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Michael AC, Borland LM, editors. Electrochemical Methods for Neuroscience. Boca Raton (FL): CRC Press/Taylor & Francis; 2007.

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# Chapter 22In Vivo Fast-Scan Cyclic Voltammetry of Dopamine near Microdialysis Probes

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## Introduction

As several of the previous chapters in this volume have explained, the various electrochemical techniques for the detection of catecholamine and indolamine neurotransmitters are finding their way into an increasing number of application areas. Electrochemical techniques offer high speed capabilities, which make them particularly useful for monitoring dynamic processes such as the clearance of neurotransmitters from extracellular fluids. This has provided for the detailed analysis of the clearance kinetics of dopamine, norepinephrine, and serotonin transporters in a number of preparations (Chapter 3 through Chapter 8). High speed electrochemical recording has enabled detailed investigations of exocytotic events at the single cell level (Chapter 14 through Chapter 16). The recent ability to record spontaneous dopamine transients in vivo is providing new insights into the functional properties of dopaminergic systems (Chapter 2).

Despite the recent advances in the capabilities of the electrochemical techniques, where in vivo measurements in living animals are concerned, many laboratories continue to employ microdialysis sampling. However, studies have suggested that the tissue trauma associated with the implantation of microdialysis probes represents a shortcoming of the technique [1–3]. If the tissue surrounding the probe is in a traumatized state, it is necessary to ask how similar the properties of the traumatized tissue are to normal, uninjured tissue. A strategy that we have pursued to address this question, in the case of dopamine measurements, is to implant carbon fiber voltammetric electrodes at various distances from microdialysis probes and compare the results. In several papers, we have demonstrated that voltammetric results vary dramatically as a function of distance from the probe, suggesting that the dopamine system in the traumatized tissue near the probe is very different from that in normal, uninjured tissue.

Given that several histological studies have pointed to probe-induced tissue trauma [4–6], it is perhaps surprising that more attention has not been paid to the effects of that trauma on the outcome of microdialysis measurements. The origin of this can be linked to the absence of procedures for the in vivo calibration of microdialysis probes. Without calibration, it is difficult to know exactly how the concentration of dopamine (as measured in a microdialysate sample) is related to the actual in vivo concentration of dopamine in the tissue surrounding the probe. For this reason, it is not particularly obvious that the dopamine is derived from traumatized tissue. It is also true, however, that procedures for the in vivo calibration of electrochemical techniques do not exist. Electrodes are calibrated on the lab bench either before or after they are used for in vivo measurements but not during the actual in vivo measurements themselves. The approach we have adopted to examining the effects of trauma on the dopamine system is to compare responses recorded simultaneously at similar electrodes at different recording locations. So, it is the comparison between similar electrodes that provides the insights into the effects of trauma, not the absolute value of the dopamine concentrations that we are measuring.

The rationale of using voltammetry as a tool for investigating the effect of trauma is based on the difference in size of the microdialysis probes and the carbon fiber microelectrodes. The probes have diameters of 225 μm or more and some studies have noticed disrupted tissue as far as 1.4 mm from the probe implantation site [6]. The carbon fiber electrodes we use for measuring dopamine have a diameter of 7 μm and the majority of the tissue disruption they cause is confined to within 3 μm of the electrode [7]. On a volume:volume basis, the probes are at least 10,000 times larger than the microelectrodes:

$πrprobe2ℓprobeπrelect2ℓelect=π(0.110 mm)2(4 mm)π(0.0035 mm)2(0.4 mm)≅10,000$

So, the concept of using a carbon fiber microelectrode as a tool for probing the trauma layer surrounding a microdialysis probe seems legitimate.

The suggestion that in vivo calibration of microdialysis probes is not routinely performed might appear surprising considering that a number of papers on the topic of quantitative microdialysis exist [4,8–11]. However, findings from voltammetry next to microdialysis probes suggest that the originally proposed approach to quantitative microdialysis has a problem. The original approach involved the determination of the quantity called the concentration of no-net-flux, which was assumed to be equal to the concentration in the fluid surrounding the probe. But, the no-net-flux concentration is only equal to the external concentration if the microdialysis extraction fraction and relative recovery are the same during the measurement. Voltammetry near microdialysis probes shows that these parameters are not the same when dopamine is measured in the striatum of chloralhydrate anesthetized rats [2,3].

