Validations of apomorphine-induced BOLD activation correlations in hemiparkinsonian rhesus macaques.

Identification of Parkinson's disease at the earliest possible stage of the disease may provide the best opportunity for the use of disease modifying treatments. However, diagnosing the disease during the pre-symptomatic period remains an unmet goal. To that end, we used pharmacological MRI (phMRI) to assess the function of the cortico-basal ganglia circuit in a non-human primate model of dopamine deficiency to determine the possible relationships between phMRI signals with behavioral, neurochemical, and histological measurements. Animals with unilateral treatments with the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), that expressed stable, long-term hemiparkinsonism were challenged with the dopaminergic receptor agonist, apomorphine, and structure-specific phMRI blood oxygen level-dependent (BOLD) activation responses were measured. Behavioral, histopathological, and neurochemical measurements were obtained and correlated with phMRI activation of structures of the cortico-basal ganglia system. Greater phMRI activations in the basal ganglia and cortex were associated with slower movement speed, decreased daytime activity, or more pronounced parkinsonian features. Animals showed decreased stimulus-evoked dopamine release in the putamen and substantia nigra pars compacta and lower basal glutamate levels in the motor cortex on the MPTP-lesioned hemisphere compared to the contralateral hemisphere. The altered neurochemistry was significantly correlated with phMRI signals in the motor cortex and putamen. Finally, greater phMRI activations in the caudate nucleus correlated with fewer tyrosine hydroxylase-positive (TH+) nigral cells and decreased TH+ fiber density in the putamen. These results reveal the correlation of phMRI signals with the severity of the motor deficits and pathophysiological changes in the cortico-basal ganglia circuit.

Methodology and effects of repeated intranasal delivery of DNSP-11 in awake Rhesus macaques.

BACKGROUND: To determine if the intranasal delivery of neuroactive compounds is a viable, long-term treatment strategy for progressive, chronic neurodegenerative disorders, such as Parkinson's disease (PD), intranasal methodologies in preclinical models comparable to humans are needed. NEW METHOD: We developed a methodology to evaluate the repeated intranasal delivery of neuroactive compounds on the non-human primate (NHP) brain, without the need for sedation. We evaluated the effects of the neuroactive peptide, DNSP-11 following repeated intranasal delivery and dose-escalation over the course of 10-weeks in Rhesus macaques. This approach allowed us to examine striatal target engagement, safety and tolerability, and brain distribution following a single 125I-labeled DNSP-11 dose. RESULTS: Our initial data support that repeated intranasal delivery and dose-escalation of DNSP-11 resulted in bilateral, striatal target engagement based on neurochemical changes in dopamine (DA) metabolites-without observable, adverse behavioral effects or weight loss in NHPs. Furthermore, a 125I-labeled DNSP-11 study illustrates diffuse rostral to caudal distribution in the brain including the striatum-our target region of interest. COMPARISON WITH EXISTING METHODS: The results of this study are compared to our experiments in normal and 6-OHDA lesioned rats, where DNSP-11 was repeatedly delivered intranasally using a micropipette with animals under light sedation. CONCLUSIONS: The results from this proof-of-concept study support the utility of our repeated intranasal dosing methodology in awake Rhesus macaques, to evaluate the effects of neuroactive compounds on the NHP brain. Additionally, results indicate that DNSP-11 can be safely and effectively delivered intranasally in MPTP-treated NHPs, while engaging the DA system.

Intraputamenal infusion of exogenous neurturin protein restores motor and dopaminergic function in the globus pallidus of MPTP-lesioned rhesus monkeys.

The neurorestorative effects of exogenous neurturin (NTN) delivered directly into the putamen via multiport catheters were studied in 10 MPTP-lesioned rhesus monkeys expressing stable parkinsonism. The parkinsonian animals were blindly assigned to receive coded solutions containing either vehicle (n = 5) or NTN (n = 5, 30 microg/day). Both solutions were coinfused with heparin using convection-enhanced delivery for 3 months. The NTN recipients showed a significant and sustained behavioral improvement in their parkinsonian features during the treatment period, an effect not seen in the vehicle-treated animals. At study termination, locomotor activity levels were increased by 50% in the NTN versus vehicle recipients. Also, DOPAC levels were significantly increased by 150% ipsilateral (right) to NTN infusion in the globus pallidus, while HVA levels were elevated bilaterally in the NTN-treated animals by 10% on the left and 67% on the right hemisphere. No significant changes in DA function were seen in the putamen. Volumetric analysis of putamenal NTN labeling showed between-subject variation, with tissue distribution ranging from 214 to 744 mm3, approximately equivalent to 27-93% of area coverage. Our results support the concept that intraparenchymal delivery of NTN protein may be effective for the treatment of PD. More studies are needed to determine strategies that would enhance tissue distribution of exogenous NTN protein, which could contribute to optimize its trophic effects in the parkinsonian brain.

