Primary Axons Research Articles

Pathfinding and growth termination of primary trigeminal sensory afferents in the embryonic rat hindbrain.

Axons of the trigeminal ganglion convey sensory information from mechanoreceptors, thermoreceptors, and nociceptors in the face and nasal mucosa, then terminate on several groups of neurons including the principal sensory nucleus and the nuclei of the spinal trigeminal tract. To understand guidance mechanisms during the development of trigeminal sensory axons (TA) in the embryonic brain, we first investigated the growth pattern of TA in relation to organization in the hindbrain using flat whole-mount preparation from rat. We found that the primary TA from the trigeminal ganglion entered the brainstem and grew longitudinally within the hindbrain. Whereas descending axons ran just medial to the primary vestibular axons to innervate the spinal nucleus, ascending axons stayed near the entry point. In flat whole-mount culture, the TA extended both ascending and descending branches as they do in vivo. Rostral hindbrain was found to be a less permissive substrate for the TA compared to caudal hindbrain. In addition, the nonpermissive property of the ventral hindbrain substrate restricted the invasion of TA along the entire length of the hindbrain. Thus, cooperation of absolute and relative permissiveness of the substrate plays important roles in the guidance of TA to their targets.

Molecular analysis of axon repulsion by the notochord

During development of the amniote peripheral nervous system, the initial trajectory of primary sensory axons is determined largely by the action of axon repellents. We have shown previously that tissues flanking dorsal root ganglia, the notochord lying medially and the dermamyotomes lying laterally, are sources of secreted molecules that prevent axons from entering inappropriate territories. Although there is evidence suggesting that SEMA3A contributes to the repellent activity of the dermamyotome, the nature of the activity secreted by the notochord remains undetermined. We have employed an expression cloning strategy to search for axon repellents secreted by the notochord, and have identified SEMA3A as a candidate repellent. Moreover, using a spectrum of different axon populations to assay the notochord activity, together with neuropilin/Fc receptor reagents to block semaphorin activity in collagen gel assays, we show that SEMA3A probably contributes to notochord-mediated repulsion. Sympathetic axons that normally avoid the midline in vivo are also repelled, in part, by a semaphorin-based notochord activity. Although our results implicate semaphorin signalling in mediating repulsion by the notochord, repulsion of early dorsal root ganglion axons is only partially blocked when using neuropilin/Fc reagents. Moreover, retinal axons, which are insensitive to SEMA3A, are also repelled by the notochord. We conclude that multiple factors act in concert to guide axons in this system, and that further notochord repellents remain to be identified.

Neurotrophic properties of olfactory ensheathing glia

Olfactory ensheathing cells (OEC) constitute a specialized population of glia that accompany primary olfactory axons and have been reported to facilitate axonal regeneration after spinal cord injury in vivo. In the present report we describe OEC neurotrophic factor expression and neurotrophic properties of OECs in vitro. Investigation of the rat olfactory system during development and adulthood by radioactive in situ hybridization revealed positive labeling in the olfactory nerve layer for the neurotrophic molecules S-100β, CNTF, BMP-7/OP-1, and artemin, as well as for the neurotrophic factor receptors RET and TrkC. Ribonuclease protection assay of cultured OEC revealed expression of NGF, BDNF, GDNF, and CNTF mRNA, while NT3 and NT4 mRNA were not detectable. In vitro bioassays of neurotrophic activity involved coculturing of adult OEC with embryonic chick ganglia and demonstrated increased neurite outgrowth from sympathetic, ciliary, and Remak’s ganglia. However, when culturing the ganglia with OEC-conditioned medium, neurite outgrowth was not stimulated to any detectable extent. Our results suggest that the neurotrophic properties of OEC may involve secretion of neurotrophic molecules but that cellular interactions are crucial.

