(2R,6R)-Hydroxynorketamine restores postsynaptic localization of AMPAR in the prelimbic cortex to provide sustained pain relief

doi:10.21203/rs.3.rs-2261014/v1

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(2R,6R)-Hydroxynorketamine restores postsynaptic localization of AMPAR in the prelimbic cortex to provide sustained pain relief

https://doi.org/10.21203/rs.3.rs-2261014/v1

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Neuropathic pain is a difficult-to-treat pain condition that can affect patients for years. (2R,6R)-hydroxynorketamine (R-HNK) is a ketamine metabolite without dissociative effects and has been evaluated as an alternative to ketamine in chronic pain management. The mechanism of action remains elusive. Here we report that repeated systemic or contra-prelimbic cortex (PrL) infusion of R-HNK in the acute stage of nerve injury produces sustained pain relief for at least 14 days in the mouse spared nerve injury (SNI) model of neuropathic pain. Transcriptomic analysis suggests that SNI is associated with increased Brain-derived neurotrophic factor (Bdnf) signaling, abnormal dendritic spine organization, and reduced α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) activity in PrL. Activity-regulated cytoskeleton-associated protein (Arc) is identified as the top gene in the leading-edge analysis of the gene set. R-HNK administration abolishes these transcriptomic changes. Further studies confirm the transcriptome findings. Finally, we show that enhancing PrL activity by R-HNK increases PrL-periaqueductal gray (PAG) connectivity, which is essential for R-HNK-mediated pain relief. Our study highlights AMPAR suppression due to continuous Bdnf/Arc elevation in PrL as a mechanism of central sensitization after SNI. R-HNK can recalibrate Bdnf/Arc/AMPAR axis and restore PrL-PAG connectivity to induce sustained alleviation of neuropathic pain.

Health sciences/Medical research/Preclinical research

Health sciences/Medical research/Translational research

Health sciences/Neurology/Neurological disorders/Neuropathic pain

Chronic pain is one of the most significant global health burdens. Neuropathic pain is the most difficult-to-treat chronic pain condition. To date, oral analgesics remain the recommended first-line therapy for neuropathic pain. However, the benefits of conventional analgesics (e.g., opioids and non-steroidal anti-inflammatory drugs) are limited due to inadequate efficacy, abuse potential, and other side effects^1,2. Modified treatment paradigms or novel analgesics are warranted to improve the management of neuropathic pain.

The effectiveness of ketamine in managing acute pain, especially acute postoperative pain management, has been well recognized by numerous randomized controlled clinical trials³. Because of its multifaceted role in the central nervous system (CNS), ketamine has been extensively studied in preclinical models to treat chronic pain, particularly chronic neuropathic pain, characterized by central sensitization⁴. Despite these advantages, the risk-benefit ratio of long-term ketamine use in terms of its abuse potential (as a party drug), neurotoxicity (via intrathecal injection), and other adverse effects remain an open issue^5,6. Ketamine derivatives are investigational candidates for improving balance. (R, S)-Ketamine is metabolized by a series of cytochrome P450 enzymes to produce (2S, 6S)-HNK (S-HNK) and (2R, 6R)-HNK (R-HNK) via N-demethylation and hydroxylation^5,7. Many side effects of ketamine dissociation and abuse are mediated by N-methyl-D-aspartate glutamate receptor (NMDAR) inhibition. Unlike ketamine, antidepressant-related concentrations of (2R, 6R)-HNK are not sufficient to block NMDAR⁸ while inducing durable changes in synaptic plasticity by modulating AMPAR signaling and function in these brain regions⁹. Recently, R-HNK has also been assessed for analgesic effects. While (R, S)-HNK administration under different protocols did not affect nociception or tolerance¹⁰, R-HNK was reported to significantly increase von Frey thresholds in the mouse models of peripheral nerve injury, complex regional pain syndrome type-1 (CRPS1), and postoperative incisional pain¹¹. Importantly, repeated administration of R-HNK (e.g., 3 ´ intraperitoneal injections) resulted in sustained analgesia for 1 to 8 days, depending on the treatment paradigm and pain model. The mechanism underlying these effects is completely absent. Nonetheless, with its sustained effects, the lack of dissociative effects, and higher oral bioavailability, R-HNK appears superior to ketamine as a potential drug for treating chronic neuropathic pain. Further molecular mechanisms and treatment protocol studies may pave the way to clinical trials.

The prefrontal cortex (PFC), especially the medial prefrontal cortex (mPFC), which contains several subregions, including the prelimbic (PrL) and infralimbic (IL) cortex in rodents, is a part of the frontal lobe that is involved in processing both the sensory-discriminative and the affective-motivational aspects of pain¹². Under physiological conditions, mPFC responds to nociception and contributes to descending pain inhibition in the spinal cord dorsal horn^13-15. On the other hand, mPFC activity is suppressed in chronic pain states. Gray matter atrophy of mPFC was observed in patients with various chronic pain conditions¹². mPFC sends information to the periaqueductal gray (PAG) and thus modulates pain processing in the spinal cord. The mPFC-PAG connection, however, is suppressed and negatively correlated to pain ratings among patients with chronic low back pain¹⁶. Also, in animal models of neuropathic pain, enhanced mPFC-PAG connectivity reverses pain hypersensitivity^17,18. Taken together, mPFC is a promising target for neuropathic pain management.

