A hallmark of the central nervous system is its spatial and functional organization in synaptic layers. During neuronal development, axons form transient contacts with potential post-synaptic elements and establish synapses with appropriate partners at specific layers. These processes are regulated by synaptic cell-adhesion molecules. In the Drosophila visual system, R7 and R8 photoreceptor subtypes target distinct layers and form en passant pre-synaptic terminals at stereotypic loci of the axonal shaft. A leucine-rich repeat transmembrane protein, Capricious (Caps), is known to be selectively expressed in R8 axons and their recipient layer, which led to the attractive hypothesis that Caps mediates R8 synaptic specificity by homophilic adhesion. Contradicting this assumption, our results indicate that Caps does not have a prominent role in synaptic-layer targeting and synapse formation in Drosophila photoreceptors, and that the specific recognition of the R8 target layer does not involve Caps homophilic axon-target interactions. We generated flies that express a tagged synaptic marker to evaluate the presence and localization of synapses in R7 and R8 photoreceptors. These genetic tools were used to assess how the synaptic profile is affected when axons are forced to target abnormal layers by expressing axon guidance molecules. When R7 axons were mistargeted to the R8-recipient layer, R7s either maintained an R7-like synaptic profile or acquired a similar profile to r8s depending on the overexpressed protein. When R7 axons were redirected to a more superficial medulla layer, the number of presynaptic terminals was reduced. These results indicate that cell-surface molecules are able to dictate synapse loci by changing the axon terminal identity in a partially cell-autonomous manner, but that presynapse formation at specific sites also requires complex interactions between pre- and post-synaptic elements.
Cell adhesion is the fundamental driving force that establishes complex cellular architectures, with the nervous system offering a striking, sophisticated case study. Developing neurons adhere to neighboring neurons, their synaptic partners, and to glial cells. These adhesive interactions are required in a diverse array of contexts, including cell migration, axon guidance and targeting, as well as synapse formation and physiology. Forward and reverse genetic screens in the fruit fly Drosophila have uncovered several adhesion molecules that are required for neural development, and detailed cell biological analyses are beginning to unravel how these factors shape nervous system connectivity. Here we review our current understanding of the most prominent of these adhesion factors and their modes of action.
drosophila; cell adhesion; nervous system; glia; axon; synapse
Glial cells play important roles in the developing brain during axon fasciculation, growth cone guidance, and neuron survival. In the Drosophila brain, three main classes of glia have been identified including surface, cortex, and neuropile glia. While surface glia ensheaths the brain and is involved in the formation of the blood-brain-barrier and the control of neuroblast proliferation, the range of functions for cortex and neuropile glia is less well understood. In this study, we use the nirvana2-GAL4 driver to visualize the association of cortex and neuropile glia with axon tracts formed by different brain lineages and selectively eliminate these glial populations via induced apoptosis. The larval central brain consists of approximately 100 lineages. Each lineage forms a cohesive axon bundle, the secondary axon tract (SAT). While entering and traversing the brain neuropile, SATs interact in a characteristic way with glial cells. Some SATs are completely invested with glial processes; others show no particular association with glia, and most fall somewhere in between these extremes. Our results demonstrate that the elimination of glia results in abnormalities in SAT fasciculation and trajectory. The most prevalent phenotype is truncation or misguidance of axon tracts, or abnormal fasciculation of tracts that normally form separate pathways. Importantly, the degree of glial association with a given lineage is positively correlated with the severity of the phenotype resulting from glial ablation. Previous studies have focused on the embryonic nerve cord or adult specific compartments to establish the role of glia. Our study provides, for the first time, an analysis of glial function in the brain during axon formation and growth in larval development.
glia; Drosophila; brain; lineage
An important goal of contemporary neuroscience research is to define the neural circuits and synaptic interactions that mediate behavior. In both mammals and Drosophila, the neuronal circuitry controlling circadian behavior has been the subject of intensive investigation, but roles for glial cells in the networks controlling rhythmic behavior have only begun to be defined in recent studies.
Here, we show that conditional, glial-specific genetic manipulations affecting membrane (vesicle) trafficking, the membrane ionic gradient or calcium signaling lead to circadian arrhythmicity in adult behaving Drosophila. Correlated and reversible effects on a clock neuron peptide transmitter (PDF) and behavior demonstrate the capacity for glia-to-neuron signaling in the circadian circuitry. These studies also reveal the importance of a single type of glial cell – the astrocyte – and glial internal calcium stores in the regulation of circadian rhythms.
