How Gastrin-Releasing Peptide Opens the Spinal Gate for Itch

Neuron, 103 (1)

section_content: The senses of pain and itch have evolved to protect organisms from potentially harmful agents and stimuli (Yosipovitch et al., 2007). While the exposure to acute painful stimuli typically evokes an almost immediate withdrawal reflex and a fast onset pain sensation, pruritogens elicit a more prolonged ''waxing and waning'' sensation (Forster and Handwerker, 2014) and a less precisely timed scratching response aimed at the removal of the irritant. 

Reponses to both types of stimuli are initiated by the activation of different types of specialized sensory nerve fibers, called nociceptors and pruritoceptors, which convey sensory information to the spinal or medullary dorsal horn. Despite the presence of neuropeptides such as substance P and calcitonin-generelated peptide (CGRP) in peripheral and spinal pain pathways, plenty of evidence indicates that the responses to acute painful stimulation depend primarily on fast glutamatergic excitation of spinal cord neurons (Ault and Hildebrand, 1993; Lagerstro ¨m et al., 2011; Liu et al., 2010; Olivar and Laird, 1999). By contrast, spinal transmission of itch signals is critically dependent on neuropeptide signaling. Several neuropeptides are expressed by peripheral pruritoceptors, including B-type natriuretic peptide (Huang et al., 2018; Mishra and Hoon, 2013) and neuromedin B (Goswami et al., 2014; Wan et al., 2017). These are likely released in the spinal cord, but it is at present unclear whether they are required for efficient itch relay to central (spinal cord) neurons. However, it is well established that the downstream relay of pruritoceptive signals from second-order to third-order dorsal horn interneurons is highly dependent on neuropeptide signaling by gastrin-releasing peptide (GRP), a 27-amino acid neuropeptide of the bombesin family (Majumdar and Weber, 2011). Mice lacking the GRP receptor (GRPR) exhibit strongly reduced responses to histamine-dependent and histamine-independent pruritogens (Sun and Chen, 2007), and local spinal ablation of the neurons that express the GRPR (GRPR neurons) almost completely protects mice from pruritus evoked by a broad variety of pruritogens (Sun et al., 2009). While a critical contribution of GRP-expressing neurons to spinal itch relay is undoubted (Albisetti et al., 2019; Sun et al., 2017), significant controversy exists about the identity and localization of the neurons that release GRP onto GRPR neurons (Gutierrez-Mecinas et al., 2014; Liu et al., 2014; Mishra and Hoon, 2013; Solorzano et al., 2015; Sun and Chen, 2007). The currently prevailing concept of spinal pruritoceptive signal transmission suggests that secondorder dorsal horn interneurons that are activated by peripheral pruritoceptive neurons release GRP and in turn excite third-order (GRPR) interneurons that finally transmit pruritoceptive signals to spinoparabrachial (fourth-order) projection neurons (Goswami et al., 2014; Huang et al., 2018; Mu et al., 2017). 

The dorsal horn neurons that express GRP comprise a population of excitatory interneurons that are located together with GRPR neurons in lamina II of the spinal dorsal horn (Albisetti et al., 2019; Gutierrez-Mecinas et al., 2016). These GRP neurons express, in addition to GRP, the vesicular glutamate transporter vGluT2 that confers a glutamatergic phenotype to these neurons (Gutierrez-Mecinas et al., 2014; Sun et al., 2017). The presence of vGluT2 hence raises the question why deletion of the GRPR gene or blockade of GRPR signaling has such a strong effect on itch behavior. In the present study, we have used optogenetics in slices and in freely behaving mice to demonstrate that GRP and GRPR neurons are indeed coupled via fast glutamatergic synapses. Yet, for efficient suprathreshold activation of GRPR neurons sufficient GRP release was found to be indispensable. Such GRP release was only achieved during repetitive burst-like activation of GRP neurons. These findings explain why itch strongly depends on neuropeptide signaling. They may also offer an explanation why itch and pain occur with strikingly different time courses.
section_name: INTRODUCTION

section_name: RESULTS

section_content: To investigate synaptic communication between GRP and GRPR neurons, we used Grp::eGFP, Grp::cre, and Grpr::eGFP bacterial artificial chromosome (BAC) transgenic mice (all from Gensat, http://www. gensat. org/index. html). Eutrophic expression of eGFP in Grp::eGFP and of cre in Grp::cre mice has been reported previously (Gutierrez-Mecinas et al., 2014; Solorzano et al., 2015; Sun et al., 2017). Since Grpr::eGFP mice have not been systematically analyzed before, we used in situ hybridization on spinal cord sections to demonstrate that eGFP mRNA was restricted to Grpr mRNA-positive neurons (Figure S1 ). We then investigated biophysical and neurochemical characteristics of GRP and GRPR neurons in Grp::eGFP and Grpr::eGFP BAC transgenic mice (Figure 1 ). eGFP-expressing GRP and GRPR neurons were both concentrated in lamina II of the spinal dorsal horn. In accordance with a previous report (Gutierrez-Mecinas et al., 2014), staining with antibodies against Lmx1b and Pax2, respective markers of excitatory and inhibitory neurons, revealed that 83% ± 4% of Grp-eGFP neurons co-expressed Lmx1b, whereas no overlap with Pax2 was detected (Figure 1A ). In situ hybridization experiments with probes directed against vGluT2 and vGAT, marker genes of excitatory and inhibitory neurons, respectively, provided further support of a virtually exclusive excitatory phenotype of GRP neurons (Figure 1B ). Consistent with other recently published data (Dickie et al., 2019), most of the Grp-eGFP neurons exhibited initial burst (Ib) firing, sometimes also referred to as transient firing (Yasaka et al., 2010), upon depolarizing current injection (Figure 1C ). In dorsal horn neurons, this firing pattern is about equally abundant in excitatory and inhibitory neurons (Yasaka et al., 2010). Only few neurons (6%) responded with tonic firing that is much more frequent in the inhibitory dorsal neurons (Punnakkal et al., 2014). Unlike GRP-neurons, GRPR neurons were rather heterogeneous. Only 62% ± 4% of them co-expressed Lmx1b, while 27% ± 3% expressed the inhibitory marker Pax2 (Figure 1D ). In situ hybridization analyses of vGluT2 and vGAT revealed that 81% of Grpr-eGFP neurons were excitatory and 19% inhibitory neurons (Figure 1E ). This heterogeneity was reflected by the firing patterns (Figure 1F ). The majority of GRPR neurons (58%) responded with delayed action potential firing, but 21% showed tonic firing. Initial burst firing, phasic firing, and gap firing were also observed and each occurred in 6%-8% of the recorded neurons. These results suggest that GRPR neurons can be divided into a more abundant delayed firing and a less abundant tonic firing subtype. To correlate these firing patterns with either an excitatory or an inhibitory phenotype, we filled delayed and tonic firing Grpr-eGFP neurons during whole-cell recording with biocytin (1. 5 mg/mL) and stained them post hoc for expression of Tlx3 (another marker of excitatory dorsal horn neurons) and Pax2 (Figure S2 ). All ten delayed firing neurons were Tlx3-positive, and 5 out of 6 tonic firing neurons expressed Pax2 (c 2 test; p = 0. 0004). In the subsequent text, we refer to the delayed and tonic firing GRPR neurons as GRPR excit and GRPR inhib neurons. 

