Journal: Cell reports

Article Title: Inappropriate Intrusion of an Axonal Mitochondrial Anchor into Dendrites Causes Neurodegeneration

doi: 10.1016/j.celrep.2019.09.012

Figure Lengend Snippet: (A–F) Immunohistochemistry technique for capturing SNPH intrusion in PC dendrites. Shown is SNPH (green), Syt2 (red), and Calbindin (blue) labeling in 3.5-month-old WT (A, C, and E) and Shi (B, D, and F) mice. Scale bar, 10 μm. (G and H) High magnification of the maximum intensity projection image from the z stack through dendritic regions of WT (G) and Shi (H). (I and J) Orthogonal (slice) view of SNPH punctum (indicated by arrows in panels G and H) in the dendritic region from WT (I) and Shi (J) in x-z and y-z orientations, respectively. (K) Quantification of percentage area occupied by SNPH within the dendritic volume from 3 mice of each group. Data are shown as mean ± SEM. *p < 0.05. (L–N) Capturing SNPH intrusion by pre-tagging dendritic mitochondria in vivo using viral transduction. (L) Technique to selectively transduce PCs with AAV-Mito-mCherry. (M) Demonstration of successful pre-tagging of mitochondria in dendrites of a single PC by Calbindin staining. Scale bar, 10 μm. (N) Demonstration of how pre-tagged dendritic mitochondria in PCs are used to capture SNPH intrusion by 3D rotation. (O and P) Single dendritic tree in WT (O) or Shi (P) pre-tagged with Mito-mCherry (red) and SNPH intrusion (green) captured by co-rotation with Mito-mCherry. Merged images show the fraction of dendritic mitochondria anchored by intruded SNPH (yellow). (Q and R) Percentage of PCs with SNPH intrusions (Q) and percentage of SNPH bound to mitochondria per dendritic tree (R) from WT (n = 73) and Shi (n = 106) PCs. Data are shown as mean ± SEM. ***p < 0.005.

Article Snippet: Polyclonal Calbindin , Boster , M03047–2.

Techniques: Immunohistochemistry, Labeling, In Vivo, Transduction, Staining

Journal: Cell reports

Article Title: Inappropriate Intrusion of an Axonal Mitochondrial Anchor into Dendrites Causes Neurodegeneration

doi: 10.1016/j.celrep.2019.09.012

Figure Lengend Snippet: (A and B) Representative images of lentivirally transduced GFP-SNPH (1–469) (A) and GFP-SNPH (B) in PCs of SNPH-KO mice injected with saline (no harmaline, vehicle only). (C-H) Effect of harmaline on GFP-SNPH (1–469)-transduced (C) and GFP-SNPH-transduced (F) PC dendrites. Degenerating dendrites in GFP-SNPH-transduced PCs can be seen in (F). Also shown is Calbindin labeling of GFP SNPH (1–469) (D) and GFP-SNPH (G) from (C) and (F). Merged images of GFP SNPH (1–469) and GFP-SNPH with Calbindin are shown in (E) and (H), respectively. (I–K) Representative image of a harmaline-induced degenerating PC (white arrow in I) transduced with GFP-SNPH. Calbindin staining from the same section is shown in (J), whereas a merged image is shown in (K). Scale bars, 20 μm. (L) Quantification of dendritic shrinkage in GFP-SNPH (1–469)- and GFP-SNPH-transduced PCs in the absence (n = 3 mice, vehicle only) or presence of harmaline (n = 5 mice). Data are shown as mean ± SEM. ***p < 0.001.

Article Snippet: Polyclonal Calbindin , Boster , M03047–2.

Techniques: Injection, Saline, Labeling, Transduction, Staining

Journal: Cell reports

Article Title: Inappropriate Intrusion of an Axonal Mitochondrial Anchor into Dendrites Causes Neurodegeneration

doi: 10.1016/j.celrep.2019.09.012

Figure Lengend Snippet:

Article Snippet: Polyclonal Calbindin , Boster , M03047–2.