In this chapter, we present some findings that extend our previous studies of voltammetry near microdialysis probes. A variable that is considered important in microdialysis protocols is the time after implantation before results are collected. Our previous work has used a 2 h wait after implantation, which is very different from the 24 h wait that is conventional. Because these experiments are performed in anesthetized animals, a 24 h wait time is difficult. But, we have examined a 16 h wait time. Also, our previous work has placed a great deal of emphasis on the ability of dopamine uptake to prevent dopamine from reaching microdialysis probes by diffusion. This is based on observations of the effect of the dopamine uptake inhibitor, nomifensine, on voltammetric recordings near microdialysis probes. So, here we report results from other drugs that increase dopamine levels by mechanisms other than uptake inhibition.

## Procedures for Voltammetry near Microdialysis Probes

### Voltammetric Electrodes and Procedures

Microcylinder voltammetric electrodes (7 μm in diameter, 400 μm long) were prepared with individual carbon fibers (T300, Amoco Performance Products, Greenville, SC). Each microelectrode was electrochemically pretreated once before use with a triangular potential waveform (0–2 V vs. Ag/AgCl, 200 V/s, 1 s). Each microelectrode was calibrated in dopamine standard solutions before and after every in vivo experiment. Dopamine detection was accomplished by fast-scan cyclic voltammetry (FSCV) performed with a computer-controlled potentiostat (Ensman Instruments, Bloomington, IN) and a software package developed in-house. Electrode pretreatment and calibration were performed in artificial cerebrospinal fluid, aCSF: 145 mM Na+, 2.7 mM K +, 1.0 mM Mg2+, 1.2 mM Ca2+, 152 mM Cl, 2.0 mM phosphate, pH 7.4.

For FSCV, the potential started at 0 V and was linearly scanned at 300 V/s to 1 V, then to −.5 V, and back to 0 V vs. Ag/AgCl. Scans were repeated at 200 ms intervals. Dopamine oxidation was monitored between 0.5 and 0.7 V on the initial potential scan. Conversion of in vivo oxidation current to dopamine concentration was based on post-calibration of each electrode. Dopamine was identified during all in vivo experiments by comparing the background-subtracted voltammograms obtained in vivo and during post-calibration of each electrode.

### Dialytrode Design

This work involves a device we call a dialytrode. It is a microdialysis probe equipped with a carbon fiber microelectrode mounted onto its outer surface [2,3]. Concentric dialysis probes (200 μm i.d., 3 mm long) were constructed with a hollow fiber membrane (SpectaPor RC, MWCO 13,000, Spectrum, Inc., Houston, TX) and a fused silica outlet line (75 μm i.d., 10 cm long, Polymicro Technology, Phoenix, AZ). A carbon fiber microelectrode (800 μm long) was mounted immediately adjacent to the outer surface of the probe. The integrated carbon fiber had no impact on probe performance, which is as expected since the cross section of the fiber is negligible (~ 0.3%) compared to the area of the probe itself [2]. For the sake of comparison, dopamine was also monitored by fast scan cyclic voltammetry with a carbon fiber microelectrode (400 μm long) mounted in the probe outlet. The probe was perfused with aCSF at 0.109 μL/min.

### Surgical Procedures

The University of Pittsburgh Institutional Animal Care and Use Committee approved the procedures involving animals. Chloral hydrate anesthetized male Sprague–Dawley rats (250–350 g) were wrapped in a homeothermic blanket (EKEG Electronics, Vancouver, BC) and placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA) with the upper incisor bar raised 5 mm above the interaural line [12]. Holes were drilled through the skull. Electrical contact between brain tissue and a Ag/AgCl reference electrode was established with a salt bridge.

Dopamine was measured at two ipsilateral sites in the striatum. The posterior site was 1.8 mm anterior to bregma and 2.5 mm lateral from midline. The anterior site was 2.8 mm anterior to bregma and 2.5 mm lateral from midline. These sites are placed 1 mm apart because several studies show that tissue within 1 mm of microdialysis probes is disrupted by traumatic injury and the depletion of dialyzable substances [4–6].

Except where noted, a microcylinder electrode was placed 4.5 mm below dura at the posterior site. Then, either a second microelectrode or a dialytrode was placed at the anterior site. A stainless steel bipolar stimulating electrode (MS303/1; Plastics One, Roanoke, VA) was placed in the ipsilateral MFB. The stimulus waveform (a constant current, biphasic square wave with a pulse width of 2 ms, a pulse height of 280 μA, a frequency of 45 Hz, and a train length of 25 s) was optically isolated from the potentiostat (Neurolog 800; Medical Systems, Greenvale, NY). Experiments were continued only when stable voltammetric responses of at least 30 nA in amplitude were obtained. The microelectrode at the posterior site remained in place throughout all experiments.

