Neurons-on-a-Chip: In Vitro NeuroTools

Neurons-on-a-Chip technology has been developed to provide diverse in vitro neuro-tools to study neuritogenesis, synaptogensis, axon guidance, and network dynamics. The two core enabling technologies are soft-lithography and microelectrode array technology. Soft lithography technology made it possible to fabricate microstamps and microfluidic channel devices with a simple replica molding method in a biological laboratory and innovatively reduced the turn-around time from assay design to chip fabrication, facilitating various experimental designs. To control nerve cell behaviors at the single cell level via chemical cues, surface biofunctionalization methods and micropatterning techniques were developed. Microelectrode chip technology, which provides a functional readout by measuring the electrophysiological signals from individual neurons, has become a popular platform to investigate neural information processing in networks. Due to these key advances, it is possible to study the relationship between the network structure and functions, and they have opened a new era of neurobiology and will become standard tools in the near future.     

Effects of neuropathic and non-neuropathic isomers of tricresyl phosphate and their microsomal activation on the production of axon-like processes by differentiating mouse N2a neuroblastoma cells

The aim of this work was to investigate the sublethal neuropathic effects of tricresyl phosphate (TCP: mixed isomers), triorthocresyl phosphate (TO:CP) and triparacresyl phosphate (TP:CP) on differentiating mouse N2a neuroblastoma cells. This was achieved by a combination of measurements of cell viability, axon outgrowth and the levels of cytoskeletal proteins detectable on western blots of extracts from cells induced to differentiate in the presence and absence of the compounds. In a time-course experiment TCP inhibited the outgrowth of axon-like processes following exposure times of 24 h or longer. Dose-response experiments indicated that TCP and TO:CP exhibited similar sustained levels of toxicity following both 24 and 48 h exposure, with no significant difference between their respective IC(50) values. By contrast, TP:CP demonstrated a transient effect on the outgrowth of axon-like processes, which was detectable after 24 but not 48 h of exposure. Isomer-specific patterns of toxicity were also evident at earlier time-points, with only the ortho isomer showing significant levels of inhibition of axon outgrowth following 4-8 h exposure. Probing of western blots with antibodies against cytoskeletal proteins indicated that the inhibition of axon outgrowth by these compounds was associated with a sustained reduction in the levels of phosphorylated neurofilament heavy chain. The inhibitory effect on axon outgrowth of TO:CP but not TP:CP was enhanced in the presence of a microsomal activation system. Since TO:CP is the most neuropathic of the isomers of TCP in vivo, differentiating N2a cells provide a useful cellular system for mechanistic studies of the neurodegenerative effects of this organophosphate.     

Calbindin D28k, parvalbumin, and calretinin immunoreactivity in the main and accessory olfactory bulbs of the gray short-tailed opossum, Monodelphis domestica

The vertebrate main and accessory olfactory bulbs (MOB and AOB) are the first synaptic sites in the olfactory pathways. The MOB is a cortical structure phylogenetically well conserved in its laminar structure and overall synaptic organization, while the AOB has significant species variation in size. In order to better understand signal processing in the two olfactory systems and the species differences, immunocytochemical staining and analysis were done of the neuronal expression patterns of the calcium-binding proteins calbindin D28k (CB), parvalbumin (PV), and calretinin (CR) in the MOB and AOB in a marsupial species, the gray short-tailed opossum, Monodelphis domestica. In the MOB, antibody to CB labeled periglomerular cells, superficial short axon cells / Van Gehuchten cells; antibody to PV labeled Van Gehuchten cells; and antibody to CR immunostained periglomerular cells, superficial short axon cells / Van Gehuchten cells, and granule cells. In the AOB, CB immunoreactivity was detected in periglomerular cells and a subpopulation of granule cells; antibody to PV labeled the superficial short axon cells / Van Gehuchten cells and granule cells; and antibody to CR labeled a small number of periglomerular cells, superficial short axon cells / Van Gehuchten cells, and granule cells. These results showed that the patterns of CB, PV, and CR expression differ in the opossum main and accessory olfactory bulbs and differ from that in other animal species. These varying patterns of neuronal immunostaining may be related to the different functions of the main and accessory olfactory bulbs and to the differing signal processing features.     

