E. López-Martín

The overall rod performance test in the MPTP-treated-mouse model of Parkinsonism

We investigated the usefulness of the Overall Rotarod Performance (ORP) test for evaluating overall locomotory ability in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-injected-mouse model of Parkinsons disease (PD). For this procedure, the mice are pretrained on the rotarod and then tested at a series of increasing speeds, recording the time that the animal remains on the rod at each speed; the overall rod performance (ORP) of each animal is then calculated as the area under the curve in a plot of time-on-the-rod against rotation speed. At 15-day intervals, C57BL/6 mice were injected (or sham-injected) with MPTP, with ORP testing 7-10 days after each injection. After the fourth injection (day 45), mice in the treated group showed clearly lower ORP than mice in the control group (70-90% reduction in ORP), and were thus considered effectively lesioned. Subsequently, we investigated the short-term effects of apomorphine and L-DOPA on ORP in MPTP-treated mice. Apomorphine (at 0.5 or 2.5 mg/kg) had no significant effect, while L-DOPA (at 80 but not at 40 mg/kg) caused almost complete short-term recovery of pretreatment ORP. By about 100 days after the last MPTP injection, MPTP-treated mice showed partial long-term recovery of ORP; at this stage the mice were killed for tyrosine hydroxylase (TH) immunohistochemistry studies. TH immunoreactivity in the striatum showed a strong positive correlation with ORP as tested on day 100. We conclude that the ORP test is useful for evaluating motor deficit in MPTP-treated mice, and the effects of subsequent treatments.

1,25-Dihydroxyvitamin D3 increases striatal GDNF mRNA and protein expression in adult rats

Glial cell line-derived neurotrophic factor (GDNF) has been postulated as a possible candidate for therapeutic treatment in Parkinsons disease (PD). Recent in vitro data suggest that 1,25-dihydroxyvitamin D3 [1,25(OH)(2)D(3)] treatment may enhance GDNF mRNA expression. In the present study, using semiquantitative RT-PCR and Western blot, we have shown that 1,25(OH)(2)D(3) administration intraperitoneally, significantly increases GDNF mRNA and protein levels in the striatum of adult rats.

Time course of striatal changes induced by 6-hydroxydopamine lesion of the nigrostriatal pathway, as studied by combined evaluation of rotational behaviour and striatal Fos expression

Changes taking place after unilateral 6-hydroxydopamine lesion of the dopaminergic nigrostriatal system have been studied by performing spontaneous, amphetamine-induced and apomorphine-induced rotational behaviour testing and tyrosine hydroxylase (TH) and Fos protein immunohistochemistry in the same rats. Apomorphine at a low dosage (0.25 mg/kg) induced contraversive rotation and supersensitive striatal Fos expression that were detected 24–48 h post-lesion and gradually increased in magnitude. Twenty-four hours after lesion, both high (5 mg/kg) and low doses (0.5 mg/kg) of D-amphetamine induced contraversive rotation and intense striatal Fos activation on the denervated side; however, only the higher dose induced Fos on the normal side. Two, 3 and 4 days after lesion, 0.5 mg/kg amphetamine induced contraversive rotation, but 5 mg/kg induced transitory contraversive rotation which switched to ipsiversive. In the normal striatum, only high doses of amphetamine induced Fos, but Fos induction in the denervated striatum was similar with both doses: areas showing severely decreased TH immunoreactivity still showed considerable Fos immunoreactivity, and some areas still showing TH immunoreactivity had higher Fos density than in the normal side. Seven and 14 days after lesion the loss of TH immunoreactivity and apomorphine-induced supersensitive Fos expression were more evenly distributed, and amphetamine induced only ipsiversive rotation and a low density of Fos-positive nuclei in the denervated striatum. These results indicate that the severe and progressive loss of dopaminergic terminals is counteracted by an early and rapidly progressing dopamine supersensitivity, together with a higher susceptibility to drug-induced dopamine release. This explains the apparently paradoxical contraversive rotation induced by amphetamine during the first week post-lesion. However, experiments involving successive drug injections indicated that only the first amphetamine injection releases dopamine from the lesioned terminals.

