fig. 2a), with the peak levels obtained during the first hour after injection, and levels then decreasing over the subsequent 90 min. There was a significant difference between

Discussion

The main aim of our study was to evaluate the use of MQ microdialysis probes for measuring absolute brain concentrations of a drug and a transmitter. These can then be related to plasma concentration of the drug, providing a better understanding of the pharmacokinetics in vivo and the central action of the drug. We determined absolute brain concentrations of dopamine in the caudate nucleus under both non-stimulated conditions and after amphetamine administration using MQ dialysis and compared

Acknowledgement

PS is funded by a BBSRC CASE studentship with Pfizer Pharmaceuticals (no. 13092).

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