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Dopamine release and uptake rates both decrease in the partially denervated striatum in proportion to the loss of dopamine terminals
Brain Research 753 Ž1997. 225–234
Research report
Dopamine release and uptake rates both decrease in the partially denervated striatum in proportion to the loss of dopamine terminals Paul A. Garris a , Q. David Walker b
b,1
, R. Mark Wightman
b, )
a Department of Biological Sciences, 102 Felmley Hall, Illinois State UniÕersity, Normal, IL 61790-4120, USA Department of Chemistry and Curriculum in Neurobiology, Venable Hall, CB 3290, UniÕersity of North Carolina, Chapel Hill, NC 27599-3290, USA
Accepted 10 December 1996
Abstract The present study tested the hypothesis that normal concentrations of extracellular dopamine are preserved in the partially denervated striatum without active compensatory changes in dopamine uptake or release. One to four weeks after adult rats were unilaterally lesioned with 6-hydroxydopamine, fast-scan cyclic voltammetry at Nafion-coated, carbon-fiber microelectrodes was used to monitor extracellular dopamine levels in vivo, under urethane anesthesia. Simultaneous voltammetric recordings were collected in the lesioned and contralateral control striata. Extracellular dopamine was elicited by bilateral electrical stimulation of the medial forebrain bundle. A 20 Hz stimulation evoked similar concentrations of extracellular dopamine in both lesioned and control striata, although tissue dopamine was decreased 30–70% in lesioned striata, as determined subsequently by HPLC-EC. However, kinetic analysis of the voltammetric recordings revealed that the concentration of dopamine released per stimulus pulse and Vmax for dopamine uptake decreased in proportion to the magnitude of the lesion. These data support the hypothesis that normal extracellular dopamine levels can be generated in the partially lesioned striatum in the absence of active neuronal compensation. These results also suggest that passive mechanisms involved in the regulation of extracellular dopamine play an important role in maintaining function during the preclinical or presymptomatic phase of Parkinson’s disease.q 1997 Elsevier Science B.V. All rights reserved. Keywords: 6-Hydroxydopamine lesion; Dopamine release and uptake; Voltammetry; Nigrostriatal dopamine neuron; Electrical stimulation; Striatum
1. Introduction Parkinson’s disease is a neurodegenerative disorder associated with the loss of dopamine neurons originating in the zona compacta of the substantia nigra and terminating in the caudate and putamen of the corpus striatum w24,46x. Symptoms include tremor, rigidity of movement, and akinesia, and are alleviated by restoration of dopaminergic tone. However, severe Parkinsonism does not present until striatal dopamine deficits reach the 90% level. It has been hypothesized that the plasticity of the nigrostriatal dopamine system provides an adaptive capacity to maintain function despite extensive neuronal loss w23x. Although both pre- and postsynaptic changes have been observed post mortum after severe lesions, the compen-
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Corresponding author. Fax: q1 Ž919. 962-2388; e-mail: [email protected] 1 Current Address: Department of Pharmacology, Duke University, Durham, NC 27710.
satory mechanisms that maintain dopaminergic function during the presymptomatic phase of Parkinson’s disease are not well understood. Nigrostriate lesions of the rat brain induced by the neurotoxicant 6-hydroxydopamine Ž6-OHDA. are a widely used experimental model of Parkinson’s disease. 6-OHDA is taken up by nigrostriatal dopamine neurons, where it produces highly reactive quinones and peroxides, ultimately leading to cell death w21,40x. Resembling the clinical syndrome, lesioned animals develop motor deficits only after extensive loss of striatal dopamine w54x. In addition, similar neurochemical changes are found in the striatum after 6-OHDA lesions and in Parkinson’s disease. A particularly intriguing finding is that concentrations of dopamine in microdialysates collected in the partially denervated striatum Žless than 80%. are similar to those in the intact striatum w1,37,52x. Zigmond and coworkers w54x postulate that a variety of processes compensate for the deficits arising from an extensive loss of dopamine in the striatum. These include increased dopamine synthesis and release from the remain-
0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 0 0 3 - 6
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ing terminals, as well as reduced dopamine clearance from extracellular fluid. Compelling evidence supports adaptive changes in dopamine synthesis and uptake in animals with extensive lesions w3,28,35,47,51,53x. However, no discernible change in synthesis is apparent with lesions that deplete tissue dopamine by 50% suggesting that another adaptive mechanismŽs. besides increased synthesis mediates compensation in this condition. Evidence for a compensatory increase in dopamine release w20,42,44,52x is largely based on indirect indices such as turnover as reflected by the ratio of dopamine metabolites to tissue dopamine, fractional dopamine efflux from brain slices, and the ratio of dialysate dopamine to tissue dopamine. The major determinants of extracellular dopamine concentration are release and uptake w50x, processes which are not measured independently in most determinations of extracellular neurotransmitter. However, real-time microsensors implanted in brain tissue can be used to resolve these dynamic processes w16,26,50x. With this approach, Nafion-coated carbon fiber microelectrodes Žmaximal dimension of 20 mm. are used with fast scan cyclic voltammetry Ž100 ms repetition rate. to measure extracellular dopamine with excellent temporal and spatial resolution w16,27,32,49x. This methodology affords an unparalleled view of the extracellular events of neurotransmission in the microenvironment surrounding synapses w2x and is currently the only technique available to determine direct release rates for any neurotransmitter in the brain. In this paper, we employ this approach to investigate compensatory changes in dopamine release and uptake in the rat striatum after partial 6-OHDA lesions of nigrostriatal dopamine neurons. By doing so we hope to understand how normal dopaminergic function is maintained in the preclinical phase of Parkinson’s disease.
mgrkg i.m.., and immobilized in a stereotaxic apparatus ŽDavid Kopf Instruments, Tajunga, CA.. Core body temperature was maintained at 378C using a Deltaphase Isothermal Pad ŽBraintree Scientific, Braintree, MA.. An incision was made along the skull midline, and skin and muscle layers were retracted but not removed. All stereotaxic coordinates are according to the brain atlas of Paxinos and Watson Ž1986. w38x. Anteroposterior ŽAP. and mediolateral ŽML. positions are referenced to bregma and dorsoventral ŽDV. positions are referenced to dura. A small hole was drilled in the skull above the lateral edge of one substantia nigra Žy5.4 AP, q3.0 ML.. A 30 gauge needle was then lowered Žy8.2 DV. and 2 mL of a 0.1% ascorbic acid solution containing 2 mg of 6-OHDA was infused at a rate of 0.2 mlrmin. After completion of the infusion, the needle was removed, the hole was sealed with bone wax and the scalp wound was sutured. Animals were administered lactated ringers Ž10 ml i.p.. to prevent postsurgical dehydration and were allowed to recover from anesthesia before their return to housing. To facilitate recovery, the diet for lesioned rats was initially supplemented with highly palatable foods such as apples and peanut butter. If adipsia developed, rats were administered lactated ringers i.p. Most rats did not require special care for more than two days following surgery. 2.3. In ÕiÕo electrochemistry Electrically evoked concentrations of extracellular dopamine were measured in unilaterally lesioned rats one to four weeks after 6-OHDA administration. Surgery was performed as described above with the exception that rats were anesthetized with urethane Ž1.5 grkg i.p... The experimental design for simultaneous measurement of electri-
2. Materials and methods 2.1. Animals Male Sprague–Dawley rats Ž250–300 g. were purchased from Charles River ŽRaleigh, NC. and housed under controlled temperature and lighting conditions. Food and water were available ad libitum. Animal care was in accordance with the Guide for Care and Use of Laboratory Animals ŽNIH Publication 86-23. and was approved by the Institutional Animal Care and Use Committee of the University of North Carolina. 2.2. 6-OHDA lesions Survival surgery was performed under aseptic conditions. Animals were pretreated with the norepinephrine uptake inhibitor, desipramine Ž25 mgrkg i.p.., anesthetized with a combination of sodium pentobarbital Ž5 mgrkg i.p.., ketamine Ž50 mgrkg i.p.. and xylazine Ž10
Fig. 1. Experimental set-up for the simulataneous measurement of extracellular dopamine in the lesioned striatum and contralateral control. See Section 2 for details. Abbreviations: CFM, carbon-fiber microelectrodes; MFB, medial forebrain bundles; SE, stimulating electrodes; SN, substantia nigra; ArD, analog to digital converter; DrA, digital to analog converter.