GRP and GRPR neurons differed not only in their neurotransmitter phenotypes but also in their biophysical properties (Table 1 ). GRP neurons had more depolarized resting membrane potentials (RMPs) (À65. 9 ± 1. 2 mV versus À72. 7 ± 0. 6 mV), more depolarized action potential thresholds (À38. 4 ± 0. 5 mV versus À42. 3 ± 0. 42 mV), higher input resistances (1682 ± 104 MU versus 1050 ± 39. 7 MU), and smaller rheobases (12. 6 ± 1. 2 pA versus 22. 9 ± 1. 6 pA) than GRPR neurons (for statistical comparisons see Table 1 ). These differences indicate a higher excitability of GRP neurons compared to GRPR neurons. In general, the differences to GRP neurons were more pronounced in the delayed firing GRPR excit neurons than in the tonic firing GRPR inhib neurons. Most strikingly, we observed a 3. 5-fold larger rheobase in delayed firing GRPR excit versus tonic firing GRPR inhib neurons, suggesting that these neurons should be much less excitable than the tonic firing GRPR inhib neurons. In addition, GRP neurons also had broader action potentials compared to GRPR delayed and tonic firing neurons.
section_name: Neurochemical and Biophysical Analysis of GRP and GRPR Neurons

section_content: We next studied synaptic communication between GRP and GRPR neurons. To this end, we made use of Grp::cre;Ai32; Grpr::eGFP triple transgenic mice (short Grp-ChR2;Grpr::eGFP mice). These mice express a channelrhodopsin2-eYFP fusion protein (ChR2-eYFP) in the cell membrane of GRP neurons and eGFP in the cytoplasm of GRPR neurons, allowing targeted recordings from GRPR neurons combined with optogenetic excitation of GRP neurons (Figures 2A and 2C ). We first verified the presence of a blue light-evoked photocurrent in Grp-ChR2 neurons. After switching to current-clamp mode, 1 s blue light exposure induced an initial burst-firing pattern similar to the one we had observed previously in response to depolarizing current injections. Shorter (4 ms) light exposure induced a single action potential (Figure 2B ). We then analyzed synaptic transmission between Grp-ChR2 and GRPR neurons (Figure 2C ). We recorded evoked excitatory postsynaptic currents (EPSCs) in GRPR neurons upon wide-field illumination of the slice with short (4 ms) blue light pulses. On average, light-evoked EPSCs had amplitudes of À62. 7 ± 6. 8 pA (n = 23). Superfusion of the slice with tetrodotoxin (TTX, 1 mM, n = 5) or NBQX (20 mM, n = 7) caused a nearly complete block of EPSCs indicating that Neuron 103, 1-16, July 3, 2019 3 the recorded EPSCs depended on presynaptic action potentials and on postsynaptic activation of ionotropic glutamate receptors. We next compared latencies and jitter of light-evoked action potentials in GRP with those of light-evoked EPSCs in GRPR neurons (Figures 2D and 2E ). Action potentials occurred with latencies of 9. 5 ± 0. 4 ms (n = 9) and with a low jitter (variability in latency: 0. 51 ± 0. 07 ms; n = 9). Light-evoked EPSCs in the postsynaptic GRPR neurons occurred with only slightly longer latencies (10. 2 ± 0. 5 ms; n = 23) than the action potentials in GRP neurons and also with a low jitter (1. 0 ± 0. 2 ms). Together with the very low failure rate, these findings suggest monosynaptic connections between the two neuron types. The monosynaptic nature of these connections was also supported by confocal microscopy performed in spinal cord sections of Grp::cre;Ai14;Grpr::eGFP (short Grp-tdTom;Grpr::eGFP) mice. These analyses revealed vGluT2-positive GRP-tdTom terminals in close opposition of Grpr-eGFP dendrites (Figure 2F ). We next investigated the postsynaptic responses to single light pulse stimulation of GRP neurons in GRPR excit and GRPR inhib neurons (Figures 2G and 2H ). Unexpectedly, none of the 15 delayed firing GRPR excit neurons that responded with an excitatory postsynaptic potential (EPSP) became sufficiently depolarized to fire action potentials. The EPSPs depolarized the recorded cells on average by only 9. 0 ± 1. 46 mV (n = 15). By contrast, in 7 out of the 11 tonic firing GRPR inhib neurons, single brief light pulse stimulation of Grp-ChR2 neurons triggered action potentials with probabilities between 10% and 100% (Figure 2I ). Five GRPR neurons exhibited firing patterns different from delayed or tonic firing. These neurons also failed to generate action potentials upon optogenetic GRP neuron stimulation.
section_name: Synaptic Communication between GRP and GRPR Neurons

section_content: The above results prompted us to question whether the single brief light stimulation of the GRP neurons faithfully recapitulated the activity of GRP neurons evoked by input from peripheral pruritoceptors. To address this question, we used transgenic mice that express the cre recombinase specifically in peripheral (legend continued on next page)

Neuron 103, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 July 3, 2019 5 pruritoceptors under the transcriptional control of the masrelated G-protein-coupled receptor A3 (MrgprA3::cre mice; Han et al., 2013). We prepared spinal cord slices from MrgprA3:: cre;Ai32;Grp::eGFP (short, MrgprA3-ChR2;Grp::eGFP) mice and recorded postsynaptic responses from GRP neurons (Figure 3A ). EPSCs were evoked by brief (4 ms) optogenetic stimulation of MrgprA3-positive pruritoceptors. Six out of 28 GRP neurons responded with EPSCs with average amplitudes of À126 ± 57 pA. 

