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Published as doi: 10.1096/fj.07-8797com.
 (The FASEB Journal. 2008;22:261-275.)
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(The FASEB Journal. 2008;22:261-275.)
© 2008 FASEB

MPTP-induced deficits in striatal synaptic plasticity are prevented by glial cell line-derived neurotrophic factor expressed via an adeno-associated viral vector

Yuan-Hao Chen*,{dagger},{ddagger}, Brandon K. Harvey{ddagger}, Alexander F. Hoffman{ddagger}, Yun Wang{ddagger}, Yung-Hsiao Chiang*,{dagger},1 and Carl R. Lupica{ddagger},1

* Program of Clinical Medicine, Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan, R.O.C.;

{dagger} Department of Neurological Surgery, Tri-Service General Hospital, Taipei, Taiwan, R.O.C.; and

{ddagger} Department of Health and Human Services, National Institutes of Health, National Institute on Drug Abuse, Intramural Research Program, Baltimore, Maryland, USA

1Correspondence: C.R.L., Electrophysiology Research Section, National Institutes of Health, National Institute on Drug Abuse, Intramural Research Program, Baltimore, MD, USA. E-mail: clupica{at}intra.nida.nih.gov; Y.H.-C., Department of Neurological Surgery, Tri-Service General Hospital, No. 325, Section 2, Cheng-Kung Road, Neihu District, Taipei 114, Taiwan, or Section of Neurosurgery, Department of Surgery, Taipei Medical University Hospital, Taipei Medical University, 252 Wu Hsing Street, Taipei 110, Taiwan. E-mail: ychiang{at}mac.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study determined the consequences of dopamine denervation of the striatum on synaptic plasticity and prevention of these changes with gene therapy using an adeno-associated viral vector (AAV) expressing glial cell line-derived neurotrophic factor (GDNF). C57BL6/J mice were injected with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP); long-term depression (LTD) or potentiation (LTP) were measured in vitro. Fast-scan cyclic voltammetry measured electrically released dopamine from a functionally relevant pool in these same striatal slices. After MPTP, dopamine release and uptake were greatly diminished, and LTP and LTD were blocked in the striatal slices. The loss of plasticity resulted directly from the loss of dopamine since its application rescued synaptic plasticity. Striatal GDNF expression via AAV, before MPTP, significantly protected against the loss of dopamine and prevented the blockade of corticostriatal LTP. These data demonstrate that dopamine plays a role in supporting several forms of striatal plasticity and that GDNF expression via AAV prevents the loss of dopamine and striatal plasticity caused by MPTP. We propose that impairment of striatal plasticity after dopamine denervation plays a role in the symptomology of Parkinson’s disease and that AAV expression of neurotrophic factors represents a tenable approach to protecting against or slowing these neurobiological deficits.—Chen, Y-H., Harvey, B. K., Hoffman, A. F., Wang, Y., Chiang, Y-H., Lupica, C. R. MPTP-induced deficits in striatal synaptic plasticity are prevented by glial cell line-derived neurotrophic factor expressed via an adeno-associated viral vector.


Key Words: cyclic voltammetry • gene therapy • LTD • LTP • Parkinson’s disease


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
AN EXTENSIVE LITERATURE DOCUMENTS that degeneration of the dopaminergic nigrostriatal pathway and loss of dopamine function in the striatum result in abnormal activity of the basal ganglia (1) . This is considered a primary causative factor in the development of the motor symptoms of Parkinson’s disease (PD). Although the precise role of dopamine in regulating striatal activity and basal ganglia output is not yet understood, it appears that an interplay between dopamine and glutamatergic corticostriatal inputs is required to maintain normal striatal function (2 3 4) . These excitatory striatal inputs regulate basal ganglia function by activating large groups of normally quiescent medium spiny neurons (MSNs), which then alter neuronal activity throughout the basal ganglia (5 , 6) . Recent studies suggest that long-term changes in the strength of corticostriatal synaptic transmission may be involved in supporting striatal functions such as motor learning, motor programming, and habit learning (5 , 7 8 9) . Therefore, modulation of this excitatory input through changes in synaptic plasticity is likely critical for the performance of this neural system. Long-term potentiation (LTP) and long-term depression (LTD) of the excitatory corticostriatal pathway have both been observed after high-frequency activation of MSNs (10 11 12 13) , and studies using experimental reductions in striatal dopamine through administration of neurotoxins demonstrate the blockade of corticostriatal LTP and LTD (2 3 4 , 10) . Since other measures of corticostriatal transmission and MSN activity remained resistant to dopamine depletion via these methods (3 , 14) , it appears that one function of striatal dopamine might be to support synaptic plasticity.

Several neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), are efficacious in the remediation of, or protection against, degeneration of the nigrostriatal dopamine pathway in models of PD. GDNF prevents depletion of striatal dopamine levels after neurotoxin exposure (15 16 17) , and substantial functional benefits are achieved when it is delivered over an extended period near dopamine axon terminals in the striatum or via single intracranial injections (15 , 16 , 18) . Several approaches have been used to administer neurotrophic factors in humans, including bolus intracranial injections, chronic infusion pumps, and nonreplicating viral vectors designed to express neurotrophic factor genes (19) . Although each method has its limitations, the viral vector approach to provide sustained local intracranial release of GDNF appears promising, but requires further clinical development. Adenovirus, AAV, and lentivirus-based vectors have been used successfully to achieve sustained expression of GDNF in animal models of PD, which can result in significant behavioral and neurochemical protection and recovery (18 , 20 21 22 23) . The advantage of this approach is in the delivery of proteins within the brain over long periods of time after a single surgery (17) .

Whereas studies have demonstrated degrees of GDNF-induced behavioral recovery in PD and in animal models of the disease, there is poor understanding of the neurobiological changes underlying this recovery. Little is known about the functional remodeling of the striatal circuitry after striatal dopamine depletion, and it is uncertain whether potentially clinically important interventions such as GDNF treatment have functional effects within the circuitry of the striatum. For these reasons we examined the synaptic changes occurring in the striatum after dopamine depletion via exposure to the neurotoxin MPTP and the ability of AAV-GDNF to protect against these changes. We find that MPTP-induced depletion of striatal dopamine was associated with the loss of several forms of corticostriatal synaptic plasticity, which was prevented by intrastriatal administration of AAV-GDNF.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and brain slice preparation
All animal procedures were developed using guidelines of the NIH Animal Research Advisory Committee and were approved by the NIDA Intramural Research Program Institutional Care and Use Committee. Male C57BL/6 mice (8–14 wk old, Charles River Laboratories, Raleigh, NC, USA) were killed by cervical dislocation and rapidly decapitated. The brains were then removed and placed into a chilled (4°C) and aerated (95% O2/5% CO2) modified (24) artificial cerebrospinal fluid (m-aCSF) comprised of (mM): NaCl, 87; KCl, 2.5; MgCl2, 7; CaCl2, 0.5; NaH2PO4, 1.25; D-glucose, 25; sucrose, 75; NaHCO3, 25. Transverse brain slices containing the cerebral cortex and the striatum were then cut (300 µm) in chilled m-aCSF using a vibrating tissue slicer (Leica VT1000S, Wetzlar, Germany). The coronal brain slices were transferred to a holding chamber containing 50% normal aCSF (mM): NaCl, 126; KCl, 3.0; MgCl2, 1.5; CaCl2, 2.4; NaH2PO4, 1.2; glucose, 11.0; NaHCO3, 26, saturated with 95% O2/5% CO2, and 50% m-aCSF, at 32°C. Before initiating electrophysiological recordings, a single brain slice was submerged in a low-volume (170 µl) recording chamber and continuously bathed with warm (30–32°C) aCSF at 2 ml/min. The aCSF was warmed using an inline solution heater (TC-324B, Warner Instruments, Hamden, CT, USA).

