Biological sex does not predict glymphatic influx in healthy young, middle aged or old mice.

Sexual dimorphism is evident in brain structure, size, and function throughout multiple species. Here, we tested whether cerebrospinal fluid entry into the glymphatic system, a network of perivascular fluid transport that clears metabolic waste from the brain, was altered between male and female mice. We analyze glymphatic influx in 244 young reproductive age (2-4 months) C57BL/6 mice. We found no male/female differences in total influx under anesthesia, or across the anterior/posterior axis of the brain. Circadian-dependent changes in glymphatic influx under ketamine/xylazine anesthesia were not altered by sex. This was not true for diurnal rhythms under pentobarbital and avertin, but both still showed daily oscillations independent of biological sex. Finally, although glymphatic influx decreases with age there was no sex difference in total influx or subregion-dependent tracer distribution in 17 middle aged (9-10 months) and 36 old (22-24 months) mice. Overall, in healthy adult C57BL/6 mice we could not detect male/female differences in glymphatic influx. This finding contrasts the gender differences in common neurodegenerative diseases. We propose that additional sex-dependent co-morbidities, such as chronic stress, protein misfolding, traumatic brain injury or other pathological mechanisms may explain the increased risk for developing proteinopathies rather than pre-existing suppression of glymphatic influx.

An ocular glymphatic clearance system removes ß-amyloid from the rodent eye.

Despite high metabolic activity, the retina and optic nerve head lack traditional lymphatic drainage. We here identified an ocular glymphatic clearance route for fluid and wastes via the proximal optic nerve in rodents. ß-amyloid (Aß) was cleared from the retina and vitreous via a pathway dependent on glial water channel aquaporin-4 (AQP4) and driven by the ocular-cranial pressure difference. After traversing the lamina barrier, intra-axonal Aß was cleared via the perivenous space and subsequently drained to lymphatic vessels. Light-induced pupil constriction enhanced efflux, whereas atropine or raising intracranial pressure blocked efflux. In two distinct murine models of glaucoma, Aß leaked from the eye via defects in the lamina barrier instead of directional axonal efflux. The results suggest that, in rodents, the removal of fluid and metabolites from the intraocular space occurs through a glymphatic pathway that might be impaired in glaucoma.

Aquaporin 1 and the Na+/K+/2Cl- cotransporter 1 are present in the leptomeningeal vasculature of the adult rodent central nervous system.

BACKGROUND: The classical view of cerebrospinal fluid (CSF) production posits the choroid plexus as its major source. Although previous studies indicate that part of CSF production occurs in the subarachnoid space (SAS), the mechanisms underlying extra-choroidal CSF production remain elusive. We here investigated the distributions of aquaporin 1 (AQP1) and Na+/K+/2Cl- cotransporter 1 (NKCC1), key proteins for choroidal CSF production, in the adult rodent brain and spinal cord. METHODS: We have accessed AQP1 distribution in the intact brain using uDISCO tissue clearing technique and by Western blot. AQP1 and NKCC1 cellular localization were accessed by immunohistochemistry in brain and spinal cord obtained from adult rodents. Imaging was performed using light-sheet, confocal and bright field light microscopy. RESULTS: We determined that AQP1 is widely distributed in the leptomeningeal vasculature of the intact brain and that its glycosylated isoform is the most prominent in different brain regions. Moreover, AQP1 and NKCC1 show specific distributions in the smooth muscle cell layer of penetrating arterioles and veins in the brain and spinal cord, and in the endothelia of capillaries and venules, restricted to the SAS vasculature. CONCLUSIONS: Our results shed light on the molecular framework that may underlie extra-choroidal CSF production and we propose that AQP1 and NKCC1 within the leptomeningeal vasculature, specifically at the capillary level, are poised to play a role in CSF production throughout the central nervous system.

Impaired Glymphatic Transport in Spontaneously Hypertensive Rats.

