Characteristics of sensory neuronal groups in CGRP-cre-ER reporter mice: Comparison to Nav1.8-cre, TRPV1-cre and TRPV1-GFP mouse lines | PLOS One

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Fig 1 — Fig 1.

Electrophysiology protocols.
a. CGRP⁺ neurons (red; marked with blue arrow) selected for recording were classified by size (C_m in pF) and staining with IB4 (green; marked with yellow arrow). b. Stimulus waveform (1nA, 0.5 msec) indicated below trace generated a single AP in small DRG neuron. We analyzed AP and AHP parameters: resting membrane potential–V_m; duration at base–dB and the time required for the AHP (measured in mV) to decay by 80%—(AHP₈₀). In addition, we measured rise time (RT) and fall time (FT) of AP (Table 1). Characteristic AP “hump” is indicated by black arrow. c. Outward current was generated by the indicated waveforms found below traces. The decay constant τ was derived from standard single exponential fits between points indicated by arrows for the final outward current trace (+20 mV). Characteristic “spike-like” peak is shown by arrow on trace generated by stepping to +0 mV.

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Table 1 — Table 1.

Sensory neuronal group cluster parameters defined by characterization DRG neurons in marker-reporters.

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Fig 2 — Fig 2.

AP traces and algesic response profile for sub-classes of CGRP-cre⁺ DRG neurons.
For each CGRP-cre⁺ sensory neuronal group recorded from mouse DRG, the AP, response to ATP (30μM), capsaicin (100nM; CAP), 5-HT (30μM) and mustard oil (10μM; MO) are presented from left to right. The AP time scale (horizontal bar) is 5 msec for each panel. I_ATP time scale is 1 sec for each panel. I_ATP magnitude (vertical bar) scales are indicated for each panel. Name of the groups are also indicated when two I_ATP traces are presented on a panel. I_CAP time scale is 10 sec for each panel. I_CAP magnitude scale is indicated. I_5-HT was recorded from S3, but not S2 neurons. I_5-HT time and magnitude scales are 1 sec and 50 pA, respectively, for each panel. An exception is the M1/M1a group that has a large I_5-HT current. MO responses were measured by Ca²⁺ imaging. CGRP-cre⁺ sensory neuronal groups are shown for each row. Drug application times are illustrated by horizontal bar above traces. More complete information on subgroups is presented in Table 2.

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Fig 3 — Fig 3.

AP traces and algesic response profiles for sub-classes of Nav1.8-cre⁺/CGRP-cre^- DRG neurons.
For each Nav1.8-cre⁺/CGRP-cre^- sensory neuronal group recorded from mouse DRG, the AP, response to ATP (30μM), CAP (100nM), 5-HT (30μM) and MO (10μM) are presented from left to right. The AP time scale (horizontal bar) is 5 ms for each panel. I_ATP time and magnitude (vertical bar) scales are 1 s and 50 pA, respectively, for each panel. I_CAP time scale is indicated, and magnitude scale is 50pA for each panel, except for S6 neuronal group. I_5-HT time and magnitude scales are 1 s and 50 pA, respectively, for each panel. MO responses were measured by Ca²⁺ imaging. Names of Nav1.8-cre⁺/CGRP-cre^- sensory neuronal groups are indicated. Drug application times are illustrated by horizontal bar above traces. More complete information on subgroups is presented in Table 3.

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Fig 4 — Fig 4.

AP differences between sensory neuronal groups, and outward currents in Nav1.8-cre⁺ neurons.
a. Comparison of AP shapes generated in S1 CGRP-cre⁺/Nav1.8-cre⁺, S4 CGRP-cre^-/Nav1.8-cre⁺ and S6 CGRP-cre^-/Nav1.8-cre⁺ neurons. AP “hump” in S1 neurons and “bow” in S4 neurons are indicated with black and red arrows, respectively. S6 neurons’ AP does not display any deflection during the falling phase of AP. b. Comparison of AP in S6 and S7 neurons. Deflection on the falling phase of S7 neuron AP is indicated by black arrow. c. Comparison of single AP in M1 and M3 CGRP-cre⁺ neuronal group. “Hump” is marked with blue arrow, while “deflection” is indicated with black arrow. d. Comparison of AP in M3 and M4 Nav1.8-cre⁺/CGRP-cre^- DRG neurons. M3 neuron’s AP “deflection” is indicated with black arrow, and M4 neuron’s AP with no “deflection” is shown with red arrow. e. Typical outward current (I) produced from Nav1.8-cre⁺/CGRP-cre^- S6 and S7 neuronal groups. f. Typical I produced from the CGRP-cre⁺ S1-S3 group neurons. g. Typical I produced from CGRP-cre⁺ M1 and M3 group neurons. Names of neuronal groups are specified above traces. The time scale (horizontal bar) is 25 ms for each panel.

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Fig 5 — Fig 5.

