Development of a glial network in the olfactory nerve: role of calcium and neuronal activity

In adult olfactory nerves of mammals and moths, a network of glial cells ensheathes small bundles of olfactory receptor axons. In the developing antennal nerve (AN) of the moth Manduca sexta, the axons of olfactory receptor neurons (ORNs) migrate from the olfactory sensory epithelium toward the antennal lobe. Here we explore developmental interactions between ORN axons and AN glial cells. During early stages in AN glial-cell migration, glial cells are highly dye coupled, dividing glia are readily found in the nerve and AN glial cells label strongly for glutamine synthetase. By the end of this period, dye-coupling is rare, glial proliferation has ceased, glutamine synthetase labeling is absent, and glial processes have begun to extend to enwrap bundles of axons, a process that continues throughout the remainder of metamorphic development. Whole-cell and perforated-patch recordings in vivo from AN glia at different stages of network formation revealed two potassium currents and an R-like calcium current. Chronic in vivo exposure to the R-type channel blocker SNX-482 halted or greatly reduced AN glial migration. Chronically blocking spontaneous Na-dependent activity by injection of tetrodotoxin reduced the glial calcium current implicating an activity-dependent interaction between ORNs and glial cells in the development of glial calcium currents.     

Activation of glial FGFRs is essential in glial migration, proliferation, and survival and in glia-neuron signaling during olfactory system development

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.     

A diffusible signal attracts olfactory sensory axons toward their target in the developing brain of the moth

The signals that olfactory receptor axons use to navigate to their target in the CNS are still not well understood. In the moth Manduca sexta, the primary olfactory pathway develops postembryonically, and the receptor axons navigate from an experimentally accessible sensory epithelium to the brain along a pathway long enough for detailed study of regions in which axon behavior changes. The current experiments ask whether diffusible factors contribute to receptor axon guidance. Explants were made from the antennal receptor epithelium and co-cultured in a collagen gel matrix with slices of various regions of the brain. Receptor axons were attracted toward the central regions of the brain, including the protocerebrum and antennal lobe. Receptor axons growing into a slice of the most proximal region of the antennal nerve, where axon sorting normally occurs, showed no directional preference. When the antennal lobe was included in the slice, the receptor axons entering the sorting region grew directly toward the antennal lobe. Taken together with the previous in vivo experiments, the current results suggest that an attractive diffusible factor can serve as one cue to direct misrouted olfactory receptor axons toward the medial regions of the brain, where local cues guide them to the antennal lobe. They also suggest that under normal circumstances, in which the receptor axons follow a pre-existing pupal nerve to the antennal lobe, the diffusible factor emanating from the lobe acts in parallel and at short range to maintain the fidelity of the path into the antennal lobe.     

Different isoforms of fasciclin II are expressed by a subset of developing olfactory receptor neurons and by olfactory-nerve glial cells during formation of glomeruli in the moth Manduca sexta

During development of the primary olfactory projection, olfactory receptor axons must sort by odor specificity and seek particular sites in the brain in which to create odor-specific glomeruli. In the moth Manduca sexta, we showed previously that fasciclin II, a cell adhesion molecule in the immunoglobulin superfamily, is expressed by the axons of a subset of olfactory receptor neurons during development and that, in a specialized glia-rich "sorting zone," these axons segregate from nonfasciclin II-expressing axons before entering the neuropil of the glomerular layer. The segregation into fasciclin II-positive fascicles is dependent on the presence of the glial cells in the sorting zone. Here, we explore the expression patterns for different isoforms of Manduca fasciclin II in the developing olfactory system. We find that olfactory receptor axons express transmembrane fasciclin II during the period of axonal ingrowth and glomerulus development. Fascicles of TM-fasciclin II+ axons target certain glomeruli and avoid others, such as the sexually dimorphic glomeruli. These results suggest that TM-fasciclin II may play a role in the sorting and guidance of the axons. GPI-linked forms of fasciclin II are expressed weakly by glial cells associated with the receptor axons before they reach the sorting zone, but not by sorting-zone glia. GPI-fasciclin II may, therefore, be involved in axon-glia interactions related to stabilization of axons in the nerve, but probably not related to sorting.     