When we switched to current-clamp mode, a single stimulation of MrgprA3 fibers triggered bursts of 2-5 action potentials in 4 out of the 5 GRP neurons. This burst-like firing corresponded well with the initial burst firing elicited by depolarizing current injection (cf. Figure 1 ). We decided to mimic this firing pattern in our subsequent experiments on GRP to GRPR neuron synaptic transmission through temporally patterned optogenetic stimulation. As most forms of itch involve a prolonged presence of the pruritic stimulus and sustained activation of pruritoceptors, we first tested whether GRP neurons would be able to sustain repeated burst-like activity over prolonged periods of time (Figure 3B ). We found that GRP neurons were able to follow lightevoked burst-like stimulation with inter-burst intervals down to 2 s (0. 5 Hz). Since pruritoceptors typically fire at similarly low rates (Ma et al., 2012; Schmelz et al., 1997), we considered that this pattern of GRP neuron stimulation would likely mimic the in vivo situation during ongoing pruritic stimulation. We then tested whether this prolonged burst-like stimulation of GRP neurons would be sufficient to render GRPR excit neurons susceptible to suprathreshold activation (Figure 3C ). All 14 GRPR neurons responded with a progressive slow depolarization that built up on average to 7. 2 ± 0. 8 mV over several minutes (n = 14; p < 0. 0001, two-tailed paired t test). Four of the 14 GRPR excit neurons showed action potentials already during the first series of burst stimulations. An additional 3 GRPR excit neurons started to fire action potentials during continued Grp-ChR2 neurons stimulation. Six out of 11 neurons fired action potentials correlated with the blue light stimulation (for a time course of light triggered and spontaneous action potentials see Figure S3 ). Seven of the 14 GRPR neurons recorded also became spontaneously active, i. e., they fired action potentials uncorrelated with the optogenetic stimulation. Three out of the 14 neurons did not receive fast glutamatergic synaptic input from Grp-ChR2 neurons but still responded with a progressive depolarization and one of them also became spontaneously active. Depolarization and spontaneous activity persisted for minutes beyond the termination of synaptic stimulation. When GRP neurons were repetitively stimulated with single light stimuli (instead of bursts) at 0. 5 Hz, depolarization of GRPR neurons amounted only to 1. 6 ± 0. 9 mV (n = 6; p = 0. 13, two-tailed paired t test) and EPSPs remained subthreshold (Figure 3D ). We also tried higher stimulation frequencies (2. 5 Hz), i. e., applied the same number of light pulses as with the burst-like stimulation but separated at regular intervals. Grp-ChR2 neurons were not able to follow this stimulation for more than a few seconds (Figure 3E ). Accordingly, GRPR excit failed to exhibit a significant depolarization (0. 96 ± 0. 49 mV, n = 6; p = 0. 11, two-tailed paired t test) and did not fire action potentials.
section_name: Repetitive Burst-like GRP Neuron Stimulation Renders Excitatory GRPR Neurons Spontaneously Active and Susceptible to Suprathreshold Excitation

section_content: The observed progressive increase in excitability might be a consequence of GRP release from the GRP neurons. However, glutamate receptor-dependent plasticity constitutes an alternative mechanism. We therefore repeated the above experiment in the presence of the AMPA and NMDA receptor antagonists NBQX and AP-5. Mean progressive depolarization remained virtually unchanged (5. 6 ± 1. 2 mV; n = 10; p = 0. 001, two-tailed paired t test), and some of the neurons started firing action potentials even in the absence of fast (phasic) glutamatergic input (Figures 4A and 4B ). When we blocked GRP signaling with the peptide GRPR antagonist D-Phe 6,Leu-NHEt 13,des-Met 14 )-bombesin (6-14) (DPDMB, 1 mM), the progressive depolarization was nearly abolished (1. 4 mV ± 0. 5 mV; n = 12; p = 0. 021, two-tailed paired t test), while light-evoked EPSPs remained unaltered (Figure 4C ). In the combined presence of AMPA, NMDA, and GRPR antagonists, both progressive depolarization (0. 4 mV ± 0. 4 mV; n = 5; p = 0. 39, twotailed paired t test) and EPSPs were no longer detected (Figure 4D ). Accordingly, exogenous GRP application mimicked the effects of sustained burst-like stimulation of Grp-ChR2 neurons (Figure 5A ). Superfusion of the slices with 300 nM GRP induced a progressive depolarization of GRPR excit neurons by 10. 0 ± 1. 5 mV (n = 15; p < 0. 0001, two-tailed paired t test) and led to the generation of action potentials in 12 out of the 15 recorded neurons (Figure 5B ). GRP-mediated depolarization was completely prevented by preincubation with the GRPR antagonist DPDMB (1 mM). Other apparent effects of GRP on GRPR excit neurons included a change in the firing pattern from delayed to tonic firing and an increase in the input resistance from 1. 04 ± 0. 08 GU to 1. 46 ± 0. 12 GU (n = 12; p = 0. 0008, two-tailed paired t test). When we repolarized the recorded neurons to their RMP measured before GRP application, the firing pattern changed back to delayed firing in five out of five neurons, indicating that the effect of GRP was primarily due to its depolarizing action (Figure 5C ), consistent with a previous study on unidentified dorsal horn neurons that showed that depolarization alone was sufficient to change firing patterns from delayed to tonic (Ruscheweyh and Sandk€ uhler, 2002). GRP not only induced spontaneous action potential firing but also rendered 7 out of 8 recorded GRPR excit neurons susceptible to suprathreshold excitation by single GRP-ChR2 neuron action potentials (Figure 5D ). Tonic firing GRPR inhib neurons did not significantly depolarize Neuron 103, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 July 3, 2019 7 upon either superfusion with GRP or repetitive burst-like stimulation of Grp-ChR2 neurons (Figures S4A and S4B ).
section_name: Progressive Depolarization Depends on GRPR but Not Glutamate Receptor Signaling

