Journal: bioRxiv

Article Title: Genetic Identification of Dopamine Neurons Required for Circadian Food Anticipatory Activity in Mice

doi: 10.64898/2026.03.27.714759

Figure Lengend Snippet: A) Confocal imaging of immunofluorescence antibody staining of Th (magenta) and DAT (green) in the midbrain of Calb1 Cre ;Th Flox cKO midbrain tissue. B) Quantification of TH staining in the SN of WT (grey) and Calb1 Cre ;Th Flox cKO (gold) midbrain tissue. C) Normalized body weight of WT and Calb1 Cre ;Th Flox cKO mice over the course of the study; weight was normalized by dividing the weekly weight by the Day 0 weight, which was taken prior to beginning CR. The weight fraction was similar between the two groups at every time point. D) Normalized food intake of WT and Calb1 Cre ;Th Flox cKO mice. Food intake was normalized by dividing average 24-hour AL intake by the animal’s body weight. E) Total daily high activity in seconds of WT (grey) and Calb1 Cre ;Th Flox cKO (gold) mice throughout the course of the study. F) High activity in seconds during the 3-hour premeal window throughout the course of the study. G) Normalized high activity during the 3-hour premeal window; data is normalized by dividing the seconds of high activity in the premeal window by the total daily high activity. H-K) Group-averaged double-plotted ethograms for WT and Calb1 Cre ;Th Flox cKO mice over a 40-day period for wheel running activity (H), pellet intake (I), rewarded left nose pokes (J), and unrewarded right nose pokes are shown. Area within brown borders indicates periods of food availability, and dark phase is indicated by gray background. L-M) Group average profiles for wheel running activity, rewarded left nose pokes, and unrewarded right nose pokes during 48 h food deprivation. Blue vertical lines indicate onset and offset of food availability during previously restricted feeding schedule. Light-dark cycle indicated as black and white at the bottom of each graph.

Article Snippet: Calb1 Cre -driver mice were obtained from The Jackson Laboratory (Bar Harbor, ME; stock #028532); this allele contains an internal ribosomal entry site (IRES).

Techniques: Imaging, Immunofluorescence, Staining, Activity Assay

Journal: Journal of the American Society of Nephrology

Article Title: Gentamicin Inhibits Ca2+ Channel TRPV5 and Induces Calciuresis Independent of the Calcium-Sensing Receptor–Claudin-14 Pathway

doi: 10.1681/asn.2021030392

Figure Lengend Snippet: Figure 1. Acute effect of gentamicin administration on urinary Ca21, K1, Mg21, and Na1 excretion in mice. (A) Proximal tubular (PT) Ca21 reabsorption occurs paracellularly via claudin-2 (CLDN2) and is driven via the electrochemical gradient that is facilitated by Na1/H1 exchange via NHE3 and basolateral Na1-K1-ATPase (NKA) activity and subsequent osmotic water reabsorption. In the TAL, the lumen-positive electrical gradient generated by transport of electrolytes via the furosemide-sensitive cotransporter NKCC2, the K1 channel ROMK, the Cl2 channel CLCNKB, and the NKA facilitates paracellular Ca21 reabsorption through a complex formed by claudin-16 (CLDN16) and claudin-19 (CLDN19). Activation of the CaSR reduces Ca21 reabsorption in the TAL and increases the expression of the pore-blocking protein, claudin-14 (CLDN14). In the DCT2/CNT, TRPV5 drives transcellular Ca21 reabsorption, where inwardly transported Ca21 is buffered by calbindin-D28k and extruded by the Na1/Ca21 exchanger (NCX1/SLC8A1) and the Ca21 pump (PMCA4). (B) Gentamicin acutely increased urinary Ca21 excretion in mice, as indicated by the significant interaction (P50.001) between treatment and time. Urinary Ca21 excretion was significantly higher after gentamicin treatment compared with vehicle 4 hours after injection (n54–6). (C) The absence of a significant interaction between treatment and time indicates that urinary Mg21 excretion was not altered by gentamicin (n53–6). (D) Gentamicin did not alter urinary Na1 excretion (n53–6). (E) Urinary K1

Article Snippet: The following antibodies were used: a mouse monoclonal antibody against CLDN14, directed against the extreme C-terminal segment of mouse CLDN14 has been characterized extensively25; mouse monoclonal antibodies against calbindin-D28k and TRPV5 have been characterized previously35; rabbit polyclonal antibodies against NCX1/SLC8A1 (55075-1-AP; Proteintech); a mouse monoclonal antibody, JA9, specifically recognizes the PMCA4 isoform (ab2783; Abcam, Cambridge, UK),36 which is the main isoform expressed in the DCT2/CNT region20,21; polyclonal rabbit anti–b-actin antibody (ab8227; Abcam, Cambridge, UK).

