Brain lymphatic endothelial cells (BLECs) constitute a group of loosely connected endothelial cells that reside within the meningeal layer of the zebrafish brain without forming a vascular tubular... Show moreBrain lymphatic endothelial cells (BLECs) constitute a group of loosely connected endothelial cells that reside within the meningeal layer of the zebrafish brain without forming a vascular tubular system. BLECs have been shown to readily endocytose extracellular cargo molecules from the brain parenchyma, however, their functional relevance in relation to microglia remains enigmatic. We here compare their functional uptake efficiency for several macromolecules and bacterial components with microglia in a qualitative and quantitative manner in 5-day-old zebrafish embryos. We find BLECs to be significantly more effective in the uptake of proteins, polysaccharides and virus particles as compared to microglia, while larger particles like bacteria are only ingested by microglia but not by BLECs, implying a clear distribution of tasks between the two cell types in the brain area. In addition, we compare BLECs to the recently discovered scavenger endothelial cells (SECs) of the cardinal vein and find them to accept an identical set of substrate molecules. Our data identifies BLECs as the first brain-associated SEC population in vertebrates, and demonstrates that BLECs cooperate with microglia to remove particle waste from the brain. Show less
Coomans, C.; Saaltink, D.J.; Deboer, T.; Tersteeg, M.; Lanooij, S.; Schneider, A.F.; ... ; Vreugdenhil, E. 2021
We have recently identified a novel plasticity protein, doublecortin-like (DCL), that is specifically expressed in the shell of the mouse suprachiasmatic nucleus (SCN). DCL is implicated in... Show moreWe have recently identified a novel plasticity protein, doublecortin-like (DCL), that is specifically expressed in the shell of the mouse suprachiasmatic nucleus (SCN). DCL is implicated in neuroplastic events, such as neurogenesis, that require structural rearrangements of the microtubule cytoskeleton, enabling dynamic movements of cell bodies and dendrites. We have inspected DCL expression in the SCN by confocal microscopy and found that DCL is expressed in GABA transporter-3 (GAT3)-positive astrocytes that envelope arginine vasopressin (AVP)-expressing cells. To investigate the role of these DCL-positive astrocytes in circadian rhythmicity, we have used transgenic mice expressing doxycycline-induced short-hairpin (sh) RNA's targeting DCL mRNA (DCL knockdown mice). Compared with littermate wild type (WT) controls, DCL-knockdown mice exhibit significant shorter circadian rest-activity periods in constant darkness and adjusted significantly faster to a jet-lag protocol. As DCL-positive astrocytes are closely associated with AVP-positive cells, we analyzed AVP expression in DCL-knockdown mice and in their WT littermates by 3D reconstructions and transmission electron microscopy (TEM). We found significantly higher numbers of AVP-positive cells with increased volume and more intensity in DCL-knockdown mice. We found alterations in the numbers of dense core vesicle-containing neurons at ZT8 and ZT20 suggesting that the peak and trough of neuropeptide biosynthesis is dampened in DCL-knockdown mice compared to WT littermates. Together, our data suggest an important role for the astrocytic plasticity in the regulation of circadian rhythms and point to the existence of a specific DCL+ astrocyte-AVP(+) neuronal network located in the dorsal SCN implicated in AVP biosynthesis. Show less
Astroglial type‐1 cannabinoid (CB1) receptors are involved in synaptic transmission, plasticity and behavior by interfering with the so‐called tripartite synapse formed by pre‐ and post‐synaptic... Show moreAstroglial type‐1 cannabinoid (CB1) receptors are involved in synaptic transmission, plasticity and behavior by interfering with the so‐called tripartite synapse formed by pre‐ and post‐synaptic neuronal elements and surrounding astrocyte processes. However, little is known concerning the subcellular distribution of astroglial CB1 receptors. In particular, brain CB1 receptors are mostly localized at cells' plasmalemma, but recent evidence indicates their functional presence in mitochondrial membranes. Whether CB1 receptors are present in astroglial mitochondria has remained unknown. To investigate this issue, we included conditional knock‐out mice lacking astroglial CB1 receptor expression specifically in glial fibrillary acidic protein (GFAP)‐containing astrocytes (GFAP‐CB1‐KO mice) and also generated genetic rescue mice to re‐express CB1 receptors exclusively in astrocytes (GFAP‐CB1‐RS). To better identify astroglial structures by immunoelectron microscopy, global CB1 knock‐out (CB1‐KO) mice and wild‐type (CB1‐WT) littermates were intra‐hippocampally injected with an adeno‐associated virus expressing humanized renilla green fluorescent protein (hrGFP) under the control of human GFAP promoter to generate GFAPhrGFP‐CB1‐KO and ‐WT mice, respectively. Furthermore, double immunogold (for CB1) and immunoperoxidase (for GFAP or hrGFP) revealed that CB1 receptors are present in astroglial mitochondria from different hippocampal regions of CB1‐WT, GFAP‐CB1‐RS and GFAPhrGFP‐CB1‐WT mice. Only non‐specific gold particles were detected in mouse hippocampi lacking CB1 receptors. Altogether, we demonstrated the existence of a precise molecular architecture of the CB1 receptor in astrocytes that will have to be taken into account in evaluating the functional activity of cannabinergic signaling at the tripartite synapse. Show less
