Granule neuron DNA damage following deafferentation in adult rats cerebellar cortex: a lesion model

Neuronal programmed cell death is regulated by a neurotrophic supply from targets and afferent inputs. The relative contribution of each component varies according to neuronal type and age. We have previously reported that primary cultures of cerebellar granule cells undergo apoptosis when deprived of depolarising KCl concentrations, suggesting a significant role of afferent inputs in the control of cerebellar granule cells survival. This issue was investigated by setting up various in vivo lesional paradigms in order to obtain partial or total deafferentation of the cerebellar granule layer in adult rats. At different times after surgery, cerebellar sections were subjected to TUNEL staining in order to detect possible DNA damage. One week after unilateral pedunculotomy, few scattered groups of apoptotic granule neurons were observed in the homolateral hemisphere. On the contrary, total deafferentation obtained by a new experimental paradigm based on an "L-cut" lesion induced massive and widespread apoptotic death in the granule layer of the deafferentated area. The time window of DNA fragmentation in granule layer was one to seven days after the "L-cut". Selective Purkinje cell deafferentation obtained by 3-acetylpyridine injection did not result in TUNEL staining in the cerebellar cortex. The current finding that mossy fiber axotomy induces granule cell apoptotic death points out for the first time the crucial role of afferent inputs in mature granule cell survival. Moreover, the in vivo lesional model described here may prove to be an useful tool for investigating cellular and molecular mechanisms of neuronal death triggered by deafferentation.     

Excitatory tonus is required for the survival of granule cell precursors during postnatal development within the cerebellum

In addition to protective effects within the adult central nervous system (CNS), in vivo application of N-methyl-d-aspartate inhibitors such as (+) MK-801 have been shown to induce neurodegeneration in neonatal rats over a specific developmental period. We have systematically mapped the nature and extent of MK-801-induced neurodegeneration throughout the neonatal murine brain in order to genetically dissect the mechanism of these effects. Highest levels of MK-801-induced neurodegeneration are seen in the cerebellar external germinal layer; while mature neurons of the internal granule layer are unaffected by MK-801 treatment. Examination of external germinal layer neurons by electron microscopy, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) and bromodeoxyuridine (BrdU) labeling, and caspase-3 activation demonstrate that these neurons die through the process of programmed cell death soon after they exit from the cell cycle. Significantly, ablation of caspase-3 activity completely inhibited the MK-801-induced (and developmental) programmed cell death of external germinal layer neurons. Similar to caspase-3, inactivation of muscarinic acetylcholine receptors in vivo using scopolamine inhibited MK-801-induced programmed cell death. By contrast, the GABAergic agonist diazepam, either alone or in combination with MK-801, enhanced programmed cell death within external germinal layer neurons. These data demonstrate that, in vivo, cerebellar granule neurons undergo a dramatic change in intracellular signaling in response to molecules present in the local cellular milieu during their first 24 h following exit from the cell cycle.     

Proliferation and migration of granule cells in the developing rat cerebellum: cisplatin effects

We evaluated the relationship among proliferation, death and migration of granule cells in lobules VI-VIII of vermis, in comparison with lobule III, during cerebellar development. To this aim, a single injection of cisplatin, i.e., a cytostatic agent that is known to induce death of proliferating granule cells, was given to 10-day-old rats. Histochemical markers of proliferating (PCNA immunoreaction) and apoptotic (TUNEL staining) cells were used; the variations of the external granular layer (EGL) thickness were evaluated in parallel. After PCNA and TUNEL reactions, evident changes of the whole EGL were found on PD11 (1 day after treatment), when a reduction of the thickness of this layer was found in treated rats, mainly in consequence of the high number of apoptotic cells in all the cerebellar lobules. On PD17 (7 days after treatment), a thick layer of proliferating cells was observed in lobules VI-VIII of treated rats, while the peculiar pattern of the normal development showed a thin EGL. At the same time, in treated rats, the number of apoptotic cells in EGL was low. In all developmental stages of treated rats, after GFAP immunoreaction, glial fibers appeared twisted, thickened, and with an irregular course; intensely labeled end-feet were present. The damage of radial glia suggests an alteration of migratory processes of granule cells, which is also evidenced by the decreased thickness of the premigratory zone of the EGL. Injured radial glia fibers were restricted to lobules VI-VIII and they persisted at PD30, leading to the presence of ectopic granule cells in the molecular layer, as we previously described.     

