Transcranial direct current stimulation of cerebellum alters spiking precision in cerebellar cortex: A modeling study of cellular responses.

Transcranial direct current stimulation (tDCS) of the cerebellum has rapidly raised interest but the effects of tDCS on cerebellar neurons remain unclear. Assessing the cellular response to tDCS is challenging because of the uneven, highly stratified cytoarchitecture of the cerebellum, within which cellular morphologies, physiological properties, and function vary largely across several types of neurons. In this study, we combine MRI-based segmentation of the cerebellum and a finite element model of the tDCS-induced electric field (EF) inside the cerebellum to determine the field imposed on the cerebellar neurons throughout the region. We then pair the EF with multicompartment models of the Purkinje cell (PC), deep cerebellar neuron (DCN), and granule cell (GrC) and quantify the acute response of these neurons under various orientations, physiological conditions, and sequences of presynaptic stimuli. We show that cerebellar tDCS significantly modulates the postsynaptic spiking precision of the PC, which is expressed as a change in the spike count and timing in response to presynaptic stimuli. tDCS has modest effects, instead, on the PC tonic firing at rest and on the postsynaptic activity of DCN and GrC. In Purkinje cells, anodal tDCS shortens the repolarization phase following complex spikes (-14.7 ± 6.5% of baseline value, mean ± S.D.; max: -22.7%) and promotes burstiness with longer bursts compared to resting conditions. Cathodal tDCS, instead, promotes irregular spiking by enhancing somatic excitability and significantly prolongs the repolarization after complex spikes compared to baseline (+37.0 ± 28.9%, mean ± S.D.; max: +84.3%). tDCS-induced changes to the repolarization phase and firing pattern exceed 10% of the baseline values in Purkinje cells covering up to 20% of the cerebellar cortex, with the effects being distributed along the EF direction and concentrated in the area under the electrode over the cerebellum. Altogether, the acute effects of tDCS on cerebellum mainly focus on Purkinje cells and modulate the precision of the response to synaptic stimuli, thus having the largest impact when the cerebellar cortex is active. Since the spatiotemporal precision of the PC spiking is critical to learning and coordination, our results suggest cerebellar tDCS as a viable therapeutic option for disorders involving cerebellar hyperactivity such as ataxia.

Intracranial Injection of an Optogenetics Viral Vector Followed by Optical Cannula Implantation for Neural Stimulation in Rat Brain Cortex.

Optogenetics is rapidly gaining acceptance as a preferred method to study specific neuronal cell types using light. Optogenetic neuromodulation requires the introduction of a cell-specific viral vector encoding for a light activating ion channel or ion pump and the utilization of a system to deliver light stimulation to brain. Here, we describe a two-part methodology starting with a procedure to inject an optogenetic AAV virus into rat cortex followed by a second procedure to surgically implant an optical cannula for light delivery to the deeper cortical layers.

Long-lasting effects of neonatal ionizing radiation exposure on spatial memory and anxiety-like behavior.

Neonatal ionizing radiation exposure has been shown to induce a cerebellar cytoarchitecture disarrangement. Since cerebellar abnormalities have been linked to an impairment of behavioral functions, the aim of the present work was to investigate whether exposure of developing rats to ionizing radiations can produce behavioral deficits in the adult. Male Wistar rats were X-irradiated with 5Gy within 48h after birth and were tested in a radial maze and in an open field at 30 and 90 days post irradiation. Irradiated rats showed significative changes in spatial, exploratory, and procedural parameters in the radial maze, as well as a significative decrease in anxiety-like behavior, assessed in the open field. These results suggest that ionizing radiations can induce long-lasting spatial memory and anxiety-related changes. A relationship with radiation-induced cerebellar cytoarchitecture abnormalities supports the hypothesis that cerebellar integrity seems to be critical to achieve spatial performance and emotional behavior establishment.

Effect of an acute 900MHz GSM exposure on glia in the rat brain: a time-dependent study.

