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Alleviation of motor impairment by aerobic exercise (AE) in Parkinson's disease (PD) patients points to activation of neurobiological mechanisms that may be targetable by therapeutic approaches. However, evidence for AE-related recovery of striatal dopamine (DA) signaling or tyrosine hydroxylase (TH) loss has been inconsistent in rodent studies. This ambiguity may be related to the timing of AE intervention in relation to the status of nigrostriatal neuron loss. Here, we replicated human PD at diagnosis by establishing motor impairment with >80% striatal DA and TH loss prior to initiating AE, and assessed its potential to alleviate motor decline and restore DA and TH loss. We also evaluated if serum levels of neurofilament light (NfL) and glial fibrillary acidic protein (GFAP), biomarkers of human PD severity, changed in response to AE. 6-hydroxydopamine (6-OHDA) was infused unilaterally into rat medial forebrain bundle to induce progressive nigrostriatal neuron loss over 28 days. Moderate intensity AE (3× per week, 40 min/session), began 8–10 days post-lesion following establishment of impaired forelimb use. Striatal tissue DA, TH protein and mRNA, and serum levels of NfL/GFAP were determined 3-wks after AE began. Despite severe striatal DA depletion at AE initiation, forelimb use deficits and hypokinesia onset were alleviated by AE, without recovery of striatal DA or TH protein loss, but reduced NfL and GFAP serum levels. This proof-of-concept study shows AE alleviates motor impairment when initiated with >80% striatal DA loss without obligate recovery of striatal DA or TH protein. Moreover, the AE-related reduction of NfL and GFAP serum levels may serve as objective blood-based biomarkers of AE efficacy. • Treadmill exercise in rats followed parameters used by most Parkinson's patients. • Established motor impairment induced by 6-OHDA was alleviated by this regimen. • Despite motor recovery, striatal dopamine and tyrosine hydroxylase were unaffected. • Motor recovery from exercise was associated with decreased serum levels of NfL and GFAP. [ABSTRACT FROM AUTHOR]

The sleep-wake cycle plays an influential role in the development and progression of repeat mild traumatic brain injury (RmTBI)-related pathology. Therefore, we first aimed to manipulate the sleep-wake cycle post-RmTBI using modafinil, a wake-promoting substance used for the treatment of narcolepsy. We hypothesized that modafinil would exacerbate RmTBI-induced deficits. Chronic behavioural analyses were completed along with a 27-plex serum cytokine array, metabolomic and proteomic analyses of cerebrospinal fluid (CSF), as well as immunohistochemical staining in structures important for sleep/wake cycles, to examine orexin, melanin-concentrating hormone, tyrosine hydroxylase, and choline acetyltransferase, in the lateral hypothalamus, locus coeruleus, and basal forebrain, respectively. Contrary to expectation, modafinil administration attenuated behavioural deficits, metabolomic changes, and neuropathological modifications. Therefore, the second aim was to determine if the beneficial effects of modafinil treatment were driven by the orexinergic system. The same experimental protocol was used; however, RmTBI rats received chronic orexin-A administration instead of modafinil. Orexin-A administration produced drastically different outcomes, exacerbating anxiety-related and motor deficits, while also significantly disrupting their metabolomic and neuropathological profiles. These results suggest that the beneficial effects of modafinil administration post-RmTBI, work independently of its wake-promoting properties, as activation of the orexinergic wake-promoting system with orexin-A was detrimental. Overall, these findings highlight the complexity of sleep-wake changes in the injured brain and showcase the potential of the arousal and sleep systems in its treatment. • The sleep-wake cycle is important to traumatic brain injury pathogenesis. • Increasing wakefulness is hypothesized to exacerbate injury-induced deficits. • Administration of modafinil attenuated injury-induced impairments. • Administration of orexin-A exacerbated traumatic brain injury pathology. • Modafinil's beneficial effects work independently of its wake-promoting properties. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) rodent models provide insight into the relationship between nigrostriatal dopamine (DA) signaling and locomotor function. Although toxin-based rat models produce frank nigrostriatal neuron loss and eventual motor decline characteristic of PD, the rapid nature of neuronal loss may not adequately translate premotor traits, such as cognitive decline. Unfortunately, rodent genetic PD models, like the Pink1 knockout (KO) rat, often fail to replicate the differential severity of striatal DA and tyrosine hydroxylase (TH) loss, and a bradykinetic phenotype, reminiscent of human PD. To elucidate this inconsistency, we evaluated aging as a progression factor in the timing of motor and non-motor cognitive impairments. Male PINK1 KO and age-matched wild type (WT) rats were evaluated in a longitudinal study from 3 to 16 months old in one cohort, and in a cross-sectional study of young adult (6–7 months) and aged (18–19 months) in another cohort. Young adult PINK1 KO rats exhibited hyperkinetic behavior associated with elevated DA and TH in the substantia nigra (SN), which decreased therein, but not striatum, in the aged KO rats. Additionally, norepinephrine levels decreased in aged KO rats in the prefrontal cortex (PFC), paired with a higher DA levels in young and aged KO. Although a younger age of onset characterizes familial forms of PD, our results underscore the critical need to consider age-related factors. Moreover, the results indicate that compensatory mechanisms may exist to preserve locomotor function, evidenced by increased DA in the SN early in the lifespan, in response to deficient PINK1 function, which declines with aging and the onset of motor decline. • Pink1 knockout (KO) rats exhibited a hyperkinetic motor phenotype vs age-matched wild-type rats at young age. • Locomotor activity decreased at a faster rate in Pink1 KO rats vs age-matched wild-type rats as a function of aging. • Aging-related differences in locomotor activity in Pink1 KO rats aligned with nigral, but not striatal, dopamine markers. • Dopamine and norepinephrine levels in prefrontal cortex were affected in Pink1 KO rats throughout the lifespan. • These results indicate that despite aging, changes in dopamine markers in Pink1 KO are dissimilar from human Parkinson's. [ABSTRACT FROM AUTHOR]

The clinical manifestation of Parkinson's disease (PD) appears when neurodegeneration is already advanced, compromising the efficacy of disease-modifying treatment approaches. Biomarkers to identify the early stages of PD are therefore of paramount importance for the advancement of the therapy of PD. In the present study, by using a mouse model of PD obtained by subchronic treatment with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the clearance inhibitor probenecid (MPTPp), we identified prodromal markers of PD by combining in vivo positron emission tomography (PET) imaging and ex vivo immunohistochemistry. Longitudinal PET imaging of the dopamine transporter (DAT) by [18F]- N -(3-fluoropropyl)-2β-carboxymethoxy-3β-(4-iodophenyl) nortropane ([18F]-FP-CIT), and brain glucose metabolism by 2-deoxy-2-[18F]-fluoroglucose ([18F]-FDG) were performed before MPTPp treatment and after 1, 3, and 10 MPTPp administrations, in order to assess relation between dopamine neuron integrity and brain connectivity. The results show that in vivo [18F]-FP-CIT in the dorsal striatum was not modified after the first administration of MPTPp, tended to decrease after 3 administrations, and significantly decreased after 10 MPTPp administrations. Post-mortem immunohistochemical analyses of DAT and tyrosine hydroxylase (TH) in the striatum showed a positive correlation with [18F]-FP-CIT, confirming the validity of repeated MPTPp-treated mice as a model that can reproduce the progressive pathological changes in the early phases of PD. Analysis of [18F]-FDG uptake in several brain areas connected to the striatum showed that metabolic connectivity was progressively disrupted, starting from the first MPTPp administration, and that significant connections between cortical and subcortical regions were lost after 10 MPTPp administrations, suggesting an association between dopamine neuron degeneration and connectivity disruption in this PD model. The results of this study provide a relevant model, where new drugs that can alleviate neurodegeneration in PD could be evaluated preclinically. • Early biomarkers of PD are required to optimize the time window for treatment. • Multi-tracer PET imaging could help to identify and validate early PD biomarkers. • In vivo DAT PET correlates with ex vivo TH and DAT immunoreactivity in the striatum. • FDG uptake and metabolic connectivity are modified at earlier time points than DAT. [ABSTRACT FROM AUTHOR]

