In vivo single branch axotomy induces GAP-43-dependent sprouting and synaptic remodeling in cerebellar cortex.

Plasticity in the central nervous system in response to injury is a complex process involving axonal remodeling regulated by specific molecular pathways. Here, we dissected the role of growth-associated protein 43 (GAP-43; also known as neuromodulin and B-50) in axonal structural plasticity by using, as a model, climbing fibers. Single axonal branches were dissected by laser axotomy, avoiding collateral damage to the adjacent dendrite and the formation of a persistent glial scar. Despite the very small denervated area, the injured axons consistently reshape the connectivity with surrounding neurons. At the same time, adult climbing fibers react by sprouting new branches through the intact surroundings. Newly formed branches presented varicosities, suggesting that new axons were more than just exploratory sprouts. Correlative light and electron microscopy reveals that the sprouted branch contains large numbers of vesicles, with varicosities in the close vicinity of Purkinje dendrites. By using an RNA interference approach, we found that downregulating GAP-43 causes a significant increase in the turnover of presynaptic boutons. In addition, silencing hampers the generation of reactive sprouts. Our findings show the requirement of GAP-43 in sustaining synaptic stability and promoting the initiation of axonal regrowth.

Role of cerebellar cortical protein synthesis in transfer of memory trace of cerebellum-dependent motor learning.

We developed a new protocol that induces long-term adaptation of horizontal optokinetic response (HOKR) eye movement by hours of spaced training and examined the role of protein synthesis in the cerebellar cortex in the formation of memory of adaptation. Mice were trained to view 800 cycles of screen oscillation either by 1 h of massed training or by 2.5 h to 8 d of training with 0.5 h to 1 d space intervals. The HOKR gains increased similarly by 20-30% at the end of training; however, the gains increased by 1 h of massed training recovered within 24 h, whereas the gains increased by spaced training were sustained over 24 h. Bilateral floccular lidocaine microinfusions immediately after the end of training recovered the gains increased by 1 h of massed training but did not affect the gains increased by 4 h of spaced training, suggesting that the memory trace of adaptation was transferred from the flocculus to the vestibular nuclei within 4 h of spaced training. Blockade of floccular protein synthesis, examined by bilateral floccular microinfusions of anisomycin or actinomycin D 1-4 h before the training, impaired the gains increased by 4 h of spaced training but did not affect the gains increased by 1 h of massed training. These findings suggest that the transfer of the memory trace of adaptation occurs within 4 h of spaced training, and proteins synthesized in the flocculus during training period may play an important role in memory transfer.

Functional recovery of neuronal activity in rat whisker-barrel cortex sensory pathway from freezing injury after transplantation of adult bone marrow stromal cells.

The effect of transplantation of adult bone marrow stromal cells (MSCs) into the freeze-lesioned left barrel field cortex in the rat was investigated by measurement of local cerebral glucose utilization (lCMR(glc)) in the anatomic structures of the whisker-to-barrel cortex sensory pathway. Bone marrow stromal cells or phosphate-buffered saline (PBS) were injected intracerebrally into the boundary zone 1 h after induction of the freezing cortical lesion. Three weeks after surgery, the 2-[(14)C]deoxyglucose method was used to measure lCMR(glc) during right whisker stimulation. The volume of the primary necrotic freezing lesion was significantly reduced (P<0.05), and secondary retrograde degeneration in the left ventral posteromedial (VPM) thalamic nucleus was diminished in the MSC-treated group. Local cerebral glucose utilization measurements showed that the freezing cortical lesion did not alter the metabolic responses to stimulation in the brain stem trigeminal nuclei, but eliminated the responses in the left VPM nucleus and periphery of the barrel cortex in the PBS-treated group. The left/right (stimulated/unstimulated) lCMR(glc) ratios were significantly improved in both the VPM nucleus and periphery of the barrel cortex in the MSC-treated group compared with the PBS-treated group (P<0.05). These results indicate that MSC transplantation in adults may stimulate metabolic and functional recovery in injured neuronal pathways.

Chronic cerebellar stimulation in the monkey. Electron microscopic and biochemical observations.

The effects of chronic electrical stimulation to the surface of cerebellum in the Macaca mulatta monkey were studied with morphologic and biochemical techniques. There was considerable damage and loss of Purkinje cells in all specimens examined, including an area without electrodes, but the greatest changes appeared in tissue beneath the cathode and anode. Despite the damage, normal appearing synapses persisted in the molecular layer of all specimens. Fibrous glial processes were more numerous beneath the cathode. There were abnormalities in gamma-amino butyric acid (GABA) and polyamine concentrations in virtually all specimens, consistent with the morphologic evidence of widespread tissue damage.

Developmental plasticity of rat cerebellar cortex after cisplatin injury: inhibitory synapses and differentiating Purkinje neurons.