Electrophysiological recordings
Extracellular recordings of striatal population spikes were performed in the dorsal striatum using described techniques (25) . Extracellular population spike recordings were used rather than whole-cell voltage clamp recordings because they reflect the simultaneous activity of a large number striatal MSNs and are not susceptible to perturbations in the intracellular environment that are often observed with whole-cell techniques. To isolate glutamate-driven population spikes from GABAA-mediated currents with a similar time course (26) , the aCSF also contained the GABAA receptor/Cl– channel blocker picrotoxin (100 µM). Electrical stimulation was performed using bipolar tungsten stimulating electrodes (Frederick Haer, Bowdoin, ME, USA) placed on the tissue either close to (< 100 µm) the intrastriatal recording electrode or 1–2 mm above the corpus callosum, at the border of primary motor and somatosensory cortices (see Fig. 1 A). Baseline population spike responses were elicited using single 0.1 ms pulses (10–30 V) delivered through a stimulating electrode at a frequency of 0.033 Hz. In each case, the maximum population spike response was defined by increasing the stimulus intensity until an asymptotic amplitude was reached. Then the stimulus intensity was adjusted to elicit a response that was 40–50% of the maximum and the stimulator was left at this setting for the remainder of the experiment. Drugs were applied at fixed concentrations to flowing aCSF using calibrated syringe pumps (Razel Research Instruments, St. Albans, VT, USA). Data were acquired and stored on a personal computer via an A/D board (National Instruments PCI 6024E, Austin, TX, USA) using a Windows-based software package (WCP, courtesy of Dr. John Dempster, University of Strathclyde, Glasgow, UK; http://spider.science.strath.ac.uk/PhysPharm/showPage.php?pageName=software_ses). Analyses of population spike amplitudes were performed off-line using the same software. Comparisons were then made between averages of at least 10 responses obtained during the baseline control (pre-HFS) period or at a fixed time (usually 60 min) after application of HFS.


Figure 1
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  		Figure 1. Baseline corticostriatal populations spikes are mediated by glutamatergic synaptic transmission, and LTP of this pathway is blocked by NMDA receptor antagonism. A) Low-power stereoscopic image of a coronal hemisected brain slice containing the striatum and cerebral cortex submerged within the recording chamber. A single bipolar stimulating electrode is shown in contact with the brain slice, just above the corpus callosum (top center). Also shown at right is an extracellular glass capillary recording electrode. B) Time course of the effects of the selective AMPA/kainate receptor antagonist NBQX on cortically evoked striatal population spikes. Population spikes are composed of a nonsynaptic N1 component and a synaptically mediated N2 component. Application of NBQX for 5 min (horizontal bar) caused a large, reversible reduction in the N2 component of the population spike amplitude without affecting the N1 component (n=6 brain slices). Scale same as in panel C. C) The NMDA receptor antagonist APV blocks LTP (Fig. 2) of corticostriatal population spikes. Note that APV did not affect baseline population spike amplitude, but blocked LTP initiated by HFS-3k stimulation (n=10 slices). Averaged population spike wave forms (n=5–7 individual sweeps) collected under each condition are shown to the right of panels B, C. C) The population spike represented by the dashed line was collected 60 min after HFS-3k stimulation; the solid line represents a population spike collected during the baseline period.

Measurement of striatal synaptic plasticity
After stable baseline population spikes were measured, synaptic plasticity was assessed by examining the effects of one of two different high-frequency trains applied through either the intrastriatal stimulating electrode or the stimulating electrode placed in the cortex. One high-frequency stimulus protocol consisted of a total of 400 pulses delivered in four trains of 1 s duration, separated by 10 s intervals, at 100 Hz (HFS-400). The other protocol consisted of a total of 3000 pulses, delivered in three trains of stimuli 10 s in duration at 100 Hz, with a 10 s interval between each train (HFS-3k). Population spikes were then monitored for at least 1 h after delivery of the high-frequency trains. These protocols were chosen because they have been used by other laboratories to investigate synaptic plasticity in the dorsal striatum (3 , 12 , 27 28 29) and because they have never been compared in the same laboratory under the same experimental conditions. The effects of MPTP on these forms of striatal synaptic plasticity have not been evaluated.

Fast scan cyclic voltammetry
Carbon fiber electrode construction
Fast-scan cyclic voltammetry (FSCV) was performed using carbon fiber electrodes as described previously (30 , 31) . Carbon fibers (7 µm diameter, Goodfellow Corp., Devon, PA, USA) were aspirated into glass micropipettes, which were then pulled using a multistage patch pipette program on a Sutter P-97 electrode puller. Carbon fibers were trimmed to allow ~ 20–50 µm to protrude from the glass capillary, then sealed in the tip of the pipette by passing it quickly over a flame. Pipettes containing the carbon fiber were filled with a solution of 4M K-acetate/150 mM KCl and attached to the head stage of a patch clamp amplifier (HEKA EVA-8, HEKA Instruments Inc., Southboro, MA, USA). Voltammetric scans, stimulus wave form generation and timing, and data collection were performed using A/D boards (PCI 6052E and PCI-6711E, National Instruments, Austin, TX, USA) and custom LabView-based software (TarHeel CV, courtesy of Drs. Joseph Cheer and Michael Heien, University of North Carolina). All carbon fiber electrodes were tested for stable background currents and responsiveness in aCSF containing dopamine (5 µM) prior to each experiment.