section_content: We next addressed the GRP signaling mechanisms that increase GRPR neuron excitability. GRP-mediated depolarization of GRPR neurons was accompanied by a 40% increase in membrane input resistance (R i ) indicating that it was due to the closure of an outward conductance, presumably carried by potassium channels. GRPR typically signals via G proteins of the Ga q family (Offermanns et al., 1994; Zachary et al., 1986). These G proteins inhibit different tonically active potassium channels, including members of the potassium channel families Kv7 (KCNQ2/3, also known as M-type currents) (Brown and Passmore, 2009), Kir2 (Hermes et al., 2013), and TASK-1/3 (KCNK-3 and KCNK-9) (Wilke et al., 2014). We used XE-991 (10 mM) (Tirko et al., 2018), ML365 (10 mM) (Zou et al., 2010), and low concentration Ba 2+ (200 mM) (Li et al., 2013) to respectively block Kv7, TASK, and Kir2 channels and to test whether they would depolarize GRPR excit neurons and occlude further depolarization by GRP (300 nM) (Figure 6A ). Of these three blockers, only Ba 2+ induced a significant depolarization (by 12. 7 ± 1. 4 mV, n = 6, p < 0. 0001; repeated-measures one-way ANOVA) and prevented further depolarization by GRP (by 2. 9 ± 1. 4mV, n = 6, p = 0. 31; repeated-measures one-way ANOVA). Like GRP, Ba 2+ caused a significant increase in R i. I n addition, it prevented further increases in R i by GRP, suggesting that Ba 2+ -sensitive Kir2-like channels mediate GRP-induced depolarization. 

We then tested whether a 7 mV depolarizing shift of the RMP (equivalent to the average depolarization induced by repetitive burst-like synaptic stimulation of GRPR neurons, see also Figure 3C ) would replicate the changes in GRPR excit neuron excitability observed with repetitive burst-like synaptic stimulation. This depolarization changed the firing pattern of GRPR excit neurons from delayed to tonic-like firing (Figure 6B ). Effects on activation of these neurons by excitatory synaptic input were tested with somatic current injections (50-400 pA amplitudes) that followed the time course of EPSCs measured in GRPR neurons (rise time of 0. 49 ms, decay time of 4. 28 ms). Depolarization of the RMP by 7 mV shifted the stimulus response curve to the left by 84 pA (Figure 6C ). Previous work has attributed a delayed firing to the presence of A-type potassium currents and showed that inhibition of these currents in dorsal horn neurons induces a switch from delayed to tonic firing (Ruscheweyh and Sandk€ uhler, 2002). Because A-type currents undergo pronounced voltage-dependent inactivation, we tested whether the depolarization observed in GRPR excit neurons with GRP application or with burst-like synaptic stimulation would reduce A-type currents. The 7 mV-depolarization of the RMP reduced the amplitude of A-type potassium currents by 61. 9% ± 5. 3% (n = 11, p < 0. 0001, two-sided paired t test) (Figure 6D ). Because A-type potassium channels are not only inactivated by prolonged depolarization but also inhibited by phosphorylation via extracellular signal-regulated kinase (Erk) (Hu et al., 2003), which can be initiated by Ga q/11 -dependent signaling (Wagner et al., 2010), we tested whether the A-type potassium currents in GRPR neurons are also directly modulated by GRP. Under voltage-clamp conditions, A-type potassium currents were not changed by GRP (300 nM) (Figure 6E ). Figure 6F summarizes this signaling cascade.
section_name: Block of Kir2-like Potassium Channels and a Subsequent Reduction in A-Type Potassium Currents Mediate GRP-Induced Depolarization of GRPR Neurons

section_content: The above results obtained in spinal cord slices provide strong support for a critical contribution of GRP signaling to effective communication between GRP and GRPR neurons. They indicate that a continuous burst-like discharge activity in GRP neurons is needed to evoke sufficient GRP release, which allows action potential generation in GRPR excit neurons and subsequently the spinal relay of pruritoceptive information. It is tempting to speculate that this particular dependence on GRP may underlie the rather slow in vivo onset and offset of itch. In order to provide further support for this idea, we performed optogenetic experiments in vivo. To this end, we chronically implanted GRP-ChR2 mice with fiber optics directed toward the right dorsal horn surface of the lumbar spinal cord segments L4/L5 (Bonin et al., 2016; Christensen et al., 2016). We then stimulated the GRP-ChR2 neurons with brief (4 ms) pulses of blue light and monitored aversive behavior (Figure 7A ). Similar to what we had done in spinal cord slices, we compared single light stimulation repeated at a frequency of 0. 5 Hz with burst-like light stimulation (bursts of 5 pulses of 4 ms duration each, applied at an intraburst frequency of 25 Hz and repeated every 2 s). Significant behavioral changes were observed in 13 out of 15 trials performed in 5 GRP-ChR2 mice during burst-like light stimulation of GRP-ChR2 neurons (Figures 7B and 7C ). By contrast, no behavioral changes occurred upon low-frequency stimulation with single light pulses (delivered every 2 s, n = 6 mice), and no changes were observed in ChR2-negative (GRP-cre -;Ai32) mice after either stimulation paradigm (n = 4 and n = 5, for single pulse and burst-like light stimulation). In line with the time course of action potential firing of GRPR excit neurons observed in slices, the onset of aversive behavior also occurred only with a certain delay of 5-25 s, corresponding to the 3rd to 12th burst (Figure 7D ). Even more striking was that aversive behavior outlasted the cessation of light stimulation by several minutes. This persistence of behavioral responses resembles the time course of GRPR neuron action potential firing observed in slices, which also extended for several minutes beyond the termination of light stimulation. These similarities are remarkable in particular as light-evoked activity of GRP neurons in vivo would add to any spontaneous ongoing activity.
section_name: Itch Behavior Elicited In Vivo by Repetitive Optogenetic Stimulation of Spinal GRP Neurons

