As part of a series of feasibility studies on culture of cryopreserved dissociated neurons from varied regions of the nervous system we report on cryopreservation and subsequent functional studies on dorsal root ganglion cells and their axonal outgrowth. Rat embryonic dorsal root ganglion cells were isolated and dissociated using standard procedures. A method was developed to cryopreserve aliquots of 200 K DRG neurons which could be subsequently thawed and plated at 5 K per well (PDL coated 96 well plates) and grown in Neurobasal medium with B27. No further supplementation was required for survival to 19+ days in vitro. In culture, cryopreserved DRG neurons showed the same size and neurochemical type distribution as freshly dissociated dorsal root ganglion neurons. Over the first few days neuronal cell bodies formed ganglionated clusters and fasiculated axon bundles, but with the addition of mitotic inhibitors DRG neurons and their axons remain dispersed. Cells were transfected with eGFP using Transmesseng...

The myelin sheath enables saltatory conduction by demarcating the axon into a small nodal region for excitation and an extended, insulated internodal region for efficient spread of passive current. This anatomical demarcation produces a dramatic heterogeneity in ionic fluxes during excitation, a classical example being the restriction of Na influx at the node. However, ionic fluxes are not restricted only to Na during excitation, as recent studies have revealed that action potentials also induce calcium influx into axons of mammalian optic nerves. Does calcium influx along a myelinated axon also show spatial heterogeneity during excitation, as in the case for sodium? To address this, we analyzed spatial profiles of axonal calcium transients during excitation by selectively staining axons with calcium indicators, and analyze the data by two models, one assuming uniform axonal influx and the other assuming discrete nodal influx, with a coefficient of axial calcium diffusion empirically determined in situ usi...

To establish the intricate neuronal network with necessary precision and reliability, one essential process is that neurons must correctly project their axons to their synaptic targets during embryonic development. In vivo, axonal tips (“growth cones”) navigate through a three-dimensional (3-D) environment, and the factors that can steer growth cones include ECM adhesion molecules, topographic features of the environment, and soluble chemotropic molecules. However, in most in vitro studies, neurons were confined on a uniform two-dimensional substrate, hence it was difficult to investigate the integration process in the presence of multiple contact cues. We have studied the growth cone 3-D navigation and axon decision-making using structured polydimethylsiloxane (PDMS) substrates combined with Matrigel. PDMS substrates with grooves/wells of various width and depth were fabricated by standard photolithography and soft lithography procedures, then coated with poly-D-lysine or laminin. Murine embryonic cortica...

Axons travel frequently in bundles to reach their target. After arriving at the target, axon terminals defasciculate, migrate to topographically defined positions, and form synapses with appropriate target neurons. Here we present evidence that the B-type receptors of the erythropoietin-producing hepatocellular (Eph) family and a ligand, ephrin-B3, influence hippocampal axon defasciculation. The EphB receptors are expressed in the hippocampus, and the ligand, ephrin-B3, is transcribed in the lateral septum, the major subcortical target of hippocampal neurons. Ephrin-B3 promotes adhesion of hippocampal neurons to the ligand-expressing substrates in vitro , and the loss of the receptor EphB2 abrogates the effects of ephrin-B3. In mice deficient in EphB2 and EphB3, many hippocampal axons remain in bundles. This phenotype was also observed in mice that were specifically deleted for the cytoplasmic domain of EphB2. These observations indicate that the EphB receptors and their ligand regulate hippocampal axon de...

CNS injury results in the formation of a glial scar consisting of reactive astrocytes, microglia, fibroblasts, and growth inhibitory proteoglycans (PG) that prevent regeneration. However, the CNS of immature animals is capable of regeneration following injury, in part because reactive gliosis is growth promoting rather than inhibitory and the glial scar does not form. This work seeks to elucidate why that difference arises by examining immature astrocytes (IA, <8 DIV) and mature astrocytes (MA, >35 DIV) on an inhibitory gradient of the PG, aggrecan, along with the growth promoting molecule, laminin. No type of mature cell, either neuron or glia, can penetrate the outermost rim in this highly inhibitory model of the scar. We have learned that IA are capable of enhanced growth, as compared to MA, on the aggrecan gradient. The IA display a more stellate, expanded morphology, while the MA assume a banded, compacted morphology similar to that in the glial scar. Additionally, IA are capable of digesting aggrecan...

NG2 cells are a novel, unique and major glial population in the mammalian central nervous system (CNS). NG2 cells have been best characterized as oligodendrocyte progenitor cells (OPCs) based on their expression of the OPC antigen, PDGF α receptor, and their ability to differentiate into oligodendrocytes in vitro. Although the NG2 proteoglycan has been shown to inhibit axonal growth, the role of NG2 cells in axonal growth has not been shown. In this study, we investigated the relationship between axons and NG2 cells during development. The effect of NG2 cells on neurite outgrowth and the spatial relationship between NG2 cells and growing axons were examined and compared with those of astrocytes in vitro and in vivo. Postnatal glia- hippocampal neuron cocultures were used for all the in vitro studies. Neurons cocultured with OPCs extended 2- to 2.5- fold longer axons than control neurons. Axonal stems and growth cones did not avoid but formed relative stable connections with NG2 cells. For in vivo studies, ...

Topographic cues are important for the orientation and growth of hippocampal neurons. Previously, we demonstrated that topographic signals could influence axon and dendrite orientation and extension at 14 DIV. The effects of topography on the growth and differentiation of dendrites and on synapse distribution were studied with time in culture. Topographical cues were presented as pillared arrays (widths = 0.5 and 2 µm; interpillar gaps = 1 - 5 µm) fabricated into silicon surfaces using standard photolithographic techniques and molded polystyrene replicas. Hippocampal neurons isolated from embryonic day 18 rats were seeded onto polylysine-treated surfaces and examined for as long as 21 DIV. Immunocytochemical detection of MAP-2 and βIII-tubulin-labeled processes was used to identify axons and dendrites. Images were imported into an automated 2-D tracing program to quantitatively describe process growth. Scanning electron microscopy (SEM) provided increased resolution for descriptions of neuronal growth rela...

The transcription factor Foxg1 (BF1) is expressed in the developing eye and brain. Mice lacking Foxg1 exhibit dramatic hypoplasia of the telencephalon and distortions to the eye. Remarkably, in spite of these defects the Foxg1 mutant retina does project axons to the brain . The aim of this study is to establish which features of retinal axon navigation are affected as a result of the absence of Foxg1. During normal development axons leave the retina and grow over the ventral surface of the hypothalamus towards the midline. At a defined point on the ventral surface of the hypothalamus (the optic chiasm) the axons either cross the midline and grow dorsally towards targets on the opposite side of the brain (crossed or contralateral projection) or do not cross and grow towards targets on the same side of the brain (uncrossed or ipsilateral projection). In rodents the ipsilateral projection is much smaller than the contralateral projection and originates from the ventral temporal part of the retina whereas the ...

BackgroundAnalogues of incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) provide various degrees of neuroprotection in sensory-motor diabetic polyneuropathy (DPN) as well as in other neurodegenerati...

The axon initial segment (AIS) is the cellular compartment where most neurons integrate synaptic inputs and generate their primary output signal: the action potential. However, it was only recently that its structure and molecular composition have been ...