AP traces and algesic response profiles for sub-classes of Nav1.8-cre^- DRG neurons.
For each Nav1.8-cre-negative (Nav1.8-cre^-) sensory neuronal group recorded from mouse DRG, the AP, response to ATP (30μM) and outward current (I; see “Method” protocol-3) are presented from left to right. “Partially developed” small spike-like feature in the M2 Nav1.8-cre^- neurons is indicated with a red arrow. The AP time scale (horizontal bar) is 5 ms for each panel. I_ATP time scale is 1 s for each panel; and magnitude scales (vertical bars) are indicated separately for each panel. I current time scale is 25 ms for each panel. Names of Nav1.8^- sensory neuronal groups are indicated. Drug application times are illustrated by horizontal bar above traces. More complete information on subgroups is presented in Tables 3 and 5.

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Table 2 — Table 2.

Properties of CGRP^cre-ER/TdTomato⁺ sensory neuronal groups.

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Table 3 — Table 3.

Properties of Nav1.8^cre/TdTomato⁺ sensory neuronal groups.

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Table 4 — Table 4.

Properties of TRPV1-GFP⁺ sensory neuronal groups.

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Table 5 — Table 5.

Properties of marker-defined groups of sensory neuros.

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Fig 6 — Fig 6.

Sensory neuronal markers expressing in CGRP-cre⁺ DRG neurons.
a. Expression of CGRP-ab (green) and TRPV1 (blue) relatively CGRP-cre⁺ neurons (red) in DRG from CGRP^cre-ER/+;Rosa26^{LSL-tDTomato/+} mice. Medium-large CGRP-cre⁺/CGRP-ab⁺ neurons are marked with yellow arrows (a and a1); small CGRP-cre⁺/CGRP-ab⁺ neurons are marked with purple arrows (a and a1); and small CGRP^-/TRPV1⁺ neurons (<20μm) are marked with sapphire arrows (a and a2). b. Expression of 5HT3a (green) relative to CGRP-cre⁺ neurons (red) in DRG. CGRP-cre⁺/5HT3a⁺ neurons are marked with yellow arrows and CGRP-cre^-/5HT3a⁺ neurons are marked with sapphire arrows. c. Expression of NPY2R (green) relative to CGRP-cre⁺ neurons (red) in DRG. Medium NPY2R⁺/CGRP-cre⁺ neurons are marked with yellow arrows (c and c1) and medium NPY2R⁺/CGRP-cre^- neurons are marked with sapphire arrows (c and c1). d. Expression of mrgD (green) relative to CGRP-cre⁺ DRG neurons (red). Yellow arrows mark CGRP-cre^-/mrgD⁺ neurons (d and d1). White horizontal bar shows 20μm scale for each panel.

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Fig 7 — Fig 7.

Sensory neuronal markers non-expressing in CGRP-cre⁺ DRG neurons.
a. Expression of TH (green) and TRPV1 (blue) relatively to CGRP-cre⁺ neurons (red) in DRG from CGRP^cre-ER/+;Rosa26^{LSL-tDTomato/+} mice. Some TRPV1⁺ DRG neurons are not expressed in CGRP-cre⁺ sensory neurons. These CGRP-cre^-/TRPV1⁺ neurons are marked with yellow arrows (a and a1). b. Expression of calbindin-28K (Calb; green) relatively to CGRP-cre⁺ DRG neurons (red). Yellow arrows mark CGRP-cre^-/Calb⁺ neurons (b and b1). c. Expression of trkB (green) relatively to CGRP-cre⁺ DRG neurons (red). Yellow arrows mark CGRP-cre^-/trkB⁺ neurons; and a blue arrow shows a rare example of the CGRP-cre⁺/trkB⁺ neuron (c and c1). d. Expression of trkC (green) relatively to CGRP-cre⁺ DRG neurons (red). Yellow arrows mark CGRP-cre^-/trkC⁺ neurons; and; and a blue arrow shows an example of the CGRP-cre⁺/trkC⁺ neuron (d and d1). White horizontal bar shows 20μm scale for each panel.

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Fig 8 — Fig 8.

Expression pattern of sensory neuronal markers in CGRP-cre⁺ and Nav1.8-cre⁺ DRG neurons.
a. Expression of parvalbumin (PV; green) relative to CGRP-cre⁺ DRG neurons (red). Yellow arrows mark CGRP-cre^-/PV⁺ neurons (a and a1). White horizontal bar shows 20μm scale for each panel. b. Percentages of total L3-L5 DRG neurons labeled in CGRP^cre-ER/+; Rosa26^{LSL-tDTomato/+} mice (i.e. CGRP-cre⁺), and with CGRP antibodies (i.e. CGRP-ab⁺). Cell counting is from three animals, 3–4 sections each. c. Percentage of CGRP-cre⁺ L3-L5 DRG neurons in CGRP^cre-ER/+;Rosa26^{LSL-tDTomato/+} mice labeled with indicated neuronal markers. Cell counting is from three animals, 4–5 sections each. d. Percentages of total L3-L5 DRG neurons labeled with a variety of indicated sensory neuronal markers. Cell counting is from three animals, 3–5 sections each. e. Expression percentages of markers in Nav1.8-cre⁺ in L3-L5 mouse DRG neurons. Cell counting is from three animals, 3–5 sections each.

