Glial Cell Processes Research Articles

Dynamics of glutamatergic signaling in the mushroom body of young adult Drosophila

BackgroundThe mushroom bodies (MBs) are paired brain centers located in the insect protocerebrum involved in olfactory learning and memory and other associative functions. Processes from the Kenyon cells (KCs), their intrinsic neurons, form the bulk of the MB's calyx, pedunculus and lobes. In young adult Drosophila, the last-born KCs extend their processes in the α/β lobes as a thin core (α/β cores) that is embedded in the surrounding matrix of other mature KC processes. A high level of L-glutamate (Glu) immunoreactivity is present in the α/β cores (α/βc) of recently eclosed adult flies. In a Drosophila model of fragile X syndrome, the main cause of inherited mental retardation, treatment with metabotropic Glu receptor (mGluR) antagonists can rescue memory deficits and MB structural defects.ResultsTo address the role of Glu signaling in the development and maturation of the MB, we have compared the time course of Glu immunoreactivity with the expression of various glutamatergic markers at various times, that is, 1 hour, 1 day and 10 days after adult eclosion. We observed that last-born α/βc KCs in young adult as well as developing KCs in late larva and at various pupal stages transiently express high level of Glu immunoreactivity in Drosophila. One day after eclosion, the Glu level was already markedly reduced in the α/βc neurons. Glial cell processes expressing glutamine synthetase and the Glu transporter dEAAT1 were found to surround the Glu-expressing KCs in very young adults, subsequently enwrapping the α/β lobes to become distributed equally over the entire MB neuropil. The vesicular Glu transporter DVGluT was detected by immunostaining in processes that project within the MB lobes and pedunculus, but this transporter is apparently never expressed by the KCs themselves. The NMDA receptor subunit dNR1 is widely expressed in the MB neuropil just after eclosion, but was not detected in the α/βc neurons. In contrast, we provide evidence that DmGluRA, the only Drosophila mGluR, is specifically expressed in Glu-accumulating cells of the MB α/βc immediately and for a short time after eclosion.ConclusionsThe distribution and dynamics of glutamatergic markers indicate that newborn KCs transiently accumulate Glu at a high level in late pupal and young eclosed Drosophila, and may locally release this amino acid by a mechanism that would not involve DVGluT. At this stage, Glu can bind to intrinsic mGluRs abundant in the α/βc KCs, and to NMDA receptors in the rest of the MB neuropil, before being captured and metabolized in surrounding glial cells. This suggests that Glu acts as an autocrine or paracrine agent that contributes to the structural and functional maturation of the MB during the first hours of Drosophila adult life.

Differential distribution of aggrecan isoforms in perineuronal nets of the human cerebral cortex

Aggrecan is a component of the CNS extracellular matrix (ECM) and we show here that the three primary alternative spliced transcripts of the aggrecan gene found in cartilage are also present in the adult CNS. Using a unique panel of core protein-directed antibodies against human aggrecan we further show that different aggrecan isoforms are deposited in perineuronal nets (PNNs) and neuropil ECM of Brodmann’s area 6 of the human adult cerebral cortex. According to their distribution pattern, the identified cortical aggrecan isoforms were subdivided into five clusters spanning from cluster 1, comprised isoforms that appeared widespread throughout the cortex, to cluster 5, which was an aggrecan-free subset. Comparison of brain and cartilage tissues showed a different relative abundance of aggrecan isoforms, with cartilage-specific isoforms characterizing cluster 5, and PNN-associated isoforms lacking keratan sulphate chains. In the brain, isoforms of cluster 1 were disclosed in PNNs surrounding small-medium interneurons of layers II–V, small-medium pyramidal neurons of layers III and V and large interneurons of layer VI. Aggrecan PNNs enveloped both neuron bodies and neuronal processes, encompassing pre-terminal nerve fibres, synaptic boutons and terminal processes of glial cells and aggrecan was also observed in continuous ‘coats’ associated with satellite, neuron-associated cells of a putative glial nature. Immunolabelling for calcium-binding proteins and glutamate demonstrated that aggrecan PNNs were linked to defined subsets of cortical interneurons and pyramidal cells. We suggest that in the human cerebral cortex, discrete, layer-specific PNNs are assembled through the participation of selected aggrecan isoforms that characterize defined subsets of cortical neurons.