The glymphatic system is a brainwide CSF transport system that uses the perivascular space for fast inflow of CSF. Arterial pulsations are a major driver of glymphatic CSF inflow, and hypertension that causes vascular pathologies, such as arterial stiffening and perivascular alterations, may impede the inflow. We used dynamic contrast-enhanced MRI to assess the effect of hypertension on glymphatic transport kinetics in male young and adult spontaneously hypertensive (SHR) rats compared with age-matched normotensive Wistar-Kyoto rats (WKY). We anesthetized the rats with dexmedetomidine/isoflurane and infused paramagnetic contrast (Gd-DOTA) into the cisterna magna during dynamic contrast-enhanced MRI to quantify glymphatic transport kinetics. Structural MRI analysis showed that cerebroventricular volumes are larger and brain volumes significantly smaller in SHR compared with WKY rats, regardless of age. We observed ventricular reflux of Gd-DOTA in SHR rats only, indicating abnormal CSF flow dynamics secondary to innate hydrocephalus. One-tissue compartment analysis revealed impeded glymphatic transport of Gd-DOTA in SHR compared with WKY rats in both age groups, implying that glymphatic transport, including solute clearance from brain parenchyma, is impaired during evolving hypertension in young SHR, an effect that worsens in states of chronic hypertension. The study demonstrates the suppression of glymphatic clearance in SHR rats and thus offers new insight into the coexistence of hypertension and concomitant vascular pathologies in Alzheimer's disease. The study further highlights the importance of considering the distribution of tracers in the CSF compartment in the analysis of the glymphatic system.SIGNIFICANCE STATEMENT The glymphatic system contributes to the removal of amyloid ß from the brain and is disrupted in Alzheimer's disease and aging. Using a rat model of hypertension, we measured gross CSF flow and tracked glymphatic influx and efflux rates with dynamic contrast-enhanced MRI, showing that glymphatic transport is compromised in both early and advanced stages of hypertension. The study provides a new perspective on the importance for brain metabolite and fluid homeostasis of maintaining healthy blood vessels, an increasingly pertinent issue in an aging population that in part may explain the link between vascular pathology and Alzheimer's disease.

Cisterna Magna Injection in Rats to Study Glymphatic Function.

The recently discovered glymphatic system, which supports brain-wide clearance of metabolic waste, has become the subject of intense research within the past few years. Its nomenclature arose due to its functionally analogous nature to the lymphatic system in combination with glial cells that are part of its anatomical boundaries. The influx of cerebrospinal fluid (CSF) from perivascular spaces into the brain interstitium acts to clear intraparenchymal solutes. CSF is produced by the choroid plexus and flows from the ventricles to the subarachnoid space via the cisterna magna, and as such the injection of tracer molecules into any one of these spaces could be used for studying CSF movement through the glymphatic system. Of these options, the cisterna magna is most favorable as it offers a route of entry that does not involve craniotomy. Herein we describe the cisterna magna (CM) injection procedure carried out in rats, essential for studying glymphatic influx and efflux dynamics.

Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull-meninges connections.

Analysis of entire transparent rodent bodies after clearing could provide holistic biological information in health and disease, but reliable imaging and quantification of fluorescent protein signals deep inside the tissues has remained a challenge. Here, we developed vDISCO, a pressure-driven, nanobody-based whole-body immunolabeling technology to enhance the signal of fluorescent proteins by up to two orders of magnitude. This allowed us to image and quantify subcellular details through bones, skin and highly autofluorescent tissues of intact transparent mice. For the first time, we visualized whole-body neuronal projections in adult mice. We assessed CNS trauma effects in the whole body and found degeneration of peripheral nerve terminals in the torso. Furthermore, vDISCO revealed short vascular connections between skull marrow and brain meninges, which were filled with immune cells upon stroke. Thus, our new approach enables unbiased comprehensive studies of the interactions between the nervous system and the rest of the body.

Cannula Implantation into the Cisterna Magna of Rodents.

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to the skull or the brain parenchyma. In anesthetized rodents, the exposure of the dura mater by blunt dissection of the neck muscles allows the insertion of a cannula into the cisterna magna (CM). The cannula, composed either by a fine beveled needle or borosilicate capillary, is attached via a polyethylene (PE) tube to a syringe. Using a syringe pump, molecules can then be injected at controlled rates directly into the CM, which is continuous with the subarachnoid space. From the subarachnoid space, we can trace CSF fluxes by convective flow into the perivascular space around penetrating arterioles, where solute exchange with the interstitial fluid (ISF) occurs. CMc can be performed for acute injections immediately following the surgery, or for chronic implantation, with later injection in anesthetized or awake, freely moving rodents. Quantitation of tracer distribution in the brain parenchyma can be performed by epifluorescence, 2-photon microscopy, and magnetic resonance imaging (MRI), depending on the physico-chemical properties of the injected molecules. Thus, CMc in conjunction with various imaging techniques offers a powerful tool for assessment of the glymphatic system and CSF dynamics and function. Furthermore, CMc can be utilized as a conduit for fast, brain-wide delivery of signaling molecules and metabolic substrates that could not otherwise cross the blood brain barrier (BBB).

