In vivo Effects of the Anatoxin-a on Striatal Dopamine Release

Anatoxin-a is an important neurotoxin that acts a potent nicotinic acetylcholine receptor agonist. This characteristic makes anatoxin-a an important tool for the study of nicotinic receptors. Anatoxin-a has been used extensively in vitro experiments, however anatoxin-a has never been studied by in vivo microdialysis studies. This study test the effect of anatoxin-a on striatal in vivo dopamine release by microdialysis.The results of this work show that anatoxin-a evoked dopamine release in a concentration-dependent way. Atropine had not any effect on dopamine release evoked by 3.5 mM anatoxin-a. However, perfusion of nicotinic antagonists mecamylamine and α-bungarotoxin induced a total inhibition of the striatal dopamine release. Perfusion of α7*-receptors antagonists, metillycaconitine or α-bungarotoxin, partially inhibits the release of dopamine stimulated by anatoxin-a. These results show that anatoxin-a can be used as an important nicotinic agonist in the study of nicotinic receptor by in vivo microdialysis technique and also support further in vivo evidences that α7*nicotinic AChRs are implicated in the regulation of striatal dopamine release.     

Microdialysis of dopamine interpreted with quantitative model incorporating probe implantation trauma

Although microdialysis is widely used to sample endogenous and exogenous substances in vivo, interpretation of the results obtained by this technique remains controversial. The goal of the present study was to examine recent criticism of microdialysis in the specific case of dopamine (DA) measurements in the brain extracellular microenvironment. The apparent steady-state basal extracellular concentration and extraction fraction of DA were determined in anesthetized rat striatum by the concentration difference (no-net-flux) microdialysis technique. A rate constant for extracellular clearance of DA calculated from the extraction fraction was smaller than the previously determined estimate by fast-scan cyclic voltammetry for cellular uptake of DA. Because the relatively small size of the voltammetric microsensor produces little tissue damage, the discrepancy between the uptake rate constants may be a consequence of trauma from microdialysis probe implantation. The trauma layer has previously been identified by histology and proposed to distort measurements of extracellular DA levels by the no-net-flux method. To address this issue, an existing quantitative mathematical model for microdialysis was modified to incorporate a traumatized tissue layer interposed between the probe and surrounding normal tissue. The tissue layers are hypothesized to differ in their rates of neurotransmitter release and uptake. A post-implantation traumatized layer with reduced uptake and no release can reconcile the discrepancy between DA uptake measured by microdialysis and voltammetry. The model predicts that this trauma layer would cause the DA extraction fraction obtained from microdialysis in vivo calibration techniques, such as no-net-flux, to differ from the DA relative recovery and lead to an underestimation of the DA extracellular concentration in the surrounding normal tissue.     

Coupled Effects of Mass Transfer and Uptake Kinetics on In Vivo Microdialysis of Dopamine

Abstract: Voltammetric microelectrodes and microdialysis probes were used simultaneously to monitor extracellular dopamine in rat striatum during electrical stimulation of the medial forebrain bundle. Microelectrodes were placed far away (1 mm) from, immediately adjacent to, and at the outlet of microdialysis probes. In drug-naive rats, electrical stimulation (45 Hz, 25 s) evoked a robust response at microelectrodes far away from the probes, but there was no response at microelectrodes adjacent to and at the outlet of the probes. After nomifensine administration (20 mg/kg i.p.), stimulation evoked robust responses at all three microelectrode placements. These results demonstrate first that evoked release in tissue adjacent to microdialysis probes is suppressed in comparison with evoked release in tissue far away from the probes and second that equilibration of the dopamine concentration in the extracellular fluid adjacent to and far away from the probes is prevented by the high-affinity dopamine transporter. Hence, models of microdialysis, which assume the properties of tissue to be spatially uniform, require modification to account for the distance that separates viable sites of evoked dopamine release from the probe. We introduce new mass transfer resistance parameters that qualitatively explain the observed effects of uptake inhibition on stimulation responses recorded with microdialysis and voltammetry.     