The principal neurons of the medial nucleus of the trapezoid body and NG2+ glial cells receive coordinated excitatory synaptic input

Glial cell processes are part of the synaptic structure and sense spillover of transmitter, while some glial cells can even receive direct synaptic input. Here, we report that a defined type of glial cell in the medial nucleus of the trapezoid body (MNTB) receives excitatory glutamatergic synaptic input from the calyx of Held (CoH). This giant glutamatergic terminal forms an axosomatic synapse with a single principal neuron located in the MNTB. The NG2 glia, as postsynaptic principal neurons, establish synapse-like structures with the CoH terminal. In contrast to the principal neurons, which are known to receive excitatory as well as inhibitory inputs, the NG2 glia receive mostly, if not exclusively, α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptor–mediated evoked and spontaneous synaptic input. Simultaneous recordings from neurons and NG2 glia indicate that they partially receive synchronized spontaneous input. This shows that an NG2+ glial cell and a postsynaptic neuron share presynaptic terminals.

Myelination of the corpus callosum in male and female rats following complex environment housing during adulthood

Myelination is an important process in brain development, and delays or abnormalities in this process have been associated with a number of conditions including autism, developmental delay, attention deficit disorder, and schizophrenia. Myelination can be sensitive to developmental experience; however, although the adult brain remains highly plastic, it is unknown whether myelination continues to be sensitive to experience during adulthood. Male and female rats were socially housed until four months of age, at which time they were moved into either a complex or “enriched” environment (EC) or an isolated condition (IC). Although the area of the splenium (posterior 20% of the callosum, which contains axons from visual cortical neurons) increased by about 10% following two months of EC housing, the area occupied by myelinated axons was not influenced by adult housing condition. Instead, it was the area occupied by glial cell processes and unmyelinated axons which significantly increased following EC housing. Neither the size nor the myelin content of the genu (anterior 15% of the callosum) was sensitive to manipulations of adult housing condition, but males had more area occupied by myelinated axons in both callosal regions. Finally, the inability of two months of complex environment housing during adulthood to impact the number of myelinated axons in the splenium was confirmed in a subset of animals using quantitative electron microscopy. We conclude that the sensitivity of myelination to experience is reduced in adulthood relative to development in both sexes.

Brain edema after intracerebral hemorrhage in rats: the role of iron overload and aquaporin 4

Brain edema formation following intracerebral hemorrhage (ICH) appears to be partly related to erythrocyte lysis and hemoglobin release. An increase of brain water content was associated with an increase of brain iron, which is an erythrocyte degradation product. Expression of AQP4 is highly modified in several brain disorders, and it can play a key role in cerebral edema formation. However, the question whether AQP4 is regulated by drugs lacks reliable evidence, and the interacting roles of iron overload and AQP4 in brain edema after ICH are unknown. The goal of this study was to clarify the relationship between iron overload and AQP4 expression and to characterize the effects of the iron chelator deferoxamine (DFO) on delayed brain edema after experimental ICH. A total of 144 Sprague-Dawley rats weighing between 250 and 300 g were used in this work. The animals were randomly divided into 4 groups. The ICH models (Group C) were generated by injecting 100 microl autologous blood stereotactically into the right caudate nucleus; surgical control rats (Group B) were generated in a similar fashion, by injecting 100 microl saline into the right caudate nucleus. Intervention models (Group D) were established by intraperitoneal injection of DFO into rats in the ICH group. Healthy rats (Group A) were used for normal control models. Brain water content, iron deposition, and AQP4 in perihematomal brain tissue were evaluated over the time course of the study (1, 3, 7, and 14 days) in each group. Iron deposition was found in the perihematomal zone as early as the 1st day after ICH, reaching a peak after 7 days and remaining at a high level thereafter for at least 14 days following ICH. Rat brain water content around the hematoma increased progressively over the time course, reached its peak at Day 3, and still was evident at Day 7 post-ICH. Immunohistochemical analysis showed that AQP4 was richly expressed over glial cell processes surrounding microvessels in the rat brain; there was upregulation of the AQP4 expression in perihematomal brain during the observation period, and it reached maximum at 3 to 7 days after ICH. The changes of brain water content were accompanied by an alteration of AQP4. The application of the iron chelator DFO significantly reduced iron overload, brain water content, and AQP4 level in the perihematomal area compared with the control group. Iron overload and AQP4 may play a critical role in the formation of brain edema after ICH. In addition, AQP4 expression was affected by iron concentration. Importantly, treatment with DFO significantly reduced brain edema in rats and inhibited the AQP4 upregulation after ICH. Deferoxamine may be a potential therapeutic agent for treating ICH.

