Regional variations in the glial influence on synapse development in the mouse CNS

There is increasing evidence that synapse function depends on interactions with glial cells, namely astrocytes. Studies on specific neurons of the central nervous system (CNS) indicated that glial signals also control synapse development, but it remained unclear whether this is a general principle that applies to other neuronal cell types. To address this question, we developed new methods to immunoisolate neurons from different brain regions of postnatal mice and to culture them in a chemically defined medium. Electrophysiological recordings and immunocytochemical staining revealed vigorous synaptogenesis in hippocampal and cerebellar neurons, but not in retinal ganglion cells (RGCs) in the absence of glial cells. Co-culture with glia promoted synapse formation in RGCs as indicated by a strong increase in the incidence and frequency of action potential-independent miniature synaptic currents, but showed no such effects in hippocampal or cerebellar neurons. On the other hand, glial signals promoted the efficacy of excitatory synapses in all regions as indicated by an increase in the size of spontaneous synaptic events in cerebellar cultures and of miniature synaptic currents in hippocampal neurons and RGCs. Inhibitory synaptic currents remained largely unaffected by glia. Our results indicate that in the mammalian CNS, the way that glial signals promote the development of excitatory synapses depends on the type of neuron.     

S-laminin and N-acetylgalactosamine located at the synaptic basal lamina of skeletal muscle are involved in synaptic recognition by growing neurites

Summary The purpose of the work reported here is to identify molecular components of the synaptic basal lamina of skeletal muscle fibres which allow recognition of original synaptic sites by regenerating motor axons. We focused on s-larninin and components recognized by the lectinDolichos biflorus agglutinin previously shown to be specifically located at the synaptic basal lamina. We used a cryoculture bioassay in which chick ciliary ganglion neurons grow on rat skeletal muscle cryostat sections. In control cultures, neurites extended over the muscle sections in close association with the muscle cell surface. It was observed that most of the neurites that extended towards the endplate zone and reached an area of 40 m around the neuromuscular junction ceased to grow when they contacted the synaptic site. Masking either lectin receptors or some s-laminin molecule epitopes prior to the culture of neurons alters the behaviour of growing neurites. On sections treated either withDolichos biflorus agglutinin or anti s-laminin monoclonal antibodies (D5 and C4) most of the neurites did not stop their growth at the synaptic regions. Moreover, treating muscle sections withDolichos biflorus agglutinin removed the gradient of substratum affinity around the endplate. These results indicate that the s-laminin andDolichos biflorus agglutinin receptors present on muscle cell surfaces may play a functional role in the interaction of growing neurites with original synaptic sites in the process of neuromuscular regeneration.     

Application of glial fibrillary acidic protein immunohistochemistry in the quantification of astrocytes in the rat brain

Immunohistochemical staining for glial fibrillary acidic protein (GFAP) was employed as a tool for quantification of astrocytes in the rat brain. One-micron-methacrylate sections were prepared from 70-μm slices stained for GFAP by using a preembedding staining procedure. Numbers/unit area of astrocytes and nonastrocytes were determined for cortex, corpus callosum, and hippocampal neuropil. In each, counts from GFAP-stained, toluidine-blue-counterstained sections were compared with counts obtained from sections stained with toluidine blue alone. Numbers of nonastrocytes and total glia in all three regions were comparable in both groups of sections. Astrocyte counts in the cortex and hippocampus also showed no significant differences between the two groups. In contrast, the number of astrocytes in the corpus callosum was significantly lower in GFAP-stained, toluidine-blue-counterstained sections than in sections stained with toluidine blue alone. GFAP immunohistochemistry is a useful tool for the quantification of astrocytes in semithin plastic sections of rat brain.     

The reorganization of synaptic connexions in the rat submandibular ganglion during post-natal development.