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cally evoked dopamine in the lesioned striatum and contralateral side is diagrammed in Fig. 1. Twisted, bipolar stimulating electrodes were positioned in each medial forebrain bundle ŽMFB, y4.6 AP, "1.4 ML, y8.5 DV. and Nafion-coated, carbon-fiber microelectrodes w4x were positioned in the partially lesioned and intact striata Žq1.2 AP, "2.0 ML, y4.5 DV.. The reference electrode was a sodium saturated calomel electrode ŽSSCE. that made contact with dura via a salt bridge. Cyclic voltammograms were simultaneously recorded at each carbon-fiber electrode every 100 ms at a scan rate of 300 Vrs Žy400 to 1000 mV vs. SCCE. using a bipotentiostat w48x; EI400, Ensman Instruments, Bloomington, IN.. Data were digitized ŽDMA Labmaster, Scientific Solutions, Solon, OH. and stored to computer files. The time-dependent current was monitored over the peak oxidation potential for dopamine in successive voltammograms and converted to concentration based on postcalibration. 2.4. Electrical stimulation Constant current stimulation pulses were computer generated and optically isolated ŽNL 800, Neurolog, Medical Systems, Great Neck, NY. from the electrochemical system. Biphasic square wave pulses were applied Žcurrent and duration of each phase were "300 mA and 2 ms, respectively. using a twisted bipolar electrode with 0.2 mm diameter tips spaced 1 mm apart ŽPlastics One, Roanoke, VA..
value for each working electrode before comparison with data w26x. Where applicable, all measurements are reported as the mean " SEM and n is the number of animals. Significance testing used the Student’s t-test w41x with the significance level set at P - 0.05. 2.6. Determination of tissue dopamine After the in vivo measurements of release and uptake, the brain was removed from the skull, placed in a cold slice block, and cut coronally into 1.2 mm sections w19x. The section containing the portion of the striatum in which voltammetric recordings were made, as evidenced by damage to the dorsal cortical surface, was cut into 1 mm wide dorsoventral strips using a locally constructed tool. Mediolateral strips were stored at y258C until assayed for dopamine. On the day of assay, striatal tissue was sonified in mobile phase and centrifuged. Dopamine concentrations in the supernatant were quantitated by reverse-phase liquid chromatography with electrochemical detection as described previously w12x with some modifications. The mobile phase Žper L. consisted of 13.4 g sodium monophosphate, 5.76 g citrate, 450 mg sodium octyl sulfate, 40 mg EDTA and 20% methanol at a pH of 4.0. The column was an Ultramex 3 C18 IP Ž10 cm = 4.6 mm; Phenomenex, Torrance, CA.. Precipitated protein was determined by an assay kit from Bio-Rad ŽRichmond, CA.. 2.7. Drugs and reagents
2.5. Data analysis Extracellular concentrations of dopamine are controlled by the dynamic balance between dopamine release and uptake w49,50x. The rate of change during electrical stimulation is described by: d w DA x rd t s f w DA x p y Vmaxr Ž K m r w DA x q 1 .
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Ž 1.
where wDAx is the instantaneous extracellular concentration of dopamine, f is the pulse frequency, wDAx p is the concentration of dopamine elicited per stimulus pulse, and Vmax and K m are Michaelis–Menten uptake parameters. The basis of this equation is that each stimulus pulse elicits an instantaneous increase in extracellular dopamine, the release event, while uptake occurs continuously. The rate of disappearance of extracellular dopamine after the stimulation follows the Michaelis–Menten equation with the same rate constants describing uptake. Best-fit values for wDAx p and Vmax were obtained by analysis of the concentration changes evoked by 20 and 60 Hz stimulus trains using a value of 0.2 mM for K m w22,33x. Curve fitting employed a nonlinear regression based on a simplex minimization algorithm w16,25x. The response time of the sensor to a concentration pulse was obtained during postcalibration. Simulated curves were convolved with a response function calculated using this
All chemicals were used as received and purchased from Sigma Chemical ŽSt. Louis, MO. unless otherwise indicated. Post-calibration of working electrodes was performed in physiological saline Ž150 mM sodium chloride. buffered with HEPES Ž25 mM. at a pH of 7.4. Aqueous solutions were prepared in doubly distilled, deionized water ŽMega Pure System, Corning Glasswork, Corning, NY..
3. Results 3.1. Partial 6-OHDA lesions Prior work has shown that a graded loss of nigrostriatal dopamine neurons is produced by microinjection of 6OHDA into the lateral or rostral regions of the substantia nigra pars compacta w6,18x. Presumably, as the neurotoxin diffuses away from the injection site, a gradient is established with the highest concentrations near the site of origin. In this manner, dopamine cells in the medial substantia nigra and ventral tegmental area are spared. Since the topography or spatial arrangement of dopamine cell bodies in the substantia nigra is conserved in their projections to the striatum w11x, the pattern of dopamine terminal
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Fig. 2. Graded, unilateral lesion of nigrostriatal dopamine neurons. Top panel: diagram of a coronal view of the rat brain containing the striatum at the level in which the measurements described in the paper were taken. For each side of the brain, the striatum was dissected into lateral ŽL., mediolateral ŽML. and medial ŽM. regions, as demarcated by the dashed lines, for tissue analysis of dopamine by HPLC-EC. The values given in each region are dopamine content expressed as ngrmg protein for a single representative rat. Lower panel: evoked concentrations of extracellular dopamine as measured by voltammetry during 60 Hz, 2 s stimulus tran in the lesioned and control striatum of the same rat. Data Žsolid circles. are the concentration of dopamine measured in voltammograms at 100 ms intervals. The duration of the stimulation is shown by the solid line underneath each curve. Scale bars refer to all responses.
loss is similar in the striatum. In preliminary experiments we replicated this result using lateral injections as illustrated by the representative results from a single animal shown in Fig. 2 Župper.. A graded loss of tissue dopamine was found only in the ipsilateral striatum. The most severe lesion was in the lateral region with dopamine content reduced to 8% of the contralateral side. The dopamine content of the medial and mediolateral regions of the lesioned striatum contained 60 and 31 ng dopaminermg protein, respectively, while in the contralateral Žunlesioned. striatum the content was 105 and 115 ng dopaminermg protein, respectively. The mediolateral region thus exhibited an intermediate dopamine reduction of content Ž27% of the intact side., and was chosen for all future experiments. Prior to tissue analysis, dopamine release was evoked by electrical stimulation of the MFB and monitored with in vivo voltammetry w12x. To position the various electrodes, the carbon-fiber microsensors were initially lowered to y4.5 DV in each mediolateral striatum. A stimulating electrode was positioned just above each MFB Žy7.5 DV. and then incrementally lowered until maximal evoked responses for dopamine were recorded at each site. The final position of each microsensor was obtained by further lowering in small increments to optimize the recorded signal. The maximal evoked dopamine concentration during a 60 Hz stimulation in this animal was lower in the lesioned side than the intact side, and appeared to vary in a manner similar to tissue content ŽFig. 2, lower..
Fig. 3. Comparison of dopamine release during unilateral and bilateral stimulation of medial forebrain bundles. Recordings shown are from a single animal. The three responses shown in the left column were measured in the control striatum ŽCON. and the three responses in the central portion were measured in the lesioned striatum ŽLES.. Each adjacent pair of recordings was collected simultaneously. Underneath each trace is a solid line demarcating the time and duration of electrical stimulation Ž60 Hz, 2-s train.. The right column indicates whether a train of pulses was applied to the medial forebrain bundle Žq. or not Žy. ipsilateral to the lesioned striatum or contralateral control. In both lesioned and contralateral control striatum, the position of the working electrode was not changed for the duration of these measurements. Post mortum analysis showed the lesioned striatum contained 80% of the dopamine of the control side.
