Abstract: Rotigotine is a novel, non-ergoline dopamine D(3)/D(2)/D(1)-receptor agonist for the treatment of Parkinson's disease that can be continuously delivered by the transdermal route to provide stable plasma levels. Continuous drug delivery should reduce the risk of dyskinesia induction in comparison to pulsatile dopaminergic treatment. Thus the aim of the study was to compare the reversal of motor disability and induction of dyskinesia produced by continuous compared to pulsatile rotigotine administration in MPTP-treated common marmosets. The study also investigated whether pulsatile or continuous rotigotine administration in combination with l-DOPA prevented l-DOPA-induced dyskinesia. Animals were treated for 28 days with vehicle or pulsatile (twice daily) or continuous delivery of rotigotine (via an osmotic minipump). Subsequently, l-DOPA was then co-administered for a further 28 days. Animals were assessed for locomotor activity, motor disability and dyskinesia induction. The study showed that both continuous and pulsatile administration of rotigotine improved motor deficits and normalized motor function in MPTP-treated monkeys. However, continuous rotigotine delivery reduced dyskinesia expression compared to pulsatile treatment. Both pulsatile and continuous rotigotine administration produced less dyskinesia than administration of l-DOPA alone. The addition of l-DOPA to either pulsatile or continuous rotigotine treatment resulted in the induction of marked dyskinesia similar to that produced by treatment with l-DOPA alone. These data further support the hypothesis that continuous delivery of a dopaminergic agent reduces the risk of dyskinesia induction. However, continuous rotigotine administration did not prevent l-DOPA from inducing dyskinesia suggesting that l-DOPA may induce dyskinesia by mechanisms different from dopamine agonist drugs.
Abstract: Systemic administration of the proteasomal inhibitor I (PSI) to rats was reported to cause progressive nigral dopaminergic neuronal loss but this is disputed. A major controversy centres over the use of manual counting of tyrosine hydroxylase (TH) positive neurons at the level of third cranial nerve as opposed to employing systematic stereological analysis of cell loss in the entire substantia nigra (SN). To provide a method of marking SN neurones independent of protein expression, fluorogold (FG) was stereotaxically injected bilaterally into the striatum of male Wistar rats to retrogradely label nigral dopaminergic neurons. After 1 week, animals were treated with six doses of PSI (8 mg/kg, s.c.) or its vehicle (dimethyl sulphoxide) on alternate days over a 2-week period. Five weeks after the last treatment, PSI-treated animals showed decreased spontaneous locomotor activity and reduced TH positive SN cell number at the level of the third cranial nerve compared to control rats. Manual cell counting showed loss of FG-labelled SN neurones at this level, with a subpopulation of surviving neurons displaying abnormal morphology. Manual counting of all FG-labelled cells in the entire SN also showed regional PSI-induced loss of neurones with both normal and compromised morphology. Stereological optical fractionator estimates of total FG-labelled cell number confirmed the manual cell counting data both at the level of the third cranial nerve and throughout the entire SN. These findings confirm that PSI does cause a persistent nigral dopaminergic neuronal loss. The reason for the lack of reproducibility between laboratories requires further investigation. We suggest that a failure to distinguish between TH-positive neurones with normal and abnormal morphology following PSI administration contributes to equivocal results.
Abstract: L-DOPA treatment of Parkinson's disease induces a high incidence of motor complications, notably dyskinesia. Longer acting dopamine agonists, e.g. ropinirole, are thought to produce more continuous dopaminergic stimulation and less severe dyskinesia. However, standard oral administration of dopamine agonists does not result in constant plasma drug levels, therefore, more continuous drug delivery may result in both prolonged reversal of motor deficits and reduced levels of dyskinesia. Therefore, we compared the effects of repeated oral administration of ropinirole to constant subcutaneous infusion in MPTP-treated common marmosets. Animals received oral administration (0.4 mg/kg, BID) or continuous infusion of ropinirole (0.8 mg/kg/day) via osmotic minipumps for 14 days (Phase I). The treatments were then switched and continued for a further 14 days (Phase II). In Phase I, locomotor activity was similar between treatment groups but reversal of motor disability was more pronounced in animals receiving continuous infusion. Dyskinesia intensity was low in both groups however there was a trend suggestive of less marked dyskinesia in those animals receiving continuous infusion. In Phase II, increased locomotor activity was maintained but animals switched from oral to continuous treatment showing an initial period of enhanced locomotor activity. The reversal of motor disability was maintained in both groups, however, motor disability tended towards greater improvement following continuous infusion. Importantly, dyskinesia remained low in both groups suggesting that constant delivery of ropinirole neither leads to priming nor expression of dyskinesia. These results suggest that a once-daily controlled-release formulation may provide improvements over existing benefits with standard oral ropinirole in Parkinson's disease patients.
Abstract: The D2/D3 dopamine receptor agonist pramipexole, protects against toxin-induced dopaminergic neuronal destruction but its mechanism of action is unknown. Inflammation following glial cell activation contributes to cell death in Parkinson's disease and we now report on the effects of acute or chronic administration of pramipexole on lipopolysaccharide (LPS) induced inflammation and nigral dopaminergic cell death in the rat. At 48 h and 30 days following supranigral administration of LPS, approximately 70% of tyrosine hydroxylase (TH) immunoreactive (-ir) cells in substantia nigra had degenerated with a corresponding loss of TH-ir terminals in the striatum. In rats acutely treated with pramipexole (2x1 mg/kg; s.c.) 48 h following LPS application, there was no difference in the number of TH-ir cells or terminals compared to LPS-treated rats receiving vehicle. However, the continuous subcutaneous infusion of pramipexole for 7 days prior to LPS and 21 days subsequently, produced a marked preservation of both TH-ir cells and terminals. At 48 h or 30 days, LPS induced an up-regulation of ubiquitin-ir within the nigral TH-ir neurones, which was reduced by pramipexole treatment. Thirty days following supranigral LPS administration (9 days after the end of infusion), (+)-amphetamine (5 mg/kg, i.p.) caused robust ipsiversive rotation. In rats treated with LPS but receiving continuous subcutaneous administration of pramipexole, (+)-amphetamine-induced rotation was markedly reduced. LPS-induced increase in the levels of inflammatory markers, were not affected by either acute administration or continuous infusion of pramipexole. Continuous infusion of pramipexole protected dopaminergic neurones against inflammation induced degeneration but without modification of the inflammatory response.
