L-3,4-dihydroxyphenylalanine Treatment Research Articles

Effects of hydrogen gas inhalation on L-DOPA-induced dyskinesia

L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesia is a side effect of Parkinson's disease treatment and it is characterized by atypical involuntary movements. A link between neuroinflammation and L-DOPA-induced dyskinesia has been documented. Hydrogen gas (H2) has neuroprotective effects in Parkinson's disease models and has a major anti-inflammatory effect. Our objective is to test the hypothesis that H2 inhalation reduces L-DOPA-induced dyskinesia. 15 days after 6-hydroxydopamine lesions of dopaminergic neurons were made (microinjection into the medial forebrain bundle), chronic L-DOPA treatment (15 days) was performed. Rats were exposed to H2 (2% gas mixture, 1 h) or air (controls) before L-DOPA injection. Abnormal involuntary movements and locomotor activity were conducted. Striatal microglia and astrocyte was analyzed and striatal and plasma samples for cytokines evaluation were collected after the abnormal involuntary movements analysis. H2 inhalation attenuated L-DOPA-induced dyskinesia. The gas therapy did not impair the improvement of locomotor activity achieved by L-DOPA treatment. H2 inhalation reduced activated microglia in the lesioned striatum, which is consistent with the observed reduced pro-inflammatory cytokines levels. Display of abnormal involuntary movements was positively correlated with plasma IL-1β and striatal TNF-α levels and negatively correlated with striatal IL-10 levels. Prophylactic H2 inhalation decreases abnormal involuntary movements in a preclinical L-DOPA-induced dyskinesia model. The H2 antidyskinetic effect was associated with decreased striatal and peripheral inflammation. This finding has a translational importance to L-DOPA-treated parkinsonian patients' well-being.

Induction of aberrant agonistic behavior by a combination of serotonergic and dopaminergic manipulation in rats.

Serotonin (5-HT) and dopamine (DA) are involved in the regulation of social behaviors. However, the effects of their interactions on social behavior are not well understood. In this study, rats received a serotonergic neurotoxin injection into the raphe nuclei and/or systemic administration of L-3, 4-dihydroxyphenylalanine (L-DOPA), and their agonistic behaviors were investigated using the resident-intruder (RI) paradigm. Rats in the DA + /5-HT-group, which were administered both monoaminergic treatments, exhibited intense jump and flight responses to intruders. These behaviors were not observed in rats that received either 5-HT lesions or L-DOPA treatment only. To address the neural basis of these aberrant behaviors, we compared c-Fos immunoreactivity in the brain among the different groups. The DA + /5-HT-group had c-Fos activation in areas related to anti-predatory defensive behaviors, such as the ventromedial hypothalamic nucleus, premammillary nucleus, and periaqueductal gray. Moreover, this group had increased c-Fos expression in the ventroposterior part of the anterior olfactory nucleus (AOVP). To test the involvement of this area in the aberrant behaviors, cytotoxic lesions were performed in the AOVP prior to the monoaminergic treatments, and subsequent behaviors were examined using the RI test. The AOVP-lesioned DA + /5-HT-rats had attenuation of the aberrant behaviors. Together, these results suggest that the AOVP is involved in the generation of the aberrant defensive behaviors, and that 5-HT/DA balance is important in the regulation of social behaviors.

Effects of L-DOPA on Gene Expression in the Frontal Cortex of Rats with Unilateral Lesions of Midbrain Dopaminergic Neurons.

The development of Parkinson’s disease (PD) causes dysfunction of the frontal cortex, which contributes to the hallmark motor symptoms and is regarded as one of the primary causes of the affective and cognitive impairments observed in PD. Treatment with L-3,4-dihydroxyphenylalanine (L-DOPA) alleviates motor symptoms but has mixed efficacy in restoring normal cognitive functions, which is further complicated by the psychoactive effects of the drug. We investigated how L-DOPA affects gene expression in the frontal cortex in an animal model of unilateral PD. We performed RNA sequencing (RNA-Seq) analysis of gene expression in the frontal cortex of rats with 6-hydroxydopamine (6-OHDA)-induced unilateral dopaminergic lesions treated with L-DOPA, for two weeks. The analysis of variance identified 48 genes with a significantly altered transcript abundance after L-DOPA treatment. We also performed a weighted gene coexpression network analysis (WGCNA), which resulted in the detection of five modules consisting of genes with similar expression patterns. The analyses led to three primary observations. First, the changes in gene expression induced by L-DOPA were bilateral, although only one hemisphere was lesioned. Second, the changes were not restricted to neurons but also appeared to affect immune or endothelial cells. Finally, comparisons with databases of drug-induced gene expression signatures revealed multiple nonspecific effects, indicating that a part of the observed response is a common pattern activated by multiple types of drugs in different target tissues. Taken together, our results identify cellular mechanisms in the frontal cortex that are involved in the response to L-DOPA treatment.