Techniques: Activity Assay, Generated, Activation Assay, Expressing, Blocking Assay, Injection

Journal: Journal of the American Society of Nephrology

Article Title: Gentamicin Inhibits Ca2+ Channel TRPV5 and Induces Calciuresis Independent of the Calcium-Sensing Receptor–Claudin-14 Pathway

doi: 10.1681/asn.2021030392

Figure Lengend Snippet: Figure 6. Expression of Ca21 transport proteins after chronic treatment with gentamicin on a normal or a high Ca21 diet. (A) Immunohistochemical staining for TRPV5 in kidney cross-sections from gentamicin-treated animals on both the control and high Ca21 diet showed a reduced intensity of immunoreactive staining for TRPV5. (B) Western blotting and (C) semiquantitative analysis showed decreased calbindin-D28k protein after gentamicin administration in both the control and high Ca21 diet (n56).

Article Snippet: The following antibodies were used: a mouse monoclonal antibody against CLDN14, directed against the extreme C-terminal segment of mouse CLDN14 has been characterized extensively25; mouse monoclonal antibodies against calbindin-D28k and TRPV5 have been characterized previously35; rabbit polyclonal antibodies against NCX1/SLC8A1 (55075-1-AP; Proteintech); a mouse monoclonal antibody, JA9, specifically recognizes the PMCA4 isoform (ab2783; Abcam, Cambridge, UK),36 which is the main isoform expressed in the DCT2/CNT region20,21; polyclonal rabbit anti–b-actin antibody (ab8227; Abcam, Cambridge, UK).

Techniques: Expressing, Immunohistochemical staining, Staining, Control, Western Blot

Journal: Cells

Article Title: Regional Variation of Gap Junctional Connections in the Mammalian Inner Retina

doi: 10.3390/cells10092396

Figure Lengend Snippet: List of key resources.

Article Snippet: mouse monoclonal anti-calbindin , Synaptic Systems, Göttingen, German , 214011.

Techniques: Plasmid Preparation, Laser-Scanning Microscopy, Software

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

Journal: bioRxiv

Article Title: Neural correlates of licking behavior modulated by target position in the striatal matrix compartment

doi: 10.64898/2026.04.18.719363

Figure Lengend Snippet: A. Schematic illustration of the behavioral apparatus. The head and body of the water-deprived mouse were fixed using a metal plate and tube. A spout, adjustable in three dimensions—anterior–posterior (AP), medial–lateral (ML), and dorsal–ventral (DV)—was placed near the mouth. An optical fiber was connected to an implanted robe in the ventrolateral striatum (VLS) for fiber photometry recordings of GCaMP6f signals. B. Diagram of the operant conditioning task. Each trial began with illumination of a light-emitting diode (LED) in a soundproof operant box. A drop of sucrose water was delivered immediately after the mouse licked the spout. After water delivery, the LED was turned off, followed by an inter-trial interval (ITI). C. Schematic illustration of GCaMP fluorescence recording from matrix neurons. GCaMP6f was selectively expressed in matrix neurons via injection of AAV5.CAG.Flex.GCaMP6f into the VLS of Calb1-IRES-Cre mice. An optical probe measured calcium-dependent fluorescence of GCaMP6f excited at 465 nm. D. Schematic illustration of GCaMP fluorescence recording from striosomal neurons. GCaMP6f was selectively expressed in striosomal neurons via injection of AAV5.CAG.Flex.GCaMP6f into the VLS of Pdyn-IRES-Cre mice. E. Histological image of Cre-dependent GCaMP6f-expressing neurons in the striatum of a Calb1-IRES-Cre mouse. Scale bar: 100 µm. F. Histological image of Cre-dependent GCaMP6f-expressing neurons in the striatum of a Pdyn-IRES-Cre mouse. Scale bar: 100 µm

Article Snippet: We used seven adult male Calb1-IRES-Cre mice (129S-Calb1tm2.1(cre)Hze/J, Jackson Laboratory Cat# 028532) and six adult male Pdyn-IRES-Cre mice (129S-Pdyn(tm1.1(Cre)/Mjkr)/LowlJ, Jackson Laboratory Cat# 027958), which express Cre recombinase selectively in striatal matrix and striosomal neurons, respectively ( ).

Techniques: Fluorescence, Injection, Expressing

Journal: Ageing and neurodegenerative diseases

Article Title: Subtype-specific roles of nigrostriatal dopaminergic neurons in motor and associative learning

doi: 10.20517/and.2025.26

Figure Lengend Snippet: Targeted expression of the chemogenetic inhibitor hM4Di in Calb1 + nigrostriatal DANs. (A) Schematic depicting the stereotactic delivery of control mCherry or hM4Di AAVs in the SNc of Th Flp ; Calb1 IRESCre (TC) double KI mice; (B) Representative images showing mCherry (red), ALDH1A1 (green), and TH (magenta) expression in the SNc of control mCherry (left panel) and hM4Di (right panel)-expressing mice. Scale bar: 1 mm; (C) Magnified images of the boxed areas in (B), with arrows indicating neurons co-expressing mCherry and ALDH1A1. Scale bar: 100 mm; (D) Representative images illustrating mCherry (red), ALDH1A1 (green), and TH (magenta) staining in the striatum of control mCherry-expressing mice. Scale bar: 1 mm. Calb1 + : Calbindin 1 -positive; DANs: dopaminergic neurons; AAVs: adeno-associated viruses; SNc: substantia nigra pars compacta ; KI: knock-in; TH: Tyrosine Hydroxylase.