A single injection of cisplatin, a cytostatic agent, (5 microg/g body weight) in 10-day old rats leads later to the reorganization of the cerebellar cortex in lobules VI-VIII of the vermis. Double immunofluorescence reaction for glutamate receptor (GluR)2/3, a ionotropic glutamate receptor that labels postsynaptically Purkinje neurons, and glutamic acid decarboxylase (GAD)65, an isoform of the GABA synthesis enzyme that labels presynaptically inhibitory terminals in the molecular layer, were employed. Less-differentiated Purkinje cells were present in rats treated on postnatal day (PD)11 at the top of lobule VI and in lobules VII-VIII, in comparison with the deep zones of the same lobules and lobule III. The changes were interpreted as due to loss of trophic factors of Purkinje cell growth, e.g. signaling molecules and granule cells. However, we have shown that a remodelling of Purkinje cell dendrites occurred on PD30 (20 days after cisplatin). In fact, despite of the GluR2/3 labeling of the entire Purkinje cell dendrites, the GAD65 immunofluorescent terminals were adjacent to the proximal parts of the dendrite, while they were scarce in the distal dendritic branchlets. The findings were discussed in relation to the changed cytoarchitecture of the cerebellar cortex, which from PD17 to PD30 includes regeneration of the external germinal layer, reorientation of the main dendritic branches and of the Purkinje cell branchlets, and the presence of ectopic cells.

Lesion-induced neuronal nitric oxide synthase in Purkinje cells of the rat cerebellar cortex: histochemical and in situ hybridization study.

Lesion-induced induction of neuronal nitric oxide synthase (nNOS) was examined in the rat cerebellum. The stab-lesioned cerebellar cortex was examined with NADPH-diaphorase (NADPH-d) histochemistry and in situ hybridization using nNOS cRNA probe at 1, 3, 7, 14, 35 days post-lesion. NADPH-d- and nNOS mRNA-positive Purkinje cells appeared adjacent to the lesion by 3 days after the lesion. The area of distribution expanded and the number of positive cells increased at 7 days after the lesion, and at 14 days post-lesion, shrunken NADPH-d-positive Purkinje cells with irregular surface appeared. NADPH-d activity and nNOS mRNA signal could not be detected in Purkinje cells after 35 days post-lesion. Combined NADPH-d histochemistry and in situ hybridization using glutamic acid decarboxylase (GAD) cRNA probe revealed that nNOS-expressing Purkinje cells showed fewer GAD mRNA signals than those in normal Purkinje cells. The atrophic contour and the lower expression of GAD mRNA signals in NADPH-d positive Purkinje cells suggest that nNOS is expressed under a degenerating process.

Long-term effects of parallel fiber loss in the cerebellar cortex of the adult and weanling rat.

Short- and long-term effects of parallel fiber deafferentation of adult and weanling cerebellar cortex were investigated following parasagittal transections of the lateral cerebellar hemisphere. Short-term electron microscopic examination revealed that parallel fibers undergo rapid electron-dense degeneration within 5 days of axotomy. These axons were the only neuronal elements immediately affected by the lesion. The continued maintenance of Purkinje cell terminal branchlets and stellate cell dendrites is dependent upon the presence of an adequate parallel fiber milieu. Morphological evidence is provided which suggests that Purkinje cell dendritic spines may be phagocytically removed by Bergmann glial cells following parallel fiber loss. Although a marked decrease was reported in the number of spines projecting from terminal branchlets following deafferentation of both adult and weanling rats, these data suggest that some spines are capable of increasing their length. The elongation of these spines may represent a form of dendritic plasticity. No evidence was found to suggest that deafferentated terminal branchlets are receptive to forming heterologous synaptic contacts. The primary response to parallel fiber deafferentation for both the adult and weanling cerebellum therefore appears to be transneuronal degeneration.

Recovery of locomotor function in cats after localized cerebellar lesions.

Cats were trained to walk on a motorized treadmill, at speeds up to a brisk walk, for food reward. A cerebellar lesion was placed in each animal, either unilateral removal of paravermal cortex, or unilateral coagulation of n. interpositus, or bilateral coagulation of the fastigial nuclei. The effects of these lesions upon locomotor activity were measured by conventional kinematic methods, and were found to be generally in agreement with prior observations. Also in agreement with prior work was the fact that these initial deficits disappeared in a relatively short time. The kinematic data suggest that this recovery of function was genuine in the sense that compensatory alterations in limb motion could not be demonstrated. In disagreement with prior studies, we failed to elicit decompensation (e.g. reinstatement of the original deficits) by subsequent pyramidal tract sections, or ablation of the 'motor' cortex. We conclude that the corticospinal system is probably not essential to the recovery observed, and also that perhaps there are substantial differences in the mechanisms of recovery of 'spontaneous' overground locomotion, compared to walking on a treadmill as a conditioned instrumental response.

Chronic implanting of electrodes in the cerebellar vermis of the cat. Morphological findings.

