NMDA receptors activated by subventricular zone astrocytic glutamate are critical for neuroblast survival prior to entering a synaptic network

Abstract

Even before integrating into existing circuitry, adult-born neurons express receptors for neurotransmitters, but the intercellular mechanisms and their impact on neurogenesis remain largely unexplored. Here, we show that neuroblasts born in the postnatal subventricular zone (SVZ) acquire NMDA receptors (NMDARs) during their migration to the olfactory bulb. Along their route, neuroblasts are ensheathed by astrocyte-like cells expressing vesicular glutamate release machinery. Increasing calcium in these specialized astrocytes induced NMDAR activity in neuroblasts, and blocking astrocytic vesicular release eliminated spontaneous NMDAR activity. Single-cell knockout of NMDARs using neonatal electroporation resulted in neuroblast apoptosis at the time of NMDAR acquisition. This cumulated in a 40% loss of neuroblasts along their migratory route, demonstrating that NMDAR acquisition is critical for neuroblast survival prior to entering a synaptic network. In addition, our findings suggest an unexpected mechanism wherein SVZ astrocytes use glutamate signaling through NMDARs to control the number of adult-born neurons reaching their final destination.

Figure 1. Neuroblasts acquire NMDARs during their migration in the rSVZ and RMS_EL

(A) Perforated patch clamp records of neuroblasts at different positions along the caudal-rostral axis en route to the olfactory bulb. The different locations including the SVZ along the lateral ventricle, the rostral SVZ (rSVZ), the RMS_EL (EL for elbow), and the RMS of the olfactory bulb (RMS_OB) are illustrated on a sagittal section in (H). Pressure application of 100 μM NMDA (5 s) induced inward currents in rSVZ and RMS but not SVZ neuroblasts (held at −64 mV). Scale: 10 pA/5 s. (B) Bar graphs illustrating NMDA-induced charge transfer divided by the cell capacitance (*, p<0.05). The numbers above the bars represent the percentage of neuroblast responding to NMDA at each location. (C) NMDA-induced currents at different holding potentials (indicated on the right). Scale: 5 pA/5 s. (D) NMDA-induced depolarizations before, during, and after wash-out of 50 μM D-APV in the bath. NMDA applications are indicated by the squares over the trace. Scale: 20 mV/60 s. (E) Confocal images of Fluo-4 AM-loaded RMS_EL cells before (control) and during NMDA application (100 μM, 10 s) in an acute sagittal slice. Overlaid images (control in green and NMDA in red) is shown on the right. The arrows point to GFP-expressing cells identifying them as astrocytes. The arrowhead points to NMDA-responding neuroblasts. Scale: 20 μm. (F) Calcium traces from several neuroblasts and astrocytes from the slices shown in (E). NMDA (10 s) induces calcium increases in neuroblasts but not astrocytes. (G) Bar graphs illustrating a significant increase in the percentage of neuroblasts responding to NMDA with Ca²⁺ increases as a function of their location (**, p<0.01 and ***, p<0.001). Per location 4–11 recordings and 8–42 neuroblasts were analyzed. (H) Reconstructed image of a sagittal section with boxed regions showing the locations of the patch clamp and Ca²⁺ recordings. The percentage of cells that responded to NMDA with a current is indicated at each location. Scale: 500 μm. Inset: 100 μM NMDA-induced current in a RMS_EL neuroblast from a P91 mouse. Scale: 2 pA/1 s.

Figure 2. Most neuroblasts display phasic NMDAR activation by the time they reach the RMS_EL

(A) Perforated patch clamp records illustrating spontaneous single channel events (sSCEs) in a neuroblast of the RMS_EL. Periods (a and b) are displayed on an expanded time-scale in the left inset. The right inset represents an amplitude histogram (bin size: 0.2 pA, 1617 events) for which part of the record is shown above. A Gaussian fit yielded a mean amplitude of −3.0 pA. Scale bars: 5 pA/4 s, 400 ms (a), 50 ms (b). (B) Perforated patch clamp records illustrating that sSCEs are sensitive to D-APV. Scale: 5 pA/30 s. (C) Bar graphs representing the frequency of single channels in rSVZ, RMS_EL, and RMS_OB neuroblasts (n=6, 17, and 20, respectively). The frequency significantly increased from the rSVZ to the RMS_EL and to the RMS_OB (p<0.05). (D) Bar graphs illustrating a significant increase in the percentage of neuroblasts displaying sSCEs from the rSVZ to the RMS_EL (p<0.05). (E) Perforated patch clamp recordings in current clamp illustrating spontaneous depolarizations sensitive to D-APV in aCSF with near-physiological Mg²⁺ concentration (0.8 mM). Scale: 10 mV/3 s.