The non-immunosuppressive immunophilin ligand GPI-1046 potently stimulates regenerating axon growth from adult mouse dorsal root ganglia cultured in Matrigel

We used explant cultures of adult mouse dorsal root ganglia with spinal nerve attached growing in Matrigel to assess the effects of the non-immunosuppressive immunophilin ligand GPI-1046 [Snyder et al. (1998) TIPS 19, 21–26] on the growth rate of regenerating sensory axons and found a potent stimulation of axon growth. In these explant cultures, naked, unfasciculated axons emerge from the cut end of the spinal nerve and continue to grow in the Matrigel for up to eight days [Tonge et al. (1996) Neuroscience 73, 541–551]. Some axons are entirely smooth whilst others show prominent varicosities. Some of the former express the phosphorylated neurofilament epitope recognised by monoclonal antibody RT97, a marker for large calibre, myelinated axons, whilst the latter express calcitonin gene-related peptide, predominantly a marker for unmyelinated, and small diameter myelinated sensory axons. Many of the axons in these cultures also express the low-affinity neurotrophin receptor p75. GPI-1046 has been shown to have striking stimulatory effects on embryonic primary sensory axons growing in vitro and it was therefore of interest to see whether it could also enhance regenerating sensory axon growth from the adult ganglia in our cultures. GPI-1046 potently stimulated axon growth in our cultures in a dose-dependent manner. The stimulatory effect was not dependent on the class of sensory axon. These observations show that GPI-1046 is a potent stimulator of regenerating axons from adult, primary sensory neurones. The cellular site of action of GPI-1046 is unknown. To distinguish between a direct effect of the drug on neurones and an indirect effect we compared the effects of GPI-1046 on explant and dissociated cultures. In confirmation of previous results, we found that GPI-1046 potently stimulated axon outgrowth from explants of embryonic chick dorsal root ganglia. However, the drug was without effect on dissociated embryonic dorsal root ganglion neurones, suggesting that non-neuronal cells are important for axon growth stimulation.

EphA receptors and ephrin-A ligands exhibit highly regulated spatial and temporal expression patterns in the developing olfactory system

The spatiotemporal expression patterns of the chemorepulsive EphA receptors, EphA4 and EphA7, and three ephrins—A2, A4 and A5, were examined in the developing rat primary olfactory system. Unlike the visual system that has simple and stable gradients of Ephs and ephrins, the olfactory system demonstrates complex spatiotemporal expression patterns of these molecules. Using immunohistochemistry, we demonstrate that expression of these molecules is dynamic and tightly regulated both within and between different cell types. We reveal restricted targeting of these proteins within subcellular compartments of some neurons. EphA4, ephrin-A2 and ephrin-A5 were expressed by primary olfactory axons during the embryonic formation of the olfactory nerve. There were no gradients in expression along the rostrocaudal or ventrodorsal axes in the nasal cavity and olfactory bulb. However, during the early neonatal period, axons expressing different levels of ephrin-A5 sorted out and terminated in a subpopulation of glomeruli that were mosaically dispersed throughout the bulb. The expression of EphA4 and ephrin-A2 was dramatically down-regulated on all axons during the early neonatal period of glomerular formation. The uniform co-expression of receptors and ligands before glomerular formation suggests they play a generic role in axon–axon interactions in the olfactory nerve and nerve fibre layer. In contrast, loss of EphA4 from axons during glomerular formation may facilitate the interaction of ephrin-A5 with Eph receptors on target cells in the bulb. While EphA4, EphA5 and EphA7 are not mosaically expressed by bulbar neurons, other Eph receptors may have expression patterns complementary to the ephrin-A5-positive subpopulation of glomeruli.

The jing gene is required for embryonic brain development in Drosophila [corrected