[Pathophysiology and therapy of L-Dopa-induced dyskinesia]. / Physiopathologie et modalités thérapeutiques des dyskinésies induites à la L-Dopa.

Involuntary movements, or dyskinesias, represent a debilitating complication of levodopa therapy for Parkinson's disease. Dyskinesia is, ultimately, experienced by the vast majority of the patients. Despite the importance of this problem, little was known about the cause of dyskinesia, a situation that has dramatically evolved in the last few years. The present review presents: 1) the current understanding of dyskinesia pathophysiology and 2) the therapeutic modalities, mainly non-dopaminergic, available or in development. We here show that the questions raised by the dyskinesia may have a clinically-driven pharmacological answer: the symptomatic treatment of dyskinesia, the prevention of the priming and the de-priming of the neural networks.

Pharmacological MRI mapping of age-associated changes in basal ganglia circuitry of awake rhesus monkeys.

While the pathophysiological changes induced by the loss of dopamine innervation in the basal ganglia by Parkinson's disease (PD) are well studied, little is known about functional changes in the neural circuitry of this area during normal aging. Here we report the first survey of age-associated changes in the basal ganglia of behaviorally characterized, awake rhesus monkeys, using pharmacological MRI to map responses to dopaminergic stimulation. Apomorphine, a mixed D(1)/D(2) dopamine receptor agonist, evoked little change in the substantia nigra (SN) of aged animals while significantly reducing activation in young adult monkeys. Compared to young animals, both apomorphine and d-amphetamine (which increases synaptic dopamine levels) significantly increased activation of the aged rhesus globus pallidus externa (GPe). In addition, the aged animals showed decreased activity in the putamen in response to d-amphetamine administration. Although the responses in the SN and putamen of the aged monkeys differed from those in animal models of PD, the apomorphine-evoked activation of their GPe corresponded with apomorphine-induced increases in neuronal activity seen in Parkinson's patients and animal models. Given the major role of the GPe in regulating motor behavior, the altered responses in the aged GPe may contribute significantly to the motor slowing and movement dysfunctions characterizing advanced age.

Effect of MPTP-induced denervation on basal ganglia GABA(B) receptors: correlation with dopamine concentrations and dopamine transporter.

We investigated the effect of MPTP-induced lesion of the substantia nigra pars compacta (SNpc) dopaminergic neurons on GABA(B) receptors in the basal ganglia of mice and monkeys using receptor autoradiography and in situ hybridization. The extent of the lesion was measured with striatal catecholamine content, striatal binding of (125)I-RTI-121 to dopamine transporter (DAT), and DAT expression in the SNpc. GABA(B) receptors in mice brain were evaluated using (3)H-CGP54626 and its expression was measured with oligonucleotides probes targeting the mRNAs of GABA(B(1a+b)), GABA(B(1a)), GABA(B(1b)), GABA(B(2)) subunits. In monkeys, (125)I-CGP64213 and selective probes for GABA(B(1a+b)) and GABA(B(2)) mRNAs were used. In mice, dopamine content, (125)I-RTI-121 binding, and DAT expression were reduced by 44%, 40%, and 39% after a dose of 40 mg/kg of MPTP and 74%, 70%, and 34% after 120 mg/kg of MPTP, respectively. In monkeys, dopamine content and DAT expression were decreased by more than 90% and 80%, respectively. In the striatum and the subthalamic nucleus, GABA(B) receptors were unchanged following MPTP in both species. In the SNpc of mice, MPTP (120 mg/kg) induced a significant decrease of (3)H-CGP54626 binding (-10%) and of the expression of GABA(B(1a+b)) mRNA (-13%). The decrease of the expression of GABA(B(1a+b)) mRNA was correlated with dopamine content, (125)I-RTI-121 binding and DAT expression. In MPTP-treated monkeys, (125)I-CGP64213 binding (-40%), GABA(B(1a+b)) mRNA (-69%) and GABA(B(2)) mRNA (-66%) were also significantly decreased in the SNpc. Our results suggest that MPTP-induced denervation is associated with a decrease of GABA(B) receptors restricted to the SNpc. These observations may be relevant to the pathophysiology of motor disorders involving dysfunction of the basal ganglia such as Parkinson disease.

Chronic Intracerebral Delivery of Trophic Factors via a Programmable Pump as a Treatment for Parkinsonism.

