Neurogenesis in the adult mammalian brain occurs mainly in two neurogenic niches, the subventricular zone (SVZ) and the subgranular zone (SGZ) of the dentate gyrus (DG). Cannabinoid type 1 and 2 receptors (CB1R and CB2R) have been shown to differently modulate neurogenesis. However, low attention has been given to the interaction between CB1R and CB2R in modulating postnatal neurogenesis (proliferation, neuronal differentiation and maturation). We focused on a putative crosstalk between CB1R and CB2R to modulate neurogenesis and cultured SVZ and DG stem/progenitor cells from early postnatal (P1-3) Sprague-Dawley rats. Data showed that the non-selective cannabinoid receptor agonist WIN55,212-2 promotes DG cell proliferation (measured by BrdU staining), an effect blocked by either CB1R or CB2R selective antagonists. Experiments with selective agonists showed that facilitation of DG cell proliferation requires co-activation of both CB1R and CB2R. Cell proliferation in the SVZ was not affected by the non-selective receptor agonist, but it was enhanced by CB1R selective activation. However, either CB1R or CB2R selective antagonists abolished the effect of the CB1R agonist in SVZ cell proliferation. Neuronal differentiation (measured by immunocytochemistry against neuronal markers of different stages and calcium imaging) was facilitated by WIN55,212-2 at both SVZ and DG. This effect was mimicked by either CB1R or CB2R selective agonists and blocked by either CB1R or CB2R selective antagonists, cross-antagonism being evident. In summary, our findings indicate a tight interaction between CB1R and CB2R to modulate neurogenesis in the two major neurogenic niches, thus contributing to further unraveling the mechanisms behind the action of endocannabinoids in the brain.

The endocannabinoid system has been implicated in the modulation of adult neurogenesis. Here, we describe the effect of type 1 cannabinoid receptor (CB1R) activation on self-renewal, proliferation and neuronal differentiation in mouse neonatal subventricular zone (SVZ) stem/progenitor cell cultures. Expression of CB1R was detected in SVZ-derived immature cells (Nestin-positive), neurons and astrocytes. Stimulation of the CB1R by (R)-(+)-Methanandamide (R-m-AEA) increased self-renewal of SVZ cells, as assessed by counting the number of secondary neurospheres and the number of Sox2+/+ cell pairs, an effect blocked by Notch pathway inhibition. Moreover, R-m-AEA treatment for 48 h, increased proliferation as assessed by BrdU incorporation assay, an effect mediated by activation of MAPK-ERK and AKT pathways. Surprisingly, stimulation of CB1R by R-m-AEA also promoted neuronal differentiation (without affecting glial differentiation), at 7 days, as shown by counting the number of NeuN-positive neurons in the cultures. Moreover, by monitoring intracellular calcium concentrations ([Ca(2+)]i) in single cells following KCl and histamine stimuli, a method that allows the functional evaluation of neuronal differentiation, we observed an increase in neuronal-like cells. This proneurogenic effect was blocked when SVZ cells were co-incubated with R-m-AEA and the CB1R antagonist AM 251, for 7 days, thus indicating that this effect involves CB1R activation. In accordance with an effect on neuronal differentiation and maturation, R-m-AEA also increased neurite growth, as evaluated by quantifying and measuring the number of MAP2-positive processes. Taken together, these results demonstrate that CB1R activation induces proliferation, self-renewal and neuronal differentiation from mouse neonatal SVZ cell cultures.