Article Snippet: Calb1 IRESCre mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA; JAX# 028532).

Techniques: Expressing, Control, Staining, Knock-In

Journal: Ageing and neurodegenerative diseases

Article Title: Subtype-specific roles of nigrostriatal dopaminergic neurons in motor and associative learning

doi: 10.20517/and.2025.26

Figure Lengend Snippet: Chemogenetic inhibition of Calb1 + nigrostriatal DANs impairs spontaneous locomotion. (A-D) Quantification of total (A) (*** P = 0.0005), ambulatory (B) (** P = 0.0016), rearing (C) (** P = 0.0014), and fine (D) (** P = 0.001) movement in TC double KI mice injected with either control mCherry [ n = 10, 3 Male (M)/7 Female (F)] or hM4Di AAVs ( n = 10, 4 M/6 F) in the SNc. Locomotor activity was measured during a 30-minute open-field test following i.p. injection of JHU37160. Data are presented as mean ± SEM; unpaired t -test; (E-H) Quantification of spontaneous locomotion: total (E) ( P = 0.540), ambulatory (F) ( P = 0.435), rearing (G) ( P = 0.616), and fine (H) ( P = 0.862) movement with the same cohort of mice as in (A-D) following saline injection. Data are presented as mean ± SEM; unpaired t -test. Calb1 + : Calbindin 1 -positive; DANs: dopaminergic neurons; TC: Th Flp ; Calb 1 IRESTCre ; KI: knock-in; AAVs: adeno-associated viruses; SNc: substantia nigra pars compacta ; SEM: standard error of the mean.

Article Snippet: Calb1 IRESCre mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA; JAX# 028532).

Techniques: Inhibition, Injection, Control, Activity Assay, Saline, Knock-In

Journal: Ageing and neurodegenerative diseases

Article Title: Subtype-specific roles of nigrostriatal dopaminergic neurons in motor and associative learning

doi: 10.20517/and.2025.26

Figure Lengend Snippet: Chemogenetic inhibition of Calb1 + nigrostriatal DANs severely impairs rotarod motor skill learning. Rotarod motor skill learning in TC double KI mice injected with either control mCherry [ n = 10, 3 Male (M)/7 Female (F)] or hM4Di AAVs ( n = 10, 4 M/6 F) in the SNc. Mice underwent one training session per day for six consecutive days, with JHU37160 administered prior to each session. Data are presented as mean ± SEM. Two-way ANOVA, mCherry vs . hM4Di effect: F (1, 18) = 22.2, *** P = 0.0002 . Calb1 + : Calbindin 1 -positive; DANs: dopaminergic neurons; TC: Th Flp ; Calb 1 IRESTCre ; KI: knock-in; AAVs: adeno-associated viruses; SNc: substantia nigra pars compacta ; SEM: standard error of the mean; ANOVA: analysis of variance.

Article Snippet: Calb1 IRESCre mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA; JAX# 028532).

Techniques: Inhibition, Injection, Control, Knock-In

Journal: Ageing and neurodegenerative diseases

Article Title: Subtype-specific roles of nigrostriatal dopaminergic neurons in motor and associative learning

doi: 10.20517/and.2025.26

Figure Lengend Snippet: Chemogenetic inhibition of Calb1 + nigrostriatal DANs affects food-seeking behavior. (A) Quantification of rewards earned by control mCherry [ n = 8, 3 Male (M)/5 Female (F)] and hM4Di ( n = 8, 4 M/4 F)-expressing TC double KI mice during a 2-hour free-feeding session on the FED3 device. Data are presented as mean ± SEM; unpaired t -test, P = 0.251; (B) Quantification of pellets earned in FR1 training on FED3 across three sessions. Two-way ANOVA with Tukey’s multiple comparison test: **** P < 0.0001 (session 1), ** P = 0.0086 (session 2), and 0.5036 (session 3); (C) Quantification of nose-poking accuracy in FR1 training on FED3 across three sessions. Two-way ANOVA with Tukey’s multiple comparison test: * P = 0.0495 (session 1), ** P = 0.0096 (session 2), and 0.6679 (session 3). Calb1 + : Calbindin 1 -positive; DANs: dopaminergic neurons; TC: Th Flp ; Calb 1 IRESTCre ; KI: knock-in; FED3: Feeding Experimentation Device 3; SEM: standard error of the mean; FR1: fixed-ratio 1; ANOVA: analysis of variance.

Article Snippet: Calb1 IRESCre mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA; JAX# 028532).

Techniques: Inhibition, Control, Expressing, Comparison, Knock-In