Presence of functional ATP and dinucleotide receptors in glutamatergic synaptic terminals from rat midbrain

Glutamatergic terminals from rat midbrain were characterized by immunolocalization of synaptophysin and the vesicular glutamate transporters, either VGLUT1 or VGLUT2. Terminals containing these markers represent about 31% (VGLUT1) and 16% (VGLUT2) of the total synaptosomal population. VGLUT1-positive glutamatergic terminals responded to ATP or P1,P 5-di(adenosine-5') pentaphosphate (Ap5A) with an increase in the intrasynaptosomal calcium concentration as measured by a microfluorimetric technique in single synaptosomes. Roughly 20% of the VGLUT1-positive terminals responded to ATP, 13% to Ap5A and 11% to both agonists. Finally 56% of the terminals labeled with the anti-VGLUT1 antibody did not show any calcium increase in response to ATP or Ap5A. A similar response distribution was also observed in the VGLUT2-positive terminals. The Ca2+ responses induced by ATP and Ap5A in the glutamatergic terminals could be selectively inhibited by pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 80 micro m) and P1,P 5-di(inosine-5') pentaphosphate (Ip5I, 100 nm), respectively. Both ATP and Ap5A, once assayed in the presence of extrasynaptosomal calcium, were able to induce a concentration-dependent glutamate release from synaptosomal populations, EC50 values being 21 micro m and 38 micro m for ATP and Ap5A, respectively. Specific inhibition of glutamate release was obtained with PPADS on the ATP effect and with Ip5I on the dinucleotide response, indicating that separate receptors mediate the secretory effects of both compounds.     

The myelin-axolemmal complex: biochemical dissection and the role of galactosphingolipids

Myelin-axolemmal interactions regulate many cellular and molecular events, including gene expression, oligodendrocyte survival and ion channel clustering. Here we report the biochemical fractionation and enrichment of distinct subcellular domains from myelinated nerve fibers. Using antibodies against proteins found in compact myelin, non-compact myelin and axolemma, we show that a rigorous procedure designed to purify myelin also results in the isolation of the myelin-axolemmal complex, a high-affinity protein complex consisting of axonal and oligodendroglial components. Further, the isolation of distinct subcellular domains from galactolipid-deficient mice with disrupted axoglial junctions is altered in a manner consistent with the delocalization of axolemmal proteins observed in these animals. These results suggest a paradigm for identification of proteins involved in neuroglial signaling.     

NF-M is an essential target for the myelin-directed "outside-in" signaling cascade that mediates radial axonal growth

Neurofilaments are essential for acquisition of normal axonal calibers. Several lines of evidence have suggested that neurofilament-dependent structuring of axoplasm arises through an "outside-in" signaling cascade originating from myelinating cells. Implicated as targets in this cascade are the highly phosphorylated KSP domains of neurofilament subunits NF-H and NF-M. These are nearly stoichiometrically phosphorylated in myelinated internodes where radial axonal growth takes place, but not in the smaller, unmyelinated nodes. Gene replacement has now been used to produce mice expressing normal levels of the three neurofilament subunits, but which are deleted in the known phosphorylation sites within either NF-M or within both NF-M and NF-H. This has revealed that the tail domain of NF-M, with seven KSP motifs, is an essential target for the myelination-dependent outside-in signaling cascade that determines axonal caliber and conduction velocity of motor axons.     

