Oscillations in neuronal networks are assumed to serve various physiological functions, from coordination of motor patterns to perceptual binding of sensory information. Here, we describe an ultra-slow oscillation (0.025 Hz) in the hippocampus. Extracellular and intracellular activity was recorded from the CA1 and subicular regions in rats of the Wistar and Sprague-Dawley strains, anesthetized with urethane. In a subgroup of Wistar rats (23%), spontaneous afterdischarges (4.7+/-1.6 s) occurred regularly at 40.8+/-15.7 s. The afterdischarge was initiated by a fast increase of population synchrony (100-250 Hz oscillation; "tonic" phase), followed by large-amplitude rhythmic waves and associated action potentials at gamma and beta frequency (15-50 Hz; "clonic" phase). The afterdischarges were bilaterally synchronous and terminated relatively abruptly without post-ictal depression. Single-pulse stimulation of the commissural input could trigger afterdischarges, but only at times when they were about to occur. Commissural stimulation evoked inhibitory postsynaptic potentials in pyramidal cells. However, when the stimulus triggered an afterdischarge, the inhibitory postsynaptic potential was absent and the cells remained depolarized during most of the afterdischarge. Afterdischarges were not observed in the Sprague-Dawley rats. Long-term analysis of interneuronal activity in intact, drug-free rats also revealed periodic excitability changes in the hippocampal network at 0.025 Hz. These findings indicate the presence of an ultra-slow oscillation in the hippocampal formation. The ultra-slow clock induced afterdischarges in susceptible animals. We hypothesize that a transient failure of GABAergic inhibition in a subset of Wistar rats is responsible for the emergence of epileptiform patterns.
Cholinergic inputs from the basal forebrain to cortex exert profound effects on cortical activities, such as a rhythmic synchronization. For these modulatory effects inhibitory interneurons could play crucial roles within the cortical circuitry. To study cholinergic modulation of GABA-mediated inhibition, we recorded inhibitory postsynaptic current (IPSC) during application of cholinergic agonists in the rat frontal cortex in vitro. Both carbachol and muscarine caused two temporally different patterns of IPSC modulation in both pyramidal cells and inhibitory interneurons: tonic or periodic increase of GABA-A receptor-mediated inhibition. The tonic pattern showed a continuous increase of IPSC frequency, while the periodic increase manifested itself as rhythmic (0.1-0.3 Hz, mean 0.2 Hz) bursts of IPSC (frequency: 6-69 Hz, mean 24 Hz; burst duration: 1.2-4.3 s, mean 2.2 s). Both types of increase were suppressed by atropine or pirenzepine, muscarinic-receptor antagonists. The periodical IPSC bursts were not affected by antagonists for ionotropic glutamate receptors. Following cholinergic stimulation, periodic IPSC bursts in nearby cells were synchronized as a whole, but individual inhibitory events within the bursts were not always temporally correlated, suggesting synchronized depolarizations of several presynaptic interneurons. It has been revealed that slow rhythmic depolarizations accompanying spike firing can be generated within the cortex. In addition to this periodic excitation of cortical circuits, these results indicate that cortical inhibitory interneurons have their own acetylcholine-dependent mechanism generating the slow rhythm independent of the excitatory circuits.
Two regions in the forebrain of domestic chicks (Gallus domesticus), the intermediate and medial hyperstriatum ventrale and the lobus parolfactorius, have previously been shown to be important centres of biochemical, pharmacological and physiological change following one-trial passive avoidance training. The purpose of the present study was to examine, at the electron microscopic level, the fine spatial re-arrangement of synaptic structures in the intermediate and medial hyperstriatum ventrale (at 30 min), and in the lobus parolfactorius (at 24 h), post-training using comprehensive biometrical designs, image analysis and stochastic approaches. In intermediate and medial hyperstriatum ventrale, no significant differences in the numerical density of synapses either between control and trained chicks, or between hemispheres, were revealed using the disector method. However, after training, a nested-ANOVA demonstrated an increase in the thickness of pre- and post-synaptic electron densities (estimated via image analysis) only in the left intermediate and medial hyperstriatum ventrale, whereas synaptic apposition zone profiles increased in length bilaterally. In presynaptic terminals from the intermediate and medial hyperstriatum ventrale, stochastic analysis revealed that training resulted in the re-distribution of synaptic vesicles between two spatial pools relative to synaptic apposition zones, in both hemispheres producing a large number of synaptic vesicles closer to synaptic apposition zones; a nearest neighbour analysis of synaptic apposition zone profiles indicated that the lateral shape of the synaptic apposition zone after training is more complex in both hemispheres. In the lobus parolfactorius at 24 h post-training the main changes in synaptic fine structure involved a shift of synaptic vesicles away from synaptic apposition zones in the right hemisphere with the distance between synaptic apposition zones decreasing; in the left lobus parolfactorius, synaptic apposition zones became more regular/round in shape with a greater distance between them after training. These data suggest that the initial acquisition of memory involves population changes in the fine spatial organization of synaptic vesicles and synaptic apposition zones in synapses in the intermediate and medial hyperstriatum ventrale, which indicate a possible tendency towards greater synaptic efficacies. These changes are as dynamics as the molecular changes which have hitherto been considered the preserve of short-term correlates of memory formation.
Responses to injury in the ageing hippocampus were assessed utilizing the synaptic markers glial fibrillary acidic protein and synaptosomal-associated protein (mol. wt 25,000) following administration of the neurotoxin, 5,7-dihydroxytryptamine, into the fimbria-fornix and cingulum bundle to denervate serotonergic afferent input to the dorsal hippocampus. Age-dependent alterations in hippocampal immunohistochemical localization of glial fibrillary acidic protein and synaptosomal-associated protein were evaluated in female Fischer 344 rats following serotonergic deafferentation with 5,7-dihydroxytryptamine. Across the lifespan, as indicated by measurements taken at three, 18, 21 and 29 months, marked increases in glial fibrillary acidic protein, but not synaptosomal-associated protein immunoreactivity, occurred throughout the hippocampus at 21 and 29 months compared to three and 18 months. Following three weeks pretreatment with 5,7-dihydroxytryptamine (20 microg total dose) or vehicle (0.1% ascorbic saline; 2 microl total volume) infused in the fimbria-fornix/cingulum bundle, immunohistochemical analysis demonstrated marked increases of glial fibrillary acidic protein, but not synaptosomal-associated protein, in the 18-month 5,7-dihydroxytryptamine group compared to the 18-month vehicle and 3-month 5,7-dihydroxytryptamine groups. Additionally, a significant increase in glial fibrillary acidic protein concentration was found by enzyme-linked immunosorbent assay in the 18-month 5,7-dihydroxytryptamine group compared to the 18-month vehicle and three-month 5,7-dihydroxytryptamine groups. These results demonstrate that selective neurotoxicant damage of the hippocampal serotonergic system differentially alters the expression of glial fibrillary acidic protein. This approach may provide a valuable tool to determine the ability of the hippocampus to respond to age-related neurodegenerative injury.
Glutamate dehydrogenase is one of the main enzymes involved in the formation and metabolism of the neurotransmitter glutamate. In the present study we investigated the enzyme ultrastructurally in the cerebellar cortex, a region rich in well defined glutamatergic neurons, by pre-embedding immunocytochemical staining (peroxidase-antiperoxidase), as well as by post-embedding immunogold labelling employing a new system for quantitation and for specificity testing under the conditions of the immunocytochemical procedure. A new antiserum against immunologically purified bovine liver glutamate dehydrogenase or antibodies isolated from this by affinity chromatography were used in rats fixed by perfusion with aldehydes.
