Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Mar 20;39(12):2238-2250.
doi: 10.1523/JNEUROSCI.2559-18.2019. Epub 2019 Jan 17.

Dorsal Horn Gastrin-Releasing Peptide Expressing Neurons Transmit Spinal Itch But Not Pain Signals

Affiliations
Free PMC article

Dorsal Horn Gastrin-Releasing Peptide Expressing Neurons Transmit Spinal Itch But Not Pain Signals

Gioele W Albisetti et al. J Neurosci. .
Free PMC article

Abstract

Gastrin-releasing peptide (GRP) is a spinal itch transmitter expressed by a small population of dorsal horn interneurons (GRP neurons). The contribution of these neurons to spinal itch relay is still only incompletely understood, and their potential contribution to pain-related behaviors remains controversial. Here, we have addressed this question in a series of experiments performed in GRP::cre and GRP::eGFP transgenic male mice. We combined behavioral tests with neuronal circuit tracing, morphology, chemogenetics, optogenetics, and electrophysiology to obtain a more comprehensive picture. We found that GRP neurons form a rather homogeneous population of central cell-like excitatory neurons located in lamina II of the superficial dorsal horn. Multicolor high-resolution confocal microscopy and optogenetic experiments demonstrated that GRP neurons receive direct input from MrgprA3-positive pruritoceptors. Anterograde HSV-based neuronal tracing initiated from GRP neurons revealed ascending polysynaptic projections to distinct areas and nuclei in the brainstem, midbrain, thalamus, and the somatosensory cortex. Spinally restricted ablation of GRP neurons reduced itch-related behaviors to different pruritogens, whereas their chemogenetic excitation elicited itch-like behaviors and facilitated responses to several pruritogens. By contrast, responses to painful stimuli remained unaltered. These data confirm a critical role of dorsal horn GRP neurons in spinal itch transmission but do not support a role in pain.SIGNIFICANCE STATEMENT Dorsal horn gastrin-releasing peptide neurons serve a well-established function in the spinal transmission of pruritic (itch) signals. A potential role in the transmission of nociceptive (pain) signals has remained controversial. Our results provide further support for a critical role of dorsal horn gastrin-releasing peptide neurons in itch circuits, but we failed to find evidence supporting a role in pain.

Keywords: chemogenetics; interneuron; neuronal tracing; nociception; optogenetics; pruritus.

