Circuit dissection of the role of somatostatin in itch and pain

Abstract

Stimuli that elicit itch are detected by sensory neurons that innervate the skin. This information is processed by the spinal cord; however, the way in which this occurs is still poorly understood. Here we investigated the neuronal pathways for itch neurotransmission, particularly the contribution of the neuropeptide somatostatin. We find that in the periphery, somatostatin is exclusively expressed in Nppb⁺ neurons, and we demonstrate that Nppb⁺somatostatin⁺ cells function as pruriceptors. Employing chemogenetics, pharmacology and cell-specific ablation methods, we demonstrate that somatostatin potentiates itch by inhibiting inhibitory dynorphin neurons, which results in disinhibition of GRPR⁺ neurons. Furthermore, elimination of somatostatin from primary afferents and/or from spinal interneurons demonstrates differential involvement of the peptide released from these sources in itch and pain. Our results define the neural circuit underlying somatostatin-induced itch and characterize a contrasting antinociceptive role for the peptide.

Figure 1. Somatostatin is co-expressed with Nppb in DRG neurons.

A, in situ hybridization of sections through DRG shows that different neuropeptides are expressed in subsets of sensory neurons; SST, somatostatin, Nppb, natriuretic polypeptide B, NMB, neuromedin B, CGRP, calcitonin gene related peptide. B, double label ISH reveals that somatostatin-expressing neurons (magenta) co-express the neuropeptide Nppb (green). Similar results were obtained from 3 mice.

Figure 2. Somatostatin-expressing primary afferent neurons are sufficient to trigger itch-behavior.

A, triple label ISH reveals that in Sst^Cre;Ai32 mice expression of ChR2-YFP (green) occurs largely in Nppb-neurons (red). MrgA3-neurons (blue) are separate from both Nppb and ChR2-YFP positive neurons. Similar results were obtained from 3 mice. B, schematic diagram illustrating the strategy employed to optogenetically activate trigeminal ganglion somatostatin-expressing neurons that innervate the face. The implanted fiber optic cannula (indicated as an outlined blue line) was passed through the brain to a position approximately 1 mm dorsal to the trigeminal ganglion and fixed in place with dental cement. C, unilateral optogenetic activation of the trigeminal ganglion of Sst^Cre;Ai32 animals with 470 nm illumination generated similar numbers of scratch bouts to that elicited by intradermal administration of histamine (100 µg), but did not induce scratching of the contralateral cheek. In addition, while optogenetic stimulation of Sst^Cre;Ai32 neurons did not evoke cheek wipes, histamine injection elicited 6.2 wipes ± 1.53 (mean ± SEM). Activation with 590 nm light evoked minimal scratching bouts. Significant differences were assessed using one-way repeated measure ANOVA with post hoc Sidak tests (t F_2,16 = 3.139, *p=0.0001 for both comparisons). Data represent means ± SEM (n=5 animals optogenetic experiments and n=7 histamine cheek assay C57BL/6 mice).

Figure 3. Modulation of Nppb- and GRP-induced itch responses by somatostatin receptor agonist and antagonist.

Scratching bouts induced by intrathecal administration of: A, Nppb (5 µg), octreotide (10 ng), a combination of Nppb and octreotide, or Nppb and the somatostatin agonist CYN154806 (1 µg); B, GRP (1 nmole), octreotide (10 ng), a combination of GRP and octreotide (10 ng), or GRP and CYN154806 (1 µg); C, histamine (100 µg), or a combination of histamine and CYN154806 (1 µg) revealed that Nppb- and GRP-evoked itch behavior is significantly potentiated by octreotide and attenuated by CYN154806 (A, B). In addition, CYN154806 significantly attenuated histamine induced scratching. Significant differences for A and B were assessed using one-way ANOVA with post hoc Sidak tests: Octreotide induced scratching was significantly changed by the addition of Nppb (*p= 0.0002) and Nppb elicited scratching was significantly reduced by CYN (*p=0.0053), F_3,19 = 0.6796. Octreotide induced scratching was significantly changed by the addition of GRP (*p= 0.0001) and GRP elicited scratching was significantly reduced by CYN (*p= 0.0036) F_3,20 = 0.5998. Data represent means ± SEM (n= 6, 5, 6, 6, 6, 6, 6, and 6). Significant differences for C were assessed using two-sided unpaired Student's t-test (*p= 0.002). Data represent means ± SEM (n= 9 and 8).

Figure 4. Spinal cord dynorphin neurons modulate itch and are downstream of the site of action of somatostatin.