Acute neuronal and vascular changes following unilateral cerebellar pedunculotomy in the neonatal rat

During development of the central nervous system (CNS) both deafferentation and axotomy induce increased neuronal death and result in a smaller brain with diminished function at maturity. Unilateral cerebellar pedunculotomy has been used as a model to study the relative importance of these 2 types of lesion on the survival of developing CNS neurons. Within the cerebellum, unilateral pedunculotomy causes deafferentation of the hemicerebellum and axotomy in the efferent pathway from the ipsilateral deep cerebellar nuclei. This results in a smaller hemicerebellum with normal cortical laminae but no extracerebellar outflow. In order to identify the sequence of events which leads to this altered structure and therefore to understand the relative importance of afferent versus target-derived trophic support, unilateral cerebellar pedunculotomy was performed on neonatal rat pups, aged between 1 and 3 days. The cerebella were analysed for histological and vascular changes after survival times of 0, 3, 6, 9, 12, 18, 21, 24 and 48 h. The results show that the effects of axotomy on the deep cerebellar nuclear neurons begin within 3 h of the lesion and apoptotic neuronal degeneration occurs within 48 h. However, the cerebellar cortical neurons continue to undergo normal histological development for at least 48 h after deafferentation. In addition, since ischaemia induces similar effects, a study of the vascular tree was made. The results indicate that the pedunculotomy does not alter the blood supply to the cerebellum, nor induce ischaemia of the cerebellar neurons. From this it may be hypothesised that target-derived trophic support is more crucial for the survival of immature neurons than is the trophic effect of afferent input.     

Different types of neural cell death in the cerebellum of the ataxia and male sterility (AMS) mutant mouse

To investigate the mechanisms(s) of age-dependent atrophy of the cerebellum of the ataxia and male sterility (AMS) mouse at young age, the morphological changes were evaluated and the nature of neural cell death was examined. Dying Purkinje cells lacked characters of classical apoptosis except for light microscopic morphology, but their death was considered to be autonomous death triggered by the direct effect of ams mutation, because of the acute and near-complete disappearance and particular change of the cytoplasm. In contrast, in the granular layer, typical apoptotic bodies were recognized by electron microscopy, and substantial numbers of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end-labeling (TUNEL)-positive cells and activated caspase-3-positive cells were observed. Granule cell death was considered to be target-related apoptosis induced after post-synaptic Purkinje cell death, because the age-dependent changes in TUNEL-positive cell counts followed that of Purkinje cell loss and the peak value was still noted 1 week after total loss of Purkinje cells. These results indicate that both total and partial losses of Purkinje cells and granule cells, respectively, contributed to the atrophy of the AMS cerebellum. Furthermore, different types of neuronal death were recognized; the granule cell death was apoptotic while Purkinje cell death was different from that of classical apoptosis.     

Deprivation of sensory inputs to the olfactory bulb up-regulates cell death and proliferation in the subventricular zone of adult mice

The main olfactory bulb (MOB) is the first relay on the olfactory sensory pathway and the target of the neural progenitor cells generated in the subventricular zone (SVZ) lining the lateral ventricles and which migrate along the rostral extension of the SVZ, also called the rostral migratory stream (RMS). Within the MOB, the neuroblasts differentiate into granular and periglomerular interneurons. A reduction in the number of granule cells during sensory deprivation suggests that neurogenesis may be influenced by afferent activity. Here, we show that unilateral sensory deafferentation of the MOB by axotomy of the olfactory receptor neurons increases apoptotic cell death in the SVZ and along the rostro-caudal extent of the RMS. The vast majority of dying cells in the RMS are migrating neuroblasts as indicated by double Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling/PSA-NCAM labeling. Counting bromodeoxyuridine-labeled cells in animals killed immediately or 4 days after tracer administration showed a bilateral increase in proliferation in the SVZ and RMS which was balanced by cell death on the operated side. These data suggest that olfactory inputs are required for the survival of newborn neural progenitors. The greatest enhancement in proliferation occurred in the extension of the RMS located in the MOB, revealing a population of local precursors mitotically stimulated following axotomy. Together, these findings indicate that olfactory inputs may strongly modulate the balance between neurogenesis and apoptosis in the SVZ and RMS and provide a model for further investigation of the underlying molecular mechanisms of this activity-dependent neuronal plasticity.     