Techniques: Virus, Plasmid Preparation, Recombinant, Software, Imaging

Journal: PLOS Pathogens

Article Title: Deciphering infected cell types, hub gene networks and cell-cell communication in infectious bronchitis virus via single-cell RNA sequencing

doi: 10.1371/journal.ppat.1012232

Figure Lengend Snippet: (a) White arrows indicate the localization of IBV N protein in AQP2-expressing collecting duct cells. Green fluorescence shows positive staining for IBV N and red fluorescence shows staining for AQP2. (b) White arrows indicate the localization of IBV N protein in CALB1-expressing distal tubule cells. Red fluorescence shows positive staining for IBV N and green fluorescence shows staining for CALB1. (c) Changes in cell communication numbers: The top network diagram shows cell clusters as nodes, with line thickness indicating changes in communication numbers. The lower heatmap details these changes, with rows representing signal-sending cells and columns indicating signal-receiving cells. The color scale reflects the inter-group differences in signal communication frequency between different cell types (number of communications in the infected group—number in control group). The bar plots at the top and right side represent the overall differences in the number of signals sent/received by specific cell clusters. (d) Changes in cell communication strength: Similar to (c), with the top network diagram displaying changes in communication strength (communication strength in the infected group—strength in the control group). In the lower heatmap, the color scale reflects the inter-group differences in signal communication strength between different cell types. The bar plots at the top and right side represent the overall differences in the strength of signals sent/received by specific cell clusters (infected group—control group). (e) Inter-group differences in the communication strength of specific signaling pathways (receptor-ligand pairs) across cell clusters. Rows represent signal pathways and columns correspond to cell clusters, with heatmap colors depicting the strength variation of signals (infect group vs. control group). Upper left triangles for signal sent and lower right triangles for signal received. The bar plot on the right side shows the overall difference in communication strength of these signaling pathways between the IBV-infected group and the control group.

Article Snippet: mouse mab calb1 , 1:200 , Boster Bio (BM0203).

Techniques: Expressing, Fluorescence, Staining, Infection, Control, Protein-Protein interactions

Journal: PLOS Pathogens

Article Title: Deciphering infected cell types, hub gene networks and cell-cell communication in infectious bronchitis virus via single-cell RNA sequencing

doi: 10.1371/journal.ppat.1012232

Figure Lengend Snippet: Antbodies and reagents used in present study.

Article Snippet: mouse mab calb1 , 1:200 , Boster Bio (BM0203).

Techniques: Blocking Assay, Immunofluorescence

Journal: bioRxiv

Article Title: A Spinal Circuit That Transmits Innocuous Cool Sensations

doi: 10.1101/2022.03.23.485555

Figure Lengend Snippet: (A) Images represent double staining of Calb1 Lbx1 -Tomato (red) and Calb1 protein (green) in Calb1 Lbx1 ;Ai65 mice. Arrow denotes Calb1 protein and Tomato double-positive cell. Arrowhead indicates a Calb1 Lbx1 -Tomato positive cell that does not colocalize with Calb1 protein. n = 17 sections. Scale bar, 100 μm. (B) Double staining of Tomato with lamina markers (NK1R, CGRP, IB4, and PKCγ), excitatory neuronal marker VGLUT2, or inhibitory neuronal markers (GAD67 plus GlyT2 or Pax2) by immunohistochemistry or in situ hybridization in the dorsal horn of Calb1 Lbx1 ;Ai65 mice. Arrows denote double-positive cells for indicated mRNA and Tomato. Arrowhead indicates a protein + cell that does not colocalize with Tomato. The percentage is calculated as double-positive neurons over total number of Calb1 Lbx1 -Tomato + neurons. Insets represent higher magnification of the boxed areas. n = 17-26 sections. Scale bar, 100 μm. (C) Double staining of c-Fos and Tomato signals in acetone-treated Calb1 Lbx1 ;Ai65 mice. Inset (middle) represents higher magnification of the boxed area (left). Arrow indicates a double-positive cell for c-Fos protein and Tomato, and arrowhead shows a cell positive for c-Fos alone. Scale bar, 100 μm. (D) Total number of c-Fos positive neurons per hemi-section in either the ipsilateral or the contralateral dorsal horn of naïve (grey) and acetone-treated (light blue) Calb1 Lbx1 ;Ai65 mice. n = 9 hemi-sections in each group; **** p < 0.0001 two-way ANOVA with Sidak post hoc analysis. (E) Quantification of c-Fos colocalization between c-Fos + and Calb1 Lbx1 -Tomato + neurons per hemi-section in either the ipsilateral or contralateral dorsal horn of naïve (grey) and acetone-treated (light blue) Calb1 Lbx1 ;Ai65 mice. n = 9 hemi-sections in each group; **** p < 0.0001; two-way ANOVA with Sidak post hoc analysis.

