Unilateral nigrostriatal 6-hydroxydopamine lesions in mice II: predicting l-DOPA-induced dyskinesia

In the 6-hydroxydopamine (6-OHDA) lesioned rodent the location of the lesion produces significantly different behavioural phenotypes, responses to the dopamine precursor l-3,4-dihydroxyphenylalanine (l-DOPA) and neuropathology. Lesion extent is commonly determined by a series of motor tests, but whether any of these tests have a relationship to the development and predictability of dyskinesia is unknown. We used mice with 6-OHDA lesions of the striatum, medial forebrain bundle and substantia nigra to examine the relationship between a range of tests used to determine motor function in the absence of l-DOPA: rotarod, cylinder, corridor, the balance beam, locomotor activity, psycho-stimulant and spontaneous rotational behaviour. The mice were subsequently treated with l-DOPA in progressively increasing doses and the development of l-DOPA-induced dyskinesia assessed. Most of these tests predict dopamine depletion but only rotarod, spontaneous rotations, apomorphine-induced rotations and locomotor activities were significantly correlated with the development of dyskinesia at 6 mg/kg and 25 mg/kg l-DOPA. The losses of dopaminergic neurons and serotonergic density in the ventral and dorsal striatum were dependent upon lesion type and were also correlated with l-DOPA-induced dyskinesia. The expression of FosB/ΔFosB was differentially affected in the striatum and nucleus accumbens regions in dyskinetic mice according to lesion type.     

The L-amino acid carrier inhibitor 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) reduces L-dopa-elicited responses in dopaminergic neurons of the substantia nigra pars compacta

It is not yet clear how l-dopa, that is the most effective drug for the treatment of Parkinson's disease, enters into the dopaminergic neurons to be transformed into dopamine. It is suggested that l-dopa is mainly transported into cells by a group of l-amino acid carriers named “System L”. Since these carriers are selectively inhibited by 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), we have applied this compound to electrophysiologically recorded dopaminergic neurons of the rat substantia nigra pars compacta to examine the possible modulation of the effects of l-dopa by System L.We have observed that BCH reduced, in a concentration-dependent manner, the membrane hyperpolarization/outward current caused by l-dopa. Interestingly, the actions of dopamine were not changed by this System L inhibitor, suggesting that the reducing effects on l-dopa are not due to a BCH-induced unspecific block of dopamine-mediated events. Therefore, our electrophysiological data that an l-type amino acid carrier, possibly System L, is involved in the transport of l-dopa into dopaminergic neurons.     

The ability of grafted human sympathetic neurons to synthesize and store dopamine: a potential mechanism for the clinical effect of sympathetic neuron autografts in patients with Parkinson's disease

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

Transient ischemia increases neuronal nitric oxide synthase, argininosuccinate synthetase and argininosuccinate lyase co-expression in rat striatal neurons

In neurodegenerative diseases, an increased number of neuronal nitric oxide synthase (nNOS)-positive neurons was reported, but nothing is known on which are the neurons induced to express nNOS. Argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL) and nNOS act in the L-arginine-NO-L-citrulline cycle permitting a correct NO production. In the brain, nNOS-positive neurons co-expressing ASS were known, while those co-expressing ASL were not demonstrated. We investigated by immunohistochemistry the presence of these types of neurons in the rat striatum to verify whether there was a correlation between their changes due to neurotoxic insults and animal survival. Transient ischemia, a neurodegenerative insult model, was induced in rat brain by 2 h of middle cerebral artery occlusion. The striatum, the core of ischemia, was examined at 24, 72 and 144 h after reperfusion and compared with that of rats in normal condition. ASS, ASL and nNOS-positive neurons, some of the latter also expressing ASS and ASL, were present both in normal and ischemic conditions. At 24 h after reperfusion, the number of the nNOS-positive neurons and the percentage of those co-expressing ASS and ASL were significantly increased in the animals with a longer survival and at 144 h after ischemia there was an almost complete restore of the number and/or percentage of these neurons. We hypothesize that the neurons induced to express nNOS were the ASS- and ASL-positive ones and that the neurons co-expressing nNOS, ASS and ASL, since having the enzymes necessary to maintain a correct NO production, might protect from neurotoxic insults.     