Deafferentation-induced caspase-3 activation and DNA fragmentation in chick cochlear nucleus neurons

Cochlea removal severs peripheral processes of cochlear ganglion cells and permanently abolishes afferent input to nucleus magnocellularis (NM) neurons. Deafferented chick NM neurons undergo a series of morphologic and metabolic changes, which ultimately trigger the death of 20%–40% of neurons. Previous studies suggested that this cell specific death involves activation of the intrinsic apoptotic pathway, including increased presence of cytochrome c and active caspase-9 in the cytoplasm of deafferented NM neurons. Interestingly, however, both markers were detected pan-neuronally, in both degenerating and surviving NM neurons [Wilkinson BL, Elam JS, Fadool DA, Hyson RL (2003) Afferent regulation of cytochrome-c and active caspase-9 in the avian cochlear nucleus. Neuroscience 120:1071–1079]. Here, we provide evidence for the increased appearance of late apoptotic indicators and describe novel characteristics of cell death in deafferented auditory neurons. Young broiler chickens were subjected to unilateral cochlea removal, and brainstem sections through NM were reacted for active caspase-3 and terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL). Caspase-3 activation is observed in the cytoplasm of both dying and surviving deafferented NM neurons 24 h to 7 days following cochlea removal, suggesting that caspase-3, usually considered an “executioner” of apoptotic death, may also function as a “modulator” of death. In addition, we find that TUNEL labeling of degraded DNA is observed in deafferented NM. In contrast to upstream apoptotic markers, however, TUNEL labeling is restricted to a subpopulation of deafferented neurons. Twelve hours following cochlea removal, TUNEL labeling is observed as punctate accumulations within nuclei. Twenty-four hours following cochlea removal, TUNEL accumulates diffusely throughout neuronal cytoplasm in those neurons likely to die. This cytoplasmic TUNEL labeling may implicate mitochondrial nucleic acid degradation in the selective death of some deafferented NM neurons. Our study examines the subcellular distributions of two prominent apoptotic mediators, active caspase-3 and TUNEL, relative to known histochemical markers, in deafferented NM; provides new insight into the apoptotic mechanism of cell death; and proposes a role for mitochondrial DNA in deafferentation-induced cell death.     

Cell proliferation and apoptosis during histogenesis of the guinea pig and rabbit cerebellar cortex

Cell proliferation and apoptosis are essential for development of the nervous system. In this study we have investigated the histogenesis of the cerebellar cortex in guinea pig (a precocial species) and rabbit (an altricial species) at different stages of pregnancy and postnatal life. Proliferating cells were identified after labeling with antibodies against the proliferating cell nuclear antigen (PCNA) and/or the Ki-67 antigen. Apoptotic cells were visualized in situ by the TUNEL method and by immunodetection of cleaved caspase 3 and 9. In guinea pigs, both proliferating and apoptotic cells were detected during pre-natal life (E0-E40). Conversely, cell proliferation and apoptosis in rabbits were temporally restricted to early postnatal weeks (P0-P20). In both species cell proliferation was mainly linked to differentiation and migration of the granule cells. In both species, the majority of cells undergoing programmed cell death likely corresponded to granule cells. They were mainly detected in the external granular layer, and were by far more common than previously reported in other locations of the postnatal brain. This study shows that apoptosis is a shared process of cell death during cerebellar development in both altricial and precocial animals, and that there is a direct spatial and temporal correlation between cell proliferation and death in two mammals with different time tables in cerebellar maturation.     