Figures

Figure 1.
Figure 1.
Characterization of GRP-cre-expressing neurons. A, Immunofluorescence staining on a transversal section of lumbar spinal cord of GRP::cre mice showed GRP-tdTom neurons to be located in laminae I and II of the spinal dorsal horn, dorsally to the PKCγ neurons. B, GRP-tdTom neurons partially colocalize with the termination area of IB4+ primary afferents. C, Double ISH shows that GRP-tdTom expression is restricted to Grp-expressing neurons. Arrowheads indicate a double-labeled neuron. D, Quantification of C. E, Double ISH shows that Grp- and Grpr-expressing neurons are two nonoverlapping neuronal populations. Full arrowheads indicate Grp-expressing neurons. Empty arrowheads indicate Grpr-expressing neurons. F, Immunostaining showing that GRP-tdTom neurons express the excitatory neuronal markers Lmx1b but not the inhibitory marker Pax2. G, Quantitative colocalization studied on spinal sections of GRP::cre; ROSA26lsl-tdTom mice stained with antibodies to Lmx1b, Pax2, Tlx3, PKCγ, and calbindin (CB). H, Section of a lumbar DRG obtained from a GRP::cre;ROSA26lsl-tdTom mouse showing no tdTom expression in primary sensory neurons. Scale bars 100 μm in overviews and 20 μm in higher magnifications (A, B, C, E, F), and 100 μm (H).
Figure 2.
Figure 2.
Morphology and MrgprA3+ primary sensory input of GRP-cre-expressing neurons. A, Examples of two GRP central neurons form a spinal cord of GRP::cre ROSA26lsl-TVA mice infected with SAD.RabiesΔG.eGFP (EnvA) virus. The morphology of the same cells is shown in the dorsoventral (left) and mediolateral view (right). Scale bars, 20 μm. B, Quantification of the morphological analysis of GRP neurons. C, Immunofluorescence staining of a lumbar spinal cord section of GRP::eGFP mice showing GRP-eGFP colocalization with the excitatory marker Lmx1b in lamina II. Arrowheads indicate examples of colocalization of GRP-eGFP and Lmx1b immunoreactivity. Scale bar, 100 μm. D, Quantitative analysis verified that GRP-eGFP neurons and GRP-cre neurons exhibit similar neurochemical characteristics (compare Fig. 1G). In bar charts, data are mean ± SEM. E, Overview of the spinal dorsal horn of an MrgprA3-cre; GRP::eGFP; ROSAlsl-tdTom mouse. The termination area of MrgprA3-positive primary sensory neurons largely overlaps with the location where the GRP-eGFP cells are located. Scale bar, 100 μm. F, High-magnification images of a GRP-eGFP cell receiving direct synaptic input from MrgprA3-positive fibers. Arrowheads indicate MrgprA3-positive excitatory synaptic contacts onto a GRP-eGFP cell. Scale bar, 20 μm. G, Schematic showing optogenetic activation of MrgprA3-expressing primary afferent terminals (MrgprA3-ChR2) and targeted recordings from eGFP-positive GRP neurons (GRP-eGFP). H, Superposition of 20 consecutive light-evoked EPSCs traces recorded from GRP-eGFP neurons. Blue area represents 473 nm, 4 ms, 0.1 Hz. Gray represents individual responses. Black represents average response. I, J, Dot plots illustrate latency (I) and jitter (J) of light-elicited EPSCs recorded from GRP-eGFP cells (n = 6, from 5 animals). Circles represent individual cells. Error bars indicate SEM.
Figure 3.
Figure 3.
DTX-mediated ablation of GRP-cre neurons. A, Strategy for targeted DTX-mediated ablation of GRP neurons. B, ISH following intrathecal injection of DTX on spinal tissue of cre-negative control and GRP::cre;ROSA26lsl-iDTR mice. Scale bar, 50 μm. C, Quantification of B, showing that Grp-expressing neurons are reduced by 29% following DTX-mediated ablation. D, Coronal brain sections from GRP::cre; ROSA26lsl-tdTom mouse showing abundant GRP-tdTom expression in several brain areas. Arrowheads indicate facial nucleus and lateral vestibular nucleus (DI), ventral posterior nucleus of the thalamus (DII), and insular cortex (DIII). Scale bar, 1 mm. E, Quantification of GRP-tdTom neurons at supraspinal sites following intrathecal injection of DTX in GRP::cre;ROSA26lsl-tdTom (tdTom+/iDTR) control mice and GRP::cre;ROSA26lsl-tdTom; ROSA26lsl-iDTR (tdTom+/iDTR+) mice. Data are normalized by setting the number of cells counted in GRP::cre; ROSA26lsl-tdTom control mice as 100%. 7D, Facial nucleus; Lve, lateral vestibular nucleus; VP, ventral posterior nucleus of the thalamus; IC, insular cortex. F, Confocal image of spinal dorsal horn following intrathecal injection of DTX in GRP::cre;ROSA26lsl-tdTom and GRP::cre;ROSA26lsl-tdTom;ROSA26lsl-iDTR mice. Scale bars, 50 μm. G, Same as in E, but for different spinal cord segments. S, Sacral; L, lumbar, T, thoracic; C, cervical spinal cord. In all bar charts, data are mean ± SEM.
Figure 4.
Figure 4.
Behavioral effects of DTX-mediated ablation of GRP-cre neurons. A–D, Reduced itch responses to chloroquine, histamine, and serotonin but not SLIGRL following DTX-mediated ablation of GRP neurons. E–I, Unaltered thermal and mechanical nociceptive responses in GRP neurons ablated mice. Circles represent measurements from individual mice. Error bars indicate mean ± SEM.
Figure 5.
Figure 5.
Chemogenetic activation of GRP-cre neurons. A, Strategy for DREADD-mediated activation of GRP-cre neurons and timeline of the experiment design. B, Spinal section showing cre-dependent hM3D-mCherry expression in GRP-cre neurons. Scale bar, 100 μm. C, Time course of itch behavior before and after DREADD-mediated activation of GRP-cre neurons. CNO induces itch response in GRP::cre mice intraspinally injected with AAV1.hSyn.flex.hM3Dq-mCherry but not in control cre-negative mice. Pruritogens histamine and chloroquine were injected intradermally 120 min after CNO treatment. Chemogenetic activation of GRP neurons increases pruritogen-induced biting behavior. Biting behavior was quantified in intervals of 5 min. Timeline on the x axis indicates the time from CNO intraperitoneal injection. D, Chemogenetic activation of spinal GRP-cre neurons induced itch response. Biting behavior was quantified for 30 min starting 75 min after CNO injection. E, F, Increased histamine- and chloroquine-induced itch behavior following chemogenetic activation of spinal GRP-cre neurons. Biting behavior was quantified from 120 min after CNO treatment, for 30 min. G–K, Unaltered nociceptive responses following chemogenetic activation of spinal GRP-cre neurons. L, M, Dose-dependent induction of spontaneous pruritoceptive (biting) responses by CNO, but no sensitization to heat in the Hargreaves test. Measurements were taken 75 min after CNO injection. Circles represent measurements from individual mice. Error bars indicate mean ± SEM.
Figure 6.
Figure 6.
HSV-based anterograde tracing initiated from spinal GRP-cre neurons. Immunostaining on coronal brain sections of GRP::cre mice intraspinally injected with HSV showing brain areas receiving polysynaptic connections from GRP neurons, including the following: (A) somatosensory cortex hindlimb region (S1HL), (B) ventral posterolateral nucleus of the thalamus (VPL), (C) dorsomedial hypothalamic nucleus (DMH), (D) central nucleus of the amygdala (CeA), (E) red nucleus magnocellular part (RMC), (F) ventrolateral periaqueductal gray (VLPAG), (G) medial parabrachial nucleus (MPB), and (H) rostral ventromedial medulla (RVM). Scale bar, 200 μm.

Similar articles

See all similar articles

Cited by 7 articles

See all "Cited by" articles

Publication types

MeSH terms

Substances

Feedback