In this study, we report that repeated R-HNK administration provides sustained pain alleviation in a mouse model of neuropathic pain, and PrL might be the potential target brain region. We examined the molecular and electrophysiological changes in PrL and proposed that reshaping PrL neurotransmission and PrL-PAG neural circuit by R-HNK administration is a promising approach for chronic pain management.

Repeated administration of R-HNK produced sustained analgesic-like effects

This study adopted a commonly used mouse model of neuropathic pain, spared nerve injury (SNI), to investigate the potential analgesic effects of R-HNK and the underlying mechanism. Mice with SNI received repeated (twice daily for 3 consecutive days after surgery) intraperitoneal injection (i.p.) of saline (vehicle control, the SNI/Saline group) or R-HNK (10 mg/kg, the R-HNK/Saline group). Mice undergoing sham surgery were injected with saline and used as sham controls (the Sham/Saline group). Compared to vehicle, R-HNK administration significantly attenuated SNI-induced mechanical allodynia at post-injury day (PID) 3 (Fig. 1a). The analgesics-like effects of R-HNK were also evident at PID 7, 14 (Fig. 1a), suggesting a sustained alleviation of mechanical allodynia. Similar effects were observed in the pinprick test (Fig. 1b, C). Compared to the Sham/Saline group, mice in the SNI/Saline group showed increased response rates and higher hyperalgesia scores to blunt needle stimulation at PID 7 and 14, while R-HNK treatment persistently and completely abolished SNI-induced mechanical hyperalgesia. Given that mPFC dysregulation is closely associated with chronic pain and that R-HNK can modulate neural activities in mPFC ⁹, we investigated whether mPFC is a site of action for R-HNK. R-HNK or saline was delivered into PrL via an implanted cannula following a similar regimen as in i.p. injection. As demonstrated in Fig. 1d, contra-PrL administration of R-HNK recapitulated the analgesic effects by i.p. injection. These findings supported that R-HNK could modulate PrL for sustained pain alleviation.

R-hnk Restores The Responsiveness Of Camkiia Neurons In Prl After Nerve Injury

The effects of R-HNK on neuronal activities in PrL were then assessed in mice at PID 14. In response to brush stimulus, the number of cFos positive (cFos⁺) neurons in the SNI/Saline group was significantly lower than that in the Sham/Saline group (Fig. 2a, b). Treatment with R-HNK resulted in a higher number of cFos⁺ neurons than with saline (SNI/Saline group) (Fig. 2a, b). Co-staining with CaMKII showed that the majority (~ 90% for all 3 groups) of cFos⁺ neurons were also positive for CaMKII (Fig. 1a-c), while the proportion of cFos⁺ CaMKII neurons varied between groups (Fig. 2d) with a similar pattern as the number of cFos⁺ cells (Fig. 2b). Our results are consistent with previous reports that peripheral nerve injury could suppress neuronal activity in PrL ¹⁹. In contrast, R-HNK treatment reversed such suppression. To monitor neuronal activities in vivo, we injected rAAV2/9-CaMKIIa-GCaMP6m which over-expresses calcium indicator under CaMKII promoter in neurons into PrL and recorded the calcium flux in CaMKII neurons using fiber photometry (Fig. 2e). In response to von Frey stimulus (1 g), vehicle-treated SNI mice displayed significantly lower amplitudes of calcium flux than the sham-operated mice. R-HNK administration fully restored the neuronal responses to the mechanical stimulus, with calcium flux amplitudes comparable to those of the Sham/Saline group (Fig. 2f-h). Furthermore, the whole-cell recording showed that the amplitudes and frequencies of sEPSCs were similar between the Sham/Saline group and the SNI/R-HNK group, while these were significantly lower in the SNI/Saline group (Fig. 2i-k). There was no significant difference in the frequencies and amplitudes of sIPSCs between groups (Fig. 2l-n). These findings suggested that R-HNK could restore neuronal responsiveness in PrL to nociceptive stimuli by enhancing postsynaptic excitatory currents.

