Mercè Bonastre

Striatal 6-hydroxydopamine induces apoptosis of nigral neurons in the adult rat.

The massive dopaminergic neuronal loss that occurs in Parkinsons disease shows features of apoptosis. In the current study we have characterised the neuronal death in an animal model of Parkinsons disease. 6-Hydroxydopamine infused in the striatum of adult rats induced progressive loss of dopamine neurons, identified as tyrosine hydroxylase immunoreactive profiles, in the ipsilateral substantia nigra starting at day 5 post-lesion (32%). Silver staining revealed the presence of apoptotic profiles with neuronal morphology in the substantia nigra ipsilateral to the intrastriatal 6-hydroxydopamine injection. These apoptotic nuclei were first observed at day 6 post-lesion, peaked between days 7 and 10 and then abruptly declined. The apoptotic morphology of 6-hydroxydopamine-induced neuronal death was confirmed by electron microscopic studies. These data show that intrastriatal 6-hydroxydopamine-induced dopaminergic neuronal death in the adult rat is apoptotic and supports the use of this lesion protocol as an animal model of Parkinsons disease.

Non-NMDA receptor-mediated mechanisms are involved in levodopa-induced motor response alterations in parkinsonian rats

Chronic dopaminomimetic administration to parkinsonian animal models or Parkinsons disease patients leads to characteristic alteration in motor response. Previous studies suggested that the nonphysiologic stimulation of dopaminergic receptors on striatal medium spiny neurons enhances the synaptic efficacy of juxtaposed glutamate receptors of the N‐methyl‐D‐aspartate (NMDA) subtype. Resultant NMDA receptor sensitization due to differential changes in subunit phosphorylation appears to favor alterations in striatal output in ways that influence motor function. To detail the involvement of NMDA receptors further as well as to determine whether similar functional changes might develop in α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA) receptors, the effects of selective antagonist of AMPA receptors (6‐nitro‐7‐sulfamoyl‐benzo[f]‐quinoxaline‐2,3 (1H,4H)‐dione sodium salt, NBQX, 10 mg/kg) on levodopa‐induced response alterations in 6‐hydroxydopamine (6‐OHDA) lesioned rats were compared with drugs which act competitively (3‐(±)‐2‐carboxypiperazin‐4‐yl)‐propyl‐1‐phosphonicacid, CPP, 6.25 mg/kg) or noncompetitively (dextromethorphan, 40 mg/kg) to block NMDA receptors, or a nonselective inhibitor of glutamatergic transmission (2‐amino‐6‐trifluoromethoxy benzothiazole, riluzole, 5 mg/kg). We found that the shortened duration of the motor response to levodopa, which underlies human wearing‐off fluctuations, was reversed to a similar degree by the acute coadministration of CPP, NBQX, or riluzole (n = 4–6) but dextromethorphan did not. These observations strengthen the possibility that a reduction in levodopa‐associated changes in motor response by inhibitors of glutamatergic transmission acting generally or selectively at the glutamate binding‐sites may relate to their ability to attenuate pathologic gain in striatal glutamatergic function. The capacity of NBQX to reverse these altered responses suggests that an enhanced synaptic efficacy of striatal AMPA receptors may also participate in the generation of these motor response changes in levodopa‐treated parkinsonian rats. Synapse 36:267–274, 2000.

Effect of subthalamic nucleus or entopeduncular nucleus lesion on levodopa-induced neurochemical changes within the basal ganglia and on levodopa-induced motor alterations in 6-hydroxydopamine-lesioned rats

Inactivation of the subthalamic nucleus (STN) or the internal segment of the pallidum (GPi)/entopeduncular nucleus (EP) by deep brain stimulation or lesioning alleviates clinical manifestations of Parkinsons disease (PD) as well as reducing the side‐effects of levodopa treatment. However, the effects of STN or entopeduncular nucleus (EP) lesion on levodopa‐related motor fluctuations and on neurochemical changes induced by levodopa remain largely unknown. The effects of such lesions on levodopa‐induced motor alterations were studied in 6‐hydroxydopamine (6‐OHDA)‐lesioned rats and were assessed neurochemically by analyzing the functional activity of the basal ganglia nuclei, using the expression levels of the mRNAs coding for glutamic acid decarboxylase and cytochrome oxidase as molecular markers of neuronal activity. At the striatal level, preproenkephalin (PPE) mRNA levels were analyzed. We found in 6‐OHDA‐lesioned rats that a unilateral STN or EP lesion ipsilateral to the 6‐OHDA lesion had no effect on either the shortening in the duration of the levodopa‐induced rotational response or the levodopa‐induced biochemical changes in the basal ganglia nuclei. In contrast, overexpression of PPE mRNA due to levodopa treatment was reversed by the STN or EP lesion. Our study thus shows that lesion of the EP or STN may counteract some of the neurochemical changes induced by levodopa treatment within the striatum.