Dopaminergic (DAergic) hypofunction in the medial prefrontal cortex (mPFC) has been implicated in the negative and cognitive symptoms of schizophrenia and is regulated by serotonergic (5-HTergic) neurons in the dorsal raphe nucleus (DRN). The lateral habenula (LHb) is a key element in controlling DRN 5-HT neurons. We investigated how the LHb impacts the activity of mPFC neurons and whether it mediates the involvement of DRN on development of symptoms in a pharmacological animal model of schizophrenia. We used immunohisochemistry to assess cytochrome-c oxidase (COX) activity of the LHb in MK-801 model rats and extracellular firing recording to compare firing rates in LHb neurons of acute MK-801-treated rats. The sucrose preference, social interaction, and radial arm maze tests were used to access schizophrenia-like behavior in rats with electrolytically lesioned LHb. Finally, we examined levels of the dopamine D1 receptor (D1R) and tyrosine hydroxylase (TH) in the mPFC, and tryptophan hydroxylase 2 (TPH2) in the DRN of rats with LHb lesions to determine the possible mechanism underlying the schizophrenia-like behavior associated with lesioned LHb. We found that COX levels and LHb neuron firing rates decreased significantly in MK-801-treated animals. The LHb lesions induced negative and cognitive, but not positive symptoms of schizophrenia. The D1R and TH levels decreased in the mPFC while TPH2 expression elevated in the DRN and mPFC of LHb-lesioned rats. These results suggest that LHb hypoactivity may contribute to the negative and cognitive symptoms of schizophrenia by downregulating D1R expression in the mPFC, which might be mediated by DRN 5-HT neurons. • LHb is hypofunction in MK801-induced schizophrenia rats • LHb lesion induces negative and cognitive symptoms of schizophrenia in rats • Downregulation of D1R in the mPFC may serve as a mechanism for schizophrenia-like symptoms after LHb lesion [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is characterized by degeneration of the dopaminergic neurons in substantia nigra (SN). The motor symptoms of PD include tremor, rigidity, bradykinesia and postural impairment. In rodents, central administration of neuropeptide-S (NPS) has been shown to induce locomotor activity, dopamine release and neuronal survival by decreasing lipid peroxidation, additionally, the NPS receptor (NPSR) was detected in SN. Accumulating findings suggest that central NPS may ameliorate the parkinsonian symptoms, however, this has been explored incompletely due to the scarcity of experimental studies. Therefore, the present study was designed to test whether central NPS treatment exerts protective and/or alleviative effects on 6-OHDA-induced rat experimental PD model. Adult male Wistar rats received acute (alleviate; 10 nmol, icv) or chronic (protective; 1 nmol, icv for 7 days) NPS treatment following the central injection of 6-OHDA in medial forebrain bundle. Motor performance tests and in vivo nigral microdialysis were performed before and 7 days after the central 6-OHDA injection. The immunoreactivities for tyrosine hydroxylase (TH), NPSR, 4-hydroxynonenal (4-HNE) and c-Fos were detected by immunohistochemistry in frozen SN sections. Our double immunofluorescence labeling studies demonstrated that NPSR is present in the nigral TH-positive neurons. Central NPS injection caused a remarkable c-Fos expression in SN; whereas, no change was observed following vehicle injection. In both chronic and acute treatment groups, the 6-OHDA-induced motor dysfunction and impaired nigral dopamine release were improved significantly. However, only chronic, but not acute treatment restored the loss of nigral TH-positive cells, while decreasing the 4-HNE immunoreactivity in SN. Our findings demonstrate that central NPS treatment not only exerts a neuroprotective action on nigral dopaminergic neurons, it also improves the striatal dopaminergic signaling. Therefore, the present study candidates the NPSR agonism as a novel therapeutic approach for PD treatment. • Centrally administered NPS improved the 6-OHDA-induced motor dysfunction. • Central treatment of NPS induced the nigral dopamine release in parkinsonian rats. • NPS receptor is present in dopaminergic neurons in substantia nigra. • Central exogenous NPS attenuated lipid peroxidation in substantia nigra. [ABSTRACT FROM AUTHOR]

Compensatory mechanisms that augment dopamine (DA) signaling are thought to mitigate onset of hypokinesia prior to major loss of tyrosine hydroxylase (TH) in striatum that occurs in Parkinson's disease. However, the identity of such mechanisms remains elusive. In the present study, the rat nigrostriatal pathway was unilaterally-lesioned with 6-hydroxydopamine (6-OHDA) to determine whether differences in DA content, TH protein, TH phosphorylation, or D 1 receptor expression in striatum or substantia nigra (SN) aligned with hypokinesia onset and severity at two time points. In striatum, DA and TH loss reached its maximum (>90%) 7 days after lesion induction. However, in SN, no DA loss occurred, despite ∼60% TH loss. Hypokinesia was established at 21 days post-lesion and maintained at 28 days. At this time, DA loss was ∼60% in the SN, but still of lesser magnitude than TH loss. At day 7 and 28, ser31 TH phosphorylation increased only in SN, corresponding to less DA versus TH protein loss. In contrast, ser40 TH phosphorylation was unaffected in either region. Despite DA loss in both regions at day 28, D 1 receptor expression increased only in lesioned SN. These results support the concept that augmented components of DA signaling in the SN, through increased ser31 TH phosphorylation and D 1 receptor expression, contribute as compensatory mechanisms against progressive nigrostriatal neuron and TH protein loss, and may mitigate hypokinesia severity. [Display omitted] • Nigral, but not striatal, dopamine (DA) tissue levels correlated with locomotor functions. • Tyrosine hydroxylase (TH) protein loss occurred at a faster rate in striatum than substantia nigra. • Loss of DA was less than TH protein only in substantia nigra. • Increased ser31 TH phosphorylation occurred with TH loss, but only in substantia nigra. • Increased DA D 1 receptor expression occurred in substantia nigra with eventual DA loss. [ABSTRACT FROM AUTHOR]

Traumatic brain injuries (TBI) sustained during peri-adolescent development produce lasting neuro-behavioral changes that render individuals at an increased risk for developing substance abuse disorders. Experimental and clinical evidence of a prolonged period of hypodopaminergia after TBI have been well documented, but the effect of juvenile TBI on dopaminergic dysfunction and its relationship with substance abuse have not been investigated. In order to determine the effect of juvenile brain injury on dopaminergic signaling, female mice were injured at 21 days of age and then beginning seven weeks later were assessed for behavioral sensitization to amphetamine, a drug that increases synaptic dopamine availability. Together with a histological analysis of tyrosine hydroxylase, dopamine transporter, and dopamine D2 receptor expression, our data are indicative of a persistent state of hypodopaminergia well into adulthood after a juvenile TBI. Further, mice that sustained a juvenile TBI exhibited a significantly reduced activation of cFos in the urocortin-positive cells of the Edinger-Westphal nucleus in response to ethanol administration. Taken together, these data provide strong evidence for the vulnerability of juveniles to the development of lasting neuro-behavioral problems following TBI, and indicate a role of injury-induced hypodopaminergia as a risk factor for substance abuse later in life. [ABSTRACT FROM AUTHOR]

A neurochemical target at which cannabinoids interact to have global effects on behavior is brain noradrenergic circuitry. Acute and repeated administration of a cannabinoid receptor synthetic agonist is capable of increasing multiple indices of noradrenergic activity. This includes cannabinoid-induced 1) increases in norepinephrine (NE) release in the medial prefrontal cortex (mPFC); 2) desensitization of cortical α2-adrenoceptor-mediated effects; 3) activation of c-Fos in brainstem locus coeruleus (LC) noradrenergic neurons; and 4) increases in anxiety-like behaviors. In the present study, we sought to examine adaptations in adrenoceptor expression and function under conditions of cannabinoid receptor type 1 (CB1r) deletion using knockout (KO) mice and compare these to wild type (WT) controls. Electrophysiological analysis of α2-adrenoceptor-mediated responses in mPFC slices in WT mice showed a clonidine-induced α2-adrenoceptor-mediated increase in mPFC cell excitability coupled with an increase in input resistance. In contrast, CB1r KO mice showed an α2-adrenoceptor-mediated decrease in mPFC cell excitability. We then examined protein expression levels of α2- and β1-adrenoceptor subtypes in the mPFC as well as TH expression in the locus coeruleus (LC) of mice deficient in CB1r. Both α2- and β1-adrenoceptors exhibited a significant decrease in expression levels in CB1r KO mice when compared to WT in the mPFC, while a significant increase in TH was observed in the LC. To better define whether the same cortical neurons express α2A-adrenoceptor and CB1r in mPFC, we utilized high-resolution immunoelectron microscopy. We localized α2A-adrenoceptors in a knock-in mouse that expressed a hemoagglutinin (HA) tag downstream of the α2A-adrenoceptor promoter. Although the α2A-adrenoceptor was often identified pre-synaptically, we observed co-localization of CB1r with α2-adrenoceptors post-synaptically in the same mPFC neurons. Finally, using receptor binding, we confirmed prior results showing that α2A-adrenoceptor is unchanged in mPFC following acute or chronic exposure to the synthetic cannabinoid receptor agonist, WIN 55,212-2, but is increased, following chronic treatment followed by a period of abstinence. Taken together, these data provide convergent lines of evidence indicating cannabinoid regulation of the cortical adrenergic system. [ABSTRACT FROM AUTHOR]

Accumulating evidence suggests that an abnormal accumulation of iron in the substantia nigra (SN) is one of the defining characteristics of Parkinson's disease (PD). Accordingly, the potential neuroprotection of Fe chelators is widely acknowledged for the treatment of PD. Although desferrioxamine (DFO), an iron chelator widely used in clinical settings, has been reported to improve motor deficits and dopaminergic neuronal survival in animal models of PD, DFO has poor penetration to cross the blood–brain barrier and elicits side effects. We evaluated whether an intranasal administration of DFO improves the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced degeneration of dopaminergic neurons in the nigrostriatal axis and investigated the molecular mechanisms of intranasal DFO treatment in preventing MPTP-induced neurodegeneration. Treatment with DFO efficiently alleviated behavioral deficits, increased the survival of tyrosine hydroxylase (TH)-positive neurons, and decreased the action of astrocytes in the SN and striatum in an MPTP-induced PD mouse model. Interestingly, we found that DFO up-regulated the expression of HIF-1α protein, TH, vascular endothelial growth factor (VEGF), and growth associated protein 43 (GAP43) and down-regulated the expression of α-synuclein, divalent metal transporter with iron-responsive element (DMT1 + IRE), and transferrin receptor (TFR). This was accompanied by a decrease in iron-positive cells in the SN and striatum of the DFO-treated group. We further revealed that DFO treatment significantly inhibited the MPTP-induced phosphorylation of the c-Jun N-terminal kinase (JNK) and differentially enhanced the phosphorylation of extracellular regulated protein kinases (ERK) and mitogen-activated protein kinase (MAPK)/P38 kinase. Additionally, the effects of DFO on increasing the Bcl-2/Bax ratio were further validated in vitro and in vivo. In SH-SY5Y cells, the DFO-mediated up-regulation of HIF-1α occurred via the activation of the ERK and P38MAPK signaling pathway. Collectively, the present data suggest that intranasal DFO treatment is effective in reversing MPTP-induced brain abnormalities and that HIF-1-pathway activation is a potential therapy target for the attenuation of neurodegeneration. [ABSTRACT FROM AUTHOR]

