Leaky Gate Model: Intensity-Dependent Coding of Pain and Itch in the Spinal Cord

Abstract

Coding of itch versus pain has been heatedly debated for decades. However, the current coding theories (labeled line, intensity, and selectivity theory) cannot accommodate all experimental observations. Here we identified a subset of spinal interneurons, labeled by gastrin-releasing peptide (Grp), that receive direct synaptic input from both pain and itch primary sensory neurons. When activated, these Grp⁺ neurons generated rarely seen, simultaneous robust pain and itch responses that were intensity dependent. Accordingly, we propose a "leaky gate" model in which Grp⁺ neurons transmit both itch and weak pain signals; however, upon strong painful stimuli, the recruitment of endogenous opioids works to close this gate, reducing overwhelming pain generated by parallel pathways. Consistent with our model, loss of these Grp⁺ neurons increased pain responses while itch was decreased. Our new model serves as an example of non-monotonic coding in the spinal cord and better explains observations in human psychophysical studies.

Keywords: gate control,Grp(+)neuron; itch; leaky gate; neural circuit; pain; spinal cord.

Figure 1. Genetic labeling of itch second order neurons in the spinal cord

(A and B) Spinal cord and DRG sections from Grp^Cre; ROSA26^LSL-tdTomato mice, tdTomato fluorescence were visualized directly without staining. (C and D) Grp^Cre; ROSA26^LSL-tdTomato;Grp^EGFP and MrgprA3^Cre; ROSA26^LSL-tdTomato; Grp^EGFP spinal sections stained with GFP antibody. (E–G) Grp^Cre; ROSA26^LSL-tdTomato spinal sections stained with antibodies to CGRP, IB4 and PKCγ respectively. (H and I) Grp^Cre; ROSA26^LSL-tdTomato; Gad1^EGFP and vGlut2^Cre; ROSA26^LSL-tdTomato; Grp^EGFP spinal sections stained with GFP antibody. White arrowheads in C, G, I indicate overlap. (J, J′) Biocytin labeled individual Grp⁺ neurons, categorized as vertical neurons. All scale bars represent 20 μm. (K) Percentage of Grp⁺ neurons expressing vGlut2 and Gad1. n= 15 hemisections from three mice per group. Data are represented as mean ± SEM. (L) Diagram summarizing the potential synaptic connections between MrgprA3⁺ DRG neurons and Grp⁺ neurons in the spinal cord. MrgprA3 also overlapped with Grp and post-synaptic marker, PSD95.

Figure 2. Grp⁺ neurons receive monosynaptic itchy input

(A) Left: diagram showing light activation of MrgprA3 peripheral fibers in behavioral tests; right: diagram showing light activation of MrgprA3 central terminals and recording of Grp⁺ neurons in the spinal cord. (B) 1Hz and 5Hz 100ms light stimulation triggered scratching bouts in five minutes. MrgprA3^Cre; ROSA26^LSL-ChR2 and ROSA26^LSL-ChR2 control mice with light delivered to shaved nape regions (n=6). Data are represented as mean ± SEM. (C) From left to right: image of Grp⁺ neurons in spinal slice with electrode (black lines), representative traces of light-induced EPSCs in Grp⁺ neurons with monosynaptic input from MrgprA3⁺ neurons and percentage of Grp⁺ neurons with monosynaptic input from MrgprA3⁺ neurons. (D) Left: representative traces of light-induced EPSCs in lamina II Grp negative neurons with monosynaptic input, polysynaptic input and no synaptic input from MrgprA3⁺ neurons. Right: percentage of lamina II Grp negative neurons with monosynaptic input, polysynaptic input and no synaptic input from MrgprA3⁺ neurons.

Figure 3. Monosynaptic retrograde tracing from Grp⁺ neurons

(A) Diagram showing monosynaptic retrograde tracing strategy from Grp⁺ neurons. (B) Rabies-labeled neurons overlap with IB4 in spinal cord. (C) Top panels: L4–6 DRG sections labeled with different markers (CGRP, IB4, NF200, MrgprC11 and TrpV1). Middle panels: rabies virus trans-synaptically labeled DRG neurons. Bottom panels: merge images. Arrowhead indicates overlap of markers and rabies-labeled DRG neurons. All scale bars represent 20 μm. (D) Percentage of rabies trans-synaptically labeled DRG neurons co-localize with different markers. Pooled results from more than 30 DRG sections of at least five mice for each marker.

