CRF regulates pain sensation by enhancement of corticoaccumbal excitatory synaptic transmission

doi:10.21203/rs.3.rs-3137231/v1

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CRF regulates pain sensation by enhancement of corticoaccumbal excitatory synaptic transmission

https://doi.org/10.21203/rs.3.rs-3137231/v1

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published 07 Mar, 2024

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Both peripheral and central CRF systems have been implicated in regulating pain sensation. However, compared with the peripheral, the mechanisms underlying central CRF system in pain modulation have not yet been elucidated, especially at the neural circuit level. The corticoaccumbal circuit, a structure rich in CRF receptors and CRF-positive neurons, plays an important role in behavioral responses to stressors including nociceptive stimuli. The present study was designed to investigate whether and how CRF signaling in this circuit regulated pain sensation under physiological and pathological pain conditions. Our studies employed the viral tracing and circuit-, and cell-specific electrophysiological methods to label mPFC^CRF-NAcS circuit and record its neuronal propriety. Combining optogenetic and chemogenetic manipulation, neuropharmacological methods, and behavioral tests, we were able to precisely manipulate this circuit and depicted its role in regulation of pain sensation. The current study found that the CRF signaling in the NAcS, but not NAc core, was necessary and sufficient for the regulation of pain sensation under physiological and pathological pain conditions. This process was involved in the CRF-mediated enhancement of excitatory synaptic transmission in the NAcS. Furthermore, we demonstrated that the mPFC^CRF neurons monosynaptically connected with the NAcS neurons. Chronic pain increased the release of CRF into NAcS, and then maintained the persistent NAcS neuronal hyperactivity through enhancement of this monosynaptic excitatory connection, and thus sustained chronic pain behavior. These findings reveal a novel cell- and circuit-based mechanistic link between chronic pain and the mPFC^CRF→NAcS circuit and provide a potential new therapeutic target for chronic pain.

Biological sciences/Neuroscience

Biological sciences/Molecular biology

Corticotropin-releasing factor (CRF), widely expressed in the central nervous system, was initially known as an essential modulator in coordinating physiological responses to stress[1–5]. In recent decades, the role of the central CRF system in the modulation of multiple psychiatric and mental disorders, such as addiction, anxiety, and depression, has received increasing attention [6–8].

Chronic pain is considered to be a persistent and intense stressor [9]. Accumulating evidence suggests that central CRF signaling has been implicated in regulating physiological and pathological pain [10–14]. For instance, in patients with fibromyalgia, pain and depressive symptoms are strongly associated with the CRF concentration in cerebrospinal fluid [12]. Preclinical studies have shown that intracerebroventricular administration of CRF can induce hyperalgesia or analgesia in different animal models [15, 16]. However, the mechanisms underlying the role of central CRF system in pain modulation have yet to be elucidated, especially at the neural circuit level.

The limbic brain, a structure rich in CRF receptors and CRF-positive neurons, is a critical neural network regulating pain and pain-related disorders [17–20]. Embedded within the center of this network is the nucleus accumbens (NAc), a complex neuropeptidergic hub that synthesizes motivation, emotion, learning, cognitive, and sensorimotor information [21–23]. The NAc also plays a vital role in pain regulation and is effective as a source of analgesia [24–27]. Functional magnetic resonance imaging (fMRI) studies have found characteristic imaging manifestations in the NAc in patients with persistent pain conditions [24, 28, 29]. Deep brain stimulation (DBS) in the NAc has provided sustained pain relief in patients undergoing chronic pain [30]. In preclinical studies, the blockade of glutamate or dopamine (DA) receptors in the NAc has been shown to relieve hyperpathia symptoms [31, 32]. As a neuropeptidergic hub, the NAc contains multiple neuropeptides, including CRF, to modulate local synaptic plasticity, neuronal activity and genetic expression [33–36]. However, depicting the roles and its potential mechanism of these neuropeptides in NAc-related pain regulation still need to be complemented by further research efforts.

CRF signaling in the NAc is involved in multiple biological functions, such as motivation and emotion [37, 38]. For example, CRF in the NAc governs the emotional response to acute stressors via the regulation of dopamine release [39], and can mediate anxiety-like behaviors [40]. A previous study indicated that chronic neuropathic pain induced CRF receptor overexpression in the NAc [41]. In addition, exogenous CRF in the NAc generated a variety of aversive behaviors, such as depression and anxiety [42]. These findings implied the connection between CRF signaling in the NAc and pain information processing, while the underlying mechanism remains unknown.