Current stem cell therapies for Parkinson's disease (PD) focus on a neurorestorative approach that aims to repair the CNS during the symptomatic phase. However, the pleiotropic and supportive effects of human neural stem cells (hNSCs) may make them effective for PD treatment during the disease's earlier stages. In the current study, we investigated the therapeutic effects of transplanting hNSCs during the early stages of PD development when most dopaminergic neurons are still present and before symptoms appear. Previous studies on hNSCs in Parkinson's disease focus on the substantia nigra and its immediate surroundings, but other brain structures are affected in PD as well. Here, we investigated the therapeutic effects of hNSCs on the entire PD-afflicted brain transcriptome using RNA sequencing (RNA-seq). PD was induced with a single intranasal infusion of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) and hNSCs were transplanted unilaterally into the striatum one week later. The timepoint for hNSC transplantation coincided with upregulation of endogenous proinflammatory cytokines in the CNS, which play a role in stem cell migration. At 3 weeks post-transplantation (4 weeks post-MPTP), we assessed motor symptoms through behavioral tests, quantified dopaminergic neurons in the substantia nigra, and performed global transcriptional profiling to understand the mechanism underlying the effect of hNSCs on dopaminergic neuron degeneration. We found that early hNSC engraftment mitigated motor symptoms induced by MPTP, and also reduced MPTP-induced loss of dopaminergic neurons. In this study, we uniquely presented the first comprehensive analysis of the effect of hNSC transplantation on the transcriptional profiling of PD mouse brains showing decreased expression of 249 and increased expression of 200 genes. These include genes implicated in mitochondrial bioenergetics, proteostasis, and other signaling pathways associated with improved PD outcome following hNSC transplantation. These findings indicate that NSC transplantation during the asymptomatic phase of PD may limit or halt the progression of this neurodegenerative disorder. Transcriptional profiling of hNSC-engrafted PD mouse brains provides mechanistic insight that could lead to novel approaches to ameliorating degeneration of dopaminergic neurons and improving behavioral dysfunction in PD. • Early transplantation of hNSCs improves motor function in the MPTP mouse PD model. • Engrafted hNSCs during the early PD stage ameliorates dopaminergic neuron degeneration. • RNA-seq shows transplanted hNSCs alter cellular energetics and protein metabolism. [ABSTRACT FROM AUTHOR]

Abstract: Different experimental and clinical strategies have been used to promote survival of transplanted embryonic ventral mesencephalic (VM) neurons. However, few studies have focused on the long-distance growth of dopaminergic axons from VM transplants. The aim of this study is to identify some of the growth and guidance factors that support directed long-distance growth of dopaminergic axons from VM transplants. Lentivirus encoding either glial cell line-derived neurotrophic factor (GDNF) or netrin-1, or a combination of lenti-GDNF with either lenti-GDNF family receptor α1 (GFRα-1) or lenti-netrin-1 was injected to form a gradient along the corpus callosum. Two weeks later, a piece of embryonic day 14 VM tissue was transplanted into the corpus callosum adjacent to the low end of the gradient. Results showed that tyrosine hydroxylase (TH+) axons grew a very short distance from the VM transplants in control groups, with few axons reaching the midline. In GDNF or netrin-1 expressing groups, more TH+ axons grew out of transplants and reached the midline. Pathways co-expressing GDNF with either GFRα-1 or netrin-1 showed significantly increased axonal outgrowth. Interestingly, only the GDNF/netrin-1 combination resulted in the majority of axons reaching the distal target (80%), whereas along the GDNF/GFRα-1 pathway only 20% of the axons leaving the transplant reached the distal target. This technique of long-distance axon guidance may prove to be a useful strategy in reconstructing damaged neuronal circuits, such as the nigrostriatal pathway in Parkinson's disease. [Copyright &y& Elsevier]

Abstract: Parkinson's disease (PD) is the most common neurodegenerative disease of the basal ganglia. Like other adult-onset neurodegenerative disorders, it is without a treatment that forestalls its chronic progression. Efforts to develop disease-modifying therapies to date have largely focused on the prevention of degeneration of the neuron soma, with the tacit assumption that such approaches will forestall axon degeneration as well. We herein propose that future efforts to develop neuroprotection for PD may benefit from a shift in focus to the distinct mechanisms that underlie axon degeneration. We review evidence from human post-mortem studies, functional neuroimaging, genetic causes of the disease and neurotoxin models that axon degeneration may be the earliest feature of the disease, and it may therefore be the most appropriate target for early intervention. In addition, we present evidence that the molecular mechanisms of degeneration of axons are separate and distinct from those of neuron soma. Progress is being made in understanding these mechanisms, and they provide possible new targets for therapeutic intervention. We also suggest that the potential for axon re-growth in the adult central nervous system has perhaps been underestimated, and it offers new avenues for neurorestoration. In conclusion, we propose that a new focus on the neurobiology of axons, their molecular pathways of degeneration and growth, will offer novel opportunities for neuroprotection and restoration in the treatment of PD and other neurodegenerative diseases. [Copyright &y& Elsevier]

Abstract: Objective: Nigrostriatal reserve refers to the threshold of neuronal injury to dopaminergic cell bodies and their terminal fields required to produce parkinsonian motor deficits. Inferential studies have estimated striatal dopamine reserve to be at least 70%. Knowledge of this threshold is critical for planning interventions to prevent symptom onset or reverse nigrostriatal injury sufficient to restore function in people with Parkinson disease. In this study, we determine the nigrostriatal reserve in a non-human primate model that mimics the motor manifestations of Parkinson disease. Methods: Fifteen macaque monkeys received unilateral randomized doses of the selective dopaminergic neuronal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. We compared blinded validated ratings of parkinsonism to in vitro measures of striatal dopamine and unbiased stereologic counts of nigral neurons after tyrosine hydroxylase immunostaining. Results: The percent of residual cell counts in lesioned nigra correlated linearly with the parkinsonism score at 2months (r =−0.87, p <0.0001). The parkinsonism score at 2months correlated linearly with the percent residual striatal dopamine (r =−0.77, p =0.016) followed by a flooring effect once nigral cell loss exceeded 50%. A reduction of about 14 to 23% of nigral neuron counts or 14% to 37% of striatal dopamine was sufficient to induce mild parkinsonism. Conclusions: The nigral cell body and terminal field injury needed to produce parkinsonian motor manifestations may be much less than previously thought. [Copyright &y& Elsevier]

Abstract: Three glutamate transporters, GLT-1, GLAST, and EAAC1, are expressed in striatum. GLT-1 and, to a lesser extent, GLAST are thought to play a primary role in glutamate reuptake and mitigate excitoxicity. Progressive tyrosine hydroxylase (TH) loss seen in Parkinson''s disease (PD) is associated with increased extracellular glutamate. Glutamate receptor antagonists reduce nigrostriatal loss in PD models. These observations suggest that excess synaptic glutamate contributes to nigrostriatal neuron loss seen in PD. Decreased GLT-1 expression occurs in neurodegenerative disease and PD models, suggesting decreased GLT-1-mediated glutamate reuptake contributes to excitotoxicity. To determine how transient GLT-1 blockade affects glutamate reuptake dynamics and a Ca2+-dependent process in nigrostriatal terminals, ser19 phosphorylation of TH, the GLT-1 inhibitor dihydrokainic acid (DHK) was delivered unilaterally to striatum in vivo and glutamate reuptake was quantified ex vivo in crude synaptosomes 3h later. Ca2+-influx is associated with excitotoxic conditions. The phosphorylation of TH at ser19 is Ca2+-dependent, and a change resulting from GLT-1 blockade may signify the potential for excitotoxicity to nigrostriatal neurons. Synaptosomes from DHK infused striatum had a 43% increase in glutamate reuptake in conjunction with decreased ser19 TH phosphorylation. Using a novel GLAST inhibitor and DHK, we determined that the GLAST-mediated component of increased glutamate reuptake increased 3-fold with no change in GLAST or GLT-1 protein expression. However, GLT-1 blockade increased EAAC1 protein expression ~20%. Taken together, these results suggest that GLT-1 blockade produces a transient increase in GLAST-mediated reuptake and EAAC1 expression coupled with reduced ser19 TH phosphorylation. These responses could represent an endogenous defense against excitotoxicity to the nigrostriatal pathway. [Copyright &y& Elsevier]

