Brain Research 872 (2000) 160–171 www.elsevier.com / locate / bres
Research report
Estrogen modulates responses of striatal dopamine neurons to MPP 1 : evaluations using in vitro and in vivo techniques Michael Arvin a , Lenka Fedorkova a , Kimberly A. Disshon b , Dean E. Dluzen b , a, Robert E. Leipheimer * b
a Department of Biological Sciences, Youngstown State University, Youngstown, OH 44555, USA Department of Anatomy, Northeastern Ohio Universities College of Medicine, Rootstown, OH 44272 -0095, USA
Accepted 16 May 2000
Abstract In vitro superfusion and in vivo electrochemistry were used to investigate the role of estrogen in modulating MPP 1 -induced dopamine output in the corpus striatum and nucleus accumbens of ovariectomized female rats. For in vitro superfusion experiments, dopamine and dihydroxyphenylacetic acid release were determined using HPLC with electrochemical detection from superfusion of corpus striatum fragments with Kreb’s ringer phosphate buffer pulsed with MPP 1 alone or MPP 1 with estrogen. The in vivo electrochemistry experiments recorded the dopamine signal from carbon fiber microelectrodes stereotaxically passed through the corpus striatum and nucleus accumbens. Dopamine release was stimulated by pressure ejection of MPP 1 alone or in combination with estrogen through glass micropipettes fastened to the electrodes. Dopamine output from superfusion chambers which received infusion of MPP 1 with estrogen showed significantly lower output of dopamine compared with chambers which received MPP 1 alone. Outputs of dihydroxyphenylacetic acid did not increase following MPP 1 infusions. Data from the electrochemistry experiments demonstrated that estrogen significantly reduced both the amplitude and clearance rates of the MPP 1 -evoked dopamine signal in both the corpus striatum and nucleus accumbens. Results of this study demonstrate that: (1) MPP 1 evokes striatal dopamine release and this effect is significantly reduced in the presence of estrogen as determined by both in vivo electrochemistry and in vitro superfusion: (2) similar, albeit attenuated effects are observed in the nucleus accumbens as determined with in vivo electrochemistry; (3) estrogen acts to inhibit the clearance of dopamine in both the striatum and nucleus accumbens; and (4) estrogen may function as a neuroprotectant by reducing the uptake of neurotoxin into dopaminergic neurons. 2000 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Degenerative disease: Parkinson’s Keywords: Electrochemistry; Superfusion; Transporter; Neurotoxicity; Nigrostriatal; Neuroprotection
1. Introduction While central nervous system responses to the gonadal steroid hormone, estrogen (E) have typically been associated with the hypothalamus as involved with reproduction, it is becoming increasingly apparent that E’s actions within the central nervous system are widespread and not exclusively related to reproduction. As one example, E exerts considerable effects upon the nigrostriatal dopaminergic
(NSDA) system [6,48]. This fact has a number of important implications since it may underlie some of the clinical and experimentally induced hormonal, estrous cycle and sex differences reported in NSDA function. For instance, administration of estrogen or estrogenic oral contraceptives have been shown to result in chorea in women [2,27] and reductions in L-DOPA-induced dyskinesia [7,34] neuroleptic-induced dyskinesias [7] and tardive dyskinesia [7,32]. Furthermore, a positive correlation between estrogen use and decreased symptom severity in women with early Parkinson’s disease was recently reported by Saunders-Pullman et al. [43]. In animal
*Corresponding author. Fax: 11-330-742-1483. E-mail address:
[email protected] (R.E. Leipheimer) 0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02511-7
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models, E treatment of ovariectomized animals increases striatal tyrosine hydroxylase activity [40], concentrations of DA metabolites [15], DA release in mice [38] and rats [3,4] and improves sensorimotor performance, a behavioral index of NSDA function [5]. When comparing NSDA functions between females and males marked differences are present with females showing more intense apomorphine and amphetamine-induced stereotypy as well as amphetamine-stimulated striatal DA release [11,44] and regional differences in amphetamine-induced striatal Fos expression [10]. There is also post-mortem evidence suggesting increased DA turnover in human females versus males [35]. The significance of these sex differences in NSDA function is that there are marked gender differences (male.female) in the incidence of Parkinson’s disease — the most notable pathology associated with the NSDA system (reviewed in Dluzen et al. [23]). Related to these gender differences in Parkinson’s disease are laboratory data which show that male mice appear to be more susceptible to agents producing neurotoxicity of the NSDA system. Specifically, male mice show greater depletions of striatal DA concentrations to MPTP [8,24] and methamphetamine [50,51] compared with females, although