Treadmill exercise delays the onset of non-motor behaviors and striatal pathology in the CAG140 knock-in mouse model of Huntington's disease.

Depression, cognitive impairments, and other neuropsychiatric disturbances are common during the prodromal phase of Huntington's disease (HD) well before the onset of classical motor symptoms of this degenerative disorder. The purpose of this study was to examine the potential impact of physical activity in the form of exercise on a motorized treadmill on non-motor behavioral features including depression-like behavior and cognition in the CAG140 knock-in (KI) mouse model of HD. The CAG140 KI mouse model has a long lifespan compared to other HD rodent models with HD motor deficits emerging after 12months of age and thus provides the opportunity to investigate early life interventions such as exercise on disease progression. Motorized treadmill running was initiated at 4weeks of age (1h per session, 3 times per week) and continued for 6months. Non-motor behaviors were assessed up to 6months of age and included analysis of depression-like behavior (using the tail-suspension and forced-swim tests) and cognition (using the T-maze and object recognition tests). At both 4 and 6months of age, CAG140 KI mice displayed significant depression-like behavior in the forced swim and tail suspension tests and cognitive impairment by deficits in reversal relearning in the T-maze test. These deficits were not evident in mice engaged in treadmill running. In addition, exercise restored striatal dopamine D2 receptor expression and dopamine neurotransmitter levels both reduced in sedentary HD mice. Finally, we examined the pattern of striatal expression of mutant huntingtin (mHTT) protein and showed that the number and intensity of immunohistochemical staining patterns of intranuclear aggregates were significantly reduced with exercise. Altogether these findings begin to address the potential impact of lifestyle and early intervention such as exercise on modifying HD progression.

The Effects of Exercise on Dopamine Neurotransmission in Parkinson's Disease: Targeting Neuroplasticity to Modulate Basal Ganglia Circuitry.

Animal studies have been instrumental in providing evidence for exercise-induced neuroplasticity of corticostriatal circuits that are profoundly affected in Parkinson's disease. Exercise has been implicated in modulating dopamine and glutamate neurotransmission, altering synaptogenesis, and increasing cerebral blood flow. In addition, recent evidence supports that the type of exercise may have regional effects on brain circuitry, with skilled exercise differentially affecting frontal-striatal related circuits to a greater degree than pure aerobic exercise. Neuroplasticity in models of dopamine depletion will be reviewed with a focus on the influence of exercise on the dorsal lateral striatum and prefrontal related circuitry underlying motor and cognitive impairment in PD. Although clearly more research is needed to address major gaps in our knowledge, we hypothesize that the potential effects of exercise on inducing neuroplasticity in a circuit specific manner may occur through synergistic mechanisms that include the coupling of an increasing neuronal metabolic demand and increased blood flow. Elucidation of these mechanisms may provide important new targets for facilitating brain repair and modifying the course of disease in PD.

Exercise modifies α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor expression in striatopallidal neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mouse.

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic-acid-type glutamate receptor (AMPAR) plays a critical role in modulating experience-dependent neuroplasticity, and alterations in AMPAR expression may underlie synaptic dysfunction and disease pathophysiology. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of dopamine (DA) depletion, our previous work showed exercise increases total GluA2 subunit expression and the contribution of GluA2-containing channels in MPTP mice. The purpose of this study was to determine whether exercise-dependent changes in AMPAR expression after MPTP are specific to the striatopallidal (D2 R) or striatonigral (D1 R) medium spiny neuron (MSN) striatal projection pathways. Drd2 -eGFP-BAC transgenic mice were used to delineate differences in AMPAR expression between striatal D2 R-MSNs and D1 R-MSNs. Striatal AMPAR expression was assessed by immunohistochemical (IHC) staining, Western immunoblotting (WB) of preparations enriched for postsynaptic density (PSD), and alterations in the current-voltage relationship of MSNs. We found DA depletion results in the emergence of GluA2-lacking AMPARs selectively in striatopallidal D2 R-MSNs and that exercise reverses this effect in MPTP mice. Exercise-induced changes in AMPAR channels observed after DA depletion were associated with alterations in GluA1 and GluA2 subunit expression in postsynaptic protein, D2 R-MSN cell surface expression, and restoration of corticostriatal plasticity. Mechanisms regulating experience-dependent changes in AMPAR expression may provide innovative therapeutic targets to increase the efficacy of treatments for basal ganglia disorders, including Parkinson's disease.