Article Snippet: To detect protein expression, immunohistochemistry was performed using rabbit anti-NK1R (1:1000, #S8305 MilliporeSigma, St. Louis, MO), rabbit anti- α -CGRP (1:500, #T-4032, Peninsula Lab, San Carlos, CA), Alexa fluor 647-conjugated isolectin GS (IB4) (10 μg/mL, #I32450, ThermoFisher Scientific, Waltham, MA), rabbit anti-PKC γ (1:500, # sc-211, Santa Cruz Biotechnology, Dallas, TX), rabbit anti-Pax2 (1:100, #71-6000,ThermoFisher, Waltham, MA), mouse anti-Calb1 (1:500,# MCA-5A9, EnCor Biotech Gainesville, FL), or rabbit anti-c-Fos (1:500, #ab190289, Abcam, Cambridge, United Kingdom) which were diluted in 0.2% of Triton X-100 and 10% normal goat serum in PBS (blocking buffer) and photographed under a fluorescent microscope.

Techniques: Double Staining, Marker, Immunohistochemistry, In Situ Hybridization

Journal: bioRxiv

Article Title: A Spinal Circuit That Transmits Innocuous Cool Sensations

doi: 10.1101/2022.03.23.485555

Figure Lengend Snippet: (A) Ablation of Calb1 Lbx1 neurons in the dorsal horn. Bar graph represents quantified data for Calb1 Cre -Tomato signals in control and ablated animals. n = 27-30 sections; **** p < 0.0001, Student’s unpaired t test. Scale bar, 100 μm. (B) The acetone evaporation assay in Calb1 Abl and control groups. Control: n = 17; Calb1 Abl : n = 14; **** p < 0.0001, Mann-Whitney test. (C) Two-temperature preference between two temperature plates. Left panel: Reference plate is set at 30 °C, and test plate temperature is set at 10 °C, 20 °C, 30 °C, 40 °C, and 50 °C. Control: n = 10-11; Calb1 Ab l : n = 10; ns, no significant difference; **** p < 0.0001 two-way ANOVA with Sidak post hoc analysis. Right panel: Reference plate is set at 20 °C, test plate temperature is set at 10 °C. Data points represent the average percentage of time spent on the reference plate across two trials over the total trial time. Control: n = 6; Calb1 Abl : n = 7; ns, no significant difference; Student’s unpaired t test. (D) Gradient temperature ranging from 5 °C to 50 °C is quantified as percentage of time spent in each temperature zone over the total trial time. Control: n = 13; Calb1 Abl : n = 18; * p < 0.05; *** p < 0.001; two-way ANOVA with Bonferroni post hoc analysis. (E) Quantified forelimb withdrawal latency to 0 °C cold plate, including forelimb lick and flinch responses. n = 10 in each group; ns, no significant difference; Student’s unpaired t test. (F) Quantitative nocifensive response to dry ice application to the hindpaw. Score represents average response across four trials per mouse. Control: n = 12; Calb1 Abl : n = 10; ns, no significant difference; Student’s unpaired t test. (G) Acute punctate mechanical pain threshold as measured by up-down von Frey withdrawal threshold was significantly increased in Calb1 Abl mice compared to controls. Control: n = 18; Calb1 Abl : n = 13; **** p < 0.0001, Student’s unpaired t test.

