A noncanonical release of GABA and glutamate modulates neuronal migration

Abstract

Immature neurons express GABA and glutamate receptors before synapse formation, and both transmitters are released at an early developmental stage. We have now tested the hypothesis that the ongoing release of GABA and glutamate modulates neuronal migration. Using 5-bromo-2'-deoxyuridine labeling and cocultures of hippocampal slices obtained from naive and green fluorescent protein-transgenic mice, we report that migration is severely affected by GABA(A) or NMDA receptor antagonist treatments. These effects were also present in munc18-1 knock-out slices in which soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent vesicular secretion of transmitters has been deleted. GABA(A) antagonists were more efficient than NMDA antagonists to reduce cell migration, in keeping with the earlier maturation of GABAergic mechanisms. We conclude that GABA and, to a lesser degree, glutamate released in a SNARE-independent mechanism exert a paracrine action on neuronal migration.

Figure 1.

Migrating neuroblasts in the hippocampal organotypic slice coculture assay. A, Left, Diagram depicting the two hippocampal organotypic slices as they are arranged in the coculture assay. The two slices, the fluorescent donor slice and the nonfluorescent host slice, are plated in very tight apposition one relative to another, at the level of the neuroepithelium of the CA1 region. Right, Higher magnification of the square shown in the left panel. Fluorescent (GFP) migrating neuroblasts, originated in the neuroepithelium of the GFP slice (GFP ne), are migrating onto the nonfluorescent slice to reach the stratum pyramidale (s. pyr.), crossing the migration area [i.e., the neuroepithelium of the nonfluorescent slice (WT ne) and the future stratum oriens (s. or.)]. s. rad., stratum radiatum. B, Images of living cocultivated hippocampal slices, as they appear 1 h after plating onto the membrane of a Millicell-CM insert, in light transmission (left) and under UV light (right). The borders of the nonfluorescent slice are shown in white. C, Immunostaining of E17 slices cocultured for 1 DIV showing migrating neuroblasts double-labeled with antibodies against GFP (green) and βIII-tubulin (red). Most neuroblasts originated in the fluorescent slice (GFP slice) are migrating radially onto the nonfluorescent slice (WT slice) toward the s. pyr., crossing the future s. or. ne, Neuroepithelium. D, Immunostaining of E17 slices cocultured for 1 DIV, showing migrating neuroblasts at larger magnification, double-labeled with antibodies against GFP (green; top) and βIII-tubulin (red; left middle) or MAP2 (red; right middle). Green and red channels are merged in bottom panels. Migrating neuroblasts are either monopolar with a single leading process or bipolar with a branched leading process (white arrowhead in the left top panel). They often display a short trailing process (gray arrowhead in the right top panel). Lamellipodia are distinguishable at the tip of the leading process (white arrows in the top panels), and few filopodia are often present along the process (gray arrows in the left top panel). E, Immunostaining of E17 slices cocultured for 1 DIV, showing GFP-positive migrating neuroblasts (green; left) and GABA-positive fibers (red; middle). Virtually all GFP-positive migrating neuroblasts are immunonegative to GABA (merged green and red channels on the right panel). Scale bars: C, 50 μm; D, 10 μm; E, 25 μm.

Figure 2.

Receptor expression and electrophysiological properties of migrating neuroblasts. A-C, Immunostaining of an E17 slice cocultured for 1 DIV showing a migrating neuroblast double-labeled with antibodies against GFP (green; A and top panels in B and C) and NR1 subunit of the NMDA receptor (red; A and middle panels in B and C). Squares surrounding the soma and the tip of the leading process are shown enlarged in B and C. Green and red channels are merged in the bottom panels in B and C. D-F, Immunostaining of E17 slices cocultured for 1 DIV showing a migrating neuroblast double-labeled with antibodies against GFP (green; D and top panels in E and F) and GABA_A receptor (red; D and middle panels in E and F). Squares surrounding the soma and the tip of the leading process are shown enlarged in E and F. Green and red channels are merged in the bottom panels in E and F. G, Photomicrograph of a GFP-positive migrating neuroblast recorded with a pipette filled with rhodamine. Left, GFP-positive neuroblast originated into the fluorescent slice (GFP slice) migrating onto the nonfluorescent hippocampal slice (WT slice). The arrow points the leading process, and the arrowhead points to the trailing process. Middle, The same neuroblast shown in light transmission (Light Transm.), impaled with the patch pipette. Right, The same neuroblast filled with rhodamine (Rhod.). H, Spikelet evoked by depolarizing pulses applied to a migrating neuroblast recorded with a KCl-filled pipette solution. The capacitance of the cell was 10 pF. The spikelet (indicated with an asterisk and shown at lower time scale on the right) is evoked at a membrane potential of -25 mV. I, Currents mediated by bath application of NMDA and isoguvacine and recorded at 30 mV in the same cell (C_m, 6 pF) with a CsGlu-filled pipette solution. Both receptor-mediated currents are blocked by their respective antagonists. J, Migrating neuroblast (C_m, 3 pF) recorded at 30 mV with a CsGlu-filled pipette solution. Bath application of GABA_A receptor antagonists generates an inward current associated with a decrease of the basal noise. Depolarizing pulses (5 mV, 10 ms, applied every 10 s) show that these effects are not associated with changes in the serial resistance. The bottom traces show representative responses to the current pulse before (a) and at the plateau (b) of the tonic current. Scale bars: A, D, 20 μm; B, E, 5 μm; C, F, 2 μm; G, 10 μm.

Figure 3.

