Schwann cell-neuron relationships in spinal cord gray matter

Schwann cells develop within the ventral gray matter following exposure of lumbosacral spinal cords to x-rays in early postnatal rats. These ventral gray matter Schwann cell aggregates occurred in about 40% of the animals 8 or more weeks following irradiation. Light microscopically these cells appeared to be apposed to somata of large motor neurons, raising a question regarding the fate of axo-somatic synapses. This study focused on neuron-Schwann cell relationships and demonstrated ultrastructurally that the intraspinal Schwann cells established a variety of relationships with the neuronal somata and primary dendrites. These relationships ranged from direct contact without an intervening basal lamina to the presence of synaptic contacts intervening between neuron and Schwann cell basal lamina. Occasionally, the Schwann cells occupied an intermediate position between neurons and blood vessels, suggesting functions similar to those carried out by astrocytes. In these instances, as in all cases of Schwann cell-blood vessel contact, the vessels lacked their normal investiture by astrocytes. Light microscopic evaluation of synaptophysin-immunostained sections revealed decreased immunoreactivity in neuropil occupied by the Schwann cells but confirmed the presence of synapses on neuronal somata. Possible mechanisms underlying Schwann cell induction in the ventral gray matter are discussed. An understanding of the interactions between Schwann cells and the cellular constituents of the gray matter is important in light of attempts to enhance repair in the central nervous system by transplanting Schwann cells into that environment. © 1996 Wiley-Liss, Inc.     

Synaptic Contacts and the Transient Dendritic Spines of Developing Retinal Ganglion Cells

The dendrites of ganglion cells in the mammalian retina become extensively remodelled during synapse formation in the inner plexiform layer. In particular, after birth in the cat, many short spiny protrusions are lost from the dendrites of ganglion cells during the time when ribbon, presumably bipolar, synapses appear in the inner plexiform layer and when conventional, presumed amacrine, synapses increase significantly in number. It has therefore been postulated that these transient spines may be the initial or preferred substrates for competitive interactions between amacrine or bipolar cell terminals that subsequently result in the formation of appropriate synapses onto the ganglion cells. If so, the majority of synapses made onto developing ganglion cells should be found on these dendritic spines. To test this hypothesis, we determined the synaptic connectivity of identified ganglion cells in the postnatal cat retina during the period of peak spine loss and synapse formation. The dendritic trees of ganglion cells were intracellularly filled with Lucifer yellow that was subsequently photo-oxidized into an electron-dense product suitable for electron microscopy. In serial reconstructions of the dendrites of a postnatal day 11 (P11) alpha ganglion cell and a P14 beta ganglion cell, conventional and ribbon synapses were found predominantly on dendritic shafts. Only three out of a total of 341 dendritic spines from the two cells received direct presynaptic input, all of which were conventional synapses. Thus, our observations suggest that the transient dendritic spines are not the preferred postsynaptic sites as previously suspected. However, it is possible that these structures play a different role in synaptogenesis, such as mediating interactions between retinal neurons that may lead to cell-cell recognition, a necessary step prior to synapse formation at the appropriate target sites (Cooper and Smith, Soc. Neurosci. Abstr. , 14 , 893, 1988).     

Glial modulation of synaptic transmission in the retina

Glial modulation of synaptic transmission and neuronal excitability in the mammalian retina is mediated by several mechanisms. Stimulation of glial cells evokes Ca(2+) waves, which propagate through the network of retinal astrocytes and Müller cells and result in the modulation of the activity of neighboring ganglion cells. Light-evoked spiking is enhanced in some ganglion cells and depressed in others. A facilitation or depression of light-evoked excitatory postsynaptic currents is also seen in ganglion cells following glial stimulation. In addition, stimulation of glial cells evokes a sustained hyperpolarizing current in ganglion cells which is mediated by ATP release from Müller cells and activation of neuronal A(1) adenosine receptors. Recent studies reveal that light-evoked activity in retinal neurons results in an increase in the frequency of Ca(2+) transients in Müller cells. Thus, there is two-way communication between neurons and glial cells, suggesting that glia contribute to information processing in the retina.     