Decarboxylation of Exogenous l-3,4-Dihydroxyphenylalanine in Rat Striatum as Studied by In Vivo Voltammetry

An in vivo voltammetric technique was used to determine whether striatal nondopaminergic neurons take up and decarboxylate exogenous L-3,4-dihydroxyphenylalanine (L-DOPA) and release it as dopamine. After the striatal serotonergic neurons of the rat had been destroyed by intraventricular injection of 5,7-dihydroxytryptamine, L-DOPA was administered intraperitoneally. It was found that changes in the dopamine concentration in the striatal extracellular fluid of the rat were the same as those in the nonlesioned rat. L-DOPA was also administered to the rat after the striatal perikarya had been destroyed by the intrastriatal injection of kainate. The striatal dopamine concentrations of the lesioned rat changed in parallel with 5,7-dihydroxytryptamine-lesioned rats, as well as the nonlesioned rats. Moreover, when normal rats were administered L-DOPA, the dopamine concentration was not increased in the cerebellum, where dopamine neurons do not exist. From these observations, it is concluded that exogenous L-DOPA is taken up, decarboxylated to dopamine, and released only in the striatal dopamine neurons.     

Extracellular Events Induced by γ-Hydroxybutyrate in Striatum: A Microdialysis Study

The modification of dopamine release and accumulation induced by gamma-hydroxybutyrate (GHB) was studied using both striatal slices and in vivo microdialysis of caudate-putamen. GHB inhibited dopamine release for approximately 5-10 min in vitro, and this was associated with an accumulation of dopamine in the tissue. Subsequently, there was an increase in dopamine release. In the microdialysis experiments, low doses of GHB inhibited dopamine release, whereas higher doses strongly increased release; the initial decrease seen in slices could not be detected in vivo. Thus, GHB had a biphasic effect on the release of dopamine: An initial decrease in the release of transmitter was followed by an increase. A time-dependent biphasic effect was observed when GHB was added to brain slices, and a dose-dependent biphasic effect was seen in dialysate after systemic administration of GHB. Naloxone blocked GHB-induced dopamine accumulation and release both in vitro and in vivo. GHB also increased the release of opioid-like substances in the striatum. A specific antagonist of GHB receptors completely blocked both the dopamine response and the release of opioid-like substances. These data suggest that GHB increases dopamine release via specific receptors that may modulate the activity of opioid interneurons.     

Evidence on extracellular dopamine level in rat striatum: implications for the validity of quantitative microdialysis

Microdialysis zero-net-flux (ZNF) method is commonly used to monitor drug-induced changes in neurotransmitter baseline and release/uptake processes. Recent studies in this field suggest that microdialysis ZNF method seriously underestimates the resting concentration of extracellular dopamine in the rat neostriatum because probe implantation preferentially damages nearby dopamine release sites and that dopamine uptake inhibition increases the relative recovery of dopamine by microdialysis. This study assessed the validity of these claims by examining current data on extracellular dopamine levels at rest and after drug application obtained by voltammetry, a technique thought to induce less tissue disruption than microdialysis. To obtain the extracellular baseline value for dopamine from the evoked overflow data, we modified the existing dopamine kinetic model to suit both the resting and stimulated circumstances. It was found that dopamine uptake inhibition did in fact decrease the microdialysis relative recovery of dopamine, implying that the average basal extracellular dopamine level is within the range of 7-20 nm in rat striatum. This study concludes that the microdialysis ZNF method indeed underestimates the extracellular dopamine concentration, although not by as much as had been thought. Chronic microdialysis damages both neurotransmitter release and uptake, but it does so in a somewhat relative and proportional way for both processes. Thus the validity of the microdialysis ZNF method is not seriously undermined.     

D2 Receptors May Modulate the Function of the Striatal Transporter for Dopamine: Kinetic Evidence from Studies In Vitro and In Vivo

Abstract: Recently it was hypothesized by others that the D₂dopamine receptor can regulate the uptake of dopamine. However, the evidence in support of this hypothesis, although compelling, was not based on observations related to direct measures of the kinetic activity of the transporter itself. Here kinetic evidence in support of this hypothesis is shown. The apparent time-resolved initial velocity of the transport of 1.0 μ M dopamine into striatal suspensions, measured using rotating disk electrode voltammetry, was found to increase in the presence of the D₂ receptor agonist, quinpirole, at 100 μ M . This effect was reversed by sulpiride. In separate studies it was shown that acute and chronic treatments with haloperidol at 0.5 mg/kg, i.p., reduced the reuptake transport of dopamine in vivo following intrastriatal stimulation of its release by K⁺. Thus, it appears that D₂ receptors may influence the functioning of the striatal transporter for dopamine. These results are consistent with a model in which presynaptically released dopamine may feed back onto the function of its transporter to increase the velocity of the clearance of synaptic dopamine following an action potential, suggesting the existence of a mechanism, in addition to release and synthesis modulation, for fine-tuning dopaminergic chemical signaling.     