Loss- and gain-of-function studies have demonstrated a crucial role for the jing zinc finger transcription factor in neuronal and glial differentiation and survival in the embryonic central nervous system midline of Drosophila. Here, we have studied the role of jing during embryonic brain development. Proper jing function is required for the formation of the primary brain axon scaffold. In homozygous jing (3) mutant embryos the preoral commissure is not pioneered and never forms. Other axon pathways are pioneered but subsequently do not form properly, including the postoral tritocerebral commissure, the circumesophageal connectives and the pathways that connect the brain with the ventral nerve cord. To understand the cellular basis of the axon phenotype the jing expression pattern in the brain was characterized using a jing-lacZ enhancer trap. jing-lacZ enhancer trap expression occurs in glia and neurons in the brain midline and lateral clusters as determined by co-localization of the lacZ gene product with Repo and Castor, respectively. In addition, the jing-lacZ enhancer trap and the basic helix-loop-helix-PAS gene, single-minded ( sim), are expressed in the only glial midline cluster present in stage-14 wild-type embryos. jing function is required for the differentiation of Repo-, Castor- and Sim-positive cells in the embryonic brain as each of these populations contain a reduced number of cells in homozygous jing (3) mutant embryos. We further find that jing is required for neuronal and glial survival as repo- and castor-expressing cells undergo cell death in homozygous jing (3) mutant embryos, as revealed by double labeling with Tunel. Expression of jing in sim-expressing cells in the brain disrupts the entire axon scaffold but most significantly results in loss of the preoral and postoral tritocerebral commissures. In addition, circumesophageal connectives are repelled after expression of two copies of UAS- jing in sim-expressing cells, suggesting the activation of axon repellent molecules. Over-expression of sim in the brain is also associated with loss of preoral and postoral tritocerebral commissures. Therefore, the proper dosage of jing and sim in the brain is critical for the formation of the primary axon scaffold. These results show that an important role for jing in the developing brain is the regulation of neuronal and glial differentiation and survival.

Mouse rudimentary diastema tooth primordia are devoid of peripheral nerve fibers.

The tooth is a well-defined peripheral target organ for trigeminal nerve fibers. However, only limited information is available regarding pioneer axon guidance to the developing tooth target field. In rodents there is a toothless diastema region between incisors and molars that in the mouse maxilla contains three rudimentary tooth anlagen. Their development stop at the early bud stage when the primary nerve axons grow towards the developing first molar tooth germs. In order to provide background information for studies of regulatory mechanisms of pioneer axon guidance to the developing tooth germs, we investigated the distribution of nerve fibers in the mouse diastema tooth buds, and compared it to the axon growth to the maxillary and mandibular first molar tooth germs by immunohistochemical localization of peripherin and PGP9.5. Analysis of serial sections showed that trigeminal nerve fibers emerging from the trigeminal maxillary and mandibular nerve trunks started to grow towards the developing molar tooth germ at the early bud stage, and subsequently they diverged into buccal and lingual branches next to the condensed dental mesenchyme. During the cap stage, nerve fibers were observed around the tooth germ in the dental follicle region. In contrast, no nerve fibers were located in the vicinity of the diastema tooth primordia at any stage studied, nor did any nerve fibers appear to grow towards this region. Our results show that the development and subsequent disappearance of the diastema tooth primordia takes place without peripheral trigeminal innervation. The diastema tooth primordia may therefore be a useful model system for future studies on molecular regulatory mechanisms of pioneer axon guidance to the tooth germs, and possibly also for evolutionary studies of peripheral axon guidance mechanisms.

Regeneration of primary sensory axons into the adult rat spinal cord via a peripheral nerve graft bridging the lumbar dorsal roots to the dorsal column.

This study investigated the feasibility of using a peripheral nerve autograft (NAG) to promote and guide regeneration of sensory axons from the caudal lumbar dorsal roots to the rostral dorsal column following a lower thoracic cordotomy in adult rats. After a left hemicordotomy at the T13 vertebra level and ipsilateral L3 and L4 rhizotomies, a peripheral NAG (peroneal nerve) was connected to the distal roots stumps, then implanted into the left dorsal column 10 mm rostral to hemicordotomy site (n = 12). After surgery, all animals of the experimental group experienced complete anesthesia in their left hindlimb. Three months later, a slight response to nociceptive stimulation reappeared in L3 and/or L4 dermatomes in 6 of the 12 experimental animals. None of these animals exhibited self-mutilation. Nine months after surgery, we performed retrograde tracing studies by injecting horseradish peroxidase (HRP) into the left dorsal column 30 mm rostral to the NAG implantation site. In eight animals, we found HRP-stained neurons in the left L3 and/or L4 dorsal root ganglia (DRG). The mean number of HRP-stained neurons per DRG was 71 +/- 92 (range 2-259). In control groups, no HRP-stained neurons were found in L3 or L4 DRG. Histological analysis of the NAG showed evidence of axonal regeneration in all 8 animals with positive retrograde labeling of DRG neurons. However, we did not find a statistical correlation between the number of HRP-stained neurons and the degree of sensory recovery. This study demonstrates that an NAG joining dorsal roots to the dorsal column, thus shunting the original CNS-PNS junction, can support regeneration of central axons from DRG primary sensory neurons into the dorsal column over distances of at least 30 mm despite the inhibitory influence of the CNS white matter.