Development of the human cervical spinal cord with reference to synapse formation in the motro nucleus

Synaptic development in the motor neuropil of the cervical spinal cord was quantitatively studied by light and electron microscopy in human embryos and fetuses ranging from five to 19 weeks of ovulation age. The numbers of axodendritic synapses increase substantially at the end of the eighth week. However, axosomatic synapses rapidly proliferate between 10.5 and 13 weeks of ovulation age. The increases in both types of synapses generally coincide with the behavioral chages in human fetuses that have been reported by other investigators. Only synaptic boutons containing spherical vesicles (S-type synapses) were found in the motor neuropil throughout the stages examined; no synapses with flattened vesicles (F-type synapses) were encountered. The majority of these synaptic boutons contain only a small number of synaptic vesicles (fewer than 20), although the number tends to increase with maturation. There is no significant maturational change in the size of synaptic vesicles. The present study suggests that synapse formation in the motor neuropil of the human fetus cervical spinal cord may continue up to 19 weeks of ovulation age because immature types of synapse are found in all fetuses. Myelin formation probably begins by the 11th week.     

Maturation of postsynaptic nicotinic structures on autonomic neurons requires innervation but not cholinergic transmission

Postsynaptic development at the neuromuscular junction depends on nicotinic transmission and secreted components from the presynaptic motor nerve terminal. Similarly, secreted components and synaptic activity are both thought to guide development of glutamatergic synapses in the CNS. Nicotinic synapses on chick ciliary neurons are structurally complex: a large presynaptic calyx engulfs the postsynaptic neuron and overlays a series of discrete mats of receptor-rich somatic spines tightly interwoven and folded against the soma. We used fluorescence imaging of alpha 7-containing nicotinic receptors and the spine constituent drebrin to monitor postsynaptic development. The results show that surgical disruption of the preganglionic input or removal of the ganglionic synaptic target tissue after synapses form in the ganglion does not disrupt the receptor-rich spine mats. Similarly, removal of the target tissue even prior to synapse formation in the ganglion does not prevent subsequent formation of the receptor clusters and associated spine constituents. Postsynaptic development is arrested, however, if normal innervation is prevented by ablating the preganglionic neurons prior to synapse formation. In this case the neurons express reduced levels of nicotinic receptors and cytoskeletal components and organize them only into early-stage clusters. Even low levels of residual innervation, however, can restore much of the normal postsynaptic receptor patterns. Chronic pharmacological blockade of cholinergic synaptic activity fails to replicate the effects of ablating the preganglionic nucleus. The results indicate that ciliary neurons are programmed to express postsynaptic components and can initiate clustering of alpha 7-containing receptors but need presynaptic guidance for maturation of the postsynaptic structure.     

Astrocyte lineage cells are essential for functional neuronal differentiation and synapse maturation in human iPSC-derived neural networks

Human astrocytes differ dramatically in cell morphology and gene expression from murine astrocytes. The latter are well known to be of major importance in the formation of neuronal networks by promoting synapse maturation. However, whether human astrocyte lineage cells have a similar role in network formation has not been firmly established. Here, we investigated the impact of human astrocyte lineage cells on the functional maturation of neural networks that were derived from human induced pluripotent stem cells (hiPSCs). Initial in vitro differentiation of hiPSC-derived neural progenitor cells and immature neurons (glia+ cultures) resulted in spontaneously active neural networks as indicated by synchronous neuronal Ca²⁺ transients. Depleting proliferating neural progenitors from these cultures by short-term antimitotic treatment resulted in strongly astrocyte lineage cell-depleted neuronal networks (glia− cultures). Strikingly, in contrast to glia+ cultures, glia− cultures did not exhibit spontaneous network activity. Detailed analysis of the morphological and electrophysiological properties of neurons by patch clamp recordings revealed reduced dendritic arborization in glia− cultures. In addition, a reduced action potential frequency upon current injection in pyramidal-like neurons was observed, whereas the electrical excitability of multipolar neurons was unaltered. Furthermore, we found a reduced dendritic density of PSD95-positive excitatory synapses, and more immature properties of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) miniature excitatory postsynaptic currents (mEPSCs) in glia− cultures, suggesting that the maturation of glutamatergic synapses depends on the presence of hiPSC-derived astrocyte lineage cells. Intriguingly, addition of the astrocyte-derived synapse maturation inducer cholesterol increased the dendritic density of PSD95-positive excitatory synapses in glia− cultures.     

