Journal: eLife

Article Title: Spinal V1 inhibitory interneuron clades differ in birthdate, projections to motoneurons, and heterogeneity

doi: 10.7554/eLife.95172

Figure Lengend Snippet: ( A ) MafB-GFP+ cells in mature (P15) mice. Most MafB-GFP+ cells in lamina VII belong to the V1 lineage (tdTomato). They are found in the most dorsal and ventral regions of the distribution of V1s. Those that are also calbindin-IR fall in the distinctive ventral region occupied by Renshaw cells. In addition, there are many dorsal horn non-V1 MafB-GFP+ neurons. The small cells throughout the white and gray matter are microglia. ( B ) Expression of transcription factors (TFs) and calbindin in MafB-GFP+ cells in neonatal (P5) mice. Top row, immunoreactivity for calbindin (Renshaw cells) and clade-specific TFs. Bottom row, superimposition with MafB-GFP (V1-tdTomato is not shown for simplicity). Compared to P15, a few more ventral horn neurons express MafB-GFP at P5, including some motoneurons, but MafB labeling is weaker in these cells. Within MafB-GFP+ V1 neurons, the two groups located at the most dorsal and most ventral regions correspond to the V1 neurons that retain MafB-GFP at P15 (see A ). These groups are indicated with rectangles in the figure. The ventral group expresses calbindin. The dorsal group expresses Pou6f2 at this age. Little-to-no Foxp2-IR or Sp8-IR is found in either dorsal or ventral groups of MafB-GFP+ V1s. ( C ) Quantitation of MafB-V1 neurons in P5 mice. Around 13% of all V1s express MafB-GFP+, and the percentages located in the Renshaw cell area (‘ventral’) or dorsal lamina VII (‘dorsal’) are evenly split (n=17 mice, 4 ventral horns each; bars show SD). ( D ) More than half of the MafB-GFP+ V1 cells have detectable levels of MafB (Sigma) immunoreactivity at P5, in both the ventral and dorsal groups (n=9 mice, 4 ventral horns each; error bars show SD). ( E ) V1-clade marker expression in dorsal and ventral P5 MafB-GFP V1 neurons. Dorsal MafB-GFP-V1s express Pou6f2, but do not express calbindin, Sp8, or Foxp2. Ventral MafB-GFP-V1s express calbindin (Renshaw cells) and do not express Pou6f2, Foxp2, or Sp8 (n=4–5 mice, 4 ventral horns each; error bars show SD). ( F ) 5-Ethynyl-2'-deoxyuridine (EdU) birthdating reveals similar proportions of EdU+ neurons at P5 for dorsal MafB-GFP vs MafB-IR neurons pulse-labeled at each embryonic time point (n=2 mice per time point per condition, 4 ventral horns each; error bars show SD). The mismatch in birthdates between MafB genetic and antibody labeling at E11 in the ventral group could arise because some E11-born ventral MafB-GFP V1s quickly downregulate MafB to levels that are undetectable with antibodies.

Article Snippet: Calbindin , Recombinant protein (calbindin D-28k from rat) , Rabbit, polyclonal , Swant , CB-38a , RRID: AB_10000340 , 1:1000.

Techniques: Expressing, Labeling, Quantitation Assay, Marker, Antibody Labeling

Journal: eLife

Article Title: Spinal V1 inhibitory interneuron clades differ in birthdate, projections to motoneurons, and heterogeneity