The present study was designed to evaluate the possible neuroprotective effects of metabotropic glutamate receptor (mGluR7) allosteric agonist N,N'-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082) on developmental sevoflurane neurotoxicity. To achieve the objective, hippocampal cultures (7 DIV, 7 day in vitro) were treated with different doses of L-(+)-2-amino-4-phosphonobutyric acid (L-AP4, an agonist of group III mGluRs), (RS)-α-Methylserine-O-phosphate (MSOP, an antagonist of group III mGluRs), AMN082 or cis-2-[[(3,5-dichlorophenyl)amino]carbonyl]cyclohexanecarboxylic acid (VU0155041, an agonist of mGluR4) before exposed to sevoflurane. Cell apoptosis were determined by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL)-staining. For in vivo study, rat pups (7 PND, 7 postnatal day) were injected with AMN082, L-AP4 or saline before sevoflurane exposure. Extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38, caspase-3, Bcl-2, and Bax were detected by Western blot. The locomotor activity and cognitive functions were evaluated by open-field test and Morris water maze (MWM), respectively. We found that L-AP4 prevented sevoflurane-induced cell apoptosis, but MSOP promoted. Specially, application of AMN082 contributed to the relief of sevoflurane-induced apoptosis in vitro, whereas VU0155041 did not. In addition, sevoflurane treatment led to a decrease of Bcl-2 and an increase of caspase-3 and Bax, which were mitigated by AMNO82 in vivo. Moreover, we showed that sevoflurane treatment resulted in a remarkable suppression of phospho-ERK1/2, which was restored by AMN082. Application of U0126 (an inhibitor of MEK) abolished the neuroprotective effects of AMN082 on sevoflurane neurotoxicity both in vitro and in vivo. In addition, sevoflurane exposure also led to an increase of phospho-JNK, but SP600125 (an inhibitor of JNK) did not attenuate sevoflurane-induced apoptosis. The total and phosphorylated p38 remained unchanged in sevoflurane-treated rat pups. Finally, AMN082 improved the learning and memory defects caused by postnatal sevoflurane exposure without alternations in emotion or locomotor activity. These preliminary data indicate that AMN082 may protect immature brain against sevoflurane neurotoxicity, and the ERK1/2 MAP kinase signaling is likely to be involved. Further studies are needed to fully assess the neuroprotective role of mGluR7 agonist AMN082 in developmental anesthetic neurotoxicity.
The aim of the present in vivo microdialysis study was to investigate whether prenatal exposure to the CB(1) receptor agonist WIN55,212-2 mesylate (WIN; (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinyl-methyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone), at a dose of 0.5 mg/kg (s.c. from the fifth to the 20th day of gestation), that causes neither malformations nor overt signs of toxicity, influences cortical glutamate extracellular levels in adult (90-day old) rats. Dam weight gain, pregnancy length and litter size at birth were not significantly affected by prenatal treatment with WIN. Basal and K(+)-evoked dialysate glutamate levels were lower in the cerebral cortex of adult rats exposed to WIN during gestation than in those born from vehicle-treated mothers. In both group of animals WIN (0.1 mg/kg, i.p.) increased dialysate glutamate levels. However, while the blockade of the CB1 receptors with the selective receptor antagonist SR141716A completely counteracted the WIN-induced increase in those rats exposed to vehicle during gestation, it failed to antagonise the increase in those born from WIN-treated dams. These findings suggest that prenatal exposure to the CB1 receptor agonist WIN, at a concentration which is not associated with gross malformations and/or overt signs of toxicity, induces permanent alterations in cortical glutamatergic function. The possibility that these effects might underlie, at least in part, some of the cognitive deficits affecting the offspring of marijuana users is discussed.
In response to injury, endogenous precursors in the adult brain can proliferate and generate new neurons, which may have the capacity to replace dysfunctional or dead cells. Although injury-induced neurogenesis has been demonstrated in animal models of stroke, Alzheimer's disease (AD) and Huntington's disease (HD), studies of Parkinson's disease (PD) have produced conflicting results. In this study, we investigated the ability of adult mice to generate new neurons in response to the parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which causes selective degeneration of nigrostriatal dopamine neurons. MPTP lesions increased the incorporation of 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdU), as well as the number of cells that co-expressed BrdU and the immature neuronal marker doublecortin (DCX), in two neuroproliferative regions-the subgranular zone of the dentate gyrus (DG) and the rostral subventricular zone (SVZ). BrdU-labeled, DCX-expressing cells were not found in the substantia nigra (SN) of MPTP-treated mice, where neuronal cell bodies are destroyed, but were present in increased numbers in the striatum, where SN neurons lost in PD normally project. Fibroblast growth factor-2 (FGF-2), which enhances neurogenesis in a mouse model of HD, also increased the number of BrdU/DCX-immunopositive cells in the SN of MPTP-treated mice. Thus, MPTP-induced brain injury increases striatal neurogenesis and, in combination with FGF-2 treatment, also stimulates neurogenesis in SN.
The effect of acute administration of 1,2,3,4-tetrahydroisoquinoline, an endogenous substance suspected of producing Parkinsonism in humans, on the levels of glutathione and reactive oxygen species and on the enzymatic activity of gamma-glutamyl transpeptidase was investigated in the substantia nigra, striatum and cortex of rat brain. Four hours after a single dose of 1,2,3,4-tetrahydroisoquinoline (100 mg/kg i.p.), a significant increase in tissue glutathione level was found in the dopaminergic structures studied. The most pronounced effect was observed in the substantia nigra and cortex, and the weakest in the striatum. At the same time, significant inhibition of gamma-glutamyl transpeptidase was observed in the substantia nigra, cortex and striatum whose extent strictly corresponded to the increase in glutathione levels in those structures. Moreover, in 1,2,3,4-tetrahydroisoquinoline-treated rats, the production of reactive oxygen species was significantly reduced in the substantia nigra, whereas it was markedly enhanced in the striatum.Our results suggest that the increase in tissue glutathione level in the dopaminergic structures studied results from inhibition of gamma-glutamyl transpeptidase and refers to the extracellular pool of this peptide. Moreover, it is likely that both the 1,2,3,4-tetrahydroisoquinoline-induced alterations in glutathione level and the enhanced production of reactive oxygen species in the striatum may have implications for the pathogenesis of Parkinson's disease.
The finding that endogenous tetrahydroisoquinolines may be involved in the etiology of Parkinson's disease suggests that their administration may cause changes resembling those observed in parkinsonian brain. We tested, using a high-performance liquid chromatography method, how single and chronic administration of 1,2, 3,4-tetrahydroisoquinoline and salsolinol affects dopamine and serotonin metabolism in the neurons of extrapyramidal and mesolimbic dopaminergic systems. We report that chronic administration of tetrahydroisoquinoline and salsolinol causes a decrease in a dopamine metabolism: the effect of tetrahydroisoquinoline was limited to the striatum, while salsolinol caused also a dramatic decline of dopamine level in the substantia nigra. The effect of both compounds on serotonin metabolism was small or absent. The tetrahydroisoquinolines produced no changes in the nucleus accumbens. The results indicate that tetrahydroisoquinoline and salsolinol specifically affect the nigrostriatal dopamine system, but only when administered chronically, and thus are compatible with the view that endogenous tetrahydroisoquinolines may participate in pathogenesis of Parkinson's disease.
Organotypic slice co-culture of the ventromedial portion of the mesencephalon and striatum was used to evaluate the neurotoxicity of 1-benzyl-1,2,3,4-tetrahydroisoquinoline, an endogenous brain amine related to Parkinson’s disease. 1-Benzyl-1,2,3,4-tetrahydroisoquinoline is specifically increased in the cerebrospinal fluid of patients with Parkinson’s disease and induces parkinsonian features in the monkey and mouse. Here, it decreased the dopamine content of the cultured mesencephalon in both dose- (10–100 μM) and time- (24 h to 7 days) dependent manners. This result suggests that the neurotoxicity of 1-benzyl-1,2,3,4-tetrahydroisoquinoline is correlated with the overall exposure (concentration multiplied by exposure time). Culture with 100 μM 1-benzyl-1,2,3,4-tetrahydroisoquinoline for 24 h irreversibly reduced the dopamine content. Furthermore, culture with 100 μM 1-benzyl-1,2,3,4-tetrahydroisoquinoline for 10 days caused morphological changes, including cell body shrinkage and distortion of dendritic morphology, in tyrosine hydroxylase-positive cells in the mesencephalon and reduced the number of cells by half. The increase in lactate dehydrogenase activity in the media produced by 1-benzyl-1,2,3,4-tetrahydroisoquinoline was significant in culture of the mesencephalon alone or its co-culture with striatum, but not in cultures of other brain regions. We suggest that 1-benzyl-1,2,3,4-tetrahydroisoquinoline is toxic to tyrosine hydroxylase-positive cells in the ventral mesencephalon and that it is correlated with the integral of the concentration by time of exposure. Thus a low concentration of 1-benzyl-1,2,3,4-tetrahydroisoquinoline may first induce a decrease in the dopamine content then shrinkage of the cell body, followed by the slow death of dopaminergic neurons over a long period. This is the first report that indicates 1-benzyl-1,2,3,4-tetrahydroisoquinoline exerts neurotoxicity at the cellular level, and reveals in part the character of its neurotoxicity.