section_content: The present study was triggered by the questions why itch, in contrast to pain, depends critically on neuropeptide signaling, specifically on signaling via the neuropeptide GRP, and whether this neuropeptide signaling might contribute to the low temporal resolution of itch sensations. We have focused our efforts on the relay of spinal itch signals from second-order GRP to third-order GRPR neurons. We found that, although both neuron types were coupled via monosynaptic glutamatergic connections, single presynaptic action potentials in GRP neurons were not sufficient to evoke suprathreshold postsynaptic excitation of GRPR excit neurons; i. e., they were unable to drive action potential firing in GRPR excit neurons. Only when GRP neurons were driven to fire in bursts, suprathreshold activation was achieved. Furthermore, in many of the GRPR excit neurons prolonged burst activity was required to elicit action potential firing, which then persisted for minutes beyond the termination of burst stimulation. A similar dependence on presynaptic burst activity and similarly delayed onset and offset of itch responses were observed in vivo when spinal GRP neurons were optogenetically stimulated, indicating that it is this dependence of GRPR excit neuron activation on conditioning depolarization that makes itch critically dependent on GRP signaling. About half of the recorded GRPR excit neurons not only became responsive to excitatory synaptic input but also became spontaneously active upon repeated burst-like GRP neuron input or exposure to exogenous GRP. Appearance of spontaneous activity indicates that GRP did not only prime GRPR excit neurons for suprathreshold activation but was also able to provoke spontaneous activity. The GRPR inhib neurons differ from their excitatory cousins not only in their firing pattern and neurochemistry but also in their immediate excitability by input from GRP neurons. The function of these neurons is currently unknown but they might potentially be elements of a pain inhibitory circuit initiated from pruritoceptive neurons.
section_name: DISCUSSION

section_content: Excitatory GRPR neurons did not fire action potentials after single or regularly spaced repetitive single light-pulse synaptic stimulations; instead, they became activated only after burst-like stimulation of GRP neurons. Repetitive burst firing induces larger and more sustained rises in presynaptic Ca 2+ to enable efficient neuropeptide release (Bruns and Jahn, 1995; Leenders et al., 1999; van den Pol, 2012). Interestingly, it has been shown that release of vasopressin arginine peptide from the neurohypophysis is optimally triggered by short phasic burst stimulation protocols, which lead to gradual built-up of residual Ca 2+ levels in the presynaptic terminal (Muschol and Salzberg, 2000). In fact, although amino acid and neuropeptide transmitters are present in the same axon terminals, they are stored in different classes of vesicles, with neuropeptides being released from so-called dense core vesicles (Johansson et al., 1980; Torrealba and Carrasco, 2004). Their location in the presynaptic terminal is more diffuse and more distant from the voltage-gated Ca 2+ channels in the active release zones, which explains why peptide release requires more sustained and widespread Ca 2+ signals (Bruns and Jahn, 1995). The initial burst-firing pattern that is found in the vast majority of GRP neurons promotes strong rises in intracellular Ca 2+ and thus fosters neuropeptide release. In addition, their relatively broad action potentials further support intracellular Ca 2+ rises (Bean, 2007). Co-release of fast amino acid transmitters with a peptide transmitter is a widespread phenomenon in the mammalian CNS (Nusbaum et al., 2017). A large body of literature also describes slow depolarization or hyperpolarization (depending on the downstream signaling cascades) induced by exogenous application of neuropeptides or by repetitive presynaptic stimulation (Strand, 1999; van den Pol, 2012). However, few, if any, reports have shown such a critical dependence of suprathreshold postsynaptic excitation on co-release of a neuropeptide. 

(B and C) Depolarization by 7 mV of the RMP changed delayed firing into tonic-like firing (B) and increased action potentials probability in response to somatic EPSC-like current injections (C). Left: voltage trace examples evoked by EPSC-like current injections of increasing amplitude (50-400 pA). Right: stimulus response curves (n = 11 from 3 mice) fitted to the Boltzmann equation. (D) A 7 mV depolarization of the RMP reduced A-type potassium current amplitudes in GRPR excit neurons by 61. 9% ± 5. 3% (n = 11, p < 0. 0001, two-tailed, paired t test). (E) GRP (300 nM) had no effects on the A-type potassium current amplitude when the membrane potential was kept constant (7. 0% ± 3. 5%, n = 8 from 3 mice; p = 0. 11; two-tailed, paired t test). (F) Schematic illustration of the intracellular signaling events triggered by GRPR activation in GRPR excit neurons. 

Neuron 103, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 July 3, 2019 11 A typical feature of neuropeptide signaling is volume transmission, which, by its underlying mechanisms, occurs rather slowly, on a timescale of seconds to minutes, and typically reaches well beyond the structural extent of synaptic connections (Fuxe et al., 2007). In agreement with this concept, our experiments showed that repetitive burst-like stimulation induced depolarization not only in GRPR neurons with direct glutamatergic input from GRP neurons but also in neurons that lacked such direct input. This spatially extended signaling may contribute to our rather poor ability to localize itch stimuli.
section_name: Presynaptic Features Supporting GRP Release

section_content: In agreement with earlier studies that analyzed the effects of exogenously applied GRP in the spinal dorsal horn (Aresh et al., 2017; Koga et al., 2011; Kusube et al., 2016), we found that GRP-mediated depolarization of GRPR neurons was accompanied by a 40% increase in R i. Our experiments with several potassium channel blockers revealed that the increase in R i and the subsequent depolarization resulted from the inhibition of tonically active Ba 2+ -sensitive potassium currents, likely mediated by inwardly rectifying potassium channels of the Kir2 family, thereby recapitulating GRP actions in thalamic neurons (Hermes et al., 2013). Members of the Kir2 family, in particular, Kir2. 2 channels, are extensively expressed in the superficial dorsal horn (Pr€ uss et al., 2005) providing further support for their involvement. Activity of Kir2 channels requires the presence of phosphatidylinositol-4,5-bisphosphate (PIP2) in the cell membrane (Lee et al., 2016), which gets depleted upon activation of phospholipase Cb (PLCb). The susceptibility of PLCb to activation by Ga q/11 links this pathway to GRPR activation. Our results are also consistent with an additional protein kinase-dependent regulation of Kir2 channels. Other potassium channels that are inhibited by Ga q/11 -dependent signaling, such as TASK1/3 and KCNQ2/3 channels (Brown and Passmore, 2009; Suh et al., 2004; Wilke et al., 2014),are also expressed in the spinal dorsal horn (Gabriel et al., 2002; Pr€ uss et al., 2005; Talley et al., 2001), but did apparently not contribute. 