this MPTP result is not universally obtained [46]. We have endeavored to isolate and identify some of the basis for this gender difference by examining the effects of NSDA neurotoxins in gonadectomized animals treated or not with E. Our data indicate that E may be critically involved with these sex differences in NSDA pathology and suggest that this gonadal steroid may function as a neuroprotectant of the NSDA system. This follows since DA contents of ovariectomized1E-treated rats receiving an intra-striatal infusion of 6-OHDA [20] or mice receiving systemic MPTP [21,22] are significantly greater than comparably treated animals receiving no E. In general, there appears to exist a variety of experimental conditions in which E can serve as a neuroprotectant of central nervous system function [23]. In an attempt to better understand the basis for these effects, we have examined the acute responses of superfused striatal DA neurons in response to a direct infusion of MPP 1 in the presence or absence of E. Results from these experiments demonstrated that a co-infusion of E with MPP 1 significantly reduced the output of DA in response to MPP 1 alone, indicating that E can modulate initial responses of striatal DA neurons to this neurotoxin [18]. The present experiments were undertaken to more clearly understand the mechanisms of these E-dependent attenuations of MPP 1 -induced DA output. To accomplish this goal, two techniques were employed to assess DA output: in vivo electrochemistry and high-speed in vitro superfusion. The application and comparisons of these two techniques allows us to achieve improved time resolutions in responsiveness as well as to contrast data obtained with in vivo versus in vitro approaches.
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2. Materials and methods
2.1. Animals Adult female rats were used as subjects in these experiments. For all experiments rats were ovariectomized and allowed a minimum of 2–3 weeks to recover from surgery prior to the experiments. Water and lab chow were available ad libitum. All treatments of the animals adhere to the NIH Guidelines for the Care and Treatment of Animals and were approved by the animal care committees at NEOUCOM and YSU.
2.2. In vitro experiments 2.2.1. In vitro superfusion Determinations of dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) outputs were performed using an in vitro superfusion. The superfusion medium was a modified Kreb’s ringer phosphate (KRP) buffer (in mM): 120 NaCl, 4.8 KCl, 0.8 CaCl 2 , 1.2 MgSO 4 , 10.2 Na 2 HPO 4 , 1.8 NaH 2 PO 4 and 0.18% glucose at pH 7.4. The superfusion chambers consisted of a 1-cm 3 tuberculin syringe cut off at the 0.3 ml level. The chamber was connected to a peristaltic pump via a 22-gauge lumbar puncture needle. The top of the chamber was sealed with a rubber stopper containing two openings, one for inflow of filtered, humidified air to oxygenate the tissue while the other served as an exit port for effluent samples. The chambers were placed in a temperature controlled water bath maintained at 378C. 2.2.2. Experimental conditions On the day of an experiment, an animal was killed and the corpus striatum (CPu) was removed. Following a midline bisection, the ventricles were pried open revealing the CPu. The entire CPu, within the perimeter of the corpus callosum, was dissected. Prior to placement into the superfusion chamber, the CPu was further chopped into 0.530.530.5-mm tissue fragments with an average (6S.E.M.) of 21.961.3 mg of tissue placed in each chamber. The tissue fragments were suspended on cellulose filter paper within approximately 100 ml of KRP buffer in the superfusion chamber. The superfusion procedure used in this experiment represents a modified version of our previously described established procedures [18,19]. In the present experiment, the flow rate was 150 ml / min which enabled the collection of effluent samples at relatively frequent intervals of 0.5 min. Such rapid collection intervals permitted an enhanced time resolution of the superfusion response profile. Following a 20-min equilibration at a flow rate of 30 ml / min, the flow rate was increased to 150 ml / min for an additional 10-min equilibration before collection of effluent samples every
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0.5 min. With the start of effluent sample collections, the KRP medium was either continued or changed to one containing 300 nM 17 b-estradiol (E). In this way, the CPu tissue fragments from each individual rat was subjected to the two different treatment conditions of the experiment: MPP 1 infusion in the presence or absence of E within the KRP medium. During collection interval three of the superfusion, KRP medium containing 10 mM 1-methyl-4phenylpyridinium iodide (MPP 1 ) was infused directly into the superfusion chambers. With the start of collection interval 4, the respective KRP superfusion medium (6E) was resumed until the end of the experiment at collection interval 12. At the end of the superfusion, tissue fragments were removed and weighed to assess the amount of tissue placed in each chamber.