Alterations in the intrinsic properties of striatal cholinergic interneurons after dopamine lesion and chronic L-DOPA.

Changes in striatal cholinergic interneuron (ChI) activity are thought to contribute to Parkinson's disease pathophysiology and dyskinesia from chronic L-3,4-dihydroxyphenylalanine (L-DOPA) treatment, but the physiological basis of these changes is unknown. We find that dopamine lesion decreases the spontaneous firing rate of ChIs, whereas chronic treatment with L-DOPA of lesioned mice increases baseline ChI firing rates to levels beyond normal activity. The effect of dopamine loss on ChIs was due to decreased currents of both hyperpolarization-activated cyclic nucleotide-gated (HCN) and small conductance calcium-activated potassium (SK) channels. L-DOPA reinstatement of dopamine normalized HCN activity, but SK current remained depressed. Pharmacological blockade of HCN and SK activities mimicked changes in firing, confirming that these channels are responsible for the molecular adaptation of ChIs to dopamine loss and chronic L-DOPA treatment. These findings suggest that targeting ChIs with channel-specific modulators may provide therapeutic approaches for alleviating L-DOPA-induced dyskinesia in PD patients.

Combination Treatment of Icariin and L-DOPA Against 6-OHDA-Lesioned Dopamine Neurotoxicity.

Until now, the dopamine (DA) precursor, L-3,4-dihydroxyphenylalanine (L-DOPA), remains the gold standard effective drug therapy for Parkinson’s disease (PD) patients. Nevertheless, long-term chronic L-DOPA administration leads to the drug efficacy loss and severe adverse effects, such as L-DOPA-induced dyskinesia (LID). Icariin (ICA), a flavonoid that is extracted from Epimedium, has been proved to evoke neuroprotection against DA neuronal loss in PD animal models. Here, the present study detected the effects of ICA combined with L-DOPA on 6-hydroxydopamine (6-OHDA)-elicited DA neurotoxicity and L-DOPA-induced motor dysfunction as well. PC12 cells were applied to investigate the combination treatment of ICA and L-DOPA against 6-OHDA-lesioned neurotoxicity. In addition, rat substantia nigral stereotaxic injection of 6-OHDA-induced DA neuronal injury was performed to explore the neuroprotective effects mediated by ICA combined with L-DOPA. The pathological movement triggered by L-DOPA was determined by the abnormal involuntary movements (AIM) scores analysis. In PC12 cells, ICA combined with L-DOPA produced better neuroprotection from 6-OHDA-induced neurotoxicity than ICA or L-DOPA alone treatment. In parkinsonian 6-OHDA lesioned rats, ICA conferred DA neuroprotection as monotherapy and an enhancement benefit of L-DOPA treatment after daily administration of L-DOPA and ICA for 21 days. Moreover, ICA ameliorated the development of LID as evidenced by the lowered AIM scores without affecting L-DOPA-mediated efficacy. Furtherly, ICA attenuated neuroinflammation in 6-OHDA-induced DA neuronal loss and the development of LID in vivo. In conclusion, these findings suggest ICA might be a potential promising adjuvant to enhance L-DOPA efficacy and attenuate L-DOPA-produced adverse effects in PD.

Nociceptive Response to L-DOPA-Induced Dyskinesia in Hemiparkinsonian Rats.