Synapse-independent and synapse-dependent apoptosis of cerebellar granule cells in postnatal rabbits occur at two subsequent but partly overlapping developmental stages

It has long been known that cells in the external granular layer die during postnatal development of the cerebellum. More recent findings indicate that at certain developmental stages, cell death occurs upon activation of an apoptotic program. We show that cerebellar granule cells in rabbits undergo programmed cell death at two different stages of maturation. At postnatal day 5 (P5), granule cell precursors and pre-migratory granule cells in the external granular layer incorporate the S-phase markers 5-bromo-2'-deoxyuridine and 5-iodo-2'-deoxyuridine with a pattern that is dependent upon the interval between the administration of the two tracers. Within 12-24 h after proliferation a significant number of labeled cells show typical ultrastructural alterations of apoptosis. DNA electrophoresis and cleavage of poly-ADP-ribose polymerase confirm the activation of the apoptotic machinery. After Southern blotting and immunodetection, incorporated 5-bromo-2'-deoxyuridine is present at the level of low size DNA oligomers as soon as 12 h after cell division. Therefore, this apoptotic phase is intrinsic to external granular layer neurons and independent of synaptic interactions with targets.Apoptotic cells, although fewer in number, are detected also in the internal granular layer and tend to increase from P5 to P10. It seems unlikely that these cells undergo DNA fragmentation in the external granular layer and subsequently migrate to their final destination, considering the data on cell cycle kinetics and the rapid tissue clearance by the glia. Parallel fiber-Purkinje spine synapses are already present in the molecular layer at P5. Therefore, the post-migratory granule cells likely undergo apoptosis as a failure to make proper synaptic contacts in the forming molecular layer.We conclude that the massive apoptosis of pre-migratory cells likely has a role in regulating the size of this rapidly expanding population of pre-mitotic neurons. The less tumultuous cell death of post-mitotic granule cells in the internal granular layer appears to be linked to the formation of the mature synaptic circuitry of the developing cerebellar cortex.     

Mild traumatic brain injury induces apoptotic cell death in the cortex that is preceded by decreases in cellular Bcl-2 immunoreactivity

Although mild traumatic brain injury is associated with behavioral dysfunction and histopathological alterations, few studies have assessed the temporal pattern of regional apoptosis following mild brain injury. Anesthetized rats were subjected to mild lateral fluid-percussion brain injury (1.1–1.3 atm), and brains were evaluated for the presence of in situ DNA fragmentation (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling, TUNEL) and morphologic characteristics of apoptotic cell death (nuclear and cytoplasmic condensation, presence of apoptotic bodies). Significant numbers of apoptotic TUNEL(+) cells were observed in the injured parietal cortex and underlying white matter up to 72 h post-injury (P<0.05 compared to sham-injured-injured), with maximal numbers present at 24 h. Apoptosis was confirmed by the presence of 180–200 bp nuclear DNA fragments in tissue homogenates. The appearance of apoptotic TUNEL(+) cells in the injured cortex was preceded by a marked decrease in immunoreactivity for the anti-cell death protein, Bcl-2, as early as 2 h post-injury. This decrease in cellular Bcl-2 staining was not accompanied by a concomitant loss of staining for the pro-cell death Bax protein, suggesting that post-traumatic neuronal death in the cortex may be dependent on altered cellular ratios of Bcl-2:Bax. In the hippocampus, no significant increase in apoptotic TUNEL(+) cells was observed compared to sham-injured-injured animals. However, selective neuronal loss was evident in the CA3 region at 24 h post-injury, that was preceded by an overt loss of neuronal Bcl-2 immunoreactivity at 6 h. No changes in either cellular Bcl-2 or Bax expression were observed in the thalamus or white matter at any time post-injury.

Taken together from these data, we suggest that apoptosis contributes to cell death in both gray and white matter, and that decreases in cellular Bcl-2 may, in part, be associated with both apoptotic and non-apoptotic cell death following mild brain trauma.     

Inhibition of cerebellar granule cell turning by alcohol

Ectopic neurons are often found in the brains of fetal alcohol spectrum disorders (FASD) and fetal alcohol syndrome (FAS) patients, suggesting that alcohol exposure impairs neuronal cell migration. Although it has been reported that alcohol decreases the speed of neuronal cell migration, little is known about whether alcohol also affects the turning of neurons. Here we show that ethanol exposure inhibits the turning of cerebellar granule cells in vivo and in vitro. First, in vivo studies using P10 mice demonstrated that a single intraperitoneal injection of ethanol not only reduces the number of turning granule cells but also alters the mode of turning at the EGL–ML border of the cerebellum. Second, in vitro analysis using microexplant cultures of P0–P3 mouse cerebella revealed that ethanol directly reduces the frequency of spontaneous granule cell turning in a dose-dependent manner. Third, the action of ethanol on the frequency of granule cell turning was significantly ameliorated by stimulating Ca²⁺ and cGMP signaling or by inhibiting cAMP signaling. Taken together, these results indicate that ethanol affects the frequency and mode of cerebellar granule cell turning through alteration of the Ca²⁺ and cyclic nucleotide signaling pathways, suggesting that the abnormal allocation of neurons found in the brains of FASD and FSA patients results, at least in part, from impaired turning of immature neurons by alcohol.     