Comparative analysis of monoaminergic cerebrospinal fluid-contacting cells in Osteichthyes (bony vertebrates).

Cerebrospinal fluid-contacting (CSF-c) cells containing monoamines such as dopamine (DA) and serotonin (5-HT) occur in the periventricular zones of the hypothalamic region of most vertebrates except for placental mammals. Here we compare the organization of the CSF-c cells in chicken, Xenopus, and zebrafish, by analyzing the expression of synthetic enzymes of DA and 5-HT, respectively, tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH), and draw an evolutionary scenario for this cell population. Due to the lack of TH immunoreactivity in this region, the hypothalamic CSF-c cells have been thought to take up DA from the ventricle instead of synthesizing it. We demonstrate that a second TH gene (TH2) is expressed in the CSF-c cells of all the three species, suggesting that these cells do indeed synthetize DA. Furthermore, we found that many CSF-c cells coexpress TH2 and TPH1 and contain both DA and 5-HT, a dual neurotransmitter phenotype hitherto undescribed in the brain of any vertebrate. The similarities of CSF-c cells in chicken, Xenopus, and zebrafish suggest that these characteristics are inherited from the common ancestor of the Osteichthyes. A significant difference between tetrapods and teleosts is that teleosts possess an additional CSF-c cell population around the posterior recess (PR) that has emerged in specific groups of Actinopterygii. Our comparative analysis reveals that the hypothalamus in mammals and teleosts has evolved in a divergent manner: placental mammals have lost the monoaminergic CSF-c cells, while teleosts have increased their relative number.

Purinergic receptor P2RY12-dependent microglial closure of the injured blood-brain barrier.

Microglia are integral functional elements of the central nervous system, but the contribution of these cells to the structural integrity of the neurovascular unit has not hitherto been assessed. We show here that following blood-brain barrier (BBB) breakdown, P2RY12 (purinergic receptor P2Y, G-protein coupled, 12)-mediated chemotaxis of microglia processes is required for the rapid closure of the BBB. Mice treated with the P2RY12 inhibitor clopidogrel, as well as those in which P2RY12 was genetically ablated, exhibited significantly diminished movement of juxtavascular microglial processes and failed to close laser-induced openings of the BBB. Thus, microglial cells play a previously unrecognized protective role in the maintenance of BBB integrity following cerebrovascular damage. Because clopidogrel antagonizes the platelet P2Y12 receptor, it is widely prescribed for patients with coronary artery and cerebrovascular disease. As such, these observations suggest the need for caution in the postincident continuation of P2RY12-targeted platelet inhibition.

A Distinct Population of Microglia Supports Adult Neurogenesis in the Subventricular Zone.

Microglia are involved in synaptic pruning both in development and in the mature CNS. In this study, we investigated whether microglia might further contribute to circuit plasticity by modulating neuronal recruitment from the neurogenic subventricular zone (SVZ) of the adult mouse striatum. We found that microglia residing in the SVZ and adjacent rostral migratory stream (RMS) comprise a morphologically and antigenically distinct phenotype of immune effectors. Whereas exhibiting characteristics of alternatively activated microglia, the SVZ/RMS microglia were clearly distinguished by their low expression of purinoceptors and lack of ATP-elicitable chemotaxis. Furthermore, the in vivo depletion of these microglia hampered the survival and migration of newly generated neuroblasts through the RMS to the olfactory bulb. SVZ and RMS microglia thus appear to comprise a functionally distinct class that is selectively adapted to the support and direction of neuronal integration into the olfactory circuitry. Therefore, this unique microglial subpopulation may serve as a novel target with which to modulate cellular addition from endogenous neural stem and progenitor cells of the adult brain. SIGNIFICANCE STATEMENT: Microglial cells are a specialized population of macrophages in the CNS, playing key roles as immune mediators. As integral components in the CNS, the microglia stand out for using the same mechanisms, phagocytosis and cytochemokine release, to promote homeostasis, synaptic pruning, and neural circuitry sculpture. Here, we addressed microglial functions in the subventricular zone (SVZ), the major postnatal neurogenic niche. Our results depict microglia as a conspicuous component of SVZ and its anterior extension, the rostral migratory stream, a pathway used by neuroblasts during their transit toward olfactory bulb layers. In addition to other unique populations residing in the SVZ niche, microglia display distinct morphofunctional properties that boost neuronal progenitor survival and migration in the mammalian brain.

