Plasticity of mouse dorsal root ganglion neurons by innate immune activation is influenced by electrophysiological activity

Plasticity of mouse dorsal root ganglion neurons by innate immune activation is influenced by electrophysiological activity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Plasticity of mouse dorsal root ganglion neurons by innate immune activation is influenced by electrophysiological activity Timothy N. Friedman, Shawn M. Lamothe, Aislinn D. Maguire, Thomas Hammond, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4094312/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background. The intricate interplay between inflammation, its effects on neuronal excitability, and the ensuing plasticity of dorsal root ganglion (DRG) sensory neurons remains to be fully explored. In this study, we have investigated the impact of inflammatory conditioned media on the excitability and activity of DRG neurons and how this relates to subsequent growth responses of these cells. Findings. We show here that an early phase of increased neuronal activity in response to inflammatory conditioned media is critical for the engagement of plastic processes, and that neuronal excitability profiles are linked through time to the structural phenotype of individual neurons. Pharmacological blockade of neuronal activity was able to abolish the growth promoting effects of inflammatory media. Our findings also demonstrate a sex specificity of these responses. Conclusions. Our results suggest that targeting the activity of DRG neurons may provide a novel therapeutic avenue to manipulate their growth status and potential for plasticity in response to inflammation. While further studies are needed to fully elucidate the underlying mechanisms of the relationship between neural activity and growth status, a more complete understanding of this relationship may ultimately lead to the development of new treatments for neuropathic pain in disorders associated with heightened immune responses such as rheumatoid arthritis and MS. DRG pain inflammation neurite extension TNFα plasticity electrophysiology Kv7 channels Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Pain is a prevalent symptom in autoimmune diseases such as multiple sclerosis and rheumatoid arthritis, and its management remains a challenge. Based on the current understanding of their pathophysiological mechanisms, these diseases involve the dysregulated activation of the innate and adaptive immune system, leading to high levels of inflammation that damage tissue in the periphery and central nervous system along with the sensitization of primary sensory neurons, culminating in pain and disability ( 1 – 5 ). Inflammation is not always detrimental, however. For instance, after injury, immune-mediated inflammation can trigger regenerative mechanisms, assisting in debris clearance or promoting regeneration ( 6 – 8 ). Inflammation is a dynamic process that can be initiated in response to various stimuli and then resolve when no longer required ( 9 – 11 ). In individuals with multiple sclerosis (MS), an inflammatory autoimmune disease characterized by periods of attack and remission, these inflammatory processes can coincide with periods of disability. Although inflammation can resolve in MS, it is not always the case, and persistent low-grade inflammation may lead to neurodegeneration, emphasizing the importance of the resolution process ( 12 , 13 ). Chronic inflammation can lead to disability and may also play a role in the induction of chronic pain. As inflammation is adaptive and designed to resolve, its persistence may indicate an impairment in the resolution process. Dorsal root ganglion (DRG) neurons have an innate capacity for growth after injury to their distal axons( 14 , 15 ). This plasticity can be demonstrated in cell culture. Dissociated DRG neurons can readily establish and send out axons in short periods of time, a process that can be encouraged by the priming of these cells with ‘conditioning lesions’( 16 – 18 ) or discouraged by inhibition of retrograde transport along the injured axon( 19 , 20 ). Furthermore, the structural plasticity of neurons can be modulated by pharmacological manipulation of ion channels. For example, inhibition of Ca 2+ channel trafficking to the membrane( 21 ) with gabapentin can modulate synaptogenesis and neural circuitry rewiring( 22 , 23 ). This effect is also observed in vivo as gabapentinoids can be used to increase regeneration of axons after spinal cord injury ( 24 – 26 ). Similarly, Baclofen reduces the amplitude of voltage-gated Ca 2+ -currents in DRG neurons ( 27 , 28 ) and promotes their regeneration after spinal cord injury. The growth state of sensory neurons can also be modulated by inflammation ( 29 – 32 ). Not only can the inflammatory response to injury, even potentially sterile injury, have effects on outgrowth, but inflammation triggered by adaptive or innate immunity can also affect outgrowth. Therefore, inflammation