R-hnk Administration Reverses Sni-induced Transcriptomic Changes In Prl

To explore possible mechanisms by which R-HNK modulates excitatory neurotransmission, transcriptome analysis was performed. Mice (n = 3 per group) underwent sham or SNI surgery and were treated with vehicle or R-HNK. The PrL tissues were collected at PID 14 for RNA sequencing. Principal component analysis (PCA) showed that the SNI/Saline group was separated from the Sham/Saline group, while convergence was observed between the SNI/R-HNK group and the Sham/Saline group (Fig. 3a), indicating that R-HNK treatment reversed nerve injury-induced transcriptome alterations. It was supported by the analysis of differentially expressed genes (DEGs) between groups, which identified 243 and 17 DEGs (fold change > 1.5 or < 1.5, adjusted p-value < 0.05) in the comparisons of Sham/Saline vs. SNI/Saline and Sham/Saline vs. SNI/R-HNK, respectively (Fig. 3b). Only two common DEGs were identified between the two pairs of comparison (Fig. 3b). We then used the list of DEGs between Sham/Saline and SNI/Saline groups to perform pathway and gene ontology (GO) annotation. GO analysis revealed a large number of biological process (BP) terms. The terms were clustered using the Leiden algorithm and plotted on the first two uniform manifold approximation and projection (UMAP) dimensions (Fig. 3c). Several neurotransmission-related BP terms were clustered (Fig. 3), including positive regulation of AMPA receptor activity, dendritic spine morphogenesis, and dendritic spine organization (Fig. 3d, only terms with an adjusted p-value < 0.05 were plotted). These annotations implied that peripheral nerve injury could specifically alter AMPAR activity in spines. It should be noted that the expression levels of SNI-associated DEGs involved in these BP terms were comparable between the Sham/Saline group and the SNI/R-HNK group. Further Bio Planet analysis revealed that the Bdnf signaling pathway was enriched in the SNI/Saline group, accompanied by a significant difference in mRNA levels of Bdnf between groups (fold change = 3 between SNI/Saline and Sham/Saline group, Fig. 3, e, f). Given the close association between Bdnf and synaptic plasticity, elevated Bdnf might act as a driver of abnormal AMPAR activity after SNI. We then performed gene interaction analysis of DEGs on STRING. This identified an interaction network consisting of tens of DEGs. The Cytoscape app MCODE, which can find highly interconnected regions within an interaction network, revealed two significant subnetworks, including the one encompassing Bdnf, Arc, Egr4, and Dusp1 (Fig. 3g). This result indicated that these genes might act in concert with Bdnf to reshape AMPAR activity. Indeed, Bdnf has been demonstrated to upregulate the Arc gene²⁰, which may modulate the shuttle of AMPAR-containing vesicles between synapses and dendrites²¹. To compare the difference between SNI/Saline vs. Sham/Saline and SNI/R-HNK vs. Sham/Saline from the GO perspective, GSEA covered all genes instead of just DEGs. Hundreds of BP terms were enriched for SNI (Supplementary file. 2) and R-HNK treatment (Supplementary file. 3). It is noted that several synapse-related BPs, including vesicle-mediated transport in the synapse, response to pain, and positive regulation of synaptic transmission, were significantly associated with the Sham/Saline vs. SNI/Saline group but not with the Sham/Saline vs. SNI/R-HNK group (Fig. 3h, i). Interestingly, Arc was again highlighted as the top 1 leading edge gene of vesicle-mediated transport in the synapse (Fig. 3j). There were significant differences in Arc mRNA expression between groups (Fig. 3k). These bioinformatic analyses indicated that Bdnf/Arc-associated synaptic plasticity might play a role in SNI-induced PrL repression. R-HNK treatment could rectify the dysregulated axis after nerve injury.

A Continuous Increase In Bdnf Expression In Prl Leads To Pain Hypersensitivity

To determine the role of Bdnf in pain hypersensitivity, the Bdnf expression in PrL was examined after pain development. Consistent with transcriptome analysis, SNI significantly increased Bdnf protein levels in PrL as early as at PID 3 and persisted for at least 14 days, as indicated by the immunofluorescence staining (Fig. 4a, b) and ELISA (Fig. 4c). In contrast, early administration of R-HNK (i.p. injection, 2 ⋅ 3 injections at PID 1, 2, and 3) blocked SNI-induced sustained upregulation of Bdnf (Fig. 4a-c). Along with behavioral tests (Fig. 1), our study indicated that Bdnf plays a role in pain hypersensitivity. We then infused recombinant Bdnf proteins into PrL via an osmotic pump for 14 days to determine the molecular function of Bdnf (Fig. 4d). As demonstrated in von Frey (Fig. 4e) and pinprick tests (Fig. 4f-g), continuous Bdnf administration was sufficient to sensitize pain-like responses in sham-operated mice, mimicking the effect of peripheral nerve injury. Moreover, while R-HNK consistently alleviated SNI-induced pain hypersensitivity, co-administration with Bdnf (contra-PrL infusion) abolished the analgesic-like effects (Fig. 4e-g). These results supported that Bdnf was essential for SNI-associated central sensitization and that R-HNK might act through Bdnf suppression after nerve injury.

Continuous Bdnf Elevation Reduced The Postsynaptic Distribution Of Ampar In Prl

Our RNA sequencing analysis suggested that peripheral nerve injury was associated with dendritic spine organization and abnormal regulation of AMPAR activity (Fig. 3). Given that Bdnf infusion causes central sensitization and that reversal of pain hypersensitivity by R-HNK. It was related to Bdnf suppression (Fig. 4), we investigated whether Bdnf might remodel synaptic organization after SNI. We first performed sparse single-color labeling with YFP to visualize the dendrites and spines of CaMKII neurons in PrL sections (Fig. 5a). The data showed no significant difference in dendrite numbers or spine densities between the three groups (Fig. 5b, c). Dendritic spines can be classified into several subtypes based on their morphology: mushroom spines, thin spines, stubby spines, and filopodia (Fig. 5d). It was noted that the density of the mushroom spine, one of the spine subtypes representing stable and mature spines and with the highest synaptic strength, was significantly decreased in the SNI/Saline group compared to the Sham/Saline group. Administration with R-HNK completely reversed nerve injury-induced loss of mushroom spine (Fig. 5d, e). Our data suggested that R-HNK might maintain excitatory transmission by preserving mushroom spines under SNI conditions.