Fig. 4. Concentrations of extracellular dopamine in the lesioned and control striatum electrically evoked by a physiological frequency. Data Žsolid circles. are the concentration of dopamine measured at 100 ms intervals. Open boxes denote inititation and termination of the electrical stimulation Ž20 Hz, 6-s train.. The time and duration of the pulse train is also indicated by the solid line underneath the bottom trace. The tissue content of dopamine in the lesioned striatum was 45% of the contralateral control. Inset: background subtracted cyclic voltammograms measured in the lesioned striatum in vivo Žopen circles. and from dopamine in vitro during postcalibration. Both voltammograms were collected at the same working electrode. Current Ž I . is 1 nA and 50 pA for in vitro and in vivo determinations, respectively.
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Fig. 5. Relationships between extracellular concentration and tissue content of dopamine in the lesioned and control striatum. Data are expressed as the mean"SEM Ž ns 5.. Tissue dopamine content in the lesioned striatum is expressed as a percent of the value in the control striatum for each animal and then averaged for the entire group. Average concentrations of dopamine elicited by 60 Hz in the lesioned striatum were significantly Ž P - 0.05. different from control. Abreviations: CON, control striatum; LES, lesioned striatum; wDAx 20 Hz , maximum concentration of extracellular dopamine elicited by 20 Hz; wDAx60 Hz , maximum concentration of extracellular dopamine elicited by 60 Hz.
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tration in the two regions are similar. The shape of the background subtracted voltammogram recorded in both striata during the 20 Hz response in vivo was identical to that measured at 60 Hz Ždata not shown. and to dopamine measured in vitro at the same microsensor during postcalibration Žinset.. In all animals examined Ž n s 5., results similar to those for the single animal described in Fig. 4 were obtained with 20 Hz stimulations. As described above, dopamine was identified by its cyclic voltammogram and maximal concentrations were 0.23 " 0.05 and 0.31 " 0.09 mM in control and lesioned striata, respectively ŽFig. 5.. This was the case despite the fact that the tissue content of dopamine, expressed as a percent of the control striatum in each animal, was 54% of control. In contrast, dopamine concentrations evoked by 60 Hz were significantly Ž P - 0.05. lower in the lesioned striatum compared to control Ž1.8 " 0.5 and 3.6 " 0.8 mM, respectively.. On a percent basis, the decrease in evoked extracellular dopamine concentration with 60 Hz stimulation Ž50%. paralleled the decrease in tissue dopamine Ž54%.. 3.3. Rate constants for dopamine release and uptake In each animal, the temporal responses of extracellular dopamine to 20 and 60 Hz stimulation were analyzed to
3.2. EÕoked concentrations of extracellular dopamine in lesioned and contralateral striata Bilateral stimulation of the MFB with a 60 Hz train elicited an increase in extracellular dopamine in the mediolateral portion of both the lesioned and intact striata ŽFig. 3, bottom panel.. As in the animal shown in Fig. 2, the maximal concentration of dopamine in the extracellular fluid is lower on the lesional side whose content was 80% of the intact side. No signal was recorded in either striatum when the stimulating electrode ipsilateral to it was disconnected Žtop and middle panels.. However, responses recorded in the striatum of the unilaterally stimulated side of the brain were identical to those recorded when both stimulating electrodes were operable. There was no evidence of stimulus artifact. Recordings collected when the stimulating electrodes were positioned just dorsal to the MFB showed no response Ždata not shown.. Fig. 4 shows concentrations of extracellular dopamine in a lesioned striatum and the contralateral intact striatum of a different animal during simultaneous electrical stimulation of the MFB bilaterally with a frequency of 20 Hz. In this rat, the tissue content of dopamine in the region of the lesioned striatum where dopamine release and uptake was monitored was 45% of the contralateral side. Note that the maximal amplitude and dynamics of the dopamine concen-
Fig. 6. Relationships between dopamine release and uptake rates, and tissue content of dopamine in the lesioned and control striata. wDAx p is the concentration of dopamine released per stimulus, whereas dopamine uptake is described by the Michaelis–Menten term for the maximum rate of uptake, Vma x . Rate constants are the averages determined in the lesioned striata ŽLES. and contralateral controls ŽCON. as described in Section 2 and averaged. Values for the tissue content of dopamine are the same as in Fig. 5. Data are expressed as the mean"S.E.M. Ž ns 5, same animals as in Fig. 5..
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Fig. 7. Theorectical curves for electrically evoked concentrations of extracellular dopamine in the lesion and control striatum. Simulations were calculated from the average rate constants for dopamine release and uptake shown in Fig. 6 and assuming a K m of 0.2 mM. The simulation resolution was 20 ms. Curves were then convoluted with an impulse response function for thin layer diffusion using a film thickness of 400 nm and a diffusion coefficient of 1P10y9 cm2 rs w26x. Electrical stimulation was initiated at time 0 s and, for both frequencies, consisted of a 120 pulse train.
determine the kinetics of dopamine release and uptake parameters. Mean values for the concentration of dopamine released per stimulus pulse, wDAx p , and the maximal rate of dopamine uptake, Vmax , obtained from the data from all animals are shown in Fig. 6. In these analyses, the value of K m was fixed at 0.2 mM in the intact and lesioned striata since results from synaptosomal preparations show that K m for dopamine uptake is similar in the partially lesioned and intact striatum w53x. Both wDAx p Ž38 " 11 versus 78 " 18 nM, respectively. and Vmax Ž1.6 " 0.4 versus 2.7 " 0.5 mM, respectively. were significantly Ž P - 0.05. decreased in the lesioned striatum compared to the contralateral control. The average correlation coefficient Ž r . for each analysis was 0.92. The percent decrease in both the release term Ž49%. and Vmax Ž59%. were similar to the decrease in tissue dopamine Ž54%.. Simulated curves for 20 and 60 Hz responses in the lesioned and control striatum are shown in Fig. 7. Simulations were calculated from the average measured parameters for dopamine release and uptake given in Fig. 6. Theoretical calculations parallel experimental results in that simulations differ in amplitude at 60 Hz and are more similar at 20 Hz. The percent decrease in the 60 Hz simulation for the lesioned striatum was 46% of control Ž1.7 versus 3.7 mM, respectively. compared to 75% Ž0.21 versus 0.28 mM, respectively. for 20 Hz.