To address these questions, we used a combination of optogenetics, slice electrophysiology, neuropharmacology, and behavioral tests to examine the role of accumbal CRF signaling in pain regulation and the underlying cellular and synaptic mechanisms. We also performed transsynaptic virus tracing, projection-specific optogenetic and slice recording experiments to examine whether the monosynaptic connections between medial prefrontal cortex (mPFC) CRF-containing neurons and NAc neurons are necessary and sufficient for the regulation of pain sensation. The results revealed a novel cell- and circuit-based mechanistic link between regulation of pain sensation and the CRF signaling in corticoaccumbal circuit.

Animals

All experiments were reviewed and approved by the Animal Care and Use Committee of Xuzhou Medical University, and performed in accordance with the National Institutes of Health Guidelines and Use of Laboratory Animals and the Committee for Research and Ethical Issues of the International Association for the Study of Pain. Mice were group-housed (maximum five mice per cage) under a 12-h light/dark cycle (light from 8:00 A.M. to 8:00 P.M.), with food and water available ad libitum. The ambient temperature was maintained at 21–22°C with 55% relative humidity. Only C57BL/6 J male mice (8–13 weeks old) of normal weight (22-24g) were used for all studies. All behavioral tests were conducted during the light period, and the investigators were blinded to experimental conditions during testing.

Data analysis and statistics

Prior to further analysis, each dataset was subjected to a normality test using the Shapiro–Wilk test. For datasets that were normally distributed, parametric tests of the variance and group differences (paired, unpaired t tests, one-way or two-way ANOVA with repeated measures followed by Bonferroni post–tests) were used. Otherwise, the non-parametric Wilcoxon matched-pairs signed rank test, Mann Whitney Rank Sum Test, and the Kruskal Wallis One Way ANOVA on ranks with Uncorrected Dunn’s test were used. Statistical analyses were conducted with GraphPad Prism 7.0 software (GraphPad Software) without specification. All data are shown ± standard error to the mean (SEM). The Detailed descriptions can be found in the figure legends. Statistical significance was set at four levels (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Detailed information about the materials and methods is described in the supplementary material.

CRF signaling in the NAcS regulates pain

Details on the expression pattern of CRF messenger RNA (mRNA) and protein in the bilateral NAc during the development of chronic pain are still lacking [3, 43–45]. Using a neuropathic pain model of chronic constrictive injury (CCI) of the sciatic nerve, we found a significant increase in CRF protein expression in the contralateral, but not ipsilateral, NAc to the injury site at 3, 7, 14, and 21 days following CCI surgery (Fig. 1A, B), while the level of Crf mRNA at these time points in the bilateral NAc did not exhibit any detected changes (Fig. 1C).

The NAc is usually divided into two functionally distinct subregions: the NAc shell (NAcS) and core (NAcC) [46]. Thus, to determine the role and possible difference in CRF signaling in the two subregions in pain regulation, we measured the change in PWTs and PWLs to assess pain sensation in CCI mice after pharmacological inhibition of CRF receptors by injecting a competitive CRF receptor antagonist (ɑ-helical CRF, 200 µM, 0.1 µL) into the NAcS or NAcC (Fig. 1D, S1A). Behavioral results showed that intra-NAcS, but not intra-NAcC, ɑ-helical CRF injection significantly reversed the established thermal and mechanical hyperalgesia in CCI mice (Fig. 1E, S1B). Repeated injection of ɑ-helical CRF into the NAcS, but not into the NAcC, for 5 consecutive days via a pre-implanted cannula produced a prolonged antinociceptive effect, which lasted at least 2 days from the last injection (Fig. 1F, G, S1C, D). In the multiple-injection experiment, behavioral tests were performed eight hours after each injection to exclude the acute anti-nociceptive effect of CRF receptor antagonist (Fig. 1F, S1C). These results suggested that CRF signaling in the NAcS is necessary for regulating the pain sensation underlying the chronic neuropathic pain state.