Abstract: The endogenous opioid peptides, met- or leu-enkephalin, and corticotropin-releasing factor (CRF) regulate noradrenergic neurons in the locus coeruleus (LC) in a convergent manner via projections from distinct brain areas. In contrast, the opioid peptide dynorphin (DYN) has been shown to serve as a co-transmitter with CRF in afferents to the LC. To further define anatomical substrates targeting noradrenergic neurons by DYN afferents originating from limbic sources, anterograde tract-tracing of biotinylated dextran amine (BDA) from the central amygdaloid complex was combined with immunocytochemical detection of DYN and tyrosine hydroxylase (TH) in the same section of tissue. Triple labeling immunocytochemistry was combined with electron microscopy in the LC where BDA was identified using an immunoperoxidase marker, and DYN and TH were distinguished by the use of sequential immunogold labeling and silver enhancement to produce different sized gold particles. Results show direct evidence of a monosynaptic pathway linking amygdalar DYN afferents with LC neurons. To determine whether DYN-containing amygdalar LC-projecting neurons colocalize CRF, retrograde tract-tracing using fluorescent latex microspheres injected into the LC was combined with immunocytochemical detection of DYN and CRF in single sections in the central amygdala. Retrogradely labeled neurons from the LC were distributed throughout the rostro-caudal extent of the central nucleus of the amygdala (CeA) as previously described. Cell counts showed that approximately 42% of LC-projecting neurons in the CeA contained both DYN and CRF. Taken with our previous studies showing monosynaptic projections from amygdalar CRF neurons to noradrenergic LC cells, the present study extends this by showing that DYN and CRF are co-transmitters in monosynaptic projections to the LC and are poised to coordinately impact LC neuronal activity. [Copyright &y& Elsevier]

Abstract: This study investigated the effect of a knockout of the caspase 2 gene on the sensitivity of murine nigral dopaminergic neurons to 1-methyl-4-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity. Female wild type (WT), heterozygous caspase 2 NL (HET) and homozygous caspase 2 null (NL) mice were treated with cumulative dosages of 0, 10, 15 or 20mg/kg MPTP free base. Without MPTP treatment, one week later dopamine (DA) levels were not significantly different in HET or NL versus WT mice. Twenty mg/kg MPTP reduced striatal DA in WT and HET (p<0.01) but not NL mice. This same MPTP dosage regimen also induced a significantly greater decrease in tyrosine hydroxylase immunopositive (TH+) protein in striata of WT compared to NL mice (p<0.001). Subsequently, WT and NL mice were treated daily with 20mg/kg MPTP for 3days and 25mg/kg MPTP for 2 additional days, and TH+ neurons in the substantia nigra (SN) were estimated using unbiased stereology. When compared to untreated WT, the numbers of TH+ neurons were significantly lower in the SN of untreated NL mice (p<0.05). Treatment with the MPTP regimen significantly reduced TH+ neurons in WT mice but not NL mice. In primary mesencephalic cultures both the cell bodies and the neuronal processes of TH immunopositive (TH+) neurons from NL embryos were significantly (p<0.001) more resistant to 10μM MPP+ compared to WT. Following MPP+ treatment, features of apoptotic cell death were also significantly (p<0.001) more prevalent in nuclei of TH+ neurons in cultures prepared from WT versus NL mouse pups. These results suggest that caspase 2 may play a role in modulating the MPTP-induced damage to the nigrostriatal dopaminergic system. [Copyright &y& Elsevier]

Abstract: The accurate and reliable counting of tyrosine hydroxylase positive (TH+) and tyrosine hydroxylase negative (TH−) neurons in the ventral midbrain is an important measure in studies related to Parkinson''s disease and many other disorders associated with this region. Despite recent advancements, the use of stereology remains limited due to a variety of challenges for many users. We implemented a real-time fluorescence detection method and the use of an antibody to the neuron specific nuclear antigen (NeuN) to overcome some challenges for users. We found that the regional value for the two different cell types (TH+ and TH−) varied with the method of detection (chromogenic versus fluorescence) and with different nuclear markers (Nissl, DAPI, or NeuN). The number of both TH+ and TH− neurons was higher using fluorescence detection. The number of TH− neurons was higher using NeuN as a neuronal nuclear marker compared to DAPI. We identified 3 types of neuronal nuclei using NeuN staining characteristics. The method is applicable for mouse and rat. We describe a practical approach for epifluorescence-based counting of these two types of neurons that may offer significant advantages over existing methods for potential users. [Copyright &y& Elsevier]

Abstract: Dystrophic axons and terminals are common in the myenteric plexus and smooth muscle of the gastrointestinal (GI) tract of aged rats. In young adult rats, alpha-synuclein in its normal state is abundant throughout the myenteric plexus, making this protein–which is prone to fibrillization–a candidate marker for axonopathies in the aged rat. To determine if aggregation of alpha-synuclein is involved in the formation of age-related enteric neuropathies, we sampled the stomach, small intestine and large intestine of adult, middle-aged, and aged virgin male Fischer 344 rats stained for alpha-synuclein in both its normal and pathological states. Alpha-synuclein-positive dystrophic axons and terminals were present throughout the GI tract of middle-aged and aged rats, with immunohistochemical double labeling demonstrating co-localization within nitric oxide synthase-, calretinin-, calbindin-, or tyrosine hydroxylase-positive markedly swollen neurites. However, other dystrophic neurites positive for each of these four markers were not co-reactive for alpha-synuclein. Similarly, a subpopulation of alpha-synuclein inclusions contained deposits immunostained with an anti-tau phospho-specific Ser262 antibody, but not all of these hyperphosphorylated tau-positive aggregates were co-localized with alpha-synuclein. The presence of heteroplastic and potentially degenerating neural elements and protein aggregates both positive and negative for alpha-synuclein suggests a complex chronological relationship between the onset of degenerative changes and the accumulation of misfolded proteins. Additionally, proteins other than alpha-synuclein appear to be involved in age-related axonopathies. Finally, this study establishes the utility of the aging Fischer 344 rat for the study of synucleopathies and tauopathies in the GI tract. [Copyright &y& Elsevier]

Abstract: Identifying guidance cues that direct axon growth to their final connections during development is of crucial interest if we aim to repair circuits damaged in adulthood following neurodegenerative disorders or common traumatic injuries. In this work, we set out to determine the ephrinA5 guidance molecule involvement in the establishment of the mouse mesostriatal pathway during development. We showed, in vitro and in vivo, that a proportion of mesencephalic dopaminergic cells express the ephrinA5 receptor, EphA5. Moreover, we observed, using stripe assays, that ephrinA5 purified protein has a repulsive effect on most of the mesencephalic dopaminergic projections. In vivo, we detected rostro-caudal and ventro-dorsal ephrinA5 protein expression gradients in the vicinity of the dopaminergic axons in the ventral telencephalon and in the striatum, during the embryonic and early postnatal development. In addition, other EphA5 ligands were also detected in the mesostriatal pathway. Together, these expression patterns suggest that, ephrinAs and more specifically ephrinA5, may be actors in the guidance of dopaminergic projections. Further studies will focus on identifying the molecular specificity of these guidance cues, taking into account the mesencephalic dopaminergic heterogeneous neuronal population. This may help increase the integration of neuronal transplants in the mature lesioned brain or provide tools to re-establish mesostriatal circuits in vivo. [Copyright &y& Elsevier]

Abstract: The pathogenesis of Parkinson''s disease is not fully understood, but there is evidence that excitotoxic mechanisms contribute to the pathology. However, data supporting a role for excitotoxicity in the pathophysiology of the disease are controversial and sparse. The goal of this study was to determine whether changes in glutamate signaling and uptake contribute to the demise of dopaminergic neurons in the substantia nigra. Mice were treated chronically with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and probenecid or vehicle (probenecid or saline alone). Extracellular levels of glutamate in the substantia nigra were substantially increased, and there was an increase in the affinity, but no change in the velocity, of glutamate transport after MPTP/probenecid treatment compared to vehicle controls. In addition, the substantia nigra showed two types of programmed death, apoptosis (type I) and autophagic (type II) cell death. These data suggest that increased glutamate signaling could be an important mechanism for the death of dopaminergic neurons and trigger the induction of programmed cell death in the chronic MPTP/probenecid model. [Copyright &y& Elsevier]

Abstract: Dopamine (DA) affects GABA neuronal function in the striatum and together these neurotransmitters play a large role in locomotor function. We recently reported that unilateral striatal administration of GDNF, a growth factor that has neurotrophic effects on DA neurons and enhances DA release, bilaterally increased striatal neuron activity related to locomotion in aged rats. We hypothesized that the GDNF enhancement of DA function and resulting bilateral enhancement of striatal neuronal activity was due to prolonged bilateral changes in DA- and GABA-regulating proteins. Therefore in these studies we assessed dopamine- and GABA-regulating proteins in the striatum and substantia nigra (SN) of 24 month old Fischer 344 rats, 30 days after a single unilateral striatal delivery of GDNF. The nigrostriatal proteins investigated were the DA transporter (DAT), tyrosine hydroxylase (TH), and TH phosphorylation and were examined by blot-immunolabeling. The striatal GABA neuron-related proteins were examined by assay of the DA D1 receptor, DARPP-32, DARPP-32 Thr34 phosphorylation, and glutamic acid decarboxylase (GAD). Bilateral effects of GDNF on TH and DAT occurred only in the SN, as 30 μg GDNF increased ser19 phosphorylation, and 100 μg GDNF decreased DAT and TH protein levels. GDNF also produced bilateral changes in GAD protein in the striatum. A decrease in DARPP-32 occurred in the ipsilateral striatum, while increased D1 receptor and DARPP-32 phosphorylation occurred in the contralateral striatum. The 30 μg GDNF infusion into the lateral striatum was confined to the ipsilateral striatum and substantia nigra. Thus, long-lasting bilateral effects of GDNF on proteins regulating DA and GABA neuronal function likely alter physiological properties in neurons, some with bilateral projections, associated with locomotion. Enhanced nigrostriatal excitability and DA release by GDNF may trigger these bilateral effects. [Copyright &y& Elsevier]