Article Snippet: To detect protein expression, immunohistochemistry was performed using rabbit anti-NK1R (1:1000, #S8305 MilliporeSigma, St. Louis, MO), rabbit anti- α -CGRP (1:500, #T-4032, Peninsula Lab, San Carlos, CA), Alexa fluor 647-conjugated isolectin GS (IB4) (10 μg/mL, #I32450, ThermoFisher Scientific, Waltham, MA), rabbit anti-PKC γ (1:500, # sc-211, Santa Cruz Biotechnology, Dallas, TX), rabbit anti-Pax2 (1:100, #71-6000,ThermoFisher, Waltham, MA), mouse anti-Calb1 (1:500,# MCA-5A9, EnCor Biotech Gainesville, FL), or rabbit anti-c-Fos (1:500, #ab190289, Abcam, Cambridge, United Kingdom) which were diluted in 0.2% of Triton X-100 and 10% normal goat serum in PBS (blocking buffer) and photographed under a fluorescent microscope.

Techniques: Evaporation, MANN-WHITNEY

Journal: bioRxiv

Article Title: A Spinal Circuit That Transmits Innocuous Cool Sensations

doi: 10.1101/2022.03.23.485555

Figure Lengend Snippet: (A) The acetone evaporation assay before and 40 minutes after CNO injection in mice with hM4Di receptors in spinal Calb1 Lbx1 -neuron silenced (Calb1 Silenced ) and control groups. Control: n = 8; Calb1 Silenced : n = 7; **** p < 0.0001, two-way ANOVA with Sidak post hoc analysis. (B) Two-temperature preference between two temperature plates before and 40 minutes after CNO injection in Calb1 Silenced and control groups. Reference plate is set at 30°C, and test plate temperature is set at 30 °C, 20 °C and 10 °C. Data points represent the average percentage of time spent on the reference plate across two trials over the total trial time. n = 6 in each group; ** p < 0.01; two-way ANOVA with Sidak post hoc analysis. (C) Gradient temperature ranging from 5 °C to 50 °C was quantified as percentage of time spent in each temperature zone before and 40 minutes after CNO injection in Calb1 Silenced and control groups. Control: n = 4; Calb1 Silenced : n = 7; *** p < 0.001; **** p < 0.0001; two-way ANOVA with Bonferroni post hoc analysis. (D) Quantified forelimb flinch and lick withdrawal latency to 0 °C cold plate before and 40 minutes after CNO injection in Calb1 Silenced and control groups. Control: n = 8; Calb1 Silenced : n = 7; ns, no significant difference; two-way ANOVA with Sidak post hoc analysis. (E) Nocifensive responses to hindpaw application of dry ice stimulus before and 40 minutes after CNO injection in Calb1 Silenced and control groups. Score represents average response across four trials per mouse. Control: n = 9; Calb1 Silenced : n = 11; two-way ANOVA with Sidak post hoc analysis. (F) Acute punctate mechanical pain measured using the up-down von Frey assay before and 40 minutes after CNO injection in Calb1 Silenced and control groups. Control: n = 8; Calb1 Silenced : n = 7; **** p < 0.0001; two-way ANOVA with Sidak post hoc analysis.