Brief mitochondrial inhibition causes lasting changes in motor behavior and corticostriatal synaptic physiology in the Fischer 344 rat.

The striatum is particularly vulnerable to mitochondrial dysfunction and this problem is linked to pathology created by environmental neurotoxins, stimulants like amphetamine, and metabolic disease and ischemia. We studied the course of recovery following a single systemic injection of the mitochondrial complex II inhibitor 3-nitropropionic acid (3-NP) and found 3-NP caused lasting changes in motor behavior that were associated with altered activity-dependent plasticity at corticostriatal synapses in Fischer 344 rats. The changes in synapse behavior varied with the time after exposure to the 3-NP injection. The earliest time point studied, 24h after 3-NP, revealed 3-NP-induced an exaggeration of D1 Dopamine (DA) receptor dependent long-term potentiation (LTP) that reversed to normal by 48 h post-3-NP exposure. Thereafter, the likelihood and degree of inducing D2 DA receptor dependent long-term depression (LTD) gradually increased, relative to saline controls, peaking at 1 month after the 3-NP exposure. NMDA receptor binding did not change over the same post 3-NP time points. These data indicate even brief exposure to 3-NP can have lasting behavioral effects mediated by changes in the way DA and glutamate synapses interact.

Isolation of a 2:1 hydrochlorothiazide-formaldehyde adduct impurity in hydrochlorothiazide drug substance by preparative chromatography and characterization by electrospray ionization LC-MS.

Hydrochlorothiazide drug substance (19 lots) from five different manufacturers and four different countries of origin (USA, Italy, Hungary, and Croatia) were analyzed for the presence of impurities using a gradient elution chromatographic system, with acetonitrile-water as the mobile phase. Two known impurities of hydrochlorothiazide, 4-amino-6-chloro-1,3-benzenedisulfonamide and chlorothiazide, were separated, as well as a late-eluting, unknown, recurring impurity. The unknown impurity was isolated by preparative liquid chromatography followed by preparative thin-layer chromatography. It was characterized by electrospray ionization LC-MS as a 2:1 hydrochlorothiazide-formaldehyde adduct of the parent drug substance. The adduct is believed to form through the double condensation reaction of hydrochlorothiazide with excess formaldehyde during the parent compound's synthesis. The concentration of this impurity ranged from 0.02 to 1.1% (area%), and was above the 0.1% USP Other Impurities threshold in 16 of the 19 lots examined.

Postnatal expression of alpha-synuclein protein in the rodent substantia nigra and striatum.

A primary goal of our research is to elucidate the mechanisms involved in neuroplasticity of the basal ganglia in both development and in response to injury. One means to this aim is through the analysis of the ontological profile of proteins in the basal ganglia and to correlate their pattern of expression with morphological development. One protein thought to be important in neuroplasticity is alpha-synuclein. The purpose of this study was to characterize and compare the pattern of expression of alpha-synuclein protein using immunocytochemistry in the substantia nigra and striatum of the rodent in early postnatal and adult life. Our results demonstrate that there is a high level of expression of alpha-synuclein protein within cell bodies of the substantia nigra pars compacta in the 1st week of postnatal life that decreases both in intensity and number of immunoreactive cells between postnatal days 7 and 14. This is in contrast to the substantia nigra pars reticulata where alpha-synuclein protein expression in the neuropil increases after postnatal day 7. In the striatum, expression in early postnatal life is distributed in a mosaic-like fashion and becomes more diffuse after postnatal day 14. Our results support the findings of others that expression of alpha-synuclein is developmentally regulated and suggest that alpha-synuclein may play an important role in establishing the function of the basal ganglia. Understanding the role of alpha-synuclein in the normal basal ganglia may provide insights into the molecular mechanisms involved in neuroplasticity in response to injury.

Reliability and validity of a new global dyskinesia rating scale in the MPTP-lesioned non-human primate.