2.2.3. Assay Levels of DA and DOPAC in effluent samples were determined using HPLC with electrochemical detection (ESA Inc.). Biogenic amines were separated on a Biophase C-18, 5 mm sphere column (Bioanalytical Systems Inc.). The mobile phase consisted of (in mM): 50 sodium acetate, 27.4 citric acid, 10 sodium hydroxide, 0.1 sodium octyl sulfate, 0.1 EDTA and 7% methanol in filtered distilled water. The final pH of 4.5 was achieved with the addition of sodium hydroxide and the mobile phase was filtered prior to use. Standards for DA and DOPAC were diluted in the KRP superfusion medium and doses of 12.5, 25, 50, 100 and 400 pg / 20 ml were used to construct a standard curve. The sensitivity of this assay, as determined by the identification of reliable peaks above baseline noise of the chromatogram, was ,12.5 pg / 20 ml. 2.2.4. Statistical analyses The data obtained from the 12 collection intervals of the DA and DOPAC output profiles were blocked into three sets of intervals for analyses. The means of collection intervals 1–2 were used to compare pre-treatment (basal) outputs between the two conditions, while collection intervals 3–7 and 8–12 were used to evaluate the initial and latter response profiles to the MPP 1 infusion in superfusion chambers containing E or not. Mann–Whitney U-tests were used to compare responses between the two treatment conditions (MPP 1 versus MPP 1 / E) for each of the three blocked segments of the superfusion. Separate analyses were performed for the DA and DOPAC data. In addition, to assess the temporal characteristics of responses to MPP 1 , Wilcoxon signed ranks tests were used to compare outputs of DA and DOPAC between collection interval 2 (immediately prior to MPP 1 infusion) with that of the following four collection intervals (3–6) to identify the point at which significant changes in output occurred. A value of P#0.05 was required for results to be considered statistically significant.