This is the first demonstration in any system that adult glial cells can physiologically modulate circadian neuronal circuitry and behavior. A role for astrocytes and glial calcium signaling in the regulation of Drosophila circadian rhythms emphasizes the conservation of cellular and molecular mechanisms that regulate behavior in mammals and insects.
Cell-cell adhesion molecules play key roles at the intercellular junctions of a wide variety of cells, including interneuronal synapses and neuron-glia contacts. Functional studies suggest that adhesion molecules are implicated in many aspects of neural network formation, such as axon-guidance, synapse formation, regulation of synaptic structure and astrocyte-synapse contacts. Some basic cell biological aspects of the assembly of junctional complexes of neurons and glial cells resemble those of epithelial cells. However, the neuron specific junctional machineries are required to exert neuronal functions, such as synaptic transmission and plasticity. In this review, we describe the distribution and function of cell adhesion molecules at synapses and at contacts between synapses and astrocytes.
synapses; cell adhesion molecules; cadherin superfamily; immunoglobulin superfamily; nerve tissue proteins; axons
Astroglia secrete factors that promote synapse formation and maintenance. In culture, glial contact has also been shown to facilitate synaptogenesis. Here, we examined whether glial contact is important for establishing circuits in vivo by simultaneously monitoring differentiation of glial cells and local synaptogenesis over time. Photoreceptor circuits of the vertebrate retina are particularly suitable for this study because of the relatively simple, laminar organization of their connectivity with their target neurons, horizontal cells (HCs) and bipolar cells. Also, individual photoreceptor terminals are ensheathed within the outer plexiform layer (OPL) by the processes of one type of glia, Müller glia (MG). We conducted in vivo time-lapse multiphoton imaging of the rapidly-developing and relatively transparent zebrafish retina to ascertain the time course of MG development relative to OPL synaptogenesis. The emergence of synaptic triads, indicative of functional photoreceptor circuits, and structural association with glial processes were also examined across ages by electron microscopy. We first show that MG processes form territories that tile within the inner and outer synaptic layers. We then demonstrate that cone photoreceptor synapses are assembled before the elaboration of MG processes in the OPL. Using a targeted cell ablation approach, we also determined whether the maintenance of photoreceptor synapses is perturbed when local MG are absent. We found that removal of MG had no appreciable effect on the stability of newly formed cone synapses. Thus, in contrast to other CNS circuits, contact from glia is not necessary for the formation or immediate stabilization of outer retinal synapses.
retina; glia; photoreceptor; live-cell imaging; horizontal cell; synaptogenesis
Tripartite (three-part) synapses are defined by physical and functional interactions of glia with pre- and post-synaptic elements. Although tripartite synapses are thought to be of widespread importance in neurological health and disease, we are only beginning to develop an understanding of glial contributions to synaptic function. In contrast to studies of neuronal mechanisms, a significant limitation has been the lack of an invertebrate genetic model system in which conserved mechanisms of tripartite synapse function may be examined through large-scale application of forward genetics and genome-wide genetic tools. Here we report a Drosophila tripartite synapse model which exhibits morphological and functional properties similar to those of mammalian synapses, including glial regulation of extracellular glutamate, synaptically-induced glial calcium transients and glial coupling of synapses with tracheal structures mediating gas exchange. In combination with classical and cell-type specific genetic approaches in Drosophila, this model is expected to provide new insights into the molecular and cellular mechanisms of tripartite synapse function.
In recent years it has becoming clear that glial cells of the central and peripheral nervous system play a crucial role from the earliest stages of development throughout adult life. Glial cells are important for neuronal plasticity, axonal conduction and synaptic transmission. In this respect, glial cells are able to produce, uptake and metabolize many factors that are essential for neuronal physiology, including classic neurotransmitters and neuroactive steroids. In particular, neuroactive steroids, which are mainly synthesized by glial cells, are able to modulate some neurotransmitter receptors affecting both glia and neurons. Among the signaling systems that are specialized for neuron-glial communication, we can include neurotransmitter GABA.