Signaling steps subsequent to inhibition of Kir2 channels probably involve depolarization-induced inactivation of the A-type potassium currents. These currents underlie the relatively long delay with which action potentials occur after a depolarizing current injection in delayed firing dorsal horn neurons (Ruscheweyh and Sandk€ uhler, 2002; Yoshimura and Jessell, 1989). Their pharmacological inhibition with 4-aminopyridine (Ruscheweyh and Sandk€ uhler, 2002; Yoshimura and Jessell, 1989) or genetic ablation of the underlying Kv4. 2 channels (Hu et al., 2006) increase the excitability of dorsal horn neurons. A-type potassium channels, in particular, Kv4. 2 channels, are not only inactivated by prolonged depolarization but also by phosphorylation via Ga q/11 -dependent extracellular signal regulated kinase (Erk) (Hu et al., 2003; Wagner et al., 2010). Furthermore, it has been suggested that GRPR-mediated itch responses occur through downstream activation of the phosphoinositid 3-kinase g (PI3Kg)/Akt pathway (Pereira et al., 2015). However, our results do not support a depolarization-independent effect of GRP on A-type currents. 

Features Underlying the Differential Susceptibility of Excitatory and Inhibitory GRPR Neurons to GRP Neuron Input Differences in two biophysical characteristics between delayed firing GRPR excit and tonic firing GRPR inhib neurons may explain their different susceptibility to suprathreshold excitation by glutamatergic input from GRP neurons. First, the rheobase (i. e., the minimum depolarizing current sufficient to trigger an action potential) is 3. 5 times higher in GRPR excit versus GRPR inhib neurons. However, the average GRP neuron-evoked EPSC in GRPR excit neurons exceeded the rheobase by more than 4-fold, questioning whether the difference in the rheobase is the major determinant. A second potentially contributing factor are A-type potassium channels, which underlie the delayed firing pattern in excitatory dorsal horn neurons but are absent from inhibitory dorsal horn neurons (Ruscheweyh and Sandk€ uhler, 2002). These A-type potassium currents become quickly activated upon depolarization and thereby effectively limit the depolarization of neurons by postsynaptic glutamatergic input. The relevance of this process for transmission across the GRP to GRPR neuron synapse is underscored by our observation that GRP neuron-evoked EPSPs depolarized GRPR excit neurons on average by only 9 mV, i. e., to about À64 mV, at the peak of the EPSP. This value is far from the action potential threshold of GRPR excit neurons (À41. 5 mV, cf. Results and Table 1 ). It is hence most likely the A-type potassium currents that limit the susceptibility of the GRPR excit neurons to suprathreshold activation by glutamatergic input from GRP neurons.
section_name: Downstream GRPR Signaling Cascades

section_content: Our results identify a cellular and neurophysiological basis for the critical contribution of spinal GRP signaling to itch behaviors. On the cellular level, GRP inhibits a tonic outward conductance (likely Kir2 potassium current) in GRPR excit neurons. This inhibition depolarizes GRPR excit neurons, partially inactivates their A-type potassium currents and renders them more excitable to synaptic input and even spontaneously active (Figure 6F ). On a circuit level, GRP-releasing and GRP-sensing (GRPR) neurons are placed at a particular strategic site between peripheral pruritoceptive input and spinoparabrachial output neurons (Figure 7E ). While fast glutamatergic signaling is apparently sufficient for signal relay at the first and last synapse of this tri-synaptic pathway (Figure 3A ; Aresh et al., 2017), the synapse between the GRP and GRPR neuron requires GRP released during repetitive burst-like presynaptic activity to open the spinal gate for itch signals. GRP signaling thus adds an additional level of sophistication to other already well-established control mechanisms of spinal itch transmission including fast inhibitory control via GABA and glycine receptors (Foster et al., 2015; Ralvenius et al., 2018; Ross et al., 2010) and via opioid peptide signaling (Huang et al., 2018; Kardon et al., 2014).
section_name: Summary and Implications for the Systems Physiology of Itch

section_content: Detailed methods are provided in the online version of this paper and include the following: 5 Mg-ATP, 0. 5 Na-GTP (pH 7. 35, 290 -300 mosm l -1 ). Membrane potentials were corrected for the liquid junction potential of +15. 2 mV. The biophysical properties of GRP neurons were investigated in spinal cord slices prepared from Grp::eGFP mice. Biophysical properties of GRPR neurons and the effect of exogenous GRP on GRPR neurons were determined with targeted patch-clamp recordings in slices prepared from Grpr::eGFP and Grp-ChR2;Grpr::eGFP mice. Additional patch-clamp recordings were performed in slices prepared from MrgprA3-ChR2;Grp::eGFP and Grp-ChR2;Grpr::eGFP mice, as reported in the figures. Passive and active biophysical properties of GRP and GRPR neurons were examined in current-clamp mode. The membrane potential recorded after switching from voltage-clamp to current-clamp mode, was considered the RMP (RMP). Input resistance (R input ) and membrane capacitance (C m ) were determined through injection of hyperpolarizing current steps (2 s, À5 pA increments, delivered every 10 s). Action potential (AP) firing was evoked by depolarizing current steps of increasing magnitude (2 s, +10 pA increments, delivered every 10 s). Single AP properties (threshold, amplitude, width and afterhyperpolarization) were determined considering the first action potential at rheobase. AP firing patterns evoked by depolarizing current injection were classified according to previously published criteria (Abraira et al., 2017; Punnakkal et al., 2014). Briefly, delayed (D) firing neurons were characterized by a prominent delay between the onset of the depolarizing step and the AP discharge. Tonic (T) neurons were characterized by persistent APs discharge. Phasic (P) neurons featured a burst of action potentials at the rheobase that became persistent injecting current of higher magnitude. Neurons with a prominent gap between series of AP discharges were classified as gap (G) firing and neurons with a burst of action potentials at the beginning as initial bursting (Ib) neurons. 

To activate ChR2 in acute slices, wide field illumination through a 40x water immersion objective (W Plan-Apochromat, Zeiss) was applied using a Polychrome V monochromator controlled using Live Acquisition Software v2. 2. 0 (TILL Photonics, Gr€ afelfing, Germany). GRP neurons or MrgprA3-expressing terminals were stimulated with pulses of blue light (473 ± 5 nm wavelength, 1. 15 mW). After identification of GFP neurons we implemented a recovery period of 10 -15 min before the patch-clamp recording to allow neurons to recover from blue light exposure. Latency, jitter and failure rate of synaptic responses were considered as criteria for monosynaptic connections. The latency was determined between light onset and the AP peak or the onset of the EPSC. We noted that light exposure followed the output trigger signal with a delay of 3. 38 ms (attributable to electronic and mechanical delay). AP and EPSC latencies were corrected for this delay. The jitter was calculated as the standard deviation of the latency values of twenty consecutive EPSCs. Light-evoked EPSCs were recorded at a holding potential of À70 mV. 