Stroke induces the recruitment of neuronal precursors from the subventricular zone (SVZ) into the ischemic striatum. In injured areas, de-routed neuroblasts use blood vessels as a physical scaffold to their migration, in a process that resembles the constitutive migration seen in the rostral migratory stream (RMS). The molecular mechanism underlying injury-induced vasculature-mediated migration of neuroblasts in the post-stroke striatum remains, however, elusive. Using adult mice we now demonstrate that endothelial cells in the ischemic striatum produce brain-derived neurotrophic factor (BDNF), a neurotrophin that promotes the vasculature-mediated migration of neuronal precursors in the RMS, and that recruited neuroblasts maintain expression of p75NTR, a low-affinity receptor for BDNF. Reactive astrocytes, which are widespread throughout the damaged area, ensheath blood vessels and express TrkB, a high-affinity receptor for BDNF. Despite the absence of BDNF mRNA, we observed strong BDNF immunolabeling in astrocytes, suggesting that these glial cells trap extracellular BDNF. Importantly, this pattern of expression is reminiscent of the adult RMS, where TrkB-expressing astrocytes bind and sequester vasculature-derived BDNF, leading to the entry of migrating cells into the stationary phase. Real-time imaging of cell migration in acute brain slices revealed a direct role for BDNF in promoting the migration of neuroblasts to ischemic areas. We also demonstrated that cells migrating in the ischemic striatum display higher exploratory behavior and longer stationary periods than cells migrating in the RMS. Our findings suggest that the mechanisms involved in the injury-induced vasculature-mediated migration of neuroblasts recapitulate, at least partially, those observed during constitutive migration in the RMS.

Cell transplantation is a promising approach for the reconstruction of neuronal circuits after brain damage. Transplanted neurons integrate with remarkable specificity into circuitries of the mouse cerebral cortex affected by neuronal ablation. However, it remains unclear how neurons perform in a local environment undergoing reactive gliosis, inflammation, macrophage infiltration, and scar formation, as in traumatic brain injury (TBI). To elucidate this, we transplanted cells from the embryonic mouse cerebral cortex into TBI-injured, inflamed-only, or intact cortex of adult mice. Brain-wide quantitative monosynaptic rabies virus (RABV) tracing unraveled graft inputs from correct regions across the brain in all conditions, with pronounced quantitative differences: scarce in intact and inflamed brain versus exuberant after TBI. In the latter, the initial overshoot is followed by pruning, with only a few input neurons persisting at 3 months. Proteomic profiling identifies candidate molecules for regulation of the synaptic yield, a pivotal parameter to tailor for functional restoration of neuronal circuits.

Neural stem cells (NSCs) from the subventricular zone (SVZ) have been indicated as a source of new oligodendrocytes to use in regenerative medicine for myelin pathologies. Indeed, NSCs are multipotent cells that can self-renew and differentiate into all neural cell types of the central nervous system. In normal conditions, SVZ cells are poorly oligodendrogenic, nevertheless their oligodendrogenic potential is boosted following demyelination. Importantly, progressive restriction into the oligodendrocyte fate is specified by extrinsic and intrinsic factors, endocannabinoids being one of these factors. Although a role for endocannabinoids in oligodendrogenesis has already been foreseen, selective agonists and antagonists of cannabinoids receptors produce severe adverse side effects. Herein, we show that hemopressin (Hp), a modulator of CB1 receptors, increased oligodendroglial differentiation in SVZ neural stem/progenitor cell cultures derived from neonatal mice. The original results presented in this work suggest that Hp and derivates may be of potential interest for the development of future strategies to treat demyelinating diseases.

Transplantation is a clinically relevant approach for brain repair, but much remains to be understood about influences of the disease environment on transplant connectivity. To explore the effect of amyloid pathology in Alzheimer's disease (AD) and aging, we examined graft connectivity using monosynaptic rabies virus tracing in APP/PS1 mice and in 16- to 18-month-old wild-type (WT) mice. Transplanted neurons differentiated within 4 weeks and integrated well into the host visual cortex, receiving input from the appropriate brain regions for this area. Unexpectedly, we found a prominent several-fold increase in local inputs, in both amyloid-loaded and aged environments. State-of-the-art deep proteome analysis using mass spectrometry highlights complement system activation as a common denominator of environments promoting excessive local input connectivity. These data therefore reveal the key role of the host pathology in shaping the input connectome, calling for caution in extrapolating results from one pathological condition to another.