doi: 10.7554/eLife.95172

Figure Lengend Snippet: ( A ) Motor column identification from lower thoracic to upper sacral spinal cord in P20 mice following labeling with choline acetyltransferase (ChAT) antibodies: PGC = preganglionic cell column; MMC = medial motor column; HMC = hypaxial motor column; LMCd/v=lateral motor column (dorsal/ventral). ( B ) Schematic representation of the rostro-caudal span of each motor column in the spinal segments studied. ( C ) Synapse quantification. Axons of Foxp2 and non-Foxp2 V1 interneurons were respectively labeled with EGFP and tdT in En1 Cre , Foxp2 flpo , R26 CE:dualGFP/Ai9-lsl-tdT mice. In en1 Cre , Ai9 R26 lsl-tdT mice we identified V1-Renshaw cell axons using calbindin antibodies. Synaptic locations were labeled with VGAT antibodies and the postsynaptic motoneurons with ChAT antibodies. Synapse densities were analyzed in a ribbon of membrane at mid-cell body level (7 optical planes, 1 µm z-step). C1, Single optical plane of an L4/5 LMCv motoneuron surrounded by genetically labeled Foxp2-V1 and non-Foxp2-V1 axons. Inhibitory synapses on ChAT-IR motoneurons are VGAT+. C2, Single optical image of an L4/5 LMCv ChAT-IR motoneuron receiving synapses from V1 Renshaw cells (genetically labeled V1 axons with calbindin-IR and VGAT). C3–4, Method for estimating synapse densities on motoneuron cell bodies using C2 as example. C3, V1-VGAT (red arrowheads) and V1-CB-VGAT synapses (yellow arrowhead) are marked (VGAT-IR is not shown for clarity), and the cell body contour annotated with regions corresponding to dendrite exits. This process was repeated in seven consecutive mid-cell body optical planes (cross-sections with well-defined nucleus and nucleolus). C4, A membrane surface slab is reconstructed in 3D (two different rotations shown). The surface area corresponding to dendrite exits is subtracted from the total surface area of the slab to calculate the available surface area on the motoneuron cell body. V1-VGAT synapses (red), V1-CB-VGAT synapses (yellow), and CB-VGAT synapses (green) are marked. A similar process was followed for calculating Foxp2-V1 synapse density. ( D ) Quantification of total V1-VGAT synapse densities on motoneuron cell bodies in different motor columns (n=21–30 motoneurons per motor column, n=5 animals with 4–9 motoneurons per animal per motor column). Each data point is one motoneuron color-coded by mouse origin. Average synaptic densities ± SD indicated to the right of scatter plots. A nested ANOVA found significant differences among motor column/segments (p<0.0001) with no inter-animal variability (p=0.4768). The table summarizes all post hoc pairwise comparisons for average V1 synaptic densities of each motor column and segment (Bonferroni-corrected t-tests) (further statistical details are found in ). Colors indicate increased (>1, red) or decreased ratios (<1, blue) of column motoneurons vs row motoneurons. PGC neurons receive significantly fewer V1 synapses than MMC or LMC motoneurons. The LMC (ventral and dorsal) in lower lumbar (L4/L5) had significantly more V1 contacts than MMC motoneurons or L6 dorsal LMC. ( E ) Comparison of synaptic densities from Foxp2-V1 and non-Foxp2-V1 neurons (top) or Renshaw cells (bottom). All motoneurons sampled in 2–3 animals for each comparison were pooled together. Densities of V1-VGAT synapses from Foxp2-V1s, non-Foxp2 V1s, or calbindin (CB)+ V1s (Renshaw cells) (n=6–17 motoneurons sampled per motor column/segments, average = 12.1 ± 2.9 SD) were compared using a two-way ANOVA for axon type vs motor column and segment. Foxp2-V1 vs non-Foxp2-V1 synapses: significant differences in density were found for type of synapse (p=0.001), motor column location (p<0.0001), and their interaction (p<0.0001). Significant differences after post hoc Bonferroni tests are indicated (*p<0.05; ****p<0.0001). In general, synapses from Foxp2-V1 axons have higher density than non-Foxp2-V1 axons on HMC and LMC columns at all spinal segments except for L1/L2 LMC. MMC motoneurons receive similar synaptic densities from both types of V1 axons, except at the sacral level in which non-Foxp2 V1 synapses predominate. PGC neurons receive very low densities of V1 axons and there are no significant differences between either type in any region. Foxp2-V1 vs CB+ V1 synapses: significant density differences were found for type of synapse (p<0.0001), motor column location (p<0.0001), and their interaction (p<0.0001). Significant differences between Foxp2-V1 and CB+ V1 synapses after post hoc Bonferroni tests are indicated (*p<0.05; ****p<0.0001). Synapses from Foxp2-V1 axons have higher density than CB+ V1 axons in HMC and LMC columns at all spinal segments except for L1/L2 LMC. MMC motoneurons receive similar synaptic densities from both types of V1 axons in upper lumbar regions, but Foxp2-V1 synapse predominate in lower lumbar. In S1 the density of CB+/V1 synapses is significantly higher. The low synaptic densities estimated in PGC neurons for Foxp2-V1s and CB+ V1s are not significantly different. Further details of statistical comparisons are in . ( F ) Comparing the numbers of Foxp2 and CB+ (Renshaw) V1 synapses to the total number of V1 synapses, we estimated their respective percentages. From these estimates we calculated that the remainder belongs to non-Foxp2 and non-CB+ Renshaw cells. The large majority of V1 synapses on the cell bodies of LMC, HMC, and MMC motoneurons are either from Renshaw cells or Foxp2-V1s. ( G ) Summary diagram of major V1 clade connectivity to motoneuron cell bodies. Foxp2-V1s and Renshaw cells form the majority of inhibitory V1 contacts on LMC and HMC motoneurons, with slightly higher density from Foxp2-V1s. The MMC receives roughly equal portions of V1 contacts from Foxp2-V1s and Renshaw cells. V1s provide only sparse inhibition on preganglionic sympathetic neurons and most originate in V1 clades other than Renshaw cells and Foxp2-V1s.