AMPA receptor antagonist LY293558 reverses preproenkephalin mRNA overexpression in the striatum of 6-OHDA-lesioned-rats treated with L-dopa.

Striatal neurons that contain GABA and enkephalin and project to the external segment of the pallidum are thought to be overactive in Parkinsons disease. Furthermore, it has been shown that the appearance of l‐dopa‐induced dyskinesias is correlated to an increase of preproenkephalin (PPE) mRNA expression and that some antagonists of glutamate receptors can prevent and reverse l‐dopa‐induced dyskinesias in parkinsonian rats. The aim of this study was therefore to analyse the effect of a systemic treatment with glutamate receptor antagonists, alone or in combination with l‐dopa, on the PPE mRNA level in rats with a 6‐hydroxydopamine‐induced unilateral lesion of the nigrostriatal pathway. In vehicle‐treated animals, PPE mRNA levels were markedly increased in the striatum on the lesioned side. Sub‐chronic l‐dopa treatment, with bi‐daily injections for 22 days, induced a further increase in PPE mRNA expression in the denervated striatum. Administration of the AMPA receptor antagonist, LY293558, partially reversed the lesion‐induced and l‐dopa‐induced increases in PPE mRNA expression. However, although the administration of the NMDA receptor antagonist MK801 showed a tendency to decrease this l‐dopa induced overexpression, it did not reach significance. This study provides evidence that glutamatergic antagonists, and particularly AMPA antagonists, tend to reverse PPE neurochemical changes at the striatal level induced by l‐dopa in hemiparkinsonian rats.

Effect of long-term haloperidol treatment on striatal neuropeptides: Relation to stereotyped behavior

Behavioral and biochemical responses to D1 and D2 dopamine (DA) agonists were used to evaluate the participation of striatal peptidergic mechanisms in the motor function alterations that attend chronic neuroleptic treatment. Rats, given haloperidol (1 mg/kg, i.c.) for 21 consecutive days, were randomly allocated to one of the following treatments: the D1 agonist SKF 38393, the D2 agonist quinpirole, their combination or saline. Stereotyped behavior and neuropeptide levels were evaluated after 5 days treatment and 4 days washout. Haloperidol increased most oral behaviors including licking, chewing and biting as well as striatal enkephalin and somatostatin levels. Subsequent treatment with SKF 38393 diminished the haloperidol-induced increase in licking and chewing; quinpirole reduced chewing behavior. The administration of both agonists together decreased chewing and biting. Neither DA agonist alone, nor their combination, reduced the haloperidol-induced increase in enkephalin levels. Both SKF 38393 and quinpirole, when given alone, tended to decrease the haloperidol-induced increase in somatostatin levels; when both D1 and D2 agonists were administered together, somatostatin levels declined significantly. These results suggest that somatostatin- but not enkephalin-containing striatal neurons contribute to the expression of haloperidol-induced stereotypies.

Narrow beneficial effect of dextromethorphan on levodopa-induced motor response alterations in an experimental model of parkinsonism.

The effects of acute and chronic dextromethorphan on levodopa-induced motor response alterations have been studied in rats with unilateral lesion of nigrostriatal pathway induced by 6-hydroxydopamine (6-OHDA). Male Sprague-Dawley rats received a 6-OHDA injection (8 microg) into the left medial forebrain bundle. To validate the effect of acute dextromethorphan administration, groups of rats were treated with levodopa (25 mg/kg, twice daily) for 22 days. On day 23, animals received dextromethorphan (20, 30 or 40 mg/kg) immediately before levodopa. In a second set of experiments, lesioned rats were concomitantly treated with levodopa plus dextromethorphan (20, 30 or 40 mg/kg, twice at day) for 22 consecutive days in order to investigate the potential effect of chronic dextromethorphan administration in preventing the decrease in the duration of motor response. As expected, the duration of the motor response to levodopa had significantly decreased by the 22nd day of levodopa in each group of treatment. Acute administration of dextromethorphan on day 23 reversed the reduction in the duration of the levodopa response only when administered at the lowest dose used in the present study (20 mg/kg) (p<0.05). Chronic administration of dextromethorphan concomitant to levodopa did not prevent levodopa effect showing a significant decrease on motor response duration (124+/-4 on day 1 vs. 88+/-16 on day 22, p<0.05, 30 mg/kg, twice a day). Our results indicate that in parkinsonian rats dextromethorphan is not a useful drug to prevent levodopa-induced motor alterations, however, low doses of dextromethorphan may be beneficial to reverse these alterations in motor response.

The metabotropic glutamate receptor antagonist 2-methyl-6-(phenylethynyl) pyridine decreases striatal VGlut2 expression in association with an attenuation of L-DOPA-induced dyskinesias.