2.3. In vivo electrochemistry experiments 2.3.1. Electrochemical methods On the day of the experiment a rat was anesthetized with urethane (1.25–1.5 g / kg, i.p.) and placed in a stereotaxic apparatus for the placement of electrochemical recording electrodes and Ag /AgCl reference electrodes. Core body temperature was monitored with a rectal thermometer and maintained at 378C with the use of a heating pad. The electrochemistry probe assembly consisting of a carbon fiber microelectrode (30 mm tip diameter) and glass micropipette (see below) was stereotaxically directed to the CPu of the rat according to the stereotaxic atlas of Paxinos and Watson [41]. Multiple passes for data collection were made bilaterally at 1.0, 1.5, and 2.0 mm anterior to Bregma and 1.5 or 2.0 mm lateral to the midline. Data were obtained using these coordinates at 0.5-mm intervals from between 3.0 and 7.5 mm from the surface of the brain in the CPu and nucleus accumbens (AcbC). Single carbonfiber electrodes were either purchased from Quanteon Limited Liability, Inc. (Denver, CO), or were constructed at YSU with carbon fibers generously provided by Textron Specialty Materials (Lowell, MA), according to methods modified from Gerhardt [28]. All recording electrodes were coated with Nafion (Aldrich Chemical Co., Milwaukee, WI) which increases their selectivity and sensitivity for monoamine neurotransmitters [26,29,30]. Highspeed chronoamperometric measurements were recorded using a computer-controlled electrochemical instrument (IVEC-10, Medical Systems Corp., Greenvale, NY). Measurements were taken at 5 Hz and then averaged over 1 s to enhance the signal-to-noise ratio of the recordings according to procedures previously described [25,31,33]. Briefly, a potential of 10.55 V versus a Ag /AgCl reference was applied to the electrode for 100 ms. The resulting oxidation current produced by the electroactive molecules near the electrode tip was digitally integrated during the last 80 ms of the pulse. After the electrode returned to resting potential, the reduction current produced by the oxidized electroactive molecules was integrated and stored by the computer in the same manner. The ratio of the reduction current to oxidation current (redox ratio) was used to identify the electroactive compounds giving rise to the electrochemical signal. Prior to implantation, each recording electrode was calibrated to determine its sensitivity to DA and selectivity for DA against ascorbic acid. For this experiment, redox ratios of electrodes calibrated with DA ranged between 0.35 and 1.14 and averaged 0.7360.5. The minimum sensitivity of electrodes to DA in these experiments was greater than 150 000 (as determined from the slope of the DA oxidation standard curve, current / mM DA). The minimum selectivity of electrodes for DA against ascorbic acid was set at 1000:1. For this experiment, electrode selectivity averaged greater than 500 000:1.
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2.3.2. Local application of drugs Glass micropipettes (tip diameter 10–15 mm i.d.) were filled with solutions of 1-methyl-4-phenylpyridinium (MPP 1 , RBI, Natick, MA) alone (1 mM), or MPP 1 with E in a concentration of 40 mM 17 b-estradiol (Sigma Chemical Co., St. Louis, MO). The solutions were passed through a 0.45-mm syringe filter (Micron Separations Inc., Westboro, MA) to remove any particulate contaminants prior to filling the glass micropipettes. The pipettes were fastened to the recording electrodes with a tip separation of 270–330 mm using sticky wax (Kerr USA, Romulus, MI). The test solutions were ejected from the glass pipettes using a Picospritzer II (General Valve Corp., Fairfield, NJ) with pressure pulses of 20–30 p.s.i. and durations of 1–6 s. Pressure and duration of the pulses were adjusted to maintain infused volumes in the range of 150–175 nl. Infused volumes were monitored by the use of a dissecting microscope equipped with an ocular micrometer positioned to observe the meniscus of the solution within the glass micropipette. The dissecting microscope micrometer was calibrated such that each micron equaled 25 nl as described in Friedemann and Gerhardt [25]. A separate test to investigate any potential inhibitory effects of estrogen on electrode sensitivity to DA was conducted in vitro. Standard curves were generated for increasing DA concentrations for electrodes under control conditions (absence of E) and after the addition of 40 mM E (the same concentration used in the present experiments) or 400 mM E for the same electrodes. The slope of the oxidation curve (current / mM DA) was used to determine electrode sensitivity to DA. Results demonstrated that E did not effect electrode sensitivity to DA (control slope averaged 190 54263396; 40 mM E averaged 191 67664762; and 400 mM E averaged 202 850615 567, values are mean6S.E.M.). 2.3.3. Data collection and statistical analysis High-speed chronoamperometry was used to record DA signals from the CPu and AcbC of two groups of animals. In one group the test solution consisted of MPP 1 alone (eight animals) and in the second group the test solution consisted of MPP 1 / E (eight animals). Experiments began after the probe assembly was stereotaxically implanted into the dorsal striatum. After a 5–10-min equilibration period, the electrode was activated and a stable baseline was established. The effects of the test drugs on the dynamics of the DA signal were assessed. For each recording the amplitude of the DA signal as well as temporal data were stored for later analysis. A total of 176 responses were obtained from rats treated with MPP 1 alone, and 180 responses obtained from rats treated with MPP 1 / E. Of the 176 MPP 1 -stimulated responses, 142 were recorded from the CPu and 34 in the AcbC. Of the 180 MPP 1 / E stimulated responses, 139 were recorded in the CPu and 41 in the AcbC. In this study, the amplitudes of DA output
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(mM concentration) and two measures of clearance were analyzed for each treatment group. The rate of clearance was determined (by the IVEC-10 software program) from the decay portion of the DA signal from 20% to 60% of peak value and expressed as nM DA / s. We also analyzed the comparable clearance times from approximately 0.1 mM concentration to baseline for both treatment groups. This second measure was conducted to evaluate the clearance times for DA over similar ranges in concentration for each treatment. Differences between groups for amplitude, clearance rate, and comparable clearance time were determined by Student’s t-test (SigmaStats program, Jandel Scientific, Corte Madera, CA) with P,0.05 set as the minimum level for significance.
