Norepinephrine transporter inhibition with desipramine ...

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Norepinephrine transporter inhibition with desipramine exacerbates L-DOPA-induced dyskinesia: role for synaptic dopamine regulation in denervated nigrostriatal terminals Tanya Chotibut, Victoria Fields, Michael F. Salvatore Department of Pharmacology, Toxicology, & Neuroscience Louisiana State University Health Sciences Center 1501 Kings Highway Shreveport, Louisiana 71130

This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302

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Running title: Norepinephrine transporter and L-DOPA dyskinesia Corresponding Author: Michael F. Salvatore, Ph.D. Department of Pharmacology, Toxicology, & Neuroscience Louisiana State University Health Sciences Center 1501 Kings Highway Shreveport, Louisiana 71130 Email: [email protected] Document statistics: number of text pages: 31 figures: 8 references: 81 words in Abstract: 245 Introduction: 801 Discussion: 1688 Abbreviations used: AIMS: abnormal involuntary movements DA: dopamine DAT: dopamine transporter DMI: desipramine ERK: extracellular signal-regulated protein kinase L-DOPA: L-dihydroxyphenylalanine LID: L-DOPA-induced dyskinesia NE: norepinephrine NET: norepinephrine transporter PD: Parkinson’s disease


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Abstract

Pharmacological dopamine (DA) replacement with Levodopa (L-DOPA) is the gold standard

treatment for Parkinson’s disease (PD). However, long term L-DOPA treatment is complicated

by eventual debilitating abnormal involuntary movements termed L-DOPA induced dyskinesia

(LID), a clinically significant obstacle for the majority of patients who rely on L-DOPA to alleviate

PD-related motor symptoms. The manifestation of LID may in part be driven by excessive

extracellular DA derived from L-DOPA, but potential involvement of DA reuptake in LID severity

or expression is unknown. We recently reported that in 6-OHDA-lesioned striatum,

norepinephrine transporter (NET) expression increases and may play a significant role in DA

transport. Furthermore, L-DOPA preferentially inhibits DA uptake in lesioned striatum.

Therefore we hypothesized that desipramine (DMI), a NET antagonist, could affect the severity

of LID in an established LID model. While DMI alone elicited no dyskinetic effects in lesioned

rats, DMI + L-DOPA treated rats gradually expressed more severe dyskinesia compared to L-

DOPA alone over time. At the conclusion of the study, we observed reduced NET expression

and norepinephrine-mediated inhibition of DA uptake in the DMI + L-DOPA group compared to

L-DOPA alone group in lesioned striatum. LID severity positively correlated with striatal ERK

phosphorylation among the three treatment groups, with increased ppERK1/2 in DMI + L-DOPA

group compared to the L-DOPA- and DMI-alone groups. Taken together, these results indicate

that the combination of chronic L-DOPA and NET-mediated DA reuptake in lesioned

nigrostriatal terminals may have a role in LID severity in experimental Parkinsonism.


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INTRODUCTION Parkinson’s Disease (PD) is the most common neurodegenerative movement disorder

and its incidence is likely to only increase with the impending growth of the aging population

(Siderowf and Stern, 2003). PD is primarily characterized by the loss of dopamine (DA)

neurons in the substantia nigra pars compacta. Therapeutically, the primary loss in nigral

dopamine necessitates its replacement through the exogenous administration of L-DOPA, a

dopamine precursor or with DA agonists (Hornykiewicz and Kish, 1987; Steiger and Quinn,

1995). L-DOPA in particular, has remained the drug of choice for treating PD for nearly half a

century (Calne and Sandler, 1970). Despite the ability of L-DOPA to significantly improve motor

function, it is not without considerable side effects that severely limit its use long-term. L-DOPA-

induced dyskinesia (LID) is a debilitating movement disorder brought on by chronic L-DOPA

use. Approximately 90% of patients within the first 10 years of treatment develop LID (Ahlskog

& Meunter 2001; Marsden, 1994; Mones et al., 1971; Olanow and Koller, 1998). Not only is the

onset of dyskinesia a significant setback for the patient, its presence is often permanent,

occurring with every subsequent exposure to L-DOPA, which limits the clinical efficacy of what

has long been considered our gold standard in PD treatment.

Evidence suggests chronic L-DOPA leads to major adaptive molecular changes

occurring within the basal ganglia that may underlie LID pathophysiology (for review see Cenci

and Konradi, 2010). Loss of nigrostriatal dopaminergic neurons not only impairs pre-synaptic

control of DA regulation, but also leads to large variances in extracellular levels of DA that

parallel L-DOPA dosing regimens (Cenci and Lundblad, 2006). It is these supra-physiological

fluctuations in extracellular DA content that are thought to underlie the induction of L-DOPA

induced dyskinesia (Chase, 1998). LID has been associated with plastic changes in post-

synaptic neuronal targets within the striatum, including abnormal trafficking of the DA D1

receptor (Berthet et al., 2009; Guigoni et al., 2007). This has also been shown clinically as well:


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positron emission tomography (PET) imaging studies reveal an association between peak-dose

dyskinesia with abnormally high levels of synaptic DA in the caudate-putamen of L-DOPA

induced dyskinetic patients (de la Fuente-Fernandez et al., 2004; Pavese et al., 2006). In line

with these studies, pre-clinical data show that dyskinetic rats exhibit higher levels of extracellular

DA after L-DOPA administration than those seen in non-dyskinetic animals (Meissner et al.,

2006; Lindgren et al., 2010). These data raise the possibility that not only does striatal

extracellular DA play a pivotal role in the onset of dyskinesia, but could be the triggering

element of the post-synaptic alterations identified in both clinical and pre-clinical models.

However, despite considerable efforts directed at delineating the role L-DOPA plays in altering

dopaminergic striatal signaling, the exact mechanism of how this occurs remains conflicting and

inconclusive (Cenci & Konradi 2010).

In PD, the loss of the dopamine transporter (DAT) likely impairs its ability to maintain DA

bioavailability, yet most studies indicate motor symptoms are seen only when ~70% of striatal

DAT is lost (Bernheimer et al., 1973; Bezard et al., 2001), indicating an alternate mechanism

through which DA may still be effective to produce normal locomotion. Indeed, many studies

have concluded that serotonergic terminals may transport L-DOPA or DA, and subsequently

release DA in order to maintain DA signaling, albeit in a dysregulated fashion (Arai et al., 1995;

Miller and Abercrombie et al., 1999; Tanaka et al., 1999; Kannari et al. 2001, Carta et al., 2007).