Next, we compared the spine localization of AMPAR between the three groups. We reconstructed spines and GluA1 (an AMPAR subunit)-associated fluorescent puncta and generated co-localization channels using Imaris software (Fig. 5f). Our results showed that the GluA1 volume in the spines was significantly lower in the SNI/Saline group than in the Sham/Saline group, while R-HNK treatment fully restored the volume reduction due to SNI (Fig. 5g). Co-localization analysis of GluA1 and PSD95, an excitatory postsynaptic marker, showed similar trends. We found that compared to sham operation, SNI significantly reduced the ratio of co-localized GluA1 and PSD95. R-HNK treatment increased GluA1/PSD95 co-localization compared to the SNI/Saline group. Remarkably, similar to SNI surgery, continuous infusion with low-dose Bdnf was sufficient to induce a decrease in postsynaptic AMPAR localization and to block the effect of R-HNK on abnormal GluA1 translocation after SNI (Fig. 5i). These data implied that persistent Bdnf elevation, rather than amplification, might impair synaptic connectivity by reducing the distribution of AMPAR in excitatory post-synapses after SNI. Blocking sustained Bdnf upregulation is a possible mechanism underlying the action of R-HNK.

Bdnf/arc Axis Suppresses Ampar Activity And Counteracts R-hnk-mediated Pain Alleviation

Previous studies demonstrated that Arc could directly modulate spine morphology and AMPAR endocytosis in the spine^21,22. Our transcriptome study highlighted Arc as a potential target that was dysregulated by SNI but rectified after R-HNK administration (Fig. 3). In addition, Arc is a potential collaborating partner of Bdnf/Bdnf signaling (Fig. 3). We examine whether Bdnf might modulate Arc to remodel the subcellular localization of AMPAR. Our immunostaining result confirmed that SNI caused an increase in Arc expression at PID 3, 7, and 14 compared to sham operation, which was attenuated after R-HNK administration (Fig. 6a, b). Accordingly, nerve injury resulted in increased postsynaptic localization of Arc in PrL, but this was reversed with R-HNK treatment (Supplementary Fig. 1a, b). On the other hand, accompanied by the induction of central sensitization (Fig. 4), continuous Bdnf infusion in PrL also led to higher Arc expression than saline in sham-operated mice and abolished the effect of R-HNK on Arc expression after SNI (Fig. 6c, d). Arc expression appeared to be directly regulated by Bdnf, as exposure of Bdnf (25 ng/mL) alone, but not with the Tropomyosin receptor kinase B (TrkB) inhibitor K252a, increased Arc mRNA expression in primary cortical neurons (Supplementary Fig. 1c). These results supported that Bdnf and Arc might act as a functional axis and that R-HNK administration inhibited Bdnf signaling to suppress Arc expression.

We then explored whether dysregulated Arc affected the postsynaptic localization and activity of AMPAR in PrL. Arc or EGFP overexpression was induced, followed by surgery and drug treatment. Consistent with the above results, SNI resulted in elevated Arc expression accompanied by a reduced ratio of GluA1/PSD95, which could be reversed by R-HNK administration. However, Arc overexpression abolished such an effect of R-HNK (Fig. 6e, f). We then assessed AMPAR activity by recording AMAPR-induced currents (Fig. 6g). Compared to sham operation, nerve injury significantly decreased AMPAR-mediated eEPSCs at different injection currents (Fig. 6h). Treatment with R-HNK fully restored AMPAR currents after SNI, which was counteracted by Arc overexpression. A similar pattern of change was observed in the ratio of AMAPR/NMDAR-associated eEPSCs (Fig. 6i), supporting that Arc elevation represses postsynaptic localization and activity of AMPAR after SNI. Finally, under AMPAR modulation, Arc overexpression also abrogated R-HNK-mediated analgesia (Fig. 6j, l). Our study demonstrated that suppressing Bdnf/Arc to replenish AMPAR activity was essential for R-HNK-mediated pain relief after SNI.

R-hnk Administration Restores Prl-vlpag Descending Pain Inhibition

CaMKII neurons in PFC can project to multiple brain regions, including the amygdala, nucleus accumbens (NAc), and PAG, forming neural circuits involved in stress, anxiety, and pain processing^17,23−25. mPFC-PAG connection is involved in PAG-spinal cord descending pain inhibition pathway. Impaired functional connectivity between PrL and vlPAG has been reported in humans with chronic pain and animal models of chronic pain^17,18. We explored whether R-HNK, which restored PrL excitability after SNI, could enhance PrL-vlPAG connectivity and thereby lead to pain relief. We found that intraperitoneal administration of R-HNK could significantly increase the number of cFos⁺ in vlPAG after SNI surgery (Supplementary Fig. 2a, b). We then injected rAAV2/9retro-CaMKIIa-Cre into vlPAG and rAAV2/9-EF1a-DIO-GCaMP6f into PrL to selectively transduce calcium sensor into vlPAG-projecting neurons in PrL, followed by recording fiber implantation in PrL. Fiber photometry showed that SNI surgery significantly decreased the amplitudes of calcium flux in response to mechanical stimuli compared to sham operation (Fig. 7a-c). R-HNK administration resulted in a comparable calcium flux between the SNI/R-HNK group and the Sham/Saline group (Fig. 7a-c). These results suggested that vlPAG-projecting neurons were included in the neuronal population affected by nerve injury. R-HNK might function by restoring this specific cluster of projection neurons. We then selectively inhibited vlPAG-projecting neurons by chemogenetic approach. rAAV2/9-CaMKIIα-EGFP-Cre and rAAV2/9retro-DIO-hM4D(Gi)-mCherry were injected into PrL and vlPAG, respectively (Fig. 7d-e). hM4D overexpression without CNO treatment did not affect R-HNK-mediated pain relief (SNI/Saline vs. SNI/R-HNK, p < 0.05, Fig. 7f). CNO administration completely abolished the pharmacological action of R-HNK (SNI/R-HNK + CNO vs. SNI/Saline, p > 0.05, SNI/R-HNK + CNO vs. SNI/R-HNK/Saline, p < 0.05, Fig. 7f). Further, it lowered the pain thresholds in the SNI group (SNI + CNO vs. SNI/Saline, p < 0.05, Fig. 7f). These results provided evidence that enhancing PrL-vlPAG connectivity was essential for pain alleviation by R-HNK.