2.3.4. Histology After each experiment the rat, under deep urethane anesthesia, was perfused transcardially with 0.9% saline, followed by 10% formalin. The brains were removed, and stored in 10% formalin until sectioned. Frozen sections were stained to verify recording sites microscopically.
3. Results
3.1. In vitro superfusion The raw data in vitro output profiles for the CPu tissue fragments infused with MPP 1 or MPP 1 / E are presented in Fig. 1 (A, DA; B, DOPAC), while summaries of the data analyses are presented in Fig. 2. Basal (pre-treatment) levels of DA, as defined by the overall mean output of collection intervals 1–2 of the superfusion, were virtually identical and not statistically different between the two treatment groups (P50.94). The output of DA from superfusion chambers which received the infusion of MPP 1 / E showed significantly lower output of DA compared with chambers receiving MPP 1 alone. Statistically significant differences were obtained during the initial (collection intervals 3–7, P,0.034) as well as during the latter (collection intervals 8–12, P,0.036) periods of the superfusion following MPP 1 infusion. No statistically significant differences were obtained for the basal output of DOPAC (P50.063). However, for both the initial (collection intervals 3–7, P,0.012) and latter (collection intervals 8–12, P,0.015) periods of MPP 1 infusion, DOPAC outputs of the MPP 1 / E conditions were significantly lower than that of the group receiving MPP 1 alone. There was a very rapid response in DA output to MPP 1 from tissue fragments with statistically significant increases obtained in interval 3 (P,0.02) and maximal DA output in interval 4 (P,0.002). By contrast, maximal DA output of the MPP 1 / E group was not observed until collection interval 5 and failed to differ significantly from
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Fig. 1. Mean and S.E.M. of in vitro release rates (pg / mg / min) from superfused striatal tissue fragments of ovariectomized rats. In the MPP 1 / E condition (n59), estradiol (E; 300 nM) was included within the superfusion medium while the MPP 1 condition (n517) was E-free throughout the entire superfusion period. For both conditions, MPP 1 (10 mM) was infused directly into the superfused tissue fragments during interval 3 of the superfusion. Dopamine levels are contained in A and corresponding DOPAC levels are presented in B. A summary of the analyses of these data is contained in Fig. 2.