Novel bridge of axon-like processes of epithelial cells in the aboral sense organ of ctenophores

We describe by light and electron microscopy a novel structure in the aboral sense organ (apical organ) of cydippid (Pleurobrachia) and lobate (Mnemiopsis) ctenophores. An elevated bundle of long, thin, microtubule-filled processes arises from the apical ends of two groups of epithelial cells located on opposite sides of the apical organ along the tentacular plane of the body. This bundle of axon-like processes arches over the epithelial floor like a bridge, with branches at both ends running toward opposing pairs of ciliary balancers that are motile pacemakers for the rows of locomotory ciliary comb plates. The bridge in Pleurobrachia is approximately 40 microm long and 3-4 microm wide and consists of approximately 60 closely packed processes, 0.2-0.8 microm thick, containing vesicles and numerous microtubules running parallel to their long axes. There are approximately 30 epithelial cells in each of the two groups giving rise to the bridge and each cell forms a single process, so roughly half of the processes in the bridge must originate from cells on one side and diverge into branches to a pair of balancers on the opposite side of the apical organ. The 150-200 cilia in each balancer arise from morphologically complex cellular projections with asymmetric lateral extensions directed towards a fork of the bridge. Presynaptic triad structures and vesicles are found in this region but clear examples of synaptic contacts between bridge processes and balancer cells have not yet been traced. Cydippid larvae of Mnemiopsis have a conspicuous bridge along the tentacular plane of the apical organ. Beroid ctenophores that lack tentacles at all stages do not have a bridge. We discuss the possibility that the bridge is an electrical conduction pathway to balancers that coordinates tentacle-evoked swimming responses of ctenophores, such as global ciliary excitation.     

Noggin is required for correct guidance of dorsal root ganglion axons

Members of the bone morphogenetic protein family of secreted protein signals have been implicated as axon guidance cues for specific neurons in Caenorhabditis elegans and in mammals. We have examined axonal pathfinding in mice lacking the secreted bone morphogenetic protein antagonist Noggin. We have found defects in projection of several groups of neurons, including the initial ascending projections from the dorsal root ganglia, motor axons innervating the distal forelimb, and cranial nerve VII. The case of the dorsal root ganglion defect is especially interesting: initial projections from the dorsal root ganglion enter the dorsal root entry zone, as normal, but then project directly into the gray matter of the spinal cord, rather than turning rostrally and caudally. Explant experiments suggest that the defect lies within the spinal cord and not the dorsal root ganglion itself. However, exogenous bone morphogenetic proteins are unable to attract or repel these axons, and the spinal cord shows only very subtle alterations in dorsal-ventral pattern in Noggin mutants. We suggest that the defect in projection into the spinal cord is likely the result of bone morphogenetic proteins disrupting the transduction of some unidentified repulsive signal from the spinal cord gray matter.     

Slit antagonizes netrin-1 attractive effects during the migration of inferior olivary neurons

Inferior olivary neurons (ION) migrate circumferentially around the caudal rhombencephalon starting from the alar plate to locate ventrally close to the floor-plate, ipsilaterally to their site of proliferation. The floor-plate constitutes a source of diffusible factors. Among them, netrin-1 is implied in the survival and attraction of migrating ION in vivo and in vitro. We have looked for a possible involvement of slit-1/2 during ION migration. We report that: (1) slit-1 and slit-2 are coexpressed in the floor-plate of the rhombencephalon throughout ION development; (2) robo-2, a slit receptor, is expressed in migrating ION, in particular when they reach the vicinity of the floor-plate; (3) using in vitro assays in collagen matrix, netrin-1 exerts an attractive effect on ION leading processes and nuclei; (4) slit has a weak repulsive effect on ION axon outgrowth and no effect on migration by itself, but (5) when combined with netrin-1, it antagonizes part of or all of the effects of netrin-1 in a dose-dependent manner, inhibiting the attraction of axons and the migration of cell nuclei. Our results indicate that slit silences the attractive effects of netrin-1 and could participate in the correct ventral positioning of ION, stopping the migration when cell bodies reach the floor-plate.