However, it is a comparatively neglected observation that in sparsely dopaminergic innervated

regions, such as the frontal cortex, the norepinephrine transporter (NET) can also transport DA

(Moron et al., 2002), and NE uptake inhibitors which can result in increased extracellular DA

levels within the prefrontal cortex (Carboni et al., 1990; Di Chiara et al., 1992; Tanda et al.,

1997; Gresch et al., 1995; Masana et al., 2012; Yamamoto and Novotney, 1998; Wayment et

al., 2001). In line with these observations, we have recently reported that DA transport still

occurs in the 6-OHDA-lesioned striatum, despite major loss of DAT, and that a potential role for


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NET in DA uptake is evident (Chotibut et al., 2012). In these experiments, L-DOPA

preferentially inhibits DA reuptake when striatal DAT loss exceeds the level necessary for PD

symptom appearance, and preferentially inhibits norepinephrine over DA uptake in striatal

tissue. Therefore, based upon these observations, chronic L-DOPA could exacerbate the L-

DOPA induced-dysregulation of synaptic DA by its influence on a NET-dominated regulation of

DA uptake that evolves with the gradual loss of DA terminals.

In an effort to further explore this possibility, we examined how the blockade of NET in a

chronic desipramine (DMI) paradigm would affect dyskinesia expression, NET expression, DA

uptake, and an established post-translational event in striatum associated with dyskinesia,

extracellular signal-regulated protein kinase phosphorylation (ppERK), in an established 6-

OHDA LID rodent model.

MATERIALS and METHODS:

Animals

Male Sprague Dawley rats purchased from Charles-River were used in all experiments.

A total of 21 test subjects were used at the start of the study, were 4-8 months of age, and were

housed 1 per cage and under controlled lighting conditions (12:12 light:dark cycle) with food and

water available ad libitum. All animals were used in compliance with federal and the institutional

Animal Care and Use Committee guidelines at LSU Health Sciences Center-Shreveport. All

behavioral testing was performed between 10:00 and 16:00 h. Behavioral data obtained from

test subjects that did not have >70% TH loss, as discovered after completion of LID

assessments and tissue analysis, was excluded from the results. We note that protein recovery

was limited in some rats due to the fact we analyzed DA uptake, TH, NET, tERK, and ppERK all

from the same striatal tissues and as such, the treatment group numbers are not of equal size.


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In all experiments, rats were rendered briefly unconscious with isoflourane and

immediately decapitated on day 40 of the experiment (1 day post last behavioral test and

treatment (L-DOPA + DMI, L-DOPA or vehicle) for dissection of striatal tissues.

6-OHDA Lesions

Rats were anesthetized with 40 mg/kg Nembutal intraperitoneal (i.p.) (pentobarbital

Lundbeck Inc, Deerfield, IL) with supplement of 9.0, 0.6, and 0.3 mg/kg ketamine, xylazine, and

acepromazine, respectively. Each animal then underwent survival surgery to deliver the

neurotoxin 6-OHDA unilaterally to the medial forebrain bundle while immobilized in a stereotaxic

frame (coordinates ML +1.5, AP -3.8, DV -8.0 relative to Bregma). All stereotaxic coordinates

are cited according to the stereotaxic atlas of Paxinos and Watson rat brain atlas, 4th ed.

(Academic Press, 1998). A total of 16 µg of 6-OHDA in a total of 4 µl in 0.02% ascorbic acid

(concentration of 4 mg/ml) was infused unilaterally at a rate of 1 µl/minute. The contralateral

medial forebrain bundle was infused with vehicle (.02% ascorbic acid) and infused at the same

rate and coordinates. The syringe was left in place for 10 min before removal to allow for

maximal diffusion of drug and to avoid further mechanical damage to the tissue. Body

temperature was maintained at 37º during surgery using a temperature monitor with probe and

heating pad (FHC, Bowdoingham, ME). Animals were kept warm after surgery and monitored

closely after anesthesia. In our hands (Chotibut et al., 2012, Fig 7)), we have previously shown

that after 6-OHDA infusion, there was no significant difference in lesioned striatal tissue NE

content compared to contralateral striatum and as such, have based these experiments from

these observations. As such, we did not pre-treat these rats with DMI.

Amphetamine Testing

The extent of the lesion was evaluated 7 days post-surgery based on the net ipsilateral

rotations measured over a 60 minute period following an injection of 2.5 mg/kg D-amphetamine


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i.p (in 0.9% saline) (Ungerstedt and Arbuthnott, 1970). 100 net full turns on the ipsilateral side

to the lesion was necessary to be included in the study. In our hands, we have established this

time frame as enough to consistently catch >60% of TH loss (Chotibut et al., 2012; 2014;

Salvatore, et al., 2014). Ultimately however, post-mortem verification of the lesion severity was

determined by assessment of tyrosine hydroxylase protein as previously reported (Chotibut et

al., 2012; 2014). And thus, any further exclusions were due to rats that did not also have >70%

TH loss after western blot analysis at the end of the study. The behavioral AIMS data reflects

test subjects with >70% TH protein loss.

Desipramine and L-DOPA Administration

9 days post lesion, rats were randomly divided into 3 groups. 2 groups received a

treatment of DMI (Tocris, cat #3067) (12mg/kg) and 1 group received Vehicle (.9% saline

12mg/kg) i.p for 30 consecutive days. Then, at 19 days post lesion, an additional treatment of

either L-DOPA (12mg/kg) and Benserazide-hydrochloride (15mg/kg) or Vehicle (.9% saline) was

given once daily for 20 consecutive days (Fig 1). In summary, 3 treatment groups were created:

(1) DMI Pretreatment + L-DOPA/Benserazide, (2) Vehicle Pretreatment + L-DOPA/Benserazide

and (3) DMI Pretreatment + Vehicle (0.9% NaCl Saline, Hospira, Lake Forest, IL). With 6-

OHDA lesion present in each group, these three groups were used to evaluate the impact of

DMI on LID behavior caused by chronic L-DOPA, with the DMI alone group controlling for

whether NET-blockade alone could produce LID.