Abstract: Dementia with Lewy bodies (DLB) accounts for 15-20% of the millions of people worldwide with dementia. In the current work we investigate the association between proteasome dysfunction and the development of cortical Lewy body pathology. Analysis of post-mortem cortical tissue indicated levels of the alpha-subunit of the 20S proteasome were significantly reduced in DLB cortex, but not Alzheimer's, in comparison to control and this reduction correlated with both the severity and duration of dementia. Application of proteasome inhibitors to rodent cortical primary neurones in vitro and by direct injection onto rodent cholinergic forebrain neurons in vivo gave rise to dose dependent neuronal death and in rodent cortex -- marked cholinergic deficits accompanied by the accumulation of inclusions that stained positive for alpha-synuclein and ubiquitin. These findings suggest that proteasomal abnormalities are present within cortical Lewy body disease and the experimental inhibition of proteasomal function mirrors the neuropathological changes seen within the disorder.
Abstract: The neurotoxin MPTP induces nigral dopaminergic cell death in primates and produces a partial model of Parkinson's disease (PD). Pramipexole is a D2/D3 dopamine receptor agonist used in the symptomatic treatment of PD, and which also protects neuronal cells against dopaminergic toxins in vitro. We now demonstrate that pramipexole partially prevents MPTP toxicity in vivo in a primate species. Common marmosets were repeatedly treated with pramipexole either before, coincidentally with, or after low-dose MPTP treatment designed to induce a partial lesion of the substantia nigra. Animals pretreated with pramipexole had a significantly greater number of surviving tyrosine hydroxylase (TH) positive neurones in the pars compacta of the substantia nigra. Pramipexole pretreatment also prevented degeneration of striatal dopamine terminals. Treatment with pramipexole concurrently with MPTP or following MPTP did not prevent TH-positive cell loss. Pramipexole pretreatment appears to induce adaptive changes that protect against dopaminergic cell loss in primates.
Abstract: The presence of 3-nitrotyrosine (3-NT) adducts in Lewy bodies in Parkinson's disease suggests a role for nitrative stress in dopaminergic cell death. Whether this is a direct effect of increased nitric oxide (NO) formation or requires its reaction with superoxide to form peroxynitrite is not clear. In the present study, we show that direct nigral administration of a NO donor, SNOG, in the rat produced only local toxicity to dopaminergic neurones pre-labeled with fluorogold with no 3-NT formation. However, administration of a peroxynitrite donor, SIN-1, caused widespread damage to dopaminergic neurones and marked expression of 3-NT immunoreactivity. Importantly, dopaminergic cell loss and the expression of 3-NT were completely prevented when SIN-1 was co-administered with the NO/peroxynitrite scavenger, carboxy-PTIO. The results suggest that increased NO formation is not inherently toxic to dopaminergic neurons, but when both oxidative and nitrative stress combine to cause peroxynitrite formation, neurotoxicity occurs.
Abstract: Dysfunction of the ubiquitin-proteasome system occurs in the substantia nigra (SN) in Parkinson's disease (PD). However, it is unknown whether this is a primary cause or a secondary consequence of other components of the pathogenic process. We have investigated in nonhuman primates whether initiating cell death through mitochondrial complex I inhibition using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) altered proteasomal activity or the proteasomal components in the SN. Chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolase (PGPH) activating of 20S proteasome were decreased in SN homogenates of MPTP-treated marmosets compared to naïve animals. Western blotting revealed a marked decrease in the expression of 20S-alpha subunits, but no change in 20S-beta subunits in the SN of MPTP-treated marmoset compared to naïve animals. There was a marked decrease in the expression of the proteasome activator 700 (PA700) and proteasome activator 28 (PA28) regulatory complexes. The 20S-alpha4 subunit immunoreactivity was decreased in the nucleus of colocalized tyrosine hydroxylase (TH)-positive cells of MPTP-treated animals compared to naïve animals but no difference in the intensity of 20S-beta1i subunit staining. Immunoreactivity for PA700-Rpt5 and PA28-alpha subunits within surviving TH-positive cells of MPTP-treated marmoset was reduced compared to naïve controls. Overall, the changes in proteasomal function and structure occurring follow MPTP-induced destruction of the SN in common marmosets were very similar to those found in PD. This suggests that altered proteasomal function in PD could be a consequence of other pathogenic processes occurring in SN as opposed to initiating cell death as previously suggested.