Monitoring the Stimulated Release of Dopamine with In Vivo Voltammetry. II: Clearance of Released Dopamine from Extracellular Fluid

Microvoltammetric electrodes implanted in the caudate nucleus of the anesthetized rat have been used to monitor dopamine released following electrical stimulation of the medial forebrain bundle. These electrodes are fabricated from unmodified carbon fibers and have been used with normal pulse voltammetry. Dopamine appears in the vicinity of the electrode when the stimulation is initiated, and disappears almost immediately when the stimulation is terminated. The data suggest that the effective diffusion distance is less than 100 micron. Postmortem analysis using liquid chromatography with electrochemical detection shows that dopamine released in this manner is metabolized to 3,4-dihydroxyphenylacetic acid (DOPAC); however, neither substance is observed electrochemically in the extracellular fluid within seconds after the stimulation. In addition, inhibitors of neuronal uptake of dopamine, amphetamine (1.8 or 15 mg X kg-1) or benztropine (25 mg X kg-1), or of dopamine metabolism, pargyline (150 mg X kg-1) or tropolone (100 mg X kg-1), do not significantly affect the rate at which dopamine disappears from extracellular fluid, although they can affect the amount released. These results suggest that dopamine cannot freely diffuse in the extracellular fluid because an extraneuronal uptake mechanism exists that clears dopamine from extracellular fluid into an extraneuronal pool where metabolism to 3,4-dihydroxyphenylacetic acid occurs. Dopamine can be observed during electrical stimulation of the ascending fibers because neuronal and extraneuronal uptake systems are unable to remove dopamine on these short time scales.     

Preferentially impaired neurotransmitter release sites not their discreteness compromise the validity of microdialysis zero-net-flux method

Intracerebral microdialysis is a popular technique for studying neurochemistry and neural circuits in various brain regions. Recent studies called into question the validity of the microdialysis zero-net-flux (ZNF) method by suggesting that this method significantly underestimates the basal level of extracellular dopamine as a result of the discreteness of dopamine release sites as well as the preferential damage to dopamine release over uptake. To identify which factor is most important in undermining the microdialysis ZNF measurements and the extent of underestimation, two mathematical models were developed to explore the influences of the discrete nature and the probe-induced impairment in the neurotransmitter release. The two models differ in their characterizations of the transmitter release as spatially discrete and homogeneous, respectively. Simulations using physiologically reasonable parameters for striatal dopamine systems indicate that the preferential release site damage surrounding the implanted probe is the most important determinant to the underestimation of the microdialysis ZNF concentration. Under normal physiological conditions, the discreteness of neurotransmitter release sites is of minor importance, except when neuronal degeneration occurs. It is concluded that homogeneous models can adequately describe microdialysis operating processes as long as the corresponding tissue damage parameters in such models are appropriately incorporated.     

Ionic Composition of Microdialysis Perfusing Solution Alters the Pharmacological Responsiveness and Basal Outflow of Striatal Dopamine

While using the technique of in vivo microdialysis, we have assessed the effect of the ionic composition of the perfusing solution on extracellular dopamine levels during resting conditions and following a pharmacological manipulation. Our results indicate that perfusion with solutions containing the ionic composition of commercially available Ringer's solution, which mimic the ionic composition of plasma as opposed to brain extracellular fluid, alters the turnover rate and basal release of dopamine. Moreover, perfusion with solutions containing higher calcium levels, i.e., 3.4 mM, than the amount we have determined to be present in the extracellular fluid of striatum (1.2 mM) alters the pharmacological responsiveness of the nigrostriatal dopamine system to synthesis inhibition.     

Amphetamine-Induced Dopamine Release in the Rat Striatum: An In Vivo Microdialysis Study