The main focus of this review is to illustrate the cross-talk between neurons and glial cells in terms of GABA neurotransmission and actions of neuroactive steroids. To this purpose, we will review the presence of the different GABA receptors in the glial cells of the central and peripheral nervous system. Then, we will discuss their modulation by some neuroactive steroids.
GABA-A receptor; GABA-B receptor; neurosteroids; microglia; macroglia
Brain morphogenesis depends on the maintenance of boundaries between populations of non-intermingling cells. We used molecular markers to characterize a boundary within the optic lobe of the Drosophila brain and found that Slit and the Robo family of receptors, well-known regulators of axon guidance and neuronal migration, inhibit the mixing of adjacent cell populations in the developing optic lobe. Our data suggest that Slit is needed in the lamina to prevent inappropriate invasion of Robo-expressing neurons from the lobula cortex. We show that Slit protein surrounds lamina glia, while the distal cell neurons in the lobula cortex express all three Drosophila Robos. We examine the function of these proteins in the visual system by isolating a novel allele of slit that preferentially disrupts visual system expression of Slit and by creating transgenic RNA interference flies to inhibit the function of each Drosophila Robo in a tissue-specific fashion. We find that loss of Slit or simultaneous knockdown of Robo, Robo2 and Robo3 causes distal cell neurons to invade the lamina, resulting in cell mixing across the lamina/lobula cortex boundary. This boundary disruption appears to lead to alterations in patterns of axon navigation in the visual system. We propose that Slit and Robo-family proteins act to maintain the distinct cellular composition of the lamina and the lobula cortex.
Glia; Neuron; Compartment boundary; Optic lobe; Drosophila
In recent years, the Drosophila olfactory system, with its unparalleled opportunities for genetic dissection of development and functional organization, has been used to study the development of central olfactory neurons and the molecular basis of olfactory coding. The results of these studies have been interpreted in the absence of a detailed understanding of the steps in maturation of glial cells in the antennal lobe. Here, we present a high-resolution study of the glia associated with olfactory glomeruli in adult and developing antennal lobes. The study provides a basis for comparison of findings in Drosophila with those in the moth Manduca sexta that indicate a critical role for glia in antennal lobe development. Using flies expressing GFP under a Nervana2 driver to visualize glia for confocal microscopy, and probing at higher resolution with the electron microscope, we find that glial development in Drosophila differs markedly from that in moths: glial cell bodies remain in a rind around the glomerular neuropil; glial processes ensheathe axon bundles in the nerve layer but likely contribute little to axonal sorting; their processes insinuate between glomeruli only very late and then form only a sparse, open network around each glomerulus; and glial processes invade the synaptic neuropil. Taking our results in the context of previous studies, we conclude that glial cells in the developing Drosophila antennal lobe are unlikely to play a strong role in either axonal sorting or glomerulus stabilization and that in the adult, glial processes do not electrically isolate glomeruli from their neighbors.
glial cells; olfactory lobe; olfactory bulb; glomeruli
Understanding the mechanisms regulating expression of retinal ganglion cell (RGC) specific and axon-guidance genes during development and in retinal stem cells will be critical for successful optic nerve regeneration. Müller glia have some characteristics of retinal stem cell properties, but in mammals have demonstrated limited potential to differentiate into RGCs. Chromatin remodeling through histone deacetylation and DNA methylation are a potential mechanism for silencing genes necessary for neuronal differentiation of glial cells. We investigated DNA methylation as a mechanism for regulating expression of mouse EphA5, one member of a large family of ephrin receptor genes that regulate patterning of the topographic connections of RGCs during visual system development. We analyzed spatial and age-related patterns of EphA5 promoter methylation by bisulfite sequencing and mRNA expression by quantitative RT-PCR in the mouse retina. The CpG island in the EphA5 promoter was hypomethylated in the retina and showed no change in overall methylation with age, despite a decline in EphA5 mRNA expression levels in the adult retina. In the nasal retina of post-natal day 0 mice, there was a modest, but statistically significant increase in methylation. Increased methylation corresponded with lower levels of receptor mRNA expression in the nasal retina. We cloned the EphA5 promoter and found that site-specific differences in methylation could preferentially activate or repress promoter activity in transient transfections of rat retinal progenitor cells (R28) using luciferase assays. In sphere cultures generated by EGF/FGF2 stimulation of conditionally immortalized mouse Müller glia (ImM10), EphA5 promoter was hypermethylated and EphA5 mRNA was not detected. Demethylation using 5-azadeoxycytidine (AzadC) resulted in a significant decrease of methylation of the EphA5 promoter and re-expression of the EphA5 mRNA. The inverse relationship between EphA5 promoter methylation and mRNA expression is consistent with a role for DNA methylation in modulating the spatial patterns of EphA5 gene expression in the retina and in silencing EphA5 expression in ImM10 cells. The robust up regulation of EphA5 in ImM10 cells following demethylation suggests that modulation of chromatin structure may be a useful approach for promoting expression of silenced developmental genes and increasing the neurogenic potential of Müller glia.