Abstract: 5-Hydroxytryptamine 1a (5-HT(1a)) receptor agonists, such as sarizotan and tandospirone, are reported to reduce levodopa-induced dyskinesia in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated macaques and in Parkinson's disease without worsening motor disability. However, these compounds are not specific for 5-HT(1a) receptors and also possess dopamine antagonist actions. We now report on the effects of (2R)-(+)-8-hydroxy-2-(di-n-propylamino)tetralin [(R)-(+)-8-OHDPAT], a selective 5-HT(1a) agonist lacking dopaminergic activity, on motor disability and dyskinesia (chorea and dystonia) in levodopa-primed MPTP-treated common marmosets. Administration of (R)-(+)-8-OHDPAT (0.2, 0.6, and 2.0 mg/kg s.c), in conjunction with levodopa/carbidopa (12.5 mg/kg each p.o.) to levodopa-primed animals, dose-dependently reduced levodopa-induced chorea but did not affect dystonic movements. However, (R)-(+)-8-OHDPAT treatment also reduced locomotor activity and the reversal of motor disability. Administration of (R)-(+)-8-OHDPAT alone had no effects of motor behaviors. The effects of (R)-(+)-8-OHDPAT on levodopa-induced motor behaviors were antagonized by the 5-HT(1a) receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate (WAY-100635) (1.0 mg/kg s.c.). Administration of (R)-(+)-8-OHDPAT (0.6 mg/kg s.c.) also reduced chorea produced by the administration of the D(2)/D(3) dopamine receptor agonist pramipexole (0.06 mg/kg p.o.) to levodopa-primed MPTP-treated animals. However, again the increase in locomotor activity and reversal of motor disability produced by pramipexole were also inhibited. These data suggest that selective 5-HT(1a) agonists do not provide an effective means of suppressing levodopa-induced dyskinesia, except with worsening of parkinsonism.
Abstract: Sustained reactive microgliosis may contribute to the progressive degeneration of nigral dopaminergic neurons in Parkinson's disease (PD), in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) exposed human and in non-human primates. However, the temporal relationship between glial cell activation and nigral cell death is relatively unexplored. Consequently, the effects of acute (24 h) and chronic (30 days) glial cell activation induced by unilateral supranigral lipopolysaccharide (LPS) administration were studied in rats. At 24 h, LPS administration caused a marked reduction in the number of tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the substantia nigra (SN) but striatal TH-ir was unaffected. By 30 days, the loss of TH-positive neurons in the LPS-treated nigra was no greater than at 24 h although a heterogeneous loss of striatal TH-ir was present. The loss of nigrostriatal neurons was of functional significance, as at 30 days, LPS-treated rats exhibited ipsiversive circling in response to (+)-amphetamine administration. At 24 h, there was a moderate increase in glial fibrillary acidic protein (GFAP)-ir astrocytes in the SN but a marked elevation of p47phox positive OX-42-ir microglia, and intense inducible nitric oxide synthase (iNOS)-ir and 3-nitrotyrosine (3-NT)-ir was present. However, by 30 days the morphology of OX-42-ir microglia returned to a resting state, the numbers were greatly reduced and no 3-NT-ir was present. At 30 days, GFAP-ir astrocytes were markedly increased in number and iNOS-ir was present in fibrillar astrocyte-like cells. This study shows that acute glial activation leading to dopaminergic neuron degeneration is an acute short-lasting response that does not itself perpetuate cell death or lead to prolonged microglial activation.
Abstract: Direct intracerebral administration of sonic hedgehog (SHH) reduces 6-OHDA and MPTP toxicity to nigral dopaminergic cells in rats and primates. To determine whether transfection of the DNA sequence for SHH using viral vectors also protects against 6-OHDA toxicity, a type 2 adeno- associated virus (AAV) incorporating 600 base pairs of N-terminal SHH DNA was generated to induce SHH expression in rat striatum.AAV-SHH was injected into the striatum, 3 weeks prior to the initiation of an unilateral partial 6-OHDA nigro-striatal lesion. Animals receiving 4x10(7) viral particles of AAV-SHH showed a reduction in (+)-amphetamine induced ipsilateral turning over 4 weeks, when compared to animals receiving vehicle or a LacZ encoding vector. Following vehicle or AAV-LacZ administration, 6-OHDA caused a marked loss of striatal dopamine content and nigral tyrosine hydroxylase (TH) immunopositive cells. Following treatment with 4x10(7) viral particles of AAV-SHH the loss of striatal dopamine content was reduced and there was marked preservation of nigral dopaminergic cells. However, administration of 4x10(8) particles of AAV-SHH did not cause a significant change in (+)-amphetamine-induced rotation, striatal dopamine levels or the number of nigral TH immunoreactive cells following 6-OHDA lesioning compared to vehicle or AAV-LacZ treated animals.The results show that SHH delivered via a viral vector can protect dopaminergic neurons against 6-OHDA toxicity and suggest that this could be developed into a novel treatment for PD. However, the effects maybe dose limited due to uncoupling of hedgehog receptor signalling at higher levels of SHH expression.
Abstract: Standard MPTP treatment regimens in primates result in > 85% destruction of nigral dopaminergic neurons and the onset of marked motor deficits that respond to known symptomatic treatments for Parkinson's disease (PD). The extent of nigral degeneration reflects the late stages of PD rather than events occurring at its onset. We report on a modified MPTP treatment regimen that causes nigral dopaminergic degeneration in common marmosets equivalent to that occurring at the time of initiation of motor symptoms in man. Subcutaneous administration of MPTP 1 mg/kg for 3 consecutive days caused a reproducible 60% loss of nigral tyrosine hydroxylase (TH)-positive cells, which occurred mainly in the calbindin-D(28k)-poor nigrosomes with a similar loss of TH-immunoreactivity (TH-ir) in the caudate nucleus and the putamen. The animals showed obvious motor abnormalities with reduced bursts of activity and the onset of motor disability. However, the loss of striatal terminals did not reflect early PD because a greater loss of TH-ir occurred in the caudate nucleus than in the putamen and a marked reduction in TH-ir occurred in striatal patches compared to the matrix. Examination of striatal fibres following a partial MPTP lesion showed a conspicuous increase in the number and the diameter of large branching fibres in the putaminal and to some extent caudatal matrix, pointing to a possible compensatory sprouting of dopaminergic terminals. In addition, these partially lesioned animals did not respond to acute treatment with L-DOPA. This primate partial lesions model may be useful for examining potential neuroprotective or neurorestorative agents for PD.