Behavioral AIMS ratings

L-DOPA-induced abnormal involuntary movements (AIMs) were then rated at 6 discrete

time points (days 19, 23, 27, 31, 35, 39) during the 20-day administration of L-DOPA starting on

day 19-post lesion until the end of the study. AIMs were assessed by an investigator blind to

treatment. Twenty minutes after L-DOPA administration, rats were placed in separate cages


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and individual dyskinetic behaviors were quantified based on severity and frequency during a 1-

minute observation period performed every 20 minutes over a period of 160 minutes. AIMs

were classified into three subtypes as follows: axial AIMs (dystonic posturing of the upper trunk

towards the side contralateral to the lesion), limb AIMs (hyperkinetic or jerky movements of the

forelimb contralateral to the lesion), and orolingual AIMs (abnormal jaw movements, facial

twitching and tongue protrusion). Each subtype was scored based on severity and frequency

from 0 to 4, with 4 being the most severe and/or occurring continuously during the entire 1-

minute observation period (for review, see Cenci et al., 1998 and Lundblad et al., 2004). Global

AIMs scores were calculated by multiplying the severity score x frequency score for each

observation period and combining the scores on all monitoring periods. Theoretically, the

highest AIMs score achievable in one behavioral test day (with 8 observation periods) would be

384.

Preparation of Synaptosomes

In order to ascertain uptake properties in conjunction with protein and protein

phosphorylation expression levels present in the rats immediately following the last LID

assessment, synaptosomes were prepared according to the protocol previously described

(Salvatore et. al. 2003) with the following modifications: Tissue dissected from dorsal striatum

was homogenized in 5 mL of 0.32 M sucrose solution using a Teflon/glass homogenizing wand

(Glas-Col, Terre Haute, IN) then spun at 1000 x g for 10 minutes in a chilled (4° C) centrifuge.

The resulting pellet was stored as the P1 fraction while the supernatant was spun further at

16,500 x g for 30 minutes at 4° C, yielding the P2 fraction. An aliquot of the P1 fraction was

saved for determination of TH protein, ppERK, and total ERK protein from the 6-OHDA-lesioned

and contralateral (control) striatum against a standard curve of TH protein standard (Salvatore

et al., 2009). We have determined in previous experiments that this fraction is sufficient for


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precise assessment of cytosolic proteins (Chotibut et al., 2012, 2014), reflecting the relative

quantities recovered in fresh frozen preparations (Salvatore et al., 2009). An aliquot of P2

fraction was saved for determination of NET protein from the 6-OHDA lesioned and contralateral

(control) striatum. The supernatant was aspirated and resuspended in 1 mL of Kreb’s buffer

(118 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 25 mM NaHCO3, 1.0 mM Na2EDTA, 1.7 mM

CaCl2, 10 mM glucose, 100 μM parglyline, 100 μM ascorbic acid). Protein concentration was

determined using a BCA colormetric assay (Thermo Scientific, Rockford, IL). All tissue was

kept on ice or at 4 °C from the moment of brain excision until the uptake assay took place.

[3H]DA uptake into Synaptosomes

Synaptosomes were distributed in ice-cold test tubes to prepare for DA uptake. The

determination of [3H]DA uptake in the crude synaptosomes from dorsal striatum harvested from

the contralateral and 6-OHDA-infused hemispheres was conducted simultaneously and included

assessments of DA uptake capacity in the presence of unlabeled 1 µM NE and DA. We

previously determined that this concentration of NE or DA had differential impact on DA uptake

(Chotibut et al., 2012) and consistent with previous observations. Each determination was done

in triplicate for each assay condition and uptake was determined comparing the lesioned

striatum with the contralateral control striatum. Non-specific uptake was determined by counts

obtained in synaptosomes incubated with 500 nM DA (all as labeled DA) on ice during the time

period of uptake. Background was determined and subtracted in the same manner as in the DA

uptake studies.

Synaptosomes (30 μg protein per replicate) were added to 4 °C oxygenated Kreb's

buffer and test ligand (if indicated) to reach a total volume of 100 μL. The synaptosomes were

then warmed to 35°C for 5 min, then 100 μL of pre-warmed 1µM 3H-dopamine, prepared from

ViTrax, [7-, 8- 3H-DA], specific activity of 25 Ci/mmol was added to the synaptosome


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preparations (giving a 500 nM final [3H]DA concentration), allowed to incubate for uptake, and

terminated after 120 seconds with an excess volume of ice-cold Kreb’s buffer and re-immersing

the tubes in the ice-bath. The uptake time for DA was chosen to be as close as technically and

practically possible to the approximately 2-minute uptake time of striatal dopamine observed in

vivo as seen by Sabeti et. al. (2002) and where differences in uptake capacity between lesioned

and intact striatum have been previously reported (Chotibut et al., 2012). Synaptosomes were

washed extensively to remove excess labeled-dopamine with equal-osmolarity PBS buffer

through a Brandel M24-TI (Gaithersburg, MD) cell harvester using Brandel GF/C filter paper

pretreated with a 2% polyethylenimine solution to reduce non-specific binding of label. The filter

paper containing the rinsed synaptosomes was transferred into scintillation vials containing 5

mL of biodegradable scintillation cocktail (Research Products International, Mount Prospect, IL)

and counted with a Beckman Coulter LS6500 scintillation counter (Brea, CA).

Calculating DA uptake

To determine the quantity of DA uptake, the percent of [3H]DA recovered in the

synaptosomes against the total amount of [3H]DA added during the uptake experiment was first

determined. The total pmole of recovered [3H]DA was then determined based upon the percent

[3H]DA recovery in the synaptosomes after subtracting the non-specific binding value, and the

result was normalized to synaptosome protein and expressed as pmole DA per mg protein per

minute.

Analyses of proteins and ERK phosphorylation

Synaptosome fraction (P1) and the processed preparatory sample (P2) were sonicated

in a 1% sodium dodecyl sulfate solution (pH ~8) using a Branson Sonifier 150 (Danbury, CT).

Protein concentration was determined using the bicinchoninic acid colormetric assay.

Following gel electrophoresis, proteins were transferred for 500 volt hours in a


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Tris/glycine/methanol buffer onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules,

CA).

