A standard Texas Crimson filtering set (excitation 560 nm/emission 630 nm) was utilized for visualization of CLCDsRed and tubulinDsRed. the plasma membrane can be an costly endeavour whose function reaches present unclear in astrocytes energetically. However, considering that intracellular CB1Rs can engage cell signalling pathways, it is likely that this process plays an important regulatory role. Keywords:actin, acutely isolated astrocyte, cannabinoid receptor, microtubule, vesicular trafficking Abbreviations:ANP, atrial natriuetic peptide; CB1R, cannabinoid receptor 1; DIC, differential interference contrast; EYFP, enhanced yellow fluorescent protein; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; GPCR, G-protein-coupled receptor; HBSS, Hanks balanced salt solution; LSD, least significant difference; -MEM, -minimum essential medium; MSD, mean square displacement; NA, numerical aperture; P1 etc., postnatal day 1 etc; PEI, TTA-Q6(isomer) polyethyleneimine; PN-1, protease nexin-1; RTPCR, reverse transcription-PCR; SEP, superecliptic pHluorin; TRITC, tetramethylrhodamine -isothiocyanate; VAMP8, vesicle-associated membrane protein 8; V-ATPase, vacuolar-type proton ATPase == INTRODUCTION == Astrocytes are a diverse population of glial cells, with functions ranging from structural support to modulation of synaptic transmission. Astrocytes possess TTA-Q6(isomer) a multitude of receptors [reviewed in (Verkhratsky, 2006)] for signalling molecules released by neurons and themselves, including the cannabinoid receptor 1, CB1R (Rodriguez et al., 2001), which is thought to mediate neuronastrocyte communication (Navarrete and Araque, 2008). Protein trafficking plays an important role in regulating CB1R TTA-Q6(isomer) expression, with receptors changing localization between the plasma membrane and intracellular compartments. In certain cell types at rest, CB1R is constitutively endocytosed, leading to a predominantly intracellular localization (Leterrier et al., 2004;McDonald et al., 2007a). Thus endocytic trafficking can be an important regulator of CB1R availability at the plasma membrane. Few studies on protein trafficking have been carried out in astrocytes. These have been limited to studies of the secreted ANP (atrial natriuetic peptide), the serpin PN-1 (protease nexin-1) and the synaptic-vesicle-associated protein synaptobrevin 2 (Giau et al., 2005;Potokar et al., 2005;Crippa et al., 2006;Potokar et al., 2007). As these molecules all function within the exocytotic pathway, they are likely to have similar trafficking dynamics and may be found within common vesicle pools. As the picture of how proteins found within the exocytotic pathway traffic in astrocytes is being elucidated, there are no comparative data as to the trafficking of receptors, such as GPCRs (G-protein-coupled receptors), in astrocytes. The trafficking of GPCRs, such as CB1R and the -2 adrenergic receptor, which are expressed by astrocytes (Aoki et al., 1987;Rodriguez et TTA-Q6(isomer) al., 2001), is better understood in other cell types, particularly with regard to endocytic trafficking (Coutts et al., 2001;Leterrier et al., 2004;Shumay et al., 2004). In the present study, we have investigated CB1R expression and intracellular trafficking within cultured astrocytes isolated from rat visual cortex. Fluorescent CB1R chimeras were used in combination with pharmacological agents to examine CB1R trafficking dynamics. Our findings indicate that within cultured astrocytes at rest there is a pronounced vesicular pool of CB1Rs, which is trafficked both by microtubule- and actin-based mechanisms; microtubule-dependent radial shuttling towards and away from the cell centre is evident. In addition, a population of CB1RGFP (green fluorescent protein) was found to be relatively immobile, with movement characteristic of passive diffusion. Astrocytes expressing a SEP (superecliptic pHluorin)-tagged CB1R (SEPCB1R) and treated with bafilomycin A1 showed the presence of CB1R within low-pH endocytic intracellular compartments. We found that the punctate CB1RGFP label mainly co-localized with the endosomal marker endobrevin [also referred to as VAMP8 (vesicle-associated membrane protein 8)]. Trafficking of receptors to and from the astrocyte plasma membrane probably shapes astrocytic responses to signalling molecules, but the nature of receptor trafficking in astrocytes has yet to be investigated in detail. To TTA-Q6(isomer) our knowledge the present study of CB1R trafficking constitutes the first investigation of receptor protein trafficking in astrocytes. This should be of additional importance when one considers the previous demonstration of preferential localization of CB1Rin situto the astrocytic end-foot (Rodriguez et al., 2001). Future studies of CB1R trafficking in more intact systems may Zfp622 help to develop our understanding of how membrane proteins are preferentially localized to distinct domains of astrocytes and how they influence local cell signalling..