Glial cells in the developing and adult olfactory lobe of the moth Manduca sexta

The antennal lobe of the moth contains several classes of glial cells that are likely to play functional roles in both the developing and mature lobe. In this study, confocal and electron microscopy were used to examine in detail the morphology of two classes of glial cells, those associated with olfactory receptor axons as they course to their targets in the lobe and those that form borders around the synaptic neuropil of the olfactory glomeruli. The former, the nerve-layer glia, have long processes with multiple expansions that enwrap axon fascicles; the latter, the neuropil glia, constitute two subgroups: complex glia with large cell bodies and branching, vellate arbors; and simple glia, with multiple, mostly unbranched processes with many lamellate expansions along their lengths. The processes of complex glia appear to be closely associated with axon fascicles as they enter the glomeruli, while those of the simple glia surround the glomeruli as part of a multi-lamellar glial envelope, their processes rarely invading the synaptic neuropil of the body of the glomerulus. The full morphological development of antennal-lobe glial cells requires more than two-thirds of metamorphic development. During this period, cells that began as cuboidal or spindle-shaped cells that were extensively dye-coupled to one another gradually assume their adult form and, at least under nonstimulated conditions, greatly reduce their coupling. These changes are only weakly dependent on the presence of olfactory receptor axons. Glial processes are somewhat shorter and less branched in the absence of these axons, but basic structure and degree of dye-coupling are unchanged.     

The L1-CAM, Neuroglian, functions in glial cells for Drosophila antennal lobe development

Although considerable progress has been made in understanding the roles of olfactory receptor neurons (ORNs) and projection neurons (PNs) in Drosophila antennal lobe (AL) development, the roles of glia have remained largely mysterious. Here, we show that during Drosophila metamorphosis, a population of midline glial cells in the brain undergoes extensive cellular remodeling and is closely associated with the collateral branches of ORN axons. These glial cells are required for ORN axons to project across the midline and establish the contralateral wiring in the ALs. We find that Neuroglian (Nrg), the Drosophila homolog of the vertebrate cell adhesion molecule, L1, is expressed and functions in the midline glial cells to regulate their proper development. Loss of Nrg causes the disruption in glial morphology and the agenesis of the antennal commissural tract. Our genetic analysis further demonstrates that the functions of Nrg in the midline glia require its ankyrin-binding motif. We propose that Nrg is an important regulator of glial morphogenesis and axon guidance in AL development.     

中华蜜蜂嗅叶胚后发育过程中的神经胶质

本研究通过形态解剖、免疫组织化学等技术,对中华蜜蜂Apis cerana cerana Fabricius工蜂嗅叶胚后发育中神经胶质的模式进行了比较研究。结果表明:神经胶质在蜜蜂嗅叶的发育过程中起着重要的作用,它们在嗅叶发育早期就划定了神经纤维网的边界;在嗅觉神经轴突进入嗅叶之前,对神经轴突进行"分选",并引导它们进入嗅叶特定的区域,形成神经纤维球;它们不仅规定了神经纤维网的边界和区域,还为神经纤维网提供内部的分隔。中华蜜蜂神经胶质增殖的高峰期集中在幼虫发育末期和预蛹期。     

Axons of olfactory receptor cells of transsexually grafted antennae induce development of sexually dimorphic glomeruli in Manduca sexta

The influence of olfactory receptor cell (ORC) axons from transsexually grafted antennae on the development of glomeruli in the antennal lobes (ALs), the primary olfactory centers, was studied in the moth Manduca sexta. Normally during metamorphic adult development, the pheromone-specific macroglomerular complex (MGC) forms only in the ALs of males, whereas two lateral female-specific glomeruli (LFGs) develop exclusively in females. A female AL innervated by ORC axons from a grafted male antenna developed an MGC with three glomeruli, like the MGC of a normal male AL. Conversely, a male AL innervated by ORC axons from a grafted female antenna lacked the MGC but exhibited LFGs. ORC axons from grafted male antenna terminated in the MGC-specific target area, even in cases when the antennal nerve (AN) entered the AL via an abnormal route. Within ectopic neuromas formed by ANs that had become misrouted and failed to enter the brain, male-specific axons were not organized and formed terminal branches in many areas. The results suggest the presence of guidance cues within the AL for male-specific ORC axons. Depending on the sex of the antennal innervation, glial borders formed in a pattern characteristic of the MGC or LFGs. The sex-specific number of projection neurons (PNs) in the medial group of AL neurons remained unaffected by the antennal graft, but significant changes occurred in the organization of PN arborizations. In gynandromorphic females, LFG-specific PNs extended processes into the induced MGC, whereas in gynandromorphic males, PNs became restricted to the LFGs. The results indicate that male-and female-specific ORC axons play important roles in determining the position, anatomical features, and innervation of sexually dimorphic glomeruli.     