The effects of acute and chronic administration of 1,2,3,4-tetrahydroisoquinoline, an endogenous substance suspected of producing parkinsonism in humans, on the muscle tone and metabolism of dopamine in the striatum, and on the number of tyrosine hydroxylase-immunoreactive cells in the substantia nigra were investigated in rats. Muscle tone was examined using a combined mechanomyographic and electromyographic method which measured simultaneously the muscle resistance of the rat's hind foot to passive extension and flexion in the ankle joint and electromyographic activity of the antagonistic muscles of that joint: gastrocnemius and tibialis anterior. 1,2,3,4-Tetrahydroisoquinoline administered at doses of 50 and 100 mg/kg intraperitoneally for 19 days increased muscle resistance 1 h after the first injection (acute treatment), 1 h after the last injection (chronic treatment) and three days after compound withdrawal. Rigidity observed on the third day of 1,2,3,4-tetrahydroisoquinoline withdrawal was accompanied by an increased tonic (resting) electromyographic activity of the gastrocnemius and tibialis anterior muscles. At the same time, a significant reduction in the number of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra and a decrease in the dopamine level in the striatum were also found. A declining number of tyrosine hydroxylase-immunoreactive neurons in the whole substantia nigra showed a significant negative correlation with the enhanced muscle resistance, as well as with the tonic electromyographic activity recorded at rest, i.e. before the start of movements, from the gastrocnemius and tibialis anterior muscles. Our results suggest that 1,2,3,4-tetrahydroisoquinoline may be one of the endogenous substances involved in the progress of Parkinson's disease.
We performed a combined neurochemical and behavioral study to determine the effects of 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1-BnTIQ) on the extracellular dopamine concentrations in the striatum. Single dose administration of 1-BnTIQ (20, 40, and 80 mg/kg i.p.) increased striatal dopamine extracellular levels in a dose-dependent manner when an in vivo microdialysis technique was used to assess dopamine levels in the striatum of rats. Enhancement of striatal dopamine levels by systemic administration of a single dose of 1-BnTIQ was suppressed by perfusion of tetrodotoxin and a calcium ion-free solution into the striatum. This 1-BnTIQ-induced increase in extracellular dopamine concentration was also inhibited by pre-treatment with a dopamine uptake inhibitor, GBR12909 (1-(2-[bis(4-Fluorophenyl)-4-(3-phenylpropyl)piperazine dihydrochloride). Local application of 1-BnTIQ into the striatum via a dialysis probe failed to enhance the extracellular concentration of dopamine. However, microinjection of 1-BnTIQ into the substantia nigra pars compacta increased the extracellular dopamine levels in the striatum. Locomotor activity was increased by systemic administration of a single dose of 1-BnTIQ in a dose-dependent manner. This 1-BnTIQ-induced locomotor activity was attenuated by pre-treatment with SCH23390 (R(+)-7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochlodride) and raclopride, D(1) and D(2) dopaminergic receptor antagonists, respectively. Moreover, 1-BnTIQ induced ipsilateral rotational behavior in 6-hydroxydopamine-lesioned rats. These results suggest that systemic administration of a single dose of 1-BnTIQ increases striatal extracellular dopamine concentration through activation of dopaminergic nigra striatal neurons via the dopamine transporter.
Parkinson's disease (PD) is a progressive neurodegenerative disorder whose etiology is thought to have environmental (toxin) and genetic contributions. The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrimidine (MPTP) induces pathological features of PD including loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and striatal dopamine (DA) depletion. We previously described the striatal transcriptional response following acute MPTP administration in MPTP-sensitive C57BL/6J mice. We identified three distinct phases: early (5h), intermediate (24h) and late (72h) and reported that the intermediate and late responses were absent in MPTP-resistant Swiss-Webster (SWR) mice. Here we show that C57BL/6J mice pre-treated with a single 40 mg/kg dose of MPTP and treated 9 days later with 4×20 mg/kg MPTP, display a striatal transcriptional response similar to that of MPTP-resistant SWR mice, i.e. a robust acute response but no intermediate or late response. Transcriptional refractoriness is dependent upon the dose of the priming challenge with as little as 10mg/kg MPTP being effective and can persist for more than 28 days. Priming of SWR mice has no effect on their response to subsequent challenge with MPTP. We also report that paraquat, another free radical producer, also elicits striatal transcriptional alterations but these are largely distinct from those triggered by MPTP. Paraquat-induced changes are also refractory to priming with paraquat. However neither paraquat nor MPTP elicits cross-attenuation. Thus exposure to specific toxins triggers distinct transcriptional responses in striatum that are influenced by prior exposure to the same toxin. The prolonged refractory period described here for MPTP could explain at the molecular level the reported discrepancies between different MPTP administration regimens and may have implications for our understanding of the relationship between environmental toxin exposure and PD.
The therapeutic potential of BL-1023, a chemical combination of L-3,4-dihydroxyphenylalanine (L-DOPA) and gamma-aminobutyric acid (GABA), was investigated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxicated mice. Such animals exhibit nigrostriatal degeneration, characteristic of human Parkinson's disease. Drug was administered during and after the development of MPTP-induced nigrostriatal lesions followed by measures of motor function and behavior, surviving nigrostriatal dopaminergic neurons and termini, and striatal dopamine levels. When administered after lesion development, BL-1023 improved motor function of MPTP-mice as measured by rotarod, total floor and vertical plane movements, and stereotypic movements in open field activity tests compared to MPTP-mice without treatment. This also paralleled modest nigral dopaminergic neuronal protection. Such significant improvements in motor function, behaviors, and dopaminergic neuronal numbers were not seen when BL-1023 was administered during MPTP-induced lesion development. The data demonstrate select abilities of BL-1023 to increase dopaminergic neuronal survival and improve motor function in MPTP-mice.
Administration of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, to primates produces an excellent behavioral model of idiopathic Parkinson's disease. In the vervet monkey, regional biochemical differences in the striatum of two 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated groups were examined one to two months after treatment and compared with controls; one group displayed no observable gross motor abnormalities after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment (asymptomatic), whereas the other group became markedly parkinsonian (symptomatic). In both 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated groups massive depletions of dopamine and homovanillic acid concentrations were observed in the striatum; generally, dopamine losses in the symptomatic group (greater than 95%) were greater than in the asymptomatic group (greater than 75%). However, in striatum, a marked heterogeneity in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine susceptibility was found; certain striatal regions having 99% depletion of dopamine even in asymptomatic monkeys. Overall, in ventromedial regions of striatum the losses of dopamine and homovanillic acid concentrations were less than in dorsolateral regions at the same coronal level. There was a significant negative correlation between control homovanillic acid/dopamine ratios and susceptibility of examined regions to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity. Unlike idiopathic, but similar to postencephalitic, Parkinson's disease, dopamine and homovanillic acid levels in caudate nucleus were not spared relative to putamen; in fact, in the asymptomatic group caudate nucleus dopamine and homovanillic acid concentrations were depleted to a greater extent than in putamen.(ABSTRACT TRUNCATED AT 250 WORDS)
Parkinson's disease is associated with the loss of dopaminergic neurons in the substantia nigra and decreased striatal dopamine levels. We now report that caffeic acid phenethyl ester (CAPE), an active component of propolis, attenuated dopaminergic neurodegeneration and dopamine loss in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model of Parkinson's disease. The neuroprotective effect of CAPE was associated with marked reductions in inducible nitric oxide synthase (iNOS) and caspase 1 expression. Additionally, CAPE inhibited MPP+-induced neurotoxicity in vitro and directly inhibited MPP+-induced release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria. Thus, CAPE may have beneficial effects in slowing or preventing the progression of Parkinson's disease and other neurodegenerative disorders.
The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces parkinsonian neurochemical and functional deficits in human and non-human primates. The utility of MPTP-induced parkinsonism in monkeys as an animal model of Parkinson's disease would be greater if it produced a persistent and stable behavioural syndrome so that the effects of novel therapeutic treatments can be accurately assessed. Further, the claim that many species including primates spontaneously recover from MPTP is a significant difference from idiopathic Parkinson's disease. This experiment focused on the long-term (six months) persistence of behavioural deficits in severely and moderately parkinsonian monkeys. The severity of the syndrome was based on a quantitative and objective measure of parkinsonism. Adult male African green (vervet) monkeys (Cercopithecus aethiops sabaeus) were treated with MPTP (cumulative dose 2.5 mg/kg over five days), and six were saline-control treated. MPTP-treated subjects were examined in two groups: those that were severely parkinsonian (“severe” group, n=11) and those that were moderately impaired (“moderate” group, n=5) the month after treatment. Summary factor scores were examined reflecting abnormal (“parkinsonian”) behaviour and normal “healthy” behaviour. Subjects that displayed severe parkinsonism the month after MPTP were found to show stable and severe parkinsonism for the time period studied. In contrast, the group of animals that initially were moderately parkinsonian did not show a stable deficit during the study.