4. Discussion The goal of the present study was to investigate alterations in extracellular dopamine folowing partial denervation in the rat striatum. Real-time microsensors were employed to monitor dynamic changes in extracellular
dopamine evoked by transient electrical stimulation of ascending dopamine fibers. In situ rate constants for dopamine release and uptake were determined from the temporally and spatially resolved chemical measurements. After partial 6-OHDA lesions of nigrostriatal dopamine neurons, we observed that concentrations of extracellular dopamine in the striatum elicited by a frequency in the physiological range are similar in the intact and lesioned striata. However, when data from a higher frequency were analyzed, both dopamine release and uptake rates were found to decrease in proportion to the loss of dopamine terminals. These results support the hypothesis that normal concentrations of extracellular dopamine are maintained in the partially denervated striatum without active compensatory changes in dopamine release or uptake. In this paper, rate constants for dopamine release and uptake are compared, on a per dopamine terminal basis, in striatal regions denervated to different degrees. The density of dopamine terminals after lesions was estimated by dopamine tissue content determined by HPLC-EC subsequent to the voltammetric experiment. The adequacy of this index is demonstrated by the excellent relationship between tissue dopamine content and the density of dopamine terminals in different regions of the rat telencephalon w9,10,29,36x. Furthermore, the technique of flow cytometry has been used to show that, after 6-OHDA lesions, the percent of synaptosomes labeled with a fluorescent probe for tyrosine hydroxylase correlates well with striatal dopamine depletion w51x. 4.1. Extracellular concentrations of dopamine after partial lesions Partial denervation of dopamine neurons in the striatum have been produced by injection of 6-OHDA into the substantia nigra, MFB or cerebral ventricles of adult rats w3,6,20,39,53x. In the present study, unilateral, partial denervation of the mediolateral striatum was accomplished by an injection of 6-OHDA into the lateral edge of one substantia nigra. The partial lesion technique reduced tissue content of dopamine in the mediolateral striatum to 30–70% of control. The spatial resolution afforded by the carbon fiber microelectrodes allowed release and uptake of dopamine, chemically identified by the cyclic voltammograms, to be probed in this discrete striatal region. Measurements were collected bilaterally in the denervated and intact striatum so that each rat served as its own control. A similar approach has previously been used by Robinson and Whishaw w39x who determined basal levels of extracellular dopamine with the microdialysis technique. An intriguing finding obtained from prior 6-OHDA lesion studies is that concentrations of dopamine in microdialysates collected in the partially denervated striatum are similar to those in the intact striatum w39,52,1,37x. The present results are in agreement when dopamine release is
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evoked by low frequency electrical stimulation. A train of pulses at a frequency at the high end of the physiological range, 20 Hz w8,5x, evokes similar concentrations of extracellular dopamine in the partially lesioned striatum and contralateral control ŽFig. 4 and Fig. 5.. In contrast, at a much higher stimulation frequency Ž60 Hz., the maximal evoked dopamine is proportionately lower in the partially lesioned striatum. 4.2. Dopamine release and uptake in partially lesioned striata Microdialysis and voltammetry provide complementary information about the regulation of extracellular dopamine. Because of superior sensitivity and selectivity, microdialysis affords an estimate of basal concentrations of extracellular dopamine. However, direct investigation of the mechanisms responsible for measured concentrations are beyond the current capability of this technique. On the other hand, voltammetry permits resolution of measured concentrations into the respective contributions of uptake and release w49,26,16x. This is because the two primary determinants of extracellular dopamine exhibit different dynamics. Since dopamine release is an exocytotic process w15x, it is evoked at the time of an individual electrical impulse and can be considered an instantaneous event given the time scale of the measurement Ž100 ms.. In contrast, uptake is both a concentration- and a time-dependent process that obeys the Michaelis–Menten equation. Release events occur more frequently as the frequency of a stimulus train increases. In contrast, the rate of uptake is independent of the rate of impulse flow but instead increases with concentration until sufficient dopamine accumulates that it becomes limited by the upper limit set by Vmax . However as the stimulation frequency increases, less time is available for uptake to occur in between stimulus pulses. For this reason, at higher frequencies of impulse flow evoked concentrations of dopamine are most affected by the rate of release, i.e., a release dominated situation. Indeed, during stimulations that resemble burst firing, release appears more intense w17x. In contrast, at a low frequency a balance between release and uptake occurs, and a lower, steady-state concentration of dopamine is obtained. The steady-state level of extracellular dopamine concentration seen during 20 Hz stimulations is a direct consequence of this balance. Steady state occurs because, at this frequency, the rates of uptake and release equal each other. Since similar concentrations are observed in both the lesioned and intact striata with this stimulation frequency, these results can either mean that there was no change in the rate of release and uptake caused by the lesion, or that both uptake and release have changed in an identical manner. This dichotomy is the same as that confronted in the interpretation of concentrations obtained by microdialysis. The use of a second, higher frequency enables the
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contributions of release and uptake to be resolved. The higher frequency leads to release dominated conditions where, in the lesioned striatum, the reduced capacity for dopamine release is readily observed. With the use of Eqn. 1 and the experimental results from the two different frequencies, the rate constants that control these dynamic events can be mathematically resolved. It was found that wDAx p , the rate constant for dopamine release, and Vmax , the maximal uptake rate, both decreased in direct proportion to the degree of the lesion ŽFig. 6.. The simulations in Fig. 7 clearly show that similar concentrations can be generated at a low frequency when there are considerable differences in the rates of uptake and release that are employed. The results in the lesioned striatum are reminiscent of our findings for dopamine release and uptake in the amygdala and prefrontal cortex w13x. Although the dopamine content of both of those regions is much lower than that of the intact striatum, similar extracellular concentrations can be evoked in all three regions at low frequencies. As in the lesioned striatum, the lower rate of release in the nonstriatal regions is balanced by a lower rate of uptake. In vitro studies measuring the uptake of radiolabeled dopamine into synaptosomes w3,53x and previous voltammetric studies w7,28,47x are in agreement with the observed decrease in dopamine uptake rate in the partially denervated striatum ŽFig. 6.. However, the observed decrease in dopamine release in the partially denervated striatum contradicts some previous interpretations. Microdialysis studies demonstrating normal levels of extracellular dopamine after partial lesions have been offered as support for a compensatory increase in dopamine release after partial lesions because the ratio of extracellular dopamine to dopamine tissue content increases with the degree of the lesion w54x. Similarly, in vitro slice experiments show that electrically evoked efflux of dopamine, when normalized to tissue content, increases with the lesion w42,44x. However, like voltammetric data obtained at a single stimulation frequency, these measures do not provide sufficient information to resolve uptake and release. Even when efflux is measured in the presence of a competitive uptake inhibitor, some uptake still occurs w25x preventing evaluation of release. 4.3. Dopamine neurotransmission in the partially denerÕated striatum Based on a wide range of biochemical and physiological studies, Zigmond and co-workers w54x have developed a working hypothesis for adaptive changes in dopamine neurotransmission following 6-OHDA lesions. With partial lesions they postulate that the loss of dopamine neurons is compensated by diffusion of dopamine from the remaining terminals to more distant receptor sites than in the intact animal. Our findings provide strong support for this hypothesis. First, we find a decrease in Vmax for
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uptake in situ. This term is the product of the density of the uptake sites and the turnover rate of the transporter w14x. Since uptake sites are primarily on dopamine neurons w34x, a reduction in Vmax is not unexpected. When the rate of uptake is decreased, the lifetime of extracellular dopamine is increased giving it greater opportunity to diffuse to more remote sites. However, we also find a decrease in the release rate constant, wDAx p . This term is the product of the density of release sites and the amount released per terminal w14x. Since the reduction in this term in partially lesioned animals is similar to the reduction in dopamine content, our data argue against an active compensatory mechanism for release. Indeed, as seen by the evoked dopamine response at a low frequency, active compensation is not required. Rather, the concurrently reduced rate of uptake provides the necessary compensation to maintain dopamine extracellular concentrations at their prelesion levels. Compensatory increases in dopamine synthesis after 6-OHDA lesions of a greater extent than studied here are well established w35,51,53x and these changes parallel increases in dopamine turnover as indexed by the ratio of dopamine metabolites to dopamine in striatal tissue w3x. However, both synthesis and turnover rates only begin to increase when lesions reach the 50% level and exponentially rise as the lesion becomes more severe. In the more severely lesioned striatum, the higher turnover and synthesis of dopamine could reflect increased release rates as earlier postulated, a hypothesis that could be tested with this technique. On the other hand, another possibility is that faster rates of turnover and synthesis are related to reduced uptake after lesions. Uptake not only terminates dopamine neurotransmission but also acts to conserve released dopamine w22x. If less dopamine is conserved, then dopamine synthesis Žand therefore turnover. must be increased to maintain terminal stores. Partial lesions of nigrostriatal dopamine neurons produce a condition that mimics the early or preclinical phase of Parkinson’s Disease w6,53,54x. The lack of symptoms in patients with this disease until the deficit in dopamine terminals becomes severe may be a consequence of the passive compensation we observe in rats with partially lesioned striata. Both dopamine uptake sites w30x and the messenger RNA for the dopamine transporter w45x have been shown to decrease in clinically diagnosed Parkinsonian patients. Thus, the consequences of a simultaneous loss of dopamine release and uptake capacity observed in this animal model may directly indicate how normal dopaminergic tone is maintained in preclinical subjects.
5. Conclusions In conclusion, we have used real-time microsensors to examine the regulation of extracellular dopamine in rats
with partial, unilateral lesions of nigrostriatal dopamine neurons. These results extend previous studies using the technique of microdialysis to assess basal levels of extracellular dopamine after lesions and demonstrate that an active, compensatory increase in dopamine release is not necessary to contribute to the maintenance of normal concentrations of extracellular dopamine in the partially denervated striatum. Simultaneous loss of release and uptake sites concurrent with lesions of dopamine neurons can provide sufficient compensation because these processes are spatially fixed to the dopamine innervation w15,31,43x. Whether this form of adaptive compensation is operable in all dopamine terminal fields with their distinct combinations of release and uptake w13x remains to be determined. These results also suggest that passive mechanisms involved in the regulation of extracellular dopamine in the striatum play an important role in maintaining function during the preclinical or presymptomatic phase of Parkinson’s Disease.