The most common treatment for Parkinson's disease (PD) aims at pharmacologically augmenting striatal dopamine (DA) using the DA precursor levodopa. Such treatment provides symptomatic relief, but does not slow or halt continued degeneration of nigral dopaminergic neurons. Considerable effort has been devoted to the search for neurotrophic factors with survival-promoting activities on dopaminergic neurons that could potentially be of therapeutic value in the treatment of PD. One such candidate is glial cell line-derived neurotrophic factor (GDNF).

Motor slowing and parkinsonian signs in aging rhesus monkeys mirror human aging.

Motor slowing is a universal feature of human aging, and parkinsonian signs are commonly expressed in human senescence. In the present study, age-associated declines in motor functions in 31 female rhesus monkeys were quantified by activity monitors and an automated test panel, and the incidence of parkinsonian signs was scored using a movement dysfunction assessment scale. Activity levels in middle-aged monkeys (12-17 years old) were less than half that of young animals (5-8 years old) and were further depressed in aged monkeys (21-27 years old). Movement dysfunction scores increased significantly with increasing age. Two or more parkinsonian signs were exhibited by 20% of the middle-aged monkeys and 36% of the aged monkeys. Slowing performance times on fine-motor hand tasks correlated significantly with increasing age. Motor learning was seen in all age groups, but improved faster in the young monkeys. The data suggest that aging rhesus monkeys provide an appropriate model to analyze the biological processes leading to motor slowing and the expression of parkinsonian signs in human senescence.

Dopamine-receptor stimulation: biobehavioral and biochemical consequences.

The MPTP monkey is a well-characterized animal model of parkinsonism and provides an exceptional tool for the study of dyskinesias induced by dopamine-like agents. Several such agents have been tested during the past 15 years, and it has been found that the duration of action of these compounds is the most reliable variable with which to predict their dyskinesiogenic profile. It is proposed that L-dopa-induced dyskinesias represent a form of pathological learning caused by chronic pulsatile (nonphysiological) stimulation of dopamine receptors, which activates a cascade of molecular and biochemical events. These events include defective regulation of Fos proteins that belong to the deltaFosB family, increased expression of neuropeptides, and defective GABA- and glutamate-mediated neurotransmission in the output structures of the basal ganglia.

Regulation by chronic treatment with cabergoline of dopamine D1 and D2 receptor levels and their expression in the striatum of Parkinsonian-monkeys.

1. Chronic treatment for one month with the long-acting dopamine D2-like agonist cabergoline (0.25 mg/kg s.c. every 48 hours), had despite partial tolerance, sustained antiparkinsonian activity in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Parkinsonian monkeys (Macaca fascicularis). 2. Cabergoline treatment decreased by half striatal D2 receptor binding density measured by [3H]spiperone autoradiography versus untreated MPTP monkeys. No change in D2 mRNA measured by in situ hybridization and D2 receptor immunostaining was observed. 3. No change in either D1 receptor binding density or D1 receptor mRNA levels was observed in cabergoline-treated MPTP-monkeys compared to untreated MPTP-monkeys, suggesting receptor subfamily specificity of cabergoline. 4. The present results suggest that the cabergoline-induced behavioral partial tolerance is accompanied by a decrease in D2 receptor binding but not due to alterations in the steady state of D2 mRNA levels.

Dopaminergic therapy improves upper limb motor performance in aged rhesus monkeys.

The potential of dopaminergic treatments to improve upper limb motor movements was tested in 7 aged rhesus monkeys using L-3,4-dihydroxyphenylalanine (L-dopa) or the selective dopamine uptake inhibitor 1-2(bis[4-fluorophenyl] methoxy]ethyl)-4-(3-phenylpropyl) piperazine hydrochloride (GBR-12909). Six young monkeys were studied for comparison. L-Dopa or GBR-12909 improved upper limb motor performance by up to 40% in the aged animals. At this point their performance was comparable to that of young adults. Dopaminergic therapy could be useful in elderly humans experiencing declines in upper limb motor functions.

Molecular basis of levodopa-induced dyskinesias.

A series of experiments were performed in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of parkinsonism for the purpose of understanding the mechanism of dopaminergic dyskinesias. Dyskinesias can be induced in this model by de novo treatment with levodopa, or selective D1 or D2 agonists, provided the drugs are short acting and administered in the pulsatile mode. Biochemical analysis of the brains revealed several alterations in dopamine receptor-binding and messenger RNA message following denervation and dopaminergic treatment, but none that clearly correlated with the presence of dyskinesias. On the other hand, gamma-aminobutyric acid (GABA)A binding was increased in the internal segment of the globus pallidus of dyskinetic MPTP monkeys. This was observed consistently and could be associated with an exaggerated response to GABAergic inhibitory inputs in this strategic structure. Increased preproenkephalin message was also found to correlate with dyskinesias and may be linked to changes in GABA receptors. Treatments that caused dyskinesias induced, in the striatum, chronic Fos proteins of the deltaFosB family which, when coupled with Jun-D, form AP-1 complexes that can affect several genes, including enkephalin and N-methyl-D-aspartate receptor. We suggest that levodopa-induced dyskinesias represent a form of pathological learning, which results from deficient gating of glutamatergic inputs to the striatum by dopamine.