Cocaine abuse is a significant problem in the United States, including its use by approximately 1% of pregnant women. Cocaine acts as an indirect agonist of dopamine at the dopamine transporter, resulting in the presence of excess dopamine in the synapse. Since synaptic dopamine can rapidly oxidize to form free radicals, it was hypothesized that exposure to this drug might produce damage in dopaminergic systems such as the substantia nigra pars compacta, damage to which is a hallmark of Parkinson's disease. To test this hypothesis we exposed mice both in utero and as adults to cocaine and examined its effects on the nigrostriatal system. We found that exposure to cocaine both in utero or as adults did not affect substantia nigra cell number, but did make these neurons more susceptible to the parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. We also found long-lasting changes in D2 receptor mRNA levels as well as changes in the monoamine transport system and several growth factors. This work suggests that use of cocaine might be a predisposing factor for development of Parkinson's disease in both adults exposed chronically as well as in individuals exposed prenatally.
The concept of neuroprotective immunity identifies a new role of autoimmune cells in the CNS pathology. Specifically, immune cells infiltrating the CNS during an injury may help in a regeneration process and prevent the secondary degeneration of neurons. The objectives of our study were to determine the role of autoimmune and peripheral immune enhancement in neurodegeneration process, and to compare the results between young adult and aging animals. C57Bl mice were immunized with either myelin oligodendrocyte glycoprotein (MOG) 35-55 combined with complete Freund adjuvant (CFA), or CFA alone. Following 6 days, the animals were injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine to produce a damage of the nigrostriatal dopaminergic system. Although immunization with MOG 35-55 combined with CFA resulted in autoimmune encephalomyelosis, it substantially enhanced neuronal survival after the toxic insult. The immunization with CFA alone was also effective in preventing neuronal cell death, but the magnitude of the neuroprotective effect was smaller. Interestingly, the neuroprotective effect of MOG 35-55 and CFA was more pronounced in aging (i.e. 10-month-old) compared with young (i.e. 2-month-old) mice. Our results indicate that an increased immune activation may be beneficial for neurodegenerative processes following the CNS injury, but the mechanisms of such immune neuroprotection and of age differences need further investigation.
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.
The sites in the mouse brain where 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine can be oxidized to the toxic metabolite 1-methyl-4-phenylpyridine were determined using a histochemical technique. The method involved the demonstration of monoamine oxidase activity using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine as the substrate by means of a sensitive coupled peroxidase technique. The distribution of neurons displaying the ability to oxidize 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine via a monoamine oxidase catalysed reaction was compared to that of various amine systems identified with immunohistochemistry. Dopamine neurons, and in particular the nigrostriatal dopamine cells, did not display the capacity to oxidize 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Noradrenergic neurons showed intense monoamine oxidase activity when 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine was used as substrate, and this activity was blocked by the monoamine oxidase-A inhibitor clorgyline. Serotonin neurons and histamine neurons were also able to oxidize 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. The reaction in these neurons was blocked by deprenyl, an inhibitor of monoamine oxidase-B. Pretreatment with inhibitors of monoamine oxidase-B has been previously shown to prevent the neurotoxic action of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine on dopaminergic neurons. Therefore, since serotonin and histamine neurons are able to oxidize 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine by means of monoamine oxidase-B, these neurons may be involved in the production of the toxic metabolites of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in vivo.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces, in both human and non-human primates, a syndrome very similar to idiopathic Parkinson's disease. The syndrome is associated with degeneration of the dopamine-containing neurons in the substantia nigra, many of which project to the neostriatum. The purpose of the present study was to quantify the regional distribution of midbrain dopamine neurons remaining after MPTP administration to the monkey (Macaca fascicularis) and to develop alternative procedures for maintaining the normal nutrition in MPTP-treated animals. Three monkeys were treated with MPTP and three served as controls. Representative sections were examined from rostral to caudal through the midbrain dopamine cell nuclei and the location of every tyrosine hydroxylase-containing cell was entered into a computer. Midbrain dopamine neuronal cell loss ranged from 36-78%, being most extensive in the two monkeys which exhibited the most severe parkinsonian syndrome. The greatest cell loss (46-93%) occurred in the substantia nigra pars compacta, or nucleus A9, and the loss was primarily in the ventral portion of the nucleus. Contrary to most previous reports, however, there was also a loss of cells in the ventral tegmental area (28-57%) and ventral reticular formation (33-87%), corresponding to nuclei A10 and A8, respectively. Since neuroanatomical tracing studies have shown that the dorsal and lateral portions of the striatum (areas showing the greatest dopamine depletion after MPTP) receive input from cells in the ventral A9 and from cells in the A8 and A10 areas, the present data suggest that MPTP preferentially destroys dopamine cells that project to the striatum (i.e. the mesostriatal cells).
Idiopathic Parkinson's disease (PD) is a neurodegenerative disorder of mature and older individuals. Since all aged individuals do not develop PD, predisposing conditions may exist that pair with the stress placed on the basal ganglia during aging to produce the symptoms of PD. In this project we used 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to test the hypothesis that a sensitization stage and a precipitating stage underlie idiopathic PD. To induce the sensitization stage, pregnant C57BL/6J mice were treated with MPTP (10 mg/kg/day) during gestation days 8-12 to target the emerging fetal nigrostriatal dopamine neurons. For the precipitating stage, the 3-months old offspring were administered MPTP for 7 days, to simulate the changes that occur during aging. The weights and motor activity of the offspring, high performance liquid chromatography (HPLC) striatal dopamine and its metabolites and Western blot for tyrosine hydroxylase (TH) were determined. Offspring exposed to prenatal MPTP showed lower birth weights that eventually recovered. Prenatal MPTP also reduced motor activity by 10-30%, striatal TH by 38%, dopamine by 14%, homovanillic acid by 16.5% and 3-methoxytyramine by 66%. The postnatal MPTP was more potent in the prenatal MPTP-exposed offspring. MPTP at 10, 20 and 30 mg/kg, dose-relatedly, reduced striatal TH by 9.4%, 48.6% and 82.4% in the prenatal-phosphate buffered saline (PBS) mice and by 48%, 78.7% and 92.7% in the prenatal-MPTP groups. More importantly, postnatal MPTP at 10 mg/kg that showed slight effects on DA, DOPAC, HVA and 3-MT in the prenatal-PBS offspring, showed 69.9%, 80.0%, 48.4% and 65.4% reductions in the prenatal-MPTP mice. The study may identify a new model for PD, and the outcome suggests that some cases of idiopathic PD may have a fetal basis in which early subtle nigrostriatal impairments occurred and PD symptoms are precipitated later by deteriorating changes in the nigrostriatum, that would not caused symptoms in individuals with normal nigrostriatal system.
Up-regulation of nestin expression was significantly induced in the caudate-putamen of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice in our previous observation [Brain Res 925 (2002) 9]. We hypothesized that the nestin-expressing cells might play an important role in the pathogenesis of parkinsonian model, and characterization of these nestin-expressing cells was studied by RT-PCR, immunohistochemistry and semi-quantitative analysis for various markers of glial fibrillary acid protein (GFAP), S-100, neuronal nuclear specific protein (NeuN), β-tubulin, Ki-67 and brain-derived neurotrophic factor (BDNF) expression in MPTP-treated C57/BL mice. Firstly, significant increasing in both nestin protein and mRNA was found in MPTP-treated mice. Up-regulation of nestin expression started at day 1, peaked at day 3, and gradually went down at days 7–21 in the neostriatum after MPTP treatment. Secondly, double immunofluorescence indicated that almost all of nestin-positive cells exhibited GFAP (98%) or S-100 (96%)-immunoreactivity, whereas NeuN or β-tubulin was hardly detected in these nestin-positive cells. Thirdly, a minor population (7.0%) of nestin-positive cells showed Ki-67 (cell proliferation marker)-immunoreactivity, showing some of them went into cell mitotic state. Finally but more interestingly, a major population (86%) of nestin-expressing cells also exhibited immunoreactivity for BDNF, one neurotrophic factor.
In humans, mutations in the alpha-synuclein gene or exposure to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produce Parkinson's disease with loss of dopaminergic neurons and depletion of nigrostriatal dopamine. alpha-Synuclein is a vertebrate-specific component of presynaptic nerve terminals that may function in modulating synaptic transmission. To test whether MPTP toxicity involves alpha-synuclein, we generated alpha-synuclein-deficient mice by homologous recombination, and analyzed the effect of deleting alpha-synuclein on MPTP toxicity using these knockout mice. In addition, we examined commercially available mice that contain a spontaneous loss of the alpha-synuclein gene. As described previously, deletion of alpha-synuclein had no significant effects on brain structure or composition. In particular, the levels of synaptic proteins were not altered, and the concentrations of dopamine, dopamine metabolites, and dopaminergic proteins were unchanged. Upon acute MPTP challenge, alpha-synuclein knockout mice were partly protected from chronic depletion of nigrostriatal dopamine when compared with littermates of the same genetic background, whereas mice carrying the spontaneous deletion of the alpha-synuclein gene exhibited no protection. Furthermore, alpha-synuclein knockout mice but not the mice with the alpha-synuclein gene deletion were slightly more sensitive to methamphetamine than littermate control mice. These results demonstrate that alpha-synuclein is not obligatorily coupled to MPTP sensitivity, but can influence MPTP toxicity on some genetic backgrounds, and illustrate the need for extensive controls in studies aimed at describing the effects of mouse knockouts on MPTP sensitivity.
