GABAergic Input From the Basal Forebrain Promotes the Survival of Adult-Born Neurons in the Mouse Olfactory Bulb

Abstract

A unique feature of the olfactory system is the continuous generation and integration of new neurons throughout adulthood. Adult-born neuron survival and integration is dependent on activity and sensory experience, which is largely mediated by early synaptic inputs that adult-born neurons receive upon entering the olfactory bulb (OB). As in early postnatal development, the first synaptic inputs onto adult-born neurons are GABAergic. However, the specific sources of early synaptic GABA and the influence of specific inputs on adult-born neuron development are poorly understood. Here, we use retrograde and anterograde viral tracing to reveal robust GABAergic projections from the basal forebrain horizontal limb of the diagonal band of Broca (HDB) to the granule cell layer (GCL) and glomerular layer (GL) of the mouse OB. Whole-cell electrophysiological recordings indicate that these projections target interneurons in the GCL and GL, including adult-born granule cells (abGCs). Recordings from birth-dated abGCs reveal a developmental time course in which HDB GABAergic input onto abGCs emerges as the neurons first enter the OB, and strengthens throughout the critical period of abGC development. Finally, we show that removing GABAergic signaling from HDB neurons results in decreased abGC survival. Together these data show that GABAergic projections from the HDB synapse onto immature abGCs in the OB to promote their survival through the critical period, thus representing a source of long-range input modulating plasticity in the adult OB.

Keywords: GABA; adult neurogenesis; basal forebrain; granule cells; olfaction.

Figure 1

Cell type-specific retrograde tracing reveals long-range GABAergic projections to the olfactory bulb (OB). (A) Schematic depicting viral injection a retro2 serotype adeno associated virus expressing Cre-dependent mVenus (rAAV-Ef1α-flex-mVenus) into the OB of Vgat-Cre-expressing mice. Retrograde labeling reveals sources of synaptic input to the OB. (B) GABAergic retrograde labeling in OB with inset showing mVenus expression in the granule cell layer (GCL). (C) GABAergic retrograde labeling in a coronal section including the anterior olfactory nucleus (AON) with inset showing mVenus expression in lateral AON. (D) GABAergic retrograde labeling in a coronal section including the horizontal diagonal band of Broca (HDB) and lateral septum (LS) with insets showing mVenus expression in LS and HDB. (E) CHAT immunolabeling overlaid with GABAergic retrograde labeling in a coronal section including HDB, with inset showing lack of colocalization between CHAT+ and mVenus+ neurons in the HDB. (F) Quantification of CHAT+, mVenus+, and colocalized neurons in the HDB normalized to DAPI from single 40 μm thick sections. Points reflect cell counts from individual animals. N = 10 mice. Error bars are SE. One sample Wilcoxon rank-sum test. *p < 0.05, **p < 0.01. (G) Retrograde labeling in a coronal section including the ventral subiculum (vSUB) with inset showing sparse mVenus expression in vSUB. (H) mVenus+ cell counts by brain region from single 40 μm thick sections normalized to the number of DAPI+ cells in each region. Points reflect cell counts from individual animals. N = 8–14 mice. Error bars are SE. One sample Wilcoxon rank-sum test. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars are 100 μm unless otherwise specified.

Figure 2

Anterograde tracing reveals GABAergic projections from HDB to GCL and glomerular layer (GL). (A) Schematic depicting viral injection of Cre-dependent synaptophysin fused to GFP (AAV-Ef1α-flex-synaptophysin::GFP) and anterogradely-labeled projections in Vgat-Cre-expressing mice. (B) Coronal section showing viral injection site and synaptophysin::GFP expression in HDB. Inset shows cell bodies expressing GFP in HDB (dashed lines) (C). Anterograde labeling in OB with yellow line demonstrating orientation of the line scan plane from the OB surface to the rostral migratory stream (RMS, dashed line) for quantification of GFP intensity (shown in E). (D) Synaptophysin::GFP expression is dense throughout the GCL and GL, highest in the internal plexiform layer (IPL) and lowest in the external plexiform layer (EPL). (E) Quantification of GFP intensity along a line scan from the OB surface to 900 μm deep toward the RMS (shown in C). The dark green trace shows GFP intensity peak-normalized by the animal, averaged across five same-sized sections from five animals. The light green band shows SE. Dashed vertical lines show approximate borders of GL, EPL, and GCL.