Dopamine measurement in striatal slices
Carbon fibers were inserted ~75–100 µm into the dorsal striatal brain slice and positioned between the separated tips of a bipolar stimulating electrode (FHC Inc., Bowdoin, ME, USA) using a stereo microscope (32) . Voltammetric scans from –0.4 to 1.0 V and back were performed at 100–400 V/s (7 to 28 ms scan duration) at a frequency of 10 Hz. A 5 s background measurement (50 scans) was taken prior to electrical stimulation of the brain slice and subtracted from the voltammetric scan obtained at the signal peak immediately after electrical stimulation. This was used to generate a voltammogram (current vs. voltage plot) for each signal. All signals matched those expected for the oxidation and reduction of dopamine (30 , 32) . Electrical stimulation consisting of a single 4 ms biphasic pulse, which did not overlap the voltammetric scans, was used to release dopamine. Constant-current stimulus intensity was varied from 0.1 to 1 mA in order to construct input-output curves for each placement in the striatal slice. In experiments performed with inhibitors of dopamine uptake, each dopamine signal served as its own control, with at least three baseline responses (30–50% of maximum) taken at 2 min intervals prior to inhibitor application. Time constants ({tau}) for the decay phase of each dopamine signal were obtained by fitting a single exponential function using a least-squares minimization algorithm: Y(t) = A–t/{tau}, where A = peak signal amplitude (nA), t = time (ms), and Y = signal amplitude at any given t. Initial comparisons of the sum of squares (F-test) between single and double exponential functions (Prism v. 4.03) confirmed that the decay phase was best fit by a single exponential in all cases. It has been demonstrated that the first-order rate constant (k, or 1/{tau}) obtained using this approach provides an index of the efficiency (Vmax/Km) of dopamine clearance mediated via the dopamine transporter at low dopamine concentrations (33) .

MPTP treatment
MPTP or saline vehicle was administered to male C57BL6/J mice (Charles Rivers Laboratories) via intraperitoneal injection. Pilot experiments determined that 100 mg/kg MPTP administered in four 25 mg/kg injections at 2 h intervals resulted in substantial depletions of dopamine in the dorsal striatum as measured 5–7 days after the last MPTP injection (n=22). However, mortality using this paradigm was high (20/42 mice=48%). Therefore, the mice used for the electrophysiological and electrochemical studies were treated with 80 mg/kg MPTP administered using four separate 20 mg/kg injections at 2 h intervals. This paradigm also resulted in a substantial reduction of dorsal striatal dopamine levels (see Results; n=20). However, the mortality rate at this dose of MPTP was much lower (4/24=16%); therefore, 80 mg/kg MPTP was used for the remainder of the experiments. Stimulus-evoked dopamine levels were measured using FSCV at several sites in each striatal slice and in several brain slices from each saline-injected control and MPTP-treated animal. Dopamine currents in brain slices were converted to concentration by extrapolating these signals to those recorded during measurement of known concentrations of dopamine during calibration. These evoked dopamine levels were then averaged for all the sites and for all of the brain slices from a single animal in which dopamine signals were measured. Thus, a single animal yielded a single dopamine concentration data point obtained from multiple sites and multiple striatal slices.

AAV packaging, purification, and titering
The construction of a self-complementing AAV vector expressing enhanced green fluorescent protein (AAV-GFP) has been described (34) . The self-complementing AAV vector expressing rat GDNF (AAV-GDNF) was made by replacing GFP with the cDNA for rat GDNF. Viral stocks were prepared using the triple transfection method with some modifications (35) . Briefly, twenty 15 cm dishes containing HEK293 cells at 85–95% confluence were transfected by the CaCl2 method with pHelper (Stratagene, San Diego, CA, USA), pdsAAV-GDNF, or pdsAAV-GFP (34) and pXR5 (36) . pdsAAV-GFP, pdsAAV-GDNF, and pXR5 were generously provided by Dr. Xiao Xiao, Gene Therapy Division, Department of Pharmacology, University of North Carolina (Chapel Hill, NC, USA). Approximately 48 h post-transfection, cells were harvested, lysed by freezing and thawing, and purified by centrifugation on a CsCl gradient. Final samples were dialyzed in PBS, aliquotted, and stored at –80°C. All vectors were titered by quantitative PCR using the cytomegalovirus virus promoter as the target sequence. Viral titers were recorded as viral genome/ml for AAV-GDNF (1x1012 vg/ml) and AAV-GFP (4x1012 vg/ml). For injections, titers were diluted to 1 x 1010 vg/ml.

AAV injections
Male C57BL6/J mice (8–10 wk old) were anesthetized with chloral hydrate (400 mg/kg, i.p.) and placed into sterotaxic apparatus. A burr hole was drilled (1 mm diameter bit) at the following sterotaxic coordinates relative to bregma: 0 mm anterior-posterior, 1.8 mm left medial-lateral. Using a 10 µl Hamilton syringe with a 33 gauge blunt hypodermic needle, 2 µl of AAV or PBS was injected at a rate of 0.5 µl/min into each site (2.5–3.2 mm ventral to the dura) using an UltraMicroPumpII (World Precision Instruments, Sarasota, FL, USA). The burr hole was sealed with bone wax after each injection; animals were kept warm and monitored during recovery.

Statistics
Group data are presented as mean ± SE. In all cases, n refers to the number of brain slices. However, the data were obtained from multiple subjects in each group to ensure that results were replicable. The plasticity data were analyzed using a repeated measures ANOVA design, and all statistical tests were performed using a critical probability of P < 0.05 (GB-Stat, Dynamic Microsystems, Silver Spring, MD, USA; or Prism v4.0, GraphPad Software, San Diego CA, USA). Post hoc analyses (Tukey-Kramer) were performed only when the ANOVA yielded a significant main effect.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The form of synaptic plasticity in the dorsal striatum is dependent on the amount and location of high-frequency stimulation
Population spike responses to single-pulse electrical stimulation were recorded in the dorsal striatum (Fig. 1A ). In the presence of the GABAA receptor blocker picrotoxin (100 µM), single-pulse stimulation of the cortex (Fig. 1A ) or within the striatum evoked a population spike wave form that was dependent on the synaptic release of glutamate and activation of the postsynaptic AMPA/kainate receptors. Corticostriatal population spikes are composed of a nonsynaptic negative (downward) component (N1) and a synaptically mediated negative component (N2). It is likely that N1 reflects ion currents generated in presynaptic axons by the stimulus, whereas N2 results from the coordinated action potential discharge of many MSNs after their activation by glutamate. In accord with this, the AMPA/kainate receptor antagonist NBQX (10 µM) nearly eliminated the synaptic N2 component without significantly affecting the nonsynaptic N1 component of the population spike (Fig. 1B ). To examine the effect of nigrostriatal pathway damage by MPTP on synaptic plasticity of these synaptic glutamatergic responses, we used two different types of high-frequency electrical stimulation. These stimulation protocols consisted of 400 (HFS-400) or 3000 pulses (HFS-3k) delivered at 100 Hz. We also examined the effects of each of these forms of stimulation on synaptic responses when applied at the border of primary motor and somatosensory cortices, above the corpus callosum, or at intrastriatal sites near the extracellular recording electrode (Fig. 2 A). The type of striatal synaptic plasticity was dependent on both the site of stimulation and the number of high-frequency pulses delivered to the tissue (Fig. 2B-D ). When applied within the striatum, the HFS-400 paradigm either generated LTP (n=6/12 brain slices) or had no long-term effect on subsequent synaptic responses evoked via this stimulation electrode (n=6/12; Fig. 2B, D ). Thus, LTP was seen in 50% of brain slices after HFS-400 stimulation within the striatum (Fig. 2D ). In contrast, when HFS-400 stimulation was applied to the cortex above the corpus callosum, long-term depression (LTD) was observed in the majority (n=12/17) of striatal brain slices (Fig. 2B, D ). No change (i.e., <10%) was observed in the population spike responses in the remaining five brain slices in which HFS-400 was used.