Pharmacological validation of a mouse model of l-DOPA-induced dyskinesia

Dyskinesia (abnormal involuntary movements) is a common complication of l-DOPA pharmacotherapy in Parkinson's disease, and is thought to depend on abnormal cell signaling in the basal ganglia. Dopamine (DA) denervated mice can exhibit behavioral and cellular signs of dyskinesia when they are treated with l-DOPA, but the clinical relevance of this animal model remains to be established. In this study, we have examined the pharmacological profile of l-DOPA-induced abnormal involuntary movements (AIMs) in the mouse. C57BL/6 mice sustained unilateral injections of 6-hydroxydopamine (6-OHDA) in the striatum. The animals were treated chronically with daily doses of l-DOPA that were sufficient to ameliorate akinetic features without inducing overt signs of dyskinesia upon their first administration. In parallel, other groups of mice were treated with antiparkinsonian agents that do not induce dyskinesia when administered de novo, that is, the D2/D3 agonist ropinirole, and the adenosine A2a antagonist KW-6002. During 3 weeks of treatment, l-DOPA-treated mice developed AIMs affecting the head, trunk and forelimb on the side contralateral to the lesion. These movements were not expressed by animals treated with ropinirole or KW-6002 at doses that improved forelimb akinesia. The severity of l-DOPA-induced rodent AIMs was significantly reduced by the acute administration of compounds that have been shown to alleviate l-DOPA-induced dyskinesia both in parkinsonian patients and in rat and monkey models of Parkinson's disease (amantadine, -47%; buspirone, -46%; riluzole, -33%). The present data indicate that the mouse AIMs are indeed a functional equivalent of l-DOPA-induced dyskinesia.     

Ropinirole versus L-DOPA effects on striatal opioid peptide precursors in a rodent model of Parkinson's disease: implications for dyskinesia

The dopamine precursor, L-3,4-dihydroxyphenylalanine (L-DOPA), remains the most common treatment for Parkinson's disease. However, following long-term treatment, disabling side effects, particularly L-DOPA-induced dyskinesias, are encountered. Conversely, D2/D3 dopamine receptor agonists, such as ropinirole, exert an anti-parkinsonian effect while eliciting less dyskinesia when administered de novo in Parkinson's disease patients. Parkinson's disease and L-DOPA-induced dyskinesia are both associated with changes in mRNA and peptide levels of the opioid peptide precursors preproenkephalin-A (PPE-A) and preproenkephalin-B (PPE-B). Furthermore, a potential role of abnormal opioid peptide transmission in dyskinesia is suggested due to the ability of opioid receptor antagonists to reduce the L-DOPA-induced dyskinesia in animal models of Parkinson's disease. In this study, the behavioural response, striatal topography and levels of expression of the opioid peptide precursors PPE-A and PPE-B were assessed, following repeated vehicle, ropinirole, or L-DOPA administration in the 6-OHDA-lesioned rat model of Parkinson's disease. While repeated administration of L-DOPA significantly elevated PPE-B mRNA levels (313% cf. vehicle, 6-OHDA-lesioned rostral striatum; 189% cf. vehicle, 6-OHDA-lesioned caudal striatum) in the unilaterally 6-OHDA-lesioned rat model of Parkinson's disease, ropinirole did not. These data and previous studies suggest the involvement of enhanced opioid transmission in L-DOPA-induced dyskinesia and that part of the reason why D2/D3 dopamine receptor agonists have a reduced propensity to elicit dyskinesia may reside in their reduced ability to elevate opioid transmission.     