Subependymal giant cell astrocytoma (SEGA): Is it an astrocytoma? Morphological, immunohistochemical and ultrastructural study

Subependymal giant-cell astrocytoma (SEGA) is a rare intra-ventricular low-grade tumor which frequently occurs as a manifestation of tuberous sclerosis complex. The histogenesis of SEGA is controversial and its astrocytic nature has been doubted. First studies suggested the astrocytic nature of SEGA while several recent reports demonstrate its glio-neuronal nature. In spite of this, in the recently revised WHO classification of the CNS tumors, SEGA has been still included in the group of astrocytomas. We studied nine tuberous sclerosis complex-associated SEGAs. Patients were 1-18 years old. Eight patients (89%) had a solitary lesion located in the lateral ventricle close to of the head of the caudate nucleus, the remaining patient (11%) had two tumors, one located close to the head of the left caudate nucleus and the other in the central part of the right lateral ventricle. Histologically, tumors were composed of three types of cells: spindle, gemistocytic and ganglion-like. Four tumors (44%) had a prominent vascularization and three (33%) showed an angiocentric pattern. Calcifications were observed in six cases (66%). By immunohistochemistry, the majority of the tumors were GFAP- (9; 100%), neurofilament- (8, 89%), neuron-specific enolase- (9, 100%), and synaptophysin- (8; 89%) positive. Ultrastructural studies were performed on four cases. In all four there were glial cell processes filled with intermediate filaments. In one case dense core putative neurosecretory granules were appreciable. Our results emphasize the glio-neuronal nature of SEGA. We suggest moving it into the group of mixed glio-neuronal tumors under the denomination of subependymal giant cell tumor.

Stimulation of the inferior olivary complex alters the distribution of the type 1 corticotropin releasing factor receptor in the adult rat cerebellar cortex

In a previous study, it was shown that populations of climbing fibers, derived from the inferior olivary complex (IOC) contain the peptide corticotropin releasing factor (CRF) and that the expression of this peptide in climbing fibers could be modulated by the level of activity in olivary afferents. The intent of this study was to determine if there was comparable plasticity in the distribution of the type 1 CRF receptor (CRF-R1) in the cerebellum of the rat. Our results indicate that CRF-R1 was localized primarily to Purkinje cell somata and their primary dendrites and granule cells. In addition, scattered immunolabeling was present over the somata of Golgi cells, basket cells and stellate cells, as well as Bergmann glial cells and their processes. IOC stimulation for 30 min at 1 Hz increased CRF-R1 expression in molecular layer interneurons and processes of Bergmann glial cells. Little to no effect on CRF receptor distribution was observed in Purkinje cells, granule cells, or Golgi cells. IOC stimulation at 5 Hz however, increased CRF-R1 expression in the processes of Bergmann glial cells while decreasing its expression in basket, stellate and, to some extent, in Purkinje cells. The present results suggest that there is activity-dependent plasticity in CRF-R1 expression that must be considered in defining the mechanism by which the CRF family of peptides modulates activity in cerebellar circuits. The present results also suggest that the primary targets of CRF released from climbing fibers are Bergmann glial cells and interneurons in the molecular layer. Further, interneurons responded with a decrease in receptor expression following more intense levels of stimulation suggesting the possibility of internalization of the receptor. In contrast, Bergmann glial cells showed an increased expression in receptor expression. These data suggest that CRF released from climbing fibers may modulate the physiological properties of basket and stellate cells as well as having a heretofore unidentified and potentially unique effect on Bergmann glia.