Figure 3. VGLUT1 and Synaptobrevin 2 are only expressed in astrocytes in the SVZ/RMS

(A) Electron micrograph of pre-embedding immunocytochemistry for L-glutamate in the rSVZ. The electron dense material for glutamate (as indicated by the arrows) is observed exclusively in astrocytes (As) surrounding neuroblasts (N). Scale: 1 μm. (B) Confocal image displaying co-immunostaining for VGLUT1 (green), doublecortin (DCX, blue), and GLAST (red) in the RMS. Scale: 10 μm. (C) Electron micrographs showing post-embedding immunocytochemistry for VGLUT1 expression in the rSVZ. The right panel represents a higher magnification image of the boxed region shown in the left panel. The secondary antibody was conjugated to 5 nm-gold particles (arrows). Scale bars: 200 nm (left)/50 nm (right). (D) Confocal image of synaptobrevin 2 (green), DCX (blue), and GLAST (red). Scale: 10 μm.

Figure 4. Ca²⁺-dependent vesicular glutamate release from RMS astrocytes contributes to NMDAR activation in neuroblasts

(A) Confocal image displaying co-immunostaining for GFP in MrgA1⁺ mice (green), GFAP (blue), and BLBP (red) in the SVZ. The inset illustrates GFP/BLBP and GFP/GFAP staining from the image in the boxed region. Scale bars: 70 μm (top image)/40 μm. (B) Top: Confocal images of GFP- and Fluo-4-fluorescent cells in the RMS before (control) and during FLRFa pressure application (5 μM, 10 s). Recordings were performed in the RMS in acute sagittal slices from a MrgA1⁺ mouse. The arrow indicates astrocytes that are outlined by GFP fluorescence. Scale: 10 μm. Bottom: Overlay of the images under control (green) and during FLRFa application (red). On the right, activity graph illustrating Ca²⁺ increases in GFP⁺ cells, i.e. astrocytes. (C) Top: Model illustrating that FLRFa is applied to induce Ca²⁺ increases in astrocytes and examine whether NMDARs are activated in adjacent neuroblasts. Bottom: Perforated patch clamp records displaying sSCEs in neuroblasts under control and in the presence of FLRFa (5 μM, bath) followed by D-APV addition in slices from MrgA1⁺ mice. Scale bars: 5 pA/1 s. (D) Bar graphs illustrating the % increase of sSCE frequency in the presence of FLRFa without and with D-APV in MrgA1⁺ mice and control single transgenic mice (tetO-MrgA1, MrgA1⁻). The asterisks indicate significant enhancement above control levels of 100% (*, p<0.05; **, p<0.01). (E) Perforated patch clamp records displaying spontaneous SCEs in neuroblasts after 15 hrs pre-incubation in BoNT/A and after addition of D-APV. Scale: 5 pA/3 s. (F) Perforated patch clamp records displaying the lack of spontaneous SCEs in neuroblasts after 11 hrs pre-incubation in BoNT/B. Application of NMDA still induced inward currents. Scale: 5 pA/4 s. (G) Bar graphs illustrating the frequency of spontaneous SCEs following pre-incubation in control aCSF (for 4 hrs or 11–15 hrs) or BoNT/A, and the absence of SCEs following pre-incubation in Con A or BoNT/B. (** p<0.01, * p<0.05).