Abstract: The free radical theory of ageing postulates that age-associated neurodegeneration is caused by an imbalance between pro-oxidants and antioxidants resulting in oxidative stress. The current study showed regional variation in brain susceptibility to age-associated oxidative stress as shown by increased lipofuscin deposition and protein carbonyl levels in male rats of age 15-16 months compared to control ones (3-5 months). The hippocampus is the area most vulnerable to change compared to the cortex and cerebellum. However, proteasomal enzyme activity was not affected by age in any of the brain regions studied. Treatment with melatonin or coenzyme Q10 for 4 weeks reduced the lipofuscin content of the hippocampus and carbonyl level. However, both melatonin and coenzyme Q10 treatments inhibited beta-glutamyl peptide hydrolase activity. This suggests that these molecules can alter proteasome function independently of their antioxidant actions.
Abstract: Pallidotomy paradoxically reduces the intensity of levodopa-induced dyskinesia without worsening motor symptoms. The reasons for this are not clear and no experimental study has investigated this phenomenon. The objective of this investigation was to evaluate the effects of unilateral pallidotomy on locomotor activity, motor disability and levodopa-induced dyskinesia in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated levodopa-primed common marmosets. Animals were primed to exhibit dyskinesia by daily administration of levodopa until stable dyskinesia was evoked by each dose. Locomotor activity, motor disability and dyskinesia were assessed weekly at baseline and following an acute levodopa challenge. Prior to pallidotomies, two distinct groups of animals emerged: poor responders to levodopa with mild dyskinesia (Group 1) and those exhibiting a marked increase in motor activity and pronounced dyskinesia (Group 2). Electrolytic lesions were placed in the left internal segment of the globus pallidus. Pallidotomy had no effect on basal or levodopa-induced motor activity in either group but significantly improved basal motor disability in Group 2. Following pallidotomy, the ability of levodopa to reduce motor disability was significantly increased in both groups. Pallidotomy improved dyskinesia in both Groups 1 and 2 but it was more effective in reducing dystonia compared with chorea. The effect of pallidotomy on dyskinesia in Group 2 was transient, with the intensity of involuntary movements reverting to presurgery levels 4 weeks later. This study shows that in levodopa-primed, parkinsonian marmosets, placement of discrete globus pallidus lesions can ameliorate levodopa-induced dyskinesia but not akinesia. This model allows the evaluation of pallidotomy-induced biochemical changes in dyskinetic primates.
Abstract: N-Methyl-d-aspartate (NMDA)-induced striatal excitotoxicity is mediated by nitric oxide (NO) but the role of inflammatory mechanisms and inducible nitric oxide synthase (iNOS) induction is not clear. Unilateral intrastriatal administration of NMDA to rats resulted in the loss of intrinsic striatal neurones and the degeneration of NADPH-diaphorase positive interneurones within 24 h. NMDA administration caused activation of glial fibrillary acidic protein positive astroglial cells and MAC-1 ir microglia. Marked iNOS immunoreactivity was expressed within both astroglial and microglial cells and there was marked cellular labelling for 3-nitrotyrosine (3-NT). One month following the NMDA lesion, administration of (+)-amphetamine (AMPH) produced a circling response in rats. Pre-treatment of rats with the iNOS inhibitor aminoguanidine (AG) decreased the extent of NMDA-induced striatal cell loss at 24 h and reduced 3-NT expression but was without effect on glial cell activation. AG pre-treatment also prevented the onset of rotation to AMPH at 30 days following NMDA lesioning. NMDA administration unexpectedly caused a loss of tyrosine hydroxylase immunoreactive (TH-ir) fibres in the striatum at 24 h and at 30 days the number of TH-ir cells were decreased in the substantia nigra. The loss of nigral cells was prevented by AG pre-treatment. This study demonstrates a role for iNOS induction in NO-mediated NMDA excitotoxicity to rat striatum and suggests that inflammatory mechanisms play a key role in this process.
Abstract: We have investigated the ability of pramipexole, a dopamine agonist used in the symptomatic treatment of Parkinson's disease (PD), to protect against cell death induced by 1-methyl-4-phenylpyridinium (MPP+) and rotenone in dopaminergic and non-dopaminergic cells. Pre-incubation with either the active (-)- or inactive (+)-enantiomer forms of pramipexole (10 microm) decreased cell death in response to MPP+ and rotenone in dopaminergic SHSY-5Y cells and in non-dopaminergic JK cells. The protective effect was not prevented by dopamine receptor blockade using sulpiride or clozapine. Protection occurred at concentrations at which pramipexole did not demonstrate antioxidant activity, as shown by the failure to maintain aconitase activity. However, pramipexole reduced caspase-3 activation, decreased the release of cytochrome c and prevented the fall in the mitochondrial membrane potential induced by MPP+ and rotenone. This suggests that pramipexole has anti-apoptotic actions. The results extend the evidence for the neuroprotective effects of pramipexole and indicate that this is not dependent on dopamine receptor occupation or antioxidant activity. Further evaluation is required to determine whether the neuroprotective action of pramipexole is translated to a disease-modifying effect in PD patients.