1. The innervation of neurones in the submandibular ganglion of neonatal and adult rats has been studied with intracellular recording, and light and electron microscopy. 2. Intracellular recordings from neurones in isolated ganglia from adult animals showed that about 75% of the ganglion cells are innervated by a single preganglionic fibre. 3. However multiple steps in the post-synaptic potential (about five on average) were elicited in ganglion cells from neonatal animals by graded stimulation of the preganglionic nerve. The same result was obtained when the preganglionic fibres were stimulated at their emergence from the brainstem, indicating that neonatal neurones are innervated by several different preganglionic nerve cells. 4. The number of preganglionic fibres innervating individual ganglion cells gradually decreased during the first few weeks of life, and by about 5 weeks each ganglion cell was generally contacted by a single preganglionic axon. 5. Synapses were made on short protuberances in the immediate vicinity of the neuronal cell bodies in both neonatal and adult ganglia as shown by staining presynaptic boutons with the zinc-iodide osmium method, injection of horseradish peroxidase into ganglion cells, and electron microscopical examination. 6. Electron microscopical counts of synaptic profiles per ganglion cell perimeter showed that the number of synaptic contacts made on ganglion cells actually increased during the first few post-natal weeks, when the number of axons innervating each neurone was decreasing. 7. These results show that in the rat submandibular ganglion there is a reorganization of neuronal connexions during the first few weeks of life which results in a transition from multiple to generally single innervation of ganglion cells.     

Immunohistochemical demonstration of glial fibrillary acidic protein in normal rat Müller glia and retinal astrocytes

The presence of glial fibrillary acidic protein (GFA)-positive Müller glia and retinal astrocytes were studied immunohistochemically in normal rat retina. Using GFA antiserum both Müller glia and separate star-shaped cells were observed in spread-preparations as well as cryostat sections. The retinal astrocytes were also visualized using two different monoclonal GFA antibodies. These cells were found to be located in the nerve fiber and ganglion cell layers. In contrast, Müller glia were not normally visualized with any of the monoclonal GFA antibodies but could be stained 4 days after an optic nerve crush. Our results demonstrate that normal rat Müller glia expresses GFA-like immunoreactivity.     

Regional heterogeneity in the distal motor axon: three zones with distinctive intrinsic components

Summary In this study we examined the distribution of cytoskeletal and other intrinsic axonal elements in motor nerve terminalsin vivo. Components of axons were visualized with immunocytochemical staining of frozen longitudinal sections of muscle. Using these methods we compared the distribution of neurofilaments, tubulin, MAP2, actin and synaptic elements in distal regions of axons at and near neuromuscular junctions in rat muscles. Our results show that three discrete regions can be defined based on the anatomy and intrinsic components of distal axons. The preterminal axon, extending from its exit from the intramuscular nerve toward the neuromuscular junction, has a cytoskeletal composition similar to the more proximal axon with abundant staining of neurofilaments, tubulin, MAP2 and actin. The terminal arborization, a branched region of the axon extending through the endplate region, contains neurofilaments but little tubulin, actin, MAP2 or synaptic elements. Finally, the synaptic zone, demonstrated with antibodies to the synaptic vesicle protein synaptophysin, contains few cytoskeletal elements. We conclude that there is considerable regional heterogeneity in the composition of distal motor axons. The distribution of neurofilaments, other cytoskeletal elements and synaptic vesicle proteins varies among different discrete zones of terminal motor axons.     