Because of the increasing use of mobile phones, the possible risks of radio frequency electromagnetic fields adverse effects on the human brain has to be evaluated. In this work we measured GFAP expression, to evaluate glial evolution 2, 3, 6 and 10 days after a single GSM exposure (15min, brain averaged SAR=6W/kg, 900MHz signal) in the rat brain. A statistically significant increase of GFAP stained surface area was observed 2 days after exposure in the frontal cortex and the caudate putamen. A smaller statistically significant increase was noted 3 days after exposure in the same areas and in the cerebellum cortex. Our results confirm the Mausset-Bonnefont et al. study [Mausset-Bonnefont, A.L., Hirbec, H., Bonnefont, X., Privat, A., Vignon, J., de Seze, R., 2004. Acute exposure to GSM 900MHz electromagnetic fields induces glial reactivity and biochemical modifications in the rat brain. Neurobiol. Dis. 17, 445-454], showing the existence of glial reactivity after a 15min GSM acute exposure at a brain averaged SAR of 6W/kg. We conclude to a temporary effect, probably due to a hypertrophy of glial cells, with a temporal and a spatial modulation of the effect. Whether this effect could be harmful remains to be studied.

The effects of electrical stimulation of the paleocerebellar cortex on penicillin-induced convulsive activity in rats.

Studies in Wistar rats in conditions of free behavior showed that low-frequency stimulation of the paleocerebellar cortex (nodulus, uvula) (10-12 Hz, 0.5 msec) was accompanied by activation of spike discharges induced by systematic application of benzylpenicillin sodium (3,000,000 IU/kg). Facilitation of the formation of ictal discharges was also seen. High-frequency electrical stimulation (100-300 Hz, 0.25 msec) of the same structure was accompanied by suppression of the generation of spike potentials and prevented the development of ictal potentials. The antiepileptic effect of electrical stimulation was seen in conditions of relatively low levels of convulsive activity. Electrical stimulation decreased the frequency and amplitude of spike potentials in the interstimulus intervals and decreased the total duration of epileptic foci. Repeated electrical stimulation of the paleocerebellum after electrocoagulation did not produce any changes in convulsive activity.

Epilepsia partialis continua of the abdominal muscles: a detailed electrophysiological study of a case.

We describe the clinical and electrophysiological features of an elderly woman presenting with persistent clonic twitching of the abdominal muscles that were considered to represent a rare manifestation of epilepsia partialis continua due to a metastatic cortical lesion.

HRP injection in lobule VI-VII of the cerebellar cortex reveals a bilateral inferior olive projection in granuloprival rats.

In immature rats, Purkinje cells receive synapses from multiple climbing fibers. During development, this multi-innervation regresses and only one climbing fiber innervates each Purkinje cell in the adult. The multi-innervation of immature rats is maintained in the adult if the precursors of the cerebellar granule cells are destroyed by early postnatal X-irradiation. The present study was undertaken to determine the origin of climbing fibers projecting to lobule VI-VII of the cerebellum in X-irradiated granuloprival rats. Olivary neurons were labelled by retrograde transport of wheat germ agglutinin conjugated to horseradish peroxidase, which was injected by iontophoresis in the right vermis of lobule VI-VII. Three-dimensional reconstructions of the inferior olive were made for granuloprival and control rats. No significant variation in the shape and dimension of the olive was observed between the two groups. Labeled cells were found in the middle part of the median accessory olive (MAO). In control rats, stained cells were found only in the contralateral MAO, whereas in the granuloprival rats they were located in both the contralateral and the ipsilateral MAO. Homologous zones were marked in control and granuloprival rats in the middle part of MAO. In granuloprival rats, there was a symmetry in the distribution of the stained cells in the ipsi- and contralateral MAO along the three axes. Therefore, polyinnervation involves homologous regions of both inferior olivary nuclei.

Effects of radiofrequency exposure on the GABAergic system in the rat cerebellum: clues from semi-quantitative immunohistochemistry.

The widespread use of cellular phones raises the problem of interaction of electromagnetic fields with the central nervous system (CNS). In order to measure these effects on neurotransmitter content in the CNS, we developed a protocol of neurotransmitter detection based on immunohistochemistry and image analysis. Gamma-vinyl-GABA (GVG), an inhibitor of the GABA-transaminase was injected in rats to increase GABA concentration in the CNS. The cellular GABA contents were then revealed by immunohistochemistry and semi-quantified by image analysis thanks to three parameters: optical density (O.D.), staining area, and number of positive cells. The increase in cerebellar GABA content induced by GVG 1200 mg/kg was reflected in these three parameters in the molecular and the granular layers. Therefore, control of immunohistochemistry parameters, together with appropriate image analysis, allowed both the location and the detection of variations in cellular neurotransmitter content. This protocol was used to investigate the effects of exposure to 900 MHz radiofrequencies on cerebellar GABA content. Both pulsed emission with a specific absorption rate (SAR) of 4 W/kg and continuous emission with high SAR (32 W/kg) were tested. We observed a selective diminution of the stained processes area in the Purkinje cell layer after exposure to pulsed radiofrequency and, in addition, a decrease in O.D. in the three cell layers after exposure to continuous waves. Whether this effect is, at least partly, due to a local heating of the tissues is not known. Overall, it appears that high energetic radiofrequency exposure induces a diminution in cellular GABA content in the cerebellum.