Ontogeny of CX3CR1-EGFP expressing cells unveil microglia as an integral component of the postnatal subventricular zone.

The full spectrum of cellular interactions within CNS neurogenic niches is still poorly understood. Only recently has the monocyte counterpart of the nervous system, the microglial cells, been described as an integral cellular component of neurogenic niches. The present study sought to characterize the microglia population in the early postnatal subventricular zone (SVZ), the major site of postnatal neurogenesis, as well as in its anterior extension, the rostral migratory stream (RMS), a pathway for neuroblasts during their transit toward the olfactory bulb (OB) layers. Here we show that microglia within the SVZ/RMS pathway are not revealed by phenotypic markers that characterize microglia in other regions. Analysis of the transgenic mice strain that has one locus of the constitutively expressed fractalkine CX3CR1 receptor replaced by the gene encoding the enhanced green fluorescent protein (EGFP) circumvented the antigenic plasticity of the microglia, thus allowing us to depict microglia within the SVZ/RMS pathway during early development. Notably, microglia within the early SVZ/RMS are not proliferative and display a protracted development, retaining a more immature morphology than their counterparts outside germinal layers. Furthermore, microglia contact and phagocyte radial glia cells (RG) processes, thereby playing a role on the astroglial transformation that putative stem cells within the SVZ niche undergo during the first postnatal days.

Fine-tuning the central nervous system: microglial modelling of cells and synapses.

Microglia constitute as much as 10-15% of all cells in the mammalian central nervous system (CNS) and are the only glial cells that do not arise from the neuroectoderm. As the principal CNS immune cells, microglial cells represent the first line of defence in response to exogenous threats. Past studies have largely been dedicated to defining the complex immune functions of microglial cells. However, our understanding of the roles of microglia has expanded radically over the past years. It is now clear that microglia are critically involved in shaping neural circuits in both the developing and adult CNS, and in modulating synaptic transmission in the adult brain. Intriguingly, microglial cells appear to use the same sets of tools, including cytokine and chemokine release as well as phagocytosis, whether modulating neural function or mediating the brain's innate immune responses. This review will discuss recent developments that have broadened our views of neuro-glial signalling to include the contribution of microglial cells.

Dye coupling and connexin expression by cortical radial glia in the early postnatal subventricular zone.

In this study, we have analyzed the specific contribution of the cortical radial glia (RG) for gap junctional communication (GJC) within the postnatal subventricular zone (SVZ). To specifically target RG as source of dye-coupling in situ, we have developed a new technique that involves direct cell loading through the processes that reach the pial surface, with a mix of gap junction permeant (Lucifer yellow, LY) and nonpermeant (rhodamine-conjugated dextran 3 KDa, RD) fluorochromes, the latter used as a marker for direct loaded cells. Tissue sections were analyzed for identification of directly loaded (LY+RD+) and coupled cells (LY+RD-) in the SVZ. Directly loaded cells were restricted to the region underlying the pial loading surface area. Coupled cells were distributed in a bistratified manner, along the outer dorsal surface of the SVZ and aligning the ventricle, leaving the SVZ core relatively free. Blocking GJC prior to pial loading greatly reduced dye coupling. Phenotypic analysis indicated that coupling by RG excludes neuroblasts and is mostly restricted to cells of glial lineage. Notwithstanding, no corresponding restriction to specific cell phenotype was found for two connexin isotypes, Cx43 and Cx45, in the postnatal SVZ. The extensive homocellular cell coupling by RG suggests an important role in the regulation of neurogenesis and functional compartmentalization of the postnatal SVZ.

Gap junctions are involved in cell migration in the early postnatal subventricular zone.

The massive migration of neuroblasts and young neurons through the anterior extension of the postnatal subventricular zone (SVZ), known as the rostral migratory stream (RMS) is still poorly understood on its molecular basis. In this work, we investigated the involvement of gap junctional communication (GJC) in the robust centrifugal migration from SVZ/RMS explants obtained from early postnatal (P4) rats. Cells were dye-coupled in homocellular and heterocellular pairings and expressed at least two connexins, Cx 43 and 45. Treatment with the uncoupler agent carbenoxolone (CBX, 10-100 microM) reversibly reduced outgrowth from SVZ explants, while its inactive analog, glycyrhizinic acid (GZA), had no effect. Consistent with a direct effect on cell migration, time-lapse video microscopy show that different pharmacological uncouplers cause an abrupt and reversible arrest of cell movement in explants. Our results indicate that GJC is positively involved in the migration of neuroblasts within the SVZ/RMS.