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Fig 9 — Fig 9.

Sensory neuronal markers expressing in Nav1.8-cre⁺ DRG neurons.
a. Expression of CGRP (green) and TRPV1 (blue) in Nav1.8-cre⁺ DRG neurons (red) in L3-L5 DRG of Nav1.8^cre/+;Rosa26^{LSL-tDTomato/+} mice. Sapphire arrows mark TRPV1⁺/Nav1.8-cre^- neurons (a2 and a3); a pink arrow marks a TRPV1⁺/CGRP⁺/Nav1.8-cre⁺ neuron (a-a3), yellow arrows mark CGRP⁺/Nav1.8-cre⁺ neurons (a and a1) and blue arrows mark CGRP^-/Nav1.8-cre⁺ neurons (a and a1). b. Co-expression of TH (green) and Nav1.8-cre⁺ (red) neurons in DRG. Blue arrows mark TH⁺/Nav1.8-cre⁺ neurons (b and b1). c. Co-expression of MrgprD (green) and Nav1.8-cre⁺ (red) neurons in DRG. Pink arrows mark mrgD⁺/Nav1.8-cre⁺ neurons. d. Co-expression of NPY2R (green) and Nav1.8-cre⁺ (red) neurons in DRG. Blue arrows mark NPY2R⁺/Nav1.8-cre^- neurons and yellow arrows mark NPY2R⁺/Nav1.8-cre⁺ neurons. White horizontal bar shows 20μm scale for each panel.

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Fig 10 — Fig 10.

Sensory neuronal markers non-expressing in Nav1.8-cre⁺ DRG neurons.
a. Expression of trkB (green) and Nav1.8-cre⁺ (red) neurons in Nav1.8^cre/+;Rosa26^{LSL-tDTomato/+} mouse L3-L5 DRG. Yellow arrows mark trkB⁺/Nav1.8-cre^- neurons, and blue arrows show few trkB⁺/Nav1.8-cre⁺ neurons (a and a1). b. Expression of trkC (green) and Nav1.8-cre⁺ (red) neurons in Nav1.8^cre/+;Rosa26^{LSL-tDTomato/+} mouse L3-L5 DRG. Yellow arrows mark trkC⁺/Nav1.8-cre^- neurons and blue arrows mark trkC⁺/Nav1.8-cre⁺ neurons (b and b1). c. Expression of calbindin (Calb; green) and Nav1.8-cre⁺ (red) neurons in Nav1.8^cre/+;Rosa26^{LSL-tDTomato/+} mouse L3-L5 DRG. Yellow arrows mark Calb⁺/Nav1.8-cre^- neurons (c and c1). d. Expression of parvalbumin (PV; green) and Nav1.8-cre⁺ (red) neurons in Nav1.8^cre/+;Rosa26^{LSL-tDTomato/+} mouse L3-L5 DRG. Yellow arrows mark PV⁺/Nav1.8-cre^- neurons and a blue arrow marks a rare example of PV⁺/Nav1.8-cre⁺ neurons. White horizontal bar shows 20μm scale for each panel.

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Fig 11 — Fig 11.

Schematic representation of CGRP-cre⁺, Nav1.8-cre⁺, TRPV1-cre⁺ and V1-GFP⁺ subsets.
Schematic representation of expression pattern for CGRP-cre⁺ in L3-L5 DRG of CGRP^cre-ER/+;Rosa26^{LSL-tDTomato/+} mice; for Nav1.8-cre⁺ in L3-L5 DRG of Nav1.8^cre/+;Rosa26^{LSL-tDTomato/+}; for TRPV1-cre⁺ in L3-L5 DRG of TRPV1^cre/+;Rosa26^{LSL-tDTomato/+} and for V1-GFP⁺ in L3-L5 DRG of TRPV1-GFP reporter mice. MO responsive subsets are indicated as TRPA1-postive. “Groups” line represents name of sensory neuronal groups presented in this study (see Table 1). “Marker” line signifies distinctive markers for each group. If marker is not defined for particular sensory neuronal group, “?” sign was used. “Sensory neurons” indicates possible functions of sensory neuronal groups in the grey box. C-PMN is C-polymodal nociceptor; C-noc is C-nociceptors (modalities are unknown); C-MN is C- mechano-nociceptors; C-LTMR is C-low threshold mechano-receptor; Aδ-HTMR is Aδ-high threshold mechano-receptor; Aδ-LTMR is Aδ-low threshold mechano-receptor; Aβ-HTMR is Aβ-high threshold mechano-receptor; Aβ-LTMR is Aβ-low threshold mechano-receptor; RA Aβ-LTMR is rapid adapting Aβ-low threshold mechano-receptor; and Prop is proprioceptors.

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