Gbx2 identifies two amacrine cell subtypes with distinct molecular, morphological, and physiological properties

Abstract

Our understanding of how the nervous sytem works is limited by our ability to identify the neuronal subtypes that comprise functional circuits. Using a genetic approach, we show that the transcription factor Gbx2 labels two amacrine cell (AC) subtypes in the mouse retina that have distinct morphological, physiological, and molecular properties. One subtype of Gbx2+ ACs are likely the previously characterized On-type GABAergic CRH-1 AC. The other Gbx2+ AC population is a previously uncharacterized non-GABAergic, non-Glycinergic (nGnG) AC subtype. Gbx2+ nGnG ACs are On-Off type cells with asymmetric dendritic arbors. Gbx2+ nGnG ACs also exhibit tracer coupling to bipolar cells (BCs) through gap junctions that are modulated by dopamine signaling. This study genetically identifies a previously uncharacterized AC subtype and reveals an unusual AC-BC connectivity through gap junctions that may provide a novel model of synaptic communication and visual circuit function. RESULTS Gbx2 CreERT2-IRES-EGFP marks two distinct amacrine cell subtypes.To begin unraveling the neuronal subtype complexity in the retina, we sought to identify Cre or CreERT2 mouse lines that could be used to selectively label and manipulate single neuronal subtypes in the retina. Using scRNAseq datasets and transgenic mouse databases as a guide (Siegert et al., 2009;Macosko et al., 2015), we identified Gbx2 CreERT2-IRES-EGFP as a mouse line predicted to label a sparse population of neurons in the retina (Chen et al., 2009). Since the CreERT2-IRES-EGFP cassette is knocked into the Gbx2 locus, labeled neurons are expected to faithfully recapitulate its endogenous expression pattern. Crossing the Gbx2 CreERT2-IRES-EGFP line to multiple Credependent reporter lines labeled neurons in the inner nuclear layer (INL, Fig. 1a) and the ganglion cell layer (GCL, Fig. 1b). Low doses of tamoxifen (<0.02mg) labeled neurons in both the INL and GCL with dendrites that selectively stratify in sublamina 3 (S3) of the inner plexiform layer (IPL). When we used a saturating dose of tamoxifen (2.0 mg/day for 2 days, Fig. S1d-i), we observed a second neuronal subtype with dendrites that stratify in sublamina 5 (S5) of the IPL (Fig. 1c, g). Importantly, the same two neuronal populations were labeled whether tamoxifen was administered at embryonic (E16), postnatal (P0-2), or adult (P28) ages ( Fig. 1 -supplement 1a-c), demonstrating that the Gbx2 CreERT2-IRES-EGFP line provides a consistent and stable method for labeling the same neuronal populations from embryonic to adult stages.Both the S3-and S5-stratifying Gbx2+ populations co-localized with pan-amacrine cell markers TFAP2 and Pax6 (Fig. 1d, h, Fig 1-supplement 2a), and lacked expression of the retinal ganglion cell marker RBPMS and bipolar cell marker Chx10 (Fig. 1h, Fig 1supplement 2b, c). GFP expression could be detected in 55.3% of the total Gbx2/tdTomato+ population (Fig. 1e, i). A second marker, calretinin, labeled 53.6% of the total Gbx2+ ACs (Fig. 1f, i) and nearly all (94.9%) of the GFP+ Gbx2+ ACs were colabeled ...

Mouse Retinal Cell Atlas: Molecular Identification of over Sixty Amacrine Cell Types

“…nGnG-4 (C36) expressed Gbx2 and Lhx9. We labeled them in a Gbx2-Creer-GFP line and confirmed that both populations were GAD À and GLYT1 À , which is further supported by an independent study (Kerstein et al, 2020) Their somata were present in both the inner nuclear layer and ganglion cell layer. We additionally observed coexpression of LHX9 by immunostaining (Fig.…”