Relative preservation of thalamic centromedian nucleus in parkinsonian patients with dystonia.

To determine whether variable thalamic degeneration in Parkinson's disease (PD) contributes to less drug responsive clinical features. Formalin-fixed thalami from longitudinally followed patients with PD and early dystonia (N = 6), early falls (N = 5) or no dystonia or falls (N = 6) and age-matched controls without neuropathology (N = 10) were serially sectioned, stained, and analyzed. Neurons in the centromedian parafascicular (CM-Pf) nucleus were quantified using the optical disector method and analysis of variance with post hoc testing used to determine variability in neurodegeneration between groups. Patients with PD were confirmed to have significant neurodegeneration in the CM-Pf complex, with no difference in the degree of neurodegeneration between patients with PD with early falls compared with patients with no history of falls or dystonia. In contrast, patients with PD with early dystonia had significantly less neurodegeneration of the CM but not the Pf than patients without this feature. Preservation of the CM in patients with PD with early dystonia would result in a relative increase in CM activity through the direct basal ganglia pathway and increased primary motor cortex activity. Overall this data provides evidence for pathway-specific neurodegeneration as an underlying feature of the clinical variability observed in patients with PD.

Oral pre-treatment with epigallocatechin gallate in 6-OHDA lesioned rats produces subtle symptomatic relief but not neuroprotection.

Much recent work is investigating the role of oxidative stress and inflammatory mechanisms in the aetiology of neurodegeneration in Parkinson's disease. The present study evaluated whether the green tea constituent epigallocatechin gallate (EGCG) which has both anti-oxidant and anti-inflammatory properties, exerts neuroprotection and symptomatic effects when administered orally as a pre-treatment prior to 6-hydroxydopamine (6-OHDA) lesions. Groups of rats were given either 1mg/kg, 2mg/kg EGCG or vehicle solution for 14 days. Sham or 6-OHDA surgery was performed on day 11 of the drug administration protocol. Behavioural analysis was conducted before drugs/vehicle solution, again during the treatment period and then repeated at fortnightly intervals for 2 months post-operatively. Whilst some subtle behavioural improvements in postural abnormalities and ability to cross a narrow beam were observed in lesioned rats after EGCG (vs. vehicle) there was no evidence of neuroprotection on post-mortem quantification of degree of nigral dopaminergic neuronal loss when comparing the lesioned groups given the various treatments.

Regulation of beta-catenin trafficking to the membrane in living cells.

Beta-catenin is a key mediator of the Wnt signaling process and accumulates in the nucleus and at the membrane in response to Wnt-mediated inhibition of GSK-3beta. In this study we used live cell photobleaching experiments to determine the dynamics and rate of recruitment of beta-catenin at membrane adherens junctions (cell adhesion) and membrane ruffles (cell migration). First, we confirmed the nuclear-cytoplasmic shuttling of GFP-tagged beta-catenin, and found that a small mobile pool of beta-catenin can move from the nucleus to membrane ruffles in NIH 3T3 fibroblasts with a t(0.5) of approximately 30 s. Thus, beta-catenin can shuttle between the nucleus and plasma membrane. The localized recruitment of beta-catenin-GFP to membrane ruffles was more rapid, and the strong recovery observed after bleaching (mobile fraction 53%, t(0.5) approximately 5 s) is indicative of high turnover and transient association. In contrast, beta-catenin-GFP displayed poor recovery at adherens junctions in MDCK epithelial cells (mobile fraction 10%, t(0.5) approximately 8 s), indicating stable retention at these membrane structures. We previously identified IQGAP1 as an upstream regulator of beta-catenin at the membrane, and this is supported by photobleaching assays which now reveal IQGAP1 to be more stably anchored at membrane ruffles than beta-catenin. Further analysis showed that LiCl-mediated inactivation of the kinase GSK-3beta increased beta-catenin membrane ruffle staining; this correlated with a faster rate of recruitment and not increased membrane retention of beta-catenin. In summary, beta-catenin displays a high turnover rate at membrane ruffles consistent with its dynamic internalization and recycling at these sites by macropinocytosis.