The nitrocellulose membrane was stained with Ponceau S to further normalize staining

in each sample lane. These lanes were scanned and quantified by Image J to normalize protein

in each sample. This relative total level then served as an additional normalizing value to

determine the quantity of each protein assayed (Salvatore et al., 2012). To continue

processing, the membranes were blocked in PVP buffer (1% polyvinylpyrrolidone and 0.05%

Tween 20) for a minimum of two hours to reduce nonspecific antibody binding. The membrane

was soaked in primary antibody for 1-3 hours. Specific primary antibodies were as follows: NET

(Alpha Diagonistics, cat NET11-A) and TH (Millipore, cat #AB152), D1 receptor (Santa Cruz, cat

#14001), ERK1/2 (Millipore cat #442704), ppERK1/2 (Sigma, cat #M8159). Protein loads for

linear detection were 50ug total protein for NET, 10ug for TH and total ERK1/2, and 40ug for D1

and ppERK1/2. After primary treatment, blots were exposed to secondary antibody (swine anti-

rabbit IgG for TH and NET) signal enhancement, followed by 1 hour incubation with [125I] protein

A (PerkinElmer, Waltham, MA).

Statistical Analysis

Data were analyzed using SPSS (Chicago, IL, USA) with correction for multiple

comparisons and p-values < 0.05 considered significant. A two-way ANOVA (time x treatment)

was used to analyze AIM scores over time within each session. Total AIM scores (axial + limb +

oral (ALO)) summed across the 160-minute sessions were analyzed by two-way ANOVA. One-

way ANOVA and t-paired student tests were used in instances with more than 3 or more groups

or 2 groups, respectively. One-way ANOVA analyses was followed by Post-Hoc Tukey multi-

comparison test.


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Surgeries were performed in triplicates with the assumption that any irregularities in

efficacy of the 6-OHDA lesion may occur in all animals lesioned with the same 6-OHDA on that

day. Specifically, surgeries were performed in multiples of three each day to accommodate for

the three treatments (L-DOPA, L-DOPA + DMI, DMI). Thus, any variances that may be

encountered on each day that may affect lesion severity is assumed to occur to each rat on that

given date of surgery. Additionally, the data was also analyzed using a paired t test when

comparing contralateral and lesioned striatum or one way ANOVA between the 3 treatment

groups.

Due to no dyskinesia elicited in DMI treatment group alone, area under the curve

dyskinesia values (Fig 3A, B) plots only included DMI + L-DOPA and L-DOPA treatment groups.

As such, a paired t-test (Fig 3A) was performed to examine for differences in dependent

measures between these two treatments as well as regression analyses to determine if LID

differed over the course of L-DOPA treatment (Fig 3B).

RESULTS:

LID severity increases with desipramine (DMI) pretreatment + L-DOPA compared to L-

DOPA alone

L-DOPA-induced abnormal involuntary movements (AIMs) were rated at 6 discrete time

points (days 19, 23, 27, 31, 35, 39 post 6-OHDA lesion, or days 1, 5, 9, 13, 17, 21 of daily L-

DOPA) during the 20-day administration of L-DOPA starting on day 1 (19 days post 6-OHDA

lesion). As L-DOPA treatment duration increased, the norepinephrine transporter inhibitor,

desipramine (DMI), exacerbated dyskinesia expression in 6-OHDA lesioned male Sprague

Dawley rats compared to L-DOPA alone beginning on day 27 and through day 39 post-6-OHDA

lesion induction (Fig 2A-F). However, on day 19, when the first dose of L-DOPA was

administered, DMI significantly attenuated LID severity (Fig 2A). However, this attenuation of


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LID was not evident after day 5 of L-DOPA (or 23 days post-lesion) (Fig. 2B). Thereafter, DMI

exacerbated LID severity (Fig. 2C-F). By the end of the study on day 39, DMI pre-treatment with

L-DOPA significantly exacerbated LID at all time periods of observation compared to L-DOPA

treatment alone.

Desipramine alone (no chronic L-DOPA, 6-OHDA lesion only) did not elicit dyskinesia,

supporting previous findings that DA replacement (with its precursor, L-DOPA) is a necessary

component of L-DOPA induced dyskinesia, despite lesion severity.

The area under the curve (AUC) reflects the accumulative LID score attained between

the two groups. L-DOPA + DMI elicited greater dyskinesia than L-DOPA alone over the course

of the 20 day treatment regimen (Fig 3A). Because dyskinesia was not observed with DMI

alone (Fig 2A-F, average abnormal involuntary movement score = 0), a comparison of L-DOPA

+ DMI versus L-DOPA alone was made.

Additionally, LID severity did not change over time in the L-DOPA alone group.

However, in the L-DOPA+DMI group there was a significant increase in dyskinesia over the 20-

day course of treatment with L-DOPA + DMI, with the severity increasing as the study

progressed (Fig. 3B).

Tyrosine Hydroxylase loss in treatment groups

There were no significant differences in tyrosine hydroxylase (TH) loss among the three

treatment groups between lesioned and contralateral control striatum (Fig. 4A, B). Therefore,

the exacerbation of LID severity in DMI + L-DOPA treated rats most likely was not due to

increased dopaminergic terminal destruction or lesion severity (L-DOPA: Average TH loss

80.2% + 4.9; DMI: 79% + 5.3; L-DOPA + DMI: 77% + 4.1).


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Relative abundance of NET: lesioned vs. contralateral striatum

Either DMI alone or DMI + L-DOPA treatment significantly decreased NET abundance in

the lesioned striatum compared to contralateral control striatum in the respective groups (Fig

5A, B, D). No difference in NET expression between control and lesioned striatum was

observed in the L-DOPA group (Fig 5C, D).

ERK phosphorylation in treatment groups

Levels of phosphorylated and total ERK1/2 were assessed in tissue fractions prepared

from the 6-OHDA lesioned striatum from rats from each treatment group. Both antibodies

revealed two bands with the expected molecular weights of ERK1 (44kDa) and ERK2 (42kDa).

Accounting for ppERK ½ in lesioned striatum, when compared to DMI alone, L-DOPA and DMI

+ L-DOPA treatment groups showed increased ppERK/ERK1 and ppERK/ERK2 (Fig 6A, B

respectively; representative western blot Fig 6E). Interestingly, these changes were blunted in

the control striatum (Fig 6C, D). ppERK/ERK1 increased compared to DMI alone in both L-

DOPA and L-DOPA + DMI treatments but there were no differences between L-DOPA and L-

DOPA + DMI groups (Fig 6C). Conversely, ppERK/ERK2 was increased only in L-DOPA

treated rats compared to DMI (Fig 6D). No differences were observed in total ERK between

treatments (data not shown).