Choline uptake in cholinergic nodose cell bodies

Isolated living cell bodies were obtained by mechanical and enzymatic dissociation from adult rabbit nodose ganglion followed by separation of fibres and cells using a Percoll gradient. A purification yield of 45% was measured. Based on previous results obtained in whole ganglion and showing the presence of cholinergic cell bodies among the afferent fibres of the vagus nerve, this preparation was used to study choline uptake by neuron cell somata. Cholinergic cells counted after choline acetyltransferase immunohistological staining showed a stained population of 2.9% among the isolated population. Two [3H]choline uptake mechanisms were detected at the cell body level. The first, with Km1 = 7 microM and Vm1 = 200 pmol/h per ganglion is sodium dependent, related to acetylcholine synthesis (43%) and has an IC50 with hemicholinium-3 equal to 50 microM. The second, with Km2 = 54 microM and Vm2 = 2235 pmol/h per ganglion is sodium independent, poorly associated to acetylcholine synthesis (12%) and exhibits an IC50 of 2 microM with hemicholinium-3. Except for their sensitivity to hemicholinium-3, the high and low affinity choline uptake mechanisms observed at the somatic level have, respectively, the same characteristics as the high and low affinity mechanisms described at the synaptic level. Their physiological role, their opposed sensitivity to hemicholinium-3 compared to the synaptic uptake systems and the relation between the somatic high affinity choline transport and an acetylcholine somatic release are discussed.     

Rapid and modifiable neurotransmitter receptor dynamics at a neuronal synapse in vivo

Synaptic plasticity underlies the adaptability of the mammalian brain, but has been difficult to study in living animals. Here we imaged the synapses between pre- and postganglionic neurons in the mouse submandibular ganglion in vivo, focusing on the mechanisms that maintain and regulate neurotransmitter receptor density at postsynaptic sites. Normally, synaptic receptor densities were maintained by rapid exchange of receptors with nonsynaptic regions (over minutes) and by continual turnover of cell surface receptors (over hours). However, after ganglion cell axons were crushed, synaptic receptors showed greater lateral mobility and there was a precipitous decline in insertion. These changes led to near-complete loss of synaptic receptors and synaptic depression. Disappearance of postsynaptic spines and presynaptic terminals followed this acute synaptic depression. Therefore, neurotransmitter receptor dynamism associated with rapid changes in synaptic efficacy precedes long-lasting structural changes in synaptic connectivity.     

Dopaminergic nerve endings in the neostriatum of the rat—2. Radioautographic study following local micro-injections of tritiated dopamine

Local micro-injections of tritiated dopamine were made into the nucleus caudatus-putarnen of rats in order to identify the dopamine-containing nerve endings by high-resolution radioautography and to correlate with our results obtained with micro-injections of 5-hydroxydopamine. The density of silver grains (number per square unit) over the different kinds of nervous elements and glia was studied in single sections, showing a rather weak accumulation over mitochondria and over numerous synaptic contacts (37%) which were of the asymmetric type. Labeling was subsequently studied using semi-serial ultra-thin sections. Only about 10% of the latter contacts remained labeled in two adjacent sections. Their presynaptic areas usually contained small crowded synaptic vesicles and belonged mainly to the Type III of Bak et al. (1975).This is in agreement with our previous findings which demonstrated that about 10% of the neostriatal synaptic contacts, belonging principally to the same type and concentrating 5-hydroxydopamine, can be considered as containing dopamine.     

Surface membrane staining of immunoglobulins in paraffin sections of non-Hodgkin's lymphomas using immunogold-silver staining technique.

The immunogold-silver staining (IGSS) method is a new immunostaining technique with much enhanced sensitivity for demonstration of antigens in paraffin sections. A series of 10 non-Hodgkin's lymphomas of B cell type were stained for surface membrane immunoglobulins by the IGSS and peroxidase-antiperoxidase (PAP) methods using paraffin sections and polyclonal primary antisera. The resulting staining patterns were compared with those obtained using frozen sections of the same tissues, monoclonal antibodies and the immunoperoxidase technique. The IGSS method gave a clear demonstration of surface membrane immunoglobulins in neoplastic lymphocytes using paraffin sections and the pattern of staining achieved was comparable to that obtained by the immunoperoxidase technique employing frozen sections and monoclonal antibodies. PAP staining of paraffin sections consistently failed to demonstrate the presence of any surface membrane immunoglobulin. The IGSS method provides a new approach to the diagnosis of B cell lymphomas in which routinely fixed and processed tissues may be employed to demonstrate monoclonality.     

