Convergence and Divergence of CRH Amacrine Cells in Mouse Retinal Circuitry

Park, Silvia J. H.; Pottackal, Joseph; Ke, Jiang-Bin; Jun, Na Young; Rahmani, Pouyan; Kim, In-Jung; Singer, Joshua H.; Demb, Jonathan B.

doi:10.1523/jneurosci.2518-17.2018

Cited by 43 publications

(70 citation statements)

References 83 publications

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“…6b, right), and was smaller than the center size measured during the Off-phase (~180µm). A similar difference is apparent in a previous study (Park et al, 2018).…”

Section: S3-and S5-gbx2+ Acs Cells Receive Center and Surround Inhibisupporting

confidence: 87%

“…S5-Gbx2+ ACs also share morphological, molecular, and physiological properties with the previously described CRH-1 AC (Zhu et al, 2014;Jacoby et al, 2015;Park et al, 2018). S5-Gbx2+ ACs and CRH-1 ACs have dendritic arbors of a similar size and shape that stratify in S5 of the IPL, they both express the neuropeptide Crh (corticotrophin-releasing hormone), and have similar physiological properties in response to ON light stimuli (Jacoby et al, 2015;Park et al, 2018). The CRH-IRES-Cre line used in these studies labels at least three different AC subtypes, which can be distinguished based on their morphological differences.…”

Section: Categorization Of Amacrine Cell Subtypessupporting

confidence: 58%

“…Unlike S5 ACs, CRH-1 ACs have been shown to lack of any surround suppression during the On-phase of the response (compare Fig. 3 of (Park et al, 2018), with Fig. 6a).…”

Section: Dendrite Morphology and Gbx2+ Ac Functionmentioning

confidence: 93%

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

Kerstein

Leffler

Sivyer

et al. 2020

Preprint

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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 ...

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“…6b, right), and was smaller than the center size measured during the Off-phase (~180µm). A similar difference is apparent in a previous study (Park et al, 2018).…”

Section: S3-and S5-gbx2+ Acs Cells Receive Center and Surround Inhibisupporting

confidence: 87%

Section: Categorization Of Amacrine Cell Subtypessupporting

confidence: 58%

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

Kerstein

Leffler

Sivyer

et al. 2020

Preprint

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“…al, 2011), VG3 ACs (C13: VGlut3 [Slc17a8]; Haverkamp and Wässle, 2004;Johnson et al, 2004;Krishnaswamy et al, 2015); and A17 ACs (C6: Prkca (PKCα), Sdk1, Calb2 negative, Dab1-negative; Grimes et al, 2010;Puthussery and Fletcher 2007;Yamagata and Sanes, 2019). Other types expressed neuropeptides known to mark specific AC types; they include Cck (C10,C17,C18,C34; Firth et al, 2002), Vip, (C22,C26,C47; Park et al, 2015;Akrouh and Kerschensteiner, 2015;Pérez de Sevilla Müller et al, 2019), Crh (C37; Park et al, 2018) and Penk (C35,C59,C63; Chen et al, 2013). For some of these types, we were able to validate new markers, such as Car3 for VG3 cells (Figure 3B,C).…”

Section: Correspondence Between Clusters and Ac Typesmentioning

confidence: 86%

Molecular identification of sixty-three amacrine cell types completes a mouse retinal cell atlas

Wang

et al. 2020

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Amacrine cells (ACs) are a diverse class of interneurons that modulate input from photoreceptors to retinal ganglion cells (RGCs), rendering each RGC type selectively sensitive to particular visual features, which are then relayed to the brain. While many AC types have been identified morphologically and physiologically, they have not been comprehensively classified or molecularly characterized. We used high-throughput single-cell RNA sequencing (scRNA-seq) to profile >32,000 ACs from mouse retina, and applied computational methods to identify 63 AC types. We identified molecular markers for each type, and used them to characterize the morphology of multiple types. We show that they include nearly all previously known AC types as well as many that had not been described. Consistent with previous studies, most of the AC types express markers for the canonical inhibitory neurotransmitters GABA or glycine, but several express neither or both. In addition, many express one or more neuropeptides, and two express glutamatergic markers. We also explored transcriptomic relationships among AC types and identified transcription factors expressed by individual or multiple closely related types. Noteworthy among these were Meis2 and Tcf4, expressed by most GABAergic and most glycinergic types, respectively. Together, these results provide a foundation for developmental and functional studies of ACs, as well as means for genetically accessing them. Along with previous molecular, physiological and morphological analyses, they establish the existence of at least 130 neuronal types and nearly 140 cell types in mouse retina. SIGNIFICANCE STATEMENTThe mouse retina is a leading model for analyzing the development, structure, function and pathology of neural circuits. A complete molecular atlas of retinal cell types provides an important foundation for these studies. We used high-throughput single-cell RNA sequencing (scRNA-seq) to characterize the most heterogeneous class of retinal interneurons, amacrine cells, identifying 63 distinct types. The atlas includes types identified previously as well as many novel types. We provide evidence for use of multiple neurotransmitters and neuropeptides and identify transcription factors expressed by groups of closely related types. Combining these results with those obtained previously, we proposed that the mouse retina contains 130 neuronal types, and is therefore comparable in complexity to other regions of the brain.