Endogenous neurotensin attenuates dopamine-dependent locomotion and stereotypy

The neuropeptide neurotensin (NT) is highly sensitive to changes in dopaminergic signaling in the striatum, and is thought to modulate dopamine-mediated behaviors. To explore the interaction of NT with the dopamine system, we utilized mice with a targeted deletion of dopamine synthesis specifically in dopaminergic neurons. Dopamine levels in dopamine-deficient (DD) mice are less than 1% of control mice, and they require daily administration of the dopamine precursor L-dihydroxyphenylalanine (L-DOPA) for survival. DD mice are supersensitive to the effects of dopamine, becoming hyperactive relative to control mice in the presence of L-DOPA. We show that 24 h after L-DOPA treatment, when DD mice are in a "dopamine-depleted" state, Nt mRNA levels in the striatum of DD mice are similar to those in control mice. Administration of L-DOPA or L-DOPA plus the L-amino acid decarboxylase inhibitor, carbidopa, (C/L-DOPA) induced Nt expression in the striatum of DD mice. The dopamine D1 receptor antagonist, SCH23390, blocked C/L-DOPA-induced Nt. To test the hypothesis that this striatal Nt expression modulated dopamine-mediated behavior in DD mice, we administered SR 48692, an antagonist of the high affinity NT receptor, together with L-DOPA or C/L-DOPA. L-DOPA-induced hyperlocomotion and C/L-DOPA-induced stereotypy were potentiated by peripheral administration of SR 48692. Furthermore, intrastriatal microinjections of SR 48692 augmented L-DOPA-induced hyperlocomotion. These results demonstrate a dynamic regulation of striatal Nt expression by dopamine via D1 receptors in DD mice, and point to a physiological role for endogenous striatal NT in counteracting motor behaviors induced by an overactive dopamine system.     

Alterations in nigral NMDA and GABAA receptor control of the striatal dopamine level after repetitive exposures to nitrogen narcosis

Nitrogen pressure exposure in rats results in decreased dopamine (DA) release at the striatal terminals of the substantia nigra pars compacta (SNc) dopaminergic neurons, demonstrating the narcotic potency of nitrogen. This effect is attributed to decreased excitatory and increased inhibitory inputs to dopaminergic neurons, involving a change in NMDA and GABA_A receptor function. We investigated whether repetitive exposures to nitrogen modify the excitatory and inhibitory control of the dopaminergic nigro-striatal pathway.We used voltammetry to measure dopamine levels in freely-moving rats, implanted with dopamine-sensitive electrodes in the striatum. NMDA/GABA_A receptor agonists (NMDA/muscimol) and antagonists (AP7/gabazine) were administered through a guide-cannula into the SNc, and their effects on striatal dopamine levels were measured under normobaric conditions, before and after five repetitive exposures to 1 MPa nitrogen.NMDA-mediated dopamine release was greater following repetitive exposures, AP7-mediated inhibition of glutamatergic input was blocked, suggesting that NMDA receptor sensitivity was increased and glutamate release reduced. Muscimol did not modify dopamine levels following repetitive exposures, whereas the effect of gabazine was greater after exposures than before. This suggested that interneuronal GABA_A receptors were desensitized, leading to an increased GABAergic input at dopaminergic cells. Thus, repetitive nitrogen exposure induced persistent changes in glutamatergic and GABAergic control of dopaminergic neurons, resulting in decreased activity of the nigrostriatal pathway.     

Activated microglia cells express argininosuccinate synthetase and argininosuccinate lyase in the rat brain after transient ischemia

Argininosuccinate-synthetase (ASS), argininosuccinate-lyase (ASL) and nitric oxide synthase (NOS) act in the l-arginine-NO-l-citrulline cycle. In the rat brain, ASS is expressed in neurons, ASL in neurons and astroglia in the striatum, both are co-expressed with nNOS in medium-sized neurons. Microglia cells express iNOS and ASS after activation but no information is available on ASL and on ASS/ASL/iNOS co-expression in this glial population. The present aim was to ascertain, by immunohistochemistry, whether the microglia cells of the rat striatum and fronto-parietal cortex express ASL and ASS in control conditions and after transient ischemia induced by middle cerebral artery occlusion, and whether ASL and ASS are co-expressed with iNOS. The study was conducted 24, 72 and 144 h after reperfusion in two groups of ischemic rats with different tissue damage and survival. ASS and ASL are not expressed by microglia cells in controls while are present in most of the activated microglia cells in the ischemic rats. In those animals with longer survival, ASS and ASL were no more detectable at 144 h, while, in the animals with shorter survival, they were co-expressed with iNOS, but only at 72 h. In the cortex, at variance with the striatum, almost all of nNOS-positive neurons co-expressed ASS and ASL. In conclusion, only activated microglia cells express ASS and ASL, this expression precedes that of iNOS and does not necessarily imply its appearance. Therefore, local factors such as the NO produced by nNOS/ASS/ASL-positive neurons, could influence ASS/ASL-positive microglia cells avoiding or allowing the induction, in these cells, of iNOS.     