Neurocircuitry of two types of neurotensin-containing amacrine cells in the turtle retina

The ultrastructural features and synaptic contacts of two types of neurotensin-containing amacrine cells in turtle retina were examined by electron immunocytochemistry, and the retinal peptides were characterized using radioimmunoassay and high-pressure liquid chromatography. The two types of cell were distinguished on the basis of their sizes, dendritic arborizations, synaptic connections and cytoplasmic staining characteristics. Type A cells had lightly labeled cytoplasm and large vertically elongated cell bodies which gave rise to a single primary process which in turn branched and ramified as smooth tapering processes in stratum 3 of the inner plexiform layer. Type A cells received approximately equal synaptic input from amacrine and bipolar cells. Type A amacrines had much more overall synaptic input than synaptic output, and they made conventional synaptic contacts onto bipolar, amacrine, and ganglion cells. Type B cells had a much darker-staining cytoplasm and a smaller cell body which gave rise to numerous delicate beaded dendrites which arborized in strata 3, 4 and 5 of the inner plexiform layer. Type B cells received primarily amacrine and some bipolar cell input. Type B cells had equal amounts of synaptic input and output and they made conventional synaptic contacts onto amacrine, bipolar, and ganglion cells. Whereas there were numerous large vesicles (120 nm diameter) that stained for neurotensin in both types of cells, conventional synaptic vesicles (60 nm diameter) were not labeled. In several cases these large labeled vesicles appeared to fuse with the cell membrane in non-synaptic regions and release their contents into extracellular space, which suggested a non-synaptic release of the neurotensin from type A neurons. Immunochemical and chromatographic studies demonstrated that the neurotensin-related material in retina was indistinguishable from neurotensin found in brain. These results are consistent with a neuroactive role for the neurotensin present within the large vesicles. The differences in the synaptic contacts and dendritic arborizations of the two amacrine cell types suggest they play distinctive functions in visual processing.     

Glial cell number and neuron/glial cell ratios in postmortem brains of bipolar individuals

BACKGROUND: There are several lines of evidence implicating a glial abnormality in the pathophysiology of bipolar disorder. Previous studies have reported a wide range of abnormalities but with little consistency between their findings. METHODS: Sixty frozen postmortem temporal cortical brain samples from normal, schizophrenic, bipolar, and depressed subjects were obtained from the Stanley Neuropathology Consortium. Nissl stain was utilized to visualize cellular structures. The sections were analyzed for neuron and glial content using the computer programs SPOT Advanced and Metamorph. Three representative fields from each subject were counted and the average numbers obtained. RESULTS: There was no critical difference in glia number across the four diagnostic groups. Compared with normal controls, area occupied by glia was reduced in bipolar subjects (P = 0.018), and the ratio of glial area to neuronal area was reduced (P = 0.028). DISCUSSION: The percent glial space was substantially lower in bipolar disorder subjects compared to normal controls, suggesting that the glia may be smaller in bipolar subjects. The size reduction may be reflective of glial dysfunction. LIMITATIONS: Postmortem interval of 29.4 +/- S.D. 13.4 h may have affected cellular structure and reduced the quality of the staining.     

Multifunctional features of a gastrodermal sensory cell in Hydra: three-dimensional study

Summary Computer-assisted, three-dimensional reconstructions of two gastrodermal sensory cells from transmission electron micrographs of serial sections ofHydra revealed a unipolar morphology with the nucleus near an apical cilium and a simple unbranched axon with a widened terminal. The sensory cells were similar in size and shape to a unipolar sensory cell isolated from macerated gastrodermis and examined with scanning electron microscopy. In thin sections, the cells were characterized by the presence of numerous dense-cored vesicles in the axon and its terminal. A few dense-cored vesicles were aligned at electron-dense synaptic foci in the axon terminal of the sensory cell, which formed an axo-axonal synapse with a nearby centrally located ganglion cell and a neuromuscular synapse with the basal myoneme of a digestive cell. The ganglion cell possessed a perikaryal cilium and a slender axon that extended adjacent to the sensory cell terminal, where it formed anen passant axo-axonal synapse in reciprocal arrangement with that of the sensory cell. In addition, the ganglion cell axon formed a neuromuscular synapse in sequence with the sensory cell axo-axonal synapse. The presence of a large number of neurosecretory-like granules, apical cilium and reciprocal interneuronal and neuromuscular synaptic loci suggests that this gastrodermal sensory cell, characterized ultrastructurally for the first time, represents a third type of multifunctional neuron inHydra. Thus,Hydra may contain primitive stem-like neurons, which are sensory-motor and also function in both neurosecretion and neurotransmission.     