Figure 4. Painful stimuli strongly activate while itchy stimuli weakly activate Grp⁺ neurons

(A) Image of DRG attached spinal cord slice. Recording electrode on right and drug application electrode on left. (B) Diagram summarizing painful stimuli from DRG can strongly activate Grp⁺ neurons while itchy stimuli can only weakly activate Grp⁺ neurons. (C–F) Top panels: representative traces of action potentials from Grp⁺ neurons in responses to drugs. Bottom panels: Grp⁺ neurons in response to capsaicin (n=5, 0.5μM; n=5, 2μM; n=6, 5μM), SLIGRL (n=7, 100μM; n=6, 500μM), chloroquine (CQ, n=9, 3mM; n=10, 10mM) and histamine (n=5, 10mM; n=9, 50mM) application on DRG. Black bar indicates duration of drug application (n=6). Data are represented as mean ± SEM.

Figure 5. Intensity dependent coding of pain and itch by Grp⁺ neurons

(A) Diagram showing the strategy of capsaicin-mediated specific activation of Grp⁺ neurons in Grp^Cre; ROSA26^LSL-TrpV1; TrpV1^−/− mice. (B and C) Pain-related licking time and itch-related scratching bouts in wild type (green bars) and TrpV1^−/− mice (red bars) triggered by intrathecal delivery of 10μl capsaicin (1μg or 3.3nmol, n=6 for both genotypes), BNP (2.5mg/ml or 7.1nmol/site, n=8) and GRP peptides (200μM or 2nmol/site, n=8). (D and E) 10μl intrathecal capsaicin-triggered pain and itch responses in Grp^Cre; ROSA26^LSL-TrpV1; TrpV1^−/− mice (black bars, from left to right: 10ng or 0.03nmol, n=9; 50ng or 0.16nmol, n=6; 0.2μg or 0.67nmol, n=5; 1μg or 3.33nmol, n=6; 5μg or 16.7nmol, n=10; 20μg or 66.7nmol, n=6) together with responses in wild-type (green bars, 1μg or 3.33nmol, n=6) and TrpV1^−/− mice (red bars, 5μg or 16.7nmol, n=6) from (B and C). (F and G) 10μl intrathecal capsaicin-triggered pain and itch responses in Grp^Cre; ROSA26^LSL-TrpV1; TrpV1^−/− mice with drugs. From left to right 1μg or 3.33nmol capsaicin without and with GRPR antagonist (Deamino-Phe¹⁹,D-Ala²⁴,D-Pro²⁶-psi(CH₂NH)Phe²⁷)-GRP (19–27), 200μM or 2nmol/site, n=6 and 5); 10μg or 33.3nmol capsaicin, n=8; 10μg or 33.3nmol capsaicin with naloxone (1μg or 3.33nmol, n=7), naltrindole (10μg or 24.1nmol, n=7), CTAP (5μg or 13.7nmol, n=6), CTOP (10μg or 9.43nmol, n=6), bicuculline (10μM or 0.1nmol, n=7), cycloSomatostatin (0.1mM or 1nmol, n=8) and for (F) 5μg/ml or 0.16nmol capsaicin without (n=6) and with naloxone (1μg or 3.33nmol, n=9). (H and I) Pain and itch dose-response curve fitting of (D and E). Data are represented as mean ± SEM. *: P < 0.05, **: P<0.01, ***: P<0.001, two-tailed unpaired Student’s t test. Abbreviations: WT short for wild type. KO short for TrpV1^−/−. Grp-V1;V1KO short for Grp^Cre; ROSA26^LSL-TrpV1; TrpV1^−/−.

Figure 6. Activation of enkephalin-expressing neurons triggered by stimulation of Grp⁺ neurons

(A) Diagram showing calcium imaging of enkephalin-expressing neurons labeled by Penk^Cre while depolarizing Grp^EGFP neurons in spinal slices. (B) Enkephalin release with different doses of capsaicin from Grp^Cre; ROSA26^LSL-TrpV1; TrpV1^−/− mice (black bars) and ROSA26^LSL-TrpV1; TrpV1^−/− control mice (blue bars) normalized to per g tissue used in ELISA. (C) Representative calcium imaging results showing enkephalin-expressing neurons before, during and after activation of Grp^EGFP neurons (1Hz 50pA current injection). White lines (in pipet shape) indicated patch-clamped Grp^EGFP neurons. Arrowheads indicated activated enkephalin-expressing neurons during Grp activation. Enkephalin-expressing neurons (Penk^Cre; Rosa26^LSL-GCaMP6) were activated when 5 out of 10 Grp⁺ neurons were depolarized. (D) Penk^Cre labeled neurons co-localized with enkephalin and GAD1. Representative neurons magnified in upper right corner (Arrowheads indicated magnified cells). Scale bars represent 20μm.