The morphological modifications of the cerebellar cortex have been evaluated in eight cats with chronic electrodes implanted in the anterior vermis. In a control animal a silastic lamina without electrodes was implanted. Four animals received intermittent stimulations up to 11 hours in one case and 100 hours in the other 3. The stimulation was carried out with pulses of 0.5 msc, 200 cps, and a charge per phase of 0.68 muc. Microscopic examination of the cerebellum showed that the experimental implanting of prostheses of the cerebellar vermis produces tissue lesions consisting of fibrous reaction, imprinting of the cortex, gliosis, and loss of Purkinje cells. These lesions are independent of the electric stimulation, and seem to be related to the pressure of foreign material against the cortex.

Tissue damage after chronic cerebellar stimulation.

Two spastic patients undergoing chronic cerebellar stimulation are studied. Biopsies taken after three months of stimulation showed various tissue changes when compared with those taken at the moment of electrode implantation. The most important alterations in the cerebellar cortex were adhesions round the electrodes, with a scarring reaction, gliosis, and loss of Purkinje cells. These findings resemble those described in various experimental reports of chronic cerebellar stimulation.

Subcellular localization of low-affinity nerve growth factor receptor-immunoreactive protein in adult rat purkinje cells following traumatic injury.

Cerebellar Purkinje cells in the rat express low-affinity nerve growth factor receptor (p75 NGFR) antigen during development, but rarely in normal adult animals. In striking contrast, re-expression of p75 NGFR-immunoreactive protein was reported by light microscopy immunocytochemistry in adult rat Purkinje cells as early as 1 day after traumatic axotomy. Characteristically, varicose axons through the infraganglionic zone were also stained. To date, however, there is no information on the subcellular location of the antigenic re-expression. To address this, a pre-embedding immunocytochemical ultrastructural study using affinity-purified monoclonal 192-IgG was carried out after an experimentally induced traumatic lesion of the rat cerebellum. At the electron microscopic level, immunostaining was intense in Purkinje cells. In these cells, the immunoreactivity was always associated with the internal face of the membranes of the rough endoplasmic reticulum, Golgi apparatus and nuclear envelope. Patches of immunoreactivity were also associated with the outer surface of the plasma membrane of the cell body, dendritic processes and axons. It is noteworthy that receptor immunoreactivity was detected in recurrent collaterals of Purkinje cell axons forming symmetric synaptic contacts with the cell body and dendrites of immunonegative local circuit neurons. Results of this study show that injury-induced re-expression of p75 NGFR antigen is restricted to Purkinje cells. Also, the relative importance of the contribution of the local circuit neurons to the production of neurotrophic substances after trauma is suggested.

Mts1 protein expression in the central nervous system after injury.

We recently showed that Mts1 is expressed in white matter astrocytes in the rat brain and spinal cord from the first postnatal day. Its expression level declined in the adult CNS, but its topographical localization was maintained. Only white matter astrocytes in the cerebellum did not express Mts1. After dorsal root or sciatic nerve injury, we observed a marked upregulation of Mts1 in the area of the dorsal funiculus undergoing Wallerian degeneration. Here we show that upregulation of Mts1 is a consistent feature of astrocytes in white matter undergoing Wallerian degeneration. In addition, Mts1 is upregulated in astrocytes outlining the lesion site of a penetrating injury to the forebrain, or cerebellum. Gray matter astrocytes did not express Mts1, even after direct injury. In injured brain, we consistently noted a close relationship between Mts1-expressing astrocytes and ED1-positive microglia/macrophages, which are known to be highly motile cells. Mts1 was expressed in the periventricular area and the rostral migratory stream, i.e., sites of ongoing neuroplasticity in adulthood, and was upregulated in these areas after injury. These data suggest that Mts1-expressing astrocytes play a significant role in degenerative events in the mature white matter, interact with phagocytic microglia/macrophages and regulate cell migration and differentiation in areas of the adult brain with a high degree of plasticity.

Efeitos do etanol sobre a migração neuronal na formação do neocórtex cerebral / Effects of ethanol on the neuronal migration in the brain neocortex formation

Os neurônios migram das zonas ventriculares e subventriculares, através das glias radial e tangencial, para formar as várias camadas corticais do neocórtex adulto. A exposição crônica de ratas grávidas ao etanol desencadeia ectopia, heterotopia e despopulação neuronial, promovendo a chamada síndrome alcóolica fetal. O tratamento agudo de ratas grávidas na data de nascimento dos neurônios também promove efeitos graves na formação do neocórtex.

The neurons migrate from the ventricular and subventricular zones, through the radial and tangential glia, to form the several cortical shells of the adult neocortex. The chronic exposure to ethanol of pregnant female rats causes ectopia, heterotopia and neuronal depopulation, producint the fetal alcoholic syndrome. The acute exposure of pregnant female rats at the neurons birth time also promotes severe effects on the neocortex formation.