Alpha-synuclein Immunoreactivity Research Articles

The role of gut-brain axis in a rotenone-induced rat model of Parkinson's disease

Parkinson's disease (PD) is a widespread neurodegenerative condition affecting millions globally. This investigation centered on the gut-brain axis in a rotenone-induced PD rat model. Researchers monitored behavioral shifts, histological modifications, neurodegeneration, and inflammation markers throughout the rats' bodies. Results revealed that rotenone-treated rats displayed reduced exploration (p = 0.004) and motor coordination (p < 0.001), accompanied by decreased Nissl staining and increased alpha-synuclein immunoreactivity in the striatum (p = 0.009). Additionally, these rats exhibited weight loss (T3, mean = 291.9 ± 23.67; T19, mean = 317.5 ± 17.53; p < 0.05) and substantial intestinal histological alterations, such as shortened villi, crypt architecture loss, and inflammation. In various regions, researchers noted elevated immunoreactivity to ionized binding adapter molecule (IBA)-1 (p < 0.05) and reduced immunoreactivity to glial fibrillary acidic protein (p < 0.05) and S100B (p < 0.001), indicating altered glial cell activity. Overall, these findings imply that PD is influenced by gut-brain axis changes and may originate in the intestine, impacting bidirectional gut-brain communication.

Post mortem evaluation of inflammation, oxidative stress, and PPARγ activation in a nonhuman primate model of cardiac sympathetic neurodegeneration.

Cardiac dysautonomia is a common nonmotor symptom of Parkinson's disease (PD) associated with loss of sympathetic innervation to the heart and decreased plasma catecholamines. Disease-modifying strategies for PD cardiac neurodegeneration are not available, and biomarkers of target engagement are lacking. Systemic administration of the catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) recapitulates PD cardiac dysautonomia pathology. We recently used positron emission tomography (PET) to visualize and quantify cardiac sympathetic innervation, oxidative stress, and inflammation in adult male rhesus macaques (Macaca mulatta; n = 10) challenged with 6-OHDA (50mg/kg; i.v.). Twenty-four hours post-intoxication, the animals were blindly and randomly assigned to receive daily doses of the peroxisome proliferator-activated receptor gamma (PPARγ) agonist pioglitazone (n = 5; 5mg/kg p.o.) or placebo (n = 5). Quantification of PET radioligand uptake showed increased oxidative stress and inflammation one week after 6-OHDA which resolved to baseline levels by twelve weeks, at which time pioglitazone-treated animals showed regionally preserved sympathetic innervation. Here we report post mortem characterization of heart and adrenal tissue in these animals compared to age and sex matched normal controls (n = 5). In the heart, 6-OHDA-treated animals showed a significant loss of sympathetic nerve fibers density (tyrosine hydroxylase (TH)-positive fibers). The anatomical distribution of markers of sympathetic innervation (TH) and inflammation (HLA-DR) significantly correlated with respective in vivo PET findings across left ventricle levels and regions. No changes were found in alpha-synuclein immunoreactivity. Additionally, CD36 protein expression was increased at the cardiomyocyte intercalated discs following PPARγ-activation compared to placebo and control groups. Systemic 6-OHDA decreased adrenal medulla expression of catecholamine producing enzymes (TH and aromatic L-amino acid decarboxylase) and circulating levels of norepinephrine, which were attenuated by PPARγ-activation. Overall, these results validate in vivo PET findings of cardiac sympathetic innervation, oxidative stress, and inflammation and illustrate cardiomyocyte CD36 upregulation as a marker of PPARγ target engagement.

Alpha-synuclein in gastric and colonic mucosa in Parkinson's disease: Limited role as a biomarker.