Behavioral rating scales for dyskinesia in the non-human primate are frequently used to assess the efficacy of new treatments and to provide a clinical correlative with neurochemical and neuropathological changes. Although a large variety of different scales have been used in non-human primate studies, there is no single standardized scale, and none have been evaluated for reliability and validity. We are reporting a new global non-human primate dyskinesia rating scale (GPDRS) for the squirrel monkey, developed in the context of an independent study of dyskinesia. In this report we demonstrate the reliability and validity of this scale. The GPDRS is a single-item scale with well-defined points and brevity allowing for rapid and easy application for assessing the overall degree of dyskinesia. In this study, seven MPTP-lesioned and four non-lesioned (control) non-human primates were videotaped following treatment with either levodopa or water. To test inter- and intra-rater reliability, three examiners rated the videotape independently at two different time points and these assessments were compared. The validity of the scale was tested in two phases. First, examiners rated the videotape using the GPDRS and the Abnormal Involuntary Movement Scale (AIMS), a scale commonly used to rate dyskinesia in the non-human primate, and the ratings from each scale were compared. Second, validity was tested in the context of an independent dyskinesia study, in which the scale was used to distinguish between two treatment groups. The GPDRS was shown to have high inter- and intra-rater reliability and to be valid for the assessment of dyskinesia in the squirrel monkey. In this report we also demonstrate the inter- and intra-rater reliability of the AIMS.

Relationship among nigrostriatal denervation, parkinsonism, and dyskinesias in the MPTP primate model.

Presynaptic denervation is likely to play an important role in the pathophysiology of dyskinesias that develop after levodopa administration to patients with Parkinson's disease. In this study, the thresholds of nigrostriatal damage necessary for the occurrence of parkinsonism and levodopa-induced involuntary movements were compared in squirrel monkeys lesioned with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Animals treated with a regimen of MPTP that caused parkinsonism displayed > or =95% striatal dopamine depletion, 90% reduction of striatal dopamine uptake sites, and 70% nigral neuronal loss. Levodopa administration ameliorated the parkinsonian signs of these monkeys but also induced dyskinesias. A separate group of animals was treated with a milder MPTP regimen that caused 60%-70% striatal dopamine depletion, a 50% decrease in dopamine transporter, and 40% loss of dopaminergic nigral neurons. While these monkeys displayed no behavioral signs of parkinsonism, they all became dyskinetic after levodopa administration. The priming effect of levodopa, that is, the recurrence of dyskinesias in animals previously exposed to the drug, was compared in severely versus mildly lesioned monkeys. When severely injured parkinsonian animals underwent a second cycle of levodopa treatment, they immediately and consistently developed involuntary movements. In contrast, the recurrence of dyskinesias in primed monkeys with a partial nigrostriatal lesion required several levodopa administrations and remained relatively sporadic. The data indicate that moderate nigrostriatal damage which does not induce clinical parkinsonism predisposes to levodopa-induced dyskinesias. Once dyskinesias have been induced, the severity of denervation may enhance the sensitivity to subsequent levodopa exposures.

Investigating levodopa-induced dyskinesias in the parkinsonian primate.

Research into the cause of dyskinesias arising from levodopa treatment has been vexingly limited, partly due to the lack of an inexpensive and widely available animal model. Rodents do not develop levodopa-induced dyskinesias in a clinically recognizable form. However, nonhuman primates with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism readily develop levodopa-induced dyskinesias that are virtually indistinguishable from those seen in patients with Parkinson's disease. We have developed and validated a five-point Global Primate Dyskinesia Rating Scale to accurately measure these dyskinesias. Monkeys with MPTP-induced parkinsonism were then investigated to evaluate the relationship between dyskinesias, parkinsonism and severity of the nigrostriatal lesion. All parkinsonian animals were responsive to levodopa, and developed dyskinesias within 2-3 days of levodopa administration. Monkeys treated with only a single injection of MPTP also developed dyskinesias, even though they were not parkinsonian. It would appear that there is a different threshold of striatal dopamine depletion for parkinsonism and dyskinesias in the monkey. Finally, three hypotheses, put forward to explain the genesis of dyskinesias, are reviewed, and various experimental approaches suggested for each.