Access resistance was continuously monitored with short hyperpolarizing voltage steps. Recordings in which the access resistance changed by more than 20% during the experiment and cells with initial RMPs more depolarized than À55 mV were excluded from the analysis. Data were acquired using an EPC9 amplifier (HEKA Elektronik, Lambrecht, Germany) controlled with Patchmaster, version 2x80 acquisition software and sampled at 20 kHz. Data were analyzed using IGOR Pro 6. 22A.
section_name: STAR+METHODS

section_content: Six to twelve week-old mice of either sex were anaesthetized with pentobarbital (160 mg kg À1, i. p. ) before transcardiac perfusion with 20 mL of ice-cold ACSF followed by 100 mL of 4% ice-cold paraformaldehyde (in 0. 1 M sodium phosphate buffer, pH 7. 4). Spinal cords tissue was post-fixated for 2 h with 4% paraformaldehyde on ice, cryoprotected in 25% sucrose solution (in 0. 1 M sodium phosphate buffer) overnight at 4 C, embedded in NEG50 frozen section medium (Richard-Allen Scientific) and stored at À80 C until use. The spinal cords were cut into 30 mm cryosections using Hyrax C60 cryostat (Carl Zeiss) and mounted onto Superfrost Plus microscope slides (Thermo Fisher Scientific). Spinal cord section were incubated at 4 C overnight in a primary antibody solution (PBS, 0. 3% Triton X-100, 10% normal donkey serum) containing combinations of the following antibodies: rabbit anti-GFP (1:1000), guinea pig anti-Lmx1b (1:10,000), goat anti-Pax2 (1:200), goat anti-tdTomato (1:1000), guinea pig anti-vGluT2 (1:1000). Three washing steps of 5 min each in PBS were performed before incubating spinal cord sections with secondary antibodies (1:800) for 1h at room temperature in PBS supplemented with 0. 3% Triton X-100. For details on the antibodies, see Key Resources Table. Immunostaining of synaptic contacts between GRP and GRPR neurons was performed on 40 mm thick free floating sections (cutting was performed using Hyrax KS 34 microtome, Carl Zeiss) and the sections were pretreated 3 times for 10 min with 50% ethanol (in ddH 2 O), washed two-times for 10 min in PBS and incubated with primary antibodies for 3 days. Images were taken with a LSM 710 or LSM 800 with Airyscan confocal microscopes (Carl Zeiss) controlled with ZEN 2011 (black edition) or ZEN 2. 3 (blue edition) software, respectively, and using, respectively, an EC Plan-Neofluar 40x/1. 30 oil-immersion objective or a Plan-Apochromat 40x/1. 4 Oil DIC M27 oil-immersion objective. Z stack images of 8 optical sections and 1. 5 mm step size were used for the analysis of fluorescence colocalization and to create maximum intensity projections images, whereas Z stack images of 32 optical sections and 0. 2 mm step size were used for the analysis of synaptic contacts. Images were processed using ImageJ software. For quantification, 3 or 5 animals and three sections per animal were analyzed. Cell counting was performed using the ImageJ Cell Counter plug-in. 

To correlate the firing patterns with either an excitatory or an inhibitory phenotype Grpr::eGFP neurons were filled during whole-cell recording with a K + -gluconate based internal solution containing biocytin (1. 5 mg/ml). Slices were transferred to a 4% paraformaldehyde fixative solution and incubated for 1 h at 4 C. Afterward they were cryo-protected overnight in 20% sucrose in PB before embedding and freezing in NEG50 for sectioning. Embedded sections were re-sectioned at 25mm and mounted on Superfrost Plus microscope slides. Antibody incubation with goat anti-Pax2 and rabbit anti-Tlx3 was carried out as described above. Streptavidin-488 conjugate was applied together with the secondary antibodies at a 1:500 dilution. 

In situ hybridization Spinal cords used for in situ hybridization were dissected from 6 -10 week-old mice of either sex in ice-cold ACSF and immediately frozen in 1. 5 mL Eppendorf tubes immersed in liquid nitrogen. Tissue was cut into 20 mm cryosections, mounted onto Superfrost Plus microscope slides (Thermo Fisher Scientific) and hybridized following RNAscope Assay guidelines (Advanced Cell Diagnostics, Newark, CA, USA), using probes designed for RNAscope Fluorescent Multiplex in situ hybridization listed in the Key Resources Table. Fiber optic cannula implantation Six to eight week-old male Grp-ChR2 (Grp::cre;Ai32 double transgenic) mice were implanted with fiber optic cannulas as was described previously (Bonin et al., 2016; Christensen et al., 2016). Control experiments were performed in Grp::cre -;Ai32 mice. Ceramic ferrules measuring ⌀ 1. 25 mm (Thorlabs) were mounted with appropriate multimode optical fiber and trimmed < 1 mm at the edges. Mice were anesthetized with 2 -5% isofluorane and maintained on a motorized stereotactic frame until end of surgical procedure in 1 -2% isofluorane anesthesia. The fur over the back of the mice was shaved and an incision was made on the skin to expose the vertebral column. Incisions were made on the muscles lateral to the tendons spanning either sides of the T13 vertebral disc. The vertebral column was clamped with spinal adaptors and the T13 vertebral disc was exposed. The tissues covering the spinous and transverse processes of the disc were removed using forceps, and a hole was drilled on the caudal-transverse process approximately 2 mm from the midline to expose the L4 -L5 spinal cord segment. A rubber aspirator was used to dry the vertebral disc. Collagen strips (Lyostypt, B. Braun) were used to minimize bleeding. Small amounts of base-coat (One Coat 7 Universal, Coltene) were carefully applied to the cannula's concave end and around the drilled hole on the spinous process as well as the rostro-caudal transverse processes. The coating was cured with UV light to provide a steady base for adherence. The fiber-optic cannula was inserted into the drilled hole. A layer of dental cement (Synergy D6 Flow, Coltene) was applied around the cannula over the base-coat, cured with UV-light for 20 s, and upon hardening, a second layer of dental cement was applied and cured to firmly secure the cannula to the vertebral disc. The muscles around the vertebral column were then sutured using absorbable sutures (Safil 5-0, B. Braun) and the skin was sutured with non-absorbable sutures (Dafilon 6-0, B. Braun). The mice were allowed to recover on a heat pad. Behavior experiments started 48 h after surgery.
section_name: Immunohistochemistry and image analysis

