GDNF and GFRalpha1 promote formation of neuronal synapses by ligand-induced cell adhesion

The establishment of synaptic connections requires precise alignment of pre- and postsynaptic terminals. The glial cell line-derived neurotrophic factor (GDNF) receptor GFRalpha1 is enriched at pre- and postsynaptic compartments in hippocampal neurons, suggesting that it has a function in synapse formation. GDNF triggered trans-homophilic binding between GFRalpha1 molecules and cell adhesion between GFRalpha1-expressing cells. This represents the first example of a cell-cell interaction being mediated by a ligand-induced cell adhesion molecule (LICAM). In the presence of GDNF, ectopic GFRalpha1 induced localized presynaptic differentiation in hippocampal neurons, as visualized by clustering of vesicular proteins and neurotransmitter transporters, and by activity-dependent vesicle recycling. Presynaptic differentiation induced by GDNF was markedly reduced in neurons lacking GFRalpha1. Gdnf mutant mice showed reduced synaptic localization of presynaptic proteins and a marked decrease in the density of presynaptic puncta, indicating a role for GDNF signaling in hippocampal synaptogenesis in vivo. We propose that GFRalpha1 functions as a LICAM to establish precise synaptic contacts and induce presynaptic differentiation.     

Transmembrane agrin regulates dendritic filopodia and synapse formation in mature hippocampal neuron cultures

The transmembrane isoform of agrin (Tm-agrin) is the predominant form expressed in the brain but its putative roles in brain development are not well understood. Recent reports have implicated Tm-agrin in the formation and stabilization of filopodia on neurites of immature central and peripheral neurons in culture. In maturing central neurons, dendritic filopodia are believed to facilitate synapse formation. In the present study we have investigated the role of Tm-agrin in regulation of dendritic filopodia and synaptogenesis in maturing cultures of rat hippocampal neurons. We did this by infecting the neurons with an RNAi lentivirus to deplete endogenous agrin during the developmental period when filopodia density on the dendritic arbor was high, and synapse formation was rapid. We found that dendritic filopodia density was markedly reduced, as was synapse density along dendrites. Moreover, synapse formation was more sharply reduced on dendrites of infected neurons contacted by uninfected axons than on uninfected dendrites contacted by infected axons. The results are consistent with a physiological role for Tm-agrin in the maturation of hippocampal neurons involving positive regulation of dendritic filopodia and consequent promotion of synaptogenesis, but also suggest a role for axonal agrin in synaptogenesis.     

Vomeronasal neurons promote synaptic formation on dendritic spines but not dendritic shafts in primary culture of accessory olfactory bulb neurons

To investigate the morphological changes of accessory olfactory bulb (AOB) neurons arising from pheromonal signals, a coculture system of AOB neurons and vomeronasal (VN) neurons had been established. Our previous study indicates that under coculture condition, the density of dendritic spines of an AOB neuron is less and the individual spine-head volume is larger than those under monoculture condition. In this study, to determine whether these differences in the dendrites of AOB neurons reflect the differences in synapse formation and synaptic properties, we observed these cultured cells by electron microscopy. Various synapses were observed under each culture condition. Synapses were classified on the basis of their postsynaptic structure and the size of postsynaptic density (PSD) was measured. Under the coculture condition with VN neurons, synapses on dendritic spines, which formed between AOB neurons, were observed frequently. In contrast, many synapses were formed on dendritic shafts under monoculture condition. The PSD of asymmetrical synapses on the spines under coculture condition was larger than that under monoculture condition. Moreover, some dendrodendritic reciprocal synapses were found only in coculture. We confirmed synapse formation between VN axons and AOB dendrites by immunohistochemical electron microscopy; thus, the characteristics of synapses between AOB neurons are considered to be modified by the synaptic contacts with VN axons.     

Axonal growth regulation of fetal and embryonic stem cell-derived dopaminergic neurons by Netrin-1 and Slits