Combined assessment of tau and neuronal thread protein in Alzheimer's disease CSF.

OBJECTIVE: Comparative study of CSF levels of tau and AD7C-neuronal thread protein (NTP) in patients with AD and control subjects. BACKGROUND: AD is characterized by neurofibrillary tangles composed of the abnormally hyperphosphorylated microtubule-associated protein tau. AD7C-NTP is a proposed AD marker expressed at early stages of neurofibrillary degeneration. METHODS: Enzyme-linked immunosorbent assays specific for tau and AD7C-NTP. CSF samples were obtained from 35 demented patients (25 with antemortem clinical diagnosis of probable AD, 5 with neuropathologic diagnosis of definite AD, 5 with Lewy body pathology), 29 nondemented patients with PD, and 16 elderly healthy control subjects. Receiver operating characteristics (ROC) and multivariate discriminant analysis for AD versus controls. Correlational analysis of CSF tau and AD7C-NTP and of each marker with Mini-Mental State Examination (MMSE) scores was performed. RESULTS: Levels of both tau and AD7C-NTP were significantly elevated in the AD patients compared with control subjects. ROC analysis showed that CSF tau distinguished between patients with AD and nondemented control subjects with 63% sensitivity and 89% specificity, AD7C-NTP with 70% sensitivity and 87% specificity. Combined evaluation of both markers with discriminant analysis raised the specificity to 93% at a 63% sensitivity level. Both markers positively correlated with each other within the AD group, but not among control subjects. CSF levels of AD7C-NTP, but not of tau, showed a small but significant inverse correlation (r = -0.43) with MMSE scores of AD patients. CONCLUSIONS: CSF levels of tau and AD7C-NTP may be useful biomarkers for AD.

Experimental models of Parkinson's disease: insights from many models.

Toxin-induced and genetic experimental models have been invaluable in investigating idiopathic Parkinson's disease (PD). The neurotoxins--reserpine, 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and methamphetamine--have been used to develop parkinsonian models in a wide variety of species. Both 6-OHDA and MPTP can replicate the neurochemical, morphologic, and behavioral changes seen in human disease. The unilateral 6-OHDA rat model is an excellent model for testing and determining modes of action of new pharmacologic compounds. The nonhuman primate MPTP-induced parkinsonian model has behavioral features that best approximate idiopathic PD. These induced and genetic models have been used to study the pathophysiology of the degenerating nigrostriatal system and to evaluate novel therapeutic strategies. Important differences within these models provide insights into various aspects of the dopaminergic phenotype and its role as a target in disease. These models provide an avenue to evaluate many anti-parkinsonian compounds, such as levodopa, which was first evaluated in an animal model and is the gold standard of parkinsonian treatment today.

Isolation and identification of process impurities in trimethoprim drug substance by high-performance liquid chromatography, atmospheric pressure chemical ionization liquid chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy.

Twenty-two lots of recently synthesized trimethoprim drug substance, from five different manufacturers, in three different countries of origin, China, Israel and the United States, were investigated for the presence of impurities. A liquid chromatographic system, using gradient elution, and a mobile phase consisting of 0.25% TEA/0.1% formic acid (pH 5.8)--acetonitrile, was used to separate and detect two significant, recurring impurities in trimethoprim drug substance. The two impurities were isolated by preparative liquid chromatography and identified, using a combination of liquid chromatography/mass spectroscopy and nuclear magnetic resonance, as 2,4-diamino-5-(4-ethoxy-3,5-dimethoxybenzyl) pyrimidine and 2,4-diamino-5-(3-bromo-4,5-dimethoxybenzyl) pyrimidine. These impurities were not detected by the compendial method and were present at significant levels in 17 of the lots tested. Total impurity concentrations were in the range of 0.1-2.1%.

The native form of alpha-synuclein is not found in the cerebrospinal fluid of patients with Parkinson's disease or normal controls.