Striatal dopaminergic afferents concentrate in GDNF-positive patches during development and in developing intrastriatal striatal grafts

Glial cell line‐derived neurotrophic factor (GDNF) has potent trophic action on fetal dopaminergic neurons. We have used a double immunocytochemical approach with antibodies that recognize GDNF and tyroxine hydroxylase (TH) or the phosphoprotein DARPP‐32, to study the developmental pattern of their interactions in the rat striatum and in intrastriatal striatal transplants. Postnatally, at one day and also at 1 week, GDNF showed a patchy distribution in the striatum, together with a high level of expression in the lateral striatal border, similar to that observed for the striatal marker DARPP‐32 and also for TH. In the adult striatum, there was diffuse, weak immunopositivity for GDNF, together with widespread expression of DARPP‐32‐positive neurons and TH‐immunoreactive (TH‐ir) fibers. In 1‐week‐old intrastriatal striatal transplants, there were some GDNF immunopositive patches within the grafts and although there was not an abundance of TH‐positive fibers, the ones that were seen were located in GDNF‐positive areas. This was clearly evident in 2‐week‐old transplants, where TH‐ir fibers appeared selectively concentrated in GDNF‐positive patches. This pattern was repeated in 3‐week‐old grafts. In co‐transplants of mesencephalic and striatal fetal tissue (in a proportion of 1:4), TH‐ir somata were located mainly at the borders of areas that were more strongly immunostained for GDNF, and TH‐ir fibers were also abundant in these areas and were found in smaller numbers in regions that were weakly positive for GDNF.

GSM radiation triggers seizures and increases cerebral c-Fos positivity in rats pretreated with subconvulsive doses of picrotoxin

This study investigated the effects of mobile-phone-type radiation on the cerebral activity of seizure-prone animals. When rats transformed into an experimental model of seizure-proneness by acute subconvulsive doses of picrotoxin were exposed to 2 h GSM-modulated 900 MHz radiation at an intensity similar to that emitted by mobile phones, they suffered seizures and the levels of the neuronal activity marker c-Fos in neocortex, paleocortex, hippocampus and thalamus increased markedly. Non-irradiated picrotoxin-treated rats did not suffer seizures, and their cerebral c-Fos counts were significantly lower. Radiation caused no such differences in rats that had not been pretreated with picrotoxin. We conclude that GSM-type radiation can induce seizures in rats following their facilitation by subconvulsive doses of picrotoxin, and that research should be pursued into the possibility that this kind of radiation may similarly affect brain function in human subjects with epileptic disorders.

A study of neurotoxic biomarkers, c-fos and GFAP after acute exposure to GSM radiation at 900 MHz in the picrotoxin model of rat brains.

The acute effects of microwave exposure from the Global System for Mobile Communication (GSM) were studied in rats, using 900MHz radiation at an intensity similar to mobile phone emissions. Acute subconvulsive doses of picrotoxin were then administered to the rats and an experimental model of seizure-proneness was created from the data. Seventy-two adult male Sprague-Dawley rats underwent immunochemical testing of relevant anatomical areas to measure induction of the c-fos neuronal marker after 90min and 24h, and of the glial fibrillary acidic protein (GFAP) 72h after acute exposure to a 900MHz electromagnetic field (EMF). The experimental set-up facilitated measurement of absorbed power, from which the average specific absorption rate was calculated using the finite-difference time-domain (FDTD) 2h after exposure to EMF radiation at 1.45W/kg in picrotoxin-treated rats and 1.38W/kg in untreated rats. Ninety minutes after radiation high levels of c-fos expression were recorded in the neocortex and paleocortex along with low hippocampus activation in picrotoxin treated animals. Most brain areas, except the limbic cortical region, showed important increases in neuronal activation 24h after picrotoxin and radiation. Three days after picrotoxin treatment, radiation effects were still apparent in the neocortex, dentate gyrus and CA3, but a significant decrease in activity was noted in the piriform and entorhinal cortex. During this time, glial reactivity increased with every seizure in irradiated, picrotoxin-treated brain regions. Our results reveal that c-fos and glial markers were triggered by the combined stress of non-thermal irradiation and the toxic effect of picrotoxin on cerebral tissues.

The Action of Pulse-Modulated GSM Radiation Increases Regional Changes in Brain Activity and c-Fos Expression in Cortical and Subcortical Areas in a Rat Model of Picrotoxin-Induced Seizure Proneness

The action of the pulse‐modulated GSM radiofrequency of mobile phones has been suggested as a physical phenomenon that might have biological effects on the mammalian central nervous system. In the present study, GSM‐exposed picrotoxin‐pretreated rats showed differences in clinical and EEG signs, and in c‐Fos expression in the brain, with respect to picrotoxin‐treated rats exposed to an equivalent dose of unmodulated radiation. Neither radiation treatment caused tissue heating, so thermal effects can be ruled out. The most marked effects of GSM radiation on c‐Fos expression in picrotoxin‐treated rats were observed in limbic structures, olfactory cortex areas and subcortical areas, the dentate gyrus, and the central lateral nucleus of the thalamic intralaminar nucleus group. Nonpicrotoxin‐treated animals exposed to unmodulated radiation showed the highest levels of neuronal c‐Fos expression in cortical areas. These results suggest a specific effect of the pulse modulation of GSM radiation on brain activity of a picrotoxin‐induced seizure‐proneness rat model and indicate that this mobile‐phone‐type radiation might induce regional changes in previous preexcitability conditions of neuronal activation.