Use of the narrow beam test in the rat, 6-hydroxydopamine model of Parkinson's disease.

Batteries of behavioural tests provide a method by which researchers may examine specific functional pathways. The narrow beam test examines the ability of a rat to cross a narrow, elevated beam of wood or other material. In order to determine the utility of the narrow beam test in the study of Parkinsonism, it was of interest to characterise the performance of animals at this task. Rats were placed at one end of a 105 cm long, elevated beam and both the time it took to begin crossing the beam, as well as the total time taken to cross the beam, were measured. The effects of training, time of day and 6-hydroxydopamine lesion on beam performance were examined. Rats reached maximal performance at the task within a single test session and time of day had no effect on beam performance. Parkinsonian rats demonstrated a four-fold increase in both the latency to initiate the task and the total time to cross the beam (p < 0.05).

Novel oral drug administration in an animal model of neuroleptic therapy.

A novel method of oral drug administration was used in a neuroleptic animal study. Seventy male Sprague-Dawley rats were randomly subdivided into four groups, which were treated with clozapine, haloperidol, diazepam or a vehicle solution (5% sucrose solution). Oral drug treatment was achieved by training the rats to drink the drug of choice mixed with five percent sucrose or vehicle solution from a syringe. Within 3-4 weeks the haloperidol group developed vacuous chewing movement, which did not disappear with discontinuation of the drug. Significant weight gain was observed for all drug groups in relation to the control group, whereas only the diazepam group showed a significant increase in response latency on the disengage test of sensorimotor function, which disappeared with drug withdrawal. A novel means of testing the motivational status showed that all drug-treated groups engaged in eating chocolate before grooming (t=11.69, p<0.001), whereas the control group showed no specific tendency towards either task. Furthermore, there was a significant delay in grooming for the haloperidol group compared to the other drug groups and controls. In conclusion, a novel method of oral drug administration with minimum stress was introduced that was sufficient to cause the described changes in behavioural parameters. Additionally, the combination of tests used provided an efficient discrimination between the behavioural effects of clozapine, haloperidol and diazepam in rodents.

A comparison of degeneration in motor thalamus and cortex between progressive supranuclear palsy and Parkinson's disease.

Changes in motor cortical activation are associated with the major symptoms observed in both Parkinson's disease and progressive supranuclear palsy (PSP). While research has concentrated on basal ganglia abnormalities as central to these cortical changes, several studies in both disorders have shown pathology in the thalamus and motor cortices. In particular, we recently reported an 88% loss of corticocortical projection neurones in the pre-supplementary motor (pre-SMA) cortex in Parkinson's disease. Further analysis of the degree of neuronal loss and pathology in motor cortices and their thalamocortical relays in Parkinson's disease and PSP is warranted. Six cases with PSP, nine cases with Parkinson's disease and nine controls were selected from a prospectively studied brain donor cohort. alpha-Synuclein, ubiquitin and tau immunohistochemistry were used to identify pathological lesions. Unbiased stereological methods were used to analyse atrophy and neuronal loss in the motor thalamus [ventral anterior, ventrolateral anterior and ventrolateral posterior (VLp) nuclei] and motor cortices (primary motor, dorsolateral premotor and pre-SMA cortices). Analysis of variance and post hoc testing was used to determine differences between groups. In Parkinson's disease, the motor thalamus and motor cortices (apart from the pre-SMA) were preserved containing only rare alpha-synuclein-positive and ubiquitin-positive Lewy bodies. In contrast, patients with PSP had significant atrophy and neuronal loss in VLp (22 and 30%, respectively), pre-SMA (21 and 51%, respectively) and primary motor cortices (33 and 54%, respectively). In the primary motor cortex of PSP cases, neuronal loss was confined to inhibitory interneurones, whereas in the pre-SMA both interneurones (reduced by 26%) and corticocortical projection neurones (reduced by 82%) were affected. Tau-positive neurofibrillary and glial tangles were observed throughout the motor thalamus and motor cortices in PSP. These non-dopaminergic lesions in motor circuits are likely to contribute to the pathogenesis of both PSP and Parkinson's disease. The selective involvement of the VLp and primary motor cortex in PSP implicates these cerebellothalamocortical pathways as differentiating this disease, possibly contributing to the early falls.