Abstract: Parkinson''s disease (PD) is characterized by loss of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNc). It is widely believed that replacing lost SNc DA neurons is a key to longer-term effective treatment of PD motor symptoms, but generating new SNc DA neurons in PD patients has proven difficult. Following loss of tyrosine hydroxylase-positive (TH+) SNc neurons in the rodent 6-hydroxy-DA (6-OHDA) model of PD, the number of TH+ neurons partially recovers and there is evidence this occurs via phenotype “shift” from TH− to TH+ cells. Understanding how this putative phenotype shift occurs may help increase SNc DAergic neurons in PD patients. In this study we characterize the electrophysiology of SNc TH− and TH+ cells during recovery from 6-OHDA in mice. Three distinct phenotypes were observed: (1) TH− were fast discharging with a short duration action potential (AP), short afterhyperpolarization (AHP) and no small conductance Ca2+-activated K+ (SK) current; (2) TH+ were slow discharging with a long AP, long AHP and prominent SK current; and (3) cells with features “intermediate” between these TH− and TH+ phenotypes. The same 3 phenotypes were present also in the normal and D2 DA receptor knock-out SNc suggesting they are more closely related to the biology of TH expression than recovery from 6-OHDA. Acute inhibition of SK channel function shifted the electrophysiological phenotype of TH+ neurons toward TH− and chronic (2 weeks) inhibition of SK channel function in normal mice shifted the neurochemical phenotype of SNc from TH+ to TH− (i.e. decreased TH+ and increased TH− cell numbers). Importantly, chronic facilitation of SK channel function shifted the neurochemical phenotype of SNc from TH− to TH+ (i.e. increased TH+ and decreased TH− cell numbers). We conclude that SK channel function bidirectionally regulates the DA phenotype of SNc cells and facilitation of SK channels may be a novel way to increase the number of SNc DAergic neurons in PD patients. [Copyright &y& Elsevier]

Abstract: Adult adipose contains stromal progenitor cells with neurogenic potential. However, the stability of neuronal phenotypes adopted by Adipose-Derived Adult Stromal (ADAS) cells and whether terminal neuronal differentiation is required for their consideration as alternatives in cell replacement strategies to treat neurological disorders is largely unknown. We investigated whether in vitro neural induction of ADAS cells determined their ability to neuroprotect or restore function in a lesioned dopaminergic pathway. In vitro-expanded naïve or differentiated ADAS cells were autologously transplanted into substantia nigra 1 week after an intrastriatal 6-hydroxydopamine injection. Neurochemical and behavioral measures demonstrated neuroprotective effects of both ADAS grafts against 6-hydroxydopamine-induced dopaminergic neuron death, suggesting that pre-transplantation differentiation of the cells does not determine their ability to survive or neuroprotect in vivo. Therefore, we investigated whether equivalent protection by naïve and neurally-induced ADAS grafts resulted from robust in situ differentiation of both graft types into dopaminergic fates. Immunohistological analyses revealed that ADAS cells did not adopt dopaminergic cell fates in situ, consistent with the limited ability of these cells to undergo terminal differentiation into electrically active neurons in vitro. Moreover, re-exposure of neurally-differentiated ADAS cells to serum-containing medium in vitro confirmed ADAS cell phenotypic instability (plasticity). Lastly, given that gene expression analyses of in vitro-expanded ADAS cells revealed that both naïve and differentiated ADAS cells express potent dopaminergic survival factors, ADAS transplants may have exerted neuroprotective effects by production of trophic factors at the lesion site. ADAS cells may be ideal for ex vivo gene transfer therapies in Parkinson''s disease treatment. [Copyright &y& Elsevier]

Abstract: In addition to rods and cones of the outer retina, a third class of photoreceptive cell has recently been described in the inner retina of mammals. These intrinsically photosensitive retinal ganglion cells (ipRGCs) have been shown to relay luminance information to the mammalian brain. In addition to their intrinsic photosensitivity, the function of ipRGCs may also be modulated by signals from within the retina itself. Such signals may emanate from classical photoreceptors in the outer retina or from the circadian activity of adjacent inner retinal neurones. Prime candidates for the latter are the retinal dopamine neurones which ramify at the border of the inner plexiform and inner nuclear layers. In order to investigate the nature of any interaction between dopamine and ipRGC populations in normal retina and to assess the impact of outer retinal degeneration on this interrelationship, we examined the retinae of normal and RCS dystrophic rats. We report a direct interaction between the dendrites of ipRGCs and dopaminergic neurones which is conserved across species. Triple immunolabelling using synaptic markers provides evidence for the unidirectionality of information transfer between the two cell types, with processes of ipRGCs being directly adjacent to sites of dopamine release. This fundamental architectural feature of the mammalian retina appears resistant to degeneration of classical photoreceptors and may provide the anatomical substrate by which dopamine cells influence the physiology of central circadian targets in the brain. [Copyright &y& Elsevier]

Abstract: Natural cell death (NCD) by apoptosis is a normal developmental event in most neuronal populations, and is a determinant of the eventual size of a population. We decided to examine the timing and extent of NCD of the midbrain dopamine system in a primate species, as dopamine deficiency or excess has been implicated in several disorders. Genetic or environmental differences may alter the extent of NCD and predispose individuals to neurological or psychiatric diseases. In developing rats, NCD in the midbrain dopamine system has been observed to start at the end of gestation and peak in the postnatal period. In fetal monkey brains, apoptosis in midbrain DA neurons was identified histologically by chromatin clumping in tyrosine hydroxylase-positive cells, and confirmed by TUNEL and active caspase-3 staining. A distinct peak of NCD occurred at about E80, midway through gestation in this species. We estimate that at least 50% of the population may be lost in this process. In other brains we determined biochemically that the onset of apoptosis coincides with the time of greatest rate of increase of striatal DA concentration. Thus, marked apoptotic NCD occurs in the primate midbrain dopamine system half-way through gestation, and appears to be associated with the rapid developmental increase in striatal dopamine innervation. [Copyright &y& Elsevier]

Abstract: An autoregulated tetracycline-inducible recombinant adeno-associated viral vector (rAAV-pTetbidiON) utilizing the rtTAM2 reverse tetracycline transactivator (rAAV-rtTAM2) was used to conditionally express the human GDNF cDNA. Doxycycline, a tetracycline analog, induced a time- and dose-dependent release of GDNF in vitro in human glioma cells infected with rAAV-rtTAM2 serotype 2 virus. Introducing the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) downstream to the rtTAM2 coding sequence, resulted in a more rapid induction and a higher basal expression level. In vivo, 8 weeks after a single injection of the rAAV-rtTAM2-GDNF vector encapsidated into AAV serotype 1 capsids in the rat striatum, the GDNF protein level was 60 pg/mg tissue in doxycycline-treated animals whereas in untreated animals, it was undistinguishable from the endogenous level (∼4 pg/mg tissue). However, a residual GDNF expression in the uninduced animals was evidenced by a sensitive immunohistochemical staining. As compared to rAAV1-rtTAM2-GDNF, the rAAV1-rtTAM2-WPRE-GDNF vector expressed a similar concentration of GDNF in the induced state (with doxycycline) but a basal level (without doxycycline) ∼2.5-fold higher than the endogenous striatal level. As a proof for biological activity, for both vectors, downregulation of tyrosine hydroxylase was evidenced in dopaminergic terminals of doxycycline-treated but not untreated animals. In conclusion, the rAAV1-rtTAM2 vector which expressed biologically relevant doses of GDNF in the striatum in response to doxycycline with a basal level undistinguishable from the endogenous striatal level, as measured by quantitative ELISA assay, constitutes an interesting tool for local conditional transgenesis. [Copyright &y& Elsevier]

Abstract: Chronic cocaine use leads to pronounced alterations in neuronal functions in brain circuits associated with reward. In the present study, we examined in the rat midbrain the effects of acute, subchronic (5 days) and chronic cocaine treatments (14 days) on the gene expression of transcription factors involved in the development and maintenance of dopaminergic neurons. We show that chronic, but not acute or subchronic, cocaine administration downregulates Nurr1 and Pitx3 transcripts whereas En1 transcripts are upregulated. Conversely, Lmx1b and En2 transcripts are not affected by the drug treatment, indicating that the modulation of the midbrain transcription factors analyzed is highly selective. Interestingly, modification of the gene expression for these transcription factors persists in midbrain as long as two weeks after the last drug administration, suggesting that it may account for some of the enduring alterations in midbrain dopaminergic circuits associated with chronic cocaine use. [Copyright &y& Elsevier]