Down-regulation of glutaredoxin by estrogen receptor antagonist renders female mice susceptible to excitatory amino acid mediated complex I inhibition in CNS

beta-N-oxalyl-amino-L-alanine, (L-BOAA), an excitatory amino acid, acts as an agonist of the AMPA subtype of glutamate receptors. It inhibits mitochondrial complex I in motor cortex and lumbosacral cord of male mice through oxidation of critical thiol groups, and glutaredoxin, a thiol disulfide oxido-reductase, helps maintain integrity of complex I. Since incidence of neurolathyrism is less common in women, we examined the mechanisms underlying the gender-related effects. Inhibition of complex I activity by L-BOAA was seen in male but not female mice. Pretreatment of female mice with estrogen receptor antagonist ICI 182,780 or tamoxifen sensitizes them to L-BOAA toxicity, indicating that the neuroprotection is mediated by estrogen receptors. L-BOAA triggers glutathione (GSH) loss in male mice but not in female mice, and only a small but significant increase in oxidized glutathione (GSSG) was seen in females. As a consequence, up-regulation of gamma-glutamyl cysteinyl synthase (the rate-limiting enzyme in glutathione synthesis) was seen only in male mouse CNS but not in females. Both glutathione reductase and glutaredoxin that reduce oxidized glutathione and protein glutathione mixed disulfides, respectively, were constitutively expressed at higher levels in females. Furthermore, glutaredoxin activity in female mice was down-regulated by estrogen antagonist indicating its regulation by estrogen receptor. The higher constitutive expression of glutathione reductase and glutaredoxin could potentially confer neuroprotection to female mice.     

Decreased expression of l-dopa-induced dyskinesia by switching to ropinirole in MPTP-treated common marmosets

Current concepts suggest that pulsatile stimulation of dopamine receptors following L-dopa administration leads to priming for dyskinesia in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP)-treated primates, while continuous dopaminergic stimulation with long-acting dopamine agonists does not. We investigated whether L-dopa-induced dyskinesia is reduced by switching to a dopamine agonist. MPTP-treated marmosets received chronic treatment with L-dopa or ropinirole in doses producing equivalent motor activity and reversal of motor deficits. Administration of L-dopa led to the rapid onset of moderate to severe dyskinesia, whereas ropinirole produced only mild dyskinesia. Animals initially treated with L-dopa were switched to an equivalent dose of ropinirole and those treated with ropinirole were switched to an equivalent dose of L-dopa for 56 days. L-dopa-primed animals that were switched to ropinirole showed a trend towards a reduction of dyskinesia intensity, whereas animals initially treated with ropinirole and switched to L-dopa showed a trend toward increased dyskinesia intensity. A subsequent, acute L-dopa challenge reversed motor deficits and induced intense dyskinesia in both groups. This suggests that L-dopa leads to the priming and expression of dyskinesia, but that expression is not maintained when switching to a long-acting dopamine agonist. In contrast, dopamine agonists may prime for dyskinesia, but do not lead to its full expression.     

Does contraversive circling in the 6-OHDA-lesioned rat indicate an ability to induce motor complications as well as therapeutic effects in Parkinson's disease?

Circling behaviour in unilateral 6-OHDA-lesioned rats is interpreted as being opposite in direction to the side of the brain with highest striatal dopaminergic activation. Ipsiversive rotation indicates an action on the intact striatum, while contraversive rotation demonstrates an effect on dopamine receptors in the denervated striatum and is taken as predictive of symptomatic benefit in Parkinson's disease. But does an equivalent behavioural outcome result from stimulating the intact and denervated striatum to the same degree? We report on the behavioural responses produced by administration of L-dopa and the monoamine uptake inhibitor BTS 74,398. These were given alone, or in combination, at doses equivalent to their ED(25), ED(50) and ED(75) for inducing circling in unilateral 6-OHDA-lesioned rats. L-dopa administered alone induced dose-dependent contraversive circling while BTS 74,398 produced ipsiversive rotation. However, L-dopa and BTS 74,398 in combination, produced the same contraversive circling response as when L-dopa was administered alone. Little or no ipsiversive rotation occurred, irrespective of the combination of doses employed. This surprising finding suggests that there are major differences in the outcome of stimulating the intact and denervated striatum with the latter dominating the behavioural response. Since repeated administration of L-dopa but not BTS 74,398, sensitises rotational responses and induces abnormal movements, it may be that contraversive rotation is predictive of both clinical response in PD and the ability to induce motor complications.     