Article Snippet: To detect protein expression, immunohistochemistry was performed using rabbit anti-NK1R (1:1000, #S8305 MilliporeSigma, St. Louis, MO), rabbit anti- α -CGRP (1:500, #T-4032, Peninsula Lab, San Carlos, CA), Alexa fluor 647-conjugated isolectin GS (IB4) (10 μg/mL, #I32450, ThermoFisher Scientific, Waltham, MA), rabbit anti-PKC γ (1:500, # sc-211, Santa Cruz Biotechnology, Dallas, TX), rabbit anti-Pax2 (1:100, #71-6000,ThermoFisher, Waltham, MA), mouse anti-Calb1 (1:500,# MCA-5A9, EnCor Biotech Gainesville, FL), or rabbit anti-c-Fos (1:500, #ab190289, Abcam, Cambridge, United Kingdom) which were diluted in 0.2% of Triton X-100 and 10% normal goat serum in PBS (blocking buffer) and photographed under a fluorescent microscope.

Techniques: Evaporation, Injection

Journal: bioRxiv

Article Title: A Spinal Circuit That Transmits Innocuous Cool Sensations

doi: 10.1101/2022.03.23.485555

Figure Lengend Snippet: (A) Double staining of SOM mRNA (green) by in situ hybridization with Calb1 Lbx1 -Tomato signals (red). The percentage is calculated as double-positive neurons over total number of Calb1 Lbx1 -Tomato + neurons (top) or double-positive neurons over total number of SOM + neurons (bottom). Inset represents higher magnification of the boxed area. Arrow indicates double-positive cells for SOM and Tomato. n = 18 sections. Scale bar, 100 μm. (B) The acetone evaporation assay in SOM Abl and control groups. Control: n = 10; SOM Abl : n = 9; ns, no significant difference; Mann-Whitney test. (C) Two-temperature preference between two temperature plates. Reference plate is set at 30°C, and test plate temperature is set at 30 °C, 20 °C and 10 °C. n = 7 in each group; ns, no significant difference; two-way ANOVA with Sidak post hoc analysis. Data points represent the average percentage of time spent on the reference plate across two trials over the total trial time. (D) Gradient temperature ranging from 5 °C to 50 °C was quantified as time spent in each temperature zone. Control: n = 7; SOM Abl : n = 5; ns, no significant differences; two-way ANOVA with Bonferroni post hoc analysis. (E) Acute punctate mechanical pain measured using the up-down von Frey assay. Control: n = 10; SOM Abl : n = 9; **** p < 0.0001; Student’s unpaired t test. (F) Schematic showing proposed Calb1 Lbx1 subpopulations for cool sensations and acute punctate mechanical pain. Calb1 + neurons (light blue) represent the entire Calb1 Lbx1 population, which can be further classified into at least two distinct subgroups: Calb1 Lbx1 ;SOM + (green) for mechanical punctate pain and Calb1 Lbx1 ;SOM - (dark blue) for cool sensing.

Article Snippet: To detect protein expression, immunohistochemistry was performed using rabbit anti-NK1R (1:1000, #S8305 MilliporeSigma, St. Louis, MO), rabbit anti- α -CGRP (1:500, #T-4032, Peninsula Lab, San Carlos, CA), Alexa fluor 647-conjugated isolectin GS (IB4) (10 μg/mL, #I32450, ThermoFisher Scientific, Waltham, MA), rabbit anti-PKC γ (1:500, # sc-211, Santa Cruz Biotechnology, Dallas, TX), rabbit anti-Pax2 (1:100, #71-6000,ThermoFisher, Waltham, MA), mouse anti-Calb1 (1:500,# MCA-5A9, EnCor Biotech Gainesville, FL), or rabbit anti-c-Fos (1:500, #ab190289, Abcam, Cambridge, United Kingdom) which were diluted in 0.2% of Triton X-100 and 10% normal goat serum in PBS (blocking buffer) and photographed under a fluorescent microscope.