Abstract: Ecstasy [3,4-methylenedioxymethamphetamine (MDMA)] was shown to prolong the action of L-3,4-dihydroxyphenylalanine (L-DOPA) while suppressing dyskinesia in a single patient with Parkinson's disease (PD). The clinical basis of this effect of MDMA is unknown but may relate to its actions on either dopaminergic or serotoninergic systems in brain. In normal, drug-naive common marmosets, MDMA administration suppressed motor activity and exploratory behavior. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated, L-DOPA-primed common marmosets, MDMA transiently relieved motor disability but over a period of 60 min worsened motor symptoms. When given in conjunction with L-DOPA, however, MDMA markedly decreased dyskinesia by reducing chorea and to a lesser extent dystonia and decreased locomotor activity to the level observed in normal animals. MDMA similarly alleviated dyskinesia induced by the selective dopamine D2/3 agonist pramipexole. The actions of MDMA appeared to be mediated through 5-HT mechanisms because its effects were fully blocked by the selective serotonin reuptake inhibitor fluvoxamine. Furthermore, the effect of MDMA on L-DOPA-induced motor activity and dyskinesia was partially inhibited by 5-HT1a/b antagonists. The ability of MDMA to inhibit dyskinesia results from its broad spectrum of action on 5-HT systems. Serotoninergic receptors appear to play an important modulatory role in l-DOPA-induced dyskinesia, and this study may provide a framework for the use of serotoninergic agents in the treatment of L-DOPA-induced dyskinesia.
Abstract: The loss of dopaminergic neurones in the substantia nigra with Parkinson's disease may result from inflammation-induced proliferation of microglia and reactive macrophages expressing inducible nitric oxide synthase (iNOS). We have investigated the effects of the supranigral administration of lipopolysaccharide on iNOS-immunoreactivity, 3-nitrotyrosine formation and tyrosine hydroxylase-immunoreactive neuronal number, and retrogradely labelled fluorogold-positive neurones in the ventral mesencephalon in male Wistar rats. Following supranigral lipopolysaccharide injection, 16-18 h previously, there was intense expression of NADPH-diaphorase and iNOS-immunoreactivity in non-neuronal, macrophage-like cells. This was accompanied by intense expression of glial fibrillary acidic protein-immunoreactive astrocytosis in the substantia nigra. There were also significant reductions in the number of tyrosine hydroxylase(50-60%)- and fluorogold (65-75%)-positive neurones in the substantia nigra. In contrast, tyrosine hydroxylase-immunoreactivity in the ventral tegmental area was not altered. Pre-treatment of animals with the iNOS inhibitor, S-methylisothiourea (10 mg kg(-1), i.p.), led to a significant reduction of lipopolysaccharide-induced cell death. Similar reduction of tyrosine hydroxylase-immunoreactivity and fluorogold-labelled neurones in the substantia nigra following lipopolysaccharide administration suggests dopaminergic cell death rather than down-regulation of tyrosine hydroxylase. We conclude that the expression of iNOS- and 3-nitrotyrosine-immunoreactivity and reduction of cell death by S-methylisothiourea suggest the effects of lipopolysaccharide may be nitric oxide-mediated, although other actions of lipopolysaccharide (independent of iNOS induction) cannot be ruled out.
Abstract: Sonic hedgehog (SHH) has trophic actions on dopaminergic cell cultures and protects them from MPP(+) toxicity but its in vivo actions have not been explored. We now investigate the effects of unilateral supranigral administration of SHH on nigro-striatal function in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated common marmosets. SHH (0.1 or 1.0 microg) or vehicle was stereotaxically injected into the region of the right substantia nigra twice with an interval of 5 weeks between administrations. The first or second administration of low dose SHH (0.1 microg) did not significantly improve motor disability or locomotor activity compared to time-matched vehicle-treated animals. There was, however, an approximately 30% improvement in both motor disability and locomotor activity following the first administration of high dose SHH (1.0 microg). No further improvements occurred following the second high dose SHH treatment. Acute oral administration of L-3,4-dihydroxyphenylalanine (L-DOPA) produced a smaller increase in locomotor activity and greater reversal of motor disability in animals treated with SHH than occurred in vehicle-treated common marmosets. In the substantia nigra pars compacta, ipsilateral to SHH administration, the number of tyrosine hydroxylase-positive neurones was increased by 21% (P > 0.05) and 57% (P < 0.05) in low and high dose SHH groups respectively compared to the untreated contralateral hemisphere. There was no difference in the number of glial fibrillary acidic protein-positive cells. SHH may improve nigro-striatal function by restoring tyrosine hydroxylase positivity. This is reflected by an improvement in basal disability and a reduction in the lesion-induced response to L-DOPA.
Abstract: Parkinson's disease (PD) is associated with a progressive loss of dopamine neurons in the substantia nigra and degeneration of dopaminergic terminals in the striatum. Although L-DOPA treatment provides the most effective symptomatic relief for PD it does not prevent the progression of the disease, and its long-term use is associated with the onset of dyskinesia. In rodent and primate studies, glial cell line-derived neurotrophic factor (GDNF) may prevent 6-OHDA- or MPTP-induced nigral degeneration and so may be beneficial in the treatment of PD. In this study, we investigate the effects of GDNF on the expression of dyskinesia in L-DOPA-primed MPTP-treated common marmosets, exhibiting dyskinesia. GDNF or saline was administered by two intraventricular injections, 4 weeks apart, to MPTP-treated, L-DOPA-treated common marmosets primed to exhibit dyskinesia. Prior to GDNF or saline administration, all animals displayed marked dyskinesia when treated with L-DOPA. GDNF administration produced a significant improvement in motor disability and, following the second injection of GDNF, a significant improvement in the locomotor activity was observed. Following the administration of L-DOPA there was a greater reversal of disability and a reduction in the intensity of L-DOPA-induced dyskinesia in GDNF-treated animals compared to saline-treated controls. However, there was no significant difference in L-DOPA's ability to increase locomotor activity between GDNF-treated and saline-treated animals. GDNF treatment caused a significant increase in the number of tyrosine hydroxylase-positive neurons in the substantia nigra, but no change in [(3)H]mazindol binding to dopamine terminals was found in the striatum of GDNF-treated animals compared to saline-treated controls. In GDNF-treated animals a small but significant reduction in enkephalin mRNA was observed in the caudate nucleus but not in the putamen or the nucleus accumbens. Substance P mRNA expression was equally reduced in the caudate nucleus and the putamen of the GDNF-treated animals but not in the nucleus accumbens. Intraventricular administration of GDNF improved MPTP-induced disability and reversed dopamine cell loss in the substantia nigra. GDNF also diminished L-DOPA-induced dyskinesia, which may relate to its ability to partly restore nigral dopaminergic transmission or to modify the activity of striatal output pathways.