Gastric and colonic alpha-synuclein immunoreactivity has been reported in patients with Parkinson's disease (PD). However, enteric alpha-synuclein also has been reported in healthy individuals. We aimed to investigate the utility of alpha-synuclein immunoreactivity from gastric and colonic mucosal tissues obtained by routine endoscopy to detect PD, and to correlate the pathological burden of alpha-synuclein with motor and nonmotor features of PD. We recruited 104 study subjects, consisting of 38 patients with PD, 13 patients with probable multiple system atrophy (MSA), and 53 healthy controls. Gastric and colonic mucosal tissues obtained by endoscopic gastroduodenoscopy and colonoscopy were assessed using alpha-synuclein immunohistochemistry. Detailed motor and nonmotor features of PD were correlated with enteric alpha-synuclein immunoreactivity. No difference was seen in the enteric α-SYN immunoreactivity among patients with PD (31.6% for stomach and 10.4% for colon), patients with MSA (40.0% for stomach and 8.0% for colon), and healthy controls (33.3% for stomach and 18.5% for colon). The frequency of positive alpha-synuclein immunoreactivity was higher in gastric biopsy tissues than in colonic biopsy tissues in all of the study groups (P < 0.05). No significant correlation was found between the presence of alpha-synuclein immunoreactivity and the motor and nonmotor features of PD. The presence of alpha-synuclein immunoreactivity in gastric and colonic mucosa was detected in a similar manner in patients with PD, patients with MSA, and controls, thus suggesting a limited role of enteric mucosal alpha-synuclein as a diagnostic biomarker for PD. Future studies are warranted to detect pathological alpha-synuclein strains.

Alpha-synuclein immunoreactivity patterns in the enteric nervous system

We aimed to compare immunoreactivity patterns of four different anti-α-syn antibodies in surgical specimens of the gastrointestinal tract of Parkinson disease and control cases.Surgical specimens from stomach, small and large bowel of 6 PD cases and 12 controls were studied. Primary antibodies: anti-α-syn clone KM51, anti-phosphorylated α-syn Ser129, anti-α-syn clone 15G7 and anti-nitrated α-syn505. We found different immunoreactivity patterns: (a) coarse, Lewy-body-like aggregates labelled by the 4 antibodies and detected in 4/6 PD cases and in 1/12 controls; (b) distinct punctate cytoplasmic staining of ganglion cells labelled by anti-phosphorylated-α-syn and detected in 3/6 PD cases and 3/12 controls; (c) fine diffuse, synaptic-type staining of neural structures labelled by anti-α-syn-15G7 and anti-nitrated-α-syn505 and detected in all subjects.We conclude that different specific and non-specific immunoreactivity patterns are detected in surgical specimens of gastrointestinal tract when using different anti-α-syn antibodies, as they recognize different epitopes and states of alpha-synuclein protein. Coarse aggregates in neural structures seem to be the most promising marker for the diagnosis of Lewy-body parkinsonism when evaluating abnormal α-syn in the gastrointestinal tract.

P.19.3 Phenotypic characterization of the autosomal recessive (Pink-1 and DJ-1) gene knockout rat models of Parkinson’s disease

Recessively inherited loss-of-function mutations in the DJ-1 or PINK1 gene are linked to familial cases of early-onset Parkinson’s disease (PD). Single-knockout mice (lacking either DJ-1 or PINK1) exhibit disruptions of dopamine homeostasis but no nigral dopaminergic degeneration. As part of the strategy to provide more tools for the research community, the MJ. Fox Foundation for Parkinson’s Research (MJFF) has funded the generation of novel rat models lacking either DJ-1 or PINK1 genes and sponsored a standardized characterization including behavioral, neurochemical and pathological measures to determine if these rats exhibit progressive PD-like phenotype. In the presentation, we will discuss PINK1 KO rats motor dysfunction and gait impairment compared to wild-type rats (WT) up to 8-months of age. The DJ-1 KO rats expressed similar motor and gait abnormalities, however these deficits were less pronounced and appeared mostly between 6 and 8 months of age. Characterizations of alpha-synuclein immunoreactivity and other biomarkers levels in these rat models are currently ongoing. These preliminary data demonstrate that loss of either the PINK1 or the DJ-1 gene in rats produces a progressive loss of dopaminergic neurons, and that this neurodegeneration may be correlated to progressive motor and gait deficits. Thus, these MJFF-generated Pink1 and DJ-1 knockout rats will help elucidate the mechanisms by which these recessive genes produce PD pathology and aid in therapeutic development.