To investigate whether the activation of CRF signaling in the NAc in naïve mice is sufficient to regulate pain sensation, exogenous CRF (0.1 µL) was injected into the NAcS or NAcC to activate CRF signaling before behavioral tests. The results showed that intra-NAcS CRF injection significantly decreased the PWLs and PWTs in the paw contralateral to the injection site in naïve mice, and this effect was observed after the 50 nM, but not the 200 nm and 1000 nm, CRF injection (Fig. 1H), while this decrement was absent after the intra-NAcC injection (Fig. S1E). A stable and high level of CRF protein expression in the NAc after CCI surgery was observed. Thus, we want to know whether continuously activated CRF signaling in the NAcS through repeated injection of CRF (50n M, once per day for 5 days) is sufficient to induce a persistent pain state. The current results indicated that this repeated activation produced a prolonged painful effect and this effect lasted at least 2 days from the last injection (Fig. 1I, J). Repeated injection of CRF into the NAcC did not affect pain sensation in naïve mice (Fig. S1F, G). These findings demonstrated that the stable and high level of CRF protein in the NAcS after CCI surgery played an important role in pain sensation regulation and the establishment of persistent pain conditions.

CRF increases neuronal excitability in the NAcS by enhancing excitatory synaptic transmission

Next, we asked how CRF signaling in the NAcS regulates pain sensation and participates in the development of chronic neuropathic pain. In the CNS, the functional changes were mainly dependent on alterations in neuronal excitability [47, 48]. Using the whole-cell patch clamp recordings in acutely isolated NAc slices (Fig. 2A, S2A) from sham and CCI mice, we measured neuronal excitability by the number of evoked action potentials by depolarizing current injections (eAPs). Compared to sham mice, CCI mice showed a significant increase in the number of eAps, suggesting an increase in neuronal excitability in the NAcS but not in the NAcC (Fig. 2. B top and C left; Fig. S2. B, C). Interestingly, the resting membrane potential (RMP) and rheobase current in NAcS neurons were unaffected by CCI surgery (Fig. S3A). The changes in both synaptic transmission and neuronal intrinsic properties can affect neuronal excitability [49]. Thus, we then examined intrinsic neuronal excitability, which was defined as its excitability in the absence of synaptic inputs by blocking both inhibitory and excitatory synaptic transmission [by blocking GABA_A receptors (GABA_ARs) with 100 µM picrotoxin, AMPA (ɑ-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors (AMPARs) with 10 µM NBQX, and NMDA (N-methyl-D-aspartate) receptors (NMDARs) with 50 µM D-AP5]. We found that the intrinsic neuronal excitability in the NAcS was not significantly different between the CCI and sham groups (Fig. 2. B bottom and C right; Fig. S3B). These data indicated that the increase in NAcS neuronal excitability in CCI mice is due to alterations of synaptic transmission.

Next, we want to explore the contribution of CRF signaling to the NAcS neuronal excitability. In isolated NAc slices from CCI mice, perfusion with ɑ-helical CRF (200 µM), but not its control Acsf, significantly decreased the number of eAPs (Fig. 2D), suggesting CRF signaling made a contribution to the increased neuronal excitability in CCI mice. Furthermore, in isolated NAc slices from naïve mice, CRF (50 nM) perfusion increased the number of eAPs (Fig. 2E). Neither ɑ-helical CRF nor CRF perfusion affected the RMP or rheobase current in NAcS neurons (Fig. S4A, B). Next, we repeated the above experiments with the blockade of inhibitory and excitatory synaptic transmission. We found that, in the absence of synaptic transmission, neither ɑ-helical CRF perfusion in CCI mice nor CRF perfusion in naïve mice influenced the excitability of NAcS neurons (Fig. 2F, G; Fig S4C, D).

The above findings suggested that the increase of NAcS neuronal excitability induced by CCI injury or exogenous CRF in naïve mice might be due to changes in synaptic transmission. Thus, we next examined changes in inhibitory and excitatory synaptic transmission in NAcS neurons in CCI mice. We found that the frequency and amplitudes of spontaneous inhibitory postsynaptic currents (sIPSCs) did not differ between CCI and sham mice (Fig. 2H, I). However, the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) of the NAcS neurons is significantly increased in CCI mice compared with sham group, while its amplitude is unchanged (Fig. 2J, K). Furthermore, ɑ-helical CRF (200 µM) perfusion decreased the frequency, but not amplitude, of sEPSCs in CCI mice (Fig. 2L, M). Consistently, CRF (50 nM) perfusion in NAc slice from sham group mice increased its frequency, but not amplitude, of sEPSCs (Fig. 2N, O). These results demonstrated that CRF signaling increased NAcS neuronal excitability through enhanced excitatory presynaptic transmission.