can be seen as a critical driver of structural plasticity and the growth status of sensory neurons ( 33 ). It is also widely recognized that inflammation can alter the excitability of neurons, and traditional mechanisms of inflammation can result in increased action potential frequencies and lowered thresholds for action potential generation (i.e., sensitization). Inflammatory mediators like TNFα and IL-1β can regulate neuronal expression of voltage-gated sodium and potassium channels, increasing neuronal excitability ( 34 – 38 ). This phenomenon has been observed in both electrophysiological recordings of single cells ( 39 ) and nerve conduction studies ( 40 , 41 ). Heightened excitability and sensitization of peripheral sensory neurons results in an increased perception of pain, although this phenomenon may resolve as inflammation subsides. While inflammation can independently affect both the excitability and growth status of neurons, increasing excitability through electrical stimulation can promote axonal growth, indicating that there may be a connection between the two processes ( 42 – 44 ). Additionally, some researchers have proposed that the inflammation-induced changes in excitability may be directly involved in changes to neuronal growth status ( 45 – 47 ). Increasing neuronal excitability using optogenetics enhances axonal growth in the presence of an inflammatory stimulus ( 48 , 49 ). This suggests that the increase in neuronal excitability may be one of the mechanisms by which inflammation promotes structural plasticity. Overall, the relationship between inflammation, excitability, and the capacity for structural plasticity of DRG neurons is complex and likely involves multiple signaling cascades. To investigate this relationship, we conducted a series of experiments examining the effect of inflammation on neuronal excitability and neurite outgrowth. Our findings indicate that an early phase of increased neuronal excitability results in an increased growth status of primary sensory neurons, and that the neuron’s excitability is intricately linked to its structural phenotype. 2. Materials and Methods 2.1 Animals Unless otherwise stated, all mice are wild-type C57/BL6, purchased from Charles River and included in experiments at 8 to 10 weeks of age (C57BL/6NCrl, Strain Code: 027, https://www.criver.com/products-services/find-model/c57bl6-mouse?region=24# ). All mice were given access to food and water ad libitum and maintained in the University of Alberta facility under 12 hour light/dark cycles. 2.2 Bone Marrow Derived Macrophage (BMDM) Cultures and Conditioned Media Bone marrow derived macrophage cultures were generated under a previously described protocol( 50 ). Briefly, female and male 8 to 10 week old C57BL/6 mice were euthanized by Euthanyl® (sodium pentobarbital) injected intraperitoneally. After euthanasia, bone marrow cells were isolated from femurs as single-cell suspensions and filtered to remove excess debris. Bone marrow cells were allowed to differentiate into macrophages (BMDMs) for 8 to 10 days in L929 media. Upon completed differentiation, bone marrow derived macrophages were harvested and resuspended into freshly prepared DMEM containing 1% each of sodium pyruvate, Glutamax, Pen/Strep, and FBS. This media is hereon referred to as ‘low-serum media’. Cells were cultured in T75 flasks and were allowed to rest for 48 hours after which a full change of media was performed. A small volume of TNFα (final concentration: 1ng/ml) or vehicle (low-serum media) was added and allowed to incubate for 24 hours. After incubation, the stimulation media was fully replaced with a final volume of low-serum media and the stimulated BMDMs were allowed to rest for 6 hours. This ‘6-hour CM’ was captured at the experimental endpoint, aliquoted, and stored at -80°C until further usage avoiding freeze-thaw cycles. 2.3 Dorsal Root Ganglia Neuron Cultures Dorsal root ganglion (DRG) neurons were acquired from male and female mice, using a modified protocol from previously described work( 51 ). Briefly, animals were euthanized by Euthanyl® (sodium pentobarbital) injected intraperitoneally. After injection, animals were monitored for level of consciousness and dissections did not proceed until no response to toe pinch or corneal contact was observed. Cardiac punctures were performed to confirm euthanization and animals were perfused with 10mL of ice-cold saline. Perfused animals underwent spinal laminectomies and gross dissection of the spinal cord to expose the DRG. DRG were micro-dissected from the spinal column, taking care to remove as much residual nerve as possible while avoiding damage to the DRG. Isolated DRG were placed in ice-cold HBSS -/- until dissections were completed. To acquire single-cell suspensions of DRG neurons, the HBSS -/- was replaced with a warmed dilution of Stemxyme I (Worthington, LS004106) and DNase (Worthington, LS002007) in HBSS -/- and incubated in a 37°C water bath for approximately 45 minutes. Following