Treatment with receptor antagonists affects the length of the leading processes of migrating neuroblasts without affecting their orientation. A, The orientation of the leading processes is evaluated with respect to the correct direction of migration (corresponding to an angular deviation of 0°). Migrating neuroblasts located in the migration area [i.e., the neuroepithelium (ne) and the stratum oriens (s. or.)] and on their way to the stratum pyramidale (s. pyr.) are distributed into three groups, depending on their leading processes orientation: (1) cells with leading process orientations that deviate from 0° to 70° of the correct direction of migration; (2) cells with leading process orientations that deviate from 70° to 110°; and (3) cells with leading process orientations that deviate from 110° to 180°. Six reconstructed migrating neuroblasts are shown (2 per group). B, Distribution of the migrated cells into the three groups explained in A, after culture for 1 DIV in the absence of any treatment, in the presence of MK801, or in the presence of bicuculline. The number of experiments is given in parentheses. C, Length of the leading processes evaluated after 1 DIV in the absence of any treatment, in the presence of MK801, or in the presence of bicuculline. Data are expressed as ±SEM, and the number of experiments is given in parentheses. ^*p < 0.005; ^**p < 0.001. D, Length of the leading processes evaluated after 1 DIV in the absence of any treatment, in the presence of MK801, or in the presence of bicuculline. Sixteen reconstructed migrating neuroblasts are shown per condition. The distance between the gray horizontal bars is 50 μm. CTRL, Control, absence of any treatment; BICU, bicuculline.

Figure 4.

GABA_A and NMDA receptor antagonists prevent the BrdU-labeled cells from migrating to the stratum pyramidale in hippocampal organotypic slice culture. A, Immunostaining for BrdU on hippocampal organotypic slices cultured for 1 DIV without any treatment (control condition; left), in the presence of 10 μm MK801 (middle), and in the presence of 50 μm bicuculline (right). In the absence of any treatment, the majority of BrdU+ cells after 1 DIV has migrated to settle into the stratum pyramidale (s. pyr.). After 1 DIV, in the presence of MK801 or bicuculline, BrdU+ cells that have failed to migrate are mainly distributed into the migration area [i.e., neuroepithelium (ne)/stratum oriens (s. or.)]. Scale bar, 20 μm. B, Histogram showing the migration indices obtained after treatment for 1 DIV with 10 μm MK801 or 50 μm bicuculline compared with the untreated control condition. The migration indices are expressed (±SEM) as the ratio between the percentage of cells that reached the stratum pyramidale after 1 DIV and the percentage of cells that were still in the migration area (i.e., from the neuroepithelium to the stratum oriens), with the average of the control values being set to 10. ^**p < 0.001. The number of experiments is given in parentheses. CTRL, Control; BICU, bicuculline.

Figure 5.

Receptor expression and hippocampal cytoarchitecture in munc18-1 mutants mice versus wild-type mice at late prenatal stages. A, B, Immunostaining for the NR1 subunit of the NMDA receptor on E17 hippocampal sections, revealing no striking differences in the NR1 subunit expression throughout the stratum oriens and the stratum pyramidale of the CA1 region, in the wild-type mice compared with the munc18-1 mutant mice. Insets, Higher magnification revealing a mainly somatic (asterisks) expression of the NR1 subunits in both groups. C, D, Immunostaining for the GluR2/3 subunit of the AMPA receptor on E17 hippocampal sections, showing similar GluR2/3 subunit expression throughout the strata oriens and pyramidale of the CA1 region, in the wild-type mice compared with the munc18-1 mutant mice. Insets, Higher magnification of double immunostaining for the GluR2/3 subunit of the AMPA receptor (green) and for MAP2 (red) showing in few pyramidal cells the presence of a short apical dendrite (arrows), decorated with GluR2/3 subunits. The asterisks indicate cell nuclei. E, F, Immunostaining for the GABA_A receptor on E17 hippocampal sections, illustrating the absence of any striking difference in the GABA_A subunit expression throughout the strata oriens, radiatum, and pyramidale of the CA1 region, in the wild-type mice compared with the munc18-1 mutant mice. Insets, Higher magnification showing a mainly somatic expression of the GABA_A receptors in both groups. The asterisks indicate cell nuclei, and arrows indicate apical dendrites. Scale bars: 20 μm; insets, 10 μm. s. or., Stratum oriens; s. pyr., stratum pyramidale; s. rad., stratum radiatum.

Figure 6.

GABA_A and NMDA receptor antagonists impaired the migration of BrdU-labeled cells in hippocampal organotypic slice culture from munc18-1 mutant mice. A, Immunostaining for BrdU on hippocampal organotypic slices from munc18-1 mutant mice cultured for 1 DIV without any treatment [control condition (munc); left], in the presence of 10 μm MK801 (middle), and in the presence of 50 μm bicuculline (right). In the absence of any treatment in munc18-1 hippocampal slices, the BrdU+ cells after 1 DIV are distributed partly into the migration area [i.e., neuroepithelium (ne)/stratum oriens (s. or.)] and partly into the stratum pyramidale (s.pyr.). After 1 DIV in the presence of MK801 and to a greater extent in the presence of bicuculline, BrdU+ cells that have failed to migrate are distributed into the migration area. Scale bar, 20 μm. B, Histogram showing the migration indices obtained after treatment for 1 DIV with 10 μm MK801 or 50 μm bicuculline compared with the untreated control (munc) condition in hippocampal organotypic slice cultures from munc18-1 mutant mice. The migration indices are expressed (± SEM) as the ratio between the percentage of cells that reached the stratum pyramidale after 1 DIV and the percentage of cells that were still in the migration area (i.e., from the neuroepithelium to the stratum oriens), with the average of the control values being set to 10. ^#p < 0.01. The number of experiments is given in parentheses. CTRL, Control; BICU, bicuculline.