ERK phosphorylation and dyskinesia severity

The levels of ppERK1 are increased in L-DOPA preclinical studies (Santini et al., 2007;

Westin et al., 2007). We also observed a correlation between the severity of dyskinesia (as

seen in the L-DOPA + DMI treatment group) and the ppERK1 signal, as measured in the

lesioned striatum in each treatment group (Fig 7), indicating that NET blockade in combination

with L-DOPA may increase DA signaling associated with LID manifestation as observed at the

end of the study (day 21 of L-DOPA). Specifically, simple regression of phospho-ERK1 levels


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on the cumulative axial, limb and orolingual global AIM scores recorded from chronically L-

DOPA, DMI + L-DOPA and DMI treated rats had a positive correlation with dyskinesia severity

(Fig 7).

DA uptake and NE inhibition

DA uptake was determined in synaptosomes from lesioned versus control striatal tissue

in the presence of a concentration of DA or NE (1 µM) previously shown to differentially inhibit

uptake of labeled [3H]-DA (Chotibut et al., 2012). In the L-DOPA group, we did not observe a

significant difference in inhibition [3H]-DA uptake by unlabelled DA between the lesioned and

control striatum (% inhibition in control ~70%) (Fig. 8A). However, NE inhibited DA uptake in

lesioned striatum to a greater extent compared to that in control striatum (% inhibition in control

~50%) (Fig. 8B). In the DMI + L-DOPA group, unlabelled DA did not differentially inhibit [3H]-DA

uptake between lesioned and control striatum (% inhibition in control ~70%) (Fig. 8C).

However, NE was significantly less effective to inhibit DA uptake in lesioned compared to

control striatum (% inhibition in control ~70%) (Fig. 8D). NE also inhibited DA uptake to a

significantly greater extent in lesioned striatum in the DMI alone group (data not shown).

DISCUSSION:

Dysregulation of extracellular (DA) in the DA-denervated Parkinsonian striatum is

associated with LID (Carta et al., 2006; 2007; Cenci and Lundblad, 2006). However, the relative

contribution of DA uptake in LID onset or severity has not been established. The 6-OHDA

lesion may increase striatal NET expression and NET may affect DA uptake therein (Chotibut et

al., 2012). In intact CNS tissue, such as prefrontal cortex, there is evidence for NET-mediated

DA reuptake (Carboni et al., 1990; Tanda et al., 1997; Moron et al., 2002). Accordingly, in

lesioned striatum, the tissue content level of NE is comparable to that of DA (Chotibut et al.,


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2012), suggesting that remaining DAT and inherent NET are potentially in comparable

abundance and NET may therefore assume a greater role in DA uptake. Our results are limited

to striatal analysis because of the relative abundance of DA neuropil (although lesioned) over

other DA regions like substantia nigra. DA uptake in the substantia nigra could also affect LID

severity (Navailles et al., 2014), given significant noradrenergic innervation in this region.

However, there is comparatively less DAT protein and DA uptake in the substantia nigra, which

precluded us from examining this possibility.

L-DOPA may inhibit DA uptake in lesioned striatum (Chotibut et al., 2012; Hashimoto et

al., 2005), which could lead to accumulation of extracellular DA, and therefore affect LID onset

or severity. Increased extracellular DA in DA-denervated striatum is observed following DMI

(Arai et al., 2008). This finding is congruent with our observations that reuptake of DA, derived

from L-DOPA, may be modulated by NET. Thus, reducing NET function, either by L-DOPA-

blockade or reduced NET expression, could reduce DA clearance and exacerbate LID severity.

Accordingly, we found that NET inhibition (with DMI) and L-DOPA, gradually exacerbated LID

severity over the 20-day course of L-DOPA administration, compared to L-DOPA alone (Fig. 3).

These differences in LID severity were unrelated to differences in lesion severity (Fig. 4). Our

previous finding that striatal NET expression increases at a comparatively earlier time point in

lesion progression (Chotibut et al., 2012), coupled with the present findings, give credence to

involvement of NET in DA uptake and regulating LID expression or severity.

Our results also indicate that extracellular signal-regulated kinases 1 and 2 (ERK)

phosphorylation was increased in the DMI + L-DOPA group compared to L-DOPA alone group

(Fig. 6). Increased signaling through D1 receptors is implicated in the molecular and synaptic

responses in striatal neurons associated with LID onset (Aubert et al. 2005; Konradi et al. 2004;

Picconi et al. 2003; Westin et al., 2007) and enhanced ERK1/2 phosphorylation in DA

denervated striatum occurs with selective agonists for D1 or D2 receptors (Cai et al. 2000;


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Pavon et al., 2006; Zhen et al. 2002). Thus, increased ERK1/2 activity in striatal neurons is at

the very least a biochemical marker of L-DOPA–induced dyskinesia through D1 or D2 over-

activation. As such, phosphorylation of ERK1/2 may provide a molecular counterpart for

increased D1 activity and be involved in LID induction. Reduced ERK1/2 phosphorylation dose-

dependently decreases LID and other molecular correlates causally linked to LID development

(Santini et al., 2007). Thus, the observation that DMI pretreatment with L-DOPA both

exacerbates LID and increases ERK1/2 phosphorylation over that of L-DOPA alone supports a

dopaminergic mechanism in LID severity wherein NET-mediated DA uptake and L-DOPA

together modulate LID expression. Given that chronic DMI also reduced NET expression in

lesioned striatum (Fig. 5), this observation suggests that increasing NET function or expression

could reduce LID severity.

Compensatory changes in DA regulation do occur during the loss of DA-regulating

proteins in 6-OHDA rodent models (Snyder et al., 1990; Sarre et al., 2004; Perez et al., 2008),

but the potential involvement of NET function is a relatively novel concept. We have reported

increased NET expression in lesioned striatum (Chotibut et al., 2012). Increased locomotion is

observed in monkeys with DAT inhibitors with high NET, but low serotonin transporter, affinity in

cases of severe DAT loss (80%) compared to those with moderate DAT loss (46%) (Madras et

al., 2006). LID severity could also be diminished by other interactions with noradrenergic inputs

to the basal ganglia (Lundblad et al., 2002; Dekundy et al., 2007; Gomez-Mancilla and Bedard

1993). Furthermore, rats with combined noradrenergic and dopaminergic lesions have greater

LID severity, compared to dopaminergic lesions alone (Fulceri et al., 2007; Shin et al., 2014).