Increased APLP1 expression and neurodegeneration in the frontal cortex of manganese‐exposed non‐human primates

Chronic manganese (Mn) exposure produces a neurological syndrome with psychiatric, cognitive, and parkinsonian features. Gene expression profiling in the frontal cortex of Cynomologous macaques receiving 3.3-5.0 mg Mn/kg weekly for 10 months showed that 61 genes were increased and four genes were decreased relative to controls from a total of 6766 genes. Gene changes were associated with cell cycle regulation, DNA repair, apoptosis, ubiquitin-proteasome system, protein folding, cholesterol homeostasis, axonal/vesicular transport, and inflammation. Amyloid-beta (Abeta) precursor-like protein 1, a member of the amyloid precursor protein family, was the most highly up-regulated gene. Immunohistochemistry confirmed increased amyloid precursor-like protein 1 protein expression and revealed the presence of diffuse Abeta plaques in Mn-exposed frontal cortex. Cortical neurons and white matter fibers from Mn-exposed animals accumulated silver grains indicative of on-going degeneration. Cortical neurons also exhibited nuclear hypertrophy, intracytoplasmic vacuoles, and apoptosis stigmata. p53 immunolabeling was increased in the cytoplasm of neurons and in the nucleus and processes of glial cells in Mn-exposed tissue. In summary, chronic Mn exposure produces a cellular stress response leading to neurodegenerative changes and diffuse Abeta plaques in the frontal cortex. These changes may explain the subtle cognitive deficits previously demonstrated in these same animals.

Inhibition of Müller glial cell division blocks regeneration of the light‐damaged zebrafish retina

The adult zebrafish retina possesses a robust regenerative response. In the light-damaged retina, Müller glial cell divisions precede regeneration of rod and cone photoreceptors. Neuronal progenitors, which arise from the Müller glia, continue to divide and use the Müller glial cell processes to migrate to the outer nuclear layer and replace the lost photoreceptors. We tested the necessity of Müller glial cell division for photoreceptor regeneration. As knockdown tools were unavailable for use in the adult zebrafish retina, we developed a method to conditionally inhibit the expression of specific proteins by in vivo electroporation of morpholinos. We determined that two separate morpholinos targeted against the proliferating cell nuclear antigen (PCNA) mRNA reduced PCNA protein levels. Furthermore, injection and in vivo electroporation of PCNA morpholinos immediately prior to starting intense light exposure inhibited both Müller glial cell proliferation and neuronal progenitor marker Pax6 expression. PCNA knockdown additionally resulted in decreased expression of glutamine synthetase in Müller glia and Müller glial cell death, while amacrine and ganglion cells were unaffected. Finally, histological and immunological methods showed that long-term effects of PCNA knockdown resulted in decreased numbers of Müller glia and the failure to regenerate rod photoreceptors, short single cones, and long single cones. These data suggest that Müller glial cell division is necessary for proper photoreceptor regeneration in the light-damaged zebrafish retina and are consistent with the Müller glia serving as the source of neuronal progenitor cells in regenerating teleost retinas.