Non-motor symptoms are increasingly identified to present clinical and diagnostic importance for Parkinson's disease (PD). The multifactorial origin of pain in PD makes this symptom of great complexity. The dopamine precursor, L-DOPA (L-3,4-dihydroxyphenylalanine), the classic therapy for PD, seems to be effective in pain threshold; however, there are no studies correlating L-DOPA-induced dyskinesia (LID) and nociception development in experimental Parkinsonism. Here, we first investigated nociceptive responses in a 6-hydroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease to a hind paw-induced persistent inflammation. Further, the effect of L-DOPA on nociception behavior at different times of treatment was investigated. Pain threshold was determined using von Frey and Hot Plate/Tail Flick tests. Dyskinesia was measured by abnormal involuntary movements (AIMs) induced by L-DOPA administration. This data is consistent to show that 6-OHDA-lesioned rats had reduced nociceptive thresholds compared to non-lesioned rats. Additionally, when these rats were exposed to a persistent inflammatory challenge, we observed increased hypernociceptive responses, namely hyperalgesia. L-DOPA treatment alleviated pain responses on days 1 and 7 of treatment, but not on day 15. During that period, we observed an inverse relationship between LID and nociception threshold in these rats, with a high LID rate corresponding to a reduced nociception threshold. Interestingly, pain responses resulting from CFA-induced inflammation were significantly enhanced during established dyskinesia. These data suggest a pro-algesic effect of L-DOPA-induced dyskinesia, which is confirmed by the correlation founded here between AIMs and nociceptive indexes. In conclusion, our results are consistent with the notion that central dopaminergic mechanism is directly involved in nociceptive responses in Parkinsonism condition.

Dopamine Deficiency Mediates Early Rod-Driven Inner Retinal Dysfunction in Diabetic Mice.

PurposeElectroretinograms (ERGs) are abnormal in diabetic retinas before the appearance of vascular lesions, providing a possible biomarker for diabetic vision loss. Previously, we reported that decreased retinal dopamine (DA) levels in diabetic rodents contributed to early visual and retinal dysfunction. In the current study, we examined whether oscillatory potentials (OPs) could serve as a potential marker for detecting early inner retinal dysfunction due to retinal DA deficiency.MethodsRetinal function was tested with dark-adapted ERGs, taken at 3, 4, and 5 weeks after diabetes induction with streptozotocin. Electrical responses were analyzed and correlations were made with previously reported retinal DA levels. The effect of restoring systemic DA levels or removing DA from the retina in diabetic mice on OPs was assessed using L-3,4-dihydroxyphenylalanine (L-DOPA) treatments and retina-specific tyrosine hydroxylase (Th) knockout mice (rTHKO), respectively.ResultsDiabetic animals had significantly delayed OPs compared to control animals in response to dim, but not bright, flash stimuli. L-DOPA treatment preserved OP implicit time in diabetic mice. Diabetic rTHKO mice had further delayed OPs compared to diabetic mice with normal retinal Th, with L-DOPA treatment also providing benefit. Decreasing retinal DA levels significantly correlated with increasing OP delays mediated by rod pathways.ConclusionsOur data suggest that inner retinal dysfunction in early-stage diabetes is mediated by rod-pathway deficits and DA deficiencies. OP delays may be used to determine the earliest functional deficits in diabetic retinopathy and to establish an early treatment window for DA therapies that may prevent progressive vision loss.

Changes in kynurenine pathway metabolism in Parkinson patients with L-DOPA-induced dyskinesia.

L-3,4-Dihydroxyphenylalanine (L-DOPA) is the most effective drug in the symptomatic treatment of Parkinson's disease, but chronic use is associated with L-DOPA-induced dyskinesia in more than half the patients after 10years of treatment. L-DOPA treatment may affect tryptophan metabolism via the kynurenine pathway. Altered levels of kynurenine metabolites can affect glutamatergic transmission and may play a role in the development of L-DOPA-induced dyskinesia. In this study, we assessed kynurenine metabolites in plasma and cerebrospinal fluid of Parkinson's disease patients and controls. Parkinson patients (n=26) were clinically assessed for severity of motor symptoms (UPDRS) and L-DOPA-induced dyskinesia (UDysRS). Plasma and cerebrospinal fluid samples were collected after overnight fasting and 1-2h after intake of L-DOPA or other anti-Parkinson medication. Metabolites were analyzed in plasma and cerebrospinal fluid of controls (n=14), Parkinson patients receiving no L-DOPA (n=8), patients treated with L-DOPA without dyskinesia (n=8), and patients with L-DOPA-induced dyskinesia (n=10) using liquid chromatography-mass spectrometry. We observed approximately fourfold increase in the 3-hydroxykynurenine/kynurenic acid ratio in plasma of Parkinson's patients with L-DOPA-induced dyskinesia. Anthranilic acid levels were decreased in plasma and cerebrospinal fluid of this patient group. 5-Hydroxytryptophan levels were twofold increased in all L-DOPA-treated Parkinson's patients. We conclude that a higher 3-hydroxykynurenine/kynurenic acid ratio in plasma may serve as a biomarker for L-DOPA-induced dyskinesia. Longitudinal studies including larger patients cohorts are needed to verify whether the changes observed here may serve as a prognostic marker for L-DOPA-induced dyskinesia.