Article Snippet: Calbindin , Recombinant protein (calbindin D-28k from rat) , Rabbit, polyclonal , Swant , CB-38a , RRID: AB_10000340 , 1:1000.

Techniques: Labeling, Membrane, Comparison, Inhibition

Journal: eLife

Article Title: Spinal V1 inhibitory interneuron clades differ in birthdate, projections to motoneurons, and heterogeneity

doi: 10.7554/eLife.95172

Figure Lengend Snippet: ( A ) Targeting strategy to generate O tp::flpo mice. Flpo was inserted into the ATG in the first exon of the Otp genomic locus. Dotted lines represent approximate regions of homology in the targeting vector. Southern blot (bottom) of BamH1-digested genomic DNA with a 5’ probe external to the targeting vector identifies a 5.2 kb wild-type fragment, and an 8.2 kb knock-in fragment. Not shown: Deletion of selectable neomycin resistance gene flanked by loxP sites by crossing to Protamine-Cre mice, which recombines the floxed PGK-Neo cassette in the male germline. ( B ) Left, P0 lumbar spinal cord of O tp::flpo, R26 RCE.fsf-GFP mice, demonstrating expression in Otp-IR interneurons. Right, 98.1 ± 1.2% (mean ± SEM, n=3 mice) of Otp-expressing cells are labeled by the reporter. ( C ) Intersection of Otp flpo/+ , En1 cre/+ using the dual-color strategy with simultaneous expression of the Ai9 tdT and RCE-DC EGFP reporters. V1 cells that express otp are labeled with EGFP (green). In these cells the Ai9-tdT reporter is effectively deleted by Flpo recombination dependent on the level of Otp expression: strong (EGFP only) and weak (EGFP and tdT). V1 cells that express tdT only (red) never express Otp . In addition, the sections were labeled with antibodies for Otp (light blue) to reveal cells that retained expression of Otp at P5 and choline acetyltransferase (ChAT) (deep blue) to localize the motor pools. Most V1s express EGFP, with tdT-only cells being a minority. Right, cell plot positions of some of the cell types identified in these sections (one mouse 6 ventral horns in L4/L5). Most cells are Otp-V1s and are shown here as green (EGFP only) and pink dots (EGFP and tdT). ( D ) Quantification of cells with EGFP only (green), tdT only (red), or both (pink) (n=12 ventral horns from 2 mice; error bars show SD). 88.6% of V1s (1278 V1 cells analyzed in total) expressed EGFP; 60.8% were EGFP only, 27.8% were ‘yellow’ and surprisingly only 11.4% were tdT only. As expected, P5 Otp expression detected with antibodies was absent in most tdT-labeled V1 cells (93.5% of cells) and ‘yellow’ tdT+eGFP V1 cells (85.4%), but also in a significant proportion of EGFP-labeled V1 cells (46.7%). ( E ) V1 cells transiently expressing Otp in embryo included cells of other clades. This was examined in En1 cre/+ , Otp flpo/+ , R26 RCE:dual-eGFP mice. Detection of Pou6f2, calbindin, and Otp in genetically labeled Otp-V1 cells at P5. Left confocal image and right cell plot (n=6 ventral horns from 1 mouse). Pou6f2-Otp-V1 cells concentrate in a dorsal band within the ventral horn. Calbindin-IR Otp1-V1 cells concentrate in the Renshaw region (ventral most region) and others are in the dorsal region of the ventral horn. Otp-V1 cells retaining Otp expression at P5 occupy all dorsoventral positions in the lateral spinal cord. ( F ) Pou6f2 or calbindin immunoreactivity (-IR) in Otp-V1 cells (n=397) in 6 sections (each dot) in L4/L5 from one animal. Pou6f2 was detected in 12.8% of Otp-V1 cells at P5. In addition, 14.4% of Otp-V1 cells were calbindin+ and this included many in the Renshaw cell ventral region and few others located more dorsally. One rare dorsal Otp-V1 cell contained both Pou6f2 and calbindin (included in both percentages above). By limiting the analysis to ventral Otp-V1 interneurons in the Renshaw area we estimated that 8.3% of them are Renshaw cells. In conclusion, the Otp-V1 lineage includes cells from several V1 clades. Many downregulate Otp expression before birth, thus at P5 all Otp-expressing cells are restricted to the Foxp2-V1 lineage. In addition, many medial non-V1 Foxp2 cells also express Otp. Therefore, to specifically target the lateral group of proprioceptive Otp-Foxp2-V1 cells tightly associated to the LMC, a triple genetic intersection or alternatively, postnatally timed Otp-dependent recombination is necessary. Raw images of the blots and corresponding labeling are found in and . Figure 7—figure supplement 1—source data 1. Raw image of gel corresponding to . Figure 7—figure supplement 1—source data 2. Annotated image of gel corresponding to .