digestion, enzyme activity was quenched with equal volumes of low ovomucoid (Worthington, LS003086) and mechanically titrated with a P1000 pipette until tissue was fully dissociated. The cell suspension was filtered through a 70 um mesh filter (Biologix, 15-1070) and gently layered on top of a 20% BSA solution. This layered gradient was spun at 300G for 10 minutes at room temperature to pellet neuronal cells and remove cellular debris (mainly myelin). Debris was gently removed, and the cell pellet was resuspended in a small volume of 0.5% BSA in HBSS -/- and quantified for neuronal yield with a hemocytometer. The cell suspension was adjusted to 1000 cells / 100 uL and 100 uL of this suspension was added to equilibrated media in a 24 well (CellVis, P24-1.5H-N), poly-D-lysine coated plate (Sigma, P6407). For electrophysiological recordings, cells were plated onto glass coverslips (Fisher, 1254583) identically coated in poly-D-lysine and transferred into the electrophysiological recording setup at experimental timepoints. Unless otherwise stated, plated neurons were incubated at 37°C with 10% CO 2 for 48 hours. A full list of reagents used in cell culture experiments can be found in Supplementary Table 1 . 2.4 Pharmacology For experiments involving retigabine (RTG), a master stock of 50mM was prepared by reconstituting 10ng of lyophilized RTG (Tocris, 6233) in DMSO, aliquoted, and stored at -20°C. On experimental timepoints, a 10x concentration (200 uM) was prepared by dilutions of thawed aliquots in DMEM. The final concentration of RTG was adjusted by direct dilution into plate wells. 2.5 Immunocytochemistry At experimental endpoints, an equivalent volume of 8% PFA was added to the media of culture plates containing the adherent cells. Samples were incubated at room temperature in the diluted fixative for 15 minutes and then washed 3 times in DPBS. Permeabilization and non-specific IgG binding was blocked by one hour incubation with 10% NDS in DPBSTx 0.2% at room temperature, followed by an overnight incubation with rabbit anti-βIII tubulin (Sigma, T2200) diluted in DPBS. On the following day, cells were washed 3 times in DPS, incubated with DAPI (Invitrogen, D1306) and 594-conjugated anti-rabbit secondary antibodies (Jackson ImmunoResearch, 711-586-152) for one hour at room temperature before a final three washes. Imaging was performed on an ImageXpress Micro system and analyzed using MetaXpress 6. 2.6 Neurite Extension Well Average Analysis Imaging was performed on an ImageXpress Micro system, using a single protocol for all experiments. Fluorescence images were collected with a 10x objective, corresponding to a field-of-view of 1406um2 per image, tiled in a 6-by-6 grid with 10% overlap. Basic neurite extension as performed using the ‘Neurite Extension’ plugin in the MetaXpress 6 software. Data was aggregated by well, averaging outgrowth of Total # of Neuronal Cell Bodies / Total # of Neurites. Experiments were replicated with internal controls for normalizing, where one replicate corresponds to a single well’s average for that condition. Additional metrics were retrieved from the ‘Neurite Extension’ analysis including: # of Neuronal Cells, Total Outgrowth, # of Branches per Cell, # of Processes per Cell, and % Significant Outgrowth (data not shown). 2.7 Whole-cell Current Clamp Electrophysiology Action potentials recordings from mouse DRG neurons were acquired in current-clamp mode using an Axopatch 200B amplifier (Molecular Devices), Digidata 1440 digitizer and Clampex10 software (Molecular Devices). Whole-cell configuration was obtained in voltage-clamp mode before manually switching to current-clamp recording mode. Recordings were filtered at 5 kHz and sampled at 10 kHz. Patch pipettes were manufactured from soda lime capillary glass (Thermo Fisher Scientific) using a Sutter P-97 (Sutter Instrument) puller. Electrodes had a tip resistance of 2–4 MΩ when filled with an internal (pipette) solution. Pipette solution was comprised of; 130 mM K-gluconate, 4 mM Mg-ATP, 0.3 mM Na-GTP, 10 mM EGTA, 2 mM CaCl2, and 10 mM HEPES (adjusted to pH 7.2 with KOH). The bath was perfused with an external solution containing: 135 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2 and 10 mM HEPES (adjusted to pH 7.3 with NaOH). Patch clamp experiments were performed at room temperature (22 ± 1°C). DRG neuron dimensions (cell size) were estimated using a microscope eyepiece reticle (27 mm, 10 mm scale). For identification of IB4 + cells, DRG neurons were labelled with isolectin GS-B4 Alexa Fluor™ 488-conjugated antibody (Thermo Fisher, I21411) at least 15 minutes prior to transferring cells to the recording chamber. Images of DRG neurons in the recording chamber were acquired using a high resolution USB2.0 CMOS, 1280 x 1024, Camera (Thorlabs, DCC1645C) and ThorCam™ software. The resting membrane potential was determined immediately following whole-cell break-in at I = 0 pA. Threshold (Rheobase) was established by the first action