Dialytrodes were mounted on a high-resolution stereotaxic manipulator and slowly lowered over a time period of 20–30 min to 7.0 mm below dura. The probes were perfused in-place for a minimum of 2 h before experiments began. Previously, we showed that this implantation procedure produces a stable, impulse dependent, nanomolar-level of dopamine in basal dialysate samples [13]. Unless noted otherwise, stimulation was delivered to the MFB at 20-min intervals.

### Dual Electrode Voltammetry

Experiments involving simultaneous voltammetry at both sites were conducted according to two designs. First, microelectrodes were placed 4.0 mm below dura at both the posterior and anterior sites and were subsequently lowered 0.5 mm between consecutive stimuli to a final depth of 6.0 mm below dura. Second, microelectrodes were placed at a fixed depth of 4.5 mm below dura. Nomifensine, l -dihydroxyphenylalanine ( l -DOPA), pargyline, or sulpiride were administered after the third stimulus and post-drug stimulus responses were recorded at the intervals indicated.

### Voltammetry near Microdialysis Probes

Experiments involving voltammetry in the presence of a dialytrode were conducted according to each of the following designs.

#### Design 1

Initially, stimulus responses were recorded with microelectrodes placed at both the posterior and anterior sites. The anterior microelectrode was then removed and replaced with a dialytrode, which was perfused in-place for 2 h before MFB stimulation was continued. Responses were recorded at the posterior, adjacent, and outlet microelectrodes. Nomifensine was administered systemically after the third stimulus, and post-drug responses were recorded 20 min later.

#### Design 2

A microelectrode was placed at the posterior site and a dialytrode was placed at the anterior site. The dialytrode was perfused in-place for 16 h, during which the rat received injections of glucose in saline (0.5 mL, 5 mM, i.p.) every 2–3 h, before MFB stimulation was continued. Responses were recorded at the posterior, adjacent, and outlet microelectrodes. Nomifensine was administered systemically after the third stimulus, and post-drug responses were recorded 20 min later.

#### Design 3

A microelectrode was placed at the posterior site and a dialytrode was placed at the anterior site. The dialytrode was perfused in-place for 2–5 h, after which MFB stimulation continued. Responses were recorded at the posterior, adjacent, and outlet microelectrodes. Nomifensine, l -DOPA, pargyline, or sulpiride was administered systemically after the third stimulus and post-drug responses were recorded at the intervals indicated. Each rat received a single drug.

All drugs were administered by intraperitoneal injection in phosphate-buffered saline vehicle at the following doses: nomifensine (20 mg/kg); l -DOPA (250 mg/kg administered 30 min after 150 mg/kg carbidopa); pargyline (75 mg/kg); sulpiride (100 mg/kg). The drugs were obtained from Sigma (St Louis, MO).

## Results

### Dual Electrode Recording

Stimulus responses at the posterior and anterior sites were characterized by simultaneous voltammetry in a total of 15 rats. The depth profiling experiment was performed in three of these rats. Figure 22.1 shows that in one rat a robust stimulus response was observed at all vertical electrode placements examined at both the posterior and anterior sites, which is typical of the results obtained in all three rats. At a fixed depth of 4.5 mm below dura, the response amplitudes observed at the posterior and anterior sites in 12 rats corresponded to 17.9± 8.76 μM and 15.9± 7.39 μM (mean± s.d.), respectively, which are not significantly different according to the t -test. The black and gray bars in Figure 22.2 show the relative effects of four drugs on the response amplitude at the posterior and anterior sites, respectively.

#### FIGURE 22.1

Stimulus responses recorded with a pair of voltammetric microelectrodes at the posterior (a) and anterior (b) sites in striatum. The open circles mark the beginning and end of the stimulus. Robust stimulus responses were observed at all vertical positions (more...)

#### FIGURE 22.2

The effect of nomifensine, l -DOPA, pargyline, and sulpiride on the relative amplitude of the stimulus response obtained at the anterior site in the absence of a dialytrode (gray bars), the posterior site in the absence of a dialytrode (black bars), and (more...)

### Probe Implantation Affects the Voltammetric Responses

Experiments were performed according to Designs 1–3 of Voltammetry Near Microdialysis Probes to examine the impact of probe implantation on the general features of the voltammetric stimulus responses at the two sites within the striatum.