Article Snippet: Calbindin , Recombinant protein (calbindin D-28k from rat) , Rabbit, polyclonal , Swant , CB-38a , RRID: AB_10000340 , 1:1000.

Techniques: Plasmid Preparation, Southern Blot, Knock-In, Expressing, Labeling

Journal: eLife

Article Title: Spinal V1 inhibitory interneuron clades differ in birthdate, projections to motoneurons, and heterogeneity

doi: 10.7554/eLife.95172

Figure Lengend Snippet: Antibodies.

Article Snippet: Calbindin , Recombinant protein (calbindin D-28k from rat) , Rabbit, polyclonal , Swant , CB-38a , RRID: AB_10000340 , 1:1000.

Techniques: Recombinant, Variant Assay, Isolation

Journal: eLife

Article Title: Spinal V1 inhibitory interneuron clades differ in birthdate, projections to motoneurons, and heterogeneity

doi: 10.7554/eLife.95172

Figure Lengend Snippet: Antibodies.

Article Snippet: Calbindin , Recombinant protein (calbindin D-28k from rat) , Rabbit, polyclonal , Swant , CB-38a , RRID: AB_10000340 , 1:1000.

Techniques: Recombinant, Variant Assay, Isolation

Journal: Sleep

Article Title: Gene expression analysis in the mouse brainstem identifies Cart and Nesfatin as neuropeptides coexpressed in the Calbindin-positive neurons of the Nucleus papilio

doi: 10.1093/sleep/zsaa085

Figure Lengend Snippet: Description of Primary Antibodies Used for Immunohistochemistry

Article Snippet: Calbindin D-28K , Mouse , Whole chicken protein from gut , Swant, Marly, Swizerland , CB300 , 1–2,000.

Techniques: Recombinant

Journal: Frontiers in Neural Circuits

Article Title: C1q Regulates Horizontal Cell Neurite Confinement in the Outer Retina

doi: 10.3389/fncir.2020.583391

Figure Lengend Snippet: Antibodies used in tissue analysis.

Article Snippet: Calbindin D-28k , Full-length recombinant human Calbindin D-28K , Horizontal cells; amacrine cells; retinal ganglion cells , Novus biologicals; chicken polyclonal; NBP2-50028; no RRID , 1:2000.

Techniques: Recombinant, Purification