potential to be elicited by a series of 3s stepwise current injections that increased from 0 pA in 10 pA increments. Action potential frequencies were calculated by the number of spikes during the 3 second stepwise current injections from 0 pA in 10 pA increments. Frequency of action potentials during acute retigabine application (RTG) was analyzed using the event detection, Threshold search feature of Clampfit 10.7. The frequency of action potentials was calculated by the number of spikes over the time exposed to a specific condition (control, RTG application, washout). The baseline was set at the resting membrane potential at the beginning of the recording. The threshold level for the inclusion criteria of an action potential was set at 0 mV. Patch clamp recordings and analysis were performed independent from DRG extraction and culture; as such, the electrophysiologist was blinded to all experimental groups. 2.8 EAE induction and behavioural assessment Experimental autoimmune encephalomyelitis (EAE) was induced in female C57BL/6 mice (n = 12; 8–10 weeks old; Charles River) by subcutaneously injecting 50 ug myelin oligodendrocyte glycoprotein (MOG 35 − 55 ) emulsified in complete Freund’s adjuvant (CFA) (Hooke Laboratories, Hook Kits™, EK-2110). Mice were given 100 ng pertussis toxin, Bordatella pertussis , (Hooke Laboratories, BT-0105) via IP injection on the day of induction and 24 hours later. Mice were monitored daily until the end of the experiment. Mice were assessed for pain hypersensitivity by measuring withdrawal thresholds to punctate mechanical stimulation using calibrated von Frey Hair monofilaments. Mechanical withdrawal thresholds were measured by the von Frey assay. Animals were repeatedly habituated to a 7.5x10x7.5 cm plexiglass box on a suspended mesh platform and allowed to explore for 30 minutes. On test days, the up-down method was used to determine the 50% withdrawal threshold ( 52 ). Briefly, filaments of various force (0.04-4 g) were applied to both hind paws and positive and negative responses were recorded. The 50% withdrawal threshold was calculated, as previously described ( 53 ). Prior to immunization, baseline withdrawal thresholds were recorded. Mice were then tested on day 7, 11 and 15 after immunization for EAE. Mice were treated with either RTG (10 mg/kg, IP, n = 6) or vehicle (DMSO is saline, 10%, n = 6) daily beginning on day 7 (after von Frey testing had been completed). DRGs from these mice were harvested on day 16 and subjected to electrophysiological analysis using the same parameters described above. All animal experiments were performed according to Canadian Council on Animal Care’s Guidelines and Policies with approval from University of Alberta Health Sciences Animal Care and Use Committee. 3. Results 3.1 DRG neuronal plasticity after stimulation with BMDM conditioned media Previous experiments from our group have shown that the conditioned media from male and female macrophages after pro-inflammatory stimulation elicit different pain phenotypes when injected into the hind paw of adult mice( 50 ). Male mice were relatively resistant to change in their tactile thresholds after injection of macrophage conditioned media while females exhibited heightened pain sensitivity ( 50 ). In the current study, we sought to establish the relationship between the growth status of sensory neurons and the conditioned media that elicited these different pain phenotypes. We first cultured DRG neurons in the base media used in macrophage conditioned media experiments to confirm if neurons would readily survive and extend neurites in a non-traditional media. Optimization experiments confirmed that DRG neurons attached and begin neurite outgrowth within 24 hours post-plating in base, ‘low-serum’ media and survive at least 96 hours in culture (data not shown). We next investigated the effect on outgrowth of neurons in the presence of conditioned media from unstimulated male or female bone marrow derived macrophages (macrophage conditioned media, MCM), or with the conditioned media from sex-matched macrophages after undergoing stimulation with tumor necrosis factor alpha (TNFα macrophage conditioned media, T-MCM). Neurons were cultured for 48 hours in the presence of these different conditioned media and structural plasticity was assessed by the pan-neuronal and cytoskeletal βIII tubulin staining (Fig. 1 B-G). Mean outgrowth of neurites from neurons was quantified at the experimental endpoint for both male (Fig. 1 H) and female (Fig. 1 I) treated neurons. Both male and female neurons incubated with the standard, unstimulated macrophage condition media (MCM) had no change in outgrowth compared to regular media treated controls. However, female DRG neurons specifically demonstrated an increase in neurite outgrowth after incubation with female T-MCM (1-way ANOVA, F 2,35 = 7.635, p = 0.0018, Fig. 1 I ) . Male neurons incubated with the male equivalent T-MCM did not exhibit a statistically significant increase in neurite outgrowth (1-way ANOVA, F 2,35 = 3.035, p = 0.0609, Fig. 1 H ) . 