Acknowledgements This research was supported by a grant from NIH ŽNS 15841..
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Research report
Dopamine release and uptake rates both decrease in the partially denervated striatum in proportion to the loss of dopamine terminals Paul A. Garris a , Q. David Walker b
b,1
, R. Mark Wightman
b, )
a Department of Biological Sciences, 102 Felmley Hall, Illinois State UniÕersity, Normal, IL 61790-4120, USA Department of Chemistry and Curriculum in Neurobiology, Venable Hall, CB 3290, UniÕersity of North Carolina, Chapel Hill, NC 27599-3290, USA
Accepted 10 December 1996
Abstract The present study tested the hypothesis that normal concentrations of extracellular dopamine are preserved in the partially denervated striatum without active compensatory changes in dopamine uptake or release. One to four weeks after adult rats were unilaterally lesioned with 6-hydroxydopamine, fast-scan cyclic voltammetry at Nafion-coated, carbon-fiber microelectrodes was used to monitor extracellular dopamine levels in vivo, under urethane anesthesia. Simultaneous voltammetric recordings were collected in the lesioned and contralateral control striata. Extracellular dopamine was elicited by bilateral electrical stimulation of the medial forebrain bundle. A 20 Hz stimulation evoked similar concentrations of extracellular dopamine in both lesioned and control striata, although tissue dopamine was decreased 30–70% in lesioned striata, as determined subsequently by HPLC-EC. However, kinetic analysis of the voltammetric recordings revealed that the concentration of dopamine released per stimulus pulse and Vmax for dopamine uptake decreased in proportion to the magnitude of the lesion. These data support the hypothesis that normal extracellular dopamine levels can be generated in the partially lesioned striatum in the absence of active neuronal compensation. These results also suggest that passive mechanisms involved in the regulation of extracellular dopamine play an important role in maintaining function during the preclinical or presymptomatic phase of Parkinson’s disease.q 1997 Elsevier Science B.V. All rights reserved. Keywords: 6-Hydroxydopamine lesion; Dopamine release and uptake; Voltammetry; Nigrostriatal dopamine neuron; Electrical stimulation; Striatum
1. Introduction Parkinson’s disease is a neurodegenerative disorder associated with the loss of dopamine neurons originating in the zona compacta of the substantia nigra and terminating in the caudate and putamen of the corpus striatum w24,46x. Symptoms include tremor, rigidity of movement, and akinesia, and are alleviated by restoration of dopaminergic tone. However, severe Parkinsonism does not present until striatal dopamine deficits reach the 90% level. It has been hypothesized that the plasticity of the nigrostriatal dopamine system provides an adaptive capacity to maintain function despite extensive neuronal loss w23x. Although both pre- and postsynaptic changes have been observed post mortum after severe lesions, the compen-
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Corresponding author. Fax: q1 Ž919. 962-2388; e-mail: [email protected] 1 Current Address: Department of Pharmacology, Duke University, Durham, NC 27710.
satory mechanisms that maintain dopaminergic function during the presymptomatic phase of Parkinson’s disease are not well understood. Nigrostriate lesions of the rat brain induced by the neurotoxicant 6-hydroxydopamine Ž6-OHDA. are a widely used experimental model of Parkinson’s disease. 6-OHDA is taken up by nigrostriatal dopamine neurons, where it produces highly reactive quinones and peroxides, ultimately leading to cell death w21,40x. Resembling the clinical syndrome, lesioned animals develop motor deficits only after extensive loss of striatal dopamine w54x. In addition, similar neurochemical changes are found in the striatum after 6-OHDA lesions and in Parkinson’s disease. A particularly intriguing finding is that concentrations of dopamine in microdialysates collected in the partially denervated striatum Žless than 80%. are similar to those in the intact striatum w1,37,52x. Zigmond and coworkers w54x postulate that a variety of processes compensate for the deficits arising from an extensive loss of dopamine in the striatum. These include increased dopamine synthesis and release from the remain-
0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 0 0 3 - 6
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ing terminals, as well as reduced dopamine clearance from extracellular fluid. Compelling evidence supports adaptive changes in dopamine synthesis and uptake in animals with extensive lesions w3,28,35,47,51,53x. However, no discernible change in synthesis is apparent with lesions that deplete tissue dopamine by 50% suggesting that another adaptive mechanismŽs. besides increased synthesis mediates compensation in this condition. Evidence for a compensatory increase in dopamine release w20,42,44,52x is largely based on indirect indices such as turnover as reflected by the ratio of dopamine metabolites to tissue dopamine, fractional dopamine efflux from brain slices, and the ratio of dialysate dopamine to tissue dopamine. The major determinants of extracellular dopamine concentration are release and uptake w50x, processes which are not measured independently in most determinations of extracellular neurotransmitter. However, real-time microsensors implanted in brain tissue can be used to resolve these dynamic processes w16,26,50x. With this approach, Nafion-coated carbon fiber microelectrodes Žmaximal dimension of 20 mm. are used with fast scan cyclic voltammetry Ž100 ms repetition rate. to measure extracellular dopamine with excellent temporal and spatial resolution w16,27,32,49x. This methodology affords an unparalleled view of the extracellular events of neurotransmission in the microenvironment surrounding synapses w2x and is currently the only technique available to determine direct release rates for any neurotransmitter in the brain. In this paper, we employ this approach to investigate compensatory changes in dopamine release and uptake in the rat striatum after partial 6-OHDA lesions of nigrostriatal dopamine neurons. By doing so we hope to understand how normal dopaminergic function is maintained in the preclinical phase of Parkinson’s disease.
mgrkg i.m.., and immobilized in a stereotaxic apparatus ŽDavid Kopf Instruments, Tajunga, CA.. Core body temperature was maintained at 378C using a Deltaphase Isothermal Pad ŽBraintree Scientific, Braintree, MA.. An incision was made along the skull midline, and skin and muscle layers were retracted but not removed. All stereotaxic coordinates are according to the brain atlas of Paxinos and Watson Ž1986. w38x. Anteroposterior ŽAP. and mediolateral ŽML. positions are referenced to bregma and dorsoventral ŽDV. positions are referenced to dura. A small hole was drilled in the skull above the lateral edge of one substantia nigra Žy5.4 AP, q3.0 ML.. A 30 gauge needle was then lowered Žy8.2 DV. and 2 mL of a 0.1% ascorbic acid solution containing 2 mg of 6-OHDA was infused at a rate of 0.2 mlrmin. After completion of the infusion, the needle was removed, the hole was sealed with bone wax and the scalp wound was sutured. Animals were administered lactated ringers Ž10 ml i.p.. to prevent postsurgical dehydration and were allowed to recover from anesthesia before their return to housing. To facilitate recovery, the diet for lesioned rats was initially supplemented with highly palatable foods such as apples and peanut butter. If adipsia developed, rats were administered lactated ringers i.p. Most rats did not require special care for more than two days following surgery. 2.3. In ÕiÕo electrochemistry Electrically evoked concentrations of extracellular dopamine were measured in unilaterally lesioned rats one to four weeks after 6-OHDA administration. Surgery was performed as described above with the exception that rats were anesthetized with urethane Ž1.5 grkg i.p... The experimental design for simultaneous measurement of electri-
2. Materials and methods 2.1. Animals Male Sprague–Dawley rats Ž250–300 g. were purchased from Charles River ŽRaleigh, NC. and housed under controlled temperature and lighting conditions. Food and water were available ad libitum. Animal care was in accordance with the Guide for Care and Use of Laboratory Animals ŽNIH Publication 86-23. and was approved by the Institutional Animal Care and Use Committee of the University of North Carolina. 2.2. 6-OHDA lesions Survival surgery was performed under aseptic conditions. Animals were pretreated with the norepinephrine uptake inhibitor, desipramine Ž25 mgrkg i.p.., anesthetized with a combination of sodium pentobarbital Ž5 mgrkg i.p.., ketamine Ž50 mgrkg i.p.. and xylazine Ž10
Fig. 1. Experimental set-up for the simulataneous measurement of extracellular dopamine in the lesioned striatum and contralateral control. See Section 2 for details. Abbreviations: CFM, carbon-fiber microelectrodes; MFB, medial forebrain bundles; SE, stimulating electrodes; SN, substantia nigra; ArD, analog to digital converter; DrA, digital to analog converter.