#### Design 1

Figure 22.3 illustrates the effect of probe implantation on evoked responses observed at the posterior and anterior site in a single rat. Robust responses were evoked at both the posterior and anterior sites prior to implantation of a dialytrode. A robust response was still observed at the posterior site 2 h after the anterior microelectrode had been replaced with a dialytrode. Nevertheless, 2 h after implantation of the dialytrode no evoked response was detectable at the anterior site, i.e., at the microelectrode placed immediately adjacent to the microdialysis probes.

#### FIGURE 22.3

Stimulus responses recorded with a voltammetric microelectrode at the posterior (a) and anterior (b) sites just before and 2 h after implantation of a dialytrode at the anterior site. At the posterior site, the stimulus response was not significantly (more...)

The response amplitude at the posterior site 2 h after dialytrode implantation (12.5± 7.51 μM, mean±s.d., n = 12) was not significantly different than the amplitudes (reported above) obtained at the posterior and anterior sites without implantation of a dialytrode (one-way ANOVA: f = 1.09; d.f. = 2,33).

In a group of three rats, the experiment of Figure 22.3 was continued after the systemic administration of nomifensine. Figure 22.4 shows that the response amplitude at the posterior site increased after nomifensine administration. Moreover, evoked responses were detectable at the adjacent and outlet microelectrodes after nomifensine administration. Equivalent results were obtained in all three rats in which this protocol was used.

#### FIGURE 22.4

Pairs of stimulus responses recorded simultaneously with voltammetric microelectrodes at the posterior site (a), adjacent to a microdialysis probe (b), and at the outlet of a microdialysis probe (c). The experiment was initiated 2 h after implantation (more...)

#### Design 2

Stimulus responses were recorded at the posterior, adjacent, and outlet microelectrodes 16 h after implantation of the dialytrodes (Figure 22.5). Before nomifensine administration, the response amplitude at the posterior site was slightly but not significantly ( t -test) smaller at 16 h after implantation (9.1± 3.1 μM, mean±s.d., n = 3) than at 2 h (12.5± 7.51 μM, mean±s.d., n = 12). The relative increase in the amplitude induced by nomifensine at the posterior site was not significantly different ( t -test) at 16 h (196± 40%, mean ± s.d., n = 3) and at 2 h (164± 50%, mean±s.d., n = 3). Prior to nomifensine, evoked responses were again not detected at the adjacent and outlet microelectrodes at 16 h post implantation. At the adjacent microelectrode, the response observed after nomifensine was significantly smaller at 16 h than at 2 h: 1.09± 0.41 μM at 16 h vs. 2.94± 0.64 μM at 2 h (mean±s.d., n = 3, p <.015, t -test). The response at the outlet electrode at 16 h was too small to reliably quantify.

#### FIGURE 22.5

Results presented in the same format as in Figure 22.5 except that the experiment was initiated 16 h after implantation of the voltammetric electrode at the posterior site and the dialytrode at the anterior site. The responses recorded at the posterior (more...)

#### Design 3

Stimulus responses were recorded simultaneously at the posterior and adjacent microelectrodes before and after the administration l -DOPA, pargyline, or sulpiride (Figure 22.6). These experiments were initiated 2 h after implantation of the dialytrodes. Each drug was examined in a different group of three rats. Even though each drug increased the evoked response at the posterior site, evoked responses at the anterior and outlet microelectrodes remained undetectable (outlet microelectrode data omitted). The white bars in Figure 22.2 show the effect of each drug on the relative amplitude of the stimulus response at the posterior site after dialytrode implantation. The results for each drug were analyzed by two-way ANOVA with a repeated measures design. The main effects were the time with respect to drug administration and the recording site. The effect of time was significant in all cases (nomifensine, f = 36.2, d.f. = 1,12, p <.001: l -DOPA, f = 34.0, d.f. = 2,18, p <.001: pargyline, f = 26.0, d.f. = 3,24, p <.001: sulpiride, f = 55.9, d.f. = 3,24, p <.001). The effect of recording site was significant only in the case of sulpiride ( f = 15.8; d.f. = 2,24; p <.001). Interactions between time and recording site were not significant.

#### FIGURE 22.6

Pairs of stimulus responses recorded simultaneously with voltammetric microelectrodes at the posterior site (a) and adjacent to a microdialysis probe at the anterior site (b). Responses were recorded before (pre) and after (post) the systemic administration (more...)