Figure 5. Single-cell knockout of functional NMDARs in neuroblasts using in vivo postnatal electroporation

(A) Diagram illustrating the effect of Cre expression in cells from R26R-YFP (control) and R26R-YFP/NR1^fl mice (NR1^fl) resulting in YFP⁺ cells expressing NMDARs and in YFP⁺ NMDAR-deficient cells, respectively. (B) Diagram illustrating the in vivo electroporation procedure in neonatal R26R-YFP mice. (C) Reconstructed confocal image of a sagittal section that contained DCX-immunopositive (red) YFP-expressing cells (green). Scale: 500 μm. (D) Confocal image of YFP (green) and DCX (red) staining from the boxed image in (C). Scale: 30 μm. (E and F) Whole cell patch clamp records of YFP⁺ neuroblasts in R26-YFP (E) and R26-YFP/NR1^fl mice. Application of 100 μM NMDA (10 s) induced inward currents in neuroblasts of control R26-YFP mice, but not in neuroblasts of R26-YFP/NR1^fl mice. Scale: 25 pA/5 s. (G) Ca²⁺ activity graph from YFP⁺ and YFP⁻ neuroblasts in the RMS from a R26R-YFP/NR1^fl mouse. (H) Mean NMDA-induced Ca²⁺ responses in YFP⁺ neuroblasts from control R26R-YFP mice. A YFP⁺/Fluo-4 AM⁺ cell before and during NMDA application is shown under the Ca²⁺ trace. (I) Bar graphs illustrating the % of YFP⁺ neuroblasts responding to NMDA in the RMS in acute sagittal slices from R26R-YFP and NR1^fl mice.

Figure 6. NMDAR does not regulate neuroblast migration

(A) Examples of migratory routes of individual neuroblasts in the RMS_EL of a R26-YFP mouse before (blue lines) and during NMDA application (10 μM, red lines). The underlaid image is the first time point of acquisition. Scale: 100 μm. (B) Bar graphs illustrating the average speed of neuroblast migration under control conditions and in the presence of D-APV (50 μM) or NMDA (10 μM) in R26-YFP mice, and under control conditions in slices from R26-YFP versus NR1^fl mice.

Figure 7. In vivo blockade and genetic removal of NMDARs decrease neuroblast survival

(A) Confocal images displaying immunostaining for activated caspase 3 (red) in the RMS from control or MK-801-treated DCX-GFP mice. Scale: 100 μm. (B) Bar graphs of the density of activated caspase 3-immunopositive DCX⁺ cells in sagittal slices containing the rSVZ and RMS_EL following in vivo injections of saline solution (Ctl) or MK-801. Mice received a single dose of MK-801 and were sacrificed at different time-points following injection (2, 6, and 24 hrs). The density was obtained by dividing the cell number by the volume of the region analyzed. The inset illustrates the experimental paradigm. (C) Bar graphs of the percentage of activated caspase 3⁺ YFP⁺/DCX⁺ neuroblasts in the rSVZ, RMS and granule cell layer (GCL) of the OB quantified at 2, 4, and 8 weeks post-electroporation. The % was calculated from the total number of YFP⁺ neuroblasts. (D) Model illustrating the predicted effect of increased apoptosis on accumulated cell loss along the migratory route. Considering that ~10,000 neuroblasts are born per day, ~200 NR1-KO neuroblasts (1–3%) will be apoptotic and cleared in ~1 day. Since its takes ~11 days for newly born neuroblasts to migrate from the SVZ to the RMS_OB, apoptosis would lead to a total loss of ~2,200 neuroblasts in the RMS_OB, corresponding to 22% of the original neuroblast population.

Figure 8. Single neuroblast knockout of NR1 results in neurogenesis deficit

(A) Confocal images of YFP⁺ cells in the SVZ, RMS and GCL in sections from R26R-YFP and R26R-YFP/NR1^fl mice. Scale: 70 μm. The dashed line indicates the location of the mitral cell layer. (B) Bar graphs of the density of YFP⁺/DCX⁺ cells (i.e. YFP⁺ neuroblasts) in the rSVZ, RMS and granule cell layer of the OB (GCL) quantified at 2, 4, and 8 weeks post-electroporation. Experiments were performed in R26R-YFP (i.e. control) and R26R-YFP/NR1^fl mice (NR1^fl, 4–5 mice for each data point) at 2, 4 and 8 weeks. (* p<0.05, ** p<0.01, *** p<0.001). (C) Bar graphs of density ratios obtained by dividing the density in NR1^fl by that in R26-YFP mice before and following normalization by the density of YFP-expressing cells in the SVZ/rSVZ of the same animal.