Gynostemma pentaphyllum Ethanolic Extract Protects Against Memory Deficits in an MPTP-Lesioned Mouse Model of Parkinson's Disease Treated with L-DOPA.

This study investigated the effects of ethanol extract from Gynostemma pentaphyllum (GP-EX) on memory deficits in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mouse model of Parkinson's disease (PD) (MPTP-lesioned mice). MPTP (30 mg/kg/day, 5 days)-lesioned mice showed deficits of habit learning memory and spatial memory, which were further aggravated by treatment with L-3,4-dihydroxyphenylalanine (L-DOPA) (25 mg/kg, 21 days). However, treatment with GP-EX (50 mg/kg, 21 days) ameliorated memory deficits in MPTP-lesioned mice treated with L-DOPA (25 mg/kg): GP-EX prevented the decreases in retention latency time in the passive avoidance test and tyrosine hydroxylase-immunopositive cells and dopamine levels in the nigrostriatum. GP-EX also reduced increases in retention transfer latency time of the elevated plus-maze test and expression of N-methyl-D-aspartate (NMDA) receptor and improved decreases in phosphorylation of extracellular signal-regulated kinase (ERK1/2) and cyclic AMP-response element binding protein (CREB) in the hippocampus in the same models. By contrast, L-DOPA treatment (10 mg/kg, 21 days) ameliorated memory deficits in MPTP-lesioned mice, which were further improved by GP-EX treatment. These results suggest that GP-EX ameliorates habit learning memory deficits by activating dopaminergic neurons and spatial memory deficits by modulating NMDA receptor-ERK1/2-CREB system in MPTP-lesioned mice treated with L-DOPA. GP-EX may serve as an adjuvant phytonutrient for memory deficits in PD.

L-DOPA Oppositely Regulates Synaptic Strength and Spine Morphology in D1 and D2 Striatal Projection Neurons in Dyskinesia.

Dopamine depletion in Parkinson's disease (PD) produces dendritic spine loss in striatal medium spiny neurons (MSNs) and increases their excitability. However, the synaptic changes that occur in MSNs in PD, in particular those induced by chronic L-3,4-dihydroxyphenylalanine (L-DOPA) treatment, are still poorly understood. We exposed BAC-transgenic D1-tomato and D2-eGFP mice to PD and dyskinesia model paradigms, enabling cell type-specific assessment of changes in synaptic physiology and morphology. The distinct fluorescence markers allowed us to identify D1 and D2 MSNs for analysis using intracellular sharp electrode recordings, electron microscopy, and 3D reconstructions with single-cell Lucifer Yellow injections. Dopamine depletion induced spine pruning in both types of MSNs, affecting mushroom and thin spines equally. Dopamine depletion also increased firing rate in both D1- and D2-MSNs, but reduced evoked-EPSP amplitude selectively in D2-MSNs. L-DOPA treatment that produced dyskinesia differentially affected synaptic properties in D1- and D2-MSNs. In D1-MSNs, spine density remained reduced but the remaining spines were enlarged, with bigger heads and larger postsynaptic densities. These morphological changes were accompanied by facilitation of action potential firing triggered by synaptic inputs. In contrast, although L-DOPA restored the number of spines in D2-MSNs, it resulted in shortened postsynaptic densities. These changes in D2-MSNs correlated with a decrease in synaptic transmission. Our findings indicate that L-DOPA-induced dyskinesia is associated with abnormal spine morphology, modified synaptic transmission, and altered EPSP-spike coupling, with distinct effects in D1- and D2-MSNs.

Co-administration of cannabidiol and capsazepine reduces L-DOPA-induced dyskinesia in mice: Possible mechanism of action

