Long-range axonal calcium sweep induces axon retraction.

Axon guidance molecules trigger a cascade of local signal in growth cones and evoke various morphologic responses, including axon attraction, repulsion, elongation, and retraction. However, little is known about whether subcellular compartments, other than axonal growth cones, control axon outgrowth. We found that in isolated dentate granule cells, local application of glutamate to the somatodendritic areas, but not the axon itself, induced rapid axon retraction, during which a calcium wave propagated from the somata to the axon terminals. The calcium wave and axon retraction were both inhibited by blockade of voltage-sensitive calcium channels and intracellular calcium dynamics. A combination of perisomatic application of calcium ionophore and depolarizing current injection induced axonal calcium sweep and axon retraction. Thus, perisomatic environments can modulate axon behavior through long-range intracellular communication.

Cryopreservation of granule cells from the postnatal rat hippocampus.

Although primary cultures of neurons are essential methods for cell biological and pharmacological researches, many animals must be sacrificed for each experiment. Here we introduce a novel system to cryopreserve hippocampal granule cells (GCs) prepared from postnatal rats. Being thawed after as long as 60 days of cryopreservation, GCs expressed the mature neuronal marker MAP-2 and elongated single tau-1-positive axons and multiple tau-1-negative dendrites. These properties closely resembled intact GCs in primary cultures, providing the advantage of being able to repeatedly prepare stable cultures with a single sacrifice of animals.

Nitric oxide/cyclic guanosine monophosphate-mediated growth cone collapse of dentate granule cells.

Controlling axon and dendrite elongation is critical in developing precise neural circuits. Using isolated cultures of dentate granule neurons, we established an experimental system that can simultaneously monitor the behaviors of axonal and dendritic outgrowth. Our previous study shows that axons and dendrites respond differentially to manipulated cyclic adenosine monophosphate signaling, but we report here that cyclic guanosine monophosphate exerts similar effects on axons and dendrites; that is, both axonal and dendritic growth cones collapsed after activation of cyclic guanosine monophosphate signaling. In addition, nitric oxide donor-induced growth-cone collapse was prevented by the inhibition of cyclic guanosine monophosphate signaling, and this effect again did not differ between axons and dendrites. Thus, unlike cyclic adenosine monophosphate, cyclic guanosine monophosphate modulates extending axons and dendrites in a similar manner.

Soluble guanylyl cyclase inhibitor prevents Sema3F-induced collapse of axonal and dendritic growth cones of dentate granule cells.

Controlling axon and dendrite elongation is critical in developing precise neural circuits. Using isolated cultures of dentate granule neurons, we succeeded in simultaneously monitoring the behaviors of axonal and dendritic outgrowth. Our previous study shows that cAMP contributes differentially to Sema3F-induced responses of axons and dendrites, but we report here that the cGMP modulation does not have such a striking axo-dendritic difference. Treatment with Sema3F induced collapse of about 90% growth cones, and pretreatment with 1 muM LY83583, an inhibitor of soluble guanylyl cyclase, partially alleviated the collapse of both axons and dendrites. Thus, unlike cAMP, cGMP modulates axonal and dendritic extension in a similar manner.

cAMP differentially regulates axonal and dendritic development of dentate granule cells.

Neurite polarity is a morphological characteristic of dentate gyrus granule cells, which extend axons to the hilar region and dendrites in the opposite direction, i.e. to the molecular layer. This remarkable polarity must require a differential system for axon and dendrite guidance. Here, we report that the axon and dendrites of a granule cell are differentially responsive to cAMP. In developing cultures of dispersed granule cells, dendritic growth cones were increased in number after pharmacological activation of cAMP signaling and decreased after blockade of cAMP signaling. Activation of cAMP signaling antagonized dendritic collapse induced by the potent repellents Sema3F and glutamate. In contrast to dendrites, axons were protected from Sema3F-induced collapse when cAMP signaling was inhibited. Axonal and dendritic growth cones both expressed type 1 adenylyl cyclase, but only axons showed a cAMP increase in response to Sema3F, and the elevated cAMP was sufficient to collapse axonal growth cones. Thus, the axons and dendrites of dentate granule cells differ in the regulation of cAMP levels as well as responsiveness to cAMP. cAMP may be crucial for shaping the information flow polarity in the dentate gyrus circuit.

Environmental control of the survival and differentiation of dentate granule neurons.

Dentate granule cells (DGCs) and their microcircuits have been implicated in hippocampus-dependent memory encoding and epileptogenesis. Little is known about how the proper maturation of DGCs is determined by their intrinsic programs or external factors during development. In order to explore this, we dispersed premature DGCs on living hippocampal slices. Here we report that the survival and network formation of DGCs are supported by local cues present in the dentate gyrus ex vivo. The density of surviving DGCs was almost uniform throughout the host slices 12 h after implantation but gradually became heterogenous across substrata, with the cells engrafted onto the stratum granulosum scoring the highest rate of survival. The mossy fiber axons arising from DGCs growing on this substratum were properly guided towards CA3, whereas other misplaced DGCs exhibited heterotopic axon projection. In particular, about half of the axons originating from the hilus were misguided into the molecular layer, which resembles the supragranular mossy fiber sprouting seen in epileptic disorders. These results suggest that local environmental factors influence the cell adhesion, neurite polarization and axon guidance of DGCs.

Activity-evoked capacitative Ca2+ entry: implications in synaptic plasticity.

The Ca2+ influx controlled by intracellular Ca2+ stores, called store-operated Ca2+ entry (SOC), occurs in various eukaryotic cells, but whether CNS neurons are endowed with SOC capability and how they may operate have been contentious issues. Using Ca2+ imaging, we present evidence for the presence of SOC in cultured hippocampal pyramidal neurons. Depletion of internal Ca2+ stores by thapsigargin caused intracellular Ca2+ elevation, which was prevented by SOC channel inhibitors 2-aminoethoxydiphenyl borate (2-APB), SKF96365, and La3+. Interestingly, these inhibitors also accelerated the decay of NMDA-induced Ca2+ transients without affecting their peak amplitude. In addition, SOC channel inhibitors attenuated tetanus-induced dendritic Ca2+ accumulation and long-term potentiation at Schaffer collateral-CA1 synapses in hippocampal slice preparations. These data suggest a novel link between ionotropic receptor-activated SOC and neuroplasticity.