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cally evoked dopamine in the lesioned striatum and contralateral side is diagrammed in Fig. 1. Twisted, bipolar stimulating electrodes were positioned in each medial forebrain bundle ŽMFB, y4.6 AP, "1.4 ML, y8.5 DV. and Nafion-coated, carbon-fiber microelectrodes w4x were positioned in the partially lesioned and intact striata Žq1.2 AP, "2.0 ML, y4.5 DV.. The reference electrode was a sodium saturated calomel electrode ŽSSCE. that made contact with dura via a salt bridge. Cyclic voltammograms were simultaneously recorded at each carbon-fiber electrode every 100 ms at a scan rate of 300 Vrs Žy400 to 1000 mV vs. SCCE. using a bipotentiostat w48x; EI400, Ensman Instruments, Bloomington, IN.. Data were digitized ŽDMA Labmaster, Scientific Solutions, Solon, OH. and stored to computer files. The time-dependent current was monitored over the peak oxidation potential for dopamine in successive voltammograms and converted to concentration based on postcalibration. 2.4. Electrical stimulation Constant current stimulation pulses were computer generated and optically isolated ŽNL 800, Neurolog, Medical Systems, Great Neck, NY. from the electrochemical system. Biphasic square wave pulses were applied Žcurrent and duration of each phase were "300 mA and 2 ms, respectively. using a twisted bipolar electrode with 0.2 mm diameter tips spaced 1 mm apart ŽPlastics One, Roanoke, VA..
value for each working electrode before comparison with data w26x. Where applicable, all measurements are reported as the mean " SEM and n is the number of animals. Significance testing used the Student’s t-test w41x with the significance level set at P - 0.05. 2.6. Determination of tissue dopamine After the in vivo measurements of release and uptake, the brain was removed from the skull, placed in a cold slice block, and cut coronally into 1.2 mm sections w19x. The section containing the portion of the striatum in which voltammetric recordings were made, as evidenced by damage to the dorsal cortical surface, was cut into 1 mm wide dorsoventral strips using a locally constructed tool. Mediolateral strips were stored at y258C until assayed for dopamine. On the day of assay, striatal tissue was sonified in mobile phase and centrifuged. Dopamine concentrations in the supernatant were quantitated by reverse-phase liquid chromatography with electrochemical detection as described previously w12x with some modifications. The mobile phase Žper L. consisted of 13.4 g sodium monophosphate, 5.76 g citrate, 450 mg sodium octyl sulfate, 40 mg EDTA and 20% methanol at a pH of 4.0. The column was an Ultramex 3 C18 IP Ž10 cm = 4.6 mm; Phenomenex, Torrance, CA.. Precipitated protein was determined by an assay kit from Bio-Rad ŽRichmond, CA.. 2.7. Drugs and reagents
2.5. Data analysis Extracellular concentrations of dopamine are controlled by the dynamic balance between dopamine release and uptake w49,50x. The rate of change during electrical stimulation is described by: d w DA x rd t s f w DA x p y Vmaxr Ž K m r w DA x q 1 .
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Ž 1.
where wDAx is the instantaneous extracellular concentration of dopamine, f is the pulse frequency, wDAx p is the concentration of dopamine elicited per stimulus pulse, and Vmax and K m are Michaelis–Menten uptake parameters. The basis of this equation is that each stimulus pulse elicits an instantaneous increase in extracellular dopamine, the release event, while uptake occurs continuously. The rate of disappearance of extracellular dopamine after the stimulation follows the Michaelis–Menten equation with the same rate constants describing uptake. Best-fit values for wDAx p and Vmax were obtained by analysis of the concentration changes evoked by 20 and 60 Hz stimulus trains using a value of 0.2 mM for K m w22,33x. Curve fitting employed a nonlinear regression based on a simplex minimization algorithm w16,25x. The response time of the sensor to a concentration pulse was obtained during postcalibration. Simulated curves were convolved with a response function calculated using this
All chemicals were used as received and purchased from Sigma Chemical ŽSt. Louis, MO. unless otherwise indicated. Post-calibration of working electrodes was performed in physiological saline Ž150 mM sodium chloride. buffered with HEPES Ž25 mM. at a pH of 7.4. Aqueous solutions were prepared in doubly distilled, deionized water ŽMega Pure System, Corning Glasswork, Corning, NY..
3. Results 3.1. Partial 6-OHDA lesions Prior work has shown that a graded loss of nigrostriatal dopamine neurons is produced by microinjection of 6OHDA into the lateral or rostral regions of the substantia nigra pars compacta w6,18x. Presumably, as the neurotoxin diffuses away from the injection site, a gradient is established with the highest concentrations near the site of origin. In this manner, dopamine cells in the medial substantia nigra and ventral tegmental area are spared. Since the topography or spatial arrangement of dopamine cell bodies in the substantia nigra is conserved in their projections to the striatum w11x, the pattern of dopamine terminal
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Fig. 2. Graded, unilateral lesion of nigrostriatal dopamine neurons. Top panel: diagram of a coronal view of the rat brain containing the striatum at the level in which the measurements described in the paper were taken. For each side of the brain, the striatum was dissected into lateral ŽL., mediolateral ŽML. and medial ŽM. regions, as demarcated by the dashed lines, for tissue analysis of dopamine by HPLC-EC. The values given in each region are dopamine content expressed as ngrmg protein for a single representative rat. Lower panel: evoked concentrations of extracellular dopamine as measured by voltammetry during 60 Hz, 2 s stimulus tran in the lesioned and control striatum of the same rat. Data Žsolid circles. are the concentration of dopamine measured in voltammograms at 100 ms intervals. The duration of the stimulation is shown by the solid line underneath each curve. Scale bars refer to all responses.
loss is similar in the striatum. In preliminary experiments we replicated this result using lateral injections as illustrated by the representative results from a single animal shown in Fig. 2 Župper.. A graded loss of tissue dopamine was found only in the ipsilateral striatum. The most severe lesion was in the lateral region with dopamine content reduced to 8% of the contralateral side. The dopamine content of the medial and mediolateral regions of the lesioned striatum contained 60 and 31 ng dopaminermg protein, respectively, while in the contralateral Žunlesioned. striatum the content was 105 and 115 ng dopaminermg protein, respectively. The mediolateral region thus exhibited an intermediate dopamine reduction of content Ž27% of the intact side., and was chosen for all future experiments. Prior to tissue analysis, dopamine release was evoked by electrical stimulation of the MFB and monitored with in vivo voltammetry w12x. To position the various electrodes, the carbon-fiber microsensors were initially lowered to y4.5 DV in each mediolateral striatum. A stimulating electrode was positioned just above each MFB Žy7.5 DV. and then incrementally lowered until maximal evoked responses for dopamine were recorded at each site. The final position of each microsensor was obtained by further lowering in small increments to optimize the recorded signal. The maximal evoked dopamine concentration during a 60 Hz stimulation in this animal was lower in the lesioned side than the intact side, and appeared to vary in a manner similar to tissue content ŽFig. 2, lower..
Fig. 3. Comparison of dopamine release during unilateral and bilateral stimulation of medial forebrain bundles. Recordings shown are from a single animal. The three responses shown in the left column were measured in the control striatum ŽCON. and the three responses in the central portion were measured in the lesioned striatum ŽLES.. Each adjacent pair of recordings was collected simultaneously. Underneath each trace is a solid line demarcating the time and duration of electrical stimulation Ž60 Hz, 2-s train.. The right column indicates whether a train of pulses was applied to the medial forebrain bundle Žq. or not Žy. ipsilateral to the lesioned striatum or contralateral control. In both lesioned and contralateral control striatum, the position of the working electrode was not changed for the duration of these measurements. Post mortum analysis showed the lesioned striatum contained 80% of the dopamine of the control side.