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“…The NOS-1 cell was described also as the CRH-2 cell (Corticotropin-releasing hormone AC-2) based on labeling in the CRH-ires-Cre retina (Zhu et al, 2014). We, however, found that Cre-expressing cells in the CRH-ires-Cre::Ai32 retina are rarely labeled by the nNOS antibody (Park et al, 2018), suggesting limited overlap in CRH+ and NOS+ AC populations. Indeed, we found no co-expression of CRH and NOS by immunohistochemistry (P12 retina) (n = 66 CRH+ cells and 85 NOS+ cells, one retina) in neurons in the GCL (Figure 6E-G).…”

Section: Resultsmentioning

confidence: 83%

Connectomic analysis reveals an interneuron with an integral role in the retinal circuit for night vision

Park

Lieberman

et al. 2020

Preprint

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18Blindness to Yale University. We thank Tim Maugel and the Laboratory for Biological 19 Ultrastructure (Department of Biology, UMD) for assistance with transmission electron 20 microscopy, Alexander Baden for assistance with 3D visualization, and Kacie Furcolo and Adit 21 Sabnis for assistance with SBEM dataset skeletonization. We thank Cole Graydon for 22 comments on the manuscript. 24 Summary 31The mammalian rod bipolar (RB) cell pathway is perhaps the best-studied circuit in the 32 vertebrate retina. Its synaptic interactions with other retinal circuits, however, remain 33 unresolved. Here, we combined anatomical and physiological analyses of the mouse retina to 34 discover that the majority of synaptic inhibition to the AII amacrine cell (AC), the central neuron 35 in the RB pathway, is provided by a single interneuron type: a multistratified, axon-bearing 36 GABAergic AC, with dendrites in both ON and OFF synaptic layers, but with a pure ON 37 (depolarizing) response to light. We used the nNOS-CreER mouse retina to confirm the identity 38 of this interneuron as the wide-field NOS-1 AC. Our study demonstrates generally that novel 39 neural circuits can be identified from targeted connectomic analyses and specifically that the 40 NOS-1 AC mediates long-range inhibition during night vision and is a major element of the RB 41 pathway. 42 43 74 remain. Most significantly, we do not know the identity of the spiking, GABAergic AC that drives 75 a receptive field surround in the AII during rod-mediated vision via synaptic inhibition of the AII 76 itself (Bloomfield and Xin, 2000); understanding mechanisms contributing to surround inhibition 77 4 is important because such inhibition tunes AII responses to spatial features of the visual 78 stimulus. 79Here, we identified the major inhibitory input to the AII by combining anatomical, genetic, 80 and electrophysiological analyses in a three-step process. One, we reconstructed ACs that 81 provided synaptic input to AIIs in a volume of mouse retina imaged by scanning block-face 82 electron microscopy [SBEM; (Denk and Horstmann, 2004)]. Two, we evaluated published 83 descriptions of reporter mouse lines to identify genetically-accessible ACs that had the 84 anatomical characteristics of the cells reconstructed from SBEM images. And three, we used 85 electrophysiological recordings and genetics-based circuit analysis to demonstrate that a 86 candidate AC, which provided the great majority of the inhibitory synaptic input to AIIs, exhibited 87 a light response predicted by its anatomy and made GABAergic synapses onto AIIs. This 88 spiking, GABAergic AC is a multistratified, ON AC denoted NOS-1 AC and identified in the 89 nNOS-CreER mouse (Zhu et al., 2014). We conclude that the NOS-1 AC is an integral 90 component of the RB pathway and a significant source of long-range inhibition during night 91 vision. More generally, our study demonstrates the utility of targeted, small-scale "connectomic" 92 analysis for identification of novel neural circuits. 94 Results 95AIIs in the ...

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Convergence and Divergence of CRH Amacrine Cells in Mouse Retinal Circuitry

Cited by 43 publications

References 83 publications

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

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

Molecular identification of sixty-three amacrine cell types completes a mouse retinal cell atlas

Connectomic analysis reveals an interneuron with an integral role in the retinal circuit for night vision

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