Journal of Neurochemistry, 2003, 86, 932–946 doi:10.1046/j.1471-4159.2003.01904.x Microdialysis of dopamine interpreted with quantitative model incorporating probe implantation trauma Peter M. Bungay,* Paige Newton-Vinson, Wanda Isele,* Paul A. Garrisà and Joseph B. Justice, Jr *Division of Bioengineering & Physical Science, National Institutes of Health, DHHS, Bethesda, Maryland, USA Department of Chemistry, Emory University, Atlanta, Georgia, USA àDepartment of Biological Science, Illinois State University, Normal, Illinois, USA Abstract identified by histology and proposed to distort measurements Although microdialysis is widely used to sample endogenous of extracellular DA levels by the no-net-flux method. To and exogenous substances in vivo, interpretation of the address this issue, an existing quantitative mathematical results obtained by this technique remains controversial. The model for microdialysis was modified to incorporate a trau- goal of the present study was to examine recent criticism of matized tissue layer interposed between the probe and sur- microdialysis in the specific case of dopamine (DA) meas- rounding normal tissue. The tissue layers are hypothesized to urements in the brain extracellular microenvironment. The differ in their rates of neurotransmitter release and uptake. A apparent steady-state basal extracellular concentration and post-implantation traumatized layer with reduced uptake and extraction fraction of DA were determined in anesthetized rat no release can reconcile the discrepancy between DA uptake striatum by the concentration difference (no-net-flux) micro- measured by microdialysis and voltammetry. The model pre- dialysis technique. A rate constant for extracellular clearance dicts that this trauma layer would cause the DA extraction of DA calculated from the extraction fraction was smaller than fraction obtained from microdialysis in vivo calibration tech- the previously determined estimate by fast-scan cyclic vol- niques, such as no-net-flux, to differ from the DA relative tammetry for cellular uptake of DA. Because the relatively recovery and lead to an underestimation of the DA extracel- small size of the voltammetric microsensor produces little lular concentration in the surrounding normal tissue. tissue damage, the discrepancy between the uptake rate Keywords: dopamine, implantation trauma, in vivo microdi- constants may be a consequence of trauma from microdialy- alysis, in vivo voltammetry, striatum. sis probe implantation. The trauma layer has previously been J. Neurochem. (2003) 86, 932–946. Microdialysis is widely used in neuroscience, pharmacology In particular, the manner and the degree to which the trauma and medicine for in vivo monitoring of endogenous and might compromise interpretation of microdialysis measure- exogenous substances. The technique is an invasive procedure ments are generally unclear. requiring the implantation into target tissue of probes that are Regulation of brain dopamine (DA), a neurotransmitter relatively large by comparison to dimensions of individual involved in the functions of cognition, motivation and motor cells and intercellular space. Trauma induced in the brain by control (Le Moal and Simon 1991; Schultz 1998), has been probe implantation is characterized by both short- and long- extensively studied by microdialysis (Robinson and Justice term histological, physiological and biochemical changes in 1991). Despite relative uniformity in the practice of the nearby neural tissue (Benveniste and Diemer 1987; Benveniste et al. 1987; Westerink and De Vries 1988; Ruggeriet al. 1990; Shuaib et al. 1990; Robinson and Camp 1991; Allen et al. Received January 15, 2003; revised manuscript received May 2, 2003; 1992; Camp and Robinson 1992; Georgieva et al. 1993; accepted May 6, 2003. Fumero et al. 1994; de Lange et al. 1995; Westergren et al. Address correspondence and reprint requests to Peter M. Bungay, NIH/DBEPS, Building 13/3N17 MSC 5766, Bethesda, MD 20892-5766, 1995; Morgan et al. 1996; Grabb et al. 1998; Groothuis et al. USA. E-mail: [email protected] 1998; Clapp-Lilly et al. 1999). The consequences of Abbreviations used: aCSF, artificial cerebrospinal fluid; DA, dopam- probe-associated trauma, however, are not well understood. ine; ECS, extracellular space; FSCV, fast scan cyclic voltammetry. 