The effects of a number of biochemical and pharmacological manipulations on amphetamine (AMPH)-induced alterations in dopamine (DA) release and metabolism were examined in the rat striatum using the in vivo brain microdialysis method. Basal striatal dialysate concentrations were: DA, 7 nM; dihydroxyphenylacetic acid (DOPAC), 850 nM; homovanillic acid (HVA), 500 nM; 5-hydroxyindoleacetic acid (5-HIAA), 300 nM; and 3-methoxytyramine (3-MT), 3 nM. Intraperitoneal injection of AMPH (4 mg/kg) induced a substantial increase in DA efflux, which attained its maximum response 20-40 min after drug injection. On the other hand, DOPAC and HVA efflux declined following AMPH. The DA response, but not those of DOPAC and HVA, was dose dependent within the range of AMPH tested (2-16 mg/kg). High doses of AMPH (greater than 8 mg/kg) also decreased 5-HIAA and increased 3-MT efflux. Depletion of vesicular stores of DA using reserpine did not affect significantly AMPH-induced dopamine efflux. In contrast, prior inhibition of catecholamine synthesis, using alpha-methyl-p-tyrosine, proved to be an effective inhibitor of AMPH-evoked DA release (less than 35% of control). Moreover, the DA releasing action of AMPH was facilitated in pargyline-pretreated animals (220% of control). These data suggest that AMPH releases preferentially a newly synthesised pool of DA. Nomifensine, a DA uptake inhibitor, was an effective inhibitor of AMPH-induced DA efflux (18% of control). On the other hand, this action of AMPH was facilitated by veratrine and ouabain (200-210% of control). These results suggest that the membrane DA carrier may be involved in the actions of AMPH on DA efflux.     

In Vivo Striatal Dopamine Release by M1 Muscarinic Receptors Is Induced by Activation of Protein Kinase C

Islet-activating protein was unilaterally microinjected into rat striatum, and a dialysis cannula was implanted into the same area under anesthesia. After 2 days, various agents were perfused continuously into the striatum through the dialysis membrane, under freely moving conditions. Islet-activating protein (2 micrograms/2 microliters) treatment alone did not change in vivo striatal dopamine (DA) release and metabolism, but completely abolished the increase of striatal DA release evoked in vivo by the M1-selective agonist McN-A-343 (10(-7) M). Forskolin (10(-5) M), an adenylate cyclase activator, increased DA release and showed an additive effect on the DA release evoked by McN-A-343. Polymyxin B, a rather selective inhibitor of protein kinase C, decreased DA release and completely blocked the effect of McN-A-343. These results suggest that in vivo striatal DA release elicited by M1 muscarinic receptors is coupled with interaction with a Go protein and is induced by activation of protein kinase C.     

Effects of Hypoxia on the Activity of the Dopaminergic Neuron System in the Rat Striatum as Studied by In Vivo Brain Microdialysis

The purpose of the present study is to clarify the effects of hypoxia on the activity of the dopaminergic neurons in the brain and its mechanism of action. For this purpose, the effects of hypoxia on the extracellular levels of 3,4-dihy-droxyphenylethylamine (dopamine) were examined in the rat Striatum using in vivo brain microdialysis in the presence or absence of pretreatment with either tetrodotoxin (a blocker of voltage-dependent sodium channels) or nomifensine (a blocker of dopamine reuptake). Exposure to various degrees of hypoxia (15, 10, and 8% O₂ in N₂) increased dopamine levels in striatal dialysates to 200, 400, and 1,100%, respectively, of the control value. On reoxygenation, dopamine levels in the dialysates rapidly returned to the control level. Reexposure to hypoxia increased the dopamine levels to the same extent as during the first exposure. After addition of tetrodotoxin (40 mUM) to the perfusion fluid or pretreatment with nomifensine (100 mg/kg, i.p.), exposure to hypoxia no longer increased the dopamine levels. These results suggest that although hypoxia induces an increase in the extracellular dopamine levels (hence, an apparent increase in the activity of the dopaminergic neurons), this increase is not the result of an increase in dopamine release itself, but rather the result of inhibition of the dopamine reuptake mechanism.     

Angiotensin-Converting Enzyme Modulates Dopamine Turnover in the Striatum

Abstract: The effect of chronic inhibition of the angiotensin-converting enzyme on dopamine content and release in the striatum was investigated using in vivo microdialysis in awake, freely moving rats. Rats were treated for 1 week with the angiotensin-converting enzyme inhibitor perindopril (1 mg/kg) via the drinking water, whereas the controls were given water alone. One week after perindopril treatment, striatal dopamine dialysate levels in the treated group were markedly elevated compared with control values: control, 233 ± 43 pg/ml; perindopril, 635 ± 53 pg/ml ( p < 0.001). These results were confirmed by a complementary study in which dopamine content was measured in striatal extracts (3.5 ± 0.4 µg of dopamine/g of tissue for controls compared with 9.2 ± 2.4 µg of dopamine/g of tissue for the treated group; p < 0.05). In the rats that were dialyzed, angiotensin-converting enzyme levels in the striatum were decreased by 50% after perindopril treatment. Levels of dopamine D₁ and D₂ receptors and of preprotachykinin and tyrosine hydroxylase mRNAs were unchanged after angiotensin-converting enzyme inhibition. A small, but significant, increase was detected in striatal preproenkephalin mRNA levels in the angiotensin-converting enzyme inhibitor-treated group. These results indicate that peripherally administered angiotensin-converting enzyme inhibitors penetrate the blood-brain barrier when given chronically and modulate extracellular dopamine and striatal neuropeptide levels.     

