Flow of cerebrospinal fluid (CSF) through perivascular spaces (PVSs) in the brain is important for clearance of metabolic waste. Arterial pulsations are thought to drive flow, but this has never been quantitatively shown. We used particle tracking to quantify CSF flow velocities in PVSs of live mice. CSF flow is pulsatile and driven primarily by the cardiac cycle. The speed of the arterial wall matches that of the CSF, suggesting arterial wall motion is the principal driving mechanism, via a process known as perivascular pumping. Increasing blood pressure leaves the artery diameter unchanged but changes the pulsations of the arterial wall, increasing backflow and thereby reducing net flow in the PVS. Perfusion-fixation alters the normal flow direction and causes a 10-fold reduction in PVS size. We conclude that particle tracking velocimetry enables the study of CSF flow in unprecedented detail and that studying the PVS in vivo avoids fixation artifacts.

BackgroundApolipoprotein E (apoE) is a major carrier of cholesterol and essential for synaptic plasticity. In brain, it’s expressed by many cells but highly expressed by the choroid plexus and the predominant apolipoprotein in cerebrospinal fluid (CSF). The role of apoE in the CSF is unclear. Recently, the glymphatic system was described as a clearance system whereby CSF and ISF (interstitial fluid) is exchanged via the peri-arterial space and convective flow of ISF clearance is mediated by aquaporin 4 (AQP4), a water channel. We reasoned that this system also serves to distribute essential molecules in CSF into brain. The aim was to establish whether apoE in CSF, secreted by the choroid plexus, is distributed into brain, and whether this distribution pattern was altered by sleep deprivation.MethodsWe used fluorescently labeled lipidated apoE isoforms, lenti-apoE3 delivered to the choroid plexus, immunohistochemistry to map apoE brain distribution, immunolabeled cells and proteins in brain, Western blot analysis and ELISA to determine apoE levels and radiolabeled molecules to quantify CSF inflow into brain and brain clearance in mice. Data were statistically analyzed using ANOVA or Student’s t- test.ResultsWe show that the glymphatic fluid transporting system contributes to the delivery of choroid plexus/CSF-derived human apoE to neurons. CSF-delivered human apoE entered brain via the perivascular space of penetrating arteries and flows radially around arteries, but not veins, in an isoform specific manner (apoE2 > apoE3 > apoE4). Flow of apoE around arteries was facilitated by AQP4, a characteristic feature of the glymphatic system. ApoE3, delivered by lentivirus to the choroid plexus and ependymal layer but not to the parenchymal cells, was present in the CSF, penetrating arteries and neurons. The inflow of CSF, which contains apoE, into brain and its clearance from the interstitium were severely suppressed by sleep deprivation compared to the sleep state.ConclusionsThus, choroid plexus/CSF provides an additional source of apoE and the glymphatic fluid transporting system delivers it to brain via the periarterial space. By implication, failure in this essential physiological role of the glymphatic fluid flow and ISF clearance may also contribute to apoE isoform-specific disorders in the long term.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0138-8) contains supplementary material, which is available to authorized users.

Information transfer within neuronal circuits depends on the balance and recurrent activity of excitatory and inhibitory neurotransmission. Chloride (Cl−) is the major central nervous system (CNS) anion mediating inhibitory neurotransmission. Astrocytes are key homoeostatic glial cells populating the CNS, although the role of these cells in regulating excitatory-inhibitory balance remains unexplored. Here we show that astrocytes act as a dynamic Cl− reservoir regulating Cl− homoeostasis in the CNS. We found that intracellular chloride concentration ([Cl−]i) in astrocytes is high and stable during sleep. In awake mice astrocytic [Cl−]i is lower and exhibits large fluctuation in response to both sensory input and motor activity. Optogenetic manipulation of astrocytic [Cl−]i directly modulates neuronal activity during locomotion or whisker stimulation. Astrocytes thus serve as a dynamic source of extracellular Cl− available for GABAergic transmission in awake mice, which represents a mechanism for modulation of the inhibitory tone during sustained neuronal activity.