In this study, we reported that repeated R-HNK administration in the acute stage of nerve injury could provide sustained pain relief for at least 14 days in a mouse model of neuropathic pain. Our study suggests that peripheral nerve injury causes a continuous elevation of Bdnf in PrL, which induces Arc transcription to decrease postsynaptic localization and currents of AMPAR and suppress the excitability of CaMKII neurons in PrL. Treatment with R-HNK abrogates the mechanism of sustained Bdnf/Arc dysregulation, thereby restoring AMPAR activity and excitability of vlPAG-projecting neurons and attenuating central sensitization after peripheral nerve injury. Our study suggested that persistent Bdnf elevation, rather than enhancement reduces synaptic strength in PrL. It represents a possible new mechanism of central sensitization. We propose that restoring mPFC-PAG connectivity by controlling the Bdnf/Arc/AMPAR axis is a promising approach for long-lasting analgesia. Finally, our study describes a mechanism of action of R-HNK that could pave the way for clinical trials of R-HNK as an alternative to ketamine in treating chronic pain.

Bdnf is a neurotrophic factor with multifaceted roles in cell survival, neuronal differentiation, neurogenesis^25,26, and synaptic plasticity^27,28. Depending on the expression patterns, Bdnf can produce opposite effects on synaptic strength. For instance, rapid and transient (within 1 hour) Bdnf stimulation could enhance synaptic connection, whereas sustained (hours to days) Bdnf exposure results in synaptic downscaling²⁹. Several studies described that ketamine and R-HNK could promote Bdnf expression within hours and then facilitate AMPAR activity to produce rapid antidepressant effects^30,31. Our study demonstrated a contrary role of Bdnf in the model of neuropathic pain. After nerve injury, Bdnf elevation can be detected at PID 3 and lasts for at least 14 days. Concomitant with sustained upregulation of Bdnf, AMPAR activity is diminished as indicated by the decreased postsynaptic localization (Fig. 6f) and AMPAR-associated eEPSCs (Fig. 6g-i). Interestingly, continuous infusion of Bdnf with an implanted osmotic pump recapitulates the suppression of AMAPR activity even in mice without nerve injury, highlighting a causal relationship between Bdnf overexposure and AMPAR decline. Furthermore, such an effect is associated with reduced excitability of excitatory neurons in PrL, including the PAG-projecting neurons. These results provide a possible explanation for the impaired PrL-PAG connectivity detected in humans with chronic pain and animal models of chronic pain^32,33. Our study establishes a new Bdnf-dependent mechanism of central sensitization.

Bdnf modulates the subcellular localization of AMPAR in a context-dependent manner. Studies have described that acute Bdnf stimulation facilitates the synaptic expression of AMPAR subunits in different brain regions, such as the anterior cingulate cortex (ACC)³⁴, NAc³⁵, and hippocampus³⁶. After BDNF-TrkB binding, several downstream kinases, including Erk, PKA, and CaMKII, are regarded as critical regulators that promote the synaptic delivery of AMPAR subunits³⁷. On the other hand, it is well established that AMPAR trafficking between the synaptic surface and dendrites is mediated by the endocytosis process³⁸. Bdnf is an immediate early gene that can be rapidly synthesized for activity-dependent dendritic modulation. By interacting with components of endocytosis machinery, Bdnf is involved in the internalization of membrane receptors or channels, including AMPAR subunits³⁹. In this study, we found continuous Bdnf infusion upregulated expression in vitro and in vivo (Fig. 4a-c and Supplementary Fig. 1c). Continuous Bdnf exposure leads to sustained elevation and reduces postsynaptic expression of AMPAR and AMPAR-associated eEPSCs. These results provide a mechanism underlying long-term Bdnf stimulation-mediated decline in synaptic connection⁴⁰ and highlight a dysregulated Bdnf/AMPAR axis in PrL as a new mechanism of central sensitization after nerve injury.