Diagnostic value of minor salivary glands biopsy for the detection of Lewy pathology

The recent demonstration of the presence of Lewy pathology in the submandibular glands of Parkinson's disease (PD) patients prompted us to evaluate the diagnostic performance of minor salivary gland biopsy for PD. Minor salivary glands were examined for Lewy pathology using phosphorylated alpha-synuclein antibody in 16 patients with clinically diagnosed PD and 11 control subjects with other neurological disorders. Abnormal accumulation of alpha-synuclein was found in 3 out of 16 PD patients. Two control subjects exhibited weak phosphorylated alpha-synuclein immunoreactivity. Our results do not support the use of minor salivary glands biopsy for the detection of Lewy pathology in living subjects.

Comparison of alpha-synuclein immunoreactivity in the spinal cord between the adult and aged beagle dog

Alpha-synuclein (α-syn) is a presynaptic protein that is richly expressed in the central and peripheral nervous systems of mammals, and it is related to the pathogenesis of Parkinson's disease and other neurodegenerative disorders. In the present study, we compared the distribution of the immunoreactivity of α-syn and its related gliosis in the spinal cord of young adult (2-3 years) and aged (10-12 years) beagle dogs. We discovered that α-syn immunoreactivity was present in many neurons in the thoracic level of the aged spinal cord, however, its protein level was not distinct inform that of the adult spinal cord. In addition, ionized calcium-binding adapter molecule-1 (a marker for microglia) immunoreactivity, and not glial fibrillary acidic protein (a marker for astrocytes) immunoreactivity, was somewhat increased in the aged group compared to the adult group. These results indicate that α-syn immunoreactivity was not dramatically changed in the dog spinal cord during aging.

First neuropathological description of a patient with Parkinson’s disease and LRRK2 p.N1437H mutation

The c.4309A>C mutation in the LRRK2 gene (LRRK2 p.N1437H) has recently been reported as the seventh pathogenic LRRK2 mutation causing monogenic Parkinson’s disease (PD). So far, only two families worldwide have been identified with this mutation. By screening DNA from seven brains of PD patients, we found one individual with seemingly sporadic PD and LRRK2 p.N1437H mutation. Clinically, the patient had levodopa-responsive PD with tremor, and developed severe motor fluctuations during a disease duration of 19 years. There was severe and painful ON-dystonia, and severe depression with suicidal thoughts during OFF. In the advanced stage, cognition was slow during motor OFF, but there was no noticeable cognitive decline. There were no signs of autonomic nervous system dysfunction. Bilateral deep brain stimulation of the subthalamic nucleus had unsatisfactory results on motor symptoms. The patient committed suicide. Neuropathological examination revealed marked cell loss and moderate alpha-synuclein positive Lewy body pathology in the brainstem. There was sparse Lewy pathology in the cortex. A striking finding was very pronounced ubiquitin-positive pathology in the brainstem, temporolimbic regions and neocortex. Ubiquitin positivity was most pronounced in the white matter, and was out of proportion to the comparatively weaker alpha-synuclein immunoreactivity. Immunostaining for tau was mildly positive, revealing non-specific changes, but staining for TDP-43 and FUS was entirely negative. The distribution and shape of ubiquitin-positive lesions in this patient differed from the few previously described patients with LRRK2 mutations and ubiquitin pathology, and the ubiquitinated protein substrate remains undefined.