The sorting behaviour of olfactory and vomeronasal axons during regeneration

Summary In order to begin to understand how primary olfactory and vomeronasal organ (VNO) axons target specific regions of the olfactory bulb, we examined the sorting behaviour of these axons following neonatal unilateral olfactory bulbectomy. Bulbectomy induced widespread ipsilateral death of the primary olfactory and VNO neurons. After 4 weeks, many new sensory axons had re-grown into the cranial cavity and established a prominent plexus with evidence of dense tufts that were similar in gross appearance to glomeruli. Axons expressing the cell adhesion molecule OCAM, which normally innervate the ventrolateral and rostral halves of the main and accessory olfactory bulbs, respectively, sorted out and segregated from those axons not expressing this molecule within the plexus. In addition, VNO axons formed large discrete bundles that segregated from main olfactory axons within the plexus. Thus, VNO and primary olfactory axons as well as discrete subpopulations of both are able to sort out and remain segregated in the absence of the olfactory bulb. Sorting and convergence of axons therefore occur independently of the olfactory bulb and are probably attributable either to inherent properties of the axons themselves or to interactions between the axons and accompanying glial ensheathing cells.     

Repair of neural pathways by olfactory ensheathing cells

Damage to nerve fibre pathways results in a devastating loss of function, due to the disconnection of nerve fibres from their targets. However, some recovery does occur and this has been correlated with the formation of new (albeit abnormal) connections. The view that an untapped growth potential resides in the adult CNS has led to various attempts to stimulate the repair of disconnectional injuries. A key factor in the failure of axonal regeneration in the CNS after injury is the loss of the aligned glial pathways that nerve fibres require for their elongation. Transplantation of cultured adult olfactory ensheathing cells into lesions is being investigated as a procedure to re-establish glial pathways permissive for the regeneration of severed axons.     

Expression of the small conductance Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> channel,SK3, in the olfactory ensheathing glial cells of rat brain

Olfactory sensory neurons are wrapped by ensheathing glial cells in the olfactory nerve layer (ONL). Neither functional roles nor electrical properties of ensheathing glial cells have been, as yet, fully clarified. Four subunits (SK1–4) of small conductance Ca²⁺-activated K⁺ (SK) channels have been cloned. In the present study, immunohistochemical analyses showed that SK3 channels are expressed in ensheathing glial cells in the rat olfactory bulb, in addition to neuronal cells in other regions. Western blotting analysis demonstrated that SK3 was predominantly expressed in the olfactory bulb, thalamus, moderately in the hippocampus and cerebellum and modestly in the cerebral cortex of the rat brain. SK3 immunoreactivity was detected in the ONL of the olfactory bulb, neural cell body and fibers of the substantia nigra and hypothalamus. SK3 immunoreactivity was quite intense in the outer (superficial) part of the ONL. SK3-immunoreactive structures were overlapped with glial fibrillary acidic protein (GFAP), but not with vimentin, markers for glial cells and olfactory sensory axons, respectively. Immunoelectron microscopy showed that SK3 immunoreactivity was localized in thin processes that enfolded fascicles of immunonegative olfactory nerve axons. These results indicate that SK3 is expressed specifically in the olfactory ensheathing glial cells in olfactory regions.This work was supported in part by a Grant-in-Aid to A.F. for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, and by scholarship from Ono Pharmaceutical Company, and by Narishige Neuroscience Research Foundation.     