Optomotor-blind expression in glial cells is required for correct axonal projection across the Drosophila inner optic chiasm

In the Drosophila adult visual system, photoreceptor axons and their connecting interneurons are tied into a retinotopic pattern throughout the consecutive neuropil regions: lamina, medulla and lobula complex. Lamina and medulla are joined by the first or outer optic chiasm (OOC). Medulla, lobula and lobula plate are connected by the second or inner optic chiasm (IOC). In the regulatory mutant In(1)omb H31 of the T-box gene optomotor-blind ( omb), fibers were found to cross aberrantly through the IOC into the neuropil of the lobula complex. Here, we show that In(1)omb H31 causes selective loss of OMB expression from glial cells within the IOC previously identified as IOC giant glia (ICg-glia). In the absence of OMB, ICg-glia retain their glial cell identity and survive until the adult stage but tend to be displaced into the lobula complex neuropil leading to a misprojection of axons through the IOC. In addition, adult mutant glia show an aberrant increase in length and frequency of glial cell processes. We narrowed down the onset of the IOC defect to the interval between 48 h and 72 h of pupal development. Within the 40 kb of regulatory DNA lacking in In(1)omb H31 , we identified an enhancer element (ombC) with activity in the ICg-glia. ombC-driven expression of omb in ICg-glia restored proper axonal projection through the IOC in In(1)omb H31 mutant flies, as well as proper glial cell positioning and morphology. These results indicate that expression of the transcription factor OMB in ICg-glial cells is autonomously required for glial cell migration and morphology and non-autonomously influences axonal pathfinding.

Diffuse glioma growth: a guerilla war

In contrast to almost all other brain tumors, diffuse gliomas infiltrate extensively in the neuropil. This growth pattern is a major factor in therapeutic failure. Diffuse infiltrative glioma cells show some similarities with guerilla warriors. Histopathologically, the tumor cells tend to invade individually or in small groups in between the dense network of neuronal and glial cell processes. Meanwhile, in large areas of diffuse gliomas the tumor cells abuse pre-existent “supply lines” for oxygen and nutrients rather than constructing their own. Radiological visualization of the invasive front of diffuse gliomas is difficult. Although the knowledge about migration of (tumor)cells is rapidly increasing, the exact molecular mechanisms underlying infiltration of glioma cells in the neuropil have not yet been elucidated. As the efficacy of conventional methods to fight diffuse infiltrative glioma cells is limited, a more targeted (“search & destroy”) tactic may be needed for these tumors. Hopefully, the study of original human glioma tissue and of genotypically and phenotypically relevant glioma models will soon provide information about the Achilles heel of diffuse infiltrative glioma cells that can be used for more effective therapeutic strategies.

Morphological Evidence for Direct Interaction Between Gonadotrophin‐Releasing Hormone Neurones and Astroglial Cells in the Human Hypothalamus

In rodents, there is compelling evidence indicating that dynamic cell-to-cell communications involving cross talk between astroglial cells (such as astrocytes and specialised ependymoglial cells known as tanycytes) and neurones are important in regulating the secretion of gonadotrophin-releasing hormone (GnRH), the neurohormone that controls both sexual maturation and adult reproductive function. However, whether such astroglial cell-GnRH neurone interactions occur in the human brain is not known. In the present study, we used immunofluorescence to examine the anatomical relationship between GnRH neurones and glial cells within the hypothalamus of five women. Double-staining experiments demonstrated the ensheathment of GnRH neurone perikarya by glial fibrillary acidic protein (GFAP)-immunoreactive astrocyte processes in the periventricular zone of the tuberal region of the hypothalamus. GFAP immunoreactivity did not overlap that of GnRH at the GnRH neurone's projection site (i.e. the median eminence of the hypothalamus). Rather, human GnRH neuroendocrine fibres were found to be closely associated with vimentin or nestin-immunopositive radial glial processes likely belonging to tanycytes. In line with these light microscopy data, ultrastructural examination of GnRH-immunoreactive neurones showed numerous glial cells in direct apposition to pre-embedding-labelled GnRH cell bodies and/or dendrites in the infundibular nucleus, whereas postembedding immunogold-labelled GnRH nerve terminals were often seen to be enwrapped by glial cell processes in the median eminence. GnRH nerve button were sometimes visualised in close proximity to fenestrated pituitary portal blood capillaries and/or evaginations of the basal lamina that delineate the pericapillary space. In summary, these data demonstrate that GnRH neurones morphologically interact with astrocytes and tanycytes in the human brain and provide evidence that glial cells may contribute physiologically to the process by which the neuroendocrine brain controls the function of GnRH neurones in humans.