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Fig. 2. Mean and S.E.M. summaries of in vitro release rates (pg / mg per min) from the data of Fig. 1. Data were collapsed over specific intervals for comparison of release rates to enable comparisons between the MPP 1 (n517) and the MPP 1 / E (n59) groups over the superfusion. Three periods of the superfusion were analyzed to evaluate pre-treatment levels (intervals 1–2), initial (intervals 3–7) and later (8–12) responses to MPP 1 infusion. The dopamine data are shown in Fig. 1A and indicate no significant difference (N.S.) between the two treatments during intervals 1–2 and significantly increased dopamine output in the MPP 1 group during intervals 3–7 (P,0.034) and 8–12 (P,0.036) following MPP 1 infusion. A similar result was obtained for DOPAC (Fig. 2B) with no differences in intervals 1–2 and significantly greater DOPAC output during intervals 3–7 (P,0.012) and 8–12 (P,0.015) of the superfusion following MPP 1 infusion.
that of collection interval 2 (P50.44). Nor were DA outputs at any of the other collection intervals significantly different from that of collection interval 2 in the MPP 1 / E group. DOPAC outputs did not increase following MPP 1 infusions and levels were significantly reduced compared to that of collection interval 2 at collection interval 5 for MPP 1 (P,0.02) and 6 for MPP 1 / E (P50.05).
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Fig. 3A,B. Oxidation currents are represented as the solid line and reduction currents appear as dashed lines. In each figure, the asterisk identifies the time when test substances were ejected. The marked reductions in the amplitude of the DA signal following MPP 1 / E treatment are evident in these representative profiles. Fig. 4A–C represent analyses of group data with respect to the amplitude of DA output, clearance rates of DA, and comparable clearance times in the CPu of rats treated with MPP 1 alone or MPP 1 / E. Values depicted in the figures represent the mean6S.E.M. The mean amplitude for MPP 1 -evoked DA release in the CPu (Fig. 4A) was 1.19860.071 mM and 0.12160.0072 mM DA for the MPP 1 / E-treated group (P,0.0001), representing a tenfold reduction in the amplitude of DA output when E was applied with MPP 1 . Fig. 4B illustrates the effects of MPP 1 and MPP 1 / E treatment on the clearance rate of DA in the CPu. Clearance rates for the DA signal were 19.1461.442 nM DA / s for MPP 1 -induced responses and 3.6360.228 nM DA / s following MPP 1 / E treatment (P,0.0001), representing an 82.4% decrease in the clearance rate of the DA signal in the presence of E. Fig. 4C shows the effects of E on the comparable clearance times of MPP 1 -evoked DA output. Clearance times for DA were 23.661.3 s for MPP 1 -induced DA output and 54.963.8 s after MPP 1 / E treatment (P,0.0001), representing a 2.3-fold increase in this clearance time when E was administered with MPP 1 . Fig. 5 illustrates typical electrochemical DA signals obtained in the AcbC following treatment by MPP 1 alone (Fig. 5A), or MPP 1 / E (Fig. 5B). The dramatic reductions in the DA response after treatment by MPP 1 / E are evident from these representative profiles. Fig. 6A–C represent analyses of group data with respect to the amplitude of the DA signal, clearance rates of DA, and comparable clearance times of DA in the AcbC of rats treated with MPP 1 alone or MPP 1 / E. Values depicted in the figures represent the mean6S.E.M. MPP 1 -induced DA release was 0.53660.0435 mM and 0.068060.0086 mM in the MPP 1 / E-treated animals, representing an 87.3% decrease in the amplitude of DA output in the presence of E (Fig. 6A). Again, a similar trend was reported for the clearance rate (Fig. 6B). The MPP 1 -treated animals demonstrated a clearance rate of 13.2061.925 nM DA / s whereas the MPP 1 / E-treated rats had a clearance rate of only 3.2060.472 nM DA / s (P,0.0001), a 75.8% decrease in clearance in the presence of E. As depicted in Fig. 6C, the comparable clearance time was 16.662.8 s after MPP 1 treatment and this value increased to 32.863.9 s in the presence of E, nearly a twofold increase in this parameter of clearance time.