Six pairs of female squirrel monkeys were given a daily intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 9-14 days, beginning the same day on which they received either a bilateral 6-hydroxydopamine lesion or a sham lesion of the locus coeruleus. Sham animals developed typical parkinsonian signs (i.e. tremor, bradykinesia, hypokinesia and reduced blink rate) which largely recovered by six to nine weeks after the start of MPTP treatment. At nine weeks, post mortem levels of striatal dopamine in these same animals were partially reduced (by 45%), and this only in the putamen, compared to values obtained from three non-operated, normal control animals. Additionally, histological examination revealed a moderate loss of neuronal cell bodies in the substantia nigra, pars compacta. In marked contrast, the locus coeruleus-lesioned monkeys exhibited little or no recovery from the parkinsonian signs induced by MPTP. Post mortem examination of these animals revealed profound decreases in caudate (by 84%) and putamen (by 91%) dopamine content, and severe neuronal cell loss in the substantia nigra pars compacta of all animals. These neurological, biochemical and histological assessments indicate that lesioning of the locus coeruleus impairs the recovery which usually occurs from the parkinsonian manifestations induced by MPTP in squirrel monkeys. The results support the hypothesis that deficient locus coeruleus noradrenergic mechanisms underlie the progression of Parkinson's disease.
Parkinson's disease is associated with a progressive loss of substantia nigra pars compacta dopaminergic neurons. The cellular and molecular mechanisms underlying Parkinson's disease neurodegeneration have not been fully determined. Clinical investigations and subacute in vivo studies using the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine have generated some observations suggesting that apoptosis is involved in neurodegeneration; however, this view remains equivocal. In this study, the substantia nigra pars compacta neurodegenerative process was examined in the chronic mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid model of Parkinson's disease treated with 10 doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (25 mg/kg) and probenecid (250 mg/kg) over five weeks. One day after chronic treatment, numerous terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells were detected specifically in the substantia nigra pars compacta displaying shrunken volume, chromatin condensation, and DNA fragmentation. The number of apoptotic cells declined over time. No terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells were found in untreated or probenecid-treated control animals. Cytomorphometric analysis of substantia nigra pars compacta nuclear loci revealed eccentric nucleoli dislocation and vesicular degranulation in all of the apoptotic neurons for the mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid model for Parkinson's disease. The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells phenotypically showed neuronal origin (NeuN-positive) with a loss of tyrosine hydroxylase immunoreactivity. While the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells were not co-localized with astroglial (GFAP-positive) cells, some apoptotic cells were clearly associated with the activated microglial (macrophage antigen complex-1 and isolectin B(4)-positive) cells suggesting an active process of dead cell removal. In the one-day and seven-day post-treated mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid model for Parkinson's disease, marked depression of tyrosine hydroxylase immunoreactivity in the substantia nigra pars compacta and striatum was observed, which was correlated with significant reductions of striatal dopamine content and uptake. These results suggest that initial neuronal apoptosis and morphological changes are involved, at least in part, in the chronic neurodegeneration of mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid model for Parkinson's disease.
Evidence from clinical and experimental studies indicates that degeneration of nigrostriatal dopaminergic neurons is a pathological hallmark of Parkinson's disease (PD). The present study was designed to investigate the neuroprotective potential of theaflavin (TF) on oxidative stress, monoamine transporters and behavioral abnormalities in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurodegeneration. TF, a black tea polyphenol, has been known to possess neuroprotective effects against ischemia, Alzheimer's disease and other neurodegenerative disorders, but the mechanisms underlying its beneficial effects on MPTP-induced dopaminergic neurodegeneration are poorly defined. Administration of MPTP (30 mg/kg bw for four consecutive days) led to increased oxidative stress and reduced behavior patterns (open field, rotarod and hang test), nigrostriatal dopamine transporter (DAT) (immunohistochemistry and Western blot) and vesicular monoamine transporter 2 (VMAT2) (Western blot) expressions. Pre-treatment with TF reduces oxidative stress, improves motor behavior and expression of DAT and VMAT2 in striatum and substantia nigra. These results indicate that TF might be beneficial in mitigating MPTP-induced damage of dopaminergic neurons, possibly via its neuroprotective and its antioxidant potential.
The loss of dopamine in idiopathic or animal models of Parkinson's disease induces synchronized low-frequency oscillatory burst-firing in subthalamic nucleus neurones. We sought to establish whether these firing patterns observed in vivo were preserved in slices taken from dopamine-depleted animals, thus establishing a role for the isolated subthalamic-globus pallidus complex in generating the pathological activity. Mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) showed significant reductions of over 90% in levels of dopamine as measured in striatum by high pressure liquid chromatography. Likewise, significant reductions in tyrosine hydroxylase immunostaining within the striatum (>90%) and tyrosine hydroxylase positive cell numbers (65%) in substantia nigra were observed. Compared with slices from intact mice, neurones in slices from MPTP-lesioned mice fired significantly more slowly (mean rate of 4.2 Hz, cf. 7.2 Hz in control) and more irregularly (mean coefficient of variation of inter-spike interval of 94.4%, cf. 37.9% in control). Application of ionotropic glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2-amino-5-phosphonopentanoic acid (AP5) and the GABA(A) receptor antagonist picrotoxin caused no change in firing pattern. Bath application of dopamine significantly increased cell firing rate and regularized the pattern of activity in cells from slices from both MPTP-treated and control animals. Although the absolute change was more modest in control slices, the maximum dopamine effect in the two groups was comparable. Indeed, when taking into account the basal firing rate, no differences in the sensitivity to dopamine were observed between these two cohorts. Furthermore, pairs of subthalamic nucleus cells showed no correlated activity in slices from either control (21 pairs) or MPTP-treated animals (20 pairs). These results indicate that the isolated but interconnected subthalamic-globus pallidus network is not itself sufficient to generate the aberrant firing patterns in dopamine-depleted animals. More likely, inputs from other regions, such as the cortex, are needed to generate pathological oscillatory activity.
The anatomic distribution of N-methyl-D-aspartate receptors was investigated in the squirrel monkey brain using quantitative autoradiography with [125I]MK-801 as the radioligand. A heterogeneous distribution of [125I]MK-801 binding sites was observed, with the most intense expression in the outer cortex, hippocampus, olfactory tubercle, caudate and putamen. High levels were also observed in the thalamus, nucleus accumbens and inner cortex, with moderate levels in the claustrum. Relatively low expression levels were detected in the subthalamic nucleus with no apparent binding in the globus pallidus and the substantia nigra. Characterization of striatal [125I]MK-801 binding yielded a B(max) of 63.5 fmol/mg tissue and K(d) of 0.53 nM in the caudate, with similar values for the putamen. Experiments were subsequently performed to compare striatal [125I]MK-801 binding in the following four experimental groups: (i) control animals injected with saline; (ii) monkeys treated with levodopa; (iii) animals rendered parkinsonian after exposure to the neurotoxicant 1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine; and (iv) dyskinetic monkeys treated with both 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and levodopa. No changes were observed in 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine-lesioned animals compared with the saline control group. However, administration of levodopa to either unlesioned or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated monkeys resulted in a significant decrease in [125I]MK-801 binding in both the caudate and putamen. The data indicate that levodopa exerts a modulatory effect on the striatal glutamatergic system and suggest that a down-regulation of N-methyl-D-aspartate receptors by levodopa, combined with a deficiency in nigrostriatal dopamine function, may play a role in the development of levodopa induced dyskinesias.
The neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was used to generate a dose-dependent cell death of dopaminergic nigral neurons in the C57B1 mouse. Mice were injected with a total cumulative dose of 150 mg/kg of MPTP delivered over five days and killed at different time points both during and after the toxin injections. Two independent histological methods were used to determine whether the dopaminergic nigral neurons died via an apoptotic mechanism. In situ end-labelling with terminal deoxynucleotidyl transferase was used on paraformaldehyde-fixed, serial, frozen sections to identity cells with double-stranded DNA breaks. Apoptotic cell death was found to be initiated within 72 h of the first injection of the neurotoxin and peaked 24 h after the final MPTP injection. The metachromatic fluorochrome, Acridine Orange, was used on alternate sections to provide structural confirmation of the nuclear chromatin "clumping" considered to be representative of apoptosis. Confocal laser imaging combined with deconvolution techniques was used to resolve the fluorescent signal emitted by the in situ Acridine Orange-DNA complexes. The number of Acridine Orange-stained nuclei demonstrating chromatin clumping was identical to that of the positive in situ end-labelled nuclei observed over a 25 day period. Based upon these two independent methods of assessing apoptosis in situ, we conclude that a 150 mg/kg dose of MPTP can elicit apoptotic cell death in nigral dopaminergic neurons of the C57B1 mouse.