125I-CGP 64213 binding to GABA(B) receptors in the brain of monkeys: effect of MPTP and dopaminomimetic treatments.

Much evidence indicates that abnormal GABA neurotransmission may be implicated in the pathophysiology of Parkinson's disease (PD) and dopaminomimetic-induced dyskinesias (DID). In this study, autoradiography using (125)I-CGP 64213 was performed to investigate GABA(B) receptor density in the brain of control monkeys as well as monkeys with MPTP-induced nigrostriatal depletion. Three MPTP monkeys received pulsatile administrations of the D1 dopamine (DA) receptor agonist (SKF 82958) whereas a long-acting D2 DA receptor agonist (cabergoline) was given to another three animals. SKF 82958 treatment relieved parkinsonian symptoms but two of three animals developed DID. Cabergoline induced a comparable motor benefit effect without persistent DID. (125)I-CGP 64213 binding to GABA(B) receptors was heterogeneous throughout the brain with the highest levels in the medial habenula of the thalamus. MPTP induced a decrease (-40%) of (125)I-CGP 64213 binding to GABA(B) receptors in the substantia nigra pars compacta (SNpc) and an increase (+29%) in the internal segment of the globus pallidus (GPi). This increase in the GPi was not affected by SKF 82958 but partly reversed by cabergoline. No change was seen in the striatum, the thalamus, the external segment of the globus pallidus, and the substantia nigra pars reticulata following MPTP and dopaminomimetic treatments. The changes of GABA(B) receptors observed in the SNpc and in the GPi suggest that alteration of GABA(B) receptors may play a role in the pathophysiology of PD and DID.

Noradrenoceptor antagonism with idazoxan improves L-dopa-induced dyskinesias in MPTP monkeys.

Treatment of Parkinson's disease with L-dopa is plagued in a majority of patients by dyskinesias. Noradrenaline/dopamine interactions are proposed on behavioral, biochemical, physiological and anatomical grounds. The aim of the study was to test the potential antidyskinetic effect of the alpha2-adrenoceptor antagonist, idazoxan, in a primate model of Parkinson's disease. Six female cynomolgus monkeys previously rendered parkinsonian by the toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and presenting an unchanged syndrome for several months were used. All responded readily to L-dopa but had developed dyskinesias which were manifested with each dose. In the first part of the study, seven doses of idazoxan (ranging from 0.25 mg/kg to 10 mg/kg, p.o.) were administered together with the vehicle or in combination with a fixed dose of L-dopa/benserazide (100/25 mg, p.o.). In the second part of the study, a fixed dose of idazoxan (7.5 mg/kg) was administered daily for 10 days and L-dopa was added to idazoxan on days 1, 4, 7 and 10. Vehicle (empty capsule) was used as control. Idazoxan, by itself (ranging from 5 mg/kg to 10 mg/kg), increased locomotor activity and improved the disability score with virtually no dyskinesias in three animals. In combination with L-dopa, idazoxan did not impair the antiparkinsonian response but significantly reduced dyskinesias in all six animals up to 65% at doses of 7.5 mg/kg and 10 mg/kg and delayed their onset, so that the "ON" state without dyskinesias was prolonged. The antidyskinetic effect of idazoxan was maintained when repeatedly administered for 10 days. On day 10, the locomotor response to L-dopa was significantly potentiated by chronic administration of idazoxan. Our results indicate that idazoxan has some antiparkinsonian effect of its own and may constitute a useful adjunct to L-dopa as it can reduce dyskinesias without impairing the relief of symptoms, this effect being maintained over time in this model.

Effect of the selective D1 antagonists SCH 23390 and NNC 01-0112 on the delay, duration, and improvement of behavioral responses to dopaminergic agents in MPTP-treated monkeys.