The adult mammalian brain can produce new neurons in a process called adult neurogenesis, which occurs mainly in the subventricular zone (SVZ) and in the hippocampal dentate gyrus (DG). Brain-derived neurotrophic factor (BDNF) signaling and cannabinoid type 1 and 2 receptors (CB1R and CB2R) have been shown to independently modulate neurogenesis, but how they may interact is unknown. We now used SVZ and DG neurosphere cultures from early (P1-3) postnatal rats to study the CB1R and CB2R crosstalk with BDNF in modulating neurogenesis. BDNF promoted an increase in SVZ and DG stemness and cell proliferation, an effect blocked by a CB2R selective antagonist. CB2R selective activation promoted an increase in DG multipotency, which was inhibited by the presence of a BDNF scavenger. CB1R activation induced an increase in SVZ and DG cell proliferation, being both effects dependent on BDNF. Furthermore, SVZ and DG neuronal differentiation was facilitated by CB1R and/or CB2R activation and this effect was blocked by sequestering endogenous BDNF. Conversely, BDNF promoted neuronal differentiation, an effect abrogated in SVZ cells by CB1R or CB2R blockade while in DG cells was inhibited by CB2R blockade. We conclude that endogenous BDNF is crucial for the cannabinoid-mediated effects on SVZ and DG neurogenesis. On the other hand, cannabinoid receptor signaling is also determinant for BDNF actions upon neurogenesis. These findings provide support for an interaction between BDNF and endocannabinoid signaling to control neurogenesis at distinct levels, further contributing to highlight novel mechanisms in the emerging field of brain repair.

Neurogenesis in the subventricular zone (SVZ) is regulated by diffusible factors and cell-cell contacts. In vivo, SVZ stem cells are associated with the abluminal surface of blood vessels and such interactions are thought to regulate their neurogenic capacity. SVZ neural stem cells (NSCs) have been described to contact endothelial-derived laminin via α6β1 integrin. To elucidate whether heterocellular contacts with brain endothelial cells (BEC) regulate SVZ cells neurogenic capacities, cocultures of SVZ neurospheres and primary BEC, both obtained from C57BL/6 mice, were performed. The involvement of laminin-integrin interactions in SVZ homeostasis was tested in three ways. Firstly, SVZ cells were analyzed following incubation of BEC with the protein synthesis inhibitor cycloheximide (CHX) prior to coculture, a treatment expected to decrease membrane proteins. Secondly, SVZ cells were cocultured with BEC in the presence of an anti-α6 integrin neutralizing antibody. Thirdly, BEC were cultured with β1-/- SVZ cells. We showed that contact with BEC supports, at least in part, proliferation and stemness of SVZ cells, as evaluated by the number of BrdU positive (+) and Sox2+ cells in contact with BEC. These effects are dependent on BEC-derived laminin binding to α6β1 integrin and are decreased in cocultures incubated with anti-α6 integrin neutralizing antibody and in cocultures with SVZ β1-/- cells. Moreover, BEC-derived laminin sustains stemness in SVZ cell cultures via activation of the Notch and mTOR signaling pathways. Our results show that BEC/SVZ interactions involving α6β1 integrin binding to laminin, contribute to SVZ cell proliferation and stemness.

One hallmark of adult neurogenesis is its adaptability to environmental influences. Here, we uncovered the epithelial sodium channel (ENaC) as a key regulator of adult neurogenesis as its deletion in neural stem cells (NSCs) and their progeny in the murine subependymal zone (SEZ) strongly impairs their proliferation and neurogenic output in the olfactory bulb. Importantly, alteration of fluid flow promotes proliferation of SEZ cells in an ENaC-dependent manner, eliciting sodium and calcium signals that regulate proliferation via calcium-release-activated channels and phosphorylation of ERK. Flow-induced calcium signals are restricted to NSCs in contact with the ventricular fluid, thereby providing a highly specific mechanism to regulate NSC behavior at this special interface with the cerebrospinal fluid. Thus, ENaC plays a central role in regulating adult neurogenesis, and among multiple modes of ENaC function, flow-induced changes in sodium signals are critical for NSC biology.