Nerve cells in the substantia nigra pars compacta (SNPC) are known to express tyrosine hydroxylase (TH). By means of light and electron microscopical immunohistochemical techniques, we have shown that the dopaminergic neurons of SNPC express also kynurenine aminotransferase (KAT-I), the enzyme taking part in the formation of kynurenic acid, a neuroprotectant which is one of the endogeneous antagonists of N-methyl-d-aspartate receptors. It was also found that microglial cells and astrocytes express KAT-I. It has been shown that the highly selective dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), widely used as a model of Parkinson's disease (PD), affects not only TH of dopaminergic neurons in the SNPC but also their KAT-I immunoreactivity as well: MPTP treatment decreased the number and optical density of KAT-I immunoreactive SNPC neurons. Decrease of KAT-I after MPTP treatment has been proved also by Western blot analysis. MPTP also reduced KAT-I immunoreactivity of microglial cells, except for those involved in reactive gliosis, which were arranged in groups surrounding affected neurons of the SNPC; also the number of KAT-I immunoreactive (IR) astroglial cells was increased in SNPC. We conclude that MPTP treatment may have a dual effect: in addition to being deleterious for neurons expressing TH and KAT-I, it also affects glial cells which could exacerbate the neurodegenerative process characterizing PD.
The effect of a chronic D2 dopamine receptor agonist (U91356A) treatment on dopamine receptor gene expression in the brain of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys was investigated using quantitative in situ hybridization histochemistry. U91356A was administered to MPTP-monkeys for 27 days in a pulsatile (n=3) or continuous (n=3) schedule. Animals treated in a pulsatile mode showed progressive sensitization and developed dyskinesia; whereas with the continuous mode behavioural tolerance was observed but no dyskinesia developed. Untreated MPTP as well as naive control animals were also studied. The efficacy and uniformity of the MPTP effect was assessed by measures of dopamine concentrations by high performance liquid chromatography with electrochemical detection in the relevant brain areas. D1 and D2 receptor messenger RNAs levels were examined by in situ hybridization histochemistry using human complementary RNA probes. Intense specific labelling for D1 and D2 receptor messenger RNAs was measured in the caudate and putamen with a rostrocaudal gradient for D2 receptors and a lower density in the cortex for D1 receptors messenger RNA. D1 receptor mRNA levels in rostral striatum and cortex decreased whereas D2 receptor messenger RNA in caudal striatum increased in MPTP-monkeys compared to control animals. Continuous administration of U91356A reversed the MPTP-induced increase of D2 receptor messenger RNA, whereas the pulsatile administration did not significantly correct these messenger RNA changes. U91356A treatment whether continuous or pulsatile partially corrected the D1 receptor messenger RNA lesion-induced decrease in the striatum, whereas no correction was observed in the cortex. All MPTP-monkeys were extensively and similarly denervated suggesting that the D1 and D2 receptor expression changes following U91356A administration were treatment related.
We have previously reported that a progressively increased dose of MPTP over the course of 4 weeks induces the gradual impairment of the nigrostriatal dopamine (DA) pathway and several behaviors [Goldberg et al. (in press) Neuroscience]. To our knowledge, this is the first report of specific behavioral deficits correlated with discrete thresholds of DA loss in this pathway. In that study, MPTP was administered 5 d/wk, with behavioral and tissue analysis being carried out 3 days following the final injection at each dose. However, in order to better represent long-term progressive neurodegeneration the present study introduced a washout period of 10 days between each increased dose of MPTP. This implementation also controlled for any transient de-activation of tyrosine hydroxylase (TH), the enzyme that catalyzes synthesis of DA, caused by MPTP-induced oxidative stress which has been suggested following acute administration of the toxin [Smeyne and Jackson-Lewis (2005) Brian Res Mol Brain Res 134:57-66]. Additionally, by the end of the previous study, there was an ultimate decrease of 62% in the mean number of TH-labeled neurons/section in the substantia nigra pars compacta (SNpc) and a 74% decrease in caudate putamen (CPu) TH optical density with continuous MPTP. In the present study, we find that the washout periods lead to a final 79% decrease in the mean number of TH-labeled SNpc neurons/section, and a similar 74% decrease in CPu TH following the 32 mg/kg MPTP dose. Additionally, a dose-dependent decrease was observed in the mean number of SNpc TH-ir neurons/section in the current study which was not seen in the continuous MPTP protocol. These results suggest that a washout period following each increased MPTP dose allows for observation of continued cell death that might occur during the week following MPTP administration, and for therapeutic interventions to be applied at any of several stages during progressive neurodegeneration.
Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease, we investigated the long-term effects of dopaminergic denervation on synaptic transmission in an in vitro slice preparation of the mouse neostriatum. In control mice, electrical stimulation elicited an antidromic potential (N1) followed by a synaptically mediated field potential (N2). In many slices, a third component (N3) was observed. Determination of the maximum stimulus intensities unveiled that in 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine-pretreated animals, the stimulus strength necessary to evoke a maximum N2 response was significantly higher compared to control mice. Furthermore, 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine-pretreatment led to a less frequent appearance and/or to a reduction in the amplitude of the N3 component. Application of glutamate receptor agonists and antagonists revealed two additional differences between normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-pretreated mice. (1) Comparison of the efficacy of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist 6-cyano-7-nitroquinoxaline-2, 3-dione demonstrated an increase in the inhibitory effect of 6-cyano-7-nitroquinoxaline-2,3-dione in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-pretreated mice. (2) In normal mice, removal of magnesium ions from the bathing solution invariably led to the appearance of late N-methyl-D-aspartate receptor-dependent synaptic components. There components were only slightly expressed or virtually absent in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-pretreated mice. The described differences between the electrophysiological and pharmacological properties of evoked field potentials in slices from normal and 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine-pretreated mice disappeared following blockade of GABAA receptor-dependent inhibition by bicuculline. In normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-pretreated mice, bicuculline did not influence the amplitude of the N2 component, but invariably unmasked late synaptic components mediated by glutamate receptors. However, the potentiating effect of bicuculline was significantly stronger in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-pretreated mice compared to the controls. In the presence of bicuculline, the frequency of occurrence of the N3 component was identical in both groups. Furthermore, the apparent efficiency of 6-cyano-7-nitroquinoxaline-2,3-dione was no longer different. Application of bicuculline in the absence of magnesium ions resulted in a similar disinhibition of N-methyl-D-aspartate receptor-dependent late components as observed in the controls in the absence of bicuculline. The data demonstrate that chronic dopaminergic denervation reduces glutamate receptor-dependent synaptic excitation in the mouse neostriatum. Since differences between normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-pretreated mice disappear in the presence of bicuculline, we conclude that this reduction in excitability is due to a potentiation of GABAA receptor-dependent inhibition.
Neurogenesis occurs during development and in the normal adult brain. Recent studies identified areas exhibiting postlesional selective neurogenesis and neuronal repair. In the olfactory bulb (OB), one of the most studied regions of the brain for neurogenesis, seizures and strong odor exposure are known to enhance neurogenesis. Here, we report enhanced neurogenesis in OB after dopaminergic neuronal loss induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a selective toxin for dopaminergic neurons. The neurogenesis has been previously confirmed mainly by the uptake of 5-bromodeoxyuridine (BrdU), a marker of proliferating cells, but methodological problems related to BrdU labeling might result in inaccurate findings with respect to specificity, toxicity and incorporation into normal/lesioned brain. For a better identification of neurogenesis, we used a retroviral vector. First, we investigated the population dynamics of newly formed neurons in different regions of OB including the glomerular layer, the most superficial layer of OB. Quantification of neurogenesis in OB revealed by our retroviral vector was substantially similar to that by BrdU-based method. One week after MPTP application and dopaminergic neuronal loss in OB, neurogenesis of dopaminergic neurons in OB increased by three-fold, but no such process was noted in non-dopaminergic neurons. Our results indicate selective dopaminergic neurogenesis in OB in response to neuronal damage/loss.