Indeed, noradrenergic lesions can produce dyskinesias through DA-mediated locomotor

impairment (Donaldson et al. 1976, Rommelfanger et al. 2007). As such, NET blockade via

DMI may not be the only factor in worsening LID behavior. For example, in LID pathology,

norepinephrine has the ability to act as a D1 dopaminergic agonist (Kubrusly et al., 2007).


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Thus, it may be possible that agents which decrease NET expression could lead to increased

extracellular norepinephrine, thereby increasing D1 activation and worsening LID.

Reduced NET expression in the lesioned striatum therefore appears to play one of two

critical components of LID and its severity. The other component of LID relates to the impact of

chronic L-DOPA. DMI alone did not produce LID, but the combination of DMI and L-DOPA

gradually worsened LID compared to L-DOPA alone. The neurobiological background of

nigrostriatal lesion is also an important component. Chronic DMI reduced NET expression in

lesioned striatum (Fig. 5), arguably offsetting any lesion-induced increase (Chotibut et al.,

2012). Chronic DMI can reduce NET expression in other CNS regions such as amygdala,

striatum (Jeannotte et al., 2009) and hippocampus (Kitayama et al., 2006). We also point out

that the serotonin terminals or transporter (SERT) can affect LID severity in preclinical models

(Carta et al., 2007; Eskow et al., 2009; Bishop et al., 2012). However, chronic DMI is not

reported to alter SERT expression or function (Hyttel,1994; Mantovani et al., 2009), thus making

it unlikely that changes in SERT expression or SERT-mediated uptake are associated with our

behavioral observations. From a clinical perspective, this leads to questions as to whether an

antidepressant with NET-affinity could produce, hasten the onset of, or worsen the severity of

LID, given the prevalence of PD-related depression and that depression commonly precedes

motor manifestations (Burn 2002; Aarsland et al., 2012; Brichta et al., 2013). DMI or other

tricyclics are often prescribed for this patient population. In small-scale clinical trials (17 test

subjects completing each study), methylphenidate (which also affects NET function (Pan et al.,

1994)), tended to increase LID (Devos et al., 2007; Espay et al., 2011). Exacerbation of

dyskinesia in a clinical setting may go unnoticed given the absence of baseline readings before

DMI administration. Therefore, further study could answer to the possibility that NET-blocking

drugs could exacerbate LID severity or its frequency.


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Desipramine may also interfere with alternate receptors such as muscarinic, adrenergic

and histamine. There is a functional interaction between the cholinergic and dopaminergic

systems (Quik and Wonnacott 2011), and DA denervation can increase acetylcholine levels in

striatum. Given that acetylcholine mediates its effects via muscarinic and nicotonic receptors

and that DMI may affect the sensitivity of post and/or pre-synaptic muscarinic receptors

(Murugaiah and Ukponmwan, 2003), it is possible DMI may be mediating its effects through

muscarinic receptors, which may contribute to development of motor signs.

Behaviorally, we observe an initial delay in LID in the L-DOPA + DMI treatment group

compared to L-DOPA, which may be related to increased NET expression with 6-OHDA lesion

(Chotibut et al., 2012). Therefore, this initial increase in NET in the lesioned compared to the

contralateral unlesioned striatum may explain why dyskinetic behavior was not worsened on the

first day of L-DOPA (day 19 post lesion) and that subsequent L-DOPA, in conjunction with DMI

treatments, offset this increase in NET expression, revealing increased LID severity. Our DA

uptake experiments (at the end of the LID assessment) also indicated that DA uptake is more

inhibited by NE in the L-DOPA alone group, but less inhibited by NE in the DMI + L-DOPA

group (Fig. 8). We speculate that these differences may be related to NET expression.

Whereas, NET expression is not changed relative to control striatum in the L-DOPA group, it is

decreased in the groups with DMI. Thus, this decrease in NE-sensitivity in DMI groups could be

due to decreased NET availability in lesioned striatum. We also speculate that the lack of

difference in NET expression in rats treated with L-DOPA alone may be due to increased NET

with 6-OHDA lesion that is offset by possibility that L-DOPA could reduce NET expression over

time. As such, this is an important unresolved question moving forward.

The cellular sources of NET are an important point of consideration. Noradrenergic

innervation of dorsal striatum is relatively sparse, although NE levels are comparable with

remaining DA in lesioned striatum (Chotibut et al., 2012). In prefrontal cortex, NET transports


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DA and conversion of DA to NE may be slower than DA uptake via NET. As such, DA could be

co-released along with NE (Ahn and Klinman 1989). Therefore, pharmacologically-induced

alteration of the noradrenergic system, such by as chronic DMI, could also influence

extracellular DA levels (Devoto and Flore, 2006). Thus our results may also be related

compensatory alterations in DA release. Another possible source of NET-mediated DA

transport may be glia, as glial express NET (Takeda et. al., 2002). Astrocyte and microglia cell

numbers may increase with DA neuron loss (Teismann and Schulz, 2004), possibly increasing

NET protein abundance and DA uptake. Future studies examining alterations in DA uptake in

glial preparations from dyskinetic rats may further delineate the cellular basis for NET-mediated

DA uptake in LID pathophysiology

CONCLUSION:

In summary, our results indicate the possibility that increasing NET expression within the

striatum may be a novel therapeutic avenue to attenuate LID severity. Our previous work has

shown an upregulation of NET expression in lesioned striatum compared to contralateral

striatum after DA denervation in the absence of L-DOPA treatment, but further work could

establish whether this increase in NET is transient or alters with L-DOPA administration. Our

results also indicate that L-DOPA must be present for any development of LID following 6-

OHDA lesion. Our results add to a growing body of literature that NET regulates dopamine

uptake dynamics in the CNS, including pathophysiological mechanisms of L-DOPA induced

dyskinesia.