The proposed Bdnf/AMPAR axis is also a target of R-HNK. Several studies have reported that R-HNK, one of the most abundant and stable metabolites of ketamine, has a half-life of approximately 1 hour in the brain⁴¹ and a plasma half-life of 0.2–0.8 hours in rodents⁴², and exerts rapid and strong antidepressant effects as ketamine⁴³. Interestingly, R-HNK does not cause dissociative behavior in rodents, which remains one of the major concerns associated with the long-term administration of ketamine⁴⁴. It is recommended to use R-HNK as an alternative to ketamine to treat depression⁴⁵. Recently, similar to ketamine, its metabolite R-HNK has been demonstrated to provide pain relief in several animal models of pain, but the mechanism remains elusive. Our study supports the analgesic-like effects of R-HNK in the model of neuropathic pain (Fig. 1). Mechanistically, R-HNK disrupts the sustained elevation of Bdnf, thus reversing upregulation and AMPAR suppression in PrL. This was supported by the findings that exogenous Bdnf completely abolished R-HNK-mediated analgesia and reorganization of AMPAR localization. Furthermore, we provide evidence that recalibration of the Bdnf/AMPAR axis by R-HNK restores the excitability of PAG-projecting neurons, as indicated by the increased calcium influx in these cells (Fig. 7a-c) and increased number of cFos⁺ cells in vlPAG (Supplementary Fig. 2). Such modulation enhances the PrL-vlPAG connectivity, which has been reported to be significantly suppressed in neuropathic pain³², leading to pain alleviation. Our study provides a molecular and neural circuit perspective for understanding the analgesic-like effects of R-HNK.

Overall, we demonstrated that peripheral nerve injury induces continuous elevation of Bdnf that promotes expression to diminish AMPAR activity in the PrL, representing a novel mechanism of central sensitization. Repeated R-HNK administration abrogates Bdnf dysregulation, disables Arc-mediated AMPAR internalization, and restores PrL-vlPAG connectivity to produce sustained pain alleviation. Our study provides a mechanistic exploration for clinical trials of R-HNK as a ketamine replacement therapy for chronic pain.

Animals

Male C57BL/6 mice, 8 weeks old, were used in this study unless otherwise noted. Mice were housed under 12-hour light/dark cycles in a pathogen-free room with clean bedding and free access to food and water. Cage and bedding changes were performed each week. All animal studies were performed following protocols approved by the Animal Experimentation Ethics Committee, the Chinese University of Hong Kong or Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Science.

Spared Nerve Injury (Sni) Surgery

Mice were anesthetized by gas anesthesia (isoflurane 1–2%). For induction of SNI neuropathy, the left sciatic nerve was exposed at the level of the trifurcation into the sural, tibial, and common peroneal nerves. The tibial and common peroneal nerves were tightly ligated and severed, leaving the sural nerve intact. For the sham group, the left sciatic nerve was exposed as in the SNI procedure but was not further manipulated.

Behavioral Assessment

For the von Frey test, mice were habituated on an elevated platform with a mesh floor for 30 min. The plantar surface of the hind paw was stimulated with a series of calibrated von Frey filaments (NC12775-99, North coast medical, Morgan Hill, CA, USA). The 50% paw withdrawal thresholds were calculated using the Up-Down method.

For the pinprick test, the plantar hind paw was stimulated by stroking using a 25G safety pin. (Ratings: 0 = no response, 1 = paw withdrawal, 2 = flicking of the paw, and 3 = licking of the paw).

For the chemogenetics experiment, behavioral tests were performed at 30–120 minutes after intraperitoneal (i.p.) administration of clozapine-n-oxide (CNO) (3 mg/kg, BrainVTA, Wuhan, Hubei, China) or saline to virus-infected mice.

Drug Administration

R-HNK (SML1873, Sigma-Aldrich, St Louis, MO, USA) or saline was administered via i.p. injection (10 mg/kg) twice daily for 3 consecutive days. For continuous Bdnf delivery (14 days), recombinant Bdnf protein (0.5 µg/90 µL, PeproTech, 450-02, USA) or saline (0.9%, 90 µL, vehicle) was loaded into ALZET osmotic pumps (1003D, RWD, Shenzhen, Guangdong, China) implanted in PrL.

Transcriptome Analysis

Mice were divided into three groups and treated accordingly (n = 3 mice/group), namely Sham/Saline (Sham), SNI/Saline (SNI), and SNI/R-HNK (R-HNK). On the 14th day after surgery, PrL tissues were collected and snap-frozen in liquid nitrogen. Total RNA was isolated from the PrL using TRIzol Reagent (15596026, Thermo Fisher Scientific, Pittsburgh, PA, USA) according to the manufacturer’s instructions. RNA was purified with RNeasy Micro Kit 50 (74004, Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Library construction and sequencing services were provided by Novogene using the Illumina Novaseq 6000 system (Novogene, Tianjin, China) following a standard protocol. The sequencing data were then pre-processed to remove low-quality reads and adaptors, hisat2 (version 2.0.5) to align to mouse genome (version GRCm38), and feature counts (version 2.0.3) to quantify gene counts. Differentially expressed genes (DEGs) were identified using the DESeq2 R-package with default settings. Pathways and gene ontology (GO) annotations were conducted with Enrichr (http://amp.pharm.mssm.edu/Enrichr/) using DEGs between sham and SNI groups. The clustering of similar GO terms under the Leiden algorithm was done using the enrichment analysis visualizer provided by Enrichr Appyters. (https://github.com/MaayanLab/appyter-catalog/tree/main/appyters/Enrichment_Analysis_Visualizer). For gene set enrichment analysis (GSEA), the entire gene list was ranked according to the values of \(-log10\left(pvalues\right) sign\left(log2Foldchange\right)\) of each gene. fGSEA R-package was used to analyze GO biological processes. The log2Foldchange and p-values were generated with DESeq2 R-package. The enrichment plot was drawn with fGSEA. The volcano plots, scatter plots, bar charts, bubble plots, and ridge plots were drawn with the ggplot2 R-package.