Laminar disorganisation of mitral cells in the olfactory bulb does not affect topographic targeting of primary olfactory axons

Primary olfactory neurons expressing the same odorant receptor protein typically project to topographically fixed olfactory bulb sites. While cell adhesion molecules and odorant receptors have been implicated in guidance of primary olfactory axons, the postsynaptic mitral cells may also have a role in final target selection. We have examined the effect of disorganisation of the mitral cell soma layer in mutant mice heterozygous for the β-subunit of platelet activating factor acetylhydrolase (Lis1 −/+) on the targeting of primary olfactory axons. Lis1 −/+ mice display abnormal lamination of neurons in the olfactory bulb. Lis1 −/+ mice were crossed with the P2-IRES-tau:LacZ line of transgenic mice that selectively expresses β-galactosidase in primary olfactory neurons expressing the P2 odorant receptor. LacZ histochemistry revealed blue-stained P2 axons that targeted topographically fixed glomeruli in these mice in a manner similar to that observed in the parent P2-IRES-tau:LacZ line. Thus, despite the aberrant organisation of postsynaptic mitral cells in Lis1 −/+ mice, primary olfactory axons continued to converge and form glomeruli at correct sites in the olfactory bulb. Next we examined whether challenging primary olfactory axons in adult Lis −/+ mice with regeneration would affect their ability to converge and form glomeruli. Following partial chemical ablation of the olfactory neuroepithelium with dichlobenil, primary olfactory neurons die and are replaced by newly differentiating neurons that project axons to the olfactory bulb where they converge and form glomeruli. Despite the aberrant mitral cell layer in Lis −/+ mice, primary olfactory axons continued to converge and form glomeruli during regeneration. Together these results demonstrate that the convergence of primary olfactory axons during development and regeneration is not affected by gross perturbations to the lamination of the mitral cell layer. Thus, these results support evidence from other studies indicating that mitral cells do not play a major role in the convergence and targeting of primary olfactory axons in the olfactory bulb.

Morphological characteristics and central projections of two types of interneurons in the visual pathway of Hermissenda.

The synaptic interactions between photoreceptors in the eye and second-order neurons in the optic ganglion of the nudibranch mollusk Hermissenda are well characterized. However, the higher-order neural circuitry of the visual system, consisting of cerebropleural interneurons that receive synaptic input from photoreceptors and project to pedal motor neurons that mediate visually guided behaviors, is only partially understood. In this report we have examined the central projections of two identified classes of cerebropleural interneurons that receive excitatory or inhibitory synaptic input from identified photoreceptors. The classification of the interneurons was based on both morphological and electrophysiological criteria. Type I interneurons received monosynaptic excitatory or inhibitory synaptic input from identified photoreceptors and projected to postsynaptic targets within the cerebropleural ganglion. Type II interneurons, characterized here for the first time, received polysynaptic excitatory or inhibitory synaptic input from identified photoreceptors and projected to postsynaptic targets in either the ipsilateral pedal ganglion or the contralateral cerebropleural ganglion. Type I interneurons exhibited unique intraganglionic projections to different regions of the cerebropleural ganglion, depending on whether they received excitatory or inhibitory synaptic input from identified photoreceptors. Type I interneurons that received monosynaptic excitatory input from identified B photoreceptors terminated near the cerebropleural commissure and had multiple regions of varicosities located at branches that projected from the primary axon. Type I interneurons that received monosynaptic inhibitory input from identified B photoreceptors projected to the anterior cerebropleural ganglion and exhibited varicosities localized to the terminal region of the primary axonal process. Type II interneurons that received polysynaptic inhibitory input from identified photoreceptors projected to the contralateral cerebropleural ganglion. Most type II interneurons that projected to the pedal ganglia received polysynaptic excitatory input from identified photoreceptors. These results indicate that there is at least one additional interneuron in the higher-order visual circuit between type I interneurons and pedal motor neurons responsible for the generation of phototactic locomotion in Hermissenda.