Abstract: Glial cell line-derived neurotrophic factor (GDNF) has previously reduced motor deficits and preserved nigral dopamine neurones in rhesus monkeys with a unilateral MPTP-induced lesion of substantia nigra. We now report on the ability of GDNF to reverse motor deficits induced by parenteral administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to common marmosets resulting in bilateral degeneration of the nigrostriatal pathway. Prior to GDNF administration, all MPTP-treated animals showed akinesia or bradykinesia, rigidity, postural instability and tremor. Intraventricular injection of GDNF (10, 100 or 500 microg) at 9 and 13 weeks post MPTP treatment resulted in a concentration dependent improvement in locomotor activity and motor disability which became significant after administration of 100 and 500 microg of GDNF. The most prominent improvements were in alertness, checking movements, and posture. It is concluded that intraventricular GDNF administration improves bilateral Parkinsonian motor disability following MPTP treatment and this may reflect an action of GDNF on remaining nigral dopaminergic neurones.
Abstract: Fast cyclic voltammetry was used to measure NO and dopamine (DA) simultaneously in rat caudate putamen (CPu) slices. Analysis of electrochemical signals obtained from mixtures of DA and NO showed that subtraction of either the DA or the NO component revealed the contribution of the other component. Application of such data manipulation to signals obtained in CPu slices indicated that DA and NO components contributed to electrochemical signals. Following electrical stimulation (>1 s), site-dependent NO-like signals were observed. Pressure ejection of NMDA yielded a biphasic electrochemical signal. The first phase (lasting 10-20 s) had electrochemical characteristics of DA and was observed only during the first application of NMDA. The second phase developed more slowly, was of longer duration (1-3 min), and had electrochemical characteristics of a mixture of DA and NO. Generation of the NO-like signal by NMDA was antagonised by L-N-monomethylarginine. Pressure ejection of NO into CPu slices caused dose- and site-dependent DA release. More DA was released in the dorsolateral than in the dorsomedial CPu. Pressure ejection of DA did not generate NO. It is concluded that electrically stimulated DA release is not mediated by prior release of NO.
Abstract: Fast cyclic voltammetry at a carbon fibre microelectrode was used to measure 5-HT signals following electrical or chemical stimulation in rat substantia nigra pars reticulata slices. Chemical stimulation with (+)-amphetamine or veratrine gave signals which were indistinguishable from those of exogenous 5-HT. Electrical stimulation of sufficient duration gave voltammetric signals which were characteristic of 5-HT. Release of dopamine was not detected following either chemical or electrical stimulation. The 5-HT signals were attenuated by TTX and enhanced by fluvoxamine. It was not possible to demonstrate regulation of 5-HT release in the SNr by 5-HT1B autoreceptors using CGS 12066A or methiothepin. Signal following electrical stimulation were not enhanced by either benztropine or GBR12909, or modified in the presence of either quinpirole or sulpiride. We conclude that 5-HT release can be detected voltammetrically in the SNr; 5-HT release is likely to be from axon terminals, but somatodendritic DA release could not be detected.
Abstract: Isolated anococcygeus muscle from male rats was examined for the presence of neuropeptide Y-immunoreactive nerves and for the effects of neuropeptide Y on its tone and its contractile/relaxant responses to electrical field stimulation, acetylcholine, guanethidine and noradrenaline. Using peroxidase anti-peroxidase immunohistochemistry in stretch preparation of the anococcygeus, neuropeptide Y-immunoreactive nerve fibres were observed, in abundance, running along both vascular as well as non-vascular smooth muscle cells. Neuropeptide Y (> 250 nM) evoked phentolamine and tetrodotoxin-resistant contractile response. Neuropeptide Y, even in subspasmogenic concentrations, potentiated contractions evoked by acetylcholine, guanethidine and noradrenaline. Electrical field stimulation (trains of 3-4 pulses, 0.1 ms, 10 Hz) of the isolated anococcygeus preparation produced robust, phentolamine and tetrodotoxin sensitive contractions. Neuropeptide Y (< 10 nM) exerted a biphasic effect on the electrical field stimulation-evoked contractions; an early potentiation was followed by a delayed and progressive inhibition. Neuropeptide Y (> 10 nM) caused a concentration-dependent potentiation of electrical field stimulation-evoked contraction alone, matching its potentiation of noradrenaline-evoked contraction. Electrical field stimulation (5 pulses, 0.1 ms, 10 Hz) of guanethidine (50 microM)-contracted anococcygeus induced a relaxant response and neuropeptide Y (1-100 nM) exerted a concentration-related slight and variable effect on the electrical field stimulation-evoked relaxant response (1 nM, augmentation; 10 nM, no effect; 100 nM, reduction). It is concluded that rat anococcygeus muscle has a rich neuropeptide Y-containing innervation and neuropeptide Y is mostly stored within adrenergic nerves. The main functional roles of neuropeptide Y in the anococcygeus muscle are likely to be post-junctionally mediated facilitation and prejunctionally mediated inhibition of adrenergic motor transmission.