CRF-containing mPFC neurons innervate the NAcS for the maintenance of chronic neuropathic pain

In general, the level of mRNA is determined by local transcription, while the protein level is determined by both local synthesis and projection-release. In the present study, the levels of CRF mRNA and protein in the NAc were inconsistently changed under chronic neuropathic pain (Fig. 1C), suggesting that the increase in CRF protein in CCI mice may be due to the release of CRF from presynaptic terminals rather than local synthesis in the NAcS. On the other hand, approximately 95% of neurons in the NAc are GABAergic medium spiny neurons, which cannot induce an increase in sEPSCs in CCI mice [50]. Therefore, we speculated that the increase in CRF protein and sEPSCs in the NAcS is due to afferents from excitatory neurons outside the NAcS. To test this hypothesis, the retrograde virus AAV-CRF-eYFP was injected into the NAcS 21 days before, and then the whole-brain examination of eYFP expression was performed to trace the NAcS-projection CRF positive neurons (Fig. S5A). Consistent with a previous report [51], among multiple pain-related regions, we found that CRF-containing neurons in the prelimbic cortex and infralimbic cortex (two important neuroanatomical subregions of the mPFC [52]) preferentially innervated the NAcS (Fig. S5B, C). Immunofluorescences staining confirmed that over 98% of these eYFP positive neurons in mPFC express CRF protein (Fig. S5D, E). In addition, 70% of NAcS-projecting CRF-containing neurons in the mPFC (called mPFC^CRF→NAcS neurons) were co-labeled with CaMKIIα, a widely used marker of excitatory neurons (Fig. S5F, G), suggesting that these neurons were mainly excitatory pyramidal neurons. These findings led us to hypothesize that the mPFC^CRF→NAcS circuit participates in the maintenance of chronic neuropathic pain.

To examine the potential role of the mPFC^CRF→NAcS circuit in chronic neuropathic pain, we first detected neuronal activities through double immunofluorescence staining for c-Fos and eYFP, and the mPFC^CRF→NAcS neurons were labeled with eYFP in the mPFC 7 days after CCI surgery (Fig. 3A). Compared with sham mice, CCI mice showed a significant increased total numbers and percentages of c-Fos expression in mPFC^CRF→NAcS neurons (Fig. 3B, C). Projection-specific slice recordings also revealed that CCI mice displayed higher excitability in mPFC^CRF→NAcS neurons than sham mice as evidenced by higher numbers of eAPs and lower rheobase current (Fig. 3D-F). Furthermore, we found that both the mRNA and protein levels of CRF in the mPFC exhibited significant increases in CCI mice (Fig. 3G, H). These results suggested that chronic neuropathic pain was accompanied by hyperactivity of the mPFC^CRF→NAcS circuit.

To further evaluate the necessity of the mPFC^CRF→NAcS circuit in mediating chronic neuropathic pain, we injected a retrograde AAV virus expressing Cre recombinase (AAV2/R-CRF-Cre) into the NAcS, and an AAV vector expressing Cre-dependent halorhodopsin (AAV-DIO-NpHR-eYFP) into the mPFC to express NpHR selectively in mPFC^CRF→NAcS neurons (Fig. 3I, J). Immunofluorescence staining confirmed the expression of eYFP in mPFC^CRF→NAcS neurons and their axon terminals in the NAcS, and further electrophysiological recordings validated that the activity of the mPFC^CRF→NAcS neurons was reliably inhibited by optical stimulation (Fig. 3K). The behavioral results showed that acute chemogenetic/optogenetic (589 nm yellow laser, 10 ms pulse, 7 mW intensity, 7 s duration) inhibition of the mPFC^CRF→NAcS circuit reversed the established hyperalgesia in CCI mice (Fig. 3L, S6A-C), whereas this inhibition did not change basal pain thresholds in sham mice (Fig. S6D, S7A, B). Furthermore, we found that repeated chemogenetic/optogenetic inhibition of the mPFC^CRF→NAcS circuit (1 h per day, for 7 consecutive days) produced a prolonged antinociceptive effect, which lasted at least 2 days from the last inhibition (Fig. 3M, N, S6E-G). These data demonstrated that chronic neuropathic pain activated the mPFC^CRF→NAcS circuit, which increased the release of CRF into the NAcS to maintain the chronic pain state through the alteration of neuronal excitability.