Abstract: TrkB receptors mediate the effects of BDNF on striatal medium spiny neurons and mesencephalic dopamine neurons. The effect of partial BDNF gene deletion on locomotor activity and the gene expression of these neurons was evaluated at 3, 12, and 24 months of age in BDNF heterozygous (BDNF LacZ/neo+ ) and wildtype mice. BDNF LacZ/neo+ mice displayed less spontaneous horizontal activity than wildtypes at 3 and 24 months of age. Whereas striatal preproenkephalin and preprodynorphin mRNA and mesencephalic tyrosine hydroxylase mRNA levels were significantly lower at all ages in BDNF LacZ/neo+ mice, GAD67 mRNA was only lower at 24 months. In contrast, BDNF LacZ/neo+ mice expressed more trkB mRNA in the striatum at 3 months and less at 24 months of age than wildtypes. Total striatal cell number in the two genotypes was not different at 12 months of age, whereas Golgi staining revealed that the spine density on distal dendrites of medium spiny neurons was less in BDNF LacZ/neo+ mice than in wildtypes at 24 months of age. These data indicate that endogenous BDNF is required to maintain the normal phenotype and functioning of striatal projection neurons and mesencephalic dopamine neurons and that exaggerated dysfunction of these neurons and a concomitant decline in locomotor behavior occurs during aging. [Copyright &y& Elsevier]

Abstract: Injection with 6-hydroxydopamine (6-OHDA) into the nigrostriatal pathway results in loss of nigrostriatal dopaminergic neurons, which has been used widely as an animal model of Parkinson''s disease. In the present study, location and extent of lesions 1 day after 6-OHDA injections (2, 4, 8, or 16 μg as a free base) in the substantia nigra (SN) were evaluated in rats by T2-weighted magnetic resonance imaging (MRI). The changes in MRI were also compared to immunohistochemical and behavioral changes. Hyperintense area in MRI was found at the region corresponding to 6-OHDA injection in a dose-dependent manner and was accompanied by a loss of tyrosine hydroxylase (TH)-positive cells. The shape of hyperintense area in the SN appeared to be composed of two components (i.e., circular and longitudinal regions). Administration of a larger dose of 6-OHDA (8–16 μg) was accompanied by an increase in hyperintense area and loss of TH-positive cells beyond the SN. The hyperintense area was observed on the first and third days after 6-OHDA injection, but the size and intensity declined to near normal levels on the ninth day. Rotational behavior induced by methamphetamine reached maximal levels at 4 μg 6-OHDA, and the behavior was maintained with doses up to 16 μg of 6-OHDA. Intrastriatal injection with 6-OHDA was less effective. These results suggest that MRI provides highly valuable information for verifying the size and location of intended lesions as well as for determining the optimal dose of neurotoxins in individual animals. [Copyright &y& Elsevier]

Previous work indicated that pig umbilical cord matrix (pUCM) cells are a type of primitive stem cell and that these cells could be recovered after central or peripheral injection into rats that did not receive immune suppression therapy. To determine the safety and proliferation potential of pUCM cells after brain transplantation, approximately 150 pUCM cells were transplanted into the brains of rats that previously received a striatal injection of the neurotoxin 6-hydroxydopamine (6-OHDA). The pUCM cells were previously engineered to express enhanced green fluorescent protein (eGFP); in this way, the graft cells were identified. The rats did not receive immune suppression therapy. There were no postsurgical complications and the animals thrived following transplantation. At 2, 4, 6, and 8 weeks after transplantation, two rats were sacrificed and the morphology, size and number of graft cells, and the percentage of tyrosine hydroxylase (TH)-positive graft cells were determined. The size distribution of the grafted pUCM cells was unimodal and normal, and the average size increased significantly over the 2- to 8-week survival period. The number of pUCM cells increased from approximately 5400 cells at the 2-week survival period post-transplantation to approximately 20,000 cells at the 8-week survival period. There was an increase in the percentage of TH-positive pUCM cells from approximately 1% at the 2-week survival period to approximately 6% at the 8-week survival period. There was no evidence of a significant host immune response at any time; for example, no accumulation of CD-4, CD-8, CD-11b, CD-161 cells in the transplantation site. These results suggest that pUCM cells engraft and proliferate without requiring immune suppression. These findings also suggest that a subset of pUCM cells can differentiate into TH-positive cells within 8 weeks after transplantation into the 6-OHDA lesioned rat brain. [Copyright &y& Elsevier]

CAD cells are a murine CNS catecholaminergic (tyrosine hydroxylase-positive; TH+) neuronal cell line that undergoes morphological differentiation to resemble CNS catecholaminergic neurons upon serum deprivation. We show here that CAD cells also express neuronal nitric oxide synthase (nNOS) mRNA and protein and produce readily measurable levels of NO. Since both NO and catecholamines (l-DOPA; dopamine; norepinephrine) are redox active molecules, their production within the same cell may affect the cell''s vulnerability to insult. Thus, we examined the regulation of NO production by CAD cells and the effect of NO on cell survival. NO is generated in a dose-dependent fashion by treatment with agents (ionomycin; A23817; KCl) known to increase calcium entry across the cell membrane. The NO level can be increased further by pretreatment with sepiapterin, a membrane permeable precursor for BH4 synthesis, suggesting that the BH4 levels or access required for nNOS activation is limited in CAD cells. Reducing mitochondrial Ca2+ uptake using ruthenium red (RuR) increased ionomycin-mediated NO production over ionomycin alone and indicates a critical role for mitochondria in nNOS regulation. Cell death was significantly increased by ionomycin treatment alone or in conjunction with reduced mitochondrial Ca2+ uptake. However, NO was not the primary mediator of cell death since NOS inhibitors rescued only less than 10% of the cells. These data suggest that endogenous NO production by nNOS is not a major factor in CAD cell death under these conditions. [Copyright &y& Elsevier]

We have investigated the potential of autologous sympathetic neurons as a donor for cell therapy of Parkinson''s disease (PD). Our recent study demonstrated that sympathetic neuron autografts increase the duration of levodopa-induced “on” periods with consequent reduction in the percent time spent in “off” phase. We also found that human sympathetic neurons grown in culture have the ability to convert exogenous levodopa to dopamine and to store the synthesized dopamine. This may explain the clinically observed prolongation in the duration of levodopa effects. To further analyze the mechanism for the graft-mediated effect, the present study investigated the metabolic function of human sympathetic ganglionic neurons xenografted into the dopamine (DA)-denervated striatum of rats by monitoring striatal levels of DA and its primary metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), after systemic administration of levodopa. We also explored whether the graft-mediated effect above may last in four PD patients who had been given the grafts and followed for 12–36 months postgrafting. Clinical evaluations showed that an increase in the duration of levodopa-induced “on” phase is detected during a follow-up period of 12–36 months postgrafting in all the four patients tested. Accordingly, the percent time spent in “off” phase exhibited a 30–40% reduction as compared to the pregrafting values. The animal experiment showed that a significant increase in striatal DA levels is noted after systemic levodopa treatment, and that the DA levels remain high for longer periods of time in the grafted rats than in control animals. When given reserpine pretreatment, the levodopa-induced rise of striatal DA levels was significantly attenuated with concomitant increase in DOPAC levels. Histological examinations demonstrated that the grafts contain some tyrosine hydroxylase (TH)-positive cells. These cells were also found to express aromatic-l-amino acid decarboxylase (AADC) and vesicular monoamine transporter-2 (VMAT), both of which are important molecules for the synthesis and the storage of DA, respectively. These results indicate that grafted sympathetic neurons can provide a site for both the conversion of exogenous levodopa to DA and the storage of the synthesized DA in the DA-denervated striatum, explaining a mechanism by which sympathetic neuron autografts can increase the duration of levodopa-induced “on” phase in PD patients. [Copyright &y& Elsevier]

Sprague–Dawley rats received a unilateral injection of 6-hydroxydopamine (6-OHDA) into the striatum and were treated daily for 6 weeks with increasing doses of monoamine oxidase type B inhibitor rasagiline [R(+)-N-propargyl-1-aminoindane] or saline (controls). Both doses of rasagiline markedly increased the survival of dopaminergic neurons in the lesioned substantia nigra, compared to controls (+97% and +119%, respectively). Treatment with the lower dose of rasagiline also abolished the motor stereotypies associated with nigrostriatal lesion. Our study supports the neuroprotective potential of chronic rasagiline administration in an experimental model of Parkinson''s disease (PD). [Copyright &y& Elsevier]

Mitochondrial toxins such as the complex 1 inhibitor rotenone are widely used as pesticides and may be present in military environments. Administration of rotenone can induce biochemical and histological alterations similar to those of Parkinson''s disease in rats. However, only a subset of animals show these effects and it is unclear whether more subtle alterations are caused by chronic administration of rotenone in those animals that appear resistant to its toxic effects on dopaminergic nerve terminals. To address this question, vehicle or rotenone (2.0, 2.5, or 3.5 mg/kg/day) was administered intravenously or subcutaneously for 21 days to adult rats, and rotenone effects on survival, motor behavior, and striatal tyrosine hydroxylase immunoreactivity (TH-IR) were examined. Both intravenous and subcutaneous rotenone induced a dose-dependent decrease in survival rates. Surviving animals showed a decrease in spontaneous rearing. Locomotor activity and movement initiation time were also altered in some of the experimental groups. Confirming previous results, TH-IR in the striatum was markedly decreased in rats that fell ill early in the study and in a few of the surviving rats with high rotenone doses. However, none of the surviving rats receiving 2.0 mg/kg/day showed TH-IR loss reminiscent of Parkinson''s disease, and loss of striatal TH-IR across doses was not correlated with motor behavior in individual rats. Thus, chronic administration of low doses of rotenone induces motor anomalies even in animals that do not develop histological signs of Parkinson''s disease, indicating a pervasive neurological effect of moderate mitochondrial dysfunction in vivo. [Copyright &y& Elsevier]