The physical restoration of dopamine circuits damaged or lost in Parkinson disease by implanting embryonic stem (ES)-derived cells may become a treatment. It is critical to understand responses of ES-derived dopamine (DA) neurons to guidance signals that determine axonal path and targeting. Using a collagen gel culture system, we examined effects of secreted molecules Netrin-1 and Slits on neurite outgrowth of fetal DA neurons and murine ES-differentiated DA neurons. We have previously shown that fetal DA neurons express DCC and Robo1/2 receptors and that Netrin-1 and Slit2 function as an attractant and a repellent for DA neurite outgrowth. In the present study, we observe that both Slit1 and Slit3 repel and inhibit neurite growth of fetal DA neurons. Here, we also demonstrate that ES-differentiated neurons including DA neurons express the Netrin receptor DCC and Slit receptor Robo proteins. In the gel culture system of ES cells, Netrin-1 promoted neurite outgrowth mediated by DCC receptor, and Slit1 and Slit3 were inhibitory for neurite outgrowth through Robo receptors. Slit2 appeared to exert inhibitory as well as repulsive effects in the coculture assay. However, unlike fetal DA neurites, no directed neurite outgrowth was observed in the cocultures of ES-derived DA neurons with Netrin-1-, Slit1-, and Slit3-producing cells. The findings suggest that ES-derived DA neurons generated by current protocols can respond to guidance cues in vitro in a similar manner to fetal cells but also exhibit distinct responses. This may result from developmental differences generated by present in vitro methods of cell patterning or conditioning during ES cell differentiation.     

Ultrastructural evidence for hippocampal target cell-mediated trophic effects on septal cholinergic neurons in reaggregating cell cultures

We have previously demonstrated at the light microscopic level that when embryonic day-15 septal neurons are co-cultured for 21 days with their target cells from the hippocampus, increased numbers of septal cholinergic neurons are present as compared with co-cultures employing cells from the non-target cerebellum. In addition, fine varicose axon-like cholinergic fibers are found to be associated with the hippocampal cells but not with cerebellar cells. We now provide ultrastructural evidence for hippocampal target cell-enhanced cholinergic neuronal survival, axonal proliferation, and synapse formation in this culture system. Dissociated cell suspensions from septal, hippocampal, and cerebellar areas were obtained from 15-day mouse embryos; and hippocampal and cerebellar cells were internally labeled with rhodamine-conjugated wheat germ agglutinin. Combinations of septal and hippocampal cells, and septal and cerebellar cells were allowed to reaggregate in rotation mediated culture for either 15 or 21 days. The reaggregates were then fixed, embedded, sectioned, and processed for acetylcholinesterase-positive acetylcholinesterase-positive cells and fibers, and under fluorescence to locate rhodamine-labeled cell populations. Representative reaggregate profiles were then re-embedded for electron microscopic examination. In both types of reaggregates, either labeled hippocampal target or cerebellar non-target cells segregated from the septal cells so that areas containing each of the respective cell populations could be studied. In sections of septal-hippocampal reaggregates from 15-day cultures, 571 out of 665 (85%) cholinergic neurons examined were intact, whereas 15% of the cells showed some ultrastructural features of degeneration. Similarly, at day 21, 297 out of 335 (88%) of the cholinergic neurons were intact. In sections of septal-cerebellar reaggregates from 15-day cultures, 473 out of 572 (83%) cholinergic neurons were intact. By day 21 of culture, however, only 15 out of 110 (14%) cholinergic neurons examined were intact from the septal-cerebellar reaggregates. In areas of septal-hippocampal reaggregates occupied by rhodamine-labeled hippocampal cells, profiles of acetylcholinesterase-labeled axons were identified, and synaptic specializations were observed between cholinergic terminals and dendrites as well as somata of hippocampal target cells. In contrast, areas of septal-cerebellar reaggregates occupied by rhodamine-labeled cerebellar cells were devoid of cholinergic fibers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Oriented Schwann Cell Monolayers for Directed Neurite Outgrowth

Schwann cells are an important component of the peripheral nervous system and participate in peripheral nerve regeneration. They create a supportive environment for neurite outgrowth by releasing trophic factors and up-regulating permissive molecules on their surface. In addition, Schwann cells are able to self-organize into linear arrays in vitro and in vivo, suggesting a possible role in neurite guidance. Previously, we showed that Schwann cell placement and orientation in subconfluent cultures can be controlled using microlithographically patterned laminin substrates (Thompson, D. M., and H. M. Buettner. Tissue Eng. 7(3):247-266, 2001). In the current study, these substrates were used to create oriented Schwann cell monolayers. Both Schwann cell orientation and coverage were quantified in response to seeding density, culture medium, and micropattern dimensions. In serum-free medium, increasing the seeding density yielded a linear increase in coverage of the substrate area but decreased cell alignment. In an alternate approach, Schwann cells were first seeded in serum-free medium at moderate seeding density, allowed to align, then expanded in serum-containing growth medium. This produced complete coverage without large seeding densities while preserving alignment to the micropattern. Alignment and coverage were unaffected by micropattern dimensions. This work provides a useful methodology for investigating Schwann cell guidance effects on growing neurites.     