Aminergic modulation of graded synaptic transmission in the lobster stomatogastric ganglion

Graded chemical synaptic transmission is important for establishing the motor patterns produced by the pyloric central pattern generator (CPG) circuit of the lobster stomatogastric ganglion (Raper, 1979; Anderson and Barker, 1981; Graubard et al., 1983). We examined the modulatory effects of the amines dopamine (DA), serotonin (5-HT), and octopamine (Oct) on graded synaptic transmission at all the central chemical synapses made by the pyloric dilator (PD) neuron onto its follower cells, using synaptic input-output curves measured from cell somata. DA strongly reduced the graded synaptic strength at all the PD synapses. DA reduction of chemical synaptic strength from PD onto the inferior cardiac (IC) neuron could change the sign of synaptic interaction between these 2 cells from inhibitory to excitatory by uncovering a weak electrical connection. 5-HT had weaker and more variable effects, reducing graded synaptic strength from the PD onto the lateral pyloric and pyloric neurons and enhancing the weak synapse from the PD to the IC cell. Oct strongly enhanced the graded synaptic strength at all the PD central synapses. Oct enhancement of graded synaptic strength between the PD and IC cells could also change the sign of the interaction: weak, excitatory electrical coupling, which was sometimes dominant before Oct, was masked by the enhanced chemical inhibitory interaction during Oct application. Measurements of electrical coupling between 2 PD cells and between 2 postsynaptic cells suggest that Oct does not change the input resistance of these cells and may act directly at the PD synapses. The effects of DA and 5-HT are most easily explained by their general reductions in pre- and postsynaptic input resistance. DA, 5-HT, and Oct each produce a distinct pyloric motor pattern (Flamm and Harris-Warrick, 1986a). These amine-induced motor patterns may be explained by the unique actions of each amine on the intrinsic membrane properties of different pyloric CPG neurons (Flamm and Harris-Warrick, 1986b) and by modulation of graded synaptic transmission between the pyloric neurons.     

Interneuronal synapses formed by motor neurons appear to be glutamatergic

Acetylcholine release at motor neuron synapses has been long established; however, recent discoveries indicate that synaptic transmission by motor neurons is more complex than previously thought. Using whole-cell patch clamp, we show that spontaneous excitatory postsynaptic currents of rat motor neurons in primary ventral horn cultures are entirely glutamatergic, although the cells respond to exogenous acetylcholine. Motor neurons in cultures express the vesicular glutamate transporter VGlut2, and culturing motor neurons for weeks with glutamate receptors blocked upregulates glutamate signaling without increasing cholinergic signaling. In spinal cord slices, motor neurons showed no decrease in spontaneous excitatory synaptic potentials after blocking acetylcholine receptors. Our results suggest that motor neuron synapses formed on other neurons are largely glutamatergic in culture and the spinal cord.     