Fig. 4. Concentrations of extracellular dopamine in the lesioned and control striatum electrically evoked by a physiological frequency. Data Žsolid circles. are the concentration of dopamine measured at 100 ms intervals. Open boxes denote inititation and termination of the electrical stimulation Ž20 Hz, 6-s train.. The time and duration of the pulse train is also indicated by the solid line underneath the bottom trace. The tissue content of dopamine in the lesioned striatum was 45% of the contralateral control. Inset: background subtracted cyclic voltammograms measured in the lesioned striatum in vivo Žopen circles. and from dopamine in vitro during postcalibration. Both voltammograms were collected at the same working electrode. Current Ž I . is 1 nA and 50 pA for in vitro and in vivo determinations, respectively.
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Fig. 5. Relationships between extracellular concentration and tissue content of dopamine in the lesioned and control striatum. Data are expressed as the mean"SEM Ž ns 5.. Tissue dopamine content in the lesioned striatum is expressed as a percent of the value in the control striatum for each animal and then averaged for the entire group. Average concentrations of dopamine elicited by 60 Hz in the lesioned striatum were significantly Ž P - 0.05. different from control. Abreviations: CON, control striatum; LES, lesioned striatum; wDAx 20 Hz , maximum concentration of extracellular dopamine elicited by 20 Hz; wDAx60 Hz , maximum concentration of extracellular dopamine elicited by 60 Hz.
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tration in the two regions are similar. The shape of the background subtracted voltammogram recorded in both striata during the 20 Hz response in vivo was identical to that measured at 60 Hz Ždata not shown. and to dopamine measured in vitro at the same microsensor during postcalibration Žinset.. In all animals examined Ž n s 5., results similar to those for the single animal described in Fig. 4 were obtained with 20 Hz stimulations. As described above, dopamine was identified by its cyclic voltammogram and maximal concentrations were 0.23 " 0.05 and 0.31 " 0.09 mM in control and lesioned striata, respectively ŽFig. 5.. This was the case despite the fact that the tissue content of dopamine, expressed as a percent of the control striatum in each animal, was 54% of control. In contrast, dopamine concentrations evoked by 60 Hz were significantly Ž P - 0.05. lower in the lesioned striatum compared to control Ž1.8 " 0.5 and 3.6 " 0.8 mM, respectively.. On a percent basis, the decrease in evoked extracellular dopamine concentration with 60 Hz stimulation Ž50%. paralleled the decrease in tissue dopamine Ž54%.. 3.3. Rate constants for dopamine release and uptake In each animal, the temporal responses of extracellular dopamine to 20 and 60 Hz stimulation were analyzed to
3.2. EÕoked concentrations of extracellular dopamine in lesioned and contralateral striata Bilateral stimulation of the MFB with a 60 Hz train elicited an increase in extracellular dopamine in the mediolateral portion of both the lesioned and intact striata ŽFig. 3, bottom panel.. As in the animal shown in Fig. 2, the maximal concentration of dopamine in the extracellular fluid is lower on the lesional side whose content was 80% of the intact side. No signal was recorded in either striatum when the stimulating electrode ipsilateral to it was disconnected Žtop and middle panels.. However, responses recorded in the striatum of the unilaterally stimulated side of the brain were identical to those recorded when both stimulating electrodes were operable. There was no evidence of stimulus artifact. Recordings collected when the stimulating electrodes were positioned just dorsal to the MFB showed no response Ždata not shown.. Fig. 4 shows concentrations of extracellular dopamine in a lesioned striatum and the contralateral intact striatum of a different animal during simultaneous electrical stimulation of the MFB bilaterally with a frequency of 20 Hz. In this rat, the tissue content of dopamine in the region of the lesioned striatum where dopamine release and uptake was monitored was 45% of the contralateral side. Note that the maximal amplitude and dynamics of the dopamine concen-
Fig. 6. Relationships between dopamine release and uptake rates, and tissue content of dopamine in the lesioned and control striata. wDAx p is the concentration of dopamine released per stimulus, whereas dopamine uptake is described by the Michaelis–Menten term for the maximum rate of uptake, Vma x . Rate constants are the averages determined in the lesioned striata ŽLES. and contralateral controls ŽCON. as described in Section 2 and averaged. Values for the tissue content of dopamine are the same as in Fig. 5. Data are expressed as the mean"S.E.M. Ž ns 5, same animals as in Fig. 5..
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Fig. 7. Theorectical curves for electrically evoked concentrations of extracellular dopamine in the lesion and control striatum. Simulations were calculated from the average rate constants for dopamine release and uptake shown in Fig. 6 and assuming a K m of 0.2 mM. The simulation resolution was 20 ms. Curves were then convoluted with an impulse response function for thin layer diffusion using a film thickness of 400 nm and a diffusion coefficient of 1P10y9 cm2 rs w26x. Electrical stimulation was initiated at time 0 s and, for both frequencies, consisted of a 120 pulse train.
determine the kinetics of dopamine release and uptake parameters. Mean values for the concentration of dopamine released per stimulus pulse, wDAx p , and the maximal rate of dopamine uptake, Vmax , obtained from the data from all animals are shown in Fig. 6. In these analyses, the value of K m was fixed at 0.2 mM in the intact and lesioned striata since results from synaptosomal preparations show that K m for dopamine uptake is similar in the partially lesioned and intact striatum w53x. Both wDAx p Ž38 " 11 versus 78 " 18 nM, respectively. and Vmax Ž1.6 " 0.4 versus 2.7 " 0.5 mM, respectively. were significantly Ž P - 0.05. decreased in the lesioned striatum compared to the contralateral control. The average correlation coefficient Ž r . for each analysis was 0.92. The percent decrease in both the release term Ž49%. and Vmax Ž59%. were similar to the decrease in tissue dopamine Ž54%.. Simulated curves for 20 and 60 Hz responses in the lesioned and control striatum are shown in Fig. 7. Simulations were calculated from the average measured parameters for dopamine release and uptake given in Fig. 6. Theoretical calculations parallel experimental results in that simulations differ in amplitude at 60 Hz and are more similar at 20 Hz. The percent decrease in the 60 Hz simulation for the lesioned striatum was 46% of control Ž1.7 versus 3.7 mM, respectively. compared to 75% Ž0.21 versus 0.28 mM, respectively. for 20 Hz.