The striatal glutamatergic hyperactivity is considered critical in the development of levodopa‐induced dyskinesias (LID) in Parkinsons disease (PD). Pharmacological antagonism of the metabotropic glutamate receptors (mGluRs), in particular, the subtype mGluR5, can inhibit the expression of dyskinesia in both rodent and nonhuman primate models of PD. However, the exact mechanisms underlying the mGluR5 antagonism effects are not completely known. The vesicular glutamate transporters (VGluts) are localized in the synaptic vesicles of the striatal glutamatergic axonal terminals. The effects of mGluR5 antagonism modulating VGlut1 and VGlut2, as selective markers for the corticostriatal and thalamostriatal pathways, respectively, are still unknown. We investigated the effects of the mGluR5 antagonist, 2‐methyl‐6‐(phenylethynyl) pyridine (MPEP) on the striatal expression of VGlut1 and VGlut2 in levodopa‐treated hemiparkinsonian rats. Male Sprague–Dawley rats received a unilateral 6‐hydroxydopamine (6‐OHDA) administration in the nigrostriatal pathway. Rats were treated with: (a) levodopa (12 mg/kg/day with benserazide 15 mg/kg, ip) + vehicle; (b) MPEP (1.5 mg/kg/day, ip) + vehicle; (c) levodopa + MPEP, or (d) saline for 10 days. Levodopa treatment induced dyskinesias and did not modify the striatal expression of either VGlut1 or VGlut2. The administration of MPEP significantly attenuated LID and decreased the levels of VGlut2, but not the VGlut1, in the striatum ipsilateral to the lesion (P < 0.05). Our results suggest that the effects of MPEP on LID might be mediated by a modulating effect on VGlut 2 expression. Synapse, 2011.

Striatal c-fos levels do not correlate with haloperidol-induced behavioral supersensitivity.

Striatal c‐fos levels and stereotyped behavior have been evaluated in chronically haloperidol‐treated rats which received subsequent subacute dopamine (DA) agonist treatment to investigate the possible relationship between striatal c‐fos and behavioral supersensitivity. Haloperidol treatment (1 mg/kg/day for 21 days) increased apomorphine‐induced stereotypies but did not modify striatal c‐fos levels. The subacute administration of the DA D‐1 agonist SKF38393 (10 mg/kg/day for 5 days) and the combination of the D‐1 agonist with the D‐2 agonist quinpirole (1 mg/kg/day for 5 days) attenuated apomorphine‐induced stereotypies after haloperidol pretreatment. The administration of quinpirole alone, however, did not modify the response to haloperidol. All DA agonists significantly increased c‐fos levels after apomorphine injection. The dissociation between haloperidol‐induced behavioral supersensitivity and striatal c‐fos levels observed in this study suggests that mechanisms different from striatal c‐fos induction might be involved, and that striatal c‐fos levels are not a good marker of behavioral supersensitivity expression.

Relevance of COX-2 gene expression in dementia with lewy bodies associated with Alzheimer pathology.

The aim of the present study was to investigate whether cyclooxygenase‐2 (COX‐2) expression is involved in the pathogenesis of neurodegeneration in dementia with Lewy bodies (DLB) by measuring COX‐2 mRNA and protein expression in frontal cortex and substantia nigra pars compacta of DLB and control human brains. DLB cases were classified as pure form or common form according to the absence or the presence of Alzheimer pathology including neurofibrillary tangles and amyloid deposits by Braak staging. Using Western Blot and Real‐time Polymerase chain reaction (PCR) analysis, we have shown that cortical COX‐2 protein levels were decreased in DLB cases (P < 0.01). However, no differences in nigral COX‐2 mRNA expression were observed between control and DLB cases. In conclusion, the present results suggest that in DLB nigral COX‐2 mRNA expression does not correlate with dopaminergic neurodegeneration and that the slight changes observed in the common type are probably due to the concomitant AD pathology.

In vivo evaluation of the dopaminergic neurotransmission system using [123I]FP-CIT SPECT in 6-OHDA lesioned rats.

The 6-hydroxydopamine (6-OHDA) rodent model of Parkinsons disease (PD) has been used to evaluate the nigrostriatal pathway. The aim of this work was to explore the relationship between the degree of 6-OHDA-induced dopaminergic degeneration and [(123)I]FP-CIT binding using single photon emission computed tomography (SPECT). Fourteen rats received a 6-OHDA injection (4 or 8 µg) into the left medial forebrain bundle. After 3 weeks, magnetic resonance imaging and scans with a small-animal SPECT system were performed. Finally, the nigrostriatal lesion was assessed by immunohistochemical analysis. Immunohistochemical analysis confirmed two levels of dopaminergic degeneration. Lesions induced by 6-OHDA diminished the ipsilateral [(123)I]FP-CIT binding by 61 and 76%, respectively. The decrease in tracer uptake between control and lesioned animals was statistically significant, as was the difference between the two 6-OHDA lesioned groups. Results concluded that [(123)I]FP-CIT SPECT is a useful technique to discriminate the degree of dopaminergic degeneration in a rat model of PD.