Studies of host–graft interactions in vitro

Progenitor and stem cell transplantation represent therapeutic strategies for retinal disorders that are accompanied by photoreceptor degeneration. The transplanted cells may either replace degenerating photoreceptors or secrete beneficial factors that halt the processes of photoreceptor degeneration. The present study analyzes whether rat retinal progenitor cells differentiated into photoreceptor phenotypic cells in neurospheres have a potential to interact with rat retinal explants. Immunocytochemistry for rhodopsin and synaptophysin indicated photoreceptor cell-like differentiation in neurospheres that were stimulated by basic fibroblast growth factor and epidermal growth factor. Differentiation into neural phenotypes including photoreceptor cells was effectively blocked by an addition of leukemia inhibitory factor. Grafting of neurospheres onto retinal explants demonstrated a consistent penetration of glial cell processes into the explanted tissue. On the other hand, the incorporation of donor cells into explants was very low. A general finding was that neurospheres grafting was associated with local decrease in Müller cell activation in the explants. Further characterization of these effect(s) could provide further insight into progenitor cell-based therapies of retinal degenerative disorders.

Formation of striatum structural and ultrastructural organization in rat postnatal ontogenesis at changes of conditions of their embryonic development

Using light microscopy (Nissl and Golgi techniques), electron microscopy and immunohistochemistry, formation of structure of the brain striatum dorsolateral part from birth to three month of age was studied in rats submitted to acute hypoxia at the period of embryogenesis. Hypoxia at the 13.5th day of pregnancy (E 13.5) was found to lead to a delay of neuronogenesis for the first two weeks of postnatal development as compared with control animals, and the majority of large neurons for this period were degenerated by the type of chromatolysis with swelling of the cell body and processes and lysis of cytoplasmic organelles. By the end of the third week, shrunken hyperchromic or pycnomorphic neurons with the electron-dense cytoplasm and enlarged tubules of endoplasmic reticulum and Golgi complex were also observed. An increased number of swollen processes of glial cells was found in neuropil around the degenerating neurons. By the 30th day as well as in adult animals, destruction of mitochondrial apparatus, an increased number of lysosomes, and blade-shaped nuclei, which are characteristics of the apoptotic cell death, were observed. This is also confirmed by an increased expression of proapoptotic protein (p53) and its co-localization with caspase-3 in a part of neurons. Morphometric analysis showed a decrease of the cell distribution density in striatum and a change of ratio of different cell types in hypoxia-exposed rats as compared with control group. The most pronounced decrease (42.3% at the 5th day, 14.2% at the 10th day, p < 0.01) of the number of large neurons (larger than 80 μm2) was revealed at early stages of postnatal ontogenesis. After 3 postnatal weeks, the number of middle-sized neurons (30–95 μm2) decreased (by 11.8–19.2% as compared with control, p < 0.05). The obtained data have shown that changes of embryogenesis conditions (hypoxia) at the period of the most intensive proliferation of telencephalon neuroblasts lead to impairment of the process of striatal nervous tissue formation. This might be the cause of delay of development and disturbances of behavior and learning, which are observed in rats exposed to prenatal hypoxia.