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Kilogram-scale synthesis of a bioactive pentapeptide in solution by "3+2" fragment coupling strategy has been successively accomplished in the development of OGP (10-14), a minimal OGP-derived sequence that retains the full proliferative activity of the osteogenic growth peptide. The synthetic scheme, coupling conditions and scaling up of the process are systematically studied, the epimerization of the tripeptide fragment and pentapeptide are also evaluated. .65 (t, J = 7.6 Hz, 3H), 7.40 (t, J = 7.4 Hz, 3H), 7. 34 -7.27 (m, 3H), 7.25 (d, J = 4.1 Hz, 3H), 7.17 (d, J = 8.5 Hz, 2H), 4.56 (s, 1H), 4.19 (d, J = 11.2 Hz, 1H), 4.16 -4.06 (m, 3H), 3.89 -3.76 (m, 3H), 3.05 (dd, J = 13.9, 4.2 Hz, 1H), 2.95 (d, J = 9.8 Hz, 1H), 2.79 -2.68 (m, 2H), 1.41 -0.85 (m, 9H). Fmoc-Tyr(tBu)-Gly-Phe-OSu (3)To a stirred solution of Fmoc-Tyr(tBu)-Gly-Phe-OH (2.76 kg, 4.16mol) and HOSu (0.67 kg, 5.83mol, 1.4eq) in dry THF (25 L), a solution of DCC (1.21 kg, 5.83mol, 1.4eq) in dry THF (5 L) was added slowly under ice-cooling within 3h, then the solution was stirred overnight at ambient temperature.After cooling for 1 h, the resulting precipitate was filtered and filtrate was used further reaction without purification. TLC: a 2 , R f =0.3. Boc-Gly-OH (4)The mixture of glycine (1 kg, 13.32mol) and NaHCO 3 (1.12 kg, 13.32mmol) in H 2 O (15 L) was added slowly to the solution of Boc 2 O (3.06 L, 13.32mol) in THF (10 L) and stirred at ambient temperature for 5h. When TLC indicated the absence of starting material, the residue was evaporated and distributed by H 2 O (20 L), EtOAc (7 L) and light petroleum (7 L). The organic layer was removed and the aqueous phase adjusted to pH 2-3 with 10% citric acid solution. After extracted with EtOAc (3×10 L), the aqueous layer was separated and the combined organic extracts were washed with saline (2×10 L), dried with magnesium sulfate and evaporated to give a white crystal (1.98 kg, 84.85%). m.p.85.7～86.5°C, Boc-Gly-Gly-OH (5)Boc-Gly-OH (1.98 kg, 11.3mol) and HONp (1.73 kg, 12.43mol, 1.1eq) or HOSu (1.43 kg, 12.43mol, 1.1eq) were dissolved in dry 1,4-dioxane (25 L), a solution of DCC (2.8 kg, 13.56mol, 1.2eq) in dry 1,4-dioxane (10 L) was added and the mixture stirred for 8-10h. After cooling for 2h, the precipitate was removed, the filtrate was used directly for next reaction without purification, TLC: a 1 , R f =0.4, 0.3.The mixture of glycine (0.94 kg, 12.43mol, 1.1eq) and NaHCO 3 (1.05 kg, 12.43mol, 1.1eq) in H 2 O (10 L) was slowly added to the solution of Boc-Gly-ONp (get by above step) in 1,4-dioxane (25 L). The solution was stirred at about 25°C for 7 h, then evaporated under reduced pressure. The residue was acidified to a pH 2-3 with 10% citric acid and extracted with EtOAc (3×20 L). The organic phase was successively washed with saline (20 L), dried, and evaporated to give Boc-Gly-Gly-OH (2.37 kg, 90.3%) as a white schistose crystals. m.p.123.5～125.5°C, TLC: a 3 , R f =0.2, HPLC: e, 6.53min, purity 93.5%, Calc. for C 7 H 13 NO 4 : C, 46.55; H, 6.94; N, 12.06. Found: C, 45.40; H, 6.02; N, 14.13...