In Parkinson''s disease (PD) and animal models of parkinsonism the destruction of nigrostriatal (NSB) system results in a marked loss of the dopamine D3 receptor and mRNA in the islands of Calleja (ICj) and the nucleus accumbens shell (NAS). In animal models, it has been reported that both measures are elevated by repeated intermittent administration of l-dopa. However, a large proportion of PD cases are resistant to l-dopa-induced elevation of D3 receptor number. The zitter mutant (Zi/Zi) rat replicates the slow progressive degeneration of the NSB observed in PD and also exhibits a loss of D3 receptor number in the NAS or ICj. To test if this could be reversed with subchronic l-dopa treatment, injections of carbidopa (10 mg/kg ip) were followed an hour later with injection of l-dopa (100 mg/kg ip) twice a day for 10 days. In control Sprague–Dawley (SD) and zitter heterozygote (Zi/−) rats that do not show a loss of D3 receptors with vehicle treatment, l-dopa produced no change in D3 receptor number or in DA terminal density as measured by dopamine transporter (DAT) binding and tyrosine hydroxylase immunoautoradiography (TH-IR). There was a marked loss of DAT and TH-IR in caudate–putamen (CPu) and NA, as well as D3 receptors in NAS and ICj in Zi/Zi rats but no further change with l-dopa treatment. To determine if the resistance to l-dopa-induced increase in D3 receptor was due to a deficiency in expression of cortical BDNF or its receptor, TrkB, in CPu and NAS, we examined BDNF mRNA by ISHH in frontal cortex and TrkB mRNA in frontal cortex, CPu, and NA. The loss of the NSB in the Zi/Zi did not alter levels of BDNF or TrkB mRNA, nor did l-dopa administration alter levels BDNF or TrkB mRNA. Thus, unlike in 6-hydroxydopamine-treated rats, in Zi/Zi rats administered l-dopa does not reverse the loss of BDNF mRNA or lead to an elevation of D3 receptor number. [Copyright &y& Elsevier]

Inhibition or ablation of the hyperactive subthalamic nucleus (STN) in Parkinson''s disease (PD) does not only reverse motor deficits, silencing the glutamatergic output of the subthalamic nucleus, but has also been implicated to have neuroprotective effects on nigral neurons in animal models of Parkinson''s disease. Ablation of the subthalamic nucleus has been shown to increase the number of tyrosinhydroxylase-immunopositive cells and partially restores behavioral deficits in animal models of Parkinson''s disease. However, it is unclear whether subthalamic nucleus ablation indeed prevents cell death or whether the effect is due to the rescue of the dopaminergic (DA) phenotype of impaired cells by upregulating tyrosine hydroxylase (TH). We therefore investigated the potential neuroprotective effects of a preceding subthalamic nucleus lesion on 6-hydroxydopamine (6-OHDA)-induced nigral cell death and compared the retrograde tracer fluorogold (FG) as a marker of cell survival with tyrosinhydroxylase immunoreactivity as a marker of the dopaminergic phenotype. In the present study, we show that ablation of the subthalamic nucleus does not affect the number of fluorogold-labeled cells but increases the number of tyrosinhydroxylase-positive neurons in subthalamic nucleus-lesioned hemiparkinsonian animals and leads to partial behavioral recovery of the rats.We conclude that subthalamic nucleus ablation exerts neuroprotective properties on the dopaminergic nigrostriatal pathway against 6-hydroxydopamine toxicity in terms of rescuing the neurotransmitter phenotype in the remaining neurons rather than enhancing the total number of nigral cells. [Copyright &y& Elsevier]

Many investigators have reported extensive microglial activation in the mouse substantia nigra and striatum following acute, high-dose 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration. Our previous work demonstrated tyrosine hydroxylase (TH)-positive fiber sprouting in the striatum in monkeys that had received a partial dopaminergic lesion using a low-dose, chronic MPTP administration paradigm. To characterize the microglial response, we utilized HLA-DR (LN3) to immunolabel the class II major histocompatibility complex (MHC II). In MPTP-treated monkeys, there was an intense microglial response in the substantia nigra, nigrostriatal tract, and in both segments of the globus pallidus. This response was morphologically heterogeneous, with commingled ramified, activated, and multicellular morphologies throughout the extent of these basal ganglia structures. Surprisingly, there was little evidence of microglial reactivity in the striatum despite evidence of neurodegeneration—by silver labeling and by loss of TH immunolabeling. Moreover, this pattern of microglial reactivity was the same in all animals that had received MPTP and seemed to be independent of the degree of neurotoxin-induced neurodegeneration. Thus, we conclude that microglial reactivity, per se, is not consistently associated with neurodegeneration, but depends on regional differences. [Copyright &y& Elsevier]

The novel naphtoxazine derivative and preferential D3 vs D2 receptor agonist, S32504, restores perturbed motor function in rodent and primate models of antiparkinsonian activity with a potency superior to those of two further, preferential D3 receptor agonists, pramipexole and ropinirole. However, potential neuroprotective properties of S32054 have not, to date, been evaluated. Herein, employing several measures of cellular integrity, we demonstrate that S32504 robustly, concentration-dependently and completely protects terminally differentiated SH-SY5Y cells against 1-methyl-4-phenylpyridinium (MPP+)-induced cell death in vitro. Further, S32504 was substantially more potent than pramipexole and ropinirole, the latter of which was neurotoxic at high concentrations. In vivo, subchronic treatment with low (0.25 mg/kg) and high (2.5 mg/kg) doses of S32504 prior to and during treatment of mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, MPTP, provided complete protection against MPTP-induced tyrosine hydroxylase immunoreactive (TH-IR) neuronal death in the substantia nigra pars compacta and ventral tegmental area. A high dose of ropinirole (2.5 mg/kg) provided some protection but statistical significance was not attained, and a low dose (0.25 mg/kg) was ineffective. Neither drug afforded protection against the MPTP-induced loss of DA fibers in the striatum, as measured by TH-IR and dopamine transporter immunoreactive fiber counts. In conclusion, the novel naphotoxazine and dopaminergic agonist, S32504, robustly protects dopaminergic neurones against the neurotoxic effects of MPP+ and MPTP in in vitro and in vivo models, respectively. The underlying mechanisms and therapeutic pertinence of these actions will be of interest to further evaluate in view of its potent actions in behavioral models of antiparkinson activity. [Copyright &y& Elsevier]

Parkinson’s disease (PD) is a chronic neurodegenerative disorder, and it has been suggested that treatments promoting survival and functional recovery of affected dopaminergic neurons could have a significant and long-term therapeutic value. In the present study, we investigated the neuroprotective effects of acupuncture on the nigrostriatal system in rat unilaterally lesioned with 6-hydroxydopamine (6-OHDA, 4 μg/μl, intrastriatal injection) using tyrosine hydroxylase (TH) and receptor for brain-derived neurotrophic factor, trkB, immunohistochemistries. Two weeks after the lesions were made, rats presented with asymmetry in rotational behavior (118.3 ± 17.5 turns/h) following injection with apomorphine, a dopamine receptor agonist (0.5 mg/kg, sc). In contrast, acupunctural treatment at acupoints GB34 and LI3 was shown to significantly reduce this motor deficit (14.6 ± 13.4 turns/h). Analysis via TH immunohistochemistry revealed a substantial loss of cell bodies in the substantia nigra (SN) (45.7% loss) and their terminals in the dorsolateral striatum ipsilateral to the 6-OHDA-induced lesion. However, acupunctural treatment resulted in the enhanced survival of dopaminergic neurons in the SN (21.4% loss) and their terminals in the dorsolateral striatum. Acupuncture also increased the expression of trkB significantly (35.6% increase) in the ipsilateral SN. In conclusion, we observed that only acupuncturing without the use of any drug has the neuroprotective effects against neuronal death in the rat PD model and these protective properties of acupuncture could be mediated by trkB. [Copyright &y& Elsevier]

The survival of rat postnatal mesencephalic dopamine (DA) neurons in dissociated cell cultures was studied by examining the combinatorial effects of dibutyryl cyclic adenosine monophosphate (db-cAMP), glial cell line-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF), as well as selective inhibitors of protein kinase A (PKA), and mitogen-activated protein kinase (MAPK). Postnatal DA neurons were maintained for 14 days in vitro, and were identified by immunohistochemistry using tyrosine hydroxylase antibody. The survival and growth of DA neurons was significantly increased by the inclusion of either >100 μM db-cAMP or 10 μM Forskolin plus 100 μM IBMX in the culture medium. Neither 10–50 ng/ml GDNF nor 50 ng/ml BDNF alone significantly increased DA neuron survival in vitro. However, the combined use of GDNF and BDNF did increase DA neuron survival, and the addition of either db-cAMP or IBMX/Forskolin to media containing these neurotrophins markedly increased DA neuron survival and growth. The cAMP inhibitor Rp-cAMP, the cAMP-dependent protein kinase A inhibitor H89, and the MAP kinase (MAPK) pathway inhibitor PD98059 significantly reduced the survival of DA neurons when applied alone in the absence of added growth factors. Application of GDNF plus BDNF, or db-cAMP significantly protected the DA neurons from the deleterious effects on survival of either 20 μM H89 or 20 μM PD 98059. The results suggest that BDNF, GDNF, and cAMP produce convergent signals to activate PKA and MAPK pathways which are involved in the survival of postnatal mesencephalic DA neurons in vitro. [Copyright &y& Elsevier]

We administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to adult, male cats to model Parkinson’s disease (PD), and utilized proton magnetic resonance imaging (MRI) and spectroscopy (MRS) at a field strength of 1.5 T to identify metabolic degenerative changes in the striatum in vivo. Neurologic status and somatosensory-evoked potentials in vivo, as well as postmortem striatal histopathological and immunohistochemical parameters, were examined. Nine cats were equally divided into three groups and treated daily for 10 days as follows: saline, MPTP, and pargyline (a monoamine oxidase inhibitor) plus MPTP. The MPTP-treated cats displayed bradykinesia, head tremor, and reduced oculovestibular reflex activity. MRI showed a diffuse increase of the T2-weighted signal in the striatum of two MPTP-treated cats. Analysis of the MRS spectra indicated significantly lower N-acetylaspartate/creatine (CR) and glutamine-glutamate complex/CR ratios than the control baseline. Two MPTP-treated cats had low choline-containing compounds/CR ratio, whereas a lactate peak was present in all MPTP-treated cats. In the striatum of the MPTP-treated cats, there was a significant decline of tyrosine hydroxylase immunoreactivity and histological evidence for a diffuse cytotoxic reaction. Pretreatment with pargyline attenuated the MPTP-induced clinical signs, MRI and MRS changes, and the histopathological and immunoreactivity alterations. We conclude that proton MRI/MRS is a sensitive, noninvasive measure of neural toxicity and biochemical alteration of the striatum in a feline model of PD. [Copyright &y& Elsevier]

Neurotrophic factors regulate a variety of cellular processes, including neuronal survival during development and after injury. For instance, brain-derived neurotrophic factor (BDNF) can prevent the death of dopaminergic substantia nigra neurons in rats. Most neurotrophic factor receptors, such as TrkB for BDNF, are tyrosine kinases whose signaling is terminated by protein tyrosine phosphatases (PTPs). We tested the idea that inhibition of PTPs, and thus potentially enhancement of the efficiency of endogenous trophic factors and their receptors, would lead to increased neuronal survival. After a 2-week infusion of the small PTP inhibitor molecule peroxovanadium (pVa, pervanadate) close to the substantia nigra of adult rats, up to 66% of axotomized substantia nigra neurons had survived, compared to only 33% in control rats infused with PBS. PVa most likely affected TrkB and/or downstream signaling molecules, as ineffective doses of BDNF and pVa had a synergistic effect when given simultaneously, rescuing 82% of the neurons. PVa stimulated tyrosine hydroxylase (TH) expression in the noninjured substantia nigra but did not prevent axotomy-induced loss of TH. These results raise the possibility that PTP inhibition can prevent neuronal death by enhancing neurotrophic factor signaling pathways in the adult mammalian nervous system, identifies an important role for PTPs in neuronal functioning, and points to a novel small molecule treatment approach for neurologic disorders [Copyright &y& Elsevier]

The ability to differentiate neural stem cells (NSCs) into dopamine neurons is fundamental to their role in cell replacement therapies for neurodegenerative disorders such as Parkinson''s disease. We show here that when a clonal line (C17.2) of undifferentiated NSCs is transplanted into the intact or 6-hydroxydopamine-lesioned striatum, cells withdraw from the cell cycle (BrdU−), migrate extensively in the host striatum, and express markers associated with neuronal (β-tubulin III+, NSE+, NeuN+) but not glial (GFAP−, MBP−, A2B5−) differentiation. Importantly, by 2–5 weeks postgrafting, in the majority of these transplants, nearly all engrafted cells express the dopamine-synthesizing enzymes tyrosine hydroxylase and aromatic l-amino decarboxylase, sometimes resulting in changes in motor behavior. In contrast, no NSCs stain for dopamine-β-hydroxylase, choline acetyltransferase, glutamic acid decarboxylase, or serotonin. We conclude that, following transplantation into the intact or 6-hydroxydopamine-lesioned rat, the adult brain contains intrinsic cues sufficient to direct the specific expression of dopaminergic traits in immature multipotential neural stem cells. [Copyright &y& Elsevier]

Neural precursors with the properties of neural stem cells can be isolated from the developing brain, can be expanded in culture, and have been suggested as a potential source of cells for neuronal replacement therapies in degenerative disorders such as Parkinson''s disease (PD). Under such conditions an improved spectrum of functional benefit may be obtained through homotypic reconstruction of degenerated neural circuitry, and to this end we have investigated the potential of expanded neural precursor cells (ENPs) to form long axonal projections following transplantation in the 6-hydroxydopamine-lesioned rat model of PD. ENPs have been isolated from the embryonic pig, since implantation in a xenograft environment is thought to favor axonal growth. These porcine ENPs possessed similar properties in vitro to those described in other species: they proliferated in response to epidermal and fibroblast growth factor-2, expressed the neuroepithelial marker nestin, and differentiated into neurons, astrocytes, and occasional oligodendrocytes on mitogen withdrawal. The use of pig-specific markers following xenotransplantion into cyclosporin A-immunosuppressed rats revealed that many cells differentiated into neurons and displayed extensive axogenesis, such that when placed in the region of the substantia nigra fibers projected throughout the striatal neuropil. These neurons were not restricted in the targets to which they could project since following intrastriatal grafting fibers were seen in the normal striatal targets of the pallidum and substantia nigra. Staining for a pig-specific synaptic marker suggested that synapses were formed in these distant sites. A small number of these cells differentiated spontaneously to express a catecholaminergic phenotype, but were insufficient to mediate behavioral recovery. Our results suggest that when the efficiency of neurochemical phenotype induction is increased, ENP-derived neurons have the potential to be a uniquely flexible source of cells for therapeutic cell replacement where anatomical reconstruction is advantageous. [Copyright &y& Elsevier]

To prepare immortalized adrenal chromaffin cells for eventual clinical use, the immortalizing oncogene must be removed. We have utilized a Cre-mediated excision of a loxP-flanked Tag sequence to test whether immortalized chromaffin cells could be disimmortalized by this method. Cultures of embryonic rat adrenal cells were immortalized with the tsA-TN retroviral vector encoding the loxP-flanked temperature-sensitive allele of SV40 large T antigen (tsA-TN) and a positive/negative neo/HSV-TK sequence for selection with either G418 or gancyclovir, respectively. These cells were then infected with the 1710-CrePR1 bicistronic retroviral vector coding for a form of Cre modulatable by the synthetic steroid RU486. These immortalized loxTsTag/CrePR1/RAD cells expressed immunoreactivities (ir) for all the catecholamine enzymes: tyrosine hydroxylase (TH), dopamine β-hydroxylase (DβH), and phenylethanolamine-N-methyltransferase (PNMT). After initial incubation at 37°C with RU486 for 3 days, followed by the addition of gancyclovir for 7 days, Tag-ir was not detectable in most of the surviving chromaffin cells, compared to 100% expression in immortalized loxTsTag/CreR1/RAD cells not treated with RU486 and gancyclovir. The expression of TH, DβH, and PNMT was increased after disimmortalization and the ability of disimmortalized cells to synthesize norepinephrine was also significantly increased compared to immortalized cells. When both types of chromaffin cells were transplanted in a model of neuropathic pain and partial nerve injury, both cell grafts were equally able to reverse the behavioral hypersensitivity induced by the injury. The use of Cre/lox site-directed disimmortalization of chromaffin cells that are able to deliver neuroactive molecules offers a novel approach to cell therapy. [Copyright &y& Elsevier]

Sonic hedgehog (Shh), a member of hedgehog (hh) family of signaling molecules, is necessary for normal axial patterning and cellular differentiation in the developing central nervous system. Shh also promotes the survival of fetal dopaminergic neurons and protects cultures of fetal midbrain dopaminergic neurons from the toxic effects of N-methyl-4-phenylpyridinium (MPP+), a neurotoxin that selectively injures nigral dopaminergic neurons. The mRNA expression of Shh and its putative receptor in the adult brain indicates an important role of Shh in the mature nervous system in addition to its roles during embryogenesis. In this study we examined the behavioral and anatomical effects of intrastriatal injection of singly myristoylated wild-type human Sonic hedgehog N-terminal fragment (Shh-M) in a rat model of Parkinson''s disease (PD). Five groups of rats received a series of four intrastriatal injections of Shh-M (180 ng, 540 ng, or 4.275 μg per injection), glial cell line-derived neurotrophic factor (GDNF) (1 μg/injection), or vehicle on days 1, 3, 5, and 8. On day 4, the animals received an intrastriatal injection of 15 μg 6-hydroxydopamine (6-OHDA) free base. Intrastriatal administration of Shh (180 ng/injection) twice before and after a single intrastriatal injection of 6-OHDA reduced apomorphine- and amphetamine-induced rotation and forelimb akinesia and partially preserved dopaminergic axons in the striatum. This is the first demonstration in vivo that Shh reduces behavioral deficits induced by intrastriatal 6-OHDA lesion and suggests that Shh may be useful in the treatment of disorders that affect the nigrostriatal system, such as PD. [Copyright &y& Elsevier]