Axo-somatic inhibition of projection neurons in the lateral nucleus of amygdala in human temporal lobe epilepsy: an ultrastructural study

Here, we report ultrastructural alterations in the synaptic circuitry of the human amygdala related to neuronal cell densities in surgical specimens of patients suffering from temporal lobe epilepsy (TLE). The neuronal cell densities quantified in the basolateral complex of amygdala were significantly reduced only in the lateral nucleus (LA) of TLE patients as compared to autopsy or non-Ammon's horn sclerosis (AHS) controls (Nissl staining, immunostaining against the neuronal marker NeuN). For this reason, we focussed on the LA to perform a more detailed quantitative ultrastructural analysis, which revealed an inverse correlation between the number of axo-somatic inhibitory synaptic profiles at the somata of glutamic acid decarboxylase (GAD)-negative projection neurons and the extent of perisomatic fibrillary gliosis. In contrast, the density of GAD-immunoreactive interneurons positively correlated with the number of axo-somatic inhibitory synaptic profiles. The fibrillary material in perisomatic glial cell processes was preferentially labeled by the astroglial marker S100B. In addition, a qualitative study of the dendrites of GAD- and parvalbumin (PARV)-containing interneurons showed that they were often contacted by asymmetrical excitatory synapses. Our results are in line with anatomical data from rodents and cats, which show that amygdalar interneurons form axo-somatic inhibitory synapses on GAD-negative projection neurons, whereas the interneurons themselves receive excitatory input from recurrent collaterals of projection neurons and from cortico- and thalamo-amygdalar afferents. The structural reorganization patterns observed in the GABAergic circuitry are compatible with a reduced feedback or feed forward inhibition of amygdalar projection neurons in human TLE.

Tracheal development in the Drosophila brain is constrained by glial cells

The Drosophila brain is tracheated by the cerebral trachea, a branch of the first segmental trachea of the embryo. During larval stages the cerebral trachea splits into several main (primary) branches that grow around the neuropile, forming a perineuropilar tracheal plexus (PNP) at the neuropile surface. Five primary tracheal branches whose spatial relationship to brain compartments is relatively invariant can be distinguished, although the exact trajectories and branching pattern of the brain tracheae are surprisingly variable. Immunohistochemical and electron microscopic studies demonstrate that all brain tracheae grow in direct contact with the glial cell processes that surround the neuropile. To investigate the effect of glia on tracheal development, embryos and larvae lacking glial cells as a result of a genetic mutation or a directed ablation were analyzed. In these animals, the tracheal branching pattern was highly abnormal. In particular, the number of secondary branches entering the central neuropile was increased. Wild-type larvae possess only two central tracheae, typically associated with the mushroom body and the antennocerebral tract. In larvae lacking glial cells, six to ten tracheal branches penetrate the neuropile in a variable pattern. This finding indicates that glia-derived signals constrained tracheal growth in the Drosophila brain and restrict the number of branches entering the neuropile.

Modulation of D-Serine Levels via Ubiquitin-dependent Proteasomal Degradation of Serine Racemase

Mammalian serine racemase is a brain-enriched enzyme that converts L- into D-serine in the nervous system. D-Serine is an endogenous co-agonist at the "glycine site" of N-methyl D-aspartate (NMDA) receptors that is required for the receptor/channel opening. Factors regulating the synthesis of D-serine have implications for the NMDA receptor transmission, but little is known on the signals and events affecting serine racemase levels. We found that serine racemase interacts with the Golgin subfamily A member 3 (Golga3) protein in yeast two-hybrid screening. The interaction was confirmed in vitro with the recombinant proteins in co-transfected HEK293 cells and in vivo by co-immunoprecipitation studies from brain homogenates. Golga3 and serine racemase co-localized at the cytosol, perinuclear Golgi region, and neuronal and glial cell processes in primary cultures. Golga3 significantly increased serine racemase steady-state levels in co-transfected HEK293 cells and primary astrocyte cultures. This observation led us to investigate mechanisms regulating serine racemase levels. We found that serine racemase is degraded through the ubiquitin-proteasomal system in a Golga3-modulated manner. Golga3 decreased the ubiquitylation of serine racemase both in vitro and in vivo and significantly increased the protein half-life in pulse-chase experiments. Our results suggest that the ubiquitin system is a main regulator of serine racemase and D-serine levels. Modulation of serine racemase degradation, such as that promoted by Golga3, provides a new mechanism for regulating brain d-serine levels and NMDA receptor activity.