Alpha‐synuclein immunoreactivity in minor salivary gland biopsies of Parkinson's disease patients

Alpha‐synuclein immunoreactivity in minor salivary gland biopsies of Parkinson's disease patients

Modeling sporadic Parkinson's disease by silencing the ubiquitin E3 ligase component, SKP1A

Large-scale transcriptomics analysis of gene expression profile of sporadic Parkinson's disease (PD) substantia nigra (SN) has identified a number of differentially expressed genes participating in the neurotoxic cascade of DA neurons death, in particular those related to handling of proteins, dopaminergic transmission and iron metabolism. One of them, SKP1A (p19, S-phase kinase-associated protein 1A), an essential component of the ubiquitin-E3 ligase Skp1, Cullin 1, F-box protein (SCF) complex, has been found to be significantly decreased in the SN pars compacta of post-mortem parkinsonian brains. Recently, a new genetic cell model of sporadic PD was developed by knocking-down SKP1A in SN-derived cell-line infected with short hairpin RNA lentiviruses. SKP1A deficiency resulted in increased susceptibility to cell death and a decline in the expression of dopaminergic phenotypic markers. SKP1A-silenced cells were unable to arrest at G(0)/G(1,) when induced to differentiate, entering into an aberrant cell cycle and progressive death. During this process the cells developed rounded aggregates with characteristics of LB-like inclusions (aggresomes) including immunoreactivity for gamma-tubulin, alpha-synuclein, ubiquitin, tyrosine hydroxylase, Hsc-70 and proteasome subunit. In conclusion, future studies should focus on a careful consideration of crucial dopaminergic interacting genes, as emerged from human sporadic PD, which will serve as a basis for the development of a slowly progressive genetic animal model of sporadic PD, with the potential of evaluating drugs with "disease modifying activity".

Dopamine D2 receptor knockout mice develop features of Parkinson disease

This study questions whether increased dopamine (DA) turnover in nigral neurons leads to formation of Lewy bodies (LBs), the characteristic alpha-synuclein-containing cytoplasmic inclusion of Parkinson disease (PD). Mice with targeted deletion of the dopamine D(2) receptor gene (D(2)R[-/-]) have higher striatal and nigral dopamine turnover and elevated oxidative stress. These mice were examined for evidence of histological, biochemical, and gene expression changes consistent with a synucleinopathy. LB-like cytoplasmic inclusions containing alpha-synuclein and ubiquitin were present in substantia nigra pars compacta (SNpc) neurons of older D(2)R(-/-) mice, and were also occasionally seen in aged wild-type mice. These inclusions displaced the nucleus of affected cells and were eosinophilic. Diffuse cytosolic alpha-synuclein immunoreactivity in SNpc neurons increased with age in both wild-type and D(2)R(-/-) mice, most likely because of redistribution of alpha-synuclein from striatal terminals to SNpc cell bodies. Gene and protein expression studies indicated endoplasmic reticulum (ER) stress and changes in trafficking and autophagic pathways in D(2)R(-/-) SNpc. These changes were accompanied by a loss of DA terminals in the dorsal striatum, although there was no evidence of progressive cell death in the SNpc. Increased sprouting and DA turnover, as observed in PD and D(2)R(-/-) mice, augments LB-like inclusions and axonal degeneration of dopaminergic neurons. These changes are associated with ER stress and autophagy.

No Lewy pathology in monkeys with over 10 years of severe MPTP Parkinsonism

The recent knowledge that 10 years after transplantation surviving human fetal neurons adopt the histopathology of Parkinson's disease suggests that Lewy body formation takes a decade to achieve. To determine whether similar histopathology occurs in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-primate models over a similar timeframe, the brains of two adult monkeys made parkinsonian in their youth with intermittent injections of MPTP were studied. Despite substantial nigral degeneration and increased alpha-synuclein immunoreactivity within surviving neurons, there was no evidence of Lewy body formation. This suggests that MPTP-induced oxidative stress and inflammation per se are not sufficient for Lewy body formation, or Lewy bodies are human specific.