Stereotaxic Injections

Stereotaxic injection of recombinant adeno-associated virus (rAAV) was performed according to a standard stereotaxic surgery procedure using an automated nanoliter syringe (Nanoject II, Drummond Scientific, Broomall, PA) with the following parameters: a total volume of 300 nL or 250 nL at 40 nL/minute for injection and hold for 5 minutes before withdrawal or the micropipette. The rAAVs used in this study were listed in the Supplementary file. S1. For PrL transduction, viral particles were injected at anteroposterior (AP): +1.8–1.9 mm; medial-lateral (ML): -0.5 mm; dorsal-ventral (DV): -2.1 mm. rAAV was infused into the ventrolateral PAG (vlPAG) at AP: +4.6 mm, ML: -0.8 mm, and DV: -3 mm.

Fiber Photometry

Fiber photometry was performed using the Multi-Channel Fiber Photometry Device (Inper, Hangzhou, Zhejiang, China). We calibrated the power of the LED lamp (λ = 465 nm) that excites GCaMP to 30 µW and the power of the LED lamp (λ = 405 nm) that records the baseline to 10 µW. We recorded CaMKII neurons in PrL of mice injected with rAAV9-CaMKIIa-GCaMP6m-WPRE-hGH-pA. To record vlPAG-projecting neurons in PrL, rAAV9retro-CaMKIIa-Cre-EGFP-WPRE-pA and rAAV9-EF1a-DIO-GCaMP6f-WPRE-hGH-pA were injected into PrL and vlPAG respectively. Mice were allowed to recover for 7 days, followed by pain model development and drug treatment (2 ⋅ 3 injections). Mice were habituated to the behavioral assessment environment three days before the recording day (postoperative day 14). On the day of the test, mice were connected to the photometric system and acclimated in a chamber (as in the mechanical threshold test) on a wire mesh for 30 minutes. The GCaMP signals were recorded in an 8-minute window divided into three slots: resting state: 3 minutes; stimuli sections (with 1 g of von Frey filament every 40 s on the left hind paw): 4 minutes, and then another resting state: 1 minute.

Whole-cell Patch Clamp Recordings

For the patch clamp study, 4-week-old mice were used for pain model development and drug treatment. Brain slices were collected for recording at 7 weeks of age. Brains were quickly harvested and transferred to ice-cold dissection solution equilibrated with 95% O₂ and 5% CO₂. Coronal sections (300 µm) were cut with Leica VT1200S (Leica Biosystems, Buffalo Grove, IL, USA) and incubated at 30℃ for 30 minutes in artificial cerebrospinal fluid (aCSF, 126 mM NaCl, 2.5 mM KCl, 125 mM NaH₂PO₄, 26 mM NaHCO₃, 10 mM D-Glucose, 0.5 mM CaCl₂, and 10 mM MgSO₄) equilibrated with 95% O₂ and 5% CO₂. Layer 5 pyramidal neurons in PrL were identified under 40 X magnification. The recording electrode was filled with intracellular solution (124 mM NaCl, 2.5 mM KCl, 1.2 mM NaH₂PO₄, 24 mM NaHCO₃, 5 mM HEPES, 12.5 mM D-Glucose, 2 mM CaCl₂, 1.5 mM MgSO₄, and 1.5 mM QX-314). The patch-pipettes have a resistance of 6–9 mΩ. We placed the tip of the glass electrode at 1/3 − 2/3 of the target neurons. For the whole-cell recording, the voltage was held at -70 mV to record the sEPSC of pyramidal neurons, and at the voltage was held at + 10 mV for the sIPSC. The total recording time for each cell was 3 minutes and data from the median 1 minute was used for the analysis.

The Ratio Of Ampar/nmdar Eepscs

Acute brain slices were prepared as above and placed in aCSF containing 50 µM bicuculine. To measure AMPAR/NMDAR eEPSC ratio, the recording electrode containing the intracellular solution was attached to the target neuron in PrL, while the stimulation electrode was placed 200–250 µm away from the target neuron. The eEPSCs were then evoked with increasing currents ranging from 0-160 µA with an increment of 20 µA at each step. The AMPAR- and NMDAR-eEPSCs were recorded with a holding potential of -70 mV and + 40 mV, respectively.