Figure 3

HDB GABAergic neurons preferentially synapse onto interneurons in the OB. (A) Coronal section shows the injection site with the inset showing dense membrane labeling of neurons in the HDB (dashed lines). The upper right panel shows schematically for injection of Cre-dependent Channelrhodopsin (AAV-Ef1α-flex-ChR2::eYFP) into the HDB of Vgat-Cre mice. (B) Pie charts showing proportions of GCs, M/TCs, and periglomerular cells (PGCs) responding to stimulation of ChR2-expressing HDB GABAergic terminals in OB slices. N = 11 mice total, 14–19 cells per group type. (C) Traces show an example of a light-evoked current from a granule cell (GC) responding to ChR2 stimulation (blue tick) in aCSF (black), and following serial bath application of tetrodotoxin (TTX, red), 4-aminopyridine (4AP, green), and bicuculline (BIC, purple). The image shows the corresponding biocytin cell fill spanning the GCL, mitral cell layer (MCL) and EPL. (D) The trace shows no response to light from a mitral/tufted cell (M/TC) in aCSF. The image shows the corresponding biocytin cell fill with the cell body and lateral dendrites in the MCL and apical dendrites in the GL. EPL and GCL labeled for orientation. (E) The trace shows a representative example of a light-evoked current from a PGC responding to ChR2 stimulation in aCSF and during serial additions of TTX, 4AP, and BIC. The image shows the corresponding cell fill with a cell body in and dendrites in the GL. (F) The trace shows no response to light from a non-responsive PGC in aCSF. The image shows the corresponding biocytin cell fill with the cell body and dendrites in the GL. All scale bars for traces are 5 pA and 100 ms.

Figure 4

Adult-born granule cells (abGCs) receive early synaptic input from HDB GABAergic neurons. (A) Schematic showing viral injections for ChR2 expression (AAV-Ef1α-flex ChR2::eYFP) and adult-born neuron birth-date labeling (lenti-CMV-tdTomato). (B) Example of birth-dated adult-born neurons in a 150 μm thick horizontal section at 14 days post-RMS injection. (C) Traces show representative examples of currents evoked by ChR2 stimulation (light blue tick) in a labeled abGC at seven DPI in aCSF (blue), and following serial bath application of tetrodotoxin (TTX, red), 4-aminopyridine (4AP, green), and bicuculline (BIC, purple). Scale bar X and Y axes are 100 ms and 10 pA. (D) Cell fill corresponding to the trace in (C) showing cell body in deep GCL and dendrite extending toward the IPL. (E) Same as (C) for abGC at 20 DPI. (F) Cell fill corresponding to the trace in (E) showing cell body in GCL and dendrite spanning IPL and EPL. (G) Amplitudes of light-evoked currents from individual birth-dated neurons at different days post RMS injection (DPI) binned by age. N = 23 mice, 60 cells. Error bars are SE. Points reflect current amplitudes (pA) from individual cells in aCSF. **p < 0.01, *p < 0.05, one-way ANOVA with Tukey’s post hoc test for multiple comparisons.

Figure 5

Conditional knockout of VGAT in the HDB reduces the survival of adult-born neurons in GCL and GL. (A) Schematic showing injection of Cre-mVenus or mVenus control virus into the HDB of Vgat^f/f mice. The timeline shows the experimental design with viral injection into HDB of Vgat^f/f mice followed 2 weeks later by I. P. injections of EdU and 6 weeks later by quantification of EdU incorporation into OB and Ki67 expression in the subventricular zone (SVZ). (B) Dual-color fluorescent in situ hybridization (FISH) confirms that VGAT is knocked out in Cre-expressing cells after viral injection of Cre into the HDB of Vgat^f/f mice (top) whereas Vgat is expressed in cells infected with the control mVenus virus injected into the HDB of Vgat^f/f mice (bottom). (C) Quantification of Vgat+ cell density in the HDB of cre-injected (magenta) and control-injected (black) Vgat^f/f mice. N = 9 mice, 18 sections. Nested t-test, ***p < 0.001. (D) Coronal section from a Vgat^f/f mouse HDB injected with Cre-mVenus (green) and stained for Ki67 (magenta) to label proliferating progenitor cells in the SVZ. The inset shows Ki67+ cells in the SVZ. (E) Quantification of Ki67+ cells in the SVZ of Vgat^f/f mice HDB-injected with Cre-mVenus or mVenus control viruses (black). Points represent values from individual mice. N = 23 mice, 37 sections. Nested t-test. (F) OB sections from Vgat^f/f mice injected into the HDB with Cre-mVenus showing EdU incorporation (magenta). White arrows mark EdU+ cells in the GCL. Yellow arrows mark EdU+ cells in the GL. ONL: olfactory nerve layer. (G) Same as (F), but in Vgat^f/f mice injected with mVenus virus (control). (H) Quantification of EdU+ cell density in GCL of 40 μm OB sections from Vgat^f/f mice HDB-injected with Cre (magenta) or mVenus (black; I). Same as (H) but quantified in the GL. Points represent values from individual mice. N = 21 total mice, 69 sections. Error bars are SE. Nested t-test, *p < 0.05.