3.2. In vivo electrochemistry
4. Discussion
Typical electrochemical DA signals in the CPu after local ejection of MPP 1 alone or MPP 1 / E are shown in
The presence of E in the KRP superfusion medium exerts a powerful effect upon in vitro DA output from
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Fig. 3. DA signal release profiles following ejection of MPP 1 alone (A), or after ejection of MPP 1 / E (B) in the CPu. The asterisk represents the time when the test substance was ejected from the micropipette. The marked reductions in the DA signal following MPP 1 / E stimulation are evident in these representative profiles.
superfused striatal tissue fragments in response to MPP 1 (Fig. 1A). Not only are there statistically significant differences in DA output throughout the entire post-MPP 1
infusion period of the superfusion between E present versus absent preparations (Fig. 2: MPP 1 .MPP 1 / E), but the modest increases in DA output of the MPP 1 / E failed
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Fig. 4. Amplitude of the DA signal expressed in mM (A), clearance rate (B) of DA expressed in nM / s, and comparable clearance times of DA (C) in the CPu. Both the amplitude (A) and clearance rate (B) of the DA responses were clearly suppressed, and the comparable clearance time (C) was significantly prolonged in the MPP 1 / E groups. Values given are the mean6S.E.M. The number of responses (n) and P values are shown in the figure.
to differ significantly from pre-MPP 1 basal levels. This latter analysis indicates that E exerted a nearly complete suppression of the MPP 1 -evoked output of DA. A second notable effect of the MPP 1 -evoked DA output in E-free preparations was the rapidity of the response, with statistically significant increases already being obtained during the interval of MPP 1 infusion (interval 3) of the superfusion. These DA results are similar to that reported previously [18]; however, the magnitude of the difference between MPP 1 versus MPP 1 / E and rapidity of response in the present report is substantially greater. We believe this may be primarily attributable to differences in collection interval times: 10 versus 0.5 min. Accordingly, the increased time resolution of the present report may provide a better index of responsiveness as 0.5-min sampling intervals provide a more accurate picture of DA secretion than do 10-min intervals. With regard to DOPAC output there is an absence of any clear increase following MPP 1 infusion and a statistically significant decline below that of basal rates over the duration of the superfusion period (Fig. 1B). An increase in DA and gradual decline in DOPAC output as seen in the present MPP 1 / 6E superfusion data represents a profile very similar to that observed from the striatum of ovariectomized rats infused with MPP 1 [18] or with amphetamine [37]. Both MPP 1 and amphetamine exert effects upon DA neurons through interaction with the DA transporter. Therefore, the changes in DA responses observed in the presence of E would suggest that this gonadal steroid hormone may involve an influence on DA transporter function. This possibility was assessed by replicating these experiments under conditions where more refined measurements of dopaminergic function can be recorded as achieved with in vivo electrochemistry. Much like that observed with in vitro superfusion, local ejection of MPP 1 induced the release of DA in both the CPu and AcbC in our in vivo experiments. The presence of E was found to dramatically reduce DA output in both
brain regions, again similar to that reported above using in vitro superfusion techniques. The use of in vivo electrochemistry has the advantage of real time resolution, which allows a more detailed examination of the release and clearance characteristics of the DA signal. Within both brain regions, the amplitudes, clearance rates and comparable clearance times of the MPP 1 -induced DA signal were markedly altered with the application of E. The amplitude of the DA signal represents the amount of DA secreted by neuronal terminals into the extracellular space. MPP 1 acutely evokes DA output by uptake into the neuronal terminals via the DA transporter and subsequent displacement of DA from the storage vesicles [12,14,47]. DA is then released via the DA transporter into the extracellular space. Thus, the amplitude of the DA signal represents the effectiveness of MPP 1 in evoking the release of the transmitter. In this study there was a greater than 80% reduction in DA amplitude in the CPu and AcbC in the presence of E. This result suggests that E is interfering with the ability of MPP 1 to induce DA secretion by actions on the dopamine transporter. DA clearance is a measure of the removal of this transmitter from the extracellular space and includes diffusion, enzymatic degradation and re-uptake into the DA presynaptic terminals. However, it is the re-uptake of DA into the presynaptic terminals (by the high-affinity DA transporter) which is thought to be the major mechanism by which DA is cleared [9,49]. Therefore, a reduction in clearance rate provides one of the more definitive indices that DA uptake has been reduced. The significant, fivefold reduction in clearance rates of the MPP 1 / E group compared with the MPP 1 alone group, would strongly suggest that E is exerting its effects through inhibiting the uptake of MPP 1 into the DA nerve terminals. However, because DA uptake is concentration dependent, it could be argued that signals with very different amplitudes could have different clearance rates even though they might have similar uptake rates. Because our results demonstrated a
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Fig. 5. DA signal release profiles following ejection of MPP 1 alone (A), or after ejection of MPP 1 / E (B) in the AcbC. The asterisk represents the time when the test substance was ejected from the micropipette. The suppression of the DA signal following MPP 1 / E is evident in these representative profiles.