Authorship Contributions:

Participated in research design: Chotibut, Salvatore Conducted experiments: Chotibut, Fields Performed data analysis: Chotibut, Salvatore Wrote or contributed to writing of the manuscript: Chotibut, Salvatore


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Footnotes:

This work was funded in part by the Ike Muslow Predoctoral Fellowship Award to TC and an award to MFS through the Edward P. Stiles Trust Fund-Louisiana State University Health Sciences Center-Shreveport and The Biomedical Research Foundation of Northwest Louisiana.

Reprint requests:

Michael F. Salvatore, Ph.D. Department of Pharmacology, Toxicology, & Neuroscience Louisiana State University Health Sciences Center 1501 Kings Highway Shreveport, Louisiana 71130 Email: [email protected]


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Legends for Figures

Figure 1. Timeline of treatment paradigm. Male Sprague Dawley rats were lesioned unilaterally with 6-

OHDA and randomly assigned a treatment group. 7 days post lesion, rats were tested with

amphetamine (2.5 mg/kg i.p) and number of turns ipsilateral to lesion turns were assessed to confirm

for a successful lesion. Treatment began on day 9 with 3 treatments: (1) Desipramine + L-DOPA, (2)

Desipramine + Vehicle, (3) Vehicle + L-DOPA. Beginning on day 19, behavioral AIMs were assessed at 6

discrete time points for the remainder of the study (days 19, 23, 27, 31, 35, 39). On Day 39, rats were

sacrificed and dopamine uptake and biochemical markers were assessed.

Figure 2. Abnormal movements induced by the chronic treatment with L-DOPA in animals lesioned

with 6-OHDA. The time course of changes in dyskinesia evaluated from the product of the frequency

and amplitude behavior (orolingual, axial, forelimb) induced by a 20-day treatment with L-DOPA (6

mg/kg plus benserazide 12 mg/kg, i.p). (A) Day 19 (first day of L-DOPA) and (B) Day 23 showed minimal

difference at timepoints in dyskinesia severity. (C) Day 27, (D) Day 31 and (E) Day 35, approximately 1

week after treatment initiation, shows DMI + L-DOPA treatment is worsening dyskinesia severity at

latter timepoints in the observation period. (F) By the end of the study on Day 39, DMI + L-DOPA

treatment worsens dyskinesia severity at all timepoints compared to L-DOPA alone. In all observation

periods, DMI alone did not elicit any dyskinesia. Data analyzed by repeated ANOVA followed by

Bonferroni post hoc test (time course) Significant difference from L-DOPA only treatment: * p<.05,

**p<.01, ***p<.001.

Figure 3. Dyskinesia over time with L-DOPA versus L-DOPA + DMI treatment. (A) Area Under the Curve

(AUC) of dyskinesia elicited between day 4 until end of 20 day treatment regimen between L-DOPA and

L-DOPA + DMI (Paired Student t-Test n = 5 * p<.05) (B) Linear regression of dyskinesia and time with L-

DOPA and L-DOPA + DMI treatment (L-DOPA r2

= .30, p = .24; L-DOPA +DMI r2 = .82, p = .0028).

Percentages reflect the difference in the AUC score between the two treatment groups as a function of

L-DOPA administrations.

Figure 4. (A) Tyrosine Hydroxylase (TH) loss with treatment between control and lesioned striatum.

There was no significant differences between TH loss among treatments (one-way ANOVA, p =.87, n = 3-

5 (DMI n = 3, DMI + L-DOPA = 4, L-DOPA = 5). DMI + L-DOPA vs L-DOPA F(2,9) = .74, DMI + L-DOPA vs DMI

F(2,9) = .42; L-DOPA vs DMI F(2,9) = .24 (B) Tyrosine Hydroxylase loss representative western blot

(Standards of TH shown in ng (0.5, 2, 3, 4); UL = unlesioned, L = lesioned).


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Figure 5. NET Expression after treatment between lesioned and intact striatum. (A) Lesioned striatum

showed less NET expression compared to intact contralateral striatum with chronic DMI + L-DOPA

treatment. (B) DMI treatment also decreased NET expression in lesioned striatum compared to control.

(C) Treatment with chronic L-DOPA however, did not show changes in striatal NET expression. Paired

Student t-test (A) t = 2.692, p = .03, (B) t = 12.92,p = .006 (C) t = 2.077, p = .06 (D) Representative

western blot (L = Lesion, UL = Unlesion)

Figure 6. ppERK1/2 in lesioned striatum after treatment. (A) ppERK/ERK1 was significantly increased in

both DMI + L-DOPA and L-DOPA compared to DMI. DMI + L-DOPA treatment also increased ppERK/ERK1

compared to L-DOPA alone in lesioned striatum. One-Way ANOVA n = 3, p = .0001, f = 58.33. (DMI + L-

DOPA vs DMI: F(2, 6) = 15, DMI + L-DOPA vs L-DOPA F(2, 6) = 10, L-DOPA vs DMI F(2, 6) = 5) (# denotes

significance compared to DMI, * compared to L-DOPA). (B) ppERK/ERK2 was significantly increased in

both DMI + L-DOPA and L-DOPA compared to DMI. DMI + L-DOPA treatment also increased ppERK/ERK2

compared to L-DOPA alone in lesioned striatum. One-Way ANOVA n = 3, p = .0007, f = 30.33 (DMI + L-

DOPA vs DMI: F(2, 6) = 5, DMI + L-DOPA vs L-DOPA F(2, 6) = 11, L-DOPA vs DMI F(2, 6) = 6 (# denotes

significance compared to DMI, * compared to L-DOPA). (C) ppERK/ERK1 was significantly increased in

both L-DOPA and L-DOPA + DMI groups compared to DMI alone in unlesioned striatum n = 3, p =.02, f =

9.0 F(2, 6) = 5.20, DMI + L-DOPA vs DMI F(2, 6) = 5.19 (* denotes significance compared to DMI alone). (D)

ppERK/ERK2 was significantly increased in L-DOPA + DMI treated rats compared to DMI alone in the

unlesioned striatum n = 3, p = .02, f = 7.8, F(2, 6) = 5.42, L-DOPA vs DMI F(2, 6) = 3.87, DMI + L-DOPA vs DMI

F(2, 6) = 1.55 (* denotes significance to DMI alone) (E) ppERK 1/2 representative western blot (L = Lesion,

UL = Unlesion)

Figure 7. Correlation analysis of the severity of dyskinesia and the ppERK/ERK1 in all treated animals.

ppERK/ERK1 values with their corresponding Global AIMs over all treatments showing a significant

correlation between ppERK1 and dyskinesia severity. Correlation analysis, n = 7, R = .86, p = .003.