Time course of striatal, pallidal and thalamic α1, α2 and β2/3 GABAA receptor subunit changes induced by unilateral 6-OHDA lesion of the nigrostriatal pathway

Abstract Immunocytochemical techniques were used to investigate the distribution and abundance of GABAA receptor subunits (α1, α2 and β2/3) in the brains of unilaterally 6-OHDA-lesioned rats. Three and 7 days after lesion, the α2-subunit was significantly more abundant in the lesion-ipsilateral striatum than in the lesion-contralateral striatum; by 4 weeks after lesion, however, no significant between-side differences were observed. Three and 7 days after lesion, the α1-subunit was significantly less abundant in the lesion-ipsilateral globus pallidus than in the lesion-contralateral side; again, this difference disappeared within 4 weeks of lesion. Similarly, α1 was initially less abundant in several relay thalamic nuclei on the lesioned side while α2 was initially more abundant in intralaminar thalamic nuclei on the lesioned side. There were no significant between-side changes for the β2/3-subunits. Comparison of non-lesioned and 6-OHDA-lesioned rats revealed significant differences in brain areas which also showed differences on comparison of the lesioned and non-lesioned sides of 6-OHDA-lesioned rats. These results suggest that there is an early adaptation to the lesion, achieved through changes in GABAA receptor abundance. That some of these changes are no longer apparent after 4 weeks is due not only to partial reversion of the changes in the lesioned side but also to compensatory changes in the non-lesioned side.

EXPOSURE TO 2.45 GHz MICROWAVE RADIATION PROVOKES CEREBRAL CHANGES IN INDUCTION OF HSP-90 fi/fl HEAT SHOCK PROTEIN IN RAT

Physical agents such as non-ionizing continuous-wave 2.45GHz radiation may cause damage that alters cellular homeostasis and may trigger activation of the genes that encode heat shock proteins (HSP). We used Enzyme-Linked ImmunoSorbent Assay (ELISA) and immunohistochemistry to analyze the changes in levels of HSP-90 and its distribution in the brain of Sprague-Dawley rats, ninety minutes and twenty-four hours after acute (30min) continuous exposure to 2.45 GHz radiation in a the Gigahertz Transverse Electromagnetic (GTEM cell). In addition, we studied further indicators of neuronal insult: dark neurons, chromatin condensation and nucleus fragmentation, which were observed under optical conventional or fluorescence microscopy after DAPI staining. The cellular distribution of protein HSP-90 in the brain increased with each corresponding SAR (0.034 ± 3.10−3, 0.069 ± 5.10−3, 0.27 ± 21.10−3 W/kg), in hypothalamic nuclei, limbic cortex and somatosensorial cortex after exposure to the radiation. At twenty-four hours post-irradiation, levels of HSP-90 protein remained high in all hypothalamic nuclei for all SARs, and in the parietal cortex, except the limbic system, HSP-90 levels were lower than in non-irradiated rats, almost half the levels in rats exposed to the highest power radiation. Non-apoptotic cellular nuclei and a some dark neurons were found ninety minutes and twenty-four hours after maximal SAR exposure. The results suggest that acute exposure to electromagnetic fields triggered an imbalance in anatomical HSP90 levels but the anti-apoptotic mechanism is probably sufficient to compensate the non-ionizing stimulus. Further studies are required to determine the regional effects of chronic electromagnetic pollution on heat shock proteins and their involvement in neurological processes and neuronal damage.

An Experimental Set-Up for Measurement of the Power Absorbed from 900 MHz GSM Standing Waves by Small Animals, Illustrated by Application to Picrotoxin-Treated Rats

We describe an experimental set-up for exposure of small animals to radiofrequency standing waves that allows direct measurement of the power absorbed by the animal. Essentially, the setup consists of a metallic box containing an antenna and experimental animal immobilized in a methacrylate holder; a signal generator feeding the antenna; and a power meter. In addition, the box can also contain a video camera to record the animal’s behaviour, and a receiving antenna (connected externally to a power meter and a spectrum analyser) to detect undesired (external) radiation and possible harmonics of the radiating system. The absorbed power measurement trivially allows calculation of whole-body mean SAR from the animal’s weight; and assuming local SARs to be proportional to whole-body mean SAR, the latter can be used to adjust organ-specific SAR predictions obtained by simulation using a commercial FDTD program with a numerical phantom. The use of the system is illustrated by application to rats given subconvulsive doses of picrotoxin to induce a seizure-prone state analogous to epilepsy: levels of the neuronal activity marker c-Fos in the frontal and piriform cortex of picrotoxin-treated rats exposed to 900 MHz GSM radiation were twice as high as those of unexposed animals.