Pharmacological manipulation of the endocannabinoid system represents a promising therapy to alleviate L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesia (LID) in Parkinson's disease (PD). Our aim was to verify whether cannabidiol (CBD), the major non-psychoactive constituent of Cannabis sativa, modifies LID. To this end, the present study employed the 6-hydroxydopamine-neurotoxin model in male C57⁄BL6 mice to reproduce the pattern of cell death present in PD patients. Unilateral striatal lesioned mice received L-DOPA for 21days, developing severe axial, limb, locomotor and orofacial abnormal involuntary movements (AIMs). Following that, the animals were treated with CBD (intraperitoneally) before L-DOPA for three days, alone or in combination with antagonists of the Transient Receptor Potential Vanniloid-1 (TRPV-1), cannabinoid type 1 (CB1) or Peroxisome Proliferator-Activated type gamma (PPARγ) receptors. Nor CBD or any of the antagonists individually were effective in decreasing AIMs. CBD administered with the TRPV-1 antagonist capsazepine (CPZ) reduced AIMs. Treatment with arachidonoyl-serotonin (AA-5-HT), an inhibitor of the enzyme responsible for anandamide metabolism fatty acid amide hydrolase (FAAH) and a TRPV-1 blocker, reproduced these findings. The CB1 receptor antagonist AM251 or the PPARγ receptor antagonist GW9662 selectively reversed the antidyskinetic effect of CPZ+CBD, with AM251 decreasing limb and orofacial AIMs and GW9662 reducing axial AIMs. The decrease of LID by CPZ+CBD was associated with a reduction in the molecular markers phospho-acetylated histone H3 and phosphorylated extracellular signal-regulated protein kinases 1 and 2. L-DOPA treatment in hemiparkinsonian mice increased the pro-inflammatory markers cyclooxygenase-2 and nuclear factor-kappa B in the lesioned striatum. These markers were also decreased by CPZ+CBD treatment. Our study indicates that CBD, together with a TRPV-1 antagonist, reduces LID by acting on CB1 and PPARγ receptors and reducing the expression of the inflammatory markers cyclooxygenase-2 and nuclear factor-kappa B.

Yokukansan, a Traditional Japanese Medicine, Enhances the L-DOPA-Induced Rotational Response in 6-Hydroxydopamine-Lesioned Rats: Possible Inhibition of COMT.

The aim of the present study was to investigate the effects of the traditional Japanese medicine yokukansan (YKS) on the function of dopamine (DA) in the rat nigrostriatal system. Unilateral 6-hydroxydopamine lesions were produced in the rat nigrostriatal system. Despite a marked loss in the striatal immunoreactivity of tyrosine hydroxylase on the lesion side, striatal serotonin (5-HT) immunoreactivity was not affected. Treatment using L-3,4-dihydroxyphenylalanine (L-DOPA) in conjunction with benserazide for 15 d induced abnormal involuntary movements (AIMs) such as locomotive (rotational response), axial, forelimb, and orolingual movements in the lesioned rats. The L-DOPA-induced locomotive and axial, but not forelimb and orolingual, AIMs were significantly increased and prolonged by the pre-administration of YKS. We next investigated the effects of YKS on the production of DA from L-DOPA in 5-HT synthetic RIN 14B cells. RIN 14B cells produced DA and its metabolite, 3-methoxytyramine (3-MT), following L-DOPA treatment. YKS significantly augmented DA production and inhibited its metabolism to 3-MT in a manner similar to the catechol-O-methyltransferase (COMT) inhibitor entacapone. YKS and some alkaloids (corynoxeine: CX, geissoschizine methyl ether: GM) in Uncaria hook, a constituent herb of YKS, also inhibited COMT activity, indicating that the augmenting effect of YKS on L-DOPA-induced DA production in 5-HT synthetic cells was due to the inhibition of COMT by CX and GM. Our results suggest that YKS facilitates the DA supplemental effect of L-DOPA, and that COMT inhibition by CX and GM contributes, at least in part, to the effects of YKS.

Targeting β-arrestin2 in the treatment of l-DOPA–induced dyskinesia in Parkinson’s disease

Parkinson's disease (PD) is characterized by severe locomotor deficits and is commonly treated with the dopamine (DA) precursor l-3,4-dihydroxyphenylalanine (L-DOPA), but its prolonged use causes dyskinesias referred to as L-DOPA-induced dyskinesias (LIDs). Recent studies in animal models of PD have suggested that dyskinesias are associated with the overactivation of G protein-mediated signaling through DA receptors. β-Arrestins desensitize G protein signaling at DA receptors (D1R and D2R) in addition to activating their own G protein-independent signaling events, which have been shown to mediate locomotion. Therefore, targeting β-arrestins in PD L-DOPA therapy might prove to be a desirable approach. Here we show in a bilateral DA-depletion mouse model of Parkinson's symptoms that genetic deletion of β-arrestin2 significantly limits the beneficial locomotor effects while markedly enhancing the dyskinesia-like effects of acute or chronic L-DOPA treatment. Viral rescue or overexpression of β-arrestin2 in knockout or control mice either reverses or protects against LIDs and its key biochemical markers. In other more conventional animal models of DA neuron loss and PD, such as 6-hydroxydopamine-treated mice or rats and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated nonhuman primates, β-arrestin2 overexpression significantly reduced dyskinesias while maintaining the therapeutic effect of L-DOPA. Considerable efforts are being spent in the pharmaceutical industry to identify therapeutic approaches to block LIDs in patients with PD. Our results point to a potential therapeutic approach, whereby development of either a genetic or pharmacological intervention to enhance β-arrestin2- or limit G protein-dependent D1/D2R signaling could represent a more mechanistically informed strategy.