Dendritic Spines Analysis

In each group, 2–4 segments per CaMKII neuron, including 10–16 neurons, from 5 animals were used to reconstruct z-stacks into 3D models for analysis. To quantify dendritic spine density and morphology, Z-stacked images were converted to maximum intensity projections and analyzed using Imaris 7 software (Bitplane, Concord, MA, USA). Images of dendritic fragments were collected from secondary to tertiary dendrites. The Filament Tracer module with default settings and the auto-path mode were used for tracing the dendritic fragments. If necessary, we manually corrected the dendritic fragments and spines detection. We calculated dendritic spine density by counting the number of spines per 10 µm of dendritic length. Co-localization of spines with GluA1 puncta was 3D-reconstructed and analyzed using the “surface” and “spot” functions of Imaris. Dendritic spines were classified into mushroom spines, thin spines, stubby spines, and filopodia spines using Imaris X Tension, and the density of each spine type was calculated. Masked surface channels were created for the dendritic shaft, filament, and each spine class. Colocalized puncta in the dendritic spines were subtracted from the total co-localized puncta in each filament to determine the co-localized voxels within the spines. The co-localized volume of the individual spine was determined by co-localizing the masked spine channel with the masked filament channel of interest.

Statistical Analysis

Data were checked for normality using the Shapiro-Wilk test. Tests of significance were conducted using one-way ANOVA, followed by Bonferroni's post hoc tests. All statistics were performed using GraphPad Prism (version 9, GraphPad Software, San Diego, CA, USA) with statistical significance set at alpha = 0.05. Data were presented as mean ± standard error of the mean (mean ± SEM).

Data availability

The RNA-seq data supporting this study's findings have been deposited in the NCBI’s Gene Expression Omnibus with the GEO Series accession number GSE217622. Source data are provided with this paper.

Code availability

The source code for RNA-seq is available at https://github.com/CUHK-AIC-lab/HNK_RNAseq.

Acknowledgments

This study was supported by the Shenzhen-Hong Kong-Macau Technology Research Programme from the Shenzhen Science and Technology Innovation Commission, project number 20210823300029 (X.L.). And Technology Innovation Commission, and the Research Matching Grant from the Hong Kong University Grants Committee, project number 8601370 (X.L). I.H.T.H. is supported by the Lee Hysan Postdoctoral Fellowship in Clinical Neurosciences.

Contributions

Conceptualization: W.K.K.W., X.L., and T.J.; methodology: L.Z., H.C.; investigation: Q.L, J. Z., and Y.Z.; visualization: Y.J., and J.W.; funding acquisition: X.L.; project administration: X.L. and W.K.W.; supervision: T.G and M.T.V.C.; writing-original draft: T.J., H.L.L.N; writing-review and editing: X.L., W.K.K.W., and I.H.T.H. All authors read and approved the final manuscript.

Ethics declarations

Competing interests

The authors declare that they have no competing interests.

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There is NO Competing Interest.

SupplementaryFile1.pdf
SupplementaryFile2.csv
RNA-seq/SNI_allGOBP_with_gene_names
SupplementaryFile3.csv
RNA-seq/HNK_allGOBP_with_gene_names
Supplementarymaterialsandmethods.pdf
Supplementaryfigure1.pdf
Supplementary Fig. 1. R-HNK reversed nerve injury-induced postsynaptic localization of Arc in PrL a Representative images of Arc and PSD95 co-localization in the PrL. Arc (red), PSD95 (green). Scale bar: 12μm. b Quantification of the percentage of GluA1 and PSD95 co-localization in Arc positive (Arc⁺) cells (p < 0.001). c Quantification of Arc mRNA level of primary cortical neurons treated with Bdnf (25ng/ml) and Trkb inhibitor (K252a, 200 nM) / Bdnf (25 ng/ml) for 24 hours. (p < 0.05, p < 0.01). Data were expressed as mean ± SEM. Group comparisons were done by One-way ANOVA with Bonferroni's post-hoc test. Source data are provided as a Source Data file.
Supplementaryfigure2.pdf
Supplementary Fig. 2. R-HNK restores the number of activated neurons in vlPAG after nerve injury a Representative images of cFos staining (red) in the vlPAG. Scale bar: 200 μm. b Quantification of overall cFos positive (cFos⁺) cells in vlPAG (^*p < 0.05, ^**p < 0.01). Data were expressed as mean ± SEM. Group comparisons were done by One-way ANOVA with Bonferroni's post-hoc test. Source data are provided as a Source Data file.

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(2R,6R)-Hydroxynorketamine restores postsynaptic localization of AMPAR in the prelimbic cortex to provide sustained pain relief

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Abstract

Figures

Introduction

Results

Repeated administration of R-HNK produced sustained analgesic-like effects

R-hnk Restores The Responsiveness Of Camkiia Neurons In Prl After Nerve Injury

R-hnk Administration Reverses Sni-induced Transcriptomic Changes In Prl

A Continuous Increase In Bdnf Expression In Prl Leads To Pain Hypersensitivity

Continuous Bdnf Elevation Reduced The Postsynaptic Distribution Of Ampar In Prl

Bdnf/arc Axis Suppresses Ampar Activity And Counteracts R-hnk-mediated Pain Alleviation

R-hnk Administration Restores Prl-vlpag Descending Pain Inhibition

Discussion

Methods

Animals

Spared Nerve Injury (Sni) Surgery

Behavioral Assessment

Drug Administration

Transcriptome Analysis

Stereotaxic Injections

Fiber Photometry

Whole-cell Patch Clamp Recordings

The Ratio Of Ampar/nmdar Eepscs

Dendritic Spines Analysis

Statistical Analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1