Retinal neurons lack an acetylcholine receptor aggregating factor

Spinal cord neurons form stable synapses on muscle cells in culture, whereas retinal neurons, an inappropriate presynaptic partner for muscle cells, form synapses that are transient. We have hypothesized that a trophic influence of neurons on muscle is involved in the stabilization of synapses. Because other neural tissues that form stable synapses on muscle cells contain factors that aggregate acetylcholine receptors (AChR) into clusters on the surface of muscle cells, it may be that these aggregation factors are necessary for stabilization of neuron-muscle synapses. Therefore, we determined the AChR-aggregating activity of retinal neurons. The results showed that cocultures of retinal neurons and muscle cells and retinal-conditioned medium do not show increases in the number of AChR on muscle cells. Conversely, spinal cord-muscle cocultures and spinal cord-conditioned medium produce increases in the number of AChR clusters. These data, along with previous studies demonstrating that retinal neurons are unable to affect the electrical membrane properties of cultured muscle cells, whereas spinal cord neurons do elicit such changes, add support to the above hypothesis of a trophic influence of neurons in synapse stabilization.     

Astrocytes are crucial for survival and maturation of embryonic hippocampal neurons in a neuron‐glia cell‐insert coculture assay

Synapses represent specialized cell–cell contact sites between nerve cells. These structures mediate the rapid and efficient transmission of signals between neurons and are surrounded by glial cells. Previous investigations have shown that astrocytes are important for the formation, maintenance, and function of CNS synapses. To study effects of glial‐derived molecules on synaptogenesis, we have established an in vitro cell‐insert coculture system for E18 rat hippocampal neurons and various glial cell types. Neurons were cultured without direct contact with glial cells for distinct time periods. First, it was confirmed that astrocytes are essential to promote survival of E18 hippocampal neurons. Beginning with 10 days in culture, the concurrent expression of pre‐ and postsynaptic proteins was observed. Moreover, the colocalization of the presynaptic marker Bassoon and the postsynaptic protein ProSAP1/Shank2 indicated the formation of synapses. A technique was developed that permits the semiautomated quantitative determination of the number of synaptic puncta per neuron. The culture system was used to assess effects of pharmacological treatments on synapse formation by applying blockers and activators of small GTPases. In particular, treatment with lysophosphatidic acid enhanced synaptogenesis in the coculture system. Synapse 65:41–53, 2011. © 2010 Wiley‐Liss, Inc.     

Transplanted astrocytes reduce synaptic density in the neuropil of cerebellar cultures

Cytosine arabinoside-treated neonatal mouse cerebellar cultures, devoid of granule cells and mature glia, demonstrate heterologous synapses between sprouted Purkinje cell recurrent axon collaterals and dendritic spines in the neuropil. Such cultures were transplanted with optic nerve as a source of glia, and the effect on neuropil synapses was investigated. There was a significant reduction in the number of synapses in the neuropil and an increase in the number of free dendritic spines. Many of these spines occurred in clusters, unapposed by glial processes. The effect on the synapse density was not due to a comparable increase in the area occupied by the added astrocytes or an increase in nerve terminal diameter. The results suggest that astrocytes alter the density of neuropil synapses and may also induce the sprouting of dendritic spines.     

胚胎脊髓细胞悬液联合自体许旺细胞联合移植损伤脊髓空洞中的突触发育

目的 观察分析胚胎脊髓细胞悬液在损伤脊髓移植区中的突触发育过程。方法42只Wistar成年大鼠以改良Allen法(50g/cm)打击脊髓,3天后将孕14天(E14)FSCS 20μl植入损伤空腔,移植后2、4、6、8、10和12周,以光、电镜、免疫组织化学观察移植物成活、分化及其与宿主之间关系。结果 移植区成神经细胞最先展示了胞质突起,随之出现了低电子密度的突触前后膜,突触前、后膜电子密度逐渐增高形成良好的致密突起。突触小泡数量和种类逐渐增多,突触小泡有圆型清亮小泡、椭圆形小泡、颗粒状小泡和把扁平小泡-f型。突触的连接方式由单个的胞体-树突突触,出现多个的胞体-树突和树突-树突突触。同时,移植成神经细胞、成少突胶质细胞、成星形细胞的细胞器日渐完善,细胞功能活跃。血脑屏障也随之出现。移植区可见NF、5—HT、CGRP、GFAP阳性纤维。结论 ①胚胎脊髓细胞悬液在成年大鼠损伤脊髓内可发育为成熟的突触; ②显示了FSCS 与宿主脊髓重建突触方式的信息交换的潜在可行性。     