Guidance of glial cell migration and axonal growth on electrospun nanofibers of poly-epsilon-caprolactone and a collagen/poly-epsilon-caprolactone blend

Our long-term goal is to develop an artificial implant as a conduit for axonal regeneration after peripheral nerve injury. In this study, biodegradable, aligned poly-epsilon-caprolactone (PCL) and collagen/PCL (C/PCL) nanofibers designed as guidance structures were produced by electrospinning and tested in cell culture assays. We compared fibers of 100% PCL with fibers consisting of a 25:75% C/PCL blend. To test their biocompatibility, assays of cell adhesion, survival, migration, effects on cell morphology, axonal growth and axonal guidance were performed. Both types of eletrospun fibers supported oriented neurite outgrowth and glial migration from dorsal root ganglia (DRG) explants. Schwann cell migration, neurite orientation, and process formation of Schwann cells, fibroblasts and olfactory ensheathing cells were improved on C/PCL fibers, when compared to pure PCL fibers. While the velocity of neurite elongation from DRG explants was higher on PCL fibers, analysis of isolated sensory neurons showed significantly better axonal guidance by the C/PCL material. The data demonstrate that electrospun fibers composed of a collagen and PCL blend represent a suitable substrate for supporting cell proliferation, process outgrowth and migration and as such would be a good material for artificial nerve implants.     

Gradient micropattern immobilization of EGF to investigate the effect of artificial juxtacrine stimulation

To investigate the concentration effects of immobilized biosignal molecules by microscopic observation, a gradient micropattern immobilization technique using a photomask was devised. Epidermal growth factor (EGF) was chosen as the biosignal molecule and was conjugated with photoreactive polyallylamine. The EGF derivative was immobilized onto a polystyrene plate by UV irradiation in the presence of a gradient-micropattern photomask. Gradient micropattern immobilization of the EGF derivative on the polystyrene plate was confirmed by immunostaining with anti-EGF antibody. Chinese hamster ovary cells overexpressing EGF receptors were cultured on the micropatterned plate. Growth enhancement was observed only in dense EGF derivative immobilized regions. The present technique is useful for the investigation of concentration-dependent effects of immobilized biosignal molecules.     

The protocadherin Flamingo is required for axon target selection in the Drosophila visual system

Photoreceptor neurons (R cells) in the Drosophila visual system elaborate a precise map of visual space in the brain. The eye contains some 750 identical modules called ommatidia, each containing eight photoreceptor cells (R1-R8). Cells R1-R6 synapse in the lamina; R7 and R8 extend through the lamina and terminate in the underlying medulla. In a screen for visual behavior mutants, we identified alleles of flamingo (fmi) that disrupt the precise maps elaborated by these neurons. These mutant R1-R6 neurons select spatially inappropriate targets in the lamina. During target selection, Flamingo protein is dynamically expressed in R1-R6 growth cones. Loss of fmi function in R cells also disrupts the local pattern of synaptic terminals in the medulla, and Flamingo is transiently expressed in R8 axons as they enter the target region. We propose that Flamingo-mediated interactions between R-cell growth cones within the target field regulate target selection.     

Observations on the migratory behaviour of Schwann cells from adult peripheral nerve expiant cultures

Summary The migration of Schwann cells from adult sciatic nerve explant cultures has been examined by time-lapse photomicrography. Analysis of Schwann cell migratory behaviour indicates that the initial outwandering by individual Schwann cells was random. Although chance cell-cell contacts resulted in temporary immobilization of pairs of cells, stable multicellular structures did not form during this initial phase. As local cell densities increased, Schwann cells assembled networks within which Schwann cell movement continued to be observed.A second form of Schwann cell outgrowth was observed from degenerating fibres in which arrays of highly oriented Schwann cells migrated away from their basal laminai tubes onto the culture dish.These observations of Schwann cell random migration, network self-assembly and coordinated extratubal migration are considered to highlight aspects of Schwann cell behaviour, independent of axonal influences, which may have relevance to their role in peripheral nerve repair following nerve section.     

The development of inhibitory synaptic specializations in the mouse deep cerebellar nuclei.