The sufficient role of the mPFC^CRF→NAcS circuit in regulating pain

To examine the sufficient role of the mPFC^CRF→NAcS circuit in pain regulation, we injected an AAV2/R-CRF-Cre virus into the NAcS and a Cre-dependent virus of channelrodopsin2 (ChR2)-mCherry into the mPFC to express ChR2 in the mPFC^CRF→NAcS circuit before pain behavioral tests (Fig. 4A, B). Electrophysiological recordings further validated its functional effectiveness, as blue pulses reliably elicited precisely timed action potentials in mCherry-positive neurons in the mPFC (Fig. 4C). We found that transient optogenetic activation of the mPFC^CRF→NAcS circuit (473 nm blue laser, 5 ms pulse, 7 mW intensity, 100 ms interval, 1h duration) did not affect the PWTs or PWLs in naïve mice (Fig. 4D). This effect was also confirmed by chemogenetic activation with a single CNO (1 mg/kg) injection in naïve mice (Fig. S8A, B). A possible explanation for this finding is that brief activation of the mPFC^CRF→NAcS circuit did not induce a sufficient increase in CRF protein levels in the NAcS.

Thus, we next detected whether repeated activation of the mPFC^CRF→NAcS circuit could mimic the pathological process of chronic neuropathic pain. We optogenetically activated the mPFC^CRF→NAcS circuit 1 h per day for 5 consecutive days, and we performed pain behavioral tests and NAc tissues extraction for western blotting 8 hours after the end of stimulation (Fig. 4E). The results showed that repeated activation significantly increased the level of CRF protein in the NAc (Fig. 4F, G) and evoked prolonged hyperalgesia in naïve mice, which lasted 2 days from the last activation (Fig. 4H).

To determine whether this hyperalgesia induced by the repeated activation of the mPFC^CRF→NAcS circuit was mediated by CRF signaling, we injected a retrograde AAV-CRF-Cre virus into the NAcS and a Cre-dependent AAV-hM3Dq-ChR2-mCherry virus into the mPFC for chemogenetic activation of this circuit, and we implanted the cannula into the NAcS 14 days for microinjection of CRF receptor antagonist before behavioral tests (Fig. 4I, J). Viral function was confirmed by cell-attached recording in the mPFC (Fig. 4K). We found that the inhibition of CRF receptors with 200 µM ɑ-helical CRF (0.1 µL) before each activation abolished the hyperalgesia induced by the repeated activation of the mPFC^CRF→NAcS circuit (Fig. 4L). Collectively, these data suggested that the repeated activation of the mPFC^CRF→NAcS circuit resulted in the increased release of CRF in the NAc, which was sufficient to induce chronic neuropathic pain-like hyperalgesia.

Identifying monosynaptic connection in the mPFC→NAcS circuit and its functional alteration in chronic neuropathic pain

To disentangle the precise mechanisms of the mPFC^CRF→NAcS circuit underlying pain regulation, we used an optogenetic approach and electrophysiological recording (Fig. 5A, B). We found that optogenetic activation (473 nm blue laser, 5 ms pulse) of terminals of the mPFC^CRF→NAcS circuit induced large light-evoked excitatory postsynaptic currents (leEPSCs) in 42.9% of NAcS neurons. Pharmacological experiments demonstrated that leEPSCs were completely abolished by the application of the AMPA receptor antagonist NBQX (Fig. 5C), indicating that synaptic responses are mediated by AMPA receptors. To eliminate polysynaptic transmission relying on action potential propagation, tetrodotoxin (TTX, 1 µM) was added to the bath solution at least 5 minutes before the start of recording. These findings suggested that these NAcS neurons which responded to the optical stimulation received the monosynaptic excitatory inputs from mPFC^CRF→NAcS neurons.

Next, to explore the functional changes in these monosynaptic connections in CCI mice, two consecutive optogenetic stimulations (473 nm blue laser, 5 ms pulse, 50 ms interval) were used to obtain the paired-pulse ratio (PPR) in these NAcS neurons. We found a decrease in the PPR (Fig. 5D) and the unchanged in the AMPAR/NMDAR amplitude ratio in CCI mice (Fig. S9A-B), indicating an increased probability of presynaptic release of the mPFC^CRF→NAcS circuit under chronic pain conditions rather than a change in postsynaptic strength. Furthermore, the application of CRF increased the PPR in sham mice, and the application of ɑ-helical CRF decreased the PPR in CCI mice (Fig. 5E), confirming that the increased synaptic efficacy in the mPFC^CRF→NAcS circuit was mediated by CRF signaling.