Chronic Pharmacological Increase of Neuronal Activity Improves Sensory-Motor Dysfunction in Spinal Muscular Atrophy Mice

Dysfunction of neuronal circuits is an important determinant of neurodegenerative diseases. Synaptic dysfunction, death, and intrinsic activity of neurons are thought to contribute to the demise of normal behavior in the disease state. However, the interplay between these major pathogenic events during disease progression is poorly understood. Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by a deficiency in the ubiquitously expressed protein SMN and is characterized by motor neuron death, skeletal muscle atrophy, as well as dysfunction and loss of both central and peripheral excitatory synapses. These disease hallmarks result in an overall reduction of neuronal activity in the spinal sensory-motor circuit. Here, we show that increasing neuronal activity by chronic treatment with the FDA-approved potassium channel blocker 4-aminopyridine (4-AP) improves motor behavior in both sexes of a severe mouse model of SMA. 4-AP restores neurotransmission and number of proprioceptive synapses and neuromuscular junctions (NMJs), while having no effects on motor neuron death. In addition, 4-AP treatment with pharmacological inhibition of p53-dependent motor neuron death results in additive effects, leading to full correction of sensory-motor circuit pathology and enhanced phenotypic benefit in SMA mice. Our in vivo study reveals that 4-AP-induced increase of neuronal activity restores synaptic connectivity and function in the sensory-motor circuit to improve the SMA motor phenotype.SIGNIFICANCE STATEMENT Spinal muscular atrophy (SMA) is a neurodegenerative disease, characterized by synaptic loss, motor neuron death, and reduced neuronal activity in spinal sensory-motor circuits. However, whether these are parallel or dependent events is unclear. We show here that long-term increase of neuronal activity by the FDA-approved drug 4-aminopyridine (4-AP) rescues the number and function of central and peripheral synapses in a SMA mouse model, resulting in an improvement of the sensory-motor circuit and motor behavior. Combinatorial treatment of pharmacological inhibition of p53, which is responsible for motor neuron death and 4-AP, results in additive beneficial effects on the sensory-motor circuit in SMA. Thus, neuronal activity restores synaptic connections and improves significantly the severe SMA phenotype.     

Quantitation of synaptic, neuronal and glial development in the intermediate and medial hyperstriatum ventrale (IMHV) of the chick Gallus domesticus, pre- and post-hatch

A quantitative analysis was made of the development of synapses, neurons and glia in both left and right intermediate and medial hyperstriatum ventrale (IMVH) of the forebrain of the domestic chick, Gallus domesticus from 16 days in ovo to 9 days post-hatch. There was a marked increase in total synapse numerical density (NVsyn), from 10 synapses per 100 microns3 at 16 days in ovo to 50 synapses per 100 microns3 at 9 days post-hatch; no significant left/right hemispheric differences were evident but there were differences between the development profiles for synapses with asymmetric as compared to symmetrical synaptic junctions. In contrast to synaptic development, the number of neurons per unit volume, NVneu, decreased by approximately 50% from the value at 16 days in ovo to that at day 0 (hatching). The neuronal population density remained unchanged to 3 days post-hatch and then, in 9-day-old birds, declined to almost a quarter of the original population size; no significant left/right hemisphere differences were evident. Glial cells declined in number from 16 days in ovo to 19 days in ovo, and then gradually increased to 9 days post-hatch. When the synapse to neuron ratio was examined, a trend was observed of a gradual increase with age, but no hemispheric differences were present. It is concluded that these changes are major events which must be considered in experiments aimed at determining the effects of behavioural, and/or environmental manipulations, on the morphology of the IMHV because they may mask other, more subtle, structural changes that occur as a result of behavioural experiences.     