Alpha-synuclein has recently been shown to be a major constituent of Lewy bodies in Parkinson's disease (PD). This observation led us to investigate the possibility that its detection in the cerebrospinal fluid (CSF) could be used as a marker for Lewy bodies in the central nervous system. In this study we determined the pattern of expression of alpha-synuclein in patients with sporadic Parkinson's disease (PD) and normal controls, using western immunoblotting in conjunction with an antibody that recognizes the carboxyl terminal of alpha-synuclein protein. The native 19 kDa band normally seen in brain homogenates was not found in the CSF of either parkinsonian patients or control subjects. However, a novel band was observed, which migrated at a position in the range of 42 kDa in CSF from both patients and controls. We conclude that alpha-synuclein cannot be used as a biomarker for Lewy bodies during life. However, further characterization of the 42 kDa protein may be of interest.

Use of intrathecal baclofen in the treatment of patients with dystonia.

BACKGROUND: Continuous infusion of intrathecal (IT) baclofen is a highly effective standard therapy for severe spasticity of spinal origin. By contrast, there is limited clinical experience regarding the use of IT baclofen in treating patients with dystonia, and little is known regarding the indications for treatment, efficacy, and safety of IT baclofen in this disorder. OBJECTIVE: To study retrospectively the effects of IT baclofen in treating 25 patients with severe segmental or generalized dystonia. SETTING: Neurological Institute, Columbia-Presbyterian Medical Center, New York, NY. PATIENTS: Twenty-five patients with severe segmental or generalized dystonia that was refractory to oral medications underwent IT baclofen test dosing. In addition to dystonia, 17 patients had spasticity or painful spasms. Thirteen of 25 patients responded to the test doses of IT baclofen, according to unblinded neurological assessments that included the patient's subjective report; all 13 underwent implantation of a pump for continuous IT baclofen infusion. RESULTS: In contrast to reports of patients with spasticity of spinal origin, those with dystonia in the present series had a lower response rate to bolus IT baclofen doses and a smaller degree of clinical improvement. For 10 of the 13 responders to the test doses of IT baclofen, dystonia rating scale scores of videotaped examinations by blinded observers detected no significant change (P < .07) in severity of dystonia. Retrospective data from 11 of 13 patients with implantable pumps, followed up for a mean interval of 21 months after pump insertion, showed continuing efficacy in 6 individuals (55%), based on a determination of patient satisfaction; however, only 3 patients (27%) reported a sustained improvement in functional capacity. Five (38%) of the 13 patients with implantable pumps experienced severe complications that required hospitalization. CONCLUSIONS: Despite recent reports that have described the benefit in small numbers of patients with dystonia, we concluded that the role of IT baclofen in treating severe dystonia remains uncertain. Intrathecal baclofen may be more effective when dystonia is associated with spasticity or pain. In the present series, we detected no significant difference in the response to IT baclofen in patients with or without spasticity or pain, perhaps owing to the small sample size.

GC/MS comparison of the West Indian aphrodisiac "Love Stone" to the Chinese medication "chan su": bufotenine and related bufadienolides.

The death of a 23-year-old man resulting from digoxin-like toxicity and heart failure was attributed to ingestion of a West Indian aphrodisiac known as "Love Stone." GC/MS analyses identified bufotenine, a controlled substance under both US and New York State statutes. In addition, a series of bufadienolides, namely resibufogenin, bufalin, and cinobufagin, were also identified. Bufadienolides, which are derived from toad venom or secretions, are cardiotonic steroids that cause symptoms similar to digoxin. GC/MS analyses of the Chinese medication "Chan Su," a product derived from toads, produced a highly similar elution profile and contained the same compounds as "Love Stone." The data demonstrate that the aphrodisiac was also derived from toads.

Gas chromatographic/mass spectrometric analyses of unknown analytical response in imported Fava beans: 4-chloro-6-methoxyindole.

A halogenated unidentified analytical response (UAR) was encountered in a number of imported Fava bean samples during the Food and Drug Administration's routine pesticide-monitoring program. Gas chromatographic/mass spectrometric (GC/MS) analyses identified the halogenated component as 4-chloro-6-methoxyindole, a naturally occurring promutagen in Fava beans that has been linked to incidents of gastric cancer. Data from electron impact, positive and negative chemical ionization, collision-induced dissociation, and deuteration studies of this compound are presented, along with GC retention time data.