Glutamate N-methyl-D-aspartate and dopamine receptors have contrasting effects on the limbic versus the somatosensory cortex with respect to amphetamine-induced neurodegeneration

The roles that glutamate N-methyl-D-aspartate (NMDA) and dopamine D1-like and D2-like receptors play in the cortical neurotoxicity occurring in rats exposed to multiple doses of amphetamine (AMPH) for 2 days was evaluated. Neurodegeneration in rats that did not become hyperthermic during AMPH exposure was quantified by counting isolectin B4-labeled phagocytic microglia and Fluoro-Jade (F-J)-labeled neurons in the somatosensory parietal cortex, piriform cortex and posterolateral cortical amygdaloid nucleus (PLCo). The NMDA receptor antagonist, dizocilpine (0.63 mg/kg day) blocked AMPH-induced neurodegeneration in the somatosensory cortex. However, it did not affect degeneration in the piriform cortex and PLCo indicating that limbic degeneration was not NMDA-mediated. The dopamine antagonists, eticlopride (D2/3, 0.25 mg/kg day) and SCH-23390 (D1, 0.25 mg/kg day), blocked the stereotypic behavior and neurodegeneration in the somatosensory cortex. However, eticlopride had a lesser protective effect in the limbic regions. As well, the dopamine D2/D3 agonist quinpirole (1.5 mg/kg day) protected against cortical neurodegeneration when it was given during AMPH exposure and continued until sacrifice. The dopamine D1 agonist (SKF-38393, 12.5 mg/kg day) had no significant effect on neurodegeneration. These data indicate that there are significant differences in NMDA and dopamine D2 modulation of AMPH-induced neurodegeneration in the somatosensory cortex compared to the limbic cortices, and limbic cortical degeneration is not necessarily dependent on excessive stimulation of NMDA receptors as it is in the somatosensory cortex. Although excessive dopamine receptor stimulation during amphetamine exposure may trigger the neurodegenerative processes, continued D2 stimulation after AMPH exposure is neuroprotective in the cortex.     

New striatal neurons form projections to substantia nigra in adult rat brain after stroke

Previous studies have demonstrated that newborn striatal neurons can functionally integrate with local neural networks in adult rat brain after injury. In the present study, we determined whether these newly generated striatal neurons can develop projections to the substantia nigra, a target of striatal projection neurons. We used 5′-bromodeoxyuridine (BrdU) and a retroviral vector expressing green fluorescent protein (GFP) combined with multiple immunostaining labels of newborn striatal neurons, and nigral microinjection of fluorogold (FG) to trace the striatonigral projection in adult rat brain at different weeks following a transient middle cerebral artery occlusion (MCAO). We found that FG positive (FG⁺) cells could be detected in newly generated neurons (BrdU⁺-NeuN⁺ and GFP⁺-NeuN⁺) in ipsilateral striatum clearly at 12, but not 2 weeks after MCAO. The data suggest that ischemia-induced newborn striatal projection neurons could form long axons that targeted the substantia nigra (striatonigral projection pathway) and that have intact axonal transport from the nerve terminal to cell body. These new striatal neurons express glutamate NR2 and dopamine D2L receptors, which form the molecular basis for responding to the inputs from cortical glutamatergic and nigral dopaminergic projection neurons. Our data provide the first morphological evidence that newborn neurons in the striatum, a non-neurogenic region, can establish new striatonigral neural circuits, important pathways for the maintenance of motor function. These results help us to understand endogenous cellular mechanisms of brain repair, and suggest that increasing adult neurogenesis could be a practical strategy for enhancing the efficacy of rehabilitative therapy in stroke patients.     