Abstract: Fast cyclic voltammetry at a carbon fibre microelectrode was used to measure dopamine release following electrical or chemical stimulation in rat brain slices incorporating either the ventral tegmental area or the core region of the nucleus accumbens. Electrical or chemical stimulation gave clear voltammetric signals which corresponded to dopamine; less dopamine was released in the ventral tegmental area than in the nucleus accumbens. In contrast to the nucleus accumbens, electrically stimulated dopamine release in the ventral tegmental area was not sensitive to tetrodotoxin, was not modified by the presence of dopamine uptake inhibitors, or agonist or blockers acting at dopamine D2 autoreceptors.
Abstract: Behavioural sensitization to the locomotor stimulating effects of (+)-amphetamine or quinpirole was induced in rats by intermittent drug administration. Following expression of sensitization, locomotor activity scores on day 9 were: vehicle 87 +/- 9, (+)-amphetamine 1441 +/- 227 and quinpirole 2078 +/- 214. Electrically stimulated dopamine release was measured on day 12 in ventral tegmental slices using fast cyclic voltammetry. Dopamine release was significantly elevated in the (+)-amphetamine- and quinpirole-treated groups when compared to vehicle-treated controls over a wide range of stimulation frequencies (5-200 Hz) and pulses (1-200). Quinpirole (1 microM) in the perfusion fluid attenuated dopamine release following 40-pulse, 200-Hz electrical stimulation, by 31.6 +/- 2.8% in the ventral tegmental area of the vehicle-treated group, by 14.8 +/- 5.6% in the (+)-amphetamine-treated group and 8 +/- 7.3% in the quinpirole-treated group. This study shows that dopamine release is increased in the ventral tegmental area following sensitization with either a direct or indirectly acting dopamine agonist. The findings that dopamine release was elevated at all stimulation frequencies in sensitized animals, and that quinpirole only attenuated this release at the highest stimulation frequency, would suggest that in addition to D2 autoreceptor subsensitivity, other mechanisms contribute to the enhanced release of dopamine in these animals.
Abstract: The functional role of N-methyl-D-aspartic acid (NMDA) glutamate receptors in the real-time regulation of single electrical pulse (1 p)-stimulated endogenous dopamine release was investigated in slices of rat caudate putamen using fast cyclic voltammetry at a carbon fibre electrode. In the presence of Mg2+, 20 microM NMDA had a weak effect on background signals but did not affect 1 p-stimulated dopamine release. Removal of Mg2+ increased the background and doubled 1 p-stimulated dopamine release. In the absence of Mg2+, 20 microM NMDA caused a transient "release" of dopamine and decreased the background signal. The 1 p-stimulated dopamine release was subsequently reduced. In the presence of 1 microM (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), superfusion with 20 microM NMDA did not cause a transient "release" of dopamine, and 1 p-stimulated dopamine release was not subsequently attenuated. In the presence of 1 microM tetrodotoxin, 1 p-stimulated dopamine release was abolished, but 20 microM NMDA still caused a transient "release" of dopamine. Removal of Ca2+ from the artificial CSF abolished 1 p-stimulated dopamine release and resulted in a decline in the baseline but did not affect dopamine "release" when 20 microM NMDA was added. The dopamine release-inducing effect of 20 microM NMDA was less pronounced in sites in the caudate putamen where dopamine release increased with frequency of electrical stimulation (hot spots) than in sites where there was little frequency-dependent dopamine release (cold spots). Subsequent 1 p-stimulated dopamine release was less attenuated in cold spots than in hot spots. We conclude that in the absence of Mg2+, NMDA induces release of dopamine by acting at CPP-sensitive NMDA receptors in a Ca(2+)-independent manner. This transient release depletes dopamine from a storage site from which dopamine is released by 1 p electrical stimulation. These real-time observations of the effects of NMDA on electrical stimulus-independent and -dependent dopamine release may explain the apparently conflicting observations of the effects of NMDA on dopamine release made in previous studies. They also indicate that dopamine release and storage are heterogeneous at different sites in the rat caudate putamen.
Abstract: The effects of (+)-amphetamine on carrier-mediated and electrically stimulated dopamine release were investigated using fast cyclic voltammetry in rat brain slices incorporating the nucleus accumbens, and in the caudate putamen. In the caudate putamen, dopamine release either increased with increasing frequency of local electrical stimulation (hot spots) or did not increase significantly (cold spots); dopamine release increased with increasing frequency of electrical stimulation in the nucleus accumbens. Local pressure application of (+)-amphetamine from a micropipette caused dopamine efflux at all sites examined, and this was not affected by sulpiride, indicating that efflux of dopamine caused by (+)-amphetamine is not regulated by dopamine D2 autoreceptors. (+)-Amphetamine reduced single-pulse electrically stimulated dopamine release at all sites; sulpiride reversed this decrease, indicating that endogenous dopamine released by (+)-amphetamine activates dopamine D2 autoreceptors. In nucleus accumbens and hot spots, (+)-amphetamine did not affect 20-pulse 50-Hz-stimulated dopamine release, whereas in cold spots it potentiated 20-pulse 50-Hz-stimulated dopamine release. We conclude that (+)-amphetamine modifies electrically stimulated dopamine release by uptake inhibition or by indirect activation of D2 autoreceptors; the precise mechanism is determined by site and duration of electrical stimulation.