Mice transgenic for exon 1 of Huntington's disease: properties of cholinergic and dopaminergic pre-synaptic function in the striatum

In Huntington's disease (HD), neuronal loss is most prominent in the striatum leading to emotional, cognitive and progressive motor dysfunction. The R6/2 mice, transgenic for exon 1 of the HD gene, develop a neurological phenotype with similarities to these features of HD. In striatal tissue, electrically evoked release of tritiated acetylcholine (ACh) and dopamine (DA) were compared in wild-type (WT) and R6/2 mice. In R6/2 mice, the evoked release of ACh, its M2 autoreceptor-mediated maximum inhibition and its dopamine D2 heteroreceptor-mediated maximum inhibition was diminished to 51%, 74% and 87% of controls, respectively. Also, the activities of choline acetyltransferase and of synaptosomal high-affinity choline uptake decreased progressively with age in these mice. In the DA release model, however, electrical stimulation elicited equal amounts of [3H]-DA both in WT and R6/2 mice. Moreover, high-affinity DA uptake into striatal slices was similar in WT and R6/2 mice. In order to confirm these findings in vivo, intrastriatal levels of extracellular DA were measured by intracerebral microdialysis in freely moving mice: striatal DA levels were found to be equal in WT and R6/2 mice. In conclusion, in the transgenic R6/2 mice changes occur mainly in striatal cholinergic neurones and their pre-synaptic modulation, but not in the dopaminergic afferent terminals. Whether similar events also contribute to the pathogenesis of HD in humans has to be established.     

Uptake of Dopamine Released by Impulse Flow in the Rat Mesolimbic and Striatal Systems In Vivo

Abstract: The release of dopamine in the striatum, nucleus accumbens, and olfactory tubercle of anesthetized rats was evoked by electrical stimulation of the mesolimbic dopaminergic pathway (four pulses at 15 Hz or four pulses at 200 Hz). Carbon fiber electrodes were implanted in these regions to monitor evoked dopamine overflow by continuous amperometry. The kinetics of dopamine elimination were estimated by measuring the time to 50% decay of the dopamine oxidation current after stimulation ceased. This time ranged from 64 ms in the striatum to 113 ms in the nucleus accumbens. Inhibition of dopamine uptake by nomifensine (2–20 mg/kg), GBR 12909 (20 mg/kg), cocaine (20 mg/kg), mazindol (10 mg/kg), or bupropion (25 mg/kg) enhanced this decay time by up to +602%. Uptake inhibition also produced an increase in the maximal amplitude of dopamine overflow evoked by four pulses at 15 Hz. This latter effect was larger in the striatum (+420%) than in mesolimbic areas (+140%). These results show in vivo that these uptake inhibitors actually slow the clearance of dopamine released by action potentials and suggest that dopaminergic transmission is both prolonged and potentiated strongly by these drugs, in particular in the striatum.     

Real-Time Measurement of Electrically Evoked Extracellular Dopamine in the Striatum of Freely Moving Rats

Abstract: The real-time measurement of electrically evoked dopamine was established in brain extracellular fluid of freely moving rats. Dopamine was monitored by fast-scan cyclic voltammetry at carbon fiber microelectrodes lowered into the striatum by means of a detachable micromanipulator. A stimulating electrode, previously implanted in the substantia nigra, was used to evoke striatal dopamine efflux. Evoked extracellular dopamine was both current and frequency dependent. When low current intensities (±125 µA) and frequencies (10–20 Hz) were applied, detectable levels of dopamine were elicited without a perceptible behavioral response. Reproducible concentrations of extracellular dopamine could be evoked in the same rat for at least 2 months. These concentrations, moreover, were significantly higher in freely moving rats compared with rats anesthetized with Equithesin. Analysis of measured curves for dopamine uptake and release rates revealed that anesthesia inhibits release but does not affect uptake. It is concluded that (a) fast-scan cyclic voltammetry at carbon fiber microelectrodes is a viable technique for the measurement of electrically evoked dopamine in brain extracellular fluid of freely moving rats, (b) it is possible to determine in situ rate constants for dopamine release and uptake from these temporally and spatially resolved measurements of levels of dopamine, and (c) transient changes in extracellular dopamine levels elicited by electrical stimulation are affected by anesthesia.     