## Discussion and Implications

Several lines of evidence suggest that the tissue surrounding a microdialysis probe is disrupted from its normal state [1–6]. Hence, it is relevant to ask how the trauma affects the dopaminergic system. Voltammetry in conjunction with carbon fiber microelectrodes presents the opportunity for direct observations, since the electrodes are small enough in their dimensions to be placed with the probe’s trauma layer. The results presented here were collected in the striatum of chloral hydrate anesthetized rats, since there is an indication that the anesthetized preparation may minimize implant trauma. The use of anesthesia makes it possible to implant microdialysis probes very slowly [14–16] and makes it possible to keep the animal completely immobilized thereafter. This produces a stable, highly impulse dependent baseline of nanomolar dopamine levels in dialysate obtained 2 h after implantation of the probe [13]. The rapid attainment of a stable, impulse dependent baseline level of dialysate dopamine strongly implies that the procedures followed during this study permit the tissue surrounding the probes to stabilize quickly after probe implantation. Nevertheless, as a consequence of the decision to use anesthetized animals for this study, the outcome of this initial investigation of the feasibility of a voltammetric approach to the in vivo calibration of microdialysis is relevant only to anesthetized conditions.

Measurements of extracellular dopamine in the striatum were performed at two sites placed 1 mm apart, a distance selected on the basis of recent estimates of the physical extent of probeinduced tissue disruption [4–6]. First, voltammetric stimulus responses were recorded with microcylinder carbon fiber electrodes at both sites. At several depths of penetration beneath the brain surface, responses were consistently detected at both sites (Figure 22.1). At a fixed depth of 4.5 mm below dura the magnitude of the pre-drug evoked responses at the two sites were not statistically different. Furthermore, there was no statistical difference between the effects of four dopaminergic drugs on the amplitude of the stimulus responses at the two sites (Figure 22.2). The similarity between the responses obtained at the two sites, despite the existence of local heterogeneity in striatal tissue [17–20], can be attributed to the use in this work of microcylinder electrodes, which by virtue of their length, measure the spatial average of extracellular dopamine concentrations [21]. Overall, voltammetric responses observed with these microcylinder electrodes at the posterior and anterior sites are statistically indistinguishable. Local heterogeneities in the evoked dopamine response, therefore, are excluded as a possible explanation for the differences in results between electrodes adjacent to the probes and 1 mm from the probe.

For the most part, stimulus responses at the posterior site 1 mm from the site of probe implantation were unaffected by implantation of a dialytrode at the anterior site. The amplitudes of the responses observed at the posterior site (before and after probe implantation) were not statistically different. Also, the effects of nomifensine, l -DOPA, and pargyline on the response amplitude were not changed by probe implantation (Figure 22.2). Implantation of the dialytrode affected the results obtained after sulpiride, however, which suggests that probe implantation may have induced some disruption at the posterior site. This effect was so slight (Figure 22.2), however, that it appears reasonable to conclude that evoked responses recorded 1 mm away from a microdialysis probes provide an adequate representation of the evoked responses expected in the absence of probeinduced tissue disruption.

Overall, the results in Figure 22.1 and Figure 22.2 confirm that evoked responses at the two sites are inherently similar. Moreover, the evoked responses observed at the posterior site 2 h after implantation of a microdialysis probe at the anterior site are inherently similar to those observed without probe implantation. Hence, the lack of a detectable response at microelectrodes placed immediately adjacent to microdialysis probes cannot be attributed to any inherent variability of evoked responses in the striatum. Rather, it seems more likely that the absence of a detectable response at microelectrodes placed immediately adjacent to microdialysis probes is a consequence of probe-induced tissue disruption. Figure 22.3, which shows that an evoked response was observed at the anterior site with a conventional voltammetric electrode just prior to implantation of a dialytrode at the same location, strongly supports this interpretation.

Consistent with previous related work [1–3], evoked responses were detectable at microelectrodes placed adjacent to microdialysis probes after the systemic administration of the dopamine uptake inhibitor, nomifensine (Figure 22.4). Moreover, after uptake inhibition evoked responses were also detectable at the microelectrode placed at the outlet of the microdialysis probe (Figure 22.4). It appears from these results that uptake inhibition, which prolongs the lifetime of dopamine in the extracellular space, enables dopamine to diffuse further from sites of evoked release and thereby increases the amount of dopamine that diffuses to the probes. The appearance of an evoked response adjacent to the probe and at the probe outlet after uptake inhibition forms the basis of our suggestion that uptake inhibition increases the relative recovery of dopamine from the striatal extracellular space.