We next employed cell-type-specific WGA-mediated transsynaptic tracings to selectively label NAcS-receiving neurons innervated by mPFC CRF-containing neurons (called NAcS-receiving neurons), and then we examined their functional changes via electrophysiological recordings. An AAV vector expressing the transsynaptic tracer wheat germ agglutinin (WGA) fused to Cre-recombinase (AAV-CRF-WGA-Cre) and a cre-dependent viral vector expressing mCherry (AAV-DIO-mCherry) were injected into the mPFC and NAcS, respectively (Fig. 5F). Notably, although WGA-mediated transsynaptic transport is bidirectional [53], a previous study and our data show that few neurons within the NAc directly innervate the mPFC (Fig. S10 A-C) [54], which means that this strategy in the mPFC^CRF→NAcS circuit can unidirectionally target the receiving NAcS neurons. The results showed that the number of eAPs was significantly increased, the rheobase current was decreased, and the RMP was not affected in these mCherry-labeled NAcS-receiving neurons in CCI mice (Fig. 5G; Fig. S11A-B). These findings suggested that the increased neuronal excitability in NAcS-receiving neurons was accompanied by a chronic neuropathic pain state. Furthermore, we found that ɑ-helical CRF (200 µM) perfusion inhibited neuronal excitability in NAcS-receiving neurons, as evidenced by fewer eAPs and an increased rheobase current in CCI mice (Fig. 5I), and CRF (50 nM) perfusion enhanced neuronal excitability in naïve mice (Fig. 5J). Neither ɑ-helical CRF nor CRF perfusion affected RMP in NAcS-receiving neurons (Fig. S12A-B).

Consistent with the overall changes in the neurons of the NAcS, chronic pain-induced changes in sIPSCs in NAcS-receiving neurons were also not detected in CCI mice (Fig. S13A-C), while an increase in the frequency, but not amplitude, of sEPSCs in NAcS-receiving neurons was observed in CCI mice (Fig. 5H). Moreover, perfusion with α-helical CRF abolished the above increase in CCI mice, and CRF perfusion increased the sEPSCs frequency in NAcS-receiving neurons in sham mice (Fig. 5K, L).

The role of NAcS-receiving neurons in chronic pain

Although we demonstrated the monosynaptic connections between NAcS neurons and mPFC CRF-containing neurons and their functional changes under chronic neuropathic pain conditions, the role of NAcS-receiving neurons in the regulation of pain behaviors was still unclear. To answer this question, we injected the AAV-CRF-WGA-Cre virus into the mPFC and AAV-DIO-hM4D(Gi)-mCherry into the NAcS, and then we could specifically inhibit the activity of NAcS-receiving neurons via intraperitoneal injection of CNO (Fig. 6A, B). The viral function was confirmed by cell-attached recording in the NAcS. (Fig. 6C). Behavioral results showed that chemogenetic inhibition of these NAcS-receiving neurons did not change the basal pain thresholds in naïve mice (Fig. 6D) and reversed the established hyperalgesia in CCI mice compared to the virus control mice (Fig. 6E).

Next, we wanted to know whether repeated inactivation of activity in these NAcS-receiving neurons could produce a prolonged antinociceptive effect in CCI mice. We performed the same repeated CNO injection strategy and behavioral tests as previous experiments (Fig. 6F). Interestingly, unlike the prolonged antinociceptive effect observed with repeated inhibition of the mPFC^CRF→NAcS circuit or CRF signaling in the NAcS, this repeated inactivation for 7 consecutive days did not produce any detectable changes of thermal and mechanical threshold in CCI mice (Fig. 6G). These data suggested that the hyperactivity of NAcS neurons innervated by the mPFC^CRF→NAcS circuit is necessary for the maintenance of chronic neuropathic pain, and persistent CRF-mediated enhancement of excitatory synaptic input is also necessary for maintaining the hyperactivity of these NAcS-receiving neurons.

Our current results demonstrated that CRF signaling in the NAcS is necessary and sufficient for the regulation of pain sensation under both physiological and pathological pain conditions, which was mediated by the enhancement of excitatory synaptic transmission in the mPFC^CRF→NAcS circuit (Fig. 6H). These findings reveal a novel cell- and circuit-based mechanistic link between chronic pain and the mPFC^CRF→NAcS circuit and provide potential therapeutic interventions for chronic pain by targeting the CRF neuronal circuit.