932 Ó 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 932–946 Trauma model in dopamine microdialysis 933 microdialysis technique, various theoretical models used to drop in DA concentration of 485 nM (Kulagina et al. 2001). interpret results have led to predicted steady-state levels of Although the electrochemical technique employed, fast scan extracellular DA differing by three orders of magnitude cyclic voltammetry (FSCV), is not capable of determining an (Lindefors et al. 1989; Benveniste and Huttemeier 1990; absolute basal level of DA, it is well suited for monitoring Justice 1993). Failure to account properly for probe-induced concentration differences occurring over short sampling times trauma may also contribute to the wide range of concentration (Garris and Wightman 1995). Thus, evidence obtained by an estimates. Indeed, microdialysis sampling of DA would be alternative analytical method to microdialysis suggests that the particularly sensitive to the trauma layer adjacent to the probe extracellular DA concentration in the striatum greatly exceeds due to the high affinity neuronal transporter clearing released the low nanomolar estimates of the no-net-flux method. neurotransmitter from extracellular space (ECS) (Horn 1990; The present study extends the mathematical model previ- Giros et al. 1996). In general, the amount of tissue that ously developed to provide a quantitative relationship donates analyte to the dialysate depends inversely upon the between dialysate and extracellular concentration in steady- avidity of extracellular clearance mechanisms such as cellular state microdialysis (Bungay et al. 1990). The revised model uptake, interstitial catabolism or efflux to the blood. For DA as applies to general situations in which the mechanisms for well as any analyte rapidly cleared from interstitium, the analyte supply and removal are abnormal in a thin layer of condition of the tissue in close proximity to the probe is tissue adjacent to the probe. By distinguishing between these therefore of primary concern. Dopamine neurotransmission functional characteristics in the abnormal layer and the appears compromised in the trauma layer, as evidenced by the surrounding tissue, the model clarifies the difference between incomplete decrease in dialysate DA levels after perfusing the extraction fraction (determinable from dialysate measure- probe with a medium containing tetrodotoxin or depleted in ments) and relative recovery (potentially inaccessible by calcium (Westerink and De Vries 1988). Delaying the dialysate measurements alone). collection of dialysate for assay to allow constancy of In the present case, the modified theory uses the extraction indicators such as neurotransmitter concentrations, further- fraction from no-net-flux measurements to calculate a rate more, is not suitable as conditions may not represent normalcy constant for DA clearance. This clearance rate constant is found but rather a different quasi-steady state or a compensatory to be smaller than the one determined from FSCV for cellular adaptation to injury (Robinson and Camp 1991). uptake (Garris et al. 1994a). Because the small voltammetric The dominant approach in the last decade for estimating microsensors produce minimal tissue damage (Allen et al. steady-state levels of extracellular DA in the brain is the so 2001), discrepancies between the two clearance measurements called concentration-difference or no-net-flux measurement might arise from various factors, such as tissue damage related (Justice 1993). In this method, the analyte concentration to probe implantation, accumulation of fluid ultrafiltered perfused into the probe (inflow) is varied, and the difference through the probe membrane or formation of an abnormal between the inflow and outflow analyte concentration is tissue layer as a reaction of the tissue to the probe. Given that plotted versus the inflow concentration. Theory predicts that measurements to be reported are restricted to acute experiments the inflow concentration resulting in the zero concentration- in anesthetized rats, the most probable cause of local abnor- difference point indicates what the analyte extracellular mality is trauma. Consistency with the rate constant measured concentration would have been in the absence of the probe by voltammetry is achieved by invoking the hypothesis that, as (Bungay et al. 1990). Another prediction is that the extraction a consequence of implantation trauma, DA release is abolished fraction, which describes the exchange of DA between the and uptake is reduced in the traumatized tissue layer. probe and the tissue, equals the relative recovery of DA from the tissue under both steady-state (Bungay et al. 1990) and transient conditions (Morrison et al. 1991; Chen et al. 2002). Methods The low nanomolar (5–10 nM) concentrations in the striatum yielded by the no-net-flux approach is thought to reflect the Probe construction high efficiency and density of the DA transporter (Justice Two fused silica tubes (100 lm o.d.; 40 lm i.d., Polymicro 1993). However, in an elegant series of studies, Michael and Technologies, Inc., Phoenix, AZ, USA) were inserted into a plastic coworkers recently proposed that a trauma