It is becoming clear that astrocytes are active participants in synaptic functioning and exhibit properties, such as the secretion of classical transmitters, previously thought to be exclusively neuronal. of organelles are differentially distributed. After activation of metabotropic glutamate receptors and the release of calcium from intracellular stores, the NPY-ir puncta fuse with the cell membrane and the peptide-containing dense cores are displayed. To determine whether peptide secretion subsequently occurred, exocytosis was monitored from astrocytes expressing NPY-red fluorescent protein. In live cells, following activation of glutamate receptors, the intensity of the NPY-RFP labeled puncta declined in a step-like manner indicating a regulated release of the granular contents. Since NPY is a widespread and potent regulator of synaptic transmission, these results suggest that astrocytes could play a role in the peptidergic modulation of synaptic signaling in the CNS. a calcium-mediated process involving the vesicular exocytosis of gliotransmitters (Innocenti et al., 2000; Pasti et al., 2001; Zonta et al., 2003). Many of the molecules that are needed for neuronal vesicular transmitter release including SNARE proteins, glutamate transporters and synaptotagmin isoforms have been buy TMC-207 found in astrocytes (Montana et al., 2004; Zhang et al., 2004a; Zhang et al., 2004b). These studies indicate that astrocytes can secrete small molecule transmitters. How far do these glial-neuronal similarities extend? Do astrocytes also synthesize and release neuropeptide co-transmitters just like the majority of neurons? A variety of astrocytic neuropeptides have been identified (Michel et al., 1986; Shinoda et al., 1989; Melner et al., 1990; McKenzie et al., 1994; Barnea et al., 1998; Buzas et al., 1998) but the synthesis, trafficking and release of these molecules has not been widely studied and fundamental questions remain. In particular are these neuropeptides synthesized and packaged similar pathways to those identified in neurons? Are astrocytic neuropeptides co-localized with the small molecule gliotransmitters? Are glial neuropeptides secreted, and if so does exocytosis occur through the regulated or constitutive secretory pathways? Here we address these questions using mouse cortical astrocytes. We find that these cells are able to synthesize both foreign and native neuropeptides, including neuropeptide Y (NPY), a peptide widely distributed throughout the mammalian nervous system (Gray and Morley, 1986). Activation of metabotropic glutamate receptors results in a calcium-dependent fusion of NPY-containing dense core granules with the cell membrane and consequent peptide secretion. Therefore astrocytes, like neurons, have a regulated secretory pathway(s) that is responsible for the release of multiple classes of transmitter molecules. Since NPY is a potent regulator of synaptic transmission (van den Pol buy TMC-207 et al., 1996) and NPY receptors are found throughout the CNS (Dumont et al., 2004), astrocytes may be involved in the peptidergic regulation of synaptic transmission. MATERIALS AND METHODS Cell culture Astrocytes were cultured from C57BL/6 mice [postnatal day 1 (P1) to P3] as described Pdgfra (McCarthy and de Vellis, 1980; Parpura et al., 1995). Briefly, cortical tissue was treated with trypsin (Sigma type XI) for 50 mins at 37C, then dispersed by trituration. Cells were plated into tissue culture flasks and maintained at 37C in culture medium (DMEM/10 % FBS) in a humidified 5% CO2/95% air atmosphere. Once cultures became confluent (7C10 d) the flasks were shaken at 300 rpm at 37C in air, initially for 2 hr and then after a complete exchange of medium for 18C20 hr (repeated 3 times). The remaining cells were detached using trypsin/EDTA and plated on coverslips coated with poly-D-lysine. Purified astrocytes were used 1C12 days after buy TMC-207 plating (1C12 DAP; see results). This procedure eliminated buy TMC-207 weakly adhesive cells including neurons and microglia. Staining with an anti-GFAP antibody (see below) indicated that 70 %70 % of the cells were intensely GFAP-ir. The lack of neurons in these cultures was confirmed by buy TMC-207 the absence of MAP-ir, a neuronal dendritic marker. As a positive control, we confirmed that the MAP antibody stained dendritic processes in glial-neuronal co-cultures (not shown). Co-cultures of astrocytes and neurons (Figure 1A) were derived from C57BL/6 mice (P1 to P3). AtT-20 cells were cultured in DMEM/10 % FBS as previously described (Whim and Moss, 2001). Open in a separate window Figure 1 Identification of neuropeptide Y in GFAP-positive astrocytes(A). In co-cultures of astrocytes and hippocampal neurons, punctate NPY-immunoreactivity was observed both in neuronal processes (filled arrow) and astrocytes (open arrow). (B). Similar NPY-ir was seen in purified astrocyte cultures. Astrocytes were double stained with antibodies for GFAP (left.