Association of α-Synuclein Immunoreactivity With Inflammatory Activity in Multiple Sclerosis Lesions

Multiple sclerosis (MS) has neurodegenerative features including neuronal and axonal loss and widespread atrophy of the brain and spinal cord. The cause of this neurodegeneration has been largely attributed to inflammation, but other mechanisms, including those associated with classic neurodegenerative diseases such as the alpha-synucleinopathies, might also be involved in MS pathogenesis. In this study, 96 brain lesions containing varying degrees of inflammatory activity from 12 autopsied MS cases were compared with corresponding regions from 6 neuropathologically normal controls; 2 cerebral biopsy lesions from an MS patient were also studied. We found alpha-synuclein immunoreactivity in the cytoplasm of cells in MS lesions with inflammatory activity but not in control samples. alpha-Synuclein-immunoreactive cells were identified in active (15/15 lesions in the brainstem, 9/13 in cerebral hemispheres) and chronic active (14/15 in the brainstem, 12/22 in cerebral hemispheres) lesions but were absent in chronic inactive lesions (0/31); the greater immunoreactivity in brainstem compared with cerebral hemisphere lesions was significant (p < 0.05). Double-immunofluorescence staining revealed localization of alpha-synuclein immunoreactivity mostly in neurons, microglia/macrophages, and oligodendrocytes, and only rarely in astrocytes. The results suggest that alpha-synuclein expression regulated by inflammatory signals may contribute to neurodegenerative processes in MS lesions.

Lewy Body Pathology Involves Cutaneous Nerves

Involvement of the peripheral autonomic nervous system is a core feature of Lewy body (LB) diseases, including Parkinson disease (PD), PD with dementia, and dementia with LBs. To investigate the potential use of skin biopsy for the diagnosis of LB diseases, we assessed anti-phosphorylated alpha-synuclein immunoreactivity in peripheral nerves in samples of skin from the abdominal wall and flexor surface of the upper arm in 279 prospectively studied consecutively autopsied patients whose data were registered at the Brain Bank for Aging Research between 2002 and 2005. Positive immunoreactivity was demonstrated in the unmyelinated fibers of the dermis in 20 of 85 patients with LB pathology in the CNS and the adrenal glands, the latter representing a substitute for peripheral autonomic nervous system sympathetic ganglia; no reactivity was seen in 194 patients without CNS LB pathology. In 142 retrospectively studied patients autopsied from 1995 onward who had subclinical or clinical LB disease, the sensitivity of the positive skin immunoreactivity was 70% in PD and PD with dementia and 40% in dementia with LBs. Skin immunoreactivity was absent in cases of multiple-system atrophy, progressive nuclear palsy, and corticobasal degeneration. We demonstrate for the first time that the skin is involved and may be a highly specific and useful biopsy site for the pathological diagnosis of LB diseases.

Pathologic modifications of alpha-synuclein in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated squirrel monkeys.

alpha-Synuclein expression is increased in dopaminergic neurons challenged by toxic insults. Here, we assessed whether this upregulation is accompanied by pathologic accumulation of alpha-synuclein and protein modifications (i.e. nitration, phosphorylation, and aggregation) that are typically observed in Parkinson disease and in other synucleinopathies. A single injection of the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to squirrel monkeys caused a buildup of alpha-synuclein but not of beta-synuclein or synaptophysin within nigral dopaminergic cell bodies. Immunohistochemistry and immunoelectron microscopy also revealed large numbers of dystrophic axons labeled with alpha-synuclein. Antibodies that recognize nitrated and phosphorylated (at serine 129) alpha-synuclein stained neuronal cell bodies and dystrophic axons in the midbrain of MPTP-treated animals. After toxicant exposure, alpha-synuclein deposition occurred at the level of neuronal axons in which amorphous protein aggregates were observed by immunoelectron microscopy. In a subset of these axons, immunoreactivity for alpha-synuclein was still evident after tissue digestion with proteinase K, further indicating the accumulation of insoluble protein. These data indicate that toxic injury can induce alpha-synuclein modifications that have been implicated in the pathogenesis of human synucleinopathies. The findings are also consistent with a pattern of evolution of alpha-synuclein pathology that may begin with the accumulation and aggregation of the protein within damaged axons.