GM1 ganglioside treatment protects against long-term neurotoxic effects of neonatal X-irradiation on cerebellar cortex cytoarchitecture and motor function.

Exposure of neonatal rats to a 5 Gy dose of X-irradiation induces permanent abnormalities in cerebellar cortex cytoarchitecture (disarrangement of Purkinje cells, reduction of thickness of granular cortex) and neurochemistry (late increase in noradrenaline levels), and motor function (ataxic gait). The neuroprotective effects of gangliosides have been demonstrated using a variety of CNS injuries, including mechanical, electrolytic, neurotoxic, ischemic, and surgical lesions. Here, we evaluated whether systemically administered GM1 ganglioside protects against the long-term CNS abnormalities induced by a single exposure to ionizing radiation in the early post-natal period. Thus, neonatal rats were exposed to 5 Gy X-irradiation, and subcutaneously injected with one dose (30 mg/kg weight) of GM1 on h after exposure followed by three daily doses. Both at post-natal days 30 and 90, gait and cerebellar cytoarchitecture in X-irradiated rats were significantly impaired when compared to age-matched controls. By contrast, both at post-natal days 30 and 90, gait in X-irradiated rats that were treated with GM1 was not significantly different from that in non-irradiated animals. Furthermore, at post-natal day 90, cerebellar cytoarchitecture was still well preserved in GM1-treated, X-irradiated animals. GM1 failed to modify the radiation-induced increase in cerebellar noradrenaline levels. Present data indicate that exogenous GM1, repeatedly administered after neonatal X-irradiation, produces a long-term radioprotection, demonstrated at both cytoarchitectural and motor levels.

Effects of low-dose X-irradiation on the development of the mouse cerebellar cortex.

We labeled proliferating cells of the cerebellum of 6-day-old mice with 5-bromo-2'-deoxyuridine (BrdU) followed by a single exposure to 0.5, 1 or 2 Gy of X-rays. We then studied the effects of low-dose irradiation on the migration and survival of granule neurons in the mouse cerebellum. The animals were killed at 4 days, or at 2, 4 or 6 weeks after irradiation. Brains were fixed and BrdU-labeled cells in the cerebella were immunohistochemically analyzed. BrdU was predominantly distributed in the superficial layer of the external granular layer soon after injection. Four days after irradiation with 0.5 or 1 Gy, labeled cells were mainly seen in the inner granular layer, which was also the case in non-irradiated mice. However, following 2 Gy irradiation BrdU was found not only in the inner granular layer, but also in the Purkinje cell layer. This distribution was also seen at 2 and 4-6 weeks after irradiation. In animals irradiated with 1 Gy 4-6 weeks after irradiation, the proportion of labeled cells present in the inner granular layer decreased, while labeled cells in the Purkinje cell layer increased. On the other hand, 0.5 Gy irradiation did not change the distribution of labeled cells, except that the proportion of labeled cells in the inner granular layer decreased at 2 weeks after irradiation. The number of labeled cells in the cerebellar cortex per unit area decreased with time and dose. These results suggest that 2 Gy irradiation induces a migratory delay, abnormal distribution, and cell death of the granule neurons of the mouse cerebellum.

Effects of prenatal exposure to low dose ionizing radiation on the development of the cerebellar cortex in the rat.

The effects of maternal exposure to a single dose of whole body irradiation (0.5 Gy) on gestational days (GD) 17, 18, 19, or 20 on the development of the cerebellar cortex was examined in the offspring of Sprague Dawley rats at 21 and 28 days postnatally. No gross cerebellar anomalies were observed in the irradiated animals. However, compared to control animals, rat irradiated on each of GD-17, 18, 19 and 20 showed a significantly higher incidence (p < 0.05) of circumscribed cerebellar lesions (CL) distributed in the inner granular layer of the anterior and posterior lobes. These lesions were characterized by a loss of granule cells and atrophied and/or reduced number of Purkinje cells. In 21 days old rats, irradiation on GD-17 resulted in more CL anteriorly (75%) and in the vermis whereas on GD-20, the CL predominated posteriorly (100%) and in the lateral hemispheres. In 28 day old rats, following irradiation on each of GD-17 and GD-20, there was an equal distribution of CL in both the anterior and posterior lobes. However, with irradiation on both GD-17 and GD-20, these CL occurred more frequently in the lateral hemispheres of the anterior lobe, whereas in the posterior lobe they predominated in the vermis. These results suggest that a direct relationship exists between the proliferation, migration, development, and maturation of granule cells and their induction by Purkinje cells. The findings also support the view that both cell death and the regulation of granule cells by Purkinje cells maximize the effective development and organization of the cerebellum.