Ephrin receptor; DNA methylation; retina; Müller glia; gene regulation
Two-way communication between neurons and nonneural cells called glia is essential for axonal conduction, synaptic transmission, and information processing and thus is required for normal functioning of the nervous system during development and throughout adult life. The signals between neurons and glia include ion fluxes, neurotransmitters, cell adhesion molecules, and specialized signaling molecules released from synaptic and nonsynaptic regions of the neuron. In contrast to the serial flow of information along chains of neurons, glia communicate with other glial cells through intracellular waves of calcium and via intercellular diffusion of chemical messengers. By releasing neurotransmitters and other extracellular signaling molecules, glia can affect neuronal excitability and synaptic transmission and perhaps coordinate activity across networks of neurons.
Axon pruning is an evolutionarily conserved strategy used to remodel neuronal connections during development. The Drosophila mushroom body (MB) undergoes neuronal remodeling in a highly stereotypical and tightly regulated manner, however many open questions remain. Although it has been previously shown that glia instruct pruning by secreting a TGF-β ligand, myoglianin, which primes MB neurons for fragmentation and also later engulf the axonal debris once fragmentation has been completed, which glia subtypes participate in these processes as well as the molecular details are unknown. Here we show that, unexpectedly, astrocytes are the major glial subtype that is responsible for the clearance of MB axon debris following fragmentation, even though they represent only a minority of glia in the MB area during remodeling. Furthermore, we show that astrocytes both promote fragmentation of MB axons as well as clear axonal debris and that this process is mediated by ecdysone signaling in the astrocytes themselves. In addition, we found that blocking the expression of the cell engulfment receptor Draper in astrocytes only affects axonal debris clearance. Thereby we uncoupled the function of astrocytes in promoting axon fragmentation to that of clearing axonal debris after fragmentation has been completed. Our study finds a novel role for astrocytes in the MB and suggests two separate pathways in which they affect developmental axon pruning.
Glia are integral participants in synaptic physiology, remodeling and maturation from blowflies to humans, yet how glial structure is coordinated with synaptic growth is unknown. To investigate the dynamics of glial development at the Drosophila larval neuromuscular junction (NMJ), we developed a live imaging system to establish the relationship between glia, neuronal boutons, and the muscle subsynaptic reticulum. Using this system we observed processes from two classes of peripheral glia present at the NMJ. Processes from the subperineurial glia formed a blood-nerve barrier around the axon proximal to the first bouton. Processes from the perineurial glial extended beyond the end of the blood-nerve barrier into the NMJ where they contacted synapses and extended across non-synaptic muscle. Growth of the glial processes was coordinated with NMJ growth and synaptic activity. Increasing synaptic size through elevated temperature or the highwire mutation increased the extent of glial processes at the NMJ and conversely blocking synaptic activity and size decreased the presence and size of glial processes. We found that elevated temperature was required during embryogenesis in order to increase glial expansion at the nmj. Therefore, in our live imaging system, glial processes at the NMJ are likely indirectly regulated by synaptic changes to ensure the coordinated growth of all components of the tripartite larval NMJ.