Using confocal laser scanning microscopy and immunohistochemistry, this study shows the complete morphological development of GABAergic synaptic contacts between Purkinje cells and neurons of the deep cerebellar nuclei of the mouse. Firstly, presynaptic varicosities visualized with antibodies against synaptophysin, synapsin or glutamic acid decarboxylase, were detected when the postsynaptic GABA(A) receptors were not yet aggregated in the membrane but had a diffuse cytoplasmic distribution, which indicated a lead in maturation of presynaptic terminals over target cells. Secondly, receptor aggregates developed suddenly after an initial week of diffuse expression and these clusters matured into more numerous and larger synaptic aggregates. During this postsynaptic maturation, the presynaptic varicosities develop into numerous and well-defined spots. As soon as both pre- and postsynaptic clusters were detectable, these sites are always colocalized. We therefore consider the aggregation of postsynaptic receptor during development as a landmark of synapse formation. Our observations are consistent with a developmental model in which the presynaptic neuron differentiates its axon before the target neuron expresses the mature form of its receptors on the membrane. The presynaptic neuron can therefore instruct the target neuron about the distribution and aggregation of the postsynaptic receptors at the synapse.     

The nigrotectal projection in the cat: an electron microscope autoradiographic study

Recent evidence indicates that the nigrotectal tract plays an important role in regulating the premotor responses of cells in the in the intermediate gray layer of the superior colliculus. The purpose of the present study was to characterize the ultrastructure of nigrotectal terminals and of their postsynaptic targets in the intermediate gray layer. Nigrotectal terminals were identified in the electron microscope by labeling them autoradiographically, following injections of tritiated proline into the substantia nigra pars reticulata. The majority of nigrotectal terminals contain a high proportion of pleomorphic vesicles and form symmetrical synaptic contacts. Most of these terminals synapse with small dendritic profiles (2.00 micron +/- 0.83 SD), which may be the distal dendrites of neurons in the intermediate gray layer. Less than 10% of the labeled contacts are made with cell bodies or initial axonal segments.     

Specificity of interneuronal connections.

A fundamental problem of neurobiological research is how specific connections between individual neurons are established and maintained. In this report different levels of neuronal specificity are described. Some neuronal populations display region specificity, but within the target region they establish synapses with a variety of neurons. A characteristic feature of the afferent innervation of hippocampal neurons is that many fibers terminate in a laminated fashion. Such a layer specificity is known for the afferents from the entorhinal cortex and for the mossy fibers. The entorhinal afferents terminate in the outer molecular layer of the fascia dentata and in the stratum lacunosum-moleculare of the hippocampus proper. The mossy fibers display both region specificity and layer specificity: they form numerous synapses in hippocampal region CA3 but never invade CA1; in CA3 they are restricted to stratum lucidum. An extremely high degree of neuronal specificity is observed in the case of the axo-axonic or chandelier cells. The axons of these neurons specifically terminate on the axon initial segments of projection neurons in the neocortex, hippocampus and fascia dentata. Thus, these cells not only display a target cell specificity but a selectivity for a distinct portion of the target cell's membrane. Some of the factors that contribute to these different levels of neuronal specificity are briefly discussed. Positional cues as well as diffusible molecules from the target region may guide the outgrowing growth cone to its target. Molecular interactions between pre- and postsynaptic membranes, the functional load of the synaptic contact, and the selective death of a number of neurons and synapses further determine the specificity of interneuronal connections.(ABSTRACT TRUNCATED AT 250 WORDS)     

The distribution of microtubule-associated protein 2 changes when dendritic growth is induced in rat sympathetic neurons in vitro

We have examined the distribution of microtubule-associated protein 2 in embryonic rat sympathetic neurons grown under culture conditions that alter morphological development. Cultures were established in serum-free medium. After 8 days some were transferred to a serum-containing medium, which promotes dendritic development. Sister cultures were maintained in serum-free medium, which inhibits dendritic growth but permits normal axonal development. After growth for 2-6 weeks in serum-containing medium, sympathetic neurons were multipolar, with short, tapering dendrites and long, thin axons. Intense immunoreactivity for microtubule-associated protein 2 was observed in the somata and dendrites of all neurons, but there was little or no staining of the network of axonal processes that ran between cell somata. When the morphology of individual cells was assessed by injection of fluorescent dye before immunostaining, we found that staining for microtubule-associated protein 2 extended to the distal tips of the dendrites while the axon was essentially unstained, even in its proximal portion. Neurons from sister cultures that were not exposed to serum were usually unipolar, having only an axon. Under these conditions microtubule-associated protein 2 was also expressed, but its distribution was altered: intense immunostaining for microtubule-associated protein 2 was present in axons as well as somata. Staining in axons could sometimes be traced for several millimeters, but, since unstained segments of axons were also common, microtubule-associated protein 2 probably was not present throughout the entire axonal arborization. These results show that the expression of microtubule-associated protein 2 is not of itself sufficient to induce the formation of dendrites. Despite the association of microtubule-associated protein 2 with the axonal cytoskeleton, the light microscopic morphology of the axons was not obviously altered.     