Figure 2
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  		Figure 2. The type of striatal synaptic plasticity is dependent on the site of electrical stimulation and the number of high-frequency pulses. A) Schematic diagram of a coronal brain slice containing the cerebral cortex and striatum, showing the locations of electrical stimulation and electrophysiological recording for the experiments described below. Although both cortical and intrastriatal stimulation sites are shown schematically, synaptic plasticity in each brain slice was evaluated using only a single stimulation electrode. B) Mean time courses demonstrating the effect of high-frequency 400 pulse stimulation (HFS-400) delivered within the striatum (Str HFS, open circles) near the site of population spike recording or delivered at the border of primary motor and somatosensory cortex (Cx HFS, filled circles). Representative averaged population spike traces (n=5–10 individual sweeps) collected during the baseline period (solid lines) and 60 min after HFS-400 stimulation (dashed lines) demonstrate both LTP (top) and LTD (bottom). C) Mean time courses demonstrating the effect of high-frequency 3000 pulse stimulation (HFS-3k) delivered within the striatum (Str HFS, open circles) or at the border of primary motor and somatosensory cortex (Cx HFS, filled circles). Representative averaged population spike traces collected during baseline (solid lines) and 60 min after HFS-3k stimulation (dashed lines) demonstrating both LTP (top) and LTD (bottom) are shown. Note that in panels B, C, the number of brain slices demonstrating plasticity is shown relative to the number of slices in which recordings were made. The remaining brain slices demonstrated <10% change from baseline 60 min after HFS and were excluded from the mean time courses. Scale bar in panel C applies to all wave forms. D) Cumulative frequency histogram demonstrating the actual percentage change from baseline for all striatal brain slices tested with the HFS-400 and HFS-3k paradigms applied to both the striatal and cortical sites. Bin size was set at 5% change from baseline, and the total number of slices plotted under each condition is as described in panels B, C, above. Note that points to the right of zero change indicate the proportion of slices exhibiting LTP 60 min after HFS; points to the left of zero demonstrate LTD. The most reliable form of synaptic plasticity was LTP observed after HFS-3k stimulation of the cortical tissue (Cx HFS-3k) as shown.

The HFS-3k stimulation applied to the striatum resulted in a modest but statistically significant degree of LTD in 10/18 brain slices (P<0.05, ANOVA, Fig. 2C ), whereas this same stimulation applied to the cortex resulted in robust striatal LTP in nearly all brain slices (14/15, Fig. 2C, D ). Using the HFS-3k stimulation paradigm, we assessed whether LTP, initiated through cortical stimulation, required the activation of NMDA receptors. Application of the selective NMDA receptor antagonist APV (40 µM) completely blocked LTP (Fig. 1C ). These results indicated that the form of synaptic plasticity was dependent on both the location of HFS and the number of high-frequency pulses applied to the brain slices (Fig. 2D ). In addition, HFS-3k-induced LTP required activation of the NMDA receptors.

Independence of cortical and intrastriatal pathways
As described above, LTP evoked by HFS-3k stimulation of the cortex was the most reliable and robust form of synaptic plasticity observed under our recording conditions. Therefore, in an attempt to confirm that this form of plasticity resulted from the activation of corticostriatal afferents and not through passive current spread to synaptic terminals located in the striatum per se, we determined the extent of independence of the afferent glutamatergic pathways activated by intrastriatal or cortical stimulation. Striatal population spikes recorded from the same population of striatal neurons were alternately evoked from a stimulator placed in the striatum near the site of the recording or from a stimulator placed in the deep cortical layers (Fig. 3 A). HFS-3k stimulation was then applied through the cortical stimulator, and population spikes evoked via the cortical and the striatal stimulators were monitored for changes in synaptic plasticity. As shown in Fig. 3 , HFS-3k stimulation to the cortex resulted in robust LTP of single population spikes elicited through this stimulation site. However, no long-term changes in the population spikes evoked via intrastriatal stimulation were observed (Fig. 3B, C ). Similarly, in separate experiments, intrastriatal HFS-3k stimulation did not elicit LTP of the cortically evoked response (not shown). These data suggested that, with regard to the initiation of LTP, cortical and intrastriatal stimulation activated largely independent sets of glutamatergic axons targeting the same population of MSNs and the cortical stimulation site activated primarily corticostriatal axons.


Figure 3
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  		Figure 3. Glutamatergic inputs activated by cortical and striatal stimulation are independent with regard to LTP. A) Diagram of the recording and stimulation setup. Striatal population spikes were alternately evoked via an electrode placed in the cortex or within the striatum. After stable baseline responses were recorded using each stimulator, HFS-3k stimulation was applied only to the cortex. Population spikes elicited through both stimulation sites were monitored for 60 min after cortical HFS-3k stimulation. B) Time course of a single experiment using the paradigm described in panel A. Note that LTP was observed only in the pathway where HFS-3k stimulation was applied (cortex). Superimposed averaged wave forms in panel B demonstrate baseline population spikes (solid lines), which averaged 60 min after HFS-3k stimulation of the cortex (dashed lines) for each stimulation site in this single experiment. The reduction in the nonsynaptic N1 component of the population spike after HFS-3k likely results from the decrease in synaptic N2 latency and an increase in its amplitude. This interaction is observed when N1 and N2 overlap in time (60) . C) Averaged responses (n=6) for all brain slices tested as described in panel A. Note the presence of LTP only in the cortical pathway.

Effects of MPTP on striatal dopamine electrochemistry
Four to 14 days after MPTP (80 mg/kg) or saline injections, mice were killed and electrophysiological and electrochemical recordings were performed in brain slices containing the striatum. Single-pulse electrical stimulation of striatal slices obtained from saline-injected control animals elicited the release of an endogenous molecule whose oxidation/reduction properties were consistent with the detection of dopamine (Fig. 4 A). Furthermore, the amplitude of this signal was increased ~5-fold and its rate of decay ({tau}) was increased ~3-fold by the dopamine transporter (DAT) inhibitor, nomifensine (Fig. 4A-C , 1 µM), and by cocaine (5 µM; Fig. 4C ). In addition, there was no correlation between the amplitude of the voltammetric current and the decay {tau}, indicating that the decay of the dopamine signal was not influenced by the amount of dopamine released by the electrical stimulus and therefore was a reliable measure of dopamine clearance via DAT uptake (Fig. 4D ; see ref. 33 ). Together, these data suggest that the predominant molecule detected by our carbon fiber electrodes after single-pulse electrical stimulation of the striatum was dopamine and that the {tau} for the decay of the dopamine signal could be used to assess dopamine presynaptic terminal function in striatal slices.