Disturbance of sensorimotor filtering in the 6-OHDA rodent model of Parkinson's disease.

Abstract Aims Parkinson's disease (PD) is a movement disorder that involves non-motor symptoms including cognitive dysfunction. L-DOPA (L-3,4-dihydroxyphenylalanine), the most effective treatment for PD, might cause the development of abnormal involuntary movements and psychotic symptoms. It has been argued that a complex interaction between drug- and intrinsic disease-related components is enrolled in PD psychotic symptoms. Prepulse inhibition (PPI) is a cross-species measure of sensorimotor gating often disrupted in disorders either with basal ganglia dysfunction or with psychotomimetic drugs. There are controversial results concerning PPI in PD patients. Nevertheless, clinical studies are difficult to interpret because of differences in disease severity, concomitant medications, and comorbidities. Our aim was to investigate the functioning of sensorimotor gating in the 6-OHDA-inducing partial or complete dopaminergic degeneration of the nigrostriatal pathway. Main methods Since several studies suggested that PD-associated psychosis results from interaction between disease-related factors and dopamine replacement, we also analyzed in rats with complete unilateral lesion of the nigrostriatal pathway the effect of L-DOPA treatment (30 mg/kg, daily) for 1, 7 or 14 days. Key findings Complete and unilateral dopaminergic striatal depletion disrupted PPI response in rats. In mice, partial dopaminergic loss in the dorsal striatum, unilateral or bilateral, did not determine PPI changes. L-DOPA treatment determined either no PPI alteration or PPI increase in the 6-OHDA-lesioned rats. Significance Complete striatal degeneration induced by 6-OHDA discreetly reproduced the impairment of PPI found in PD patients. Additionally, L-DOPA at a therapeutical dose, despite adverse motor effects, should not induce an impairment of sensorimotor response.

Perinatal 6-Hydroxydopamine to Produce a Lifelong Model of Severe Parkinson's Disease.

The classic rodent model of Parkinson's disease (PD) is produced by unilateral lesioning of pars compacta substantia nigra (SNpc) in adult rats, producing unilateral motor deficits which can be assessed by dopamine (DA) D2 receptor (D2-R) agonist induction of measurable unilateral rotations. Bilateral SNpc lesions in adult rats produce life-threatening aphagia, adipsia, and severe motor disability resembling paralysis-a PD model that is so compromised that it is seldom used. Described in this paper is a PD rodent model in which there is bilateral 99% loss of striatal dopaminergic innervation, produced by bilateral intracerebroventricular or intracisternal 6-hydroxydopamine (6-OHDA) administration to perinatal rats. This procedure produces no lethality and does not shorten the life span, while rat pups continue to suckle through the pre-weaning period; and eat without impairment post-weaning. There is no obvious motor deficit during or after weaning, except with special testing, so that parkinsonian rats are indistinguishable from control and thus allow for behavioral assessments to be conducted in a blinded manner. L-DOPA (L-3,4-dihydroxyphenylalanine) treatment increases DA content in striatal tissue, also evokes a rise in extraneuronal (i.e., in vivo microdialysate) DA, and is able to evoke dyskinesias. D2-R agonists produce effects similar to those of L-DOPA. In addition, effects of both D1- and D2-R agonist effects on overt or latent receptor supersensitization are amenable to study. Elevated basal levels of reactive oxygen species (ROS), namely hydroxyl radical, occurring in dopaminergic denervated striatum are suppressed by L-DOPA treatment. Striatal serotoninergic hyperinnervation ensuing after perinatal dopaminergic denervation does not appear to interfere with assessments of the dopaminergic system by L-DOPA or D1- or D2-R agonist challenge. Partial lesioning of serotonin fibers with a selective neurotoxin either at birth or in adulthood is able to eliminate serotoninergic hyperinnervation and restore the normal level of serotoninergic innervation. Of all the animal models of PD, that produced by perinatal 6-OHDA lesioning provides the most pronounced destruction of nigrostriatal neurons, thus representing a model of severe PD, as the neurochemical outcome resembles the status of severe PD in humans but without obvious motor deficits.