Techniques: Double Staining, In Situ Hybridization, Evaporation, MANN-WHITNEY

Journal: bioRxiv

Article Title: A Spinal Circuit That Transmits Innocuous Cool Sensations

doi: 10.1101/2022.03.23.485555

Figure Lengend Snippet: (A) The number of wet-dog shakes in response to an agonist of TRPM8 channels, icilin, is abolished in Calb1 Abl mice compared to controls. Control: n = 7; Calb1 Abl : n = 9; **** p < 0.0001; Student unpaired t test. (B) Schematic representing the experimental approach to virally label individual Calb1 + neurons in the spinal cord and TRPM8 GFP afferent neurons in the DRG using Calb1 Cre ;TRPM8 GFP mice. Spinal cord tissue is embedded in a gel and expanded to enable reconstruction of morphology of Calb1 Brainbow neurons and identify the location of synaptic pairs. (C) Left: Overview of the dorsal horn of Calb1 Cre ;TRPM8 GFP mice following Brainbow labeling and immunostaining excitatory pre- and post-synaptic markers Bassoon and Homer, respectively. Right three panels: Higher magnification of insets depicting representative images of quadruple-positive interaction. Cyan: The dendritic branch of a Calb1 Brainbow neuron; purple: the axon terminal of a TRPM8 + sensory neuron; red: the presynaptic marker Bossoon; green: the postsynaptic marker Homer. Arrow shows a quadruple-positive synaptic connection. Scale bar, 30 μm estimated based on anticipated expansion factor. (D) Representative image showing the location of identified quadruple-positive synaptic connections (yellow dots) and cell morphology of Calb1 Brainbow neurons (grey) across the superficial dorsal horn. Scale bar, 30 μm estimated based on anticipated expansion factor. (E) Higher magnification of inset depicting representative image showing the morphology of a Calb1 Brainbow neuron that forms synaptic pairs with TRPM8 + afferents. Green: TRPM8 GFP ; grey: Calb1 Brainbow neuron; yellow: identified synaptic pairs between TRPM8 + afferents and this Calb1 + neurons. Scale bar, 15 μm estimated based on anticipated expansion factor. (F) Schematic summarizing the morphology of Calb1 Brainbow neurons that form synaptic connections with TRPM8 + primary sensory neurons. Green: Calb1 Brainbow neurons with local dendritic arborization in TRPM8-innervation zone; magenta: Calb1 Brainbow neurons that contain at least one dendritic arbor outside of the TRPM8-innervating zone. n = 6 sections.

Article Snippet: To detect protein expression, immunohistochemistry was performed using rabbit anti-NK1R (1:1000, #S8305 MilliporeSigma, St. Louis, MO), rabbit anti- α -CGRP (1:500, #T-4032, Peninsula Lab, San Carlos, CA), Alexa fluor 647-conjugated isolectin GS (IB4) (10 μg/mL, #I32450, ThermoFisher Scientific, Waltham, MA), rabbit anti-PKC γ (1:500, # sc-211, Santa Cruz Biotechnology, Dallas, TX), rabbit anti-Pax2 (1:100, #71-6000,ThermoFisher, Waltham, MA), mouse anti-Calb1 (1:500,# MCA-5A9, EnCor Biotech Gainesville, FL), or rabbit anti-c-Fos (1:500, #ab190289, Abcam, Cambridge, United Kingdom) which were diluted in 0.2% of Triton X-100 and 10% normal goat serum in PBS (blocking buffer) and photographed under a fluorescent microscope.