Figure 4
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  		Figure 4. Measurement of electrically evoked dopamine release and uptake in striatal brain slices. A) Background-subtracted voltammogram demonstrating the signal recorded from a carbon fiber electrode placed in the dorsolateral striatum immediately after single-pulse electrical stimulation in the striatum. The recorded voltammetric signal is consistent with the oxidation and reduction of dopamine and the DAT inhibitor, nomifensine (1 µM in each experiment), causes a large increase in the voltammetric signal (dashed line). B) Effect of nomifensine on the dopamine signal. Note the large increase in the amplitude of the dopamine signal, and the large increase in the time constant for the decay of the signal ({tau}). The dashed line represents the fitted exponential decay of the signal (Material and Methods). In this and subsequent figures, the dopamine signals represent single responses to electrical stimulation. C) Mean effects of the dopamine uptake inhibitors nomifensine and cocaine (5 µM) on the {tau} of signals evoked with single-pulse electrical stimulation at several sites within several striatal brain slices. Note the large increase in {tau} caused by nomifensine and cocaine, which is consistent with their ability to inhibit the DAT. D) Independence of {tau} and peak amplitude of the dopamine signal. The lack of correlation in these measures suggests that {tau} is a reliable measure of DAT function, even at low levels of stimulus-evoked dopamine.

The concentration of stimulus-evoked dopamine was enhanced by increasing the intensity of constant-current electrical stimulation of the striatal tissue, and this dopamine concentration reached an asymptotic level at ~300 µA (Fig. 5 C, control). Voltammetric recordings from striatal brain slices obtained from MPTP-treated mice (n=6) demonstrated a large reduction in the amplitude of the dopamine signal across the full range of electrical stimulation intensities (Fig. 5B, C ); the time constant for the decay of this signal was significantly increased in these brain slices (Fig. 5D ). These data indicated that MPTP caused a large reduction in the functional pool of dopamine from axon terminals and greatly reduced the degree of dopamine uptake via the DAT.


Figure 5
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  		Figure 5. MPTP treatment reduces electrically evoked striatal dopamine content. A) Left: pseudocolor plot showing a typical evoked dopamine current measured using FSCV. The x axis represents time, the y axis represents the applied potential, and the current in green. Dashed white lines represent the potential and time at which the current vs. time plot and background-subtracted cyclic voltammogram (right) were obtained. B) Dopamine signals obtained in C57BL/6 mouse striatal brain slices 7 days after either control saline injections or MPTP. At far right, the dopamine signal obtained from the MPTP-treated animal has been scaled to match the amplitude of the control signal to illustrate the difference in the time constants ({tau}) obtained by fitting single exponential decays to each response (gray, control=260 ms, MPTP=496 ms). Vertical scale for control is 0.5 µM, MPTP is 0.25 µM. C) Dopamine release is dependent on the strength of electrical stimulation and is reduced at all stimulus intensities in slices from MPTP-treated mice (n=6). D) MPTP treatment significantly increases the {tau} for decay of the voltammetric dopamine signal (***P<0.0001, t test).

MPTP blocks striatal synaptic plasticity initiated via cortical or intrastriatal stimulation
As described above, striatal slices obtained from control animals demonstrated significant levels of LTP or LTD, depending on the type of high-frequency stimulation paradigm (HFS-3k, or HFS-400) and the site of this stimulation (cortex or striatum; Fig. 2 ). We examined each of these forms of synaptic plasticity after MPTP treatment. Regardless of the stimulation site (cortex or striatum) or HFS protocol (HFS-400 or HFS-3k), neither LTP nor LTD were observed in striatal brain slices obtained from MPTP-treated mice (Fig. 6 ). Thus, all forms of in vitro striatal synaptic plasticity examined in this study were sensitive to the neurobiological changes initiated by in vivo treatment with MPTP. We reasoned that if dopamine itself were necessary to observe striatal synaptic plasticity during HFS, then the application of dopamine or the selective activation of dopamine receptors should restore this physiological end point. Since LTP elicited via HFS-3k stimulation of the cortex was the most reliable and robust form of synaptic plasticity observed in this study (Fig. 2 , Fig. 6A ) and, like the other forms of plasticity, it was sensitive to MPTP treatment, this paradigm was used for the remainder of the electrophysiological studies.


Figure 6
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  		Figure 6. MPTP treatment eliminates striatal synaptic plasticity. A) Mean time course of corticostriatal LTP observed after HFS-3k stimulation of the cortex in striatal brain slices obtained from saline- or MPTP-injected mice. Note the complete absence of LTP in brain slices from the MPTP-injected animals. Wave forms in 1 and 2 are representative population spike averages obtained during baseline, prior to HFS (solid line), or 60 min after HFS (dashed line) in striatal slices obtained from saline-injected control mice (1) or from MPTP-treated mice (2). This numerical convention is the same for all wave forms under each experimental paradigm described in panels B–D. B) Mean time course of LTD observed after HFS-3k stimulation of the striatum in brain slices obtained from saline- or MPTP-injected mice. Note the block of LTD in the brain slices from the MPTP-injected animals. C) Mean time course of LTD observed after HFS-400 stimulation of the cortex in brain slices obtained from saline- or MPTP-injected mice. Note the absence of LTD in the brain slices from MPTP-treated animals. D) Mean time course of the LTP after HFS-400 stimulation applied intrastriatally near the site of population spike recording. Note that LTP is blocked by the MPTP treatment.

We found that application of dopamine (10 µM) to the brain slices obtained from the MPTP-treated animals beginning 10 min before and throughout the HFS-3k stimulation of the cortex significantly restored LTP (P<0.001 compared with no change, ANOVA, and Tukey’s post hoc analysis; Fig. 7 A). To determine whether dopamine D1 receptors were involved in this restoration of LTP, the selective D1 agonist SKF 38393 (10 µM) was applied for 10 min prior to HFS-3k stimulation of the cortex in brain slices obtained from MPTP-treated mice. Like dopamine, SKF 38393 restored LTP in the brain slices obtained from MPTP-treated mice (P<0.001; Fig. 7B ), suggesting that D1 receptor activation by dopamine was necessary to observe HFS-3k-induced LTP. If D1 receptor activation was necessary to observe initiation or expression of corticostriatal LTP caused by HFS-3k stimulation, then blockade of D1 receptors with SCH 23390 during this stimulation should also disrupt this plasticity in brain slices from drug-naive mice. Although SCH 23390 alone (10 µM) had no effect on striatal population spikes (Fig. 7C ), it completely blocked HFS-3k-induced LTP in the striatum (Fig. 7C ). Finally, to determine whether dopamine D2 receptors influenced this form of synaptic plasticity, we examined the effect of the selective D2 antagonist eticlopride (10 µM) in naive striatal brain slices. Significant LTP (P<0.001) was observed in these slices in the presence of eticlopride, suggesting that dopamine D2 receptors are not involved in initiating or maintaining this form of LTP (Fig. 7D ). Collectively, these data suggest that dopamine was necessary to observe LTP in the corticostriatal pathway and that the effects of dopamine were mediated primarily by the D1 receptor. This suggests that the MPTP neurotoxin blocked striatal plasticity through a reduction in nigrostriatal dopamine, and not through other nondopaminergic mechanisms.