Development of the adult olfactory system of the moth Manduca sexta depends on reciprocal interactions between olfactory receptor neuron (ORN) axons growing in from the periphery and centrally-derived glial cells. Early-arriving ORN axons induce a subset of glial cells to proliferate and migrate to form an axon-sorting zone, in which later-arriving ORN axons will change their axonal neighbors and change their direction of outgrowth in order to travel with like axons to their target areas in the olfactory (antennal) lobe. These newly fasciculated axon bundles will terminate in protoglomeruli, the formation of which induces other glial cells to migrate to surround them. Glial cells do not migrate unless ORN axons are present, axons fail to fasciculate and target correctly without sufficient glial cells, and protoglomeruli are not maintained without a glial surround. We have shown previously that Epidermal Growth Factor receptors and the IgCAMs Neuroglian and Fasciclin II play a role in the ORN responses to glial cells. In the present work, we present evidence for the importance of glial Fibroblast Growth Factor receptors in glial migration, proliferation, and survival in this developing pathway. We also report changes in growth patterns of ORN axons and of the dendrites of olfactory (antennal lobe) neurons following blockade of glial FGFR activation that suggest that glial FGFR activation is important in reciprocal communication between neurons and glial cells.
Nervous system function requires that neurons within neural circuits are connected together precisely. These connections form during the process of axon guidance whereby each neuron extends an axon that migrates, often large distances, through a complex environment to reach its synaptic target. This task can be simplified by utilising intermediate targets to divide the route into smaller sections. This requires that axons adapt their behaviour as they migrate towards and away from intermediate targets. In the central nervous system the midline acts as an intermediate target for commissural axons. In Drosophila commissural axons switch from attraction towards to extension away from the midline by regulating the levels of the Roundabout receptor on their cell surface. This is achieved by Commissureless which directs Roundabout to an intracellular compartment in the soma prior to reaching the midline. Once across the midline Roundabout is allowed to reach the surface and acts as a receptor for the repellent ligand Slit that is secreted by cells at the midline. Here we investigated candidate intracellular mechanisms that may facilitate the intracellular targeting of Commissureless and Roundabout within the soma of commissural neurons. Using modified forms of Commissureless or Rabs we show that neither ubiquitination nor Rab activity are necessary for the intracellular targeting of Commissureless. In addition we reveal that axon outgrowth of many populations of neurons within the Drosophila central nervous system is also independent of Rab activity.
A model of glial–neuronal interactions is proposed that could be explanatory for the demyelination identified in brains with schizophrenia. It is based on two hypotheses: (1) that glia–neuron systems are functionally viable and important for normal brain function, and (2) that disruption of this postulated function disturbs the glial categorization function, as shown by formal analysis. According to this model, in schizophrenia receptors on astrocytes in glial–neuronal synaptic units are not functional, loosing their modulatory influence on synaptic neurotransmission. Hence, an unconstrained neurotransmission flux occurs that hyperactivates the axon and floods the cognate receptors of neurotransmitters on oligodendrocytes. The excess of neurotransmitters may have a toxic effect on oligodendrocytes and myelin, causing demyelination. In parallel, an increasing impairment of axons may disconnect neuronal networks. It is formally shown how oligodendrocytes normally categorize axonic information processing via their processes. Demyelination decomposes the oligodendrocyte–axonic system making it incapable to generate categories of information. This incoherence may be responsible for symptoms of disorganization in schizophrenia, such as thought disorder, inappropriate affect and incommunicable motor behavior. In parallel, the loss of oligodendrocytes affects gap junctions in the panglial syncytium, presumably responsible for memory impairment in schizophrenia.
glial–neuronal interactions; demyelination; schizophrenia; synaptic imbalance
During gastrulation in Drosophila, ventral cells change shape, undergoing synchronous apical constriction, to create the ventral furrow (VF). This process is affected in mutant embryos lacking zygotic function of the folded gastrulation (fog) gene, which encodes a putative secreted protein. Fog is an essential autocrine signal that induces cytoskeletal changes in invaginating VF cells. Here we show that Fog is also required for nervous system development. Fog is expressed by longitudinal glia in the central nervous system (CNS), and reducing its expression in glia causes defects in process extension and axon ensheathment. Glial Fog overexpression produces a disorganized glial lattice. Fog has a distinct set of functions in CNS neurons. Our data show that reduction or overexpression of Fog in these neurons produces axon guidance phenotypes. Interestingly, these phenotypes closely resemble those seen in embryos with altered expression of the receptor tyrosine phosphatase PTP52F. We conducted epistasis experiments to define the genetic relationships between Fog and PTP52F, and the results suggest that PTP52F is a downstream component of the Fog signaling pathway in CNS neurons. We also found that Ptp52F mutants have early VF phenotypes like those seen in fog mutants.