Because many studies report that microdialysis results are affected by the post implantation interval [22–26], the experiment with nomifensine was also performed with a post implantation interval of 16 h. Without uptake inhibition, evoked responses remained undetectable at the microelectrodes adjacent to the probe and at the probe outlet. After uptake inhibition, the evoked responses at the adjacent microelectrodes observed 16 h after probe implantation were substantially smaller than those observed 2 h after probe implantation. Moreover, at 16 h after probe implantation, no postnomifensine response was detected at the probe outlet. Together, these observations show that, despite the combined use of slow probe implantation and anesthetized animals, extending the post implantation interval does not eliminate the disparity between evoked responses recorded immediately adjacent to microdialysis probes and those recorded 1 mm from microdialysis probes. Indeed, the disparity after uptake inhibition is greater, leading us to wonder if the 24 h wait after probe implantation really constitutes a recovery period as it is often described.

Also, the systemic administration of drugs that increase the evoked response at conventional microelectrodes (Figure 22.6) was not effective at promoting voltammetric stimulus responses at the microelectrodes adjacent to microdialysis probes. This is consistent with the concept that uptake plays a central role in determining the distance that dopamine can diffuse from sites of evoked release. Although the other drugs studied here increase the amount of dopamine released during stimulation, they do not act to increase the extracellular lifetime of dopamine, and hence, do not increase the likelihood that dopamine will diffuse to the probe.

Overall, the results in Figure 22.1 through Figure 22.6 extend our earlier suggestion that due to the combined effects of tissue trauma and dopamine uptake, the microdialysis recovery of dopamine from the striatal extracellular space is inherently low. Dopamine release is suppressed in the tissue immediately adjacent to the probe, so normal dopamine release amplitude is observed only at several hundred micrometers from the probe [3]. Nevertheless, dopamine uptake appears to remain relatively intact, compared to release, next to the probe. Uptake inhibition has a far greater effect on the amplitude of evoked dopamine release when the microelectrode is in the trauma layer (Figure 22.1 and Ref. [ [3]]). Once dopamine is released, it has to diffuse through the trauma layer in order to be recovered by the probe. It is highly likely that dopamine will be taken up before it reaches the probe unless the transporter is inhibited [27]. So, uptake inhibition increases dopamine recovery.

The conclusion that uptake inhibition increases dopamine recovery is supported by our observations of stimulus responses after the administration of drugs that increase the response amplitude in the absence of the probe. Three drugs that we examined, l -DOPA, pargyline, and sulpiride, all increased the response amplitude in the absence of the probe, and 1 mm from the probe, but none of these drugs caused detectable responses adjacent to the probe or at the probe outlet. These drugs affect the evoked response by enhancing dopamine release, in contrast to nomifensine, which slows dopamine uptake. Enhancing evoked release did not appear to have the ability to boost dopamine recovery, supporting our suggestion that uptake kinetics are a key determinant of recovery.

The results presented here lend further support to the idea that uptake lowers dopamine recovery, which is exactly the opposite of its effect on dopamine extraction [28]. Under conditions where the recovery is smaller than the extraction fraction, the concentration of no-net-flux underestimates extracellular concentrations [3]. The exact extent to which extracellular dopamine concentrations are underestimated remains unknown, but in recent studies we found evidence for a resting striatal dopamine concentration in the micromolar range [29,30]. At present, however, our micromolar estimate of extracellular concentration is difficult to resolve with the previous work of Gonon and Buda [31], who estimated 25 nM in pargyline-treated rats. A potentially more important finding is that the recovery factor, which is key to the quantitative performance of the microdialysis probe, is sensitive to drugs that inhibit dopamine uptake.

Finally, it is interesting to note that the voltammetric recordings performed during this work at the outlet of the microdialysis probe correlated best with the voltammetric recordings performed immediately adjacent to the probe. The recordings at the outlet did not correlate with the recordings performed 1 mm away or in the absence of the probe. This observation strongly suggests that the dopamine content of the microdialysate samples most often reflects the dopamine content in the extracellular fluid in the tissue immediately adjacent to the probe, i.e., that tissue most profoundly traumatized by the probe. This emphasizes one of the key attributes of the carbon fiber microelectrode for in vivo measurements, which is that their small dimensions inflict minimal trauma at the implantation site [7].

## Acknowledgments

This work was supported by NIH (Grant NS 31442) and by a grant from the National Parkinson’s Foundation Center of Excellence at the University of Pittsburgh. Hua Yang was the recipient of a Graduate Summer Fellowship from the Division of Analytical Chemistry of the American Chemical Society.

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