Heterogeneity in olfactory neurons in mouse revealed by differential expression of glycoconjugates.

Cell surface glycoconjugates have been implicated in the growth and guidance of subpopulations of primary olfactory axons. While subpopulations of primary olfactory neurons have been identified by differential expression of carbohydrates in the rat there are few reports of similar subpopulations in the mouse. We have examined the spatiotemporal expression pattern of glycoconjugates recognized by the lectin from Wisteria floribunda (WFA) in the mouse olfactory system. In the developing olfactory neuroepithelium lining the nasal cavity, WFA stained a subpopulation of primary olfactory neurons and the fascicles of axons projecting to the target tissue, the olfactory bulb. Within the developing olfactory bulb, WFA stained the synaptic neuropil of the glomerular and external plexiform layers. In adults, strong expression of WFA ligands was observed in second-order olfactory neurons as well as in neurons in several higher order olfactory processing centres in the brain. Similar, although distinct, staining of neurons in the olfactory pathway was detected with Dolichos biflorus agglutinin. These results demonstrate that unique subpopulations of olfactory neurons are chemically coded by the expression of glycoconjugates. The conserved expression of these carbohydrates across species suggests they play an important role in the functional organization of this region of the nervous system.

A Dual Role for the Zebrafish unplugged Gene in Motor Axon Pathfinding and Pharyngeal Development

On their way toward their synaptic targets, motor growth cones encounter multiple choice points, where they are confronted with trajectory choices. We have previously shown that the zebrafish unplugged gene acts as a somite-derived cue controlling pathway choice of primary motor axons. Here, we demonstrate that this trajectory choice is not exclusively controlled by a single unplugged-dependent process, but depends on the coordinated function of additional cues. We also show that secondary motor neurons, most similar to those in birds and mammals, depend on the unplugged gene to navigate a choice point, suggesting that primary and secondary motor neurons share common mechanisms controlling axonal path selection. Moreover, we show that the unplugged gene plays an additional role guiding secondary motor axons through a single segmental nerve. Finally, we report that unplugged larvae display a striking pharyngeal arch defect, consistent with a dual function of the unplugged gene in axonal guidance and cell motility.

A single cell analysis of engrailed expression in the early embryonic brain of the grasshopper Schistocerca gregaria: ontogeny and identity of the secondary headspot cells

The expression pattern of the segment polarity gene engrailed was studied at the single cell level in the protocerebrum of the early embryonic brain of the grasshopper Schistocerca gregaria, the neuromere containing the secondary headspot cells. The engrailed protein is first expressed in the protocerebrum at about 22% of embryogenesis by a group of identified neuroblasts bordering the antennal lobe. The number of immunoreactive neuroblasts increases up to 26% of embryogenesis and then rapidly declines so that by 30% only the three most posterior remain immunoreactive. These three neuroblasts become incorporated into the developing antennal lobe of the deutocerebrum. Subsequently, there is a progressive re-expression of the engrailed protein in the protocerebrum by the so-called six secondary headspot cells. These are the first born sibling progeny of three identified protocerebral neuroblasts which themselves expressed the engrailed protein prior to generating their lineages, and so represents a reacquisition of engrailed expression within identified clones. The secondary headspot cells are neurons which direct axonal processes into the developing optic tract and so contribute to the primary axon scaffold of the brain. From our analysis of their ontogeny, we conclude that the secondary headspot cells do not represent a segmental border in the brain.

Defining classes of spinal interneuron and their axonal projections in hatchling Xenopus laevis tadpoles.

Neurobiotin was injected into individual spinal interneurons in the Xenopus tadpole to discern their anatomical features and complete axonal projection patterns. Four classes of interneuron are described, with names defining their primary axon projection: Dorsolateral ascending and commissural interneurons are predominantly multipolar cells with somata and dendrites exclusively in the dorsal half of the spinal cord. Ascending interneurons have unipolar somata located in the dorsal half, but their main dendrites are located in the ventral half of the spinal cord. Descending interneurons show bigger variance in their anatomy, but the majority are unipolar, and they all have a descending primary axon. Dorsolateral commissural interneurons are clearly defined using established criteria, but the others are not, so cluster analysis was used. Clear discriminations can be made, and criteria are established to characterize the three classes of interneuron with ipsilateral axonal projections. With identifying criteria established, the distribution and axonal projection patterns of the four classes of interneuron are described. By using data from gamma-aminobutyric acid immunocytochemistry, the distribution of the population of ascending interneurons is defined. Together with the results from the axonal projection data, this allows the ascending interneuron axon distribution along the spinal cord to be estimated. By making simple assumptions and using existing information about the soma distributions of the other interneurons, estimates of their axon distributions are made. The possible functional roles of the four interneuron classes are discussed.