Drosophila nervous system development; receptor tyrosine phosphatase; glia; motor axon guidance; epistasis; ventral furrow development; autocrine signaling; Folded Gastrulation
The ensheathment of neurons and their axons creates an ion-sensitive microenvironment that allows rapid conduction of nerve impulses. One of the fundamental questions about axonal ensheathment is how insulating glial cells wrap around axons. The mechanisms that underlie insulation of axons in invertebrates and vertebrates are not fully understood. In the present article we address cellular aspects of axonal ensheathment in Drosophila by taking advantage of glial mutants that illustrate a range of phenotypic defects including ensheathment of axons. From the findings of these mutant studies, we summarize that loss of glial cells, defects in glial membrane wrapping, failure of glial migration, and loss of specialized ladderlike septate junctions between ensheathing glial membranes result in axon-glial functional defects. These studies provide a broad perspective on glial ensheathment of axons in Drosophila and key insights into the anatomical and cellular aspects of axonal insulation. Given the powerful genetic approaches available in Drosophila, the axonal ensheathment process can be dissected in great detail to reveal the fundamental principles of ensheathment. These observations will be relevant to understanding the very similar processes in vertebrates, where defects in glial cell functions lead to devastating neurological diseases.
peripheral glia; exit glia; glial migration; septate junctions; actin cytoskeleton
Histaminergic transmission in the first synapse of the visual system in Drosophila melanogaster is mediated by two types of histamine receptors: 1) encoded by the gene hclA (HCLA), which is expressed in the second-order neurons—the large monopolar cells of the lamina, and is absolutely required for forward signal transmission; and 2) encoded by the gene hclB (HCLB), which is expressed in epithelial glia, and is involved in modulation of synaptic transmission from photoreceptors to large monopolar cells. The aim of our study was to establish whether the HCLB receptor–mediated modulation of synaptic transmission 1) contributes to the process of light adaptation, and 2) is involved in the control of the dynamics of sensitivity recovery after short-term light adaptation.
The effects of mutations in the gene hclB, encoding the subunits of the histamine receptor HCLB, were studied on 1) the intensity-response (V/logI) function of electroretinographic (ERG) responses under dark adaptation, as well as under three levels of background illumination; and 2) the dynamics of the dark sensitivity recovery after short-term light adaptation.
The amplitude of the photoreceptor component in the electroretinogram (ERG) was not significantly different between the hclB mutants and the wild-type flies, while the amplitude of the ERG ON and OFF transients, representing the activity of the second-order visual cells, was increased in the hclB mutants under both dark and light adaptation. The ON responses were affected to a greater degree. Under a given background, the ON response V/logI function was steeper and the response dynamic range was narrowed. The absolute sensitivity of the two transients was increased, as revealed by the decrease of their thresholds. The relative sensitivity of the transients, assessed by the semisaturation points of their V/logI functions, was decreased in ON responses to long (2 s) stimuli under dark and moderate light adaptation, being unchanged under bright backgrounds. Thus, the shift of the ON response V/logI function along the stimulus intensity axis during light adaptation occurred within a narrower range. The peak latencies of the ERG transients were delayed. The slower kinetics of the ERG transients was also indicated by their lower sensitivity to low-pass filtering, the effect being more pronounced under light adaptation. In wild-type flies, an instant dark sensitivity recovery or postadaptational potentiation of the ERG transients was usually observed after short-term light adaptation. In the hclB mutants the dark sensitivity recovery in similar conditions was significantly delayed.
The glial histamine receptor HCLB participates in visual sensitivity control at the level of the first synapse of the Drosophila visual system under a wide range of ambient illumination conditions and contributes to the process of light adaptation. The HCLB receptor-mediated modulation of synaptic gain helps avoid response saturation and increases the range of stimulus intensities within which dynamic responses can be generated. The HCLB receptors also speed up the sensitivity recovery after short-term light adaptation and contribute to the mechanism of postadaptational potentiation. They modulate the temporal characteristics of visual responses in a way that improves the temporal resolution of the visual system and reduces redundant (low-frequency) information.