Techniques: Labeling, Immunostaining, Marker

Journal: bioRxiv

Article Title: A Spinal Circuit That Transmits Innocuous Cool Sensations

doi: 10.1101/2022.03.23.485555

Figure Lengend Snippet: (A) Schematic demonstrating the location of Calb1 Lbx1 ;SOM - neurons in lamina I-II o . Red dots: recorded Calb1 Lbx1 ;SOM - neurons. n = 57 from 10 naïve Calb1 Lbx1 ;Ai65 mice. (B) Firing properties of Calb1 Lbx1 ;SOM - neurons in lamina I-II o . (C) Schematic demonstrating the location of Calb1 Lbx1 ;SOM + neurons in lamina II. Red dots: recorded Calb1 Lbx1 ;SOM + neurons. n = 47 from 10 naïve Calb1 Lbx1 ;Ai65 mice. (D) Firing properties of Calb1 Lbx1 ;SOM + neurons in lamina II. (E) Summary of A β -, A δ - or C-evoked EPSCs, IPSCs, and APs in Calb1 Lbx1 ;SOM - neurons in lamina I-II o (top) and Calb1 Lbx1 ;SOM + neurons in lamina II (bottom) under normal conditions, and after strychnine and bicuculline application. Table represents a summary of sensory inputs in 35 Calb1 Lbx1 ;SOM - neurons in lamina I-II o under normal condition, and 32 Calb1 Lbx1 ;SOM - neurons in lamina I-II o upon strychnine and bicuculline application; 30 Calb1 Lbx1 ;SOM + neurons in lamina II under normal condition, and 27 Calb1 Lbx1 ;SOM + neurons in lamina II upon strychnine and bicuculline application. M, monosynaptic inputs. P, polysynaptic inputs. (F) Representative traces of icilin-induced eEPSC (left) and icilin-induced eAPs (right) in Calb1 Lbx1 ;SOM - neurons in lamina I-II o . Horizontal blue line indicates 1μM icilin application to the DRG chamber. (G) Representative traces of icilin-induced eEPSC (left) and icilin-induced eAPs (right) in Calb1 Lbx1 ;SOM + neurons in lamina II. Horizontal blue line indicates 1μM icilin application to the DRG chamber. (H) Summarized table of icilin-induced eEPSCs and icilin-induced eAPs at 1 μM concentration in Calb1 Lbx1 ;SOM - neurons in lamina I-II o (top) and Calb1 Lbx1 ;SOM + neurons in lamina II (bottom) from 10 naïve Calb1 Lbx1 ;Ai65 mice. Right panel: All icilin-responsive Calb1 Lbx1 ;SOM - neurons demonstrate an initial bursting firing pattern. (I) Representative trace showing a Calb1 Lbx1 ;SOM - neuron recorded upon administration of icilin (before), during co-administration of icilin and AMTB (middle), and upon administration of icilin (after). Diagonal lines indicate passage of time between chemical administrations. Blue lines represent 1 μM icilin application. Yellow line represents 100 μM AMTB administration. (J) Quantification of icilin-induced eEPSCs in Calb1 Lbx1 ;SOM - neurons. n = 13 from 10 naïve Calb1 Lbx1 ;Ai65 mice; **** p < 0.0001 two-way ANOVA with Tukey post hoc analysis.

Article Snippet: To detect protein expression, immunohistochemistry was performed using rabbit anti-NK1R (1:1000, #S8305 MilliporeSigma, St. Louis, MO), rabbit anti- α -CGRP (1:500, #T-4032, Peninsula Lab, San Carlos, CA), Alexa fluor 647-conjugated isolectin GS (IB4) (10 μg/mL, #I32450, ThermoFisher Scientific, Waltham, MA), rabbit anti-PKC γ (1:500, # sc-211, Santa Cruz Biotechnology, Dallas, TX), rabbit anti-Pax2 (1:100, #71-6000,ThermoFisher, Waltham, MA), mouse anti-Calb1 (1:500,# MCA-5A9, EnCor Biotech Gainesville, FL), or rabbit anti-c-Fos (1:500, #ab190289, Abcam, Cambridge, United Kingdom) which were diluted in 0.2% of Triton X-100 and 10% normal goat serum in PBS (blocking buffer) and photographed under a fluorescent microscope.