Antibody to the HNK-1 glycoepitope affects fasciculation and axonal pathfinding in the developing posterior lateral line nerve of embryonic zebrafish.

The HNK-1 glycoepitope, carried by many cell recognition molecules, is present in the developing posterior lateral line nerve and on other primary axons of zebrafish. To elucidate the function of HNK-1 in vivo, the antibody 412 to HNK-1 was injected into zebrafish embryos at 16 h post fertilization (hpf). The injected antibody bound specifically to axons carrying HNK-1. This treatment selectively affected the growth of either one or both posterior lateral line nerves in 39% of the experimental cases (13 of 33 animals), which was significantly more (P<0.0002) than in uninjected, vehicle injected, and non-immune IgG injected controls (1.2% of the animals; one of 85 animals), as assessed at 27 or 33 hpf. Other HNK-1 immunoreactive nerves, such as the ventral motor nerves were unaffected, indicating that antibody binding per se did not interfere with axon growth. The primordium of the posterior lateral line was not affected in its caudal migration and in depositing differentiating neuromasts along the trunk, showing that injections did not retard development and that initial formation of lateral line organs is probably independent of contact with nerve fibers. We suggest that the HNK-1 glycoepitope is an important modulator of embryonic nerve growth.

CRMP-2 induces axons in cultured hippocampal neurons.

In cultured hippocampal neurons, one axon and several dendrites differentiate from a common immature process. Here we found that CRMP-2/TOAD-64/Ulip2/DRP-2 (refs. 2-4) level was higher in growing axons of cultured hippocampal neurons, that overexpression of CRMP-2 in the cells led to the formation of supernumerary axons and that expression of truncated CRMP-2 mutants suppressed the formation of primary axon in a dominant-negative manner. Thus, CRMP-2 seems to be critical in axon induction in hippocampal neurons, thereby establishing and maintaining neuronal polarity.

Chemically and morphologically identifiable glomeruli in the rat olfactory bulb.

Primary olfactory neurons that express the same odorant receptor are distributed mosaically throughout the olfactory neuroepithelium lining the nasal cavity, yet their axons converge and form discrete glomeruli in the olfactory bulb. We previously proposed that cell surface carbohydrates mediate the sorting out and selective fasciculation of primary olfactory axons en route to glomeruli. If this were the case, then axons that terminate in the same glomerulus would express the same complement of cell surface carbohydrates. In this study, we examined the expression of a novel carbohydrate (NOC-3) on neural cell adhesion molecule in the adult rat olfactory system. NOC-3 was expressed by a subset of neurons distributed throughout the olfactory neuroepithelium. The axons of these neurons entered the nerve fiber layer and terminated in a subset of glomeruli. It is interesting to note that we identified three unusually large glomeruli in the lateral, ventrolateral, and ventromedial olfactory bulb that were innervated by axons expressing NOC-3. NOC-3-expressing axons sorted out and fasciculated into discrete fascicles prior to entering these glomeruli. Each of these glomeruli was in a topographically fixed position in the olfactory bulbs of the same animal as well as in different animals, and their lengths were approximately 10% of the total length of the bulb. They could be identified reliably by both their topographical position and their unique morphology. These results reveal that axons expressing the same cell surface carbohydrates consistently target the same topographically fixed glomeruli, which supports a role for these molecules in axon navigation in the primary olfactory nerve pathway.