Postural changes after lesions of the substantia nigra pars reticulata in hemiparkinsonian monkeys.

Current neurosurgical strategies target overactive brain regions including the subthalamic nucleus, globus pallidus and thalamus to control various symptoms of Parkinson's disease. Subthalamotomy improves akinesia and can induce postural deficits in both parkinsonian humans and animals, pallidotomy improves limb dyskinesia and more variably, distal bradykinesia whilst thalamotomy improves tremor. Because the SNr also becomes overactive in PD and there are few surgical studies in parkinsonian primates, we therefore evaluated the effects of lesioning the SNr in hemiparkinsonian marmosets to establish the effects on symptomatology. Nine monkeys received unilateral 6-hydroxydopamine (6-OHDA) lesions. Seven weeks later, four received kainic acid lesions of the SNr. Behavioural tests were performed prior to 6-OHDA surgery and then fortnightly for 14 weeks. Unilateral 6-OHDA lesions induced ipsilateral postural bias, ipsilateral rotation after amphetamine injection and bradykinesia. Whilst, SNr lesions significantly altered the direction of head position and amphetamine-induced rotation relative to 6-OHDA lesions, there was no improvement in 6-OHDA-induced reaching deficits or sensorimotor neglect. Unbiased quantitation of the nigral lesions showed on average 88% loss of dopaminergic neurons after 6-OHDA lesions and 77% loss of non-dopaminergic neurons after SNr lesions. Our results demonstrate that the SNr is important in body orientation changes in parkinsonism.

Experimental therapeutics of Parkinson's disease.

1. The loss of central dopamine, which characterises Parkinson's disease, led to the main pharmacological strategy for treatment, namely levodopa, a dopamine-replacement therapy. Several years after treatment, the majority of patients experience dose-limiting side-effects and loss of symptom control. There is a resurgence of interest in neurosurgery for treating the Parkinson's disease, particularly in new techniques targeting the subthalamic nucleus (STN), which is overactive in Parkinson's disease and contributes to symptom development. 2. We performed unilateral subthalamotomy (lesioning the subthalamic nucleus via the toxin N-methyl-d-aspartate) in marmosets and rats with experimentally induced parkinsonism (induced using the toxin 6-hydroxydopamine). A range of similar behaviours common to both rodents and primates were evaluated before and after each type of surgery. Post-mortem histology was used to confirm the lesions. We also provide details of a case with Parkinson's disease who underwent high-frequency bilateral stimulation of the STN and in whom we analysed the STN post-mortem. 3. Unilateral subthalamotomy improved akinesia in parkinsonian primates. However, both monkeys and rodents showed postural abnormalities. The patient who underwent bilateral high-frequency stimulation showed improvement of akinesia and other disease symptoms and no postural abnormalities. Post-mortem analysis did not demonstrate substantial damage of the STN as a result of the electrodes. 4. Although unilateral subthalamotomy improves some aspects of parkinsonism, it causes postural abnormalities in animal models of Parkinson's disease. Because bilateral high-frequency STN stimulation improves disease symptoms, is reversible and is not reported to induce postural side-effects, it may be a better surgical therapy for Parkinson's disease than lesioning the STN.

Convulsive and postural effects of lesioning the mid-substantia nigra pars reticulata in naïve and 6-hydroxydopamine lesioned rats.

The subthalamic nucleus is targeted for the treatment of Parkinson's disease. Unilateral lesions improve some aspects of parkinsonism but produce postural abnormalities in animal models but the exact pathways producing these effects remain to be defined. Using a battery of tests we evaluated the effects of lesioning one of the two major subthalamic targets, the substantia nigra pars reticulata in naïve and 6-OHDA lesioned rats. Lesions targeting the mid-substantia nigra pars reticulata resulted in acute tonic-clonic seizures and intense contralateral rotational asymmetry. During the first month after substantia nigra pars reticulata lesions there was normalisation of the ipsilateral head position bias induced by unilateral 6-OHDA lesions, significant contralateral body axis bias but no significant alteration of apomorphine induced rotation and sensorimotor neglect in 6-OHDA lesioned rats. Combined with our previous data, this suggests that subthalamic projections via the substantia nigra pars reticulata are important in seizures and postural behaviours. Therefore unilateral subthalamotomy probably induces postural deficits in hemiparkinsonian animals via projections involving the substantia nigra pars reticulata. This has implications for patients undergoing subthalamotomy for treatment of Parkinson's disease.