Aromatic L-amino acid decarboxylase is present in serotonergic fibers of the striatum of the rat. A double-labeling immunofluorescence study

The aim of the present study is to examine whether serotonergic fibers of the striatum of the rat contain aromaticl-amino acid decar?ylase (AADC). By use of a double-labeling immunofluorescence method, we showed that AADC was localized in serotonergic fibers of the striatum and cerebral cortex as well as in serotonergic cell bodies of the midbrain raphe nuclei. We previously demonstrated that serotonergic fibers of the rat striatum contained dopamine after intraperitoneal injection ofl-dopa. These findings suggest that dopamine is produced from the injectedl-dopa in serotonergic fibers of the rat striatum.     

A role for dopamine in feeding responses produced by orexigenic agents

Dopamine-deficient (DD) mice become hypophagic and die of starvation by 3 to 4 weeks of age unless dopamine is restored by daily treatment with l-3-4-dihydroxyphenylalanine (l-dopa). We demonstrate here that DD mice mount qualitatively normal counter-regulatory blood glucose responses to insulin and 2-deoxy-d-glucose (2-DG). However, unlike control mice, DD mice fail to eat in response to acute glucoprivation induced by insulin or 2-DG. They also have a severely blunted response to central administration of peptide YY (PYY). Viral-mediated restoration of dopamine synthesis to the central caudate putamen (CPu) of DD mice rescues feeding and survival. However, this treatment fails to restore insulin- and 2-DG-induced feeding despite normalizing feeding in response to food deprivation and PYY. Since dopamine signaling in the CPu is not sufficient for glucoprivation-induced feeding, we propose that this feeding behavior may be mediated by dopamine in an anatomically distinct brain region.     

Pentobarbital inhibits L-DOPA-induced dopamine increases in the rat striatum: An in vivo microdialysis study

Pentobarbital is reported to inhibit ketamine-induced dopamine (DA) release in the rat nucleus accumbens. The accumbens is a part of the limbic dopaminergic system in the brain, and the dopaminergic neural activity of other components may also be sensitive to pentobarbital. We investigated the effect of pentobarbital administration on DA release in the striatum known as DA-rich basal ganglia, and the interaction between pentobarbital and L-DOPA, using in vivo microdialysis techniques. Male SD rats were implanted microdialysis probe into the right striatum. The probe was perfused with modified Ringer's solution and dialysate was directly injected to an HPLC. Every group of rats was consisted of six to seven animals. In the first experiment, rats were given saline, 25 and 50 mg kg(-1) pentobarbital. The second, each rat was given a local administration of 2 and 5 microg ml(-1) of L-DOPA with perfusate. Finally, other sets of rats were given 5 microg ml(-1) of L-DOPA and 25, 50, or 100 mg kg(-1) pentobarbital. Pentobarbital anaesthesia decreased the extracellular concentration of DA, and local administration of L-DOPA significantly increased DA concentration. Pretreatment with pentobarbital diminished the L-DOPA-induced DA increase. The results of the present investigation demonstrate that administration of pentobarbital might inhibit dopaminergic neural activity not only in the nucleus accumbens but also in the rat striatum. Pentobarbital anaesthesia antagonizes DA increase induced by L-DOPA and suggests the inhibition of metabolism of L-DOPA. The results of some animal experiments on dopaminergic activity under pentobarbital anaesthesia should be reconsidered.     

Tandospirone, a 5-HT1A agonist, ameliorates movement disorder via non-dopaminergic systems in rats with unilateral 6-hydroxydopamine-generated lesions