We assessed the antiparkinsonian response in MPTP-treated monkeys after acute or repeated treatment with oral L-Dopa, subcutaneous administration of L-Dopa methyl ester (LDME) or apomorphine, alone and in combination with D1 antagonists SCH 23390 (SCH) or NNC 01-0112 (NNC). When given alone, the L-Dopa effect occurred within the first hour after treatment. Coadministration of SCH or NNC with L-Dopa significantly delayed the onset of action. The response duration remained unchanged, as did the extent of the antiparkinsonian effect, after SCH, whereas the former became shorter at the higher doses of NNC tested. Bypass of the gastrointestinal tract using parenteral injections of LDME and apomorphine allowed the rapid turning "on" of the animals. Both D1 antagonists administered with LDME delayed the onset and shortened the duration of the therapeutic effect as the dose increased. Pretreatment with SCH failed to block the antiparkinsonian effect induced by apomorphine, but reduced the response duration markedly in a dose-related fashion. Repeated treatment of one monkey with SCH combined with the same dopaminergic drugs produced results similar to those obtained after acute treatment in four animals. The results obtained with parenteral administration of LDME and apomorphine most probably involve pharmacodynamic actions resulting in increased threshold of response. The delay observed with L-Dopa suggests pharmacokinetic interference possibly mediated via dopamine receptors located at the level of the gut.

Dopamine D1 receptor mRNA and receptor levels in the striatum of MPTP monkeys chronically treated with SKF-82958.

The density of dopamine D1 receptor antagonist sites was measured by autoradiography and dopamine D1 receptor mRNA levels were measured by in situ hybridization in the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-exposed monkeys chronically treated with the dopamine D1 receptor agonist 6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benza zep ine hydrobromide (SKF-82958) administered in intermittent or continuous mode for a month. Normal and MPTP-exposed but otherwise untreated animals were used for comparison. Intermittent treatment with SKF-82958 relieved parkinsonian features and induced dyskinesias whereas given continuously this drug induced behavioral tolerance without dyskinesias. On the one hand, MPTP treatment tended to increase dopamine D1 receptor density in the putamen whereas treatment of MPTP monkeys with SKF-82958, intermittent or continuous, produced a significant increase compared to control animals. On the other hand, dopamine D1 receptor mRNA levels in the putamen appeared to decrease after MPTP lesion and agonist treatment as compared to dopamine D1 receptor density. In contrast, an apparent decrease in dopamine D1 receptor density and mRNA levels was observed in the nucleus accumbens of untreated MPTP monkeys whereas treatment of MPTP monkeys with SKF-82958, intermittent or continuous, produced a significant decrease compared to control animals. Thus, neither dyskinesias nor tolerance can be exclusively related to an increase or decrease in striatal dopamine D1 receptors, respectively.

Chronic D1 and D2 dopaminomimetic treatment of MPTP-denervated monkeys: effects on basal ganglia GABA(A)/benzodiazepine receptor complex and GABA content.

The effect of various chronic dopaminergic treatments in 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkeys on the brain gamma-aminobutyric acid type A (GABA(A)) /benzodiazepine receptor complex and GABA content was investigated in order to assess the GABAergic involvement in dopaminomimetic-induced dyskinesia. Three MPTP monkeys received for one month pulsatile administrations of the D1 dopamine (DA) receptor agonist SKF 82958 whereas three others received the same dose of SKF 82958 by continuous infusion. A long acting D2 DA receptor agonist, cabergoline, was given to another three animals. Untreated MPTP as well as naive control animals were also included. Pulsatile SKF 82958 relieved parkinsonian symptoms but was also associated with dyskinesia in two of the three animals whereas animals treated continuously with SKF 82958 remained as untreated MPTP monkeys. Chronic cabergoline administration improved motor response with no persistent dyskinesia. MPTP treatment induced a decrease of 3H-flunitrazepam binding in the medial anterior part of caudate-putamen and an increase in the internal segment of globus pallidus (GPi) which was in general unchanged by pulsatile or continuous SKF 82958 administration. Throughout the striatum, binding of 3H-flunitrazepam remained reduced in MPTP monkeys treated with cabergoline but was not significantly lower than untreated MPTP monkeys. Moreover, cabergoline treatment reversed the MPTP-induced increase in 3H-flunitrazepam binding in the GPi. GABA concentrations remained unchanged in the striatum, external segment of globus pallidus and GPi following MPTP denervation. Pulsatile but not continuous SKF 82958 administration decreased putamen GABA content whereas cabergoline treatment decreased caudate GABA. No alteration in GABA levels were observed in the GPe and GPi following the experimental treatments. These results suggest that: (1) D2-like receptor stimulation with cabergoline modulates GABA(A) receptor density in striatal subregions anatomically related to associative cortical afferent and (2) the absence of dyskinesia in dopaminomimetic-treated monkeys might be associated with the reversal of the MPTP-induced upregulation of the GABA(A)/benzodiazepine receptor complex in the Gpi.