Schwann cell guidance of nerve growth between synaptic sites explains changes in the pattern of muscle innervation and remodeling of synaptic sites following peripheral nerve injuries

Terminal Schwann cells (SCs) are nonmyelinating glia that are a prominent component of the neuromuscular junction (NMJ) where motor neurons form synapses onto muscle fibers. These cells play important roles not only in development and maintenance of the neuromuscular synapse but also restoring synaptic function after nerve damage. In response to muscle denervation, terminal SCs undergo dramatic changes in their gene expression patterns as well as in their morphology, such as extending elaborate processes into inter-junctional space. These SC processes serve as a path to guide axon terminal sprouts from nearby innervated junctions, promoting rapid reinnervation of denervated fibers. We studied the role of terminal SCs in synapse reformation by using two different fluorescent proteins to simultaneously label motor axons and SCs; we examined these junctions repeatedly in living animals using a fluorescence microscope. Here, we show that alterations in the patterns of muscle innervation following recovery from nerve injury can be explained by SC guidance of regenerating axons. In turn, this guidance leads to remodeling of the NMJ itself.     

Astrocyte‐mediated synapse remodeling in the pathological brain

Astrocytes, a major type of glia, reciprocally influence synaptic transmission and connectivity, forming the “tripartite synapses”. Astrocytic metabotropic glutamate receptor (mGluR)‐mediated Ca²⁺ waves and release of gliotransmitters or synaptogenic molecules mediate this neuron‐glia interaction in the developing brain, but this signaling has been challenged for adult brain. However, cumulative evidence has suggested that mature astrocytes exhibit re‐awakening of such immature phenotype in the pathological adult brain. This phenotypic change in astrocytes in response to injury may induce neural circuit and synapse plasticity. In this review article, we summarize astrocyte‐mediated synapse remodeling during physiological development, discuss re‐emergence of immature astrocytic signaling in adult pathological brain, and finally highlight its contribution to significant modification of synaptic connections correlating with functional progress of brain pathology.     

Intraretinal differentiation in the synaptic organization of the inner plexiform layer of the pigeon retina

The inner plexiform layer at ten retinal loci in pigeon was examined by electron microscopy. Photomontages of the entire depth of the inner plexiform layer at each locus were analyzed with respect to the number of amacrine and bipolar synapses, their respective ratios, synaptic densities, percent amacrine synapses in serial configuration, synaptic layering patterns, and the effect of staining procedures on these quantities. The results show that the pigeon retina is not homogeneous regarding the structural complexity of the inner plexiform layer, but may be divided into four general areas in decreasing order of complexity: red field, temporal yellow field, nasal yellow field, and the area centralis. Significant differences in the amacrine synapse to bipolar synapse ration and amacrine synaptic density were observed across the retina, while bi-polar synaptic density and the percent of serial synapses were rather constant. Amacrine synapses displayed a layering pattern which was consistent throughout the retina; while bipolar synapses showed two patterns. It was further observed that the density of amacrine and bipolar synapses bears little relationship to the density of amacrine and bipolar cells in the immediately overlying inner nuclear layer. This suggests that the various retinal loci may be characterized by different proportions of the morphological types of amacrine and bipolar cells present in the pigeon retina. Based on recent studies which have shown that a relationship exists between the complexity of ganglion cell receptive fields and the synaptic complexity of the inner plexiform layer, it is suggested that the ganglion cells of pigeon would show a physiological differentiation among retinal loci consistent with the observed differences in the anatomical complexity of the inner plexiform layer.     

Tripartite synapses: glia, the unacknowledged partner.

According to the classical view of the nervous system, the numerically superior glial cells have inferior roles in that they provide an ideal environment for neuronal-cell function. However, there is a wave of new information suggesting that glia are intimately involved in the active control of neuronal activity and synaptic neurotransmission. Recent evidence shows that glia respond to neuronal activity with an elevation of their internal Ca2+ concentration, which triggers the release of chemical transmitters from glia themselves and, in turn, causes feedback regulation of neuronal activity and synaptic strength. In view of these new insights, this article suggests that perisynaptic Schwann cells and synaptically associated astrocytes should be viewed as integral modulatory elements of tripartite synapses.