Release of ATP from astrocytes is required for Ca2+ wave propagation among astrocytes and for feedback modulation of synaptic functions. However, the mechanism of ATP release and the source of ATP in astrocytes are still not known. Here we show that incubation of astrocytes with FM dyes leads to selective labelling of lysosomes. Time-lapse confocal imaging of FM dye-labelled fluorescent puncta, together with extracellular quenching and total-internal-reflection fluorescence microscopy (TIRFM), demonstrated directly that extracellular ATP or glutamate induced partial exocytosis of lysosomes, whereas an ischaemic insult with potassium cyanide induced both partial and full exocytosis of these organelles. We found that lysosomes contain abundant ATP, which could be released in a stimulus-dependent manner. Selective lysis of lysosomes abolished both ATP release and Ca2+ wave propagation among astrocytes, implicating physiological and pathological functions of regulated lysosome exocytosis in these cells.

Distinct molecules are segregated into somatodendritic and axonal compartments of polarized neurons, but mechanisms underlying the development and maintenance of such segregation remain largely unclear. In cultured hippocampal neurons, we observed an ankyrin G- and F-actin-dependent structure that emerged in the cytoplasm of the axon initial segment (AIS) within 2 days after axon/dendrite differentiation, imposing a selective filter for diffusion of macromolecules and transport of vesicular carriers into the axon. Axonal entry was allowed for KIF5-driven carriers of synaptic vesicle protein VAMP2, but not for KIF17-driven carriers of dendrite-targeting NMDA receptor subunit NR2B. Comparisons of transport rates between chimeric forms of KIF17 and KIF5B, with the motor and cargo-binding domains switched, and between KIF5 loaded with VAMP2 versus GluR2 suggest that axonal entry of vesicular carriers depends on the transport efficacy of KIF-cargo complexes. This selective AIS filtering may contribute to preferential trafficking and segregation of cellular components in polarized neurons.

A critical step in neuronal development is the formation of axon/ dendrite polarity, a process involving symmetry breaking in the newborn neuron. Local self-amplifying processes could enhance and stabilize the initial asymmetry in the distribution of axon/dendrite determinants, but the identity of these processes remains elusive. We here report that BDNF, a secreted neurotrophin essential for the survival and differentiation of many neuronal populations, serves as a self-amplifying autocrine factor in promoting axon formation in embryonic hippocampal neurons by triggering two nested positivefeedback mechanisms. First, BDNF elevates cytoplasmic cAMP and protein kinase A activity, which triggers further secretion of BDNF and membrane insertion of its receptor TrkB. Second, BDNF/TrkB signaling activates PI3-kinase that promotes anterograde transport of TrkB in the putative axon, further enhancing local BDNF/TrkB signaling. Together, these self-amplifying BDNF actions ensure stable elevation of local cAMP/protein kinase A activity that is critical for axon differentiation and growth.axon initiation and growth | cAMP/protein kinase A elevation | neurotrophin autocrine loops | Trk redistribution T he most distinct cellular feature of the neuron is its polarized structure, consisting of a single long axon and many short and highly branched dendrites. This polarized structure is essential for two primary neuronal functions: the reception and integration of synaptic inputs at the dendrite, and the conduction and delivery of output signals to other cells via the axon. For the formation of this polarity, the postmitotic neuron may have inherited an asymmetry in the activity or distribution of specific cellular components that could trigger local axon/dendrite differentiation. Alternatively, extracellular environment may provide an asymmetric signal in the neuron that specifies its polarity. Such a polarization process may also occur spontaneously, as shown by isolated embryonic hippocampal neurons cultured on a uniform substrate (1). These neurons undergo polarization through distinct stages-the formation of dynamic lamellipodia around the cell periphery (stage 1) within a few hours after plating, the appearance of several neurites of similar morphology (stage 2) after 12-16 h in culture, and differentiation of one axon and multiple dendrites (stage 3) within 1-1.5 d. Recent studies have shown that local elevation of cAMP and protein kinase A (PKA) activity, acting through downstream activation of LKB1 and SAD kinases (2-4), is a critical early event in axon initiation. In cultured developing hippocampal neuron, we also showed that extracellular applied BDNF promotes axon differentiation by elevating cAMP in the undifferentiated neurites (5). In the present study, we further showed that endogenous BDNF in developing hippocampal neurons promotes axon initiation and growth by locally elevating and stabilizing cAMP/PKA activity through self-amplifying autocrine actions.As a member of the neurotrophin family, BDNF was initiall...