Deeply located granule cells and mitral cells undergo apoptosis after transection of the central connections of the main olfactory bulb in the adult rat

The main olfactory bulb (MOB) is the first relay station of the olfactory system: it receives afferents from sensory neurons and sends efferents to the primary olfactory cortex. The MOB also receives many centrifugal afferents from various regions. Transection of peripheral afferents to the MOB has been reported to induce cell death in granule cells. However, little is known about the effect of transection of these central connections of the MOB in adult rats. Here, we used a unilateral olfactory peduncle transection model in the adult rat to examine neuronal degeneration in the MOB. In the MOB ipsilateral to the surgery, the granule cell layer (GCL) was smaller, and the number of mitral cells was decreased compared with the contralateral MOB at 7 days after surgery. Many degenerating cells were present in both the mitral cell layer (MCL) and GCL in the ipsilateral MOB at 3 days after surgery, although there were no obvious changes in the gross morphology. We also found terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labeling (TUNEL)-positive cells in the MCL and GCL in the ipsilateral MOB at 3 days after surgery. The majority of the degenerating and TUNEL-positive cells were located in the deep, rather than the superficial, GCL. Immunohistochemistry for activated caspase-9 further supported the occurrence of apoptotic cell death in the mitral and deeply located granule cells. These results indicate that not only axotomized mitral cells, but also deeply located granule cells that were not directly injured, underwent apoptosis after transection of the central connections, and suggest that sensitivities to transection of the central connections differ among granule cells according to their depth in the GCL.     

3-Amino thioacridone,a selective cyclin-dependent kinase 4 inhibitor,attenuates kainic acid-induced apoptosis in neurons

The mechanisms underlying selective neuronal cell death in kainic acid-mediated neurodegeneration are not fully understood. We have recently demonstrated that in cerebellar granule neurons, kainic acid induces the expression of proteins associated with cell-cycle progression. In the present study we show that 3-amino thioacridone (3-ATA), a selective cyclin-dependent kinase 4 inhibitor, attenuates kainic acid-induced apoptosis in cerebellar granule neurons. When neurons were pre-treated with 3-ATA 10 microM for 24 h, they were less susceptible to damage induced by kainic acid 500 microM, since the number of dead cells decreased significantly. In flow cytometry studies using propidium iodide staining, 3-ATA also reduced the ratio of apoptotic cells induced by kainic acid. Moreover, 3-ATA decreased the proportion of cells with a condensed nucleus from 55% to 22%. Our data suggest that the cell cycle pathway is involved in the mechanism of apoptosis mediated by kainic acid and that cyclin-dependent kinase 4 plays a prominent role in this process. 3-ATA may to prevent the apoptosis associated with neurodegenerative disorders without the over-activation of excitatory amino acid receptors.     

TUNEL方法结合图像分析技术检测缺血诱导的神经元损伤

目的 缺血性神经元损伤的性质(凋亡或坏死)是神经研究领域的热点.但目前用于区分凋亡与坏死细胞的TUNEL技术,较易受主观因素的影响.方法 本实验运用图像分析技术对缺血大鼠脑的TUNEL染色切片进行多视场多参数形态定量分析,即细胞形态定量参数(核面积、周长、直径、形状因子)和含量光密度(平均光密度、积分光密度).结果 局灶性脑缺血可引起神经元凋亡和坏死.尾壳核以发生凋亡为主,且凋亡神经元的出现先于坏死神经元.缺血0.5h,尾壳核开始出现凋亡神经元.缺血3h达高峰.缺血6h,出现选择性细胞丢失.结论 图像分析技术能客观而准确地鉴别1:TUNEL染色切片上神经元损伤的性质,由此为神经领域的研究提供了一种敏感准确的形态学定量方法.     