Purkinje and granule cells distribution in the cerebellum of the rat following prenatal exposure to low dose ionizing radiation.

The effects of maternal exposure to a single dose of whole body irradiation (0.5 Gy) on gestational days (GD) 17, 18, 19, or 20 on the number of Purkinje and granule cells in the pyramis of the cerebellar cortex was examined in the offspring of Sprague Dawley rats at 21 and 28 days postnatally. The laterolateral distribution of both granule and Purkinje cells in the pyramis were significantly reduced (p < 0.001) from controls in rats irradiated on each of GD-17, 18, 19 and 20. There was a greater deficit in granule cell number with irradiation on GD-20 than on GD17 (p < 0.05). Purkinje cells were reduced in number with irradiation on GD-17 and GD-20; however, the decrease did not correspond to the degree of reduction in the number of granule cells. There was a greater reduction of both granule and Purkinje cells in the vermis with irradiation on GD-17, whereas on GD-20, both granule (p < 0.05) and Purkinje cells (p < 0.001) were more reduced in the lateral hemispheres. The GC/PC ratio was smaller in rats irradiated on GD-20 than on GD-17. The GC/PC ratio between the irradiated animals and the controls were relatively similar. The findings show that irradiation does not affect the population of granule cells directly, but rather indirectly. The reduction in the number of granule cells could be an indirect consequence of reduced critical interaction with Purkinje cells. These results suggest that a direct relationship exists between the proliferation, migration, development, and maturation of granule cells and their induction by Purkinje cells.

Noradrenergic innervation of ectopic granule cells following low-level X-irradiation.

The distribution pattern of the noradrenergic system within the cerebellar cortex following low-level X-irradiation was studied using immunocytochemistry. Following one X-irradiation exposure on postnatal day 1, the laminar distribution of tyrosine hydroxylase-like immunoreactive (THIR) fibers was similar to controls at postnatal day 30, but the orientation of the fibers in the molecular layer (ML) was slightly changed. Successive daily doses, however, produced alterations in both the pattern and concentration of THIR fibers within the ML of the cerebellar cortex. In addition, THIR fibers were found among ectopic cell clusters within the ML. This relationship suggests a potential role for the noradrenergic system in the development and/or maintenance of the ectopic cell clusters.

Noradrenergic innervation of the external granular layer of the rat following low-level X-irradiation.

The distribution of the noradrenergic system within the rat cerebellar cortex following low-level X-irradiation was studied using tyrosine hydroxylase immunocytochemistry. X-Irradiation treatments consisted of 1, 3, or 5 successive daily dose beginning on postnatal day 1. When studied 24 h after the last exposure, a dense plexus of tyrosine hydroxylase-like immunoreactive (THIR) fibers, not found in age-matched controls, was observed in the reduced external granular layer (EGL) in each treatment group. At 10 days of age, the THIR fibers were found in association with the reconstituted cells of the premigratory zone of the EGL. The rapid and abnormal innervation of the EGL by the THIR fibers suggests that the noradrenergic system could play a role in the regeneration of this layer.

Motor abnormalities and changes in the noradrenaline content and the cytoarchitecture of developing cerebellum following X-irradiation at birth.