In recent decades, an increasing number of studies have suggested the role of extrahypothalamic CRF signaling including some brain regions such as the amygdala, hypothalamus, and periaqueductal gray in the regulation of pain sensation and pain-related affective behaviors [11–14, 55–62]. However, a comprehensive understanding of the brain mechanisms underlying CRF-mediated pain regulation is still lacking. We found that CCI-induced chronic neuropathic pain was accompanied with a persistent high CRF protein level in the NAc, which was in agreement with a previous study showing that CCI surgery evoked the overexpression of CRF receptors in the NAc [41]. Furthermore, we also demonstrated that CRF signaling in the NAcS was necessary and sufficient for the regulation of pain sensation under both physiological and pathological pain conditions. However, only low concentrations of CRF (50 nM) caused hyperpathia in naïve mice. In previous studies, a narrow effective dose range of CRF to evoke hyperalgesia/analgesia in the central nervous system was also observed [13, 16, 63]. CRF shows the highest affinity for binding to CRF receptor type Ⅰ (CRFR1) but may also bind to CRF receptor type Ⅱ (CRFR2) at very high concentrations [64, 65]. The action of activating the two receptors is thought to be antagonistic in previous pharmacological experiments [2, 66]. Thus, the concentration-dependent effect in our experiments may be attributed to the distinct constitution of two receptor subtypes activated by the different concentrations of CRF[49, 63, 67].

Interestingly, we found that repeated pharmacological activation of CRF receptors in the NAcS for 5 days resulted in chronic pain-like nociceptive responses that persisted for several days after the termination of pharmacological activation, and prolonged inhibition of CRF receptors induced persistent pain relief. Post-injury hyperpathia has been attributed to the sensitization and increased excitability of peripheral nociceptors or central neurons, with neuropeptides playing important modulatory roles throughout the process [68–73]. Our results suggest that the enhanced CRF signaling in NAc is a critical contributor to pain sensation regulation under physiological conditions and to the development of chronic neuropathic pain.

Growing evidence implied that the subregions of the NAc play distinct roles in processing pain information [28, 74]. In agreement with this view, we found that CRF signaling in the NAcS, but not in the NAcC, is involved in pain regulation. Similar subregion-specific effect in NAc has also been observed in the CRF-mediated oral motor activity [74]. Differential distributions of CRF receptors and CRF fiber innervating the NAcS or NAcC may account for these functional heterogeneities of NAc CRF signaling [75–77].

Several lines of evidence have indicated a link between enhanced neural activity in the NAc and painful experiences in humans and rodents [28, 41, 68, 69]. Consistently, the present study also indicated that neuronal activity and excitability in the NAcS, but not in the NAcC, were increased in CCI mice. Neuronal activity or excitability is affected by local intrinsic molecular mechanisms and presynaptic inputs from the innervating neurons [78, 79]. The transmission of glutamate in NAc has been involved in the neuronal activity and function [80–82]. The present study found that 1) the frequency, but not the amplitude, of sEPSC in NAcS neurons was increased in CCI mice and 2) the CCI-induced increase in excitability in NAcS neurons was reversed by blocking of excitatory synaptic transmission. The pain-related changes of excitatory input into NAc were also observed in several previous experiments [83, 84], while the mechanisms underlying these changes remains unclear. Our current data revealed that exogenous CRF enhances the excitatory synaptic transmission and inhibition of accumbal CRF signaling decreased presynaptic glutamate release. Combined with the previous researches, which revealed the crucial role of CRF in the modulation of presynaptic release [39, 42, 85], current results suggest that CRF-mediated enhancement of excitatory synaptic transmission accounted for neuronal hyperexcitability in NAcS neurons in CCI mice.

In line with the findings from previous whole-brain mapping studies in mice [51], our virally mediated retrograde tracing study determined that NAcS neurons were densely innervated by CRF-containing neurons in the mPFC. Animal and human study findings have implicated the neuronal connection between the mPFC and the NAc in the development of chronic pain [86–88]. However, the detailed mechanisms of this pain-related association reinforcement are still lacking. Our results indicate that the mPFC^CRF→NAcS circuit was activated following CCI surgery, as evidenced by increased c-Fos expression and neuronal excitability in NAcS-projecting mPFC CRF-containing neurons. Real-time optogenetic inhibition of the mPFC^CRF→NAcS circuit relieved hyperalgesia in CCI mice. In line with our pharmacological results, previous study which preferentially activating the NAcC-projecting mPFC CRF-positive neurons did not observed the influence of pain sensation [41]. These findings suggested that the persistent activation of the mPFC^CRF→NAcS circuit is essential for the maintenance of chronic pain.

