GABA and glutamate signaling: homeostatic control of adult forebrain neurogenesis

Abstract

The neurotransmitter GABA exerts a strong negative influence on the production of adult-born olfactory bulb interneurons via tightly regulated, non-synaptic GABAergic signaling. After discussing some findings on GABAergic signaling in the neurogenic subventricular zone (SVZ), we provide data suggesting ambient GABA clearance via two GABA transporter subtypes and further support for a non-vesicular mechanism of GABA release from neuroblasts. While GABA works in cooperation with the neurotransmitter glutamate during embryonic cortical development, the role of glutamate in adult forebrain neurogenesis remains obscure. Only one of the eight metabotropic glutamate receptors (mGluRs), mGluR5, has been reported to tonically increase the number of proliferative SVZ cells in vivo, suggesting a local source of glutamate in the SVZ. We show here that glutamate antibodies strongly label subventricular zone (SVZ) astrocytes, some of which are stem cells. We also show that some SVZ neuroblasts express one of the ionotropic glutamate receptors, AMPA/kainate receptors, earlier than previously thought. Collectively, these findings suggest that neuroblast-to-astrocyte GABAergic signaling may cooperate with astrocyte-to-neuroblast glutamatergic signaling to provide strong homeostatic control on the production of adult-born olfactory bulb interneurons.

Figure 1. Organizations of the adult subventricular zone

(A) Diagram of a sagittal rodent brain section illustrating the two neurogenic regions, the SVZ/olfactory system and the hippocampal subgranular zone (SGZ). In the SVZ, neuroblasts are continually generated and migrate as chains (not illustrated) throughout the SVZ, join the RMS and differentiate into interneurons in the olfactory bulb (OB), including granule cells (green) and periglomerular cells (red). The different stages of neurogenesis are marked along the migratory path of the neuroblasts. The SGZ is located at the base of the granule cell layer in the hilus of the dentate gyrus (DG). (B) Diagram illustrating the cellular organization in the SVZ/RMS. SVZ/RMS neuroblasts (blue) that express doublecortin (DCX) and migrate to the olfactory bulb are ensheathed by astrocytes (red). Ependymal cells (purple) line the wall of the lateral ventricle in the SVZ. Scattered transit amplifying progenitors (grey) are present along the SVZ and RMS. Astrocytes express GFAP and constitute a pool of neural stem cells. GABA released from neuroblasts diffuses and activates GABA_A receptors (green rectangles) in neuroblasts as well as astrocytes. GABA is also taken up by GABA transporters (mouse GAT4) in astrocytes. (C) Confocal microscopy photograph illustrating the cellular organization in the RMS: astrocytes labeled by GLAST antibodies (red) ensheath clusters of neuroblasts labeled by DCX antibodies (blue) in a coronal section. Scale bar: 20 μm.

Figure 2. Expression of GABA signaling molecules in the SVZ

(A) GABA immunostaining (green) in the SVZ shows the presence of GABA in some SVZ cells, but not in GLAST-positive cells (red, i.e. astrocytes). (B) Immunolabeling for vesicular GABA transporters, VGATs (green), is sparse or nonexistent in SVZ cells. Prominent punctate labeling, by contrast, is observed in the striatum in the left of the panel. (C) Positive labeling for the GABA transporter GAT1 (green) in the SVZ is observed mainly in cells that do not stain positive for GLAST (red). Scale bars: 10 μm.

Figure 3. Neuroblasts display spontaneous Ca²⁺ transients

(A) Photograph of a confocal section displaying Oregon Green loading in an acute coronal slice containing the RMS. Cells denoted by arrows (red, blue, and green) exhibit spontaneous Ca²⁺ activity as shown in (B). (B) Ca²⁺ activity graphs of cells shown in (A). (C-D) Bar graphs of the percentage of cells displaying spontaneous Ca²⁺ transients (C) and of the frequency of these transients (D) in 3 slices. 31 to 65 cells were analyzed per slice (mean of 51.3, n=3).

Figure 4. Astrocytes contain glutamate while neuroblasts express AMPA-type glutamate receptors

(A) Photograph of a confocal section displaying co-immunostaining for glutamate (green), GLAST (red), and DCX (blue) in the SVZ. Scale bar: 10 μm. (B) Photographs of an acute slice containing the RMS-OB and loaded with fluo 4-AM. Image before (left panel) and after (right panel) application of kainate (300 μM, 10 s). In this slice, 85% of the cell responded to kainate suggesting the functional expression of AMPA receptors in RMS cells. The slice was made from a postnatal day 25 mouse. (C) Ca² activity traces of three representative cells showing some spontaneous activity and Ca²⁺ increases in response to kainate application. (D) Bar graphs of the percentage (%) of cells responding to kainate application as a function of their location along the rostro-caudal axis, i.e. SVZ, RMS, and RMS of the OB (RMS-OB). An increasing percentage of neuroblasts acquire functional AMPA receptors during migration to the OB.

Figure 5. Hypothetical model illustrating a homeostatic control of neuroblast production by GABA and glutamate

Neuroblasts express GABA_A receptors and AMPA receptors in the SVZ and RMS. Neuroblasts release GABA that activates GABA_A receptors on themselves and surrounding astrocytes. In turn, astrocytes surrounding neuroblasts release glutamate which may activate AMPA receptors on migrating neuroblasts. mGluRs are expressed in the SVZ but the identity of the cell bearing these receptors is unknown. GABA_A receptor activation by ambient GABA leads to a decrease in the number of proliferative astrocytes and neuroblasts while mGluR activation by ambient glutamate induces an increase in the number of proliferative cells in the SVZ. The function of AMPA receptors in neuroblasts remains unknown.