The enteric nervous system (ENS) is a network of neurons and glia within the wall of the gastrointestinal tract that is able to control many aspects of digestive function independently from the central nervous system. Enteric glial cells share several features with astrocytes and are closely associated with enteric neurons and their processes both within enteric ganglia, and along interconnecting fiber bundles. Similar to other parts of the nervous system, there is communication between enteric neurons and glia; enteric glial cells can detect neuronal activity and have the machinery to intermediate neurotransmission. However, due to the close contact between these two cell types and the particular characteristics of the gut wall, the recording of enteric glial cell activity in live imaging experiments, especially in the context of their interaction with neurons, is not straightforward. Most studies have used calcium imaging approaches to examine enteric glial cell activity but in many cases, it is difficult to distinguish whether observed transients arise from glial cells, or neuronal processes or varicosities in their vicinity. In this technical report, we describe a number of approaches to unravel the complex neuron-glia crosstalk in the ENS, focusing on the challenges and possibilities of live microscopic imaging in both animal models and human tissue samples.
enteric neuron; enteric glia; calcium; synaptic; GCaMP
A gene network involving Notch and Pros underlies the glial regenerative response to injury in the Drosophila central nervous system.
Organisms are structurally robust, as cells accommodate changes preserving structural integrity and function. The molecular mechanisms underlying structural robustness and plasticity are poorly understood, but can be investigated by probing how cells respond to injury. Injury to the CNS induces proliferation of enwrapping glia, leading to axonal re-enwrapment and partial functional recovery. This glial regenerative response is found across species, and may reflect a common underlying genetic mechanism. Here, we show that injury to the Drosophila larval CNS induces glial proliferation, and we uncover a gene network controlling this response. It consists of the mutual maintenance between the cell cycle inhibitor Prospero (Pros) and the cell cycle activators Notch and NFκB. Together they maintain glia in the brink of dividing, they enable glial proliferation following injury, and subsequently they exert negative feedback on cell division restoring cell cycle arrest. Pros also promotes glial differentiation, resolving vacuolization, enabling debris clearance and axonal enwrapment. Disruption of this gene network prevents repair and induces tumourigenesis. Using wound area measurements across genotypes and time-lapse recordings we show that when glial proliferation and glial differentiation are abolished, both the size of the glial wound and neuropile vacuolization increase. When glial proliferation and differentiation are enabled, glial wound size decreases and injury-induced apoptosis and vacuolization are prevented. The uncovered gene network promotes regeneration of the glial lesion and neuropile repair. In the unharmed animal, it is most likely a homeostatic mechanism for structural robustness. This gene network may be of relevance to mammalian glia to promote repair upon CNS injury or disease.
The process of tissue regeneration has long been studied as a route to understanding what promotes structural robustness of cellular networks in animals. In the central nervous system (CNS), neurons and glia interact throughout adult life and during learning, at the same time accommodating functional changes while preserving the structural integrity necessary for function. The mechanisms that confer this combination of structural robustness and functional plasticity in the CNS are unknown, but they may be shared with the cellular responses to injury, which also require structural changes while retaining function. The glial cells that enwrap axons respond to injury by dividing and re-enwrapping them, leading to partial recovery of function. Here, we use Drosophila genetics to uncover a gene network underlying this glial regenerative response. This gene network enables glia to divide upon injury, prevent uncontrolled proliferation, and differentiate. We find that the network also has homeostatic properties: two cell-cycle activators (Notch and NFκB) promote the expression of a cell cycle inhibitor (Pros), providing negative feedback on cell division. Pros is also essential for glial differentiation, enabling the clearance of cellular debris and axonal enwrapment, and priming glia for further responses. By removing these genes or adding them in excess, we can shift the response to injury from prevention to promotion of lesion repair. This gene network is thus a homeostatic mechanism for structural robustness. Our findings from Drosophila may also help manipulation of glia to repair the damaged human CNS.