Fine structure of neuronal and glial processes in neuropathology

The cells of the nervous system are characterized by their well-formed cell processes and by cell-to-cell relationships that they form. The neuron reveals essentially cylindrical processes, which form synaptic junctions. On the other hand, the peripheral parts of the glial cells are mainly sheet-like in nature. Thus, the oligodendroglial cell elaborates many sheet-like processes, each of which forms a segment of the myelin sheath. Unique cell junction, transverse bands are present at the interface of oligodendroglial processes and the axon. Finally, the astrocytes also form elaborate sheet-like processes, which separate most of the CNS from the mesodermal tissue as well as surrounding certain neuronal surfaces, including synapses. Punctate adhesions, gap junctions and other adhesive devices are present between astrocytic processes. Defects or anomalies in the neuronal and glial cell processes characterize numerous pathological conditions.

Expression of connexins 36, 43, and 45 during postnatal development of the mouse retina

Gap junction channels formed by connexins (Cx) may play essential roles in some processes that occur during retinal development, such as apoptosis and calcium wave spread. The present study was undertaken to determine the distribution pattern of Cx36, Cx43, and Cx45 by immunofluorescence, as well as their gene expression levels by quantitative PCR during postnatal development of the mouse retina. Our results showed an increased expression of neuronal Cx36 from P1 until P10, when this Cx reached adult levels, and it was mainly distributed in the outer and inner plexiform layers. In turn, Cx43 was almost absent in retinal progenitor cells at P1, it became more prominent in glial cell processes about P10, and did not change until adulthood. Double-labeling studies in situ and in vitro with antivimentin, a Müller cell marker, confirmed that Cx43 was expressed by these cells. In addition, quantitative PCR showed that Cx43 and vimentin shared very similar temporal expression patterns. Finally, in contrast to Cx36 and Cx43, Cx45 mRNA was strongly down-regulated during development. In early postnatal days, Cx45 was seen ubiquitously distributed throughout the retina in cells undergoing proliferation and differentiation, as well in differentiated neurons. In adult retina, this protein had a more restricted distribution both in neurons and glial cells, as confirmed in situ and in vitro. In conclusion, we observed a distinct temporal expression pattern for Cx36, Cx43, and Cx45, which is probably related to particular roles in retinal function and maintenance of homeostasis during development of the mouse retina.

Oligodendroglial Dysplasia in Two Bullmastiff Dogs

Leukodystrophies are inherited neurological disorders involving central nervous system white matter. They are uncommon in animals but a few, breed-specific entities have been described. In 2002, two young-adult, purebred Bullmastiff dogs from central New York State presented to their referring veterinarians displaying moderate to severe ataxia of all limbs, spastic tetraparesis that was worse in the pelvic limbs, and a diffuse, action-related, whole-body tremor. Clinical signs were insidious in onset and slowly progressive. Anatomic diagnoses considered were a C1-C5 lesion or, based on the whole-body tremor, a diffuse central nervous system disorder. No gross lesions were apparent in the brain or spinal cord. Histopathologically, numerous, multifocal, sharply demarcated, small, ovoid to angular areas of myelin pallor (plaques) were present throughout the major white matter tracts of the brainstem and spinal cord. These plaques, which often were traversed by axons, did not stain with luxol fast blue for myelin and were associated with minimal astrocytosis. Ultrastructural findings include occasional hypertrophic glia in white matter, rare unmyelinated segments of axons, and focal proliferation of tubule-containing cytoplasmic glial cell processes (oligodendroglial). The described clinical and morphological findings and age of onset are similar to the well-characterized, presumably hereditary, bovine syndrome known as Charolais ataxia or oligodendroglial dysplasia. This article presents the first description of a leukodystrophy in the Bullmastiff breed and the first report of oligodendroglial dysplasia in animals other than Charolais cattle.