Astrocytes communicate with synapses by means of intracellular calcium ([Ca(2+)](i)) elevations, but local calcium dynamics in astrocytic processes have never been thoroughly investigated. By taking advantage of high-resolution two-photon microscopy, we identify the characteristics of local astrocyte calcium activity in the adult mouse hippocampus. Astrocytic processes showed intense activity, triggered by physiological transmission at neighboring synapses. They encoded synchronous synaptic events generated by sparse action potentials into robust regional (∼12 μm) [Ca(2+)](i) elevations. Unexpectedly, they also sensed spontaneous synaptic events, producing highly confined (∼4 μm), fast (millisecond-scale) miniature Ca(2+) responses. This Ca(2+) activity in astrocytic processes is generated through GTP- and inositol-1,4,5-trisphosphate-dependent signaling and is relevant for basal synaptic function. Thus, buffering astrocyte [Ca(2+)](i) or blocking a receptor mediating local astrocyte Ca(2+) signals decreased synaptic transmission reliability in minimal stimulation experiments. These data provide direct evidence that astrocytes are integrated in local synaptic functioning in adult brain.

Spreading depression (SD) is a propagating wave of neuronal depolarization and ionic shifts] waves does not depress SD propagation. This suggests that astrocytes do not drive SD propagation but are responsible for the hemodynamic failure seen deep in the cortex. Similar waves occur in anoxic depolarizations (AD), supporting the notion that SD and AD are related processes.

Background-Accumulating evidence demonstrates a critical involvement of tissue-type plasminogen activator (tPA) in pathological and physiological brain conditions. Determining whether and how vascular tPA can cross the blood-brain barrier (BBB) to enter the brain is thus important, not only during stroke but also in physiological conditions. Methods and Results-In the present work, we provide evidence in vivo that intravenous injection of tPA increases NMDA-induced striatal lesion in the absence of BBB leakage. Accordingly, we show that tPA crosses the BBB both after excitotoxic lesion and in control conditions. Indeed, vascular injected tPA can be detected within the brain parenchyma and in the cerebrospinal fluid. By using an in vitro model of BBB, we have confirmed that tPA can cross the intact BBB. Its passage was blocked at 4°C, was saturable, and was independent of its proteolytic activity. We have shown that tPA crosses the BBB by transcytosis, mediated by a member of the LDL receptor-related protein family. Conclusions-We demonstrate that blood-derived tPA can reach the brain parenchyma without alteration of the BBB. The molecular mechanism of the passage of tPA from blood to brain described here could represent an interesting target to improve thrombolysis in stroke (Circulation. 2005;111:2241-2249.)

Glucose is the primary energetic substrate of the brain, and measurements of its metabolism are the basis of major functional cerebral imaging methods. Contrary to the general view that neurons are fueled solely by glucose in proportion to their energetic needs, recent in vitro and ex vivo analyses suggest that glucose preferentially feeds astrocytes. However, the cellular fate of glucose in the intact brain has not yet been directly observed. We have used a real-time method for measuring glucose uptake in astrocytes and neurons in vivo in male rats by imaging the trafficking of the nonmetabolizable glucose analog 6-deoxy-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-aminoglucose (6-NBDG) using two-photon microscopy. During resting conditions we found that astrocytes and neurons both take up 6-NBDG at the same rate in the barrel cortex of the rat. However, during intense neuronal activity triggered by whisker stimulation, astrocytes rapidly accelerated their uptake, whereas neuronal uptake remained almost unchanged. After the stimulation period, astrocytes returned to their preactivation rates of uptake paralleling the neuronal rate of uptake. These observations suggest that glucose is taken up primarily by astrocytes, supporting the view that functional imaging experiments based on glucose analogs extraction may predominantly reflect the metabolic activity of the astrocytic network.

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26RFa, a novel RFamide neuropeptide, is the endogenous ligand of the former orphan receptor GPR103. Intracerebroventricular injection of 26RFa and its C-terminal heptapeptide, 26RFa((20-26)), stimulates food intake in rodents. To develop potent, stable ligands of GPR103 with low molecular weight, we have designed a series of aza-β(3)-containing 26RFa((20-26)) analogues for their propensity to establish intramolecular hydrogen bonds, and we have evaluated their ability to increase [Ca(2+)](i) in GPR103-transfected cells. We have identified a compound, [Cmpi(21),aza-β(3)-Hht(23)]26RFa((21-26)), which was 8-fold more potent than 26RFa((20-26)) in mobilizing [Ca(2+)](i). This pseudopeptide was more stable in serum than 26RFa((20-26)) and exerted a longer lasting orexigenic effect in mice. This study constitutes an important step toward the development of 26RFa analogues that could prove useful for the treatment of feeding disorders.

Background and Purpose The neuropeptide 26RFa and its cognate receptor GPR103 are involved in the control of food intake and bone mineralization. Here, we have tested, experimentally, the predicted ligand‐receptor interactions by site‐directed mutagenesis of GPR103 and designed point‐substituted 26RFa analogues. Experimental Approach Using the X‐ray structure of the β2‐adrenoceptor, a 3‐D molecular model of GPR103 has been built. The bioactive C‐terminal octapeptide 26RFa(19–26), KGGFSFRF‐NH2, was docked in this GPR103 model and the ligand‐receptor complex was submitted to energy minimization. Key Results In the most stable complex, the Phe‐Arg‐Phe‐NH2 part was oriented inside the receptor cavity, whereas the N‐terminal Lys residue remained outside. A strong intermolecular interaction was predicted between the Arg25 residue of 26RFa and the Gln125 residue located in the third transmembrane helix of GPR103. To confirm this interaction experimentally, we tested the ability of 26RFa and Arg‐modified 26RFa analogues to activate the wild‐type and the Q125A mutant receptors transiently expressed in CHO cells. 26RFa (10−6 M) enhanced [Ca2+]i in wild‐type GPR103‐transfected cells, but failed to increase [Ca2+]i in Q125A mutant receptor‐expressing cells. Moreover, asymmetric dimethylation of the side chain of arginine led to a 26RFa analogue, [ADMA25]26RFa(20–26), that was unable to activate the wild‐type GPR103, but antagonized 26RFa‐evoked [Ca2+]i increase. Conclusion and Implications Altogether, these data provide strong evidence for a functional interaction between the Arg25 residue of 26RFa and the Gln125 residue of GPR103 upon ligand‐receptor activation, which can be exploited for the rational design of potent GPR103 agonists and antagonists.