Effects of dopamine uptake inhibitor MRZ-9547 in animal models of Parkinson's disease.

MRZ-9547 (d-(2-(2-oxo-4(R)-phenylpyrrolidin-1-yl)-acetamide) is a drug acting at the dopamine transporter (DAT). In the present study, effects of MRZ-9547 alone and in combination with L-3,4-dihydroxyphenylalanine (L-DOPA) were investigated in rodent models predictive for efficacy in Parkinson's disease (PD) and L-DOPA-induced dyskinesia (LID). In rats pre-treated with haloperidol (0.2 mg/kg i.p.), MRZ-9547 (25-100 mg/kg i.p.) dose-dependently attenuated decrease in horizontal locomotion, activity in central zone, and rearings starting at 50 mg/kg i.p. In rats depleted of monoamines by α-methyl-p-tyrosine and reserpine treatment, MRZ-9547 attenuated hypolocomotion starting at 100 mg/kg i.p. At the doses 25-100 mg/kg i.p. the drug induced dose-dependent ipsilateral rotations in rats with unilateral 6-hydroxydopamine (6-OHDA)-induced nigrostriatal system lesions. However, MRZ-9547 enhanced contralateral rotation produced by L-DOPA given at an effective (25 mg/kg i.p.), but not at a sub-effective (6.25 mg/kg i.p.) dose. Microdialysis experiments revealed that MRZ-9547 penetrated well to the brain and did not show any pharmacokinetic interaction with L-DOPA. In unilaterally 6-OHDA-lesioned rats having developed abnormal involuntary movements (AIMs, a rodent correlate of LID) after chronic L-DOPA treatment, MRZ-9547 (50 mg/kg i.p.) did not significantly affect the AIMs expression. The results indicate that MRZ-9547 may by itself have antiparkinsonian activity at early stages of the disease, when some dopaminergic terminals are still intact. It may also enhance antiparkinsonian effect of L-DOPA. MRZ-9547 does not seem to influence the expression of LID in 6-OHDA-lesioned rats. The results support the use of MRZ-9547 in PD patients treated with L-DOPA.

Spontaneous locomotor activity and L-DOPA-induced dyskinesia are not linked in 6-OHDA parkinsonian rats.

Bradykinesia (slowness of movement) and other characteristic motor manifestations of Parkinson’s disease (PD) are alleviated by treatment with L-dihydroxyphenylalanine (L-DOPA). Long-term L-DOPA treatment, however, is associated with complications such as motor fluctuations and dyskinesia that severely impair the quality of life. It is unclear whether the effect of L-DOPA on spontaneous motor activity and its dyskinesia-inducing effect share a common mechanism. To investigate the possible connection between these two effects, we analyzed the spontaneous locomotor activity of parkinsonian rats before surgery (unilateral injection of 6-OHDA in the right medial forebrain bundle), before treatment with L-DOPA, during L-DOPA treatment (the “ON” phase), and after the end of L-DOPA treatment (the “OFF” phase). We correlated the severity of dyskinesia (AIM scores) with locomotor responses in the ON/OFF phases of chronic L-DOPA treatment at two different doses. We treated three groups of parkinsonian animals with chronic injections of 8 mg/kg L-DOPA, 6 mg/kg L-DOPA, and saline solution and one group of non-lesioned animals with 8 mg/kg L-DOPA. At the end of the experiment, tyrosine hydroxylase (TH) immunoreactivity was analyzed in the striatum of all parkinsonian rats. We found no correlation between the severity of dyskinesia and spontaneous locomotor activity in the ON or OFF phase of L-DOPA treatment. The only observed correlation was between the pathological rotation induced by L-DOPA at the highest dose and locomotor activity in the ON phase of L-DOPA treatment. In addition, a L-DOPA withdrawal effect was observed, with worse motor performance in the OFF phase than before the start of L-DOPA treatment. These findings suggest that different neural mechanisms underlie the effect of L-DOPA on spontaneous motor activity and its dyskinesia-inducing effect, with a different dose-response relationship for each of these two effects.

Norepinephrine transporter inhibition with desipramine exacerbates L-DOPA-induced dyskinesia: role for synaptic dopamine regulation in denervated nigrostriatal terminals.