Nigral burden of α‐synuclein correlates with striatal dopamine deficit

Aggregated alpha-synuclein is the hallmark of Parkinson's disease (PD), diffuse Lewy body disease (DLBD), and multiple system atrophy (MSA). Physiologically, alpha-synuclein ensures normal functions of dopamine transporter (DAT) and tyrosine hydoxylase. In alpha-synucleinopathies, it accumulates in neuronal cytoplasm and neurites through several stages. It is unclear whether the accumulation of pathological alpha-synuclein in the substantia nigra in PD correlates with the dopaminergic deficit in the striatal target. We evaluated the impact of the nigral burden of pathological alpha-synuclein immunoreactivity in 27 alpha-synucleinopathy brains by morphometric immunohistochemistry. DAT immunoreactivity in the striatum inversely correlates with the total alpha-synuclein burden in the substantia nigra but not with cytoplasmic inclusion counts only. This result has implications for imaging, clinicopathological correlative studies, and staging of the disease process.

Olfactory deficits in mice overexpressing human wildtype alpha-synuclein.

Accumulation of alpha-synuclein in neurons of the central and peripheral nervous system is a hallmark of sporadic Parkinson's disease (PD) and mutations that increase alpha-synuclein levels cause familial PD. Transgenic mice overexpressing alpha-synuclein under the Thy1 promoter (Thy1-aSyn) have high levels of alpha-synuclein expression throughout the brain but no loss of nigrostriatal dopamine neurons up to 8 months, suggesting that they may be useful to model pre-clinical stages of PD. Olfactory dysfunction often precedes the onset of the cardinal motor symptoms of PD by several years and includes deficits in odor detection, discrimination and identification. In the present study, we measured olfactory function in 3- and 9-month-old male Thy1-aSyn mice with a buried pellet test based on latency to find an exposed or hidden odorant, a block test based on exposure to self and non-self odors, and a habituation/dishabituation test based on exposure to non-social odors. In a separate group of mice, alpha-synuclein immunoreactivity was assessed in the olfactory bulb. Compared with wildtype littermates, Thy1-aSyn mice could still detect and habituate to odors but showed olfactory impairments in aspects of all three testing paradigms. Thy1-aSyn mice also displayed proteinase K-resistant alpha-synuclein inclusions throughout the olfactory bulb. These data indicate that overexpression of alpha-synuclein is sufficient to cause olfactory deficits in mice similar to that observed in patients with PD. Furthermore, the buried pellet and block tests provided sufficient power for the detection of a 50% drug effect, indicating their usefulness for testing novel neuroprotective therapies.

Striatal and nigral pathology in a lentiviral rat model of Machado-Joseph disease

Machado-Joseph disease (MJD) is a fatal, dominant neurodegenerative disorder. MJD results from polyglutamine repeat expansion in the MJD-1 gene, conferring a toxic gain of function to the ataxin-3 protein. In this study, we aimed at overexpressing ataxin-3 in the rat brain using lentiviral vectors (LV), to generate an in vivo MJD genetic model and, to study the disorder in defined brain regions: substantia nigra, an area affected in MJD, cortex and striatum, regions not previously reported to be affected in MJD. LV encoding mutant or wild-type human ataxin-3 was injected in the brain of adult rats and the animals were tested for behavioral deficits and neuropathological abnormalities. Striatal pathology was confirmed in transgenic mice and human tissue. In substantia nigra, unilateral overexpression of mutant ataxin-3 led to: apomorphine-induced turning behavior; formation of ubiquitinated ataxin-3 aggregates; alpha-synuclein immunoreactivity; and loss of dopaminergic markers (TH and VMAT2). No neuropathological changes were observed upon wild-type ataxin-3 overexpression. Mutant ataxin-3 expression in striatum and cortex, resulted in accumulation of misfolded ataxin-3, and within striatum, loss of neuronal markers. Striatal pathology was confirmed by observation in MJD transgenic mice of ataxin-3 aggregates and substantial reduction of DARPP-32 immunoreactivity and, in human striata, by ataxin-3 inclusions, immunoreactive for ubiquitin and alpha-synuclein. This study demonstrates the use of LV encoding mutant ataxin-3 to produce a model of MJD and brings evidence of striatal pathology, suggesting that this region may contribute to dystonia and chorea observed in some MJD patients and may represent a target for therapies.