Consistent with pharmacological inhibition of CRF signaling in the NAcS, repeated optogenetic inhibition of this circuit produced significant pain sensation relief in CCI mice, and this effect lasted for days after the termination of optical stimulation. These results indicate that the mPFC^CRF→NAcS circuit also governs the development of neuropathic pain. Prolonged inhibition of mPFC^CRF-NAcS circuit may reduce the release of CRF, relieve CRF-mediated excitation in the mPFC^CRF-NAcS circuit and prevent the hyperexcitability of NAcS neurons induced by CCI surgery. The result that acute optogenetic activation of mPFC^CRF→NAcS circuit did not affect the paw-withdrawal response in naïve mice is in sharp contrast to the effect of pharmacological manipulation. One possibility for the discrepancy between the result of optogenetic and pharmacological activation may be that the released CRF which is evoked by transitory activation is not sufficient for inducing hyperpathia, as CRF is stored in large dense core granules [89] and likely released by a prolonged period of activation [90, 91]. This hypothesis is supported by the following results that repeated activation of the mPFC^CRF→NAcS circuit induced a significant chronic pain-like response, which was accompanied by an increase in CRF protein levels in the NAc.

Interestingly, repeated inhibition of the mPFC^CRF→NAcS circuit or its NAcS-receiving neurons produced different effects on the regulation of pain sensation. Unlike the prolonged antinociceptive effect seen after the inhibition of the mPFC^CRF→NAcS circuit, the same manipulation on NAcS neurons innervated by the mPFC CRF-containing neurons induced only a transient antinociceptive effect. The NAc is known as one pivotal relay station for pain signaling as it receives innervation from both the prefrontal cortex and limbic regions, and projects forward to motor regions [25, 30]. Our findings suggested that 1) the regulation of pain sensation by NAcS-receiving neurons requires persistent inputs from the mPFC^CRF→NAcS circuit; and 2) targeting the mPFC^CRF→NAcS circuit may be more effective than interfering only with its receiving neurons.

In conclusion, the present findings revealed a novel cellular, synaptic and circuitry mechanism for pain regulation by central CRF signaling, and targeting CRF signaling in the corticoaccumbal circuit may be a potential therapeutic avenue for chronic pain.

Acknowledgements

The present study was supported by the National Key R&D Program of China—the Sci-Tech Innovation 2030 Major Project (2021ZD0203100), the National Natural Science Foundation of China (82130033, 82293641, 31970937, 82271255, 82101315 and 82271263); Jiangsu Province Innovative

and Entrepreneurial Team Program, the Key Project of Nature Science Foundation of Jiangsu Education Department (22KJA320006); the Innovation and Entrepreneurship Program of Xuzhou Medical University (2021CXFUZX002), China Postdoctoral Science Foundation (2022M710771 and 2022M722676), the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX21_2704).

The authors thank Li Yang, Dong-Yu Zhou, Wei Zheng and Prof. Cheng Xiao

for technical supports.

Conflict of Interest

All authors reported no biomedical financial interests or potential conflicts of interest.

Supplementary information is available at MP’s website.

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CRF regulates pain sensation by enhancement of corticoaccumbal excitatory synaptic transmission

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Journal Publication

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Abstract

Figures

INTRODUCTION

METHODS AND MATERIALS

Animals

Data analysis and statistics

RESULTS

CRF signaling in the NAcS regulates pain

CRF increases neuronal excitability in the NAcS by enhancing excitatory synaptic transmission

CRF-containing mPFC neurons innervate the NAcS for the maintenance of chronic neuropathic pain

The sufficient role of the mPFC^CRF→NAcS circuit in regulating pain

Identifying monosynaptic connection in the mPFC→NAcS circuit and its functional alteration in chronic neuropathic pain

The role of NAcS-receiving neurons in chronic pain

DISCUSSION

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

CRF regulates pain sensation by enhancement of corticoaccumbal excitatory synaptic transmission

Status:

Journal Publication

Version 1

Abstract

Figures

INTRODUCTION

METHODS AND MATERIALS

Animals

Data analysis and statistics

RESULTS

CRF signaling in the NAcS regulates pain

CRF increases neuronal excitability in the NAcS by enhancing excitatory synaptic transmission

CRF-containing mPFC neurons innervate the NAcS for the maintenance of chronic neuropathic pain

The sufficient role of the mPFCCRF→NAcS circuit in regulating pain

Identifying monosynaptic connection in the mPFC→NAcS circuit and its functional alteration in chronic neuropathic pain

The role of NAcS-receiving neurons in chronic pain

DISCUSSION

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

The sufficient role of the mPFC^CRF→NAcS circuit in regulating pain