Cultured olfactory ensheathing cells express nerve growth factor, brain-derived neurotrophic factor, glia cell line-derived neurotrophic factor and their receptors

In the primary olfactory pathway axons of olfactory neurons (ONs) are accompanied by ensheathing cells (ECs) as the fibres course towards the olfactory bulb. Ensheathing cells are thought to play an important role in promoting and guiding olfactory axons to their appropriate target. In recent years, studies have shown that transplants of ECs into lesions in the central nervous system (CNS) are able to stimulate the growth of axons and in some cases restore functional connections. In an attempt to identify a possible mechanism underlying EC support for olfactory nerve growth and CNS axonal regeneration, this study investigated the production of growth factors and expression of corresponding receptors by these cells. Three techniques immunohistochemistry, enzyme linked immunosorbent assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to assess growth factor expression in cultured ECs. Immunohistochemistry showed that ECs expressed nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and glial cell-line derived neurotrophic factor (GDNF). ELISA confirmed the intracellular presence of NGF and BDNF and showed that, compared to BDNF, about seven times as much NGF was secreted by ECs. RT-PCR analysis demonstrated expression of mRNA for NGF, BDNF, GDNF and neurturin (NTN). In addition, ECs also expressed the receptors trkB, GFRα-1 and GFRα-2. The results of the experiments show that ECs express a number of growth factors and that BDNF in particular could act both in a paracrine and autocrine manner.

EphB2 and two of its ligands have dynamic protein expression patterns in the developing olfactory system

Primary olfactory neurons are located in the olfactory neuroepithelium lining the nasal cavity. Their axons converge and form glomeruli with the dendrites of second-order neurons in the olfactory bulb. The molecular basis of primary olfactory axon guidance, targeting and subsequent arborisation is largely unknown. In this study we examined the spatio-temporal expression of the Eph receptor EphB2 and its ligands, ephrin-B1 and ephrin-B2, during development of the rat primary olfactory system. Unlike in other regions of the nervous system where receptor and ligand expression patterns are usually non-overlapping, EphB2, ephrin-B1 and ephrin-B2 were all expressed by primary and second-order olfactory neurons. In the embryonic animal we found that these three proteins had distinct and different expression patterns. EphB2 was first expressed at E18.5 by the perikarya of primary olfactory neurons. In contrast, ephrin-B1 was expressed from E13.5 and was localised to the axons of these cells up to E18.5 but was then restricted to the perikarya. Ephrin-B2, however, was expressed by olfactory ensheathing cells. EphB2, ephrin-B1 and ephrin-B2 were also expressed in the prenatal olfactory bulb and were restricted to the perikarya of mitral cells. In the post-natal olfactory bulb there was a shift in the localisation of both EphB2 and ephrin-B1 to the dendritic arborisations of mitral cells. The dynamic and tightly regulated spatio-temporal expression patterns of EphB2, ephrin-B1 and ephrin-B2 by specific olfactory cell populations suggest that these molecules have the potential to regulate important developmental events in the olfactory system.

Axons of sacral preganglionic neurons in the cat: II. Axon collaterals.

Axon collaterals were identified in 21 of 24 preganglionic neurons in the lateral band of the sacral parasympathetic nucleus of the cat. Following the intracellular injection of HRP or neurobiotin the axons from 20 of these neurons were followed and 53 primary axon collaterals were found to originate from unmyelinated segments and from nodes of Ranvier. Detailed mapping done in the five best labeled cells showed bilateral axon collaterals distributions up to 25,000 microm in length with 950 varicosities and unilateral distributions up to 12,561 microm with 491 varicosities. The axon collaterals appeared to be unmyelinated, which was confirmed at EM, and were small in diameter (average 0.3 microm). Varicosities were located mostly in laminae I, V, VII, VIII and X and in the lateral funiculi. Most varicosities were not in contact with visible structures but some were seen in close apposition to Nissl stained somata and proximal dendrites. Varicosities had average minor diameters of 1.3 microm and major diameters of 2.3 microm. Most were boutons en passant while 10-20% were boutons termineaux. EM revealed axodendritic and axoaxonic synapses formed by varicosities and by the axons between varicosities. It is estimated that the most extensive of these axon collaterals systems may contact over 200 spinal neurons in multiple locations. These data lead to the conclusion that sacral preganglionic neurons have multiple functions within the spinal cord in addition to serving their target organ. As most preganglionic neurons in this location innervate the urinary bladder, it is possible that bladder preganglionic neurons have multiple functions.