Tonic activation of GLUK5 kainate receptors decreases neuroblast migration in whole-mounts of the subventricular zone

Abstract

In the postnatal subventricular zone (SVZ), neuroblasts migrate in chains along the lateral ventricle towards the olfactory bulb. AMPA/kainate receptors as well as metabotropic glutamate receptors subtype 5 (mGluR5) are expressed in SVZ cells. However, the cells expressing these receptors and the function of these receptors remain unexplored. We thus examined whether SVZ neuroblasts express mGluR5 and Ca(2+)-permeable kainate receptors in mouse slices. Doublecortin (DCX)-immunopositive cells (i.e. neuroblasts) immunostained positive for mGluR5 and GLU(K5-7)-containing kainate receptors. RT-PCR from approximately 10 GFP-fluorescent cell aspirates obtained in acute slices from transgenic mice expressing green fluorescent protein (GFP) under the DCX promoter showed mGluR5 and GLU(K5) receptor mRNA in SVZ neuroblasts. Patch-clamp data suggest that approximately 60% of neuroblasts express functional GLU(K5)-containing receptors. Activation of mGluR5 and GLU(K5)-containing receptors induced Ca(2+) increases in 50% and 60% of SVZ neuroblasts, respectively, while most neuroblasts displayed GABA(A)-mediated Ca(2+) responses. To examine the effects of these receptors on the speed of neuroblast migration, we developed a whole-mount preparation of the entire lateral ventricle from postnatal day (P) 20-25 DCX-GFP mice. The GABA(A) receptor (GABA(A)R) antagonist bicuculline increased the speed of neuroblast migration by 27%, as previously reported in acute slices. While the mGluR5 antagonist MPEP did not affect the speed of neuroblast migration, the homomeric and heteromeric GLU(K5) receptor antagonists, NS3763 and UB302, respectively, increased the migration speed by 38%. These data show that although both GLU(K5) receptor and mGluR5 activations increase Ca(2+) in neuroblasts, only GLU(K5) receptors tonically reduce the speed of neuroblast migration along the lateral ventricle.

Figure 1. SVZ neuroblasts express mGluR5 and GLU_K5–7-containing kainate receptors

A and B, photographs of immunostaining for mGluR5 (green), DCX (red) and GLAST (blue) in a coronal section from a P30 mouse brain at low (A) and high magnification (B). mGluR5 (green) is expressed by neuroblasts (red, yellow arrow) and ependymal cells (white arrow in A), but not by GLAST-positive cells, i.e. astrocytes (blue, yellow arrowhead). C and D, photographs of immunostaining for GLU_K5–7-containing receptors (green), DCX (red) and GFAP (blue) in a coronal section from a P30 mouse brain at low (C) and high magnification (D). GLU_K5–7-containing receptors are expressed by neuroblasts (red, white arrow) and in a few GFAP cells (blue, yellow arrow). Some neuroblasts do not stain for GLU_K5–7-containing receptors (white arrowhead). Scale bars, 20 μm. E and F, agarose gel electrophoresis (F) demonstrating RT-PCR amplification of GLU_K5 (lane ‘1’) and mGluR5 (lane ‘2’) mRNA isolated during pipette aspiration of 10 GFP-fluorescent cells, i.e. neuroblasts, shown in E. GLU_K5 and mGluR5 were not detected in bath solution (lane ‘3’ for GLU_K5 and lane ‘4’ for mGluR5) controls.

Figure 2. Kainate receptor channel activity in SVZ neuroblasts

A–C, representative traces from perforated patch-clamp records of SVZ neuroblasts. A, a broad-spectrum kainate receptor agonist domoate (10 μm, 10 s) induces an inward current in a SVZ neuroblast. B and C, the GLU_K5-containing receptor agonist ATPA (25 μm) increases baseline noise (B) or induces single-channel activity (C) in two different neuroblasts. D, higher resolution of ATPA-induced single channels in a neuroblast. E, amplitude histogram (bin size: 0.12 pA) of ATPA-induced single channels pooled from 5 neuroblasts.

Figure 3. Activation of GLU_K5 receptors, mGluR5 and GABA_ARs increases Ca²⁺ in overlapping subpopulations of SVZ neuroblasts

A, DIC photograph of the lateral SVZ along the lateral ventricle (LV). Scale bar, 20 μm. St, striatum. B–C, photographs of fluo4 fluorescence in the same SVZ region as shown in A taken before (B) and after 10 μm ATPA application (C). Fluo4-AM was pressure-applied onto SVZ cells. D, a representative trace of normalized fluo4 fluorescence (from B and C) over time illustrates that ATPA-induced Ca²⁺ increases in SVZ cells were blocked by the non-competitive, homomeric GLU_K5 inhibitor NS3763. E, bar graph illustrating the percentage of SVZ cells responding to ATPA, the mGluR5 agonist DHPG (50 μm), and the GABA_AR agonist isoguvacine (iso, 50 μm). F, bar graph illustrating the percentage inhibition of ATPA-, DHPG- and isoguvacine-induced Ca²⁺ responses by NS3763, UBP302, MPEP and bicuculline (Bic). UBP302 is a competitive blocker of GLU_K5-containing receptors. G, representative ATPA-, DHPG- and isoguvacine-induced Ca²⁺ increases in the same neuroblast. H, a Venn diagram illustrating the percentage of cells expressing one or a combination of the following receptors: GLU_K5, mGluR5 and GABA_ARs.

Figure 4. Tonic activation of GABA_ARs and homomeric GLU_K5 receptors decreases the speed of neuroblast migration in whole-mounts of the lateral ventricle

A, composite images of a fixed whole-mount from a P60 DCX-GFP mouse taken at ×10 magnification. The yellow lines indicate the region where the migration movies were acquired. Scale bar, 70 μm. B, a representative Z-stack image (25 Z sections spaced by 2 μm) of SVZ chains in a DCX-GFP whole-mount preparation. C, migratory routes of individual cells from the Z-stack image shown in B before (white lines) and during NS3763 application (red lines). Scale bar, 50 μm. D, bar graph illustrating the percentage change in the migration speed in the presence of bicuculline (50 μm), MPEP (50 μm), NS3763 (25 μm) or UBP302 (10 μm). *P < 0.05. E, bar graph illustrating the percentage change in the maximum speed of neuroblasts. In the plots in D and E: box, s.e.m.; whisker, s.d.; middle line, median.