A, schematic diagram of the viral-based strategy employed to chemogenetically activate spinal cord neurons expressing dynorphin or nNOS. B, sagittal sections stained for mCherry showing that intraspinal injection of the AAV in Pdyn^Cre and nNOS^Cre mice produced expression with the expected distribution in spinal cord segments L3-L5, which innervate the hind-limb including the calf. Scale = 200 μm. C, Chemogenetic activation in the Pdyn^Cre mouse. A transverse section of spinal cord taken from a Pdyn^Cre mouse that had been injected with AAV2-flex-hM3Dq-mCherry and treated with CNO 2 hours prior to perfusion fixation. The section was immunostained to reveal mCherry (mCh, red), the somatostatin receptor Sst_2a (blue), Pax2 (gray) and Fos (green). Asterisks (*) show the cell bodies of 3 neurons that express hM3Dq-mCherry, Sst_2a receptor, Pax2 and Fos, indicating chemogenetic activation of inhibitory (Sst_2a-expressing) dynorphin cells. Similar results were obtained in experiments on 3 CNO-treated animals (see Figure S3 for numbers). Arrowhead points to a Pax2⁺ (inhibitory) Sst_2a-expressing neuron that lacks mCherry and this cell was not Fos-positive. Scale = 10 μm. D, Chemogenetic activation in the nNOS^CreERT2 mouse. Transverse section of spinal cord taken from a nNOS^CreERT2 mouse injected with AAV2-flex-hM3Dq-mCherry and treated with CNO 2 hours prior to perfusion fixation. The section was immunostained to reveal mCherry (red), Sst_2a (blue), nNOS (gray) and Fos (green). Five cells showing varying levels of nNOS-immunoreactivity are visible. Two of these (asterisks) are stained for mCherry and Sst_2a (inhibitory nNOS cells) and these are Fos-positive. Of the 3 cells with weak nNOS-immunoreactivity, one (arrow) is positive for mCherry and Fos, but lacks Sst_2a, and is therefore likely an excitatory interneuron. The other two are not labelled with either mCherry or Fos: one of these is an Sst_2a-positive inhibitory neuron (single arrowhead), while the other lacks Sst_2a and is therefore likely to be an excitatory neuron (double arrowhead). This shows chemogenetic activation of nNOS cells, including inhibitory (Sst_2a-expressing) interneurons. Similar results were obtained in experiments on 3 CNO-treated animals (see Figure S3 for numbers). Scale = 20 μm. E. The time spent biting the calf in response to intradermal injection of chloroquine (100 μg) was reduced following chemogenetic activation (CNO) in Pdyn^Cre mice, but there was no effect on itch responses in nNOS^CreERT2 animals. Significant differences were assessed using two-sided unpaired Student t-tests (t₂₁ = 2.92, *p = 0.0082; and t₂₃ = 0.875, p = 0.391 ns not significant). Data represent means ± SEM (n=11, 12, 12, and 13 animals, for Pdyn^Cre mice treated with CNO and vehicle and for nNOS^CreERT2 mice treated with CNO and vehicle, respectively). mCherry-labelled injection sites (as shown in B) were verified in all of these experiments. F, DREADDq activation following intrathecal injection of AAV2-flex-hM3Dq significantly reduced numbers of itch bouts in Pdyn^Cre mice injected into the nape of the neck with histamine (100 µg) and chloroquine (100 µg), and also when octreotide (100 ng) was administered intrathecally. Significant differences were assessed using two-sided unpaired Student t-tests (t₁₀ = 3.017, 3.053, 4.861, *p = 0.013, 0.0122, and 0.0007). Data represent means ± SEM (n= 6 animals).

Figure 5. Chemogenetic activation of Pdyn^Cre and nNOS^CreERT2 neurons modulates responses to heat and mechanical stimuli and the pro-nociceptive effect of dynorphin neuron activation likely involves excitatory interneurons.

A, Hargreaves assays revealed that while responses to heat stimulation were unaffected in mice in which dynorphin neurons were chemogenetically activated (CNO), sensitivity was significantly reduced in animals in which nNOS neurons were activated. B, von-Frey tests showed that chemogenetic activation (CNO) of dynorphin neurons elicited mechanical hyperalgesia, while activation of nNOS neurons caused significantly reduced sensitivity to mechanical stimulation. In all cases behavioral results of testing the paw ipsilateral to spinal AAV injection in vehicle- and CNO-treated mice (post-surgery) were compared with results from the same animals obtained before intraspinal injection of the AAV (pre-surgery). Significant differences were assessed using 2-way ANOVA with post hoc Sidak tests (F_1,25 = 4.566, *p = 0.0117, F_1,25 = 9.855, **p = 0.0039, F_1,25 = 19.25, ***p = 0.000012). Data represent means ± SEM (n=13, 11, 14, and 13 animals, for Pdyn^Cre mice treated with CNO and vehicle and for nNOS^CreERT2 mice treated with CNO and vehicle, respectively). C-G. Plots of the distribution of excitatory and inhibitory dynorphin-expressing cells show that the excitatory cells are highly concentrated in the region innervated from glabrous skin. C. Immunostaining for VGLUT3 was used to reveal the extent of innervation from hairy skin, since C-low threshold mechanoreceptors (which express VGLUT3) are largely absent from glabrous skin. The VGLUT3 band occupies the whole mediolateral extent of the superficial dorsal horn at L3, but is absent from the medial part of the L4 and L5 segments, which are innervated by afferents from glabrous skin. The junction between these regions is marked (arrowheads). Similar results were obtained from 3 mice. D The distribution of preprodynorphin (PPD)-immunoreactive cells that are inhibitory (Pax2-positive, blue circles) and excitatory (Pax2-negative, red circles) plotted onto outlines of the L3-L5 segments (data pooled from 3 mice). The junction between hairy and glabrous skin territories is marked by a dashed line. Note that excitatory PPD cells are concentrated in the glabrous skin territory, and are much less numerous in regions innervated from hairy skin, including the L3 segment. E-G. Examples of immunostaining for PPD (magenta), NeuN (blue) and Pax2 (green) in the medial (Med) and lateral (Lat) parts of the L3, L4 and L5 segments, respectively. Examples of Pax2-positive PPD-immunoreactive neurons are indicated with arrows, and some Pax2-negative PPD-immunoreactive cells are shown with arrowheads. Similar results were obtained from 3 mice. Scale bars C = 100 μm, E-G = 20 μm.