In an attempt to define neurochemically the part played by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as a potential Parkinson's disease-inducing neurotoxin, we measured the tissue concentrations of the monoamines dopamine, noradrenaline and serotonin in 45 brain regions in nine rhesus monkeys (Macaca mulatta) receiving repeated intramuscular injections of a total amount of 2.1-7.5 mg/kg MPTP-HCl. Four monkeys treated with MPTP during a period of one to five weeks developed permanent Parkinsonism, and five animals receiving the neurotoxin during a period of two to seven months remained asymptomatic. We found that, compared with the distribution pattern established in the brain of seven normal (drug-free) rhesus monkeys, in the MPTP-treated monkeys none of the three major brain monoamine neuron systems was completely resistant to the neurotoxin. In addition, each brain monoamine had a characteristic regional pattern of MPTP-induced changes. As expected, the most significant alterations were found within the nigrostriatal dopamine system, i.e. profound dopamine loss in caudate nucleus, putamen and substantia nigra. However, many extrastriatal regions of the subcortex and brainstem also suffered significant loss of dopamine, with the noradrenaline loss in the regionally subdivided brainstem being less widespread, and the serotonin levels least affected. Thus, in subcortex/brainstem the ranking order of sensitivity to MPTP was: dopamine greater than noradrenaline much greater than serotonin. In the cerebral (neo- and limbic) cortex, all three monoamine neuron systems suffered widespread statistically significant losses. The ranking order of MPTP sensitivity of the cortical monoamines was: noradrenaline greater than serotonin greater than dopamine. In the cerebellar cortex, dopamine and noradrenaline concentrations were significantly reduced, whereas the serotonin level remained unchanged. A remarkable observation was that many of the subcortical and cortical changes found in the symptomatic monkeys were also found in the asymptomatic animals. Our data are compatible with several possible mechanisms by which MPTP may have produced the observed patterns of monoamine loss in the brain of the rhesus monkey. Our study demonstrates that in the rhesus monkey MPTP mimicked, in addition to the profound striatal dopamine loss, some of the extrastriatal dopamine, noradrenaline and serotonin changes often seen in the brain of patients with idiopathic Parkinson's disease. However, using our treatment regimen, we have not been able to reproduce in the rhesus monkey the inter-regional pattern of striatal dopamine loss typical of idiopathic Parkinson's disease, i.e. a significantly greater loss of dopamine in the putamen compared with the caudate nucleus.
The study was aimed at investigating the expression and the activity of neuronal nitric oxide synthase, and of soluble guanylyl cyclase and phosphodiesterase activities that regulate guanosine 3',5'-cyclic monophosphate level in the midbrain, in a mouse model of PD using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine injections. Adult male mice of the C57/BL strain were given three i.p. injections of physiological saline or three i.p. injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine solution in physiological saline at 2 h intervals (summary 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine dose: 40 mg/kg), and were killed 3, 7, or 14 days later. mRNA, protein level, and/or activities of neuronal nitric oxide synthase, soluble guanylyl cyclase, phosphodiesterase and guanosine 3',5'-cyclic monophosphate were determined. Immunohistochemistry showed about 75% decrease in the number of tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta. Mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine showed increased midbrain guanylyl cyclase and total nitric oxide synthase activities at 3, 7, and 14 days post-treatment. The specific neuronal nitric oxide synthase inhibitor 7-nitroindazole (10 microM) and the specific inducible nitric oxide synthase inhibitor 1400W (10 microM) inhibited the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced excess in nitric oxide synthase activity by 63-70 and 13-25%, respectively. The increases in total midbrain nitric oxide synthase activity were accompanied by elevated guanosine 3',5'-cyclic monophosphate, enhanced expression of neuronal nitric oxide synthase and of the beta1 subunit of guanylyl cyclase at both mRNA and protein levels that persisted up to the end of the observation period, and by enhanced neuronal nitric oxide synthase and guanylyl cyclase beta1 immunoreactivities in substantia nigra pars compacta 7 and 14 days after the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment. The increases in guanylyl cyclase activity were found to occur exclusively due to increased maximal enzyme activity. No 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced change in phosphodiesterase activity has been detected in any brain region studied. 7-Nitroindazole prevented a significant increase in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced midbrain guanosine 3',5'-cyclic monophosphate level and neurodegeneration of dopaminergic neurons. These results raise the possibility that the nitric oxide/guanylyl cyclase/guanosine 3',5'-cyclic monophosphate signaling pathway may play a role in maintaining dopaminergic neurons function in substantia nigra pars compacta.
C57 BL/6 mice were rendered severely parkinsonian by exposure to high doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. The fluorescent retrograde tracer Fast Blue was injected into the neostriatum one (group A) or five weeks (group B) following exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Neurons located in the substantia nigra pars compacta and in the centre median-parafascicular complex were analysed. There was no variation in the number and distribution of Fast Blue-labelled perikarya located in the centre median-parafascicular complex, which are insensitive to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. No variation was seen in the number of Nissl-stained perikarya located in the substantia nigra pars compacta, indicating that neurons had not degenerated. The number and the density of Fast Blue retrogradely-labelled neurons located in the same region were decreased in group A by 41% and in group B by 55%. Fast Blue labelling provided a measure of functional impairment in viable neurons. The Fast Blue-to-Nissl cell ratio was 55% in controls and declined to 20% in group A and to 17% in group B mice. The present study shows that (1) functional inactivation of viable neurons can be measured by using a fluorescent retrograde tracer following exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and (ii) inactivation of retrograde axonal transport progresses from one to five weeks following withdrawal of the toxin. Fluorescent retrograde probes may be used to measure the anatomical substrate of recovery induced by drugs or by brain grafts in parkinsonian animals.
Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). With the exception of a few rare familial forms of the disease, the precise molecular mechanisms underlying PD are unknown. Inflammation is a common finding in the PD brain, but due to the limitation of postmortem analysis its relationship to disease progression cannot be established. However, studies using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD have also identified inflammatory responses in the nigrostriatal pathway that precede neuronal degeneration in the SNpc. To assess the pathological relevance of these inflammatory responses and to identify candidate genes that might contribute to neuronal vulnerability, we used quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to measure mRNA levels of 11 cytokine and chemokine encoding genes in the striatum of MPTP-sensitive (C57BL/6J) and MPTP-insensitive (Swiss Webster, SWR) mice following administration of MPTP. The mRNA levels of all 11 genes changed following MPTP treatment, indicating the presence of inflammatory responses in both strains. Furthermore, of the 11 genes examined only 3, interleukin 6 (Il-6), macrophage inflammatory protein 1 alpha/CC chemokine ligand 3 (Mip-1alpha/Ccl3) and macrophage inflammatory protein 1 beta/CC chemokine ligand 4 (Mip-1beta/Ccl4), were differentially regulated between C57BL/6J and SWR mice. In both mouse strains, the level of monocyte chemoattractant protein 1/CC chemokine ligand 2 (Mcp-1/Ccl2) mRNA was the first to increase following MPTP administration, and might represent a key initiating component of the inflammatory response. Using Mcp-1/Ccl2 knockout mice backcrossed onto a C57BL/6J background we found that MPTP-stimulated Mip-1alpha/Ccl3 and Mip-1beta/Ccl4 mRNA expression was significantly lower in the knockout mice; suggesting that Mcp-1/Ccl2 contributes to MPTP-enhanced expression of Mip-1alpha/Ccl3 and Mip-1beta/Ccl4. However, stereological analysis of SNpc neuronal loss in Mcp-1/Ccl2 knockout and wild-type mice showed no differences. These findings suggest that it is the ability of dopaminergic SNpc neurons to survive an inflammatory insult, rather than genetically determined differences in the inflammatory response itself, that underlie the molecular basis of MPTP resistance.
Oxidative damage in the dopaminergic neurons of substantia nigra pars compacta (SNpc) plays an important role in the pathogenesis of Parkinson's disease (PD). Heat shock proteins 70 kDa (HSP70s) are a sub-family of molecular chaperones involved in not only protein folding and degradation but also antioxidant defense and anti-apoptotic pathways. Here, a transgenic mice over-expressing an inducible form of Hsp70 was used to determine whether HSP70 affects 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigrostriatal degeneration, an experimental model of PD. The Hsp70 transgenic animals exhibited a high level of expression of HSP70 protein in ventral mesencephalon. Dopaminergic cell death in the SNpc was similar between wild-type and Hsp70 transgenic mice with either acute (40 mg/kg, single dose) or chronic (20 mg/kg, three times/week during 1 month) MPTP treatment. In addition, striatal dopamine loss was not different between wild-type and transgenic animals. Three months after the acute MPTP treatment, dopamine loss was partially recovered into a similar level between wild-type and transgenic groups. In conclusion, over-expression of Hsp70 does not suppress dopaminergic neuronal damage at either the somata or the axon terminals of dopaminergic neurons. Hsp70 over-expression does not help axon terminal regeneration either. These results indicate that HSP70 alone is not sufficient to reduce MPTP-induced dopaminergic neuronal damage.