Clinical correlates of selective pathology in the amygdala of patients with Parkinson's disease.

The amygdala exhibits significant pathological changes in Parkinson's disease, including atrophy and Lewy body (LB) formation. Amygdala pathology has been suggested to contribute to some clinical features of Parkinson's disease, including deficits of olfaction and facial expression. The degree of neuronal loss in amygdala subnuclei and the relationship with LB formation in non-demented Parkinson's disease cases have not been examined previously. Using stereological methods, the volume of neurones and the number of neurones in amygdala subdivisions were estimated in 18 prospectively studied, non-demented patients with Parkinson's disease and 16 age- and sex-matched controls. Careful exclusion (all cortical disease) and inclusion (non-demented, levodopa-responsive, idiopathic Parkinson's disease or controls) criteria were applied. Seven Parkinson's disease cases experienced well-formed visual hallucinations many years after disease onset, while nine Parkinson's disease cases and three controls were treated for depression. Anatomically, the amygdala was subdivided into the lateral nucleus, the basal (basolateral and basomedial) nuclei and the corticomedial (central, medial and cortical nuclei) complex. LB and Lewy neurites were identified by immunohistochemistry for alpha-synuclein and ubiquitin and were assessed semiquantitatively. LB were found throughout the amygdala in Parkinson's disease, being present in approximately 4% of neurones. Total amygdala volume was reduced by 20% in Parkinson's disease (P = 0.02) and LB concentrated in the cortical and basolateral nuclei. Lewy neurites were present in most cases but did not correlate with any structural or functional variable. Amygdala volume loss was largely due to a 30% reduction in volume (P = 0.01) and the total estimated number of neurones (P = 0.007) in the corticomedial complex. The degree of neurone loss and the proportion of LB-containing neurones in the cortical nucleus within this complex were constant across Parkinson's disease cases and neither variable was related to disease duration (R(2 )< 0.03; P > 0.5). The cortical nucleus has major olfactory connections and its degeneration is likely to contribute to the early selective anosmia common in Parkinson's disease. There was a small reduction in neuronal density in the basolateral nucleus in all Parkinson's disease cases, but no consistent volume or cell loss within this region. However, the proportion of LB-containing neurones in the basolateral nucleus was nearly doubled in cases that exhibited visual hallucinations, suggesting that neuronal dysfunction in this nucleus contributes to this late clinical feature. Detailed quantitation of the other amygdala subdivisions failed to reveal any other substantial anomalies or any associations with depression. Thus, the impact of Parkinson's disease on the amygdala is highly selective and correlates with both early and late clinical features.

Postmortem analysis of bilateral subthalamic electrode implants in Parkinson's disease.

This is the second neuropathological report detailing bilateral electrodes targeting the subthalamic nucleus (STN) in idiopathic Parkinson's disease (PD). The patient presented with unilateral tremor-dominant parkinsonism. Bilateral STN stimulation was carried out 7 years later due to significant disease progression and severe motor fluctuations. The patient exhibited bilateral improvements in rigidity and bradykinesia both intraoperatively and postoperatively. The patient died 2 months later from aspiration pneumonia. Neuropathological examination confirmed both the diagnosis of PD and the electrode placements. The tip of the left electrode was located medially and posteriorly in the left STN and the tip of the right electrode entered the base of the thalamus/zona incerta immediately above the right STN. Tissue changes associated with the subthalamic electrode tracts included mild cell loss, astrogliosis, and some tissue vacuolation. Our postmortem analysis indicates little tissue damage associated with STN stimulation for PD.