Bax, reactive oxygen, and cytochrome c release in neuronal apoptosis

Half of all neurons produced during embryogenesis undergo apoptotic death shortly before birth or soon thereafter. Two cell culture models have been used extensively to investigate the cellular and molecular mechanisms underlying apoptosis during neuronal development: (a) sympathetic neurons deprived of their required neurotrophic factor, nerve growth factor, and (b) cerebellar granule neurons deprived of serum in low-potassium medium. A dramatic increase in mitochondrial-derived reactive oxygen species (ROS) occurs during the apoptotic death of both of these cell types. These ROS lie downstream from the proapoptotic protein, Bax. Bax normally resides in the cytoplasm, but translocates to the outer mitochondrial membrane during apoptosis. Once associated with mitochondria, Bax causes release of apoptogenic factors from the mitochondria into the cytoplasm, thus inducing or augmenting the apoptotic cascade. Although there is much controversy about the exact mechanism by which Bax causes release of these factors, recent evidence suggests that the Bax-induced ROS are critical for this release to occur in both sympathetic and cerebellar granule neurons. Because Bax is critical for the apoptotic death of many other types of neurons, it is likely that increased ROS is important for the death of these cells as well.     

Cell death after dorsal root injury

Flow cytometry and terminal deoxynucleotidyl transferase-mediated biotinylated uridine triphosphate nick end-labelling (TUNEL) immunohistochemistry have been used to assess cell death in the dorsal root ganglia (DRG) or spinal cord 1, 2 or 14 days after multiple lumbar dorsal root rhizotomy or dorsal root avulsion injury in adult rats. Neither injury induced significant cell death in the DRG compared to sham-operated or naïve animals at any time point. In the spinal cord, a significant increase in death was seen at 1–2 days, but not 14 days, post injury by both methods. TUNEL staining revealed that more apoptotic cells were present in the dorsal columns and dorsal horn of avulsion animals compared to rhizotomised animals. This suggests that avulsion injury, which can often partially damage the spinal cord, has more severe effects on cell survival than rhizotomy, a surgical lesion which does not affect the spinal cord. The location of TUNEL positive cells suggests that both neuronal and non-neuronal cells are dying.     

Histochemical characterization of lectin binding in mouse cerebellum

Eight fluorescein-isothiocyanate-conjugated lectins differing from each other in carbohydrate binding specificities have been used to reveal qualitative differences in carbohydrate composition among the various cell types of the early postnatal and adult cerebellar cortex, cerebellar deep nuclei and medulla. By treating unfixed frozen tissue sections with unconjugated lectin in the presence of excess hapten sugar and subsequently staining with fluorescein-isothiocyanate-conjugated lectin in the presence of bovine serum albumin, carbohydrate specific staining was observed for all of the lectins studied. Lectin binding was selectively inhibited by appropriate hapten sugars. For several lectins (wheat germ agglutinin, Ricinus communis agglutinins and Lens culinaris agglutinins) complex carbohydrates were required for inhibition of staining.In the adult cerebellum, concanavalin A intensely stained the soma of granule cells, synaptic glomeruli and the cytoplasm of Purkinje cells, while the molecular layer and white matter were relatively unstained. Wheat germ agglutinin heavily labeled the soma of granule cells, synaptic glomeruli, parallel fibers of the molecular layer, and the nuclear membrane of Purkinje cells, and weakly labeled the white matter. Ricinus communis agglutins (m.w. 60,000 and 120,000) intensely stained the soma of granule cells, synaptic glomeruli, parallel fibers in the molecular layer, the cytoplasm of Purkinje cells, and the white matter. None of the above lectins stained Purkinje cell dendrites. Lens culinaris lectins A and B stained the soma of granule cells, synaptic glomeruli, the cytoplasm of Purkinje cells, Purkinje cell dendrites, and white matter.In the 5- and 12-day-old postnatal cerebellum, concanavalin A stained the molecular layer more strongly than in the adult. Concanavalin A, wheat germ agglutinin and Lens culinaris lectins moderately stained cell cytoplasm in the external and internal granule layers, but the external granule cell layer was heavily labeled with both Ricinus communis lectins. With the soybean agglutinin, weak staining of the molecular layer and synaptic glomeruli was detected in young cerebellum, in contrast to adult tissue where no staining was observed. No labeling of the young or adult cerebellum was observed with Ulex europaeus agglutinin I.The specific patterns of staining with different lectins probably reflect the different carbohydrate compositions of the various cell types and perhaps of the intercellular matrix.     