Reorganization of intrinsic components in the distal motor axon during outgrowth

Summary We have studied the changes in the distribution of three intrinsic axonal components during the growth and maturation of sproutsin vivo. Neurofilaments, tubulin and synaptophysin, a synaptic vesicle protein, were visualized in motor axons and their sprouts using immunocytochemical staining of frozen longitudinal sections of muscle. We examined changes in these elements in sprouts regenerating after axonal crush injury and in those evoked from intact axons by denervation changes in muscle. Our results show that intrinsic axonal components move into newly formed motor axon sprouts in different temporal patterns. Based on the patterns of reorganization of staining of intrinsic axonal components, two types of outgrowth can be distinguished. One type, synaptic elaboration, is manifest by short, broad axonal processes that produce enlargement of the synaptic zone (synaptophysin staining) with little change in the distribution of intrinsic cytoskeletal elements. A second type of outgrowth, axonal elongation, occurs during axonal regeneration and ultraterminal sprouting and is longitudinal in form. In these sprouts there is a sequential appearance of neurofilament and then, several days later, tubulin immunostaining. Synaptophysin only accumulates in these sprouts after two weeks at points of synaptic contact with a muscle fibre.     

In situ hybridisation in herpetic lesions using a biotinylated DNA probe.

In situ hybridisation was performed with a biotinylated DNA probe for herpes simplex virus (HSV) using high temperature denaturation on formalin fixed, paraffin wax sections of lung, brain, ganglion and keratinising and non-keratinising squamous epithelia. Eosinophilic viral nuclear inclusions or characteristically moulded multiple nuclei with altered chromatin, which were present in two cases of HSV encephalitis and one case of viral pneumonitis, all showed complete hybridisation visualised by an alkaline phosphatase/nitroblue tetrazolium detector system. HSV encephalitis and trigeminal ganglionitis, which were confirmed serologically or clinicopathologically but lacked nuclear changes, also gave positive dense nuclear signal in neurons, glias and satellite cells. No staining was present in the ganglion cells in trigeminal zoster, the glia in progressive multifocal leucoencephalopathy, or in a variety of cells in a lung coinfected with cytomegalovirus. In 10 herpetic blisters of squamous epithelia, infected cells hybridised strongly, while morphologically similar herpes zoster lesions remained negative. In neural tissues non-hybridisation staining was most obtrusive in corpora amylacea and seemed to reflect nonspecific probe adherence. In squamous epithelium, major non-hybridisation staining was caused by probe and antibody possibly adhering to intracellular keratin. The HSV probe permits specific detection of virus in the absence of characteristic nuclear changes and allows varicella zoster virus to be differentiated from HSV, provided that the aforementioned problems with non-hybridisation staining are borne in mind.     