Figure 7
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  		Figure 7. Dopamine and D1 receptors regulate corticostriatal LTP in vitro. A) Mean time course of the effects of dopamine on LTP in striatal brain slices from MPTP-treated mice. Dopamine application (horizontal bar) was started 10 min prior to HFS-3k stimulation of the cerebral cortex and restored LTP. Averaged population spikes are also shown during baseline conditions and 60 min after HFS-3k stimulation here and in panels B–D. B) Mean time course of the effect of the D1 agonist SKF 38393 on corticostriatal LTP in brain slices obtained from MPTP-treated mice. SKF 38393 application 10 min prior to HFS-3k stimulation of the cortex restored LTP in these brain slices. C) Mean time course of the effect of the D1 antagonist SCH 23390 on HFS-3k-induced LTP in striatal brain slices from naive mice. Note that the D1 antagonist blocked corticostriatal LTP. D) Mean time course of the effect of the D2 receptor antagonist eticlopride on corticostriatal LTP in brain slices from naive mice. Note the absence of a significant effect of the D2 antagonist on LTP. **P < 0.001, ANOVA, and Tukey’s post hoc test vs. no change in baseline 50–60 min after HFS-3k. There was no statistically significant difference between the amount of LTP observed in panels A, B (P>0.05).

The preceding experiments point to a critical role of dopamine in initiating corticostriatal LTP. However, a critical question that remains to be answered is whether LTP requires a transient increase in dopamine released during high-frequency stimulation (phasic) or whether tonic, baseline levels of dopamine are sufficient. To address this, we examined whether dopamine levels were altered in the dorsal striatum during electrical stimulation of the cortical or intrastriatal sites described in the electrophysiological experiments. We found that single-pulse and high-frequency (100 pulses at 100 Hz) stimulation of the cortex at intensities sufficient to elicit striatal population spikes did not elicit a detectable increase in the extracellular concentration of dopamine in the striatum (Fig. 8 A2, B2). In contrast, intrastriatal stimulation at single pulses or at high frequency elicited substantial increases in dopamine concentration (Fig. 8 A1, B1). However, there was no significant difference either in amplitude or time course of the dopamine signals elicited by single-pulse or high-frequency stimulation at the intrastriatal site (Fig. 8C ). The absence of cortically evoked striatal dopamine in vitro is consistent with an earlier study (37) and suggests that tonic, rather than stimulus-evoked phasic dopamine, is critical for the initiation of LTP in the dorsal striatum.


Figure 8
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  		Figure 8. Comparison of intrastriatal and cortical stimulation on striatal dopamine release in brain slices. A) Schematic diagram of the placement of the stimulating electrode in the dorsal striatum (A1) or in the cortex above the corpus callosum (A2) and recording using a carbon fiber electrode placed in the dorsal striatum. Population spikes recorded in these sites verified physiological activation of glutamatergic afferents as well (not shown). B) Voltammetric dopamine signals resulting from single pulses (4 ms, 300 µA) or high-frequency stimulation (HFS; 100 Hz, 1 s duration, 0.2 ms pulses, 300 µA) applied either intrastriatally (B1) or in the cortex (B2). HFS did not produce a detectable dopamine signal when applied to the cortical site (B2), whereas both single-pulse and HFS elicited large dopamine releases during intra-striatal stimulation (B1). C1) Summary of the mean amplitude of dopamine signals obtained under each of the conditions tested (n=8 slices, 4 mice). No significant differences in signal amplitude were observed (P=0.57, paired t test) between striatal single-pulse and HFS protocols. The limit of detection (in µM, determined as 3-fold the background recording noise) is shown with a dashed line. Note that the signals observed using HFS in cortical tissue were below the mean detection limit (dashed line), indicating no additional dopamine overflow caused by stimulation. C2) Mean decay time constants ({tau}) for the striatal dopamine signals using single-pulse and HFS protocols. No significant differences were observed between these stimulus protocols (P=0.2391, paired t test).

MPTP treatment shifts the input-output relationship of cortico-striatal glutamatergic synaptic transmission
To determine whether the impairment in corticostriatal LTP seen in brain slices from the MPTP-treated animals resulted from altered excitability of this projection, we examined the relationship between stimulation intensity in the cortex or striatum and the amplitude of population spikes in slices obtained from control and MPTP-treated mice. Input-output relationships are sensitive measures of a change in the efficacy of synaptic transmission (38) . In striatal slices taken from MPTP-treated mice, we found a significant (P<0.0001, ANOVA) increase in the amplitudes of population spikes evoked at a given stimulus intensity when the stimulus was applied to the cerebral cortex (Fig. 9 A). However, there was no change in the relationship between stimulus intensity and population spike amplitude for responses elicited by intrastriatal stimulation in brain slices obtained from MPTP-treated mice (P>0.05; Fig. 9B ). This observation is consistent with studies demonstrating increased MSN activity in response to activation of corticostriatal afferents after 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal pathway in vivo (39 , 40) .


Figure 9
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  		Figure 9. The input-output relationship is altered for cortical stimulation sites in brain slices from MPTP-treated mice. Mean input-output curves were generated by plotting population spike amplitudes across a range of stimulation intensities. A) Input-output relationships for population spikes evoked via cortical stimulation in brain slices from saline- or MPTP-treated mice. Note that after MPTP treatment, a larger population spike was evoked at a given stimulus intensity, suggesting an increase in excitability of the corticostriatal pathway (P<0.0001, ANOVA). B) Input-output relationship for intrastriatally evoked population spikes in brain slices obtained from saline- and MPTP-treated mice. There was no difference between brain slices from saline or MPTP-treated mice.