4. Discussion The goal of the present study was to investigate alterations in extracellular dopamine folowing partial denervation in the rat striatum. Real-time microsensors were employed to monitor dynamic changes in extracellular
dopamine evoked by transient electrical stimulation of ascending dopamine fibers. In situ rate constants for dopamine release and uptake were determined from the temporally and spatially resolved chemical measurements. After partial 6-OHDA lesions of nigrostriatal dopamine neurons, we observed that concentrations of extracellular dopamine in the striatum elicited by a frequency in the physiological range are similar in the intact and lesioned striata. However, when data from a higher frequency were analyzed, both dopamine release and uptake rates were found to decrease in proportion to the loss of dopamine terminals. These results support the hypothesis that normal concentrations of extracellular dopamine are maintained in the partially denervated striatum without active compensatory changes in dopamine release or uptake. In this paper, rate constants for dopamine release and uptake are compared, on a per dopamine terminal basis, in striatal regions denervated to different degrees. The density of dopamine terminals after lesions was estimated by dopamine tissue content determined by HPLC-EC subsequent to the voltammetric experiment. The adequacy of this index is demonstrated by the excellent relationship between tissue dopamine content and the density of dopamine terminals in different regions of the rat telencephalon w9,10,29,36x. Furthermore, the technique of flow cytometry has been used to show that, after 6-OHDA lesions, the percent of synaptosomes labeled with a fluorescent probe for tyrosine hydroxylase correlates well with striatal dopamine depletion w51x. 4.1. Extracellular concentrations of dopamine after partial lesions Partial denervation of dopamine neurons in the striatum have been produced by injection of 6-OHDA into the substantia nigra, MFB or cerebral ventricles of adult rats w3,6,20,39,53x. In the present study, unilateral, partial denervation of the mediolateral striatum was accomplished by an injection of 6-OHDA into the lateral edge of one substantia nigra. The partial lesion technique reduced tissue content of dopamine in the mediolateral striatum to 30–70% of control. The spatial resolution afforded by the carbon fiber microelectrodes allowed release and uptake of dopamine, chemically identified by the cyclic voltammograms, to be probed in this discrete striatal region. Measurements were collected bilaterally in the denervated and intact striatum so that each rat served as its own control. A similar approach has previously been used by Robinson and Whishaw w39x who determined basal levels of extracellular dopamine with the microdialysis technique. An intriguing finding obtained from prior 6-OHDA lesion studies is that concentrations of dopamine in microdialysates collected in the partially denervated striatum are similar to those in the intact striatum w39,52,1,37x. The present results are in agreement when dopamine release is
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evoked by low frequency electrical stimulation. A train of pulses at a frequency at the high end of the physiological range, 20 Hz w8,5x, evokes similar concentrations of extracellular dopamine in the partially lesioned striatum and contralateral control ŽFig. 4 and Fig. 5.. In contrast, at a much higher stimulation frequency Ž60 Hz., the maximal evoked dopamine is proportionately lower in the partially lesioned striatum. 4.2. Dopamine release and uptake in partially lesioned striata Microdialysis and voltammetry provide complementary information about the regulation of extracellular dopamine. Because of superior sensitivity and selectivity, microdialysis affords an estimate of basal concentrations of extracellular dopamine. However, direct investigation of the mechanisms responsible for measured concentrations are beyond the current capability of this technique. On the other hand, voltammetry permits resolution of measured concentrations into the respective contributions of uptake and release w49,26,16x. This is because the two primary determinants of extracellular dopamine exhibit different dynamics. Since dopamine release is an exocytotic process w15x, it is evoked at the time of an individual electrical impulse and can be considered an instantaneous event given the time scale of the measurement Ž100 ms.. In contrast, uptake is both a concentration- and a time-dependent process that obeys the Michaelis–Menten equation. Release events occur more frequently as the frequency of a stimulus train increases. In contrast, the rate of uptake is independent of the rate of impulse flow but instead increases with concentration until sufficient dopamine accumulates that it becomes limited by the upper limit set by Vmax . However as the stimulation frequency increases, less time is available for uptake to occur in between stimulus pulses. For this reason, at higher frequencies of impulse flow evoked concentrations of dopamine are most affected by the rate of release, i.e., a release dominated situation. Indeed, during stimulations that resemble burst firing, release appears more intense w17x. In contrast, at a low frequency a balance between release and uptake occurs, and a lower, steady-state concentration of dopamine is obtained. The steady-state level of extracellular dopamine concentration seen during 20 Hz stimulations is a direct consequence of this balance. Steady state occurs because, at this frequency, the rates of uptake and release equal each other. Since similar concentrations are observed in both the lesioned and intact striata with this stimulation frequency, these results can either mean that there was no change in the rate of release and uptake caused by the lesion, or that both uptake and release have changed in an identical manner. This dichotomy is the same as that confronted in the interpretation of concentrations obtained by microdialysis. The use of a second, higher frequency enables the
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contributions of release and uptake to be resolved. The higher frequency leads to release dominated conditions where, in the lesioned striatum, the reduced capacity for dopamine release is readily observed. With the use of Eqn. 1 and the experimental results from the two different frequencies, the rate constants that control these dynamic events can be mathematically resolved. It was found that wDAx p , the rate constant for dopamine release, and Vmax , the maximal uptake rate, both decreased in direct proportion to the degree of the lesion ŽFig. 6.. The simulations in Fig. 7 clearly show that similar concentrations can be generated at a low frequency when there are considerable differences in the rates of uptake and release that are employed. The results in the lesioned striatum are reminiscent of our findings for dopamine release and uptake in the amygdala and prefrontal cortex w13x. Although the dopamine content of both of those regions is much lower than that of the intact striatum, similar extracellular concentrations can be evoked in all three regions at low frequencies. As in the lesioned striatum, the lower rate of release in the nonstriatal regions is balanced by a lower rate of uptake. In vitro studies measuring the uptake of radiolabeled dopamine into synaptosomes w3,53x and previous voltammetric studies w7,28,47x are in agreement with the observed decrease in dopamine uptake rate in the partially denervated striatum ŽFig. 6.. However, the observed decrease in dopamine release in the partially denervated striatum contradicts some previous interpretations. Microdialysis studies demonstrating normal levels of extracellular dopamine after partial lesions have been offered as support for a compensatory increase in dopamine release after partial lesions because the ratio of extracellular dopamine to dopamine tissue content increases with the degree of the lesion w54x. Similarly, in vitro slice experiments show that electrically evoked efflux of dopamine, when normalized to tissue content, increases with the lesion w42,44x. However, like voltammetric data obtained at a single stimulation frequency, these measures do not provide sufficient information to resolve uptake and release. Even when efflux is measured in the presence of a competitive uptake inhibitor, some uptake still occurs w25x preventing evaluation of release. 4.3. Dopamine neurotransmission in the partially denerÕated striatum Based on a wide range of biochemical and physiological studies, Zigmond and co-workers w54x have developed a working hypothesis for adaptive changes in dopamine neurotransmission following 6-OHDA lesions. With partial lesions they postulate that the loss of dopamine neurons is compensated by diffusion of dopamine from the remaining terminals to more distant receptor sites than in the intact animal. Our findings provide strong support for this hypothesis. First, we find a decrease in Vmax for
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uptake in situ. This term is the product of the density of the uptake sites and the turnover rate of the transporter w14x. Since uptake sites are primarily on dopamine neurons w34x, a reduction in Vmax is not unexpected. When the rate of uptake is decreased, the lifetime of extracellular dopamine is increased giving it greater opportunity to diffuse to more remote sites. However, we also find a decrease in the release rate constant, wDAx p . This term is the product of the density of release sites and the amount released per terminal w14x. Since the reduction in this term in partially lesioned animals is similar to the reduction in dopamine content, our data argue against an active compensatory mechanism for release. Indeed, as seen by the evoked dopamine response at a low frequency, active compensation is not required. Rather, the concurrently reduced rate of uptake provides the necessary compensation to maintain dopamine extracellular concentrations at their prelesion levels. Compensatory increases in dopamine synthesis after 6-OHDA lesions of a greater extent than studied here are well established w35,51,53x and these changes parallel increases in dopamine turnover as indexed by the ratio of dopamine metabolites to dopamine in striatal tissue w3x. However, both synthesis and turnover rates only begin to increase when lesions reach the 50% level and exponentially rise as the lesion becomes more severe. In the more severely lesioned striatum, the higher turnover and synthesis of dopamine could reflect increased release rates as earlier postulated, a hypothesis that could be tested with this technique. On the other hand, another possibility is that faster rates of turnover and synthesis are related to reduced uptake after lesions. Uptake not only terminates dopamine neurotransmission but also acts to conserve released dopamine w22x. If less dopamine is conserved, then dopamine synthesis Žand therefore turnover. must be increased to maintain terminal stores. Partial lesions of nigrostriatal dopamine neurons produce a condition that mimics the early or preclinical phase of Parkinson’s Disease w6,53,54x. The lack of symptoms in patients with this disease until the deficit in dopamine terminals becomes severe may be a consequence of the passive compensation we observe in rats with partially lesioned striata. Both dopamine uptake sites w30x and the messenger RNA for the dopamine transporter w45x have been shown to decrease in clinically diagnosed Parkinsonian patients. Thus, the consequences of a simultaneous loss of dopamine release and uptake capacity observed in this animal model may directly indicate how normal dopaminergic tone is maintained in preclinical subjects.
5. Conclusions In conclusion, we have used real-time microsensors to examine the regulation of extracellular dopamine in rats
with partial, unilateral lesions of nigrostriatal dopamine neurons. These results extend previous studies using the technique of microdialysis to assess basal levels of extracellular dopamine after lesions and demonstrate that an active, compensatory increase in dopamine release is not necessary to contribute to the maintenance of normal concentrations of extracellular dopamine in the partially denervated striatum. Simultaneous loss of release and uptake sites concurrent with lesions of dopamine neurons can provide sufficient compensation because these processes are spatially fixed to the dopamine innervation w15,31,43x. Whether this form of adaptive compensation is operable in all dopamine terminal fields with their distinct combinations of release and uptake w13x remains to be determined. These results also suggest that passive mechanisms involved in the regulation of extracellular dopamine in the striatum play an important role in maintaining function during the preclinical or presymptomatic phase of Parkinson’s Disease.
Acknowledgements This research was supported by a grant from NIH ŽNS 15841..
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