Figure 6. Somatostatin acts upstream of dynorphin-expressing inhibitory neurons, which interact with the Nppb-itch pathway at the level of GRPR-neurons.

A, itch-responses to intradermally injected histamine (100 µg), and intrathecally administered Nppb (5µg) and octreotide (100ng) were significantly attenuated when the kappa opioid receptor (KOR) agonist ICI199441 (100ng) was co-administered. Significant differences were assessed using two-sided unpaired Student t-tests (t₉ = 7.059, t₁₀ = 10.14, t₉ = 11.78, *p = 0.0001 for all). Data represent means ± SEM (n= 6, 5, 6, 6, 5, and 6 animals). B, The somatostatin receptor Sst2 antagonist CYN154806 (1 µg) does not affect itch-behavior induced by the kappa opioid receptor antagonist norBinaltrorphimine (100 µg), differences were assessed using two-sided unpaired Student t-tests (t₁₂ = 2.708, ns p= 0.06). Data represent means ± SEM (n= 7 animals). C histamine (100 µg), D octreotide (100 ng), and E the kappa opioid receptor antagonist norBinaltrorphimine (100 µg) induced scratching bouts were reduced in mice treated with GRP-saporin, or GRPR antagonist, significant differences were assessed using one-way ANOVA with post hoc Sidak tests (F_2,15 = 42.48, F_2,15 = 37.87, F_2,16 = 29, *p = 0.0001 for all). Data represent means ± SEM (n= 6, 6, 6, 6, 6, 6, 6, 7, and 6 animals), Nppb-saporin (F-H) treatment reduced scratching bouts to histamine, but not to octreotide or norBinaltrorphimine, differences were assessed using two-sided unpaired Student t-tests (t₅ = 4.953, *p=0.004, t₅ = 1.184, ns p= 0.29 and t₅ = 1.466, p = 0.203). Data represent means ± SEM (n= 6 animals). I, schematic diagram of proposed model of the somatostatin-mediated itch microcircuit. Broken red arrows indicate incompletely defined pathways, and blue and green circles are neurons that are identified by either the receptor, or the neuropeptide(s) they express; Sst, somatostatin, Nppb, natriuretic polypeptide b, Npr1, Natriuretic polypeptide receptor 1, Sst_2a, somatostatin receptor 2a, GRP, gastrin releasing peptide, and GRPR, gastrin releasing peptide receptor.

Figure 7. Somatostatin-null mice exhibit itch-related behavioral deficits to pruritogens.

A, schematic diagram depicting the genetic strategy employed to conditionally eliminate the expression of somatostatin. B, ISH of section through DRG (top row) and dorsal spinal cord (bottom row) demonstrates that Sst^f/f;Trpv1^Cre mice lack expression of somatostatin in DRG, Sst^f/f;Lbx1^Cre mice lack expression of somatostatin in the spinal cord, and that Sst^f/f;Wnt1^Cre mice lack expression of somatostatin in DRG and spinal cord. Similar results were obtained from 3 mice. C, Sst^f/f;Wnt1^Cre mice are much less sensitive to intradermal injection of a variety of compounds that induce itch than normal littermate controls. Significant differences were assessed using two-sided unpaired Student t-tests (t₁₀ = 4.082, t₁₂ = 3.967, t₁₀ = 2.83, t₁₀ = 3.836, t₁₃ = 2.368, t₁₀ = 2.279, *p = 0.0022, 0.0019, 0.0179, 0.0033, 0.0034, and 0.0458). Data represent means ± SEM (n= 6, 6, 7, 7, 6, 6, 6. 6, 8, 7, 6, and 6 animals).

Figure 8. Elimination of somatostatin expression from primary afferent neurons increases pain sensitivity.

Sst^f/f;Trpv1^Cre mice are much more sensitive to noxious heat stimulation than normal littermate controls, A. In contrast, Sst^f/f;Lbx1^Cre mice exhibit similar withdrawal latency to noxious heat as normal littermate controls, B. Sst^f/f;Wnt1^Cre mice also display reduced latencies to noxious heat stimulation compared to normal littermate controls, C. Significant differences were assessed using two-sided unpaired Student t-tests (t₂₀ = 4.156, t₈ = 1.01, t₁₂ = 4.059, *p = 0.0005, 0.3421, and 0.0016). Data represent means ± SEM (n= 11, 11, 5, 5, 7, and 7 animals).