AAV-mediated expression of GDNF prevents the electrochemical and electrophysiological changes produced by MPTP
Intracranial bolus infusions GDNF have been reported to provide a significant degree of protection to midbrain dopamine neurons that are impaired by neurotoxins, such as 6-OHDA or MPTP, which are used in animals models of Parkinson’s disease (15 , 16 , 41 , 42) . However, it is not known whether GDNF affects the synaptic changes in the striatal circuitry described above. Therefore, we injected either AAV-GDNF or AAV-GFP as a control unilaterally into the dorsal striatum 7 days prior to MPTP or saline injections. We then assessed dopaminergic and synaptic function in vitro 4–14 days after exposure to the neurotoxin or saline. Intracranial injection of the AAV-GFP construct resulted in extensive expression of GFP in neuronal and non-neuronal elements (Fig. 10 A). In striatal brain slices obtained from animals exposed to MPTP that received intracranial injections of AAV-GFP (n=8), there was a substantial depletion of extracellular dopamine levels that did not differ from slices obtained from mice injected with only MPTP (P<0.0001, ANOVA; Fig. 10B, C ). In addition, {tau} for the decay of the dopamine signal was increased in the MPTP-treated animals that received AAV-GFP to the same degree as that measured in slices from animals receiving MPTP alone (Fig. 10D ). In contrast, brain slices obtained from animals treated with AAV-GDNF prior to MPTP (n=12) demonstrated significantly higher levels of stimulus-evoked dopamine release compared with striatal slices obtained from AAV-GFP- and MPTP-injected animals (P<0.001, repeated measures ANOVA; Fig. 10C ). In addition, brain slices obtained from these mice also demonstrated significantly faster dopamine clearance rates than those that had not received AAV-GDNF (P<0.01, ANOVA; Fig. 10D ). Together, these data indicate that AAV-GDNF treatment greatly reduced the MPTP-induced loss of striatal dopamine.


Figure 10
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  		Figure 10. Expression of GDNF via AAV protects against the reduction in dopaminergic function in the striatum after MPTP treatment. A) Fluorescence photomicrograph demonstrating the extent of GFP expression using the AAV-GFP vector in a representative brain slice. The location of the fluorescence signal and the approximate location of a cannula track are depicted by the box and vertical line in the schematic diagram of a coronal brain slice in the upper left corner. Numerous neuronal (arrowheads) and non-neuronal cellular elements were found to express GFP after intracranial injection of the construct. B) Voltammetric traces of single-pulse, electrically stimulated dopamine release in the dorsolateral striatum in brain slices obtained from mice treated with either AAV-GFP or AAV-GDNF prior to MPTP injections. C) Stimulus-dependent dopamine release in striatal slices from each experimental group. Treatment of mice with AAV-GDNF protected against the loss of dopamine at a range of electrical stimulus intensities. Note that the group of mice treated with AAV-GFP demonstrated reduced dopamine concentrations that did not differ significantly from brain slices taken from mice treated with MPTP alone. D) Group effects on striatal dopamine signal decay rates ({tau}) under control conditions (saline injected) after treatment with MPTP alone (no AAV) or after AAV-GFP or AAV-GDNF injections in vivo. Note that MPTP treatment (no AAV) caused a significant lengthening of mean {tau} values (P<0.001 vs. control, ANOVA, Tukey post hoc test); this measure was unaffected by AAV-GFP pretreatment (P>0.05 vs. no AAV), and significantly shortened by AAV-GDNF pretreatment (P<0.01 vs. no AAV). In addition, the mean {tau} value obtained in brain slices from MPTP-treated animals that received AAV-GDNF was not significantly different from control animals (control=dashed line; P>0.05). This indicates that DAT function was compromised in the brain slices from MPTP-treated mice and that this function was protected by AAV-GDNF treatment.

As described above, striatal slices obtained from MPTP-treated mice did not demonstrate corticostriatal LTP after HFS-3k stimulation of the cortical tissue (Fig. 6B ). Similarly, LTP was not observed in brain slices obtained from mice treated with MPTP, preceded by intrastriatal control injections with AAV-GFP (P>0.05, ANOVA; Fig. 11 A2, B). In contrast, brain slices obtained from mice treated with AAV-GDNF prior to MPTP administration demonstrated a robust corticostriatal LTP (P<0.0001, ANOVA; Fig 11A1, B ) which did not differ from that observed in naive control brain slices (P>0.05, ANOVA). Together, these data suggest that the AAV-mediated expression of GDNF in the striatum protected against the loss of dopamine as well as changes in the striatal synaptic circuitry responsible for the impairment of LTP after MPTP exposure.


Figure 11
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  		Figure 11. Treatment with AAV-GDNF protects against MPTP-induced loss of LTP in mice. A1) Averaged wave forms demonstrating corticostriatal LTP recorded 60 min after HFS-3k stimulation of the cortex in a single striatal brain slice obtained from a mouse given an intracranial injection of AAV-GDNF prior to MPTP treatment. A2) Averaged wave forms from a striatal brain slice obtained from a mouse intracranially injected with AAV-GFP before MPTP treatment. B) Mean group effects of intracranial injections with AAV-GDNF (open circles) or AAV-GFP (solid triangles) prior to MPTP treatment on corticostriatal LTP induced with HFS-3k. Control corticostriatal LTP (solid circles) is shown for comparison. Note that AAV-GDNF significantly protected against the loss of corticostriatal LTP seen after MPTP treatment.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The behavioral and neurochemical consequences of nigrostriatal dopamine neuron degeneration in humans and animals are relatively well understood (1) . Less well understood, however, are the consequences of the loss of dopaminergic function on the functional synaptic properties of the striatal circuitry. The present study demonstrates that damage to the nigrostriatal pathway by the neurotoxin MPTP reduced dopamine release in the striatum and eliminated the capacity of the striatal circuitry to demonstrate long-term changes in synaptic strength elicited through high-frequency activation of glutamatergic inputs. In addition, the expression of GDNF through the AAV vector protected against this decrease in the functional pool of dopamine and prevented the loss of corticostriatal LTP in vitro. These results indicate that the AAV vector is effective for delivering GDNF to critical sites in the brain and that this strategy can protect against physiological changes in striatal circuitry resulting from the loss of dopamine, which may also be involved in the symptomology of PD.

The present data support the idea that dopamine was necessary to activate D1 receptors during high-frequency activation of corticostriatal afferents to observe LTP. This conclusion is derived from observations that exposure to dopamine or a D1 agonist during HFS-3k restored LTP in brain slices from MPTP-treated animals and that LTP was blocked by administration of a D1 antagonist in striatal slices from naive animals. In contrast, blockade of D2 receptors did not alter HFS-3k-induced LTP. The dependence of striatal LTP on D1 receptor activation is consistent with another study in which a different form of LTP was evoked in striatal slices maintained in Mg2+-deficient aCSF (14) . The D1 receptor can activate adenylyl cyclase, thereby increasing intracellular levels of cyclic AMP (43) . Since an increase in cyclic AMP via direct stimulation in cultured striatal cells (44) or via D1 receptors in brain slices can enhance postsynaptic sensitivity to glutamate (45) , it is possible that these mechanisms are involved in the ability of D1 stimulation to restore LTP (14) . In addition, this strengthens the idea that a primary deficit resulting from the loss of striatal dopamine is corticostriatal glutamatergic plasticity and that D1 receptors are involved in supporting this form of plasticity in the intact striatal circuit (3 , 14) .