The neurite growth promoting protease nexin 1 in glial cells of the olfactory bulb of the gerbil: An ultrastructural study

The glia-derived serine protease inhibitor and neurite outgrowth promotor protease nexin-1 (PN-1) is expressed in Schwann cell precursors and astroblasts during embryogenesis. In the adult nervous system, PN-1 persists in the Schwann cells and olfactory glia only. Light-microscopic immunohistochemistry has revealed the presence of PN-1 in the olfactory mucosa and in the nerve fiber layer of the olfactory bulb. The present electron-microscopic study of the gerbil olfactory bulb confirms the occurrence of PN-1 in ensheathing cells of the olfactory nerve fiber layer, a special type of glia which envelops olfactory axons. In addition, PN-1 is contained in typical astrocytes of the nerve fiber layer and of the glomerular layer. It is inferred that synthesis of PN-1 in the olfactory bulbs is maintained throughout adulthood because its neurite outgrowth promoting action is required for the continuous renewal of olfactory receptor neurons.     

Development of an identified serotonergic neuron in the antennal lobe of the moth and effects of reduction in serotonin during construction of olfactory glomeruli

Each olfactory (antennal) lobe of the moth Manduca sexta contains a single serotonin (5-HT) immunoreactive neuron whose processes form tufted arbors in the olfactory glomeruli. To extend our present understanding of the intercellular interactions involved in glomerulus development to the level of an individual, identified antennal lobe neuron, we first studied the morphological development of the 5-HT neuron in the presence and absence of receptor axons. Development of the neuron's glomerular tufts depends, as it does in the case of other multiglomerular neurons, on the presence of receptor axons. Processes of the 5-HT neuron are excluded from the region in which the initial steps of glomerulus construction occur and thus cannot provide a physical scaffolding on which the array of glomeruli is organized. Because the neuron's processes are present in the antennal lobe neuropil throughout postembryonic development, 5-HT could provide signals that influence the pattern of development in the lobe. By surgically producing 5-HT-depleted antennal lobes, we also tested the importance of 5-HT in the construction of olfactory glomeruli. Even in the apparent absence of 5-HT, the glomerular array initiated by the receptor axons was histologically normal, glial cells migrated to form glomerular borders, and receptor axons formed terminal branches in their normal region within each glomerulus. In some cases, 5-HT-immunoreactive processes from abnormal sources entered the lobe and formed the tufted intraglomerular branches typical of most antennal lobe neurons, suggesting that local cues strongly influence the branching patterns of developing antennal lobe neurons. © 1995 John Wiley & Sons, Inc.     

Mechanisms underlying olfactory neuronal connectivity in Drosophila-the atonal lineage organizes the periphery while sensory neurons and glia pattern the olfactory lobe

Patterning of the antennal lobe of adult Drosophila occurs through a complex interaction between sensory neurons, glia, and central neurons of larval and adult origin. Neurons from the olfactory sense organs are organized into distinct fascicles lined by glial cells. The glia originate from one of the three types of sensory lineages-specified by the proneural gene atonal. Gain-of-function as well as loss-of-function analysis validates a role for cells of the Atonal lineage in the ordered fasciculation of sensory neurons. Upon entry of the antennal nerve to central regions, sensory neurons at first remain closely associated with central glia which lie around the periphery of the lobe anlage. Coincident with the arrival of sensory neurons into the brain, glial precursors undergo mitosis and neural precursors expressing Dachshund appear around the lobe. Sensory neurons and glial cells project into the lobe at around the same time and are likely to coordinate the correct localization of different glomeruli. The influence of sensory neurons on the development of the olfactory lobe could serve to match and lock peripheral and central properties important for the generation of olfactory behavior.     

Developmental distribution of CaM kinase II in the antennal lobe of the sphinx moth <Emphasis Type="Italic">Manduca sexta</Emphasis>

The antennal lobe (primary olfactory center of insects) is completely reorganized during metamorphosis. This reorganization is accompanied by changing patterns of calcium signaling in neurons and glial cells. In the present study, we investigated the developmental distribution of a major calcium-dependent protein, viz., calcium/calmodulin-dependent protein kinase II (CaM kinase II), in the antennal lobe of the sphinx moth Manduca sexta by using a monoclonal antibody. During synaptogenesis (developmental stages 6–10), we found a redistribution of CaM kinase II immunoreactivity, from a homogeneous distribution in the immature neuropil to an accumulation in the neuropil of the glomeruli. CaM kinase II immunoreactivity was less intense in olfactory receptor axons of the antennal nerve and antennal lobe glial cells. Western blot analysis revealed a growing content of CaM kinase II in antennal lobe tissue throughout metamorphosis. Injection of the CaM kinase inhibitor KN-93 into pupae resulted in a reduced number of antennal lobe glial cells migrating into the neuropil to form borders around glomeruli. The results suggest that CaM kinase II is involved in glial cell migration.This work was supported by the DFG LO779/2.     