section_content: To measure optogenetically-evoked behavior, mice were placed in cylinders and the fiber-optic cannula was connected via a mating sleeve to a 400 mm, 0. 39 NA multimode fiber-optic patch cable (Thorlabs, Inc) that could rotate to allow free movement of the mouse. After coupling of the cannula to the patch cable under brief isoflurane anesthesia, the animals were habituated for 30 min. Light was delivered to the spinal cord from a 473 nm laser (Laserglow Technologies) connected to the fiber-optic patch cable. Timing of light stimulation was controlled by custom-written scripts in LKTerm software (Loksoft). Aversive behavior (biting or scratching) was recorded for 5 min prior light stimulation. This was followed by periods of 5 min during which the mouse was stimulated with single light pulses (4 ms, 473 nm, 0. 5 Hz) or with bursts of light pulses (bursts of 5 pulses of 4 ms duration each at 25 Hz, repeated at a frequency of 0. 5 Hz) and for a 10 min post-stimulus period. The experimenters were blind to the genotype of the mice. Analysis was done offline in slow motion, at 1/4 normal speed. Aversive behavior elicited by the optogenetic stimulation consisted mainly of fast small amplitude movements of the head directed to the ipsilateral hindlimbs. This behavior is considered a typical itch response (LaMotte et al., 2011). Each animal was tested over three trials run on consecutive days. After the experiments, mice were sacrificed and the correct placement of the fiber-optic cannulas was verified. The fiber-optic cannulae were then removed and coupled to the patch cable for measurement of output light intensities ex-vivo. Light intensities ranged from 0. 7 -2. 0 mW (on average 1. 4 ± 0. 1 mW).
section_name: In vivo optogenetic stimulation and behavior

section_content: NBQX (20 mM, Bio Trend), XE-991 (10 mM) and ML 365 (10 mM) were dissolved in DMSO (end concentration 0. 02%). GRP (final concentration 300 nM, Anaspect), DPDMB (D-Phe 6,Leu-NHEt 13,des-Met 14 )-bombesin (6-14) trifluoroacetate salt (1 mM), TTX citrate (1 mM), AP-5 (50 mM, Tocris) and BaCl 2 were dissolved in water.
section_name: Drugs and Chemicals

section_content: All data are given as mean ± standard error of mean (sem). The number of animals used per experiment is described in the figure legends. Statistical comparisons were made using paired t test to compare measurement from two groups. Where independent multiple groups were compared in a single condition, one-way ANOVA followed by Bonferroni multiple comparisons post-test e4 Neuron 103, 1-16. e1-e5, July 3, 2019

Please cite this article in press as: Pagani et al., How Gastrin-Releasing Peptide Opens the Spinal Gate for Itch, Neuron (2019), https://doi. org/ 10. 1016/j. neuron. 2019. 04. 022 was used. Where related multiple groups were compared in a single condition, repeated-measurements ANOVA followed by post hoc Bonferroni correction was used. Where multiple groups tested with multiple conditions were compared, a two-way ANOVA followed by post hoc Bonferroni correction was applied. Where appropriate, a D'Agostino & Pearson normality test was conducted to assess if the data fit a normal distribution. All statistics were performed using Prism 5 (GraphPad, La Jolla, CA).
section_name: QUANTITATION AND STATISTICAL ANALYSIS

section_name: ACKNOWLEDGMENTS

section_content: The work has partly been supported by grants from the Swiss National Science Foundation ( SNSF, grant number 176398 ) and an Advanced Investigator Grant from the European Research Council ( ERC, grant number AdvG 250128 ) to H. U. Z., and by a grant from the Olga Mayenfisch Stiftung to H. W. M. S. was supported by an AMBIZIONE fellowship of the Swiss National Science Foundation ( PP00P3_176838 ). We thank Rebecca Das Gupta for help with some of the in situ hybridization experiments, Isabelle Kellenberger for genotyping the mice and for the coordination of their breeding, and Harald Osswald for technical support of the in vivo optogenetic experiments. The authors thank Dr. Xinzhong Dong, Johns Hopkins University, for breeding pairs of the MrgprA3::cre mice.
section_name: ACKNOWLEDGMENTS

section_name: DATA AND SOFTWARE AVAILABILITY

section_content: The raw data obtained in the study and the settings file used to control the light source in the in vivo optogenetic experiments are available at: https://doi. org/10. 17632/9p3tb2j2nf. 1. Neuron 103, 1-16. e1-e5, July 3, 2019 e5 Please cite this article in press as: Pagani et al., How Gastrin-Releasing Peptide Opens the Spinal Gate for Itch, Neuron (2019), https://doi. org/ 10. 1016/j. neuron. 2019. 04. 022
section_name: DATA AND SOFTWARE AVAILABILITY

section_name: AUTHOR CONTRIBUTIONS

section_content: Supplemental Information can be found online at https://doi. org/10. 1016/j. neuron. 2019. 04. 022.

section_content: M. P. conducted and analyzed all electrophysiological experiments. G. W. A. and H. W. performed and analyzed the neurochemical analyses. N. S., M. P., and H. C. J. performed the in vivo optogenetic experiments. M. P. analyzed the in vivo optogenetic experiments. M. P., H. C. J., H. W., and H. U. Z. conceptualized the study. M. P. and H. U. Z. wrote the manuscript. All authors commented on the manuscript.
section_name: AUTHOR CONTRIBUTIONS

section_content: The authors declare no competing interests. Targeted whole-cell patch-clamp recordings from Grp-eGFP and Grpr-eGFP neurons were performed at room temperature using epifluorescence for neuron identification followed by infrared gradient contrast for placing of the recording pipette. During recordings, slices were continuously superfused with ASCF at a rate of 1 -2 mL min -1. Patch pipettes (borosilicate glass; 3. 5 -4. 5 MU; Harvard Apparatus) were filled with intracellular solution containing (in mM): 130 K + gluconate, 5 NaCl, 1 EGTA, 10 HEPES,
section_name: DECLARATION OF INTERESTS

section_name: STAR+METHODS KEY RESOURCES