Detection of high molecular weight DNA fragments characteristic of early stage apoptosis in cerebellar granule cells exposed to glutamate

In cerebellar granule cells a rapid necrotic cell death has been observed during and immediately after glutamate exposure, followed by a delayed apoptotic type of neuronal death in a subpopulation of the surviving neurons. In some experimental models the DNA fragmentation characteristic of apoptosis is readily detected. In other systems apoptosis may occur only in a limited number of cells, rendering DNA fragmentation undetectable using conventional DNA-staining techniques (e.g., ethidium bromide). We have used a sensitive and non-radioactive method for labeling, detection, and quantification of high molecular weight (HMW) DNA fragments. This method is based on the introduction of thymine dimers into DNA after separation by pulse field gel electrophoresis, followed by detection with thymine dimer specific antibodies. Applying this method to cerebellar granule cells in culture, we detected an increase in the amount of HMW DNA fragments characteristic of apoptosis as early as 4 h after glutamate exposure. The N-methyl-D-aspartic acid (NMDA)-receptor antagonist MK801 protected against the fragmentation, whereas no protection was observed using the non-NMDA-receptor antagonist CNQX.     

Mechanisms underlying reorganization of fractured tactile cerebellar maps after deafferentation in developing and adult rats

Our previous studies showed that fractured tactile cerebellar maps in rats reorganize after deafferentation during development and in adulthood while maintaining a fractured somatotopy. Several months after deafferentation of the infraorbital branch of the trigeminal nerve, the missing upper lip innervation is replaced in the tactile maps in the granule cell layer of crus IIa. The predominant input into the denervated area is always the upper incisor representation. This study examined whether this reorganization was caused by mechanisms intrinsic to the cerebellum or extrinsic, i.e., occurring in somatosensory structures afferent to the cerebellum. We first compared normal and deafferented maps and found that the expansion of the upper incisor is not caused by a preexisting bias in the strength or abundance of upper incisor input in normal animals. We then mapped tactile representations before and immediately after denervation. We found that the pattern of reorganization observed in the cerebellum several months later is not caused by unmasking of a silent or weaker upper incisor representation. Both results indicate that the reorganization is not a result of subsequent growth or sprouting mechanism within the cerebellum itself. Finally, we compared postlesion maps in the cerebellum and the somatosensory cortex. We found that the upper incisor representation significantly expands in both regions and that this expansion is correlated, suggesting that reorganization in the cerebellum is a passive consequence of reorganization in afferent cerebellar pathways. This result has important developmental and functional implications.     

An enhanced expression of the immediate early gene, Egr-1, is associated with neuronal apoptosis in culture.

Cultured cerebellar granule cells grown in medium containing 10 mM K+ (K10) underwent apoptosis after four to five days in vitro, unless they were rescued by the addition of insulin-like growth factor-I. The few GABAergic neurons present in the cultures were more resistant to apoptotic degeneration, as indicated by double fluorescent staining with the chromatin dye Hoechst 33258 and with glutamate decarboxylase-67 antibodies. As compared with sister cultures grown in 25 mM K+, K10 cultures showed an increased expression of the Egr-1 protein and a reduced expression of the Fos protein. The increase in Egr-1 was more substantial in granule cells than in GABAergic neurons, and was not observed in K10 cultures chronically exposed to insulin-like growth factor-I. To examine the temporal relationship between the increase in Egr-1 and the development of programmed cell death, we induced apoptosis in K25 cultures at six days in vitro by replacing their medium with serum-free K10 medium. A substantial, but transient, increase in Egr- expression was observed in granule cells 6 h after switching the medium, a time that preceded the appearance of the phenotypical markers of apoptotic death. An early reduction in the Fos protein was observed after switching the medium from K25 into serum-free K10, but also after switching the medium into serum-free K25, a condition which was not associated with the development of apoptosis nor with the increase in Egr-1. We suggest that a transient induction of Egr-1 contributes to the chain of events leading to the execution phase of neuronal apoptosis in culture.