(Equation 1)
Introduction
The interpretation of in vivo microdialysis and voltammetry
results is a challenge because neither technique can be calibrated in a
fashion that directly determines their sensitivity towards endogenous dopamine.
Consequently, interpretations of in vivo results have depended heavily
on theoretical models of the kinetic and transport processes that determine
the fate of dopamine in the extracellular space. Various models have been
applied to either microdialysis or voltammetry but not so often to both.
Rationale
Our goal was to develop a common basis for the interpretation of both
microdialysis and voltammetry. Towards this goal, we have developed a kinetic
and transport model that qualitatively agrees with the major aspects of
results recently collected during simultaneous in vivo microdialysis
and voltammetry experiments.
Methods
Voltammetry and microdialysis were used simultaneously in chloral hydrate-anesthetized
rats to monitor striatal extracellular dopamine during electrical stimulation
of the medial forebrain bundle. Voltammetric electrodes were constructed
with single carbon fibers with a 3.5-mm radius.
Both microdisk and microcylinder electrodes (100-800 mm
in length) were used. A small number of experiments have also been conducted
with pairs of 1-mm diameter microdisk electrodes
that were separated from each other by ca. 10 mm.
Microdialysis probes, with an outer radius of 100 mm
and a length of 3 mm, were of the concentric design. These devices were
acutely implanted in the striatum of chloral hydrate-anesthetized rats.
The microdialysis probes were lowered slowly and were perfused in-place
for up to 8 hrs before experiments were started. MFB simulation was applied
at 45 Hz, with a current level of 50 mA rms,
and for a period of either 10 or 25 s.
Results and Discussion
Results obtained with microdisk electrodes demonstrate that dopamine
release occurs in a spatially non-uniform manner that gives rise to a microheterogeneous
spatial distribution of extracellular dopamine concentration. Furthermore,
the microheterogeneity is a direct consequence of the uptake-limited distance
that dopamine molecules can diffuse from the sites at which they are released
to the extracellular space.
The microheterogeneity of extracellular dopamine levels is not apparent when microcylinder electrodes are used, because these devices report the spatial average of the concentration.
Stimulation of the MFB does not elicit a detectable response at microcylinder electrodes implanted immediately adjacent to microdialysis probes, even when a large response is observed at microcylinders electrode placed 1 mm away from the probes. The evoked response at electrodes 1 mm from the probe is at least 200 times larger than the response at a similar electrode in the immediate vicinity of the probe. This large difference is eliminated by administration of nomifensine (20 mg/kg i.p), but not by administration of L-DOPA (150 mg/kg, 30 min after pretreatment with carbidopa).
These results show that a wide range of responses is obtained with analytical devices of different sizes and shapes, even though each device is used under identical in vivo experimental conditions. This clearly shows that the geometry of the analytical device is an important parameter to consider in the interpretation of in vivo results. Geometry would not be important if the concentration of dopamine in the extracellular space were spatially uniform. So, we have developed a model of extracellular dopamine that does not presume a spatially uniform concentration.
Our model uses an array of small volume elements distributed in three dimensions to serve as the diffusion source. Once the diffusable substance is released, it is cleared by a chemical process that is active only within the array of small volume elements. The model qualitatively replicates both voltammetry and microdialysis results generated in this laboratory as well as results described in the recent literature. The model provides theoretical justification for the following discussion.
Recent approaches to quantitative microdialysis, e.g. the no-net-flux method, require that the extraction fraction, E, and relative recovery, R, be the same because the following relationship exists between the extracellular, Cext, and no-net-flux, CNNF, concentrations: 1
(Equation 1)
In a spatially uniform system where kinetic processes are not at work, e.g. in a beaker, E and R values can be directly measured and are found to be equal. Our results, however, demonstrate that this condition does not exist for dopamine in the brain.
To make use of Equation 1, we have estimated R for dopamine from the ratio of the amplitudes of stimulus responses recorded with microcylinder electrodes placed in brain tissue, immediately adjacent to, and at the outlet of microdialysis probes. The results show that the R value is no greater than ~5·10-3, which in combination with Equation 1 and available values for E (0.7)2 and CNNF (6 nM)2 leads to an estimated Cext value of 840 nM for striatal dopamine.
The major source of the ~2 order-of-magnitude difference between the basal extracellular dopamine concentration estimated here and previously (e.g., Ref. 2) is attributed to the uptake-imposed limitation on the distance that dopamine can diffuse through the extracellular space. We envision microdialysis as sampling from a region largely beyond the uptake-limited diffusion distance from dopamine terminals. Results obtained with microelectrodes placed immediately adjacent to microdialysis probes provide direct experimental evidence to support this view. (Supported by NIH: Grant NS 31442).
References
1. Yang H., Peters J.L. and Michael A.C. (1998) Coupled effects of mass transfer and uptake kinetics on in vivo microdialysis of dopamine. J. Neurochem. 71:684-692.
2. Sam P.M and Justice J.B. Jr., (1996) Effect of general microdialysis-induced depletion on extracellular dopamine. Anal. Chem. 68: 724-728.