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: A Spinal Circuit That Transmits Innocuous Cool Sensations

doi: 10.1101/2022.03.23.485555

Figure Lengend Snippet: (A) Schematic demonstrating bilateral injection of fluorescent fluorophore conjugated cholera toxin subunit B (CTB) into the lateral parabrachial nucleus to label SPB neurons (green) and acetone co-administration onto hindpaw to label cooling-sensitive neurons in Calb1 Lbx1 ;Ai65 mice (red). (B) Representative image of the injection site in the lateral parabrachial nucleus. Scale bar, 100 μm. (C) Out of 68 labeled SPB neurons, no triple-positive (red: Calb1 Lbx1 -Tomato + ; green: CTB; blue: Fos + ) cool SPB neurons were identified (0%, 0/68) in the ipsilateral dorsal horn of the spinal cord following acetone treatment. Inset represents high magnification of the boxed area. Arrowhead indicates a c-Fos and CTB double-positive cooling-sensitive SPB neuron that is Tomato - . n = 9 sections from 3 mice. Scale bar, 100 μm. (D-E) Representative firing properties, C fiber inputs, and icilin-induced eEPSCs and eAPs in CTB + SPB neurons in control (D) and Calb1 Abl (E) mice. The majority of CTB + SPB neurons in control (96%, 24/25) and Calb1 Abl (91.0%, 20/22) animals display an initial bursting firing pattern upon current injection. Horizontal blue line: Icilin 1μM. Red triangle: presentation of stimulus. (F) Summary of A β -, A δ -, and C-evoked EPSC, IPSC, and APs, and icilin-induced eEPSCs and APs in CTB + SPB neurons in control and Calb1 Abl mice. Control: 25 neurons from 5 mice; Calb1 Abl : 22 neurons from 4 mice. M, monosynaptic inputs. P, polysynaptic inputs. Icilin: 1 μM. (G) Dose response curve of icilin (μM)-induced eEPSCs in CTB + SPB neurons in control and Calb1 Abl mice. Control: 13 neurons from 5 mice; Calb1 Abl : 8 neurons from 4 mice; **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; two-way ANOVA with Sidak post hoc analysis. (H) Quantification of icilin-evoked EPSCs in CTB + SPB neurons at different concentrations in control and Calb1 Abl mice. Control: 13 neurons from 5 mice; Calb1 Abl : 8 neurons from 4 mice; * p < 0.05; two-way ANOVA with Tukey post hoc analysis. (I) Quantification of icilin-evoked APs in CTB + SPB neurons at different concentrations in Control and Calb1 Abl mice. Control: 13 neurons from 5 mice; Calb1 Abl : 8 neurons from 4 mice; ** p < 0.01; * p < 0.05; two-way ANOVA with Tukey post hoc analysis. (J) Schematic showing proposed neural pathways that transmits innocuous cool sensations and acute punctate mechanical pain. Calb1 Lbx1 ;SOM - interneurons in laminae I-II o receive monosynaptic inputs from TRPM8 + primary sensory neurons and then innervate to cooling-sensitive SPB neurons via monosynaptic or polysynaptic connections; whereas Calb1 + ;SOM + interneurons in lamina II are proposed to receive inputs from mechanosensitive C-nociceptors and then connect to unknown SPB neurons for acute punctate mechanical pain. However, whether TRPM8 + fibers or C-nociceptor fibers synapse onto SPB neurons in lamina I remains unknown.

Article Snippet: To detect protein expression, immunohistochemistry was performed using rabbit anti-NK1R (1:1000, #S8305 MilliporeSigma, St. Louis, MO), rabbit anti- α -CGRP (1:500, #T-4032, Peninsula Lab, San Carlos, CA), Alexa fluor 647-conjugated isolectin GS (IB4) (10 μg/mL, #I32450, ThermoFisher Scientific, Waltham, MA), rabbit anti-PKC γ (1:500, # sc-211, Santa Cruz Biotechnology, Dallas, TX), rabbit anti-Pax2 (1:100, #71-6000,ThermoFisher, Waltham, MA), mouse anti-Calb1 (1:500,# MCA-5A9, EnCor Biotech Gainesville, FL), or rabbit anti-c-Fos (1:500, #ab190289, Abcam, Cambridge, United Kingdom) which were diluted in 0.2% of Triton X-100 and 10% normal goat serum in PBS (blocking buffer) and photographed under a fluorescent microscope.

Techniques: Injection, Labeling