Sodium-dependent potassium channels of a Slack-like subtype contribute to the slow afterhyperpolarization in lamprey spinal neurons

Synaptic vesicles aggregate at the presynaptic terminal during synapse formation via mechanisms that are poorly understood. Here we have investigated the role of the putative calcium sensor synaptotagmin I in vesicle aggregation during the formation of soma–soma synapses between identified partner cells using a simple in vitro synapse model in the mollusc Lymnaea stagnalis . Immunocytochemistry, optical imaging and electrophysiological recording techniques were used to monitor synapse formation and vesicle localization. Within 6 h, contact between appropriate synaptic partner cells up-regulated global synaptotagmin I expression, and induced a localized aggregation of synaptotagmin I at the contact site. Cell contacts between non-synaptic partner cells did not affect synaptotagmin I expression. Application of an human immunodeficiency virus type-1 transactivator (HIV-1 TAT)-tagged peptide corresponding to loop 3 of the synaptotagmin I C2A domain prevented synaptic vesicle aggregation and synapse formation. By contrast, a TAT-tagged peptide containing the calcium-binding motif of the C2B domain did not affect synaptic vesicle aggregation or synapse formation. Calcium imaging with Fura-2 demonstrated that TAT–C2 peptides did not alter either basal or evoked intracellular calcium levels. These results demonstrate that contact with an appropriate target cell is necessary to initiate synaptic vesicle aggregation during nascent synapse formation and that the initial aggregation of synaptic vesicles is dependent on loop 3 of the C2A domain of synaptotagmin I.     

Corticostriatal combinatorics: the implications of corticostriatal axonal arborizations

The complete striatal axonal arborizations of 16 juxtacellularly stained cortical pyramidal cells were analyzed. Corticostriatal neurons were located in the medial agranular or anterior cingulate cortex of rats. All axons were of the extended type and formed synaptic contacts in both the striosomal and matrix compartments as determined by counterstaining for the mu-opiate receptor. Six axonal arborizations were from collaterals of brain stem-projecting cells and the other 10 from bilaterally projecting cells with no brain stem projections. The distribution of synaptic boutons along the axons were convolved with the average dendritic tree volume of spiny projection neurons to obtain an axonal innervation volume and innervation density map for each axon. Innervation volumes varied widely, with single axons occupying between 0.4 and 14.2% of the striatum (average = 4%). The total number of boutons formed by individual axons ranged from 25 to 2,900 (average = 879). Within the innervation volume, the density of innervation was extremely sparse but inhomogeneous. The pattern of innervation resembled matrisomes, as defined by bulk labeling and functional mapping experiments, superimposed on a low background innervation. Using this sample as representative of all corticostriatal axons, the total number of corticostriatal neurons was estimated to be 17 million, about 10 times the number of striatal projection neurons.     

Axonal sprouting following lesions of the rat substantia nigra

Parkinson's disease is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Symptoms do not appear until most nigral neurons are lost, implying that compensatory mechanisms are present. Sprouting has been proposed as one of these mechanisms. This study quantified the extent of compensatory axonal sprouting following injury of dopaminergic neurons within the substantia nigra pars compacta. Specifically, the extent of the axonal arbour and axonal varicosity morphology was measured after partial destruction (with 6-hydroxydopamine) of the substantia nigra of the adult male rat. Four months later, the substantia nigra was injected with the anterograde neuronal tracer dextran-biotin to trace the full extent of individual axons. An unbiased estimate of neuron number was performed in each animal. This demonstrated nigral neuronal loss ranging from 10 to 90% on the side that received the injection whilst a 7% reduction was observed in the side contralateral to the lesion. Coincident with this loss, some nigral neurons lose tyrosine hydroxylase expression. Vigorous axonal sprouting was observed in the terminal arbours of lesioned animals and was associated with an increased axonal varicosity size. Axonal varicosities and branching points were primarily confined to the dorsal 1.5mm of the caudate-putamen, an area predominantly innervated by nigral neurons. It appears that dopaminergic neurons were responsible for this sprouting because the density of dopamine transporter immunoreactive varicosities in the caudate-putamen was maintained until about a 70% loss of neurons. It was concluded that substantial compensation in the form of sprouting and new dopaminergic synapse formation occurs following lesions of the substantia nigra pars compacta.