During development, neurons extend axons to different brain areas and produce stereotypical patterns of connections. The mechanisms underlying this process have been intensively studied in the visual system, where retinal neurons form retinotopic maps in the thalamus and superior colliculus. The mechanisms active in map formation include molecular guidance cues, trophic factor release, spontaneous neural activity, spike-timing dependent plasticity (STDP), synapse creation and retraction, and axon growth, branching and retraction. To investigate how these mechanisms interact, a multi-component model of the developing retinocollicular pathway was produced based on phenomenological approximations of each of these mechanisms. Core assumptions of the model were that the probabilities of axonal branching and synaptic growth are highest where the combined influences of chemoaffinity and trophic factor cues are highest, and that activity-dependent release of trophic factors acts to stabilize synapses. Based on these behaviors, model axons produced morphologically realistic growth patterns and projected to retinotopically correct locations in the colliculus. Findings of the model include that STDP, gradient detection by axonal growth cones and lateral connectivity among collicular neurons were not necessary for refinement, and that the instructive cues for axonal growth appear to be mediated first by molecular guidance and then by neural activity. Although complex, the model appears to be insensitive to variations in how the component developmental mechanisms are implemented. Activity, molecular guidance and the growth and retraction of axons and synapses are common features of neural development, and the findings of this study may have relevance beyond organization in the retinocollicular pathway.
Neural development is a process that involves a wide range of behaviors. As a result of these behaviors, neurons are able to extend axons to different brain areas and produce stereotypical patterns of innervation. One of the most commonly studied of these projections is in the visual system, where retinal axons project to multiple brain regions and produce retinotopic maps. This study examines the relative roles and interactions of different neural mechanisms in guiding axon growth and generating retinotopic order. We did this by producing a computational model of retinotopic development that represented many of the neural mechanisms thought to be involved, including axon and synapse growth, molecular guidance and synapse plasticity. Our results suggest that synaptic plasticity is realized by variation in the number of synapses between neurons, not through alteration of individual synaptic weights; that lateral connectivity between collicular neurons is not required for organization; and that axon arbor development does not require the gradient tracking abilities of growth cones. The mechanisms underlying neuronal development in the visual system are also observed in many other brain areas, so the findings here should apply more generally.
Radial glia are highly polarized cells that serve as neuronal progenitors and as scaffolds for neuronal migration during construction of the cerebral cortex. How radial glial cells establish and maintain their morphological polarity is unknown. Using conditional gene targeting in mice, we demonstrate that Adenomatous Polyposis Coli (APC) serves an essential function in the maintenance of polarized radial glial scaffold during brain development. In the absence of APC, radial glial cells lose their polarity and responsiveness to the extracellular polarity maintenance cues, such as neuregulin-1. Elimination of APC further leads to marked instability of the radial glial microtubule cytoskeleton. The resultant changes in radial glial function and loss of APC in radial glial progeny lead to defective generation and migration of cortical neurons, severely disrupted cortical layer formation, and aberrant axonal tract development. Thus APC is an essential regulator of radial glial polarity and is critical for the construction of cerebral cortex in mammals.
Radial glia; Cortical development; APC
Parvalbumin-expressing fast-spiking (FS) cells are interconnected via GABAergic and electrical synapses and represent a major class of inhibitory interneurons in the neocortex. Synaptic connections among FS cells are critical for regulating network oscillations in the mature neocortex. However, it is unclear whether synaptic connections among FS interneurons also play a central role in the generation of patterned neuronal activity in the immature brain, which is thought to underlie the formation of neocortical circuits. Here, we investigated the developmental time course of synaptogenesis of FS cell in mouse visual cortex. In layer 5/6 (L5/6), we recorded from two or three FS and/or pyramidal neurons (P) to study the development of electrical and chemical synaptic interactions from postnatal day 3 (P3) to P18. We detected no evidence for functional connectivity for FS-FS or FS-P pairs at P3–4. However, by P5–6, we found 20% of FS pairs were electrically coupled and 24% of pairs were connected via GABAergic synapses and by P15–18, 42% of FS pairs had established functional electrical synapses and 47% of FS pairs were connected via GABAergic synapses. FS cell GABAergic inhibition of pyramidal cells showed a similar developmental time line, but no electrical coupling was detected for FS-P pairs. We found that synaptogenesis of electrical and GABAergic connection of FS cells takes place in the same period. Together, our results suggest that chemical and electrical connections among FS cells can contribute to patterned neocortical activity only by the end of the first postnatal week.
electrical synapses; Gap junctions; FS-cells; Parvalbumin; Cortex; Development