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Figure 8. Uptake of DA in presence of unlabelled DA or NE. DA uptake was measured in preparations

of crude striatal synaptomes from the contralateral control and lesioned (>70% TH loss) striatum.

Results are presented as the percent inhibition of tritiated DA uptake by 1 µM DA or NE. Note, the

assessments of inhibition by unlabelled monoamine were conducted simultaneously in lesioned and

control synaptosome preparations, one test subject per group at a given time. (A) DA inhibition of DA

uptake in L-DOPA group. DA uptake (2 min) was determined by the parameters described in methods

(ns, Student’s two-tailed t-test, t=2.36; df=4). (B) NE inhibition of DA uptake in L-DOPA group. DA

uptake (2 min) was determined by the parameters described in methods. There was a significant

increase (37%) in inhibition of DA uptake in the lesioned striatum by NE (*p<0.05, Student’s two-tailed

t-test, t=3.38; df=4). (C) DA inhibition of DA uptake in DMI + L-DOPA group. DA uptake (2 min) was

determined by the parameters described in methods (ns, Student’s two-tailed t-test, t=3.03; df=3). (D)

NE inhibition of DA uptake in DMI + L-DOPA group. DA uptake (2 min) was determined by the

parameters described in methods. There was a significant decrease (45%) in inhibition of DA uptake in

the lesioned striatum by NE (**p<0.01, Student’s two-tailed t-test, t=8.33; df=3).


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Figure 1


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0 20 40 60 80 100 120 140 1600

10

20

30

40

50

Desipramine + Vehicle

Vehicle + L-DOPA

Desipramine + L-DOPA

*

D19:Time (min) post-L-DOPA injection

Glo

bal A

IMS

0 20 40 60 80 100 120 140 1600

10

20

30

40

50

Vehicle + L-DOPADesipramine + Vehicle


D23:Time (min) post-L-DOPA injection

Glo

bal A

IMS

0 20 40 60 80 100 120 140 1600

10

20

30

40

50 Desipramine + VehicleVehicle + L-DOPADesipramine + L-DOPA

*

**

**

***

D27: Time (min) post-L-DOPA injection

Glo

bal A

IMS

0 20 40 60 80 100 120 140 1600

10

20

30

40

50Vehicle + L-DOPA

Desipramine + Vehicle


*

D31:Time post-L-DOPA injection

Glo

bal A

IMS

0 20 40 60 80 100 120 140 1600

10

20

30

40

50 Vehicle + L-DOPADesipramine + Vehicle


*

**

******

****

D35: TIme (min) post L-DOPA injection

Glo

bal A

IMS

(Mea

sure

of D

yski

nesi

a)

0 20 40 60 80 100 120 140 1600

10

20

30

40

50Vehicle + L-DOPADesipramine + Vehicle


********* ***

***

***

**

*

D39:TIme (min) post L-DOPA injection

Glo

bal A

IMS

(Mea

sure

of D

yski

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a)

Figure 2

A B

C D

E F


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Figure 3

A

B

-1 -1

0

1000

2000

3000

4000

5000Ar

ea U

nder

the

Cur

ve

L-DOPA L-DOPA + DMI

*

0 3 6 9 12 15 18 210

1000

2000

3000

4000

5000

6000

L-DOPAL-DOPA + DMI

29%

18%

52% 45%68% 115%

days of L-DOPA

AU

C L

ID s

core


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Figure 4

A

B

TH Loss in Lesioned Striatum Compared to Unlesioned Striatum

-1 -1 -1

0

20

40

60

80

100TH

% L

oss

DMI + L-DOPA L-DOPA DMI

.5 ng 2ng 3ng 4ng UL / L UL / L


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Figure 5

A

B

C

D

NET expression in chronically treated DMI and L-DOPA: Lesioned vs Control Striatum

DMI w/DOPA Control DMI w/L-DOPA Lesion0

10

20

30

40

50

*

NE

T E

xpre

ssio

n (u

g)

NET expression in chronically treated DMI alone:Lesioned vs Control Striatum

Contralaterol Control DMI Alone Lesion0

10

20

30

40

*

NET

Exp

ress

ion

(ug)

NET Expression in chronically treated L-DOPA: Lesioned vs Control Striatum

L-DOPA Contralateral Control L-DOPA Lesion0

10

20

30

40

50

NET

Exp

ress

ion

(ug)

DMI L-‐DOPA L-‐DOPA + DMI UL / L UL / L UL / L

80 kDA


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Figure 6

A B

C D

E

ppERK/ERK1 in Lesioned Striatum

DMI + L-DOPA L-DOPA DMI 0.00

0.02

0.04

0.06

0.08

0.10

#

#

*

pp

ER

K / E

RK

1

ppERK/ERK2 in Lesioned Striatum

DMI + L-DOPA L-DOPA DMI 0.00

0.02

0.04

0.06 #

#

*

pp

ER

K/E

RK

2

ppERK/ERK1 in Control Striatum

-1 -1 -1

0.00

0.02

0.04

0.06* *

pp

ER

K /

ER

K1


L / UL L / UL L / UL

DMI L-DOPA + DMI L-DOPA

44 kDA ERK1

42 kDA ERK2


-1 -1 -1

0.00

0.01

0.02

0.03

0.04

0.05

*

pp

ER

K / E

RK

2


-1 -1 -1

0.00

0.02

0.04

0.06* *

pp

ER

K /

ER

K1



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Figure 7


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L-DOPA + Vehicle with DA

Con

trol

Lesion

ed

0

10

20

30

40

50

60

70

80

90

% inhib

itio

n o

f [3

H]-

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A

L-DOPA + Vehicle with NE

Con

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Lesion

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0

10

20

30

40

50

60

70

80

90*

% inhib

itio

n o

f [3

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E

Desip + L-DOPA with DA

Con

trol +

DA

Lesion

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DA

0

10

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90

% inhib

itio

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A Desip + L-DOPA with NE

Con

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NE

Lesion

ed +

NE

0

10

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80

**

% inhib

itio

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f [3

H]-

DA

upta

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E

Figure 8

A. B.

C. D.


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