In Vivo Electrochemical Measurement of the Long-Lasting Release of Dopamine and Serotonin Induced by Intrastriatal Kainic Acid

Abstract: Intrastriatal injection of the glutamate agonist kainic acid (KA) in rats has been used to produce an animal model to investigate the mechanism of acetylcholine and GABA cell death associated with Huntington's disease. In the present study, the time course of low (10⁻⁵ M ) and high (5 × 10⁻³ M ) concentrations of KA on striatal dopamine and serotonin release was studied in freely moving rats by using in vivo voltammetry. The response to low concentrations of KA varied between animals, either increasing dopamine release during the injection or increasing dopamine and serotonin after the injection for an extended time, suggesting that 10⁻⁵ KA is near the threshold for KA toxicity in the striatum in rats. High concentrations of KA suppressed dopamine release during injection, with both dopamine and serotonin release increasing and remaining elevated for 1–4 and 7–21 days, respectively. KA-induced changes were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione and bicuculline increased the release of dopamine but not serotonin. These findings suggest that KA-induced changes in dopamine release resulted from a disinhibition of dopamine neurons due to KA-mediated toxicity of striatal GABA neurons. An alternate possibility is that the change in dopamine and serotonin release may have arisen from a functional modification or degeneration of presynaptic terminals.     

Nitric oxide and potassium chloride-facilitated striatal dopamine efflux in vivo: role of calcium-dependent release mechanisms

Previous studies investigating the calcium-dependency of nitric oxide-facilitated striatal dopamine efflux have produced conflicting results. In the current study, we have investigated the role of extracellular calcium in nitric oxide and potassium chloride-evoked striatal dopamine efflux in vivo using microdialysis. Dialysis probes were implanted in the anterior dorsal striatum of chloral hydrate-anesthetized rats. Intrastriatal infusion (20 min fraction) of the nitric oxide generators sodium nitroprusside (200 μM, 500 μM, or 1 mM) and 3-morpholinosydnonimine (1 mM) increased extracellular dopamine levels. The facilitatory effects of 3-morpholinosydnonimine and potassium chloride on dopamine efflux were attenuated following pretreatment (100 min) and co-infusion of calcium free artificial cerebral spinal fluid containing magnesium chloride. Local potassium chloride infusion (100 mM) administered alone elevated striatal dopamine efflux to a similar degree as potassium chloride (100 mM) delivered 60 min after 3-morpholinosydnonimine infusion. These results demonstrate that like potassium chloride, nitric oxide facilitates striatal dopamine efflux in vivo via a mechanism largely dependent on extracellular calcium. Also, as intrastriatal potassium chloride infusion evoked similar increases in extracellular dopamine levels in controls and subjects receiving pretreatment with the NO-generator 3-morpholinosydnonimine, it is unlikely that the functional integrity of DA nerve terminals is compromised via a neurotoxic disruption of plasma membrane potential following enhanced striatal NO production. © 1999 Elsevier Science Ltd. All rights reserved.     

Striatal Tissue Preparation Facilitates Early Sampling in Microdialysis and Reveals an Index of Neuronal Damage

Abstract: Injury-induced efflux of dopamine was compared between two microdialysis preparations. Rats were implanted with guide cannulae 5–10 days prior to microdialysis experiments. In one group, ventral striatal tissue was punctured with stainless steel obturators that remained in place until the day of the experiment. In the other group, the tissue was not punctured until the microdialysis probes were inserted. Rats from each group were dialyzed with calcium-free artificial extracellular fluid or tetrodotoxin 4 h after probe insertion. In the rats with previously punctured tissue, calcium depletion reduced dialysate dopamine concentrations to 8% of baseline. Dialysis with tetrodotoxin reduced dopamine concentrations to less than 1% of baseline. In the rats with freshly punctured tissue, dopamine concentrations were reduced only to 50% of baseline levels by calcium depletion and to 30% during dialysis with tetrodotoxin. Thus, penetration of the tissue prior to testing can significantly reduce the acute injury-induced efflux of dopamine. Further, a significant correlation was found between baseline 3,4-dihydroxy-phenylacetic acid/dopamine ratios and the efficacy of tetrodotoxin in reducing dialysate dopamine concentrations. Thus, basal 3,4-dihydroxyphenylacetic acid/dopamine ratios appear to provide an index of the amount of injury-induced dopamine efflux following probe insertion.