Abstract: 1. Immunohistochemical and isolated organ bath techniques were used to detect the presence of neuropeptide Y (NPY) in the rat urinary bladder and to determine its effect on tone, spontaneous activity and contractile responses of the detrusor muscle to electrical field stimulation, acetylcholine and alpha,beta-methylene ATP (alpha,beta-MeATP). 2. A very rich presence of NPY-immunoreactive nerve fibres was found mainly within the bundles of detrusor muscle cells. Chronic treatment with 6-hydroxydopamine did not affect the density of NPY-positive nerve fibres. 3. NPY (> 1 nM) enhanced the force and frequency of spontaneous contractions and generated a rise in the resting tone of the detrusor. These effects of NPY on the tone and the spontaneous activity remained unaffected by atropine (3 microM), indomethacin (10 microM) and aspirin (100 microM) but were abolished by Ca(2+)-withdrawal from the bathing medium. 4. The enhancing effects of NPY on the spontaneous contractions and the resting tone were not prevented by the induction of purinoceptor desensitization. 5. NPY (1-250 nM) potentiated electrical field stimulation (EFS, 1-64 Hz, 0.1 ms pulses duration, 10s train duration)-evoked, tetrodotoxin (0.5 microM)-sensitive contractions. The atropine (3 microM)-resistant component of EFS-evoked contractions was also potentiated by NPY. By contrast, the nifedipine (1 microM)-resistant but atropine-sensitive component of EFS-evoked contraction was inhibited by NPY. 6. NPY (250 nM) did not affect acetylcholine-evoked contractions, but potentiated alpha,beta-MeATP-evoked contractions. 7. It is concluded that NPY-innervation of rat urinary bladder is largely confined to the detrusor muscle and is abundant and mainly non-adrenergic. It is further concluded that the enhancing effect of NPY on detrusor spontaneous activity and tone is caused by Ca2+ influx through nifedipine-sensitive Ca2+ channels and is not mediated through acetylcholine or cyclo-oxygenase-sensitive eicosanoids or ATP.8. The results are consistent with the hypothesis that intrinsic NPY in the rat detrusor innervation contributes to the motor transmission in two ways: by promoting non-cholinergic motor transmission and by inhibiting prejunctionally the cholinergic transmission.
Abstract: 1. Immunohistochemical and functional studies have been performed to localize and determine the effects of neuropeptide Y (NPY) in the rat seminal vesicle. 2. An abundant presence of NPY-immunoreactive nerves, mainly concentrated in the smooth muscle layer of the seminal vesicle was found. Chronic 6-hydroxydopamine treatment (four doses of 50 mg kg-1 i.p. on days 1, 2, 4 and 6; rats killed one week after the last injection) led to a large reduction but not abolition of the NPY-immunoreactivity. 3. NPY (1-250 nM) did not affect the resting tone of the seminal vesicle. 4. The seminal vesicle was contracted by electrical field stimulation (EFS) and by exposure to 5 microM noradrenaline (NA). These contractions were abolished by phentolamine (1 microM). Tetrodotoxin (0.5 microM) abolished EFS-evoked contractions but did not affect NA-evoked contractions. 5. Seminal vesicles, from animals chronically-treated with reserpine (5 mg kg-1 i.p. on days 1 and 2; rats killed on day 3), were contracted by NA but not by EFS. 6. NPY (0.25-250 nM), concentration-dependently, inhibited EFS-evoked contractions by up to 70% maximum inhibition. Contractions evoked by EFS with short trains of pulses were inhibited by NPY to a greater degree than those with longer trains. 7. NPY had no significant effect on NA-evoked contractions. 8. These data provide strong evidence that the motor transmission in rat seminal vesicle is predominantly if not exclusively, adrenergic. It is further concluded that a rich NPY-containing innervation is present in the smooth muscle layer of rat seminal vesicle. The primary effect of NPY is modulation of adrenergic motor transmission by a prejunctional inhibition of NA release.
Abstract: In rat anococcygeus muscle the inhibitory effect of nifedipine (0.01, 0.1, 1.0 and 10 microM) was determined on adrenergic twitches in response to electrical field stimulation (trains of 4 pulses, 0.1 ms pulse duration, 10 Hz) and on twitch-matching contractions evoked by noradrenaline. Nifedipine concentration-dependently reduced the neurogenic twitch with an IC50 of 0.083 microM. Nifedipine reduced the noradrenaline-evoked contraction to a markedly lesser degree (IC50 > 10 microM). The difference in the magnitude of inhibition of electrically evoked twitch and twitch-matching noradrenaline-evoked contraction was statistically significant at every concentration of nifedipine. It is concluded that inhibition of the twitch by nifedipine involves some other mechanism(s) in addition to its Ca2+ channel blocking property in smooth muscle.
Abstract: The effect of the calcium channel blocker nifedipine on the motor transmission in isolated preparations of rat detrusor smooth muscle has been studied. Nifedipine blocked the major part (75 to 80%) of the contractile response to electrical field stimulation, while atropine only blocked 20 to 25%. In preparations pretreated with atropine, the response to electrical field stimulation was completely abolished by nifedipine. The converse was also true; in preparations pretreated with nifedipine the response was fully blocked by atropine. The nifedipine-resistant response was greatly potentiated by the anticholinesterase eserine. The blocking action of nifedipine on motor transmission was partially antagonised by raising Ca2(+)-concentration. Acetylcholine concentration-response curve was shifted to the right by nifedipine. It is concluded that the non-cholinergic motor neurotransmitter evokes contraction of the rat detrusor smooth muscle by activating external Ca2(+)-transport channels whereas the cholinergic contraction is mediated partly or wholly by alternative mechanisms.