tenfold reduction in the amplitude of DA in the presence of E, we also determined the clearance time of the DA signal from similar concentration points for both treatment groups. These comparable clearance times were calculated from approximately 0.1 mM concentration to baseline for
the DA signal. E significantly prolonged these comparable clearance times for DA in the MPP1 / E-treated rats which supports our clearance rate data that E is acting to inhibit DA uptake. It is known that a critical mechanism of action of MPP 1 is its uptake through the DA transporter. The
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Fig. 6. Amplitude of the DA signal expressed in mM (A), Clearance Rate (B) of DA expressed in nM / s, and comparable clearance times for DA (C) in the AcbC. Both the amplitude (A) and the clearance rate (B) of the DA responses were markedly reduced, and the comparable clearance time was significantly lengthened following MPP 1 / E treatment. Values given are the mean6S.E.M. The number of responses (n) and P values are reported in the figure.
competition of MPP 1 with DA for transporter sites has been demonstrated by several investigators [12,36,47]. Taken together, results from our in vitro and in vivo experiments strongly suggest that E is acting at the site of the DA transporter to inhibit entry of MPP 1 into the presynaptic neurons, thus inhibiting both the output and re-uptake of DA. There is evidence that supports such a role for E. For example, Michel et al. [39] reported a decrease in dopamine uptake following treatment with estrogen or estrogen agonists. E competitively inhibits DA uptake into striatal synaptosomes via an increase in the Km of the DA transporter [16] and we have observed that E increases DA recovery following exogenously infused DA in a manner similar to that obtained with the putative DA uptake blocker nomifensine [19]. In addition, ovariectomy increases striatal DA uptake, while in vivo E treatment decreases this process [1]. Furthermore, Thompson [45] demonstrated that DA re-uptake was inhibited in ovariectomized, estrogen-treated rats as determined by in vivo voltammetry. In this way, E could function to reduce the clearance rate of DA at least in part by direct actions on the DA transporter. Also, Disshon and Dluzen [17] reported that E treatment decreased the recovery of MPP 1 evoked DA from the corpus striatum of ovariectomized rats in in vivo microdialysis experiments. These results also suggest that E may function to inhibit DA uptake in this tissue. The capacity for E to reduce the uptake may then be similar to other uptake inhibiting drugs such as amfonelic acid, benztropine, buproprion and mazindol which have been shown to completely block the DA depleting effects of MPTP [42], and nomifensine which has been demonstrated to decrease MPP 1 -induced DA release [13]. In conclusion, results of the present study demonstrate that: (1) MPP 1 evokes striatal DA output and this effect is significantly reduced in the presence of estrogen as de-
termined by both in vivo electrochemistry and in vitro superfusion; (2) similar, albeit attenuated effects are observed in the nucleus accumbens as determined with in vivo electrochemistry; (3) estrogen acts to inhibit the clearance of DA in both the striatum and nucleus accumbens; and (4) estrogen may function as a neuroprotectant by reducing the uptake of neurotoxin into dopaminergic neurons.
Acknowledgements We would like to thank Drs Greg Gerhardt and Alain Gratton for their assistance in setting up the in vivo electrochemistry technique at YSU. This work was supported by a Research Challenge Grant from NEOUCOM awarded to D.E.D. and R.E.L.
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