Serotonin 1A (5-HT1A) receptors are distributed throughout the brain with their highest concentrations in the frontal cortex, subthalamic nucleus and entopeduncular nucleus as well as the dorsal and median raphe nucleus. There is growing evidence that 5-HT1A receptor agonists have an antidepressant effect in individuals with major depressive disorders. Recent clinical studies suggest that tandospirone, a highly potent and selective 5-HT1A receptor agonist used clinically as an antidepressant in Japan and China, may act as an antiparkinsonian drug. In the present study, we investigated the effect of tandospirone on contralateral rotational behavior in a unilateral hemiparkinsonian rat model produced with 6-hydroxydopamine (6-OHDA). Tandospirone, as well as 8-hydroxy-2-(di-n-propylamino) tetralin (8-OHDPAT), significantly increased contralateral turnings in a dose-dependent manner (0.5-10 mg/kg). Tandospirone also remarkably potentiated the contralateral turning induced by 0.025 mg/kg of apomorphine. Pretreatment with WAY-100635, a 5-HT1A receptor antagonist, almost completely blocked the contralateral turning behavior evoked by tandospirone and 8-OHDPAT, but not that by apomorphine. SCH-23390, a selective dopamine D1 receptor antagonist, did not affect on the tandospirone-induced rotational behavior. These results suggested that tandospirone could act on postsynaptic 5-HT1A receptors and modulate excitatory amino acid pathways in the basal ganglia. Thus, tandospirone could have therapeutic potential for the treatment of Parkinson's disease by modulating neuronal activities of non-dopaminergic pathways.     

Stepping test in mice: A reliable approach in determining forelimb akinesia in MPTP-induced Parkinsonism

Currently existing behavioral measures for motor impairments in rodent models with bilateral dopamine depletion have demonstrated to be difficult to assess due to the degree of task complexity. There is clearly a need for a behavioral test that is simplistic in design and does not require the animal to learn a specific task, in particular for mice. Here we adapted the stepping test, originally designed for assessing asymmetric motor deficits in rats (Olsson, M., Nikkhah, G., Bentlage, C., Bjorklund, A., 1995. Forelimb akinesia in the rat Parkinson model: differential effects of dopamine agonists and nigral transplants as assessed by a new stepping test. J. Neurosci. 15, 3863–3875; Schallert, T., De Ryck, M., Whishaw, I.Q., Ramirez, V.D., Teitelbaum, P., 1979. Excessive bracing reactions and their control by atropine and l-DOPA in an animal analog of Parkinsonism. Exp. Neurol. 64, 33–43), into a mouse-friendly version for bilateral dopamine lesion induced by subacute MPTP injection. We found that MPTP-treated mice exhibit a significant and persistent reduction in the number of adjusting steps when compared to saline-treated animals. Typically, MPTP-induced stepping deficit becomes apparent by the fourth MPTP injection. The number of adjusting steps continues to decline throughout the injections, and by day 10 from the last MPTP injection, the stepping deficit observed is associated with  65% TH positive cells loss in the SN. Importantly, l-DOPA administration significantly improved stepping performance in MPTP-treated mice. Thus, stepping test in mice is a reliable and simple behavioral measure for assessing forelimb akinesia induced by systemic MPTP.     

Automated gait analysis in bilateral parkinsonian rats and the role of L-DOPA therapy

Gait disturbances and postural instability represent major sources of morbidity in Parkinson's disease (PD), and respond poorly to current treatment options. Some aspects of gait disturbances can be observed in rodent models of PD; however, knowledge regarding the stability of rodent gait patterns over time is lacking. Here we investigated the temporal constancy and reproducibility of gait patterns in neurologically intact and bilaterally 6-hydroxydopamine (6-OHDA) lesioned rats, by using an automated quantitative gait analysis method (CatWalk). The bilateral neurotoxin injections into the medial forebrain bundle resulted in an average dopamine (DA) loss of 70% in striatum, which corresponds to the DA levels observed in moderate-mid stage human PD. Rats were tested weekly during one month, and we found that in intact rats all parameters investigated remained constant over multiple tests. The 6-OHDA lesioned rats were impaired in several aspects of gait, such as stride length, swing speed, stance duration, step cycle duration, and base of support. However the stance and step cycle deficits were transient, the performance of 6-OHDA lesioned rats were indistinguishable from control rats by the last test session with regard to these parameters. Finally, we found that administration of a single dose of levodopa (l-DOPA) to the 6-OHDA lesioned rats could counteract all but one observed deficits. Based on these findings we conclude that the gait pattern of intact rats is highly reproducible, 6-OHDA lesioned rats display impairments in gait, and l-DOPA can counteract most deficits seen in this model of experimental PD.