Although LTD is often the most frequently reported type of plasticity reported in vitro, there is considerable heterogeneity in the form of striatal synaptic plasticity that can be initiated by high-frequency stimulation of the corticostriatal pathway when using varying lengths of 100 Hz stimulation (12 , 13) . A portion of this heterogeneity has been explained by the age of the animals used in these studies, as well as by the location of the intracellular or extracellular recordings of MSNs and the populations of activated axon terminals (3 , 12) . Our study suggests that the site and amount of HFS can also determine the type of synaptic plasticity. Thus, we observed both LTD and LTP after high-frequency activation of corticostriatal afferents to the dorsal striatum in brain slices. When the HFS-400 paradigm was used, LTD was more frequently observed with stimulation of the cortex whereas LTP or no change was observed when this same stimulation was applied within the striatum near the site of recording (Fig. 2B, D ). However, when 3000 pulses were applied at the same frequency (HFS-3k) at the same cortical site, LTP was observed in nearly 100% of the brain slices (Fig. 2C, D ). Striatal HFS-3k, on the other hand, resulted in a modest degree of LTD in the majority of brain slices. Since in vivo expression of striatal synaptic plasticity is likely to be dependent on activation of glutamatergic inputs arising in the cortex and since HFS-3k-LTP generated by cortical stimulation was the most robust and reliable form of plasticity, we chose to assess the effects of MPTP, and AAV-GDNF on this physiological parameter.

In vivo administration of MPTP greatly reduced the amount of dopamine released via single-pulse electrical stimulation of the striatal tissue in vitro. In addition, the time constants for the decay of the evoked dopamine signals were increased 5-fold in slices obtained from MPTP-treated animals, indicating that clearance of dopamine via the DAT was also diminished. This is consistent with the localization of the DAT to dopamine axon terminals in the striatum (46) and with the reduction of immunohistochemically identified DAT after MPTP treatment in mice (1 , 47) . The reduction in striatal dopamine was associated with a complete absence of all forms of synaptic plasticity described in this study. Thus, both LTP and LTD were absent in the striatal slices from MPTP-treated animals whether plasticity was elicited through HFS-3k or HFS-400 applied within the striatum or to the cortical tissue. This is consistent with more limited studies of the effects of 6-OHDA lesions on LTP and LTD (3 , 4) , and suggests that the presence of dopamine in the striatum is a critical factor in the capacity of this circuitry to undergo enduring synaptic changes. The present study extends these findings by demonstrating that striatal dopamine levels were not increased by electrical stimulation of the cortex, but were robustly increased by intrastriatal stimulation (Fig. 8 ; see also ref. 37 ). This, together with the demonstration of dependence on dopamine for corticostriatal LTP, suggests that it is a tonically available pool of dopamine, and not dopamine phasically elevated by HFS, that was involved in initiating this form of plasticity (48) .

Despite a clear pivotal role for dopamine in the establishment of striatal glutamatergic synaptic plasticity, it is not yet understood how the loss of dopamine through degeneration of the nigrostriatal dopaminergic projection results in this diminished capacity. This might occur through anatomical remodeling of the corticostriatal circuitry involving a loss of dendritic spines and changes in the density of excitatory asymmetric synapses on MSNs. This has been described for the brains of 6-OHDA-lesioned rodents (49 50 51) and in the postmortem striata of PD patients (52) . Since glutamate synapses are preferentially located on dendritic spines, the loss of these structures after MPTP may represent an explanation for the loss of synaptic plasticity in the present study. However, we also found that direct activation of dopamine receptors by acute dopamine or D1 agonists in striatal slices obtained from MPTP-treated mice could restore striatal LTP (see also ref. 14 ). Since these acute pharmacological actions would presumably be too rapid to alter spine density, our data suggest that coincident stimulation of dopamine receptors with high-frequency corticostriatal activity is sufficient to restore LTP and that the morphological changes in medium spiny dendrites can not entirely explain the deficits in striatal plasticity. Furthermore, input-output curves generated in brain slices from MPTP-treated mice showed a shift toward larger amplitude population spikes only for the cortical stimulation site. Thus, our in vitro electrophysiological data are consistent with microdialysis studies demonstrating an increase in glutamate levels after dopamine denervation of the striatum (50 , 51 , 53 , 54) and with in vivo electrophysiological studies demonstrating increased responsiveness of striatal MSNs to cortical stimulation after 6-OHDA lesions (39 , 40) . This suggests that rather than diminish corticostriatal glutamatergic innervation, dopamine depletion is associated with an increase in the strength of this pathway under baseline conditions. Other explanations for the blockade of synaptic plasticity after dopamine depletion of the striatum include changes in glutamate receptor density or subtype, which also occur in the 6-OHDA lesion model of PD (55 56 57 58) . Additional experiments are needed to determine the precise mechanism through which degeneration of the dopaminergic nigrostriatal pathway alters glutamate signaling so as to preclude striatal synaptic plasticity.

The expression of GDNF via the AAV vector prior to the MPTP injections significantly protected against the reduction in striatal dopamine levels, the decreased rate of dopamine clearance, and prevented the blockade of LTP. Thus, these data provide evidence that GDNF can prevent not only the neurochemical alterations that occur in this experimental model of PD but also the physiological synaptic changes that may underlie the shift in striatal function. This suggests that the expression of corticostriatal LTP may represent a means to assess PD models and interventions designed to remediate the neurodegeneration observed in these models.

Earlier studies demonstrated that GDNF expression via viral vectors in rodents and primates lesioned with 6-OHDA significantly protected against the loss of dopamine neuronal markers (tyrosine hydroxylase) or restored them, as well as motor behavior following these lesions (17 , 18 , 20 , 21 , 23) . In addition, there have been promising clinical trials with GDNF and related neurotrophic factors in treating PD. Although several means are available to deliver neurotrophic factors to critical sites in the brain, each poses risks and limitations to their use in humans. The primary concern for viral-mediated gene expression is the lack of regulation of this expression and the possibility of regionally nonspecific effects of the neurotrophic factor. However, the primary advantage of this approach is the prolonged, sustained release of neurotrophic factor at a clinically relevant concentration (19 , 59) . Although we did not directly compare different routes of GDNF administration, our study demonstrated that the protection against MPTP-induced nigrostriatal degeneration by AAV-GDNF was associated not only with the preservation of dopaminergic innervation, but also with protection against changes in striatal synaptic plasticity that likely are deleterious to normal striatal function. One might also predict that a similar loss of glutamatergic synaptic plasticity would occur in the striata of patients suffering from PD and that this impairment may contribute to the symptomology of the Parkinsonian syndrome in experimental animals and humans. Although further study is necessary to more firmly establish whether a causal relationship exists between these synaptic changes and PD, it appears that the present results and those of previous studies collectively demonstrate that GDNF can prevent the neurochemical, behavioral, structural, and physiological changes that occur in the striatal circuitry resulting from the loss of nigral dopamine input to the striatum.


   ACKNOWLEDGMENTS
 
We thank Mr. Doug Howard for technical assistance with the AAV vectors. This work was funded by the U.S. Department of Health and Human Services, The National Institutes of Health, and the National Institute on Drug Abuse Intramural Research Program.

Received for publication April 13, 2007. Accepted for publication July 5, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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