We have studied the developmental time-course of changes in the noradrenaline (NA) content of cerebellum (CE), cytoarchitecture of the cerebellar cortex, and motor abnormalities induced by the exposure of the cephalic end of rats to a single dose (5 Gy) of X-irradiation immediately after birth. At all ages examined, i.e., from postnatal (PN) d 5 to 90, CE from exposed animals show a marked atrophy, with an agranular cortex that has lost its layered structure. Purkinje cells are scattered at all depths in the cortex, and their primary dendrite is randomly oriented. The motor syndrome includes dystonia-like movements, a fine tremor, and an ataxic gait. Being progressive, the abnormal movements are evident from PN d 10, and fully developed by d 30. On the other hand, no differences in cerebellar NA content between X-irradiated rats and age-matched nonirradiated controls were detected from PN d 5 to 60. However, at PN d 90 a significant increase in NA content of CE from exposed animals is found when compared to either age-matched controls (+36%, p < 0.01), or data from irradiated rats obtained at PN d 5 to 60 (p < 0.01). These results indicate a temporal dissociation between the motor and cytoarchitectural abnormalities and the increase in cerebellar NA content produced by a single dose of X-rays at birth. The late increase in cerebellar NA content might represent a compensatory response of noradrenergic terminals to an altered information flow out of the cerebellar cortex induced by the ionizing noxa.

Gamma-radiation produces abnormal Bergmann fibers and ectopic granule cells in mouse cerebellar cortex.

Morphological changes in Bergmann glial fibers in the developing cerebellar cortex produced by exposure to gamma-rays were investigated in association with ectopic granule cells. Six-day-old mice that had been exposed to 3 Gy of gamma-radiation were killed 6 hours after exposure or at 7 through 30 days of age. Their cerebella were examined histologically and immunohistochemically for glial fibrillary acidic protein in Bergmann fibers. Extensive cell death took place in the external granular layer (EGL) of the cerebellum from 6 through 24 hours after exposure. This led to the thinning of the EGL and a decrease in the number of migrating cells in the molecular layer. The number of Bergmann cells was not decreased, but the fibers in the molecular layer were distorted; whereas, in the control these fibers were straight and perpendicular to the pial surface. The EGL began to recover 2 days after exposure, and abnormally oriented migrating cells were seen. At 17 days of age, some cell clustering was observed in the molecular layer of the irradiated cerebellum. Distortion of the Bergmann fibers was marked in regions where ectopic granule cells appeared at 30 days of age. These findings suggest that the distortion of Bergmann fibers leads to the production of ectopic granule cells after exposure to gamma-radiation.

Microtubule-associated protein 2 (MAP2) in Purkinje cell dendrites: evidence that factors other than binding to microtubules are involved in determining its cytoplasmic distribution.

We have studied the distribution of microtubule-associated protein 2 (MAP2) in the Purkinje cell dendrites of rats whose cerebella were exposed to X-irradiation during the second postnatal week. The Purkinje cells of such animals have abnormally elongated apical primary processes that branch in the other molecular layer rather than close to the cell body as in normal tissue. The results show that in these distorted dendrites the MAP2 distribution is "shifted" distally relative to the normal pattern, in which MAP2 is distributed evenly throughout the dendritic tree. Tubulin and other microtubule-associated proteins, such as MAP1, are not affected and remain evenly distributed throughout the dendritic tree despite the anatomical distortion. We conclude that the distribution of MAP2 in Purkinje cells is not determined solely by its binding to tubulin. Other factors must be involved and these appear to be related to dendritic morphology and possibly to branching.

[Synaptic localization of 5'-nucleotidase activity in the cerebellar cortex in adult rats irradiated by X rays after birth]. / Localisation synaptique de l'actitivé 5'-nucléotidase dans le cortex cérébelleux du rat adulte irradié aux rayons X après la naissance.

5'-nucleotidase cytochemistry was performed in the cerebellum of adult rats exposed previously to a single postnatal X-irradiation which maintains until adulthood the normally transient multiple innervation of cerebellar Purkinje cells by climbing fibers. 5'-nucleotidase activity persists in climbing fiber synapses and other asymmetrical synapses while it is transient during normal development. Therefore, in the X-irradiated rat, an immature stage of the excitatory synapses persists which, for the climbing fibers, coïncides with or precedes their involution during normal development.

Radiation-induced DNA strand breaks and cell pycnosis in the developing rat cerebellum.

DNA strand breaks and cell pycnosis induced by 60Co gamma radiation were studied in the developing rat cerebellum. Rats 2, 5 or 12 days old were irradiated and the repair of DNA strand breaks was studied at various intervals up to seven hours. DNA strand breaks were repaired in 30 min and post-repair DNA degradation was observed about 90 min after irradiation in 12 day old rats and some time later in the younger ones. The pycnotic activity in the external granular layer of the cerebellar cortex seen in histological preparations developed in a similar way, the older rats showing earlier signs of cell pycnosis and a faster degradation of DNA. A correspondence was found between DNA degradation and cell pycnosis.