Sequential pictorial presentation of neural interaction in the retina. 2. The depolarizing and hyperpolarizing bipolar cells at rod terminals

Each rod is connected to one depolarizing and one hyperpolarizing bipolar cell. The synaptic connections of cone processes to each bipolar cell and presynaptically to the two rod-bipolar cell synapses establishes conditions for lateral interaction at this level. Thus, the cones raise the threshold for bipolar cell depolarization which is the basis for spatial brightness contrast enhancement and consequently for high visual acuity (Sj?strand, 2001a). The cones facilitate ganglion cell depolarization by the bipolar cells and cone input prevents horizontal cell blocking of depolarization of the depolarizing bipolar cell, extending rod vision to low illumination. The combination of reduced cone input and transient hyperpolarization of the hyperpolarizing bipolar cell at onset of a light stimulus facilitates ganglion cell depolarization extensively at onset of the stimulus while no corresponding enhancement applies to the ganglion cell response at cessation of the stimulus, possibly establishing conditions for discrimination between on- vs. off-signals in the visual centre. Reduced cone input and hyperpolarization of the hyperpolarizing bipolar cell at onset of a light stimulus accounts for Granit's (1941) 'preexcitatory inhibition'. Presynaptic inhibition maintains transmitter concentration low in the synaptic gap at rod-bipolar cell and bipolar cell-ganglion cell synapses, securing proportional and amplified postsynaptic responses at these synapses. Perfect timing of variations in facilitatory and inhibitory input to the ganglion cell confines the duration of ganglion cell depolarization at onset and at cessation of a light stimulus to that of a single synaptic transmission.     

Constant and flickering light stimulations produce similar effects on RNA content in visual cells.

Adult male rats were illuminated for 2 h with a constant or flickering light of 40 Lx intensity; frequency of flickering was 2 Hz. By means of two-wave-length visible cytospectrophotometry of gallocyanin-stained sections, it was shown that the light stimulation resulted in a marked RNA accumulation in retina ganglion neurons and in the neurons of all the cell layers of visual cortex (with the only exception of the layer VI). In the cells of perineuronal glia, a decrease of the RNA content per cell was found in the retina while no changes were observed in the visual cortex. Effects of constant and flickering light stimulations were qualitatively and quantitatively similar.     

NKCC1 does not accumulate chloride in developing retinal neurons

GABA excites immature neurons due to their relatively high intracellular chloride concentration. This initial high concentration is commonly attributed to the ubiquitous chloride cotransporter NKCC1, which uses a sodium gradient to accumulate chloride. Here we tested this hypothesis in immature retinal amacrine and ganglion cells. Western blotting detected NKCC1 at birth and its expression first increased, then decreased to the adult level. Immunocytochemistry confirmed this early expression of NKCC1 and localized it to all nuclear layers. In the ganglion cell layer, staining peaked at P4 and then decreased with age, becoming undetectable in adult. In comparison, KCC2, the chloride extruder, steadily increased with age localizing primarily to the synaptic layers. For functional tests, we used calcium imaging with fura-2 and chloride imaging with 6-methoxy-N-ethylquinolinium iodide. If NKCC1 accumulates chloride in ganglion and amacrine cells, deleting or blocking it should abolish the GABA-evoked calcium rise. However, at P0-5 GABA consistently evoked a calcium rise that was not abolished in the NKCC1-null retinas, nor by applying high concentrations of bumetanide (NKCC blocker) for long periods. Furthermore, intracellular chloride concentration in amacrine and ganglion cells of the NKCC1-null retinas was approximately 30 mM, same as in wild type at this age. This concentration was not lowered by applying bumetanide or by decreasing extracellular sodium concentration. Costaining for NKCC1 and cellular markers suggested that at P3, NKCC1 is restricted to Müller cells. We conclude that NKCC1 does not serve to accumulate chloride in immature retinal neurons, but it may enable Müller cells to buffer extracellular chloride.     

Regional specialization of the quail retina: ganglion cell density and oil droplet distribution

The ganglion cell density of the quail's retina was studied in sections and whole mounts. Two regions of high ganglion cell density were found, corresponding to an afoveate area centralis and an area dorsalis. Oil droplets were found to be isotropically distributed throughout the retina. It is proposed that the significance of such retinal regional specialization, in comparison to similar studies in the pigeon and the chick, is that regional specialization in the avian retina is more closely related to feeding habits than to phylogenetic descendence.