Pharmacological dopamine (DA) replacement with Levodopa [L-dihydroxyphenylalanine (L-DOPA)] is the gold standard treatment of 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-hydroxydopamine (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. Whereas DMI alone elicited no dyskinetic effects in lesioned rats, DMI + L-DOPA-treated rats gradually expressed more severe dyskinesia compared with 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 with L-DOPA-alone group in lesioned striatum. LID severity positively correlated with striatal extracellular signal-regulated protein kinase phosphorylation among the three treatment groups, with increased ppERK1/2 in DMI + L-DOPA group compared with 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.

Effects of L-DOPA on striatal iodine-123-FP-CIT binding and behavioral parameters in the rat

PurposeThe effect of clinical l-3,4-dihydroxyphenylalanine (l-DOPA) doses on the binding of [123I]N-Ω-fluoropropyl-2β-carbomethoxy-3β-(4-iodophenyl)nortropane ([123I]FP-CIT) to the rat dopamine transporter (DAT) was investigated using small animal single-photon emission computed tomography.Materials and methodsDAT binding was measured at baseline, after challenge with the aromatic l-amino acid decarboxylase inhibitor benserazide, and after challenge with either 5 or 10 mg/kg l-DOPA plus benserazide. For baseline and challenges, striatal equilibrium ratios (V3′′) were computed as an estimation of the binding potential. Moreover, striatal V3′′ values were correlated with parameters of motor and exploratory behavior.ResultsV3′′ differed significantly between baseline and either dose of l-DOPA/benserazide. Moreover, V3′′ differed significantly between l-DOPA treatment groups. After 5 mg/kg l-DOPA/benserazide, DAT binding was inversely correlated with sitting duration (1–5 min) and sitting frequency (10–15 min). After 10 mg/kg l-DOPA/benserazide, an inverse correlation was found between DAT binding and sitting duration (1–30 min), whereas DAT binding and duration of ambulatory activity (1–30 min) as well as head and shoulder motility (10–15 min) exhibited a positive correlation.ConclusionChallenge with 5 and 10 mg/kg l-DOPA/benserazide led to mean reductions in DAT binding by 34 and 20%, respectively. Results indicate a biphasic response with a higher effect on DAT after the lower dose of l-DOPA. The reduction in DAT binding may be interpreted in terms of competition between [123I]FP-CIT and endogenous dopamine. Moreover, there is preliminary evidence of an association between striatal DAT and motor and exploratory parameters.

L-DOPA Treatment Selectively Restores Spine Density in Dopamine Receptor D2–Expressing Projection Neurons in Dyskinetic Mice

L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesia is an incapacitating complication of L-DOPA therapy that affects most patients with Parkinson's disease. Previous work indicating that molecular sensitization to dopamine receptor D1 (D1R) stimulation is involved in dyskinesias prompted us to perform electrophysiological recordings of striatal projection "medium spiny neurons" (MSN). Moreover, because enhanced D1R signaling in drug abuse induces changes in spine density in striatum, we investigated whether the dyskinesia is related to morphological changes in MSNs. Wild-type and bacterial artificial chromosome transgenic mice (D1R-tomato and D2R-green fluorescent protein) mice were lesioned with 6-hydroxydopamine and subsequently treated with L-DOPA to induce dyskinesia. Functional, molecular, and structural changes were assessed in corticostriatal slices. Individual MSNs injected with Lucifer-Yellow were detected by immunohistochemistry for three-dimensional reconstructions with Neurolucida software. Intracellular current-clamp recordings with high-resistance micropipettes were used to characterize electrophysiological parameters. Both D1R-MSNs and D2R-MSNs showed diminished spine density in totally denervated striatal regions in parkinsonian mice. Chronic L-DOPA treatment, which induced dyskinesia and aberrant FosB expression, restored spine density in D2R-MSNs but not in D1R-MSNs. In basal conditions, MSNs are more excitable in parkinsonian than in sham mice, and excitability decreases toward normal values after L-DOPA treatment. Despite this normalization of basal excitability, in dyskinetic mice, the selective D1R agonist SKF38393 increased the number of evoked action potentials in MSNs, compared with sham animals. Chronic L-DOPA induces abnormal spine re-growth exclusively in D2R-MSNs and robust supersensitization to D1R-activated excitability in denervated striatal MSNs. These changes might constitute the anatomical and electrophysiological substrates of dyskinesia.