A long-term induction of Fos-related antigens has been shown in neurons after brain injury, suggesting that Fos-related antigens are involved in enhancing the transcription of genes related to the process of regeneration and repair. In the present study, we report that levels of Fos-related antigen-2 are elevated in several models of chemically induced brain injury. Trimethyltin, which causes degeneration of neurons primarily in the hippocampus and other limbic regions, results in a five-fold induction of Fos-related antigen-2 immunoreactivity in neurons in the pyramidal and dentate layers of the hippocampus starting at seven days post-treatment and persisting for 60days. Methamphetamine and methylenedioxymethamphetamine, agents which cause degeneration of dopaminergic nerve terminals in the striatum of the mouse, cause an increase in Fos-related antigen-2 immunoreactivity which begins at three days post-treatment and returns to basal levels by days 5 and 15, respectively. Treatment with 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine elevated levels of Fos-related antigen-2 in the mouse striatum at three days post-treatment. This abbreviated time-course of Fos-related antigen-2 induction is consistent with less severe insult (terminal damage) relative to trimethyltin (cell death), but induction occurs during the period of regeneration and repair in both models. Dexfenfluramine, a non-neurotoxic amphetamine, does not induce Fos-related antigen-2 expression. Decreasing core temperature of the mouse, which blocks amphetamine-induced neurotoxicity, also blocks Fos-related antigen-2 induction. In summary, Fos-related antigen-2 is induced in models of both cell death and terminal degeneration, suggesting that this transcription factor may serve as a universal signal transduction molecule involved in the regulation of genes related to regeneration and repair in the CNS.
We have reported that 1 month following acute (20mg/kg x 4) or subchronic (30 mg/kg/day x 7d) administration of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, there is an increase or decrease, respectively, in the extracellular level of striatal glutamate as determined by in vivo microdialysis [Robinson S, Freeman P, Moore C, Touchon JC, Krentz L, Meshul CK (2003) Acute and subchronic MPTP administration differentially affects striatal glutamate synaptic function. Exp Neurol 180:73-86]. The goal of this study was to determine the effects of treatment with l-dopa (15 mg/kg) for 21 days on striatal glutamate starting on day 8 after the first dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine was administered to mice. Following acute administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, the increase in extracellular striatal glutamate due to lesion of the nigrostriatal pathway was completely reversed to a level below that found in the vehicle-treated group after l-dopa treatment. Subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment resulted in a decrease in striatal extracellular glutamate that was reversed to the level close to that observed in the vehicle-treated group. There was no change in the density of nerve terminal glutamate immunolabeling associated with the synaptic vesicle pool, suggesting that the alterations in extracellular glutamate most likely originated from the calcium-independent pool. There was a similar decrease in the relative density of tyrosine hydroxylase immunolabeling, a marker for dopamine terminals, within the dorsolateral striatum in both the acute and subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated groups that had been administered l-dopa. There was a decrease in the relative density of immunolabeling within the dorsolateral striatum for the glutamate transporter, GLT-1, following acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment in the groups administered either vehicle or l-dopa. There was no change in GLT-1 immunolabeling following subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. The results demonstrate that the reversal in the extracellular level of striatal glutamate following l-dopa treatment in both the acute and subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated groups is not due to changes in either striatal dopamine nerve terminals or in the density of the glutamate transporter, GLT-1.
A dopamine transporter-radioligand binding study demonstrated a dopaminergic innervation around the pallidal complex in the normal monkey (n=5), i.e. where a subpopulation of pallidal neurons known as "border cells" is classically identified. Surprisingly, this peripallidal binding persists in monkeys rendered parkinsonian (n=5) with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment. The border cell electrophysiological activity was then analysed in normal and parkinsonian monkeys (n=2), either in the untreated state or following administration of levodopa. Pallidal border cell firing frequency was significantly decreased after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment (8.9+/-0.7 vs 31.4+/-1.6Hz, P<0.05). This decrease was partly corrected by levodopa administration (19.2+/-1.0Hz, P<0.05 vs both normal and parkinsonian situations). The peripallidal dopaminergic innervation suggests that pallidal border cells are under a direct dopaminergic control, arising from the ventral tegmental area and/or the basal forebrain magnocellular complex, the role of which remains unknown. Moreover, the relative sparing of these dopaminergic fibers in parkinsonian monkeys suggests that they would exhibit specific adaptive properties totally different from those described in the nigrostriatal pathway.
The neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine on dopamine neurons in monkeys were found to be reduced when the catecholamine uptake inhibitor nomifensine was administered during several weeks after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. The obtained protection was partial, leading to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced changes in dopamine levels to 8, 16, 52 and 59% of control values in the caudate nucleus and to 10, 16, 101 and 99% in the putamen of four animals, respectively. At the same doses, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine alone is known to deplete striatal dopamine levels to 0.5–7% of control values. Extra-nigrostriatal monoamine neurons were generally well protected by nomifensine. Neurological examinations revealed modest hypokinesia for a maximum of 10 days after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in the two more severely affected animals. Reaction times of arm and eye movements were measured in a formal task in two of the monkeys having a moderate and a more important depletion of striatal dopamine, respectively. Only moderate impairments were seen during the initial 2 weeks after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in both animals. All parameters recovered to control levels thereafter. At 3.5 and 5.5 months after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, task performance was significantly better than control. The speed of arm movement remained largely unaffected during all periods of experimentation. Spontaneous eye movements were reduced in frequency and amplitude during the initial 1–2 weeks after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and recovered completely thereafter.
The behavioural, biochemical and morphological effects of a chronic administration of low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were studied in the common marmoset. Monkeys received the toxin (l mg/kg i.p.) twice a week for four months. Group A monkeys were studied one week after the last injection of MPTP; group B monkeys were studied eight months after the last toxic injection. The monkey behaviour was observed throughout the experiment; the biochemical and morphological correlates were studied post mortem in the neostriatum and in the substantia nigra, respectively. Data collected from MPTP-treated marmosets were compared to those obtained from sham-injected control monkeys. The results can be summarized as follows. (1) In all MPTP-treated marmosets a progressive Parkinsonism occurred. In group B monkeys, a gradual behavioural recovery was observed after MPTP was discontinued. (2) Biochemical analysis of group A marmosets showed a depletion of dopamine, of 3,4-hydroxyphenylacetic acid and of homovanillic acid, and no variations in dopamine turnover in the neostriatum of MPTP-treated marmosets. In group B, biochemical analysis showed no differences between controls and MPTP-treated animals. (3) Morphological analysis showed that the density of midbrain dopaminergic neurons located in the substantia nigra was unchanged in group A monkeys, but was reduced by 6.8% in MPTP-treated monkeys of group B. The measurement of cross-sectional area showed that midbrain dopaminergic neurons were swollen in MPTP-treated monkeys of group A, with a 11.0% increase of cell size as compared to controls. In group A the nuclei were also swollen, being 304.8% larger in MPTP-treated monkeys, with a nucleus-to-cytoplasm ratio of 65.9% (as compared to 34.0% of controls). In group B monkeys cell size was increased by 18.4% in MPTP-treated marmosets, but the nuclei were of comparable size.
Six monkeys treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine developed a Parkinsonian syndrome (rigidity, akinesia, flexed posture and tremor). In both high and low dose groups, neurons in the substantia nigra were selectively damaged. At high dose levels, nigral neurons were severely damaged, but because the monkeys died, the evolution of the pathology could not be studied. At low dose levels, some nigral neurons survived, and a significant number of these nerve cells showed reductions in the immunoreactivity of tyrosine hydroxylase. Axonal pathology was conspicuous in the nigrostriatal pathway. Loss of the immunoreactivity of tyrosine hydroxylase in perikarya may represent a retrograde axonal reaction, a potentially reversible response. The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model should prove useful for investigating abnormalities occurring as a consequence of dysfunction of the nigrostriatal system, for examining processes associated with repair of damaged neuronal systems, and for developing and testing therapeutic approaches designed to prevent or ameliorate the Parkinsonian syndrome.
Fourteen macaque monkeys were injected intravenously with N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. All developed the cardinal signs of parkinsonism (akinesia, rigidity, etc.) in varying degrees; some required repeated series of injections of the drug, while others developed the syndrome readily after the first series. Most of the subjects that were kept for longer than 4 weeks after the first dose of the drug showed complete or partial recovery after that time. Measurement, in some of the subjects, of the neostriatal levels of dopamine and dihydroxyphenylacetic acid showed the expected depletion of these substances at the peak of the behavioral action of the drug, but no recovery when the animals had returned to, or near, pre-drug behavioral status. No firm conclusion can be reached at this time as to the reasons for the behavioral recovery or the variability of the effects of the drug across subjects.