The exploration of rotenone as a toxin for inducing Parkinson's disease in rats, for application in BBB transport and PK–PD experiments

Introduction In search for a suitable rat model to study potentially affected blood-brain barrier (BBB) transport mechanisms in the course of Parkinsons disease (PD) progression, experiments were performed to characterise Parkinsons disease markers following subcutaneous (SC) and intracerebral (IC) infusion of the toxin rotenone in the rat. Methods Studies were performed using Male Lewis rats. SC infusion of rotenone (3 mg/kg/day) was performed via an osmotic minipump. IC infusion of rotenone occurred directly into the right medial forebrain bundle at three different dosages. At different times following rotenone infusion, behaviour, histopathology (tyrosine hydroxylase and alpha-synuclein immunocytochemistry), peripheral organ pathology (adrenals, heart, kidney, liver, lung, spleen and stomach) were assessed. In part of the SC and IC rats, BBB transport profiles of the permeability marker sodium fluorescein were determined using microdialysis. Results SC rotenone failed to produce dopaminergic lesions and led to extensive peripheral organ toxicity. BBB permeability for fluorescein following SC rotenone was changed, however due peripheral toxicity. In contrast, IC rotenone produced a progressive lesion of the nigrostrial dopaminergic pathway over 28 days with no associated peripheral toxicity. IC rotenone also exhibited a large increase in amphetamine induced rotational behaviour. In addition, a few IC rats showed alpha-synuclein immunoreactivity and aggregation. Following IC rotenone, no changes in BBB permeability were detected after 14 days. Discussion SC rotenone only produced peripheral toxicity. IC rotenone appeared to create a progressive lesion of the rat nigrostrial pathway, and may therefore be a more appropriate model of Parkinson's disease progression, compared with the most commonly used 6-OH-DA rat model.

Depletion of putative chemosensitive respiratory neurons in the ventral medullary surface in multiple system atrophy

Multiple system atrophy (MSA) is a disorder that may manifest with reduced respiratory chemosensitivity and central sleep apnoea. Chemosensitive glutamatergic and serotonergic neurons located just beneath the ventral medullary surface, corresponding to the human arcuate nucleus (ArcN), have recently been implicated in control of automatic breathing in response to hypercapnia and hypoxia. We sought to determine whether these neurons were affected in MSA. Medullae were obtained at post-mortem from 11 patients (8 men, 3 women, age 64 +/- 3 years) with neuropathologically confirmed MSA and 11 control subjects (6 men and 5 women, age 66 +/- 4 years). Fifty micrometre sections obtained throughout the medulla were processed for vesicular glutamate transporter-2 (VGLUT-2), tryptophan-hydroxylase (TrOH), glial fibrillary acid protein (GFAP) and alpha-synuclein immunoreactivity. Cell counts, GFAP immunoreactivity and presence of glial cytoplasmic inclusions (GCIs) were assessed in the ArcN. In MSA, compared with controls, there was a marked depletion of ArcN neurons immunoreactive for either VGLUT-2 (74 +/- 21 versus 342 +/- 84 cells/section, P < 0.004) or TrOH (5 +/- 1 versus 16 +/- 2 cells/section, P < 0.001). There was also marked astrocytic gliosis and accumulation of alpha-synuclein immunoreactive GCIs in the ventral medullary surface in all cases. Our results indicate that there is severe loss of putative chemosensitive glutamatergic and serotonergic neurons as well as marked astrocytic gliosis in the ventral medullary surface in MSA. This may provide a possible morphological basis for impaired respiratory chemosensitivity and central sleep apnoea in this disorder.