Figure 7. “Leaky gate” model in pain and itch transmission

(A) Comparison of “leaky gate” model (left) and the gate control theory (right). Grp⁺ neurons directly code for pain and itch while inhibiting pain through enkephalin-expressing interneurons. Aβ fibers activate pain transmission neurons and also indirectly inhibit pain transmission neurons via inhibitory interneurons. Yellow rectangles indicate type I incoherent feed forward loop formed by Grp⁺ neurons and Aβ fibers respectively. (B) Diagram summarizing the role of Grp⁺ neurons in pain and itch coding. Grp⁺ neurons receive weak input from itchy stimuli and strong input from painful stimuli, and positively code for itch while negatively regulate pain transmission. Enke (Enkephalin), T (pain transmission neurons), IN (inhibitory interneurons).

Figure 8. Increased pain and decreased itch responses after the ablation of Grp⁺ neurons

(A) Diagram showing ablation of Grp⁺ neurons in the spinal cord. (B) Representative images of Grp^Cre; ROSA26^{LSL-DTR; LSL-tdTomato} spinal slices with and without diphtheria toxin treatments. All scale bars represent 20 μm. (C) Quantification of Grp⁺ neurons per five 20μm hemisections in Grp^Cre; ROSA26^{LSL-DTR; LSL-tdTomato} (red) and ROSA26^{LSL-DTR; LSL-tdTomato} control mice (blue) after diphtheria toxin treatments (n=5 mice). (D) Scratching bouts induced by histamine (100mM or 5μmol, n=6 vs. 8), serotonin (1mM or 50nmol, n=7 vs. 8), SLIGRL (1mM or 50nmol, n=7) and chloroquine (4mM or 200nmol, n=11 vs. 9) injection in the nape region (50μl) in Grp^Cre; ROSA26^{LSL-DTR; LSL-tdTomato} (red) and ROSA26^{LSL-DTR; LSL-tdTomato} control mice (blue). (E) Scratching bouts induced by injections of histamine (100mM or 5μmol, n=6) and chloroquine (4mM or 200nmol, n=6) in the nape region (50μl), with saline (50μl) or GRPR antagonist (50μl, Deamino-Phe¹⁹,D-Ala²⁴,D-Pro²⁶-psi(CH₂NH)Phe²⁷)-GRP (19–27), 200μM or 2nmol/site) pretreatments 10 minutes before injection and saline (50μl) induced scratching bouts (n=6) in Grp^Cre; ROSA26^{LSL-DTR; LSL-tdTomato} (red) and ROSA26^{LSL-DTR; LSL-tdTomato} control mice (blue). (F) Pain responses from capsaicin cheek injections (1mg/ml or 33nmol, n=8; 0.5mg/ml or 16.7nmol, n=7 vs. 6), capsaicin intraplantar injections (0.1mg/ml or 3.3nmol, n=6; 0.05mg/ml or 1.67nmol, n=6) and immersion assay (50°C, n=7 vs. 6; 52°C, n=7 vs. 6) in Grp^Cre; ROSA26^{LSL-DTR; LSL-tdTomato} (red) and ROSA26^{LSL-DTR; LSL-tdTomato} control mice (blue). (G and H) Pain responses from hot plate test (52°C, n=10; 55°C, n=7 vs. 6), Hargreaves test (n=8 vs. 7) and Von Frey test responses (n=11) in Grp^Cre; ROSA26^{LSL-DTR; LSL-tdTomato} (red) and ROSA26^{LSL-DTR; LSL-tdTomato} control mice (blue). Yellow shaded regions represent responses with strong stimuli. Data are represented as mean ± SEM. *: P < 0.05, **: P<0.01, ***: P<0.001, extended Welch’s t test for response ratio comparison between different temperatures in hot plate and tail immersion test and two-tailed unpaired Student’s t test for the rest. Abbreviations: Grp/DTR/tdt short for Grp^Cre; ROSA26^{LSL-DTR; LSL-tdTomato} and DTR/tdt short for ROSA26^{LSL-DTR; LSL-tdTomato}, CQ short for chloroquine, 5-HT short for serotonin.