Drosophila cortex and neuropile glia influence secondary axon tract growth, pathfinding, and fasciculation in the developing larval brain

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.     

Transregulation of erbB expression in the mouse olfactory bulb.

Previously, we have shown that erbB-3 expression is restricted to the ensheathing cells of the olfactory nerve layer, while erbB-4 is found in the periglomerular and mitral/tufted cells of the olfactory bulb and in cells coming out from the rostral migratory stream of the subependymal layer. In the present work, we have treated adult mice with zinc sulfate intranasal irrigation and analyzed erbB-3 and erbB-4 expression in the deafferented olfactory bulb. Following treatment, olfactory axons undergo degeneration, as indicated by the loss of OMP expression in the deafferented olfactory bulb. The thickness of the olfactory nerve layer is reduced, but the specific intensity of erbB-3 labeling in the remaining olfactory nerve layer is increased with respect to control. Interestingly, following deafferentation, erbB-4 immunoreactivity decreases specifically in cell types that normally make synaptic contacts with primary olfactory neurons in the glomeruli, i.e. periglomerular and mitral/tufted cells. Partial lesion of the olfactory epithelium allows regenerative axon growth of olfactory neurons to the olfactory bulb. Following olfactory axon regeneration, erbB-3 and erbB-4 immunoreactivity in the olfactory bulb is similar to control. Thus, like tyrosine hydroxylase, the down regulation of erbB-4 expression in the periglomerular cells is reversible.     

Expression of muscarinic acetylcholine receptors and choline acetyltransferase enzyme in cultured antennal sensory neurons and non-neural cells of the developing moth Manduca sexta

Antennal sensory neurons of Manduca sexta emerge from epidermal cells that also give rise to sheath cells surrounding the peripheral parts of the neurons and to glial cells that enwrap the sensory axons in the antennal nerve. Reciprocal interactions between sensory neurons and glial cells are believed to aid in axon growth and guidance, but the exact nature of these interactions is not known. We investigated the possibility of cholinergic interactions in this process by locating muscarinic acetylcholine receptors (mAChRs) and choline acetyltransferase (ChAT) enzyme in cultured antennal sensory neurons and non-neural cells. ChAT and mAChRs were present in the sensory neurons from the first day in culture. Therefore, the sensory neurons are probably cholinergic, as previously suggested, but they may also be controlled by ACh. In 7-day-old cultures a subgroup of small non-neural cells with processes expressed ChAT activity, and in 14-day-old cultures non-neural cells that formed lamellipodia and scaffoldlike structures on the culture substrate were labeled with ChAT antibody. mAChR activity was detected in similar non-neural cells but only in areas surrounding the nuclei. In addition, mAChRs were found in flat lamellipodia and filopodia forming cells that were present in 1-day-old cultures and grew in size during the 2 week investigation period. These findings suggest muscarinic cholinergic interactions between the neural and non-neural cells during the development of Manduca antenna.     

Patterning of olfactory sensory connections is mediated by extracellular matrix proteins in the nerve layer of the olfactory bulb

In early rat embryos when axons from sensory neurons first contact the olfactory bulb primordium, lactosamine-containing glycans (LCG) are detected on neurons that are broadly distributed within the olfactory epithelium, but that project axons to a very restricted region of the ventromedial olfactory bulb. LCG(+) axons extend through channels defined by the coexpression of galectin-1 and beta2-laminin. These two extracellular matrix molecules are differentially expressed, along with semaphorin 3A, by subsets of ensheathing cells in the ventral nerve layer of the olfactory bulb. The overlapping expression of these molecules creates an axon-sorting domain that is capable of promoting and repelling subsets of olfactory axons. Specifically, LCG(+) axons preferentially grow into the region of the nerve layer that expresses high amounts of galectin-1, beta2-laminin, and semaphorin 3A, whereas neuropilin-1(+) axons grow in a complementary pattern, avoiding the ventral nerve layer and projecting medially and laterally. These studies suggest that initial patterning of olfactory epithelium to olfactory bulb connections is, in part, dependent on extracellular components of the embryonic nerve layer that mediate convergence and divergence of specific axon subsets.