3.2 Electrophysiological assessment of DRG neurons early after culturing The function of neurons is dictated by their structure and excitability. We next assessed the excitability profiles of neurons treated with the different macrophage-conditioned medias using whole-cell current clamp recordings, incubated under identical conditions to previous experiments ( 54 ). We decided to focus on female DRG neurons due to previous work indicating a specific mechanism of pain in our autoimmune inflammatory mouse model ( 55 – 57 ) as well as the female bias of autoimmunity and chronic pain in the human population ( 58 ). We analyzed general metrics of neuronal excitability including the minimum electric current required to elicit an action potential (rheobase) and the resting membrane potential (RMP) after 6 hours of incubation in the different conditioned medias. We found that all conditions displayed varying degrees of spontaneous activity and quiescence, with the T-MCM treated neurons exhibiting the most spontaneous activity (Fig. 2 C). While neurons treated with MCM had more negative RMP compared to media only control (two-tailed t-test, MCM RMP: t = 2.156, p < 0.05, Fig. 2 E ) , the rheobase of these MCM treated neurons was unchanged (Fig. 2 D). Neurons treated with T-MCM however, exhibited a lower rheobase and had less negative resting membrane potential (RMP) relative to media only treated neurons (two-tailed t-test, T-MCM Rheobase: t = 2.019, p < 0.05; T-MCM RMP: t = 2.115, p < 0.05, Fig. 2 D, E). Interestingly, the T-MCM treated neurons that had a higher excitability profile overall (i.e. most spontaneous activity, lowered rheobase and less negative resting membrane potential) at 6 hours, correlated with the neurons exhibiting the greatest structural outgrowth at 48 hours described in Fig. 1 . These findings suggest a potential positive relationship between increased early excitability of the sensory neuron and later increased structural plasticity. 3.3 Electrophysiological assessment of DRG neurons in established cultures To evaluate neurons at later time points in culture, we conducted an identical electrophysiological assessment 48-hours after plating. Notably, we observed an overall shift in the excitability profiles in all treatment conditions. Specifically, the MCM condition exhibited no spontaneous activity at this later timepoint, also exhibiting an elevated rheobase and the most negative resting membrane potential (two-tailed t-test, MCM Rheobase: t = 2.410, p < 0.05; MCM RMP: t = 0.639, p = 0.53, Fig. 3 C, D, E ) . In contrast, the T-MCM treated neurons still displayed some spontaneous activity but were identical in rheobase and resting membrane potential compared to their media only controls (Fig. 3 C, D, E). 3.4 Growth status and electrophysiological characteristics of DRG neurons As we were interested in relating electrical excitability to structural plasticity, we noted that neurons adopt distinct morphologies 48-hours after plating consistent with observations from Fig. 1 . At this time point, neurons were consistently observed to fall into three categories: a population of neurons yet to extend any neurites ( no outgrowth ); a population that display a highly complex, ‘arborizing’ phenotype characterized by numerous branches from the primary outgrowth ( arborizing ); and a population that extend ‘elongating’ neurites characterized by low amounts of branching and a high displacement of primary growth ( elongating ) (Fig. 4 A-C). Identifying and subdividing these morphological populations by visual inspection at the time of patch clamp recording, we found that the spontaneous activity seen in the T-MCM condition was restricted to neurons with no outgrowth (Fig. 4 Ai) . Furthermore, neurons in the ‘arborizing’ and ‘elongating’ categories were primarily quiescent regardless of treatment condition (Fig. 4 Bi, Ci ). Overall, these data suggest that incubation of neurons with different types of inflammatory-conditioned media convey unique excitability profiles in neurons that have yet to display outgrowth and that this can inform the extent of outgrowth. 3.5 Structural plasticity is modulated by Kv7 channel activity To assess the direct impact that electrical activity/excitability has on structural plasticity, we turned to pharmacological manipulation of these neurons in our in vitro system. Retigabine (RTG) is an anticonvulsant drug that was initially developed as a treatment for epilepsy. It acts as a positive allosteric modulator of voltage-gated potassium channels (Kv7), which stabilizes the resting membrane potential and reduces neuronal excitability ( 59 – 61 ). We repurposed this drug to act as a general inhibitor of neuronal activity to mimic the quiescent phenotype we identified in vitro . The effect of RTG is near instantaneous as repetitively evocable neurons exhibit a complete loss of electrical activity upon wash-in of RTG during recording (Fig. 5 A, B). Repetitive firing of neurons is then restored when RTG is washed off (1-way ANOVA, F 2,14 = 4.40, p < 0.05, Control vs. RTG, p = 0.0584, RTG vs. Washout, p < 0.05, Fig. 5 B ) . Basal neurite outgrowth of both male and female DRG neurons cultured in the presence of retigabine is also significantly reduced after 48 hours in culture (2-way ANOVA, treatment effect F 1,92 = 15.54, p < 0.05, Fig. 5 H). Although the quiescent phenotype was primarily shown in the later stages of outgrowth, we additionally tested the addition of RTG at a later timepoint (24 hours post plating), but this had an identical effect to the early addition (data not shown). These data indicate that neurons are dependent on some level of spontaneous activity for axonal outgrowth. 3.6 Plasticity driven by BMDM conditioned media is dependent on excitability. To assess whether the neurite growth promoting effects of T-MCM are on dependent on changes to the excitability profile of the neurons, we treated DRG neurons for 48 hours with T-MCM in the presence or absence of RTG. As seen previously (see Fig. 1 ), compared to neurons treated with media only, T-MCM promoted a significant increase in mean outgrowth (Fig. 6 A, B, D). This effect was abrogated by co-incubation of T-MCM with RTG (1-way ANOVA, treatment effect F 2,33 = 194, p < 0.0001, Fig. 6 C, D ) . 3.7 Attenuating DRG excitability prevents pain in mice with EAE Given the robust effects on neural activity and outgrowth from DRG neurons treated with RTG in the presence of inflammatory macrophage conditioned media, we next wanted to determine how this might impact pain in the context of neuroinflammatory disease. We have previously described the emergence of pain hypersensitivity to tactile stimulation in the hind paws of mice with experimental autoimmune encephalomyelitis (EAE) that is accompanied by significant increases in DRG neuron excitability and activity ( 55 ). Mice were immunized for EAE and seven days after immunization we began treatment with either RTG (10 mg/kg, IP) or vehicle-control. Vehicle treated mice exhibited well characterized, significant reductions in paw withdrawal thresholds indicative of pain hypersensitivity that became statistically different from baseline values on day 11 post immunization. In contrast, mice that began treatment with RTG on day 7 displayed a complete reversal of this behavioural hypersensitivity (Fig. 7 A, 2 -way RM ANOVA, treatment effect F 1,10 = 6.035, p < 0.05). RTG treatment effectively supressed action potential firing from DRG nociceptors compared to vehicle treated mice (Fig. 7 B) and altered the overall excitability of these neurons by lowering the resting membrane potential and significantly elevating the rheobase ( Fig. 7 C, D ) . Collectively, reducing DRG excitability with RTG limits DRG axon outgrowth in vitro and pain hypersensitivity following EAE in vivo . 4. Discussion 4.1 BMDM Conditioned Media conveys plastic potential to DRG neurons Building upon previous research indicating sex differences in innate immune inflammatory activity, we have investigated the sex-specific changes in DRG sensory neuron structural plasticity and excitability when exposed to inflammatory mediators from innate immune cells (BMDMs). Here, we demonstrate that incubating peripheral neurons with sex-matched inflammatory conditioned media from innate immune cells can impact both the structural plasticity and the excitability profile of these neurons. We find that excitability parameters of neurons that exhibit high amounts of structural outgrowth correspond to the greatest shift in excitability, becoming quiescent after an initial period of high spontaneous activity. Furthermore, we demonstrate that a pharmacological intervention limiting neuronal activation can prevent this capacity for structural plasticity in the inflammatory conditions. 4.2 DRG neurons are highly plastic cells In early neuronal development, the maturation of neurons involves complex signaling cascades that are crucial for the transcriptional profile and development of the functional component of these cells ( 62 – 64 ). Maintenance of neuronal circuitry in adult organisms is tightly regulated and these plastic processes can result in adaptive or maladaptive consequences depending on the context. For instance, heightened pain sensitivity during inflammation may trigger acute plasticity that is adaptive for preventing further injury, but intense or prolonged inflammation also has the capacity to promote signalling within sensory neurons that leads to long term changes and chronic pain syndromes ( 65 – 67 ). Pain management is a critical aspect of first-line treatments for nervous system injury. However, strategies that directly address pain without considering the structural consequences have proven to be ineffective in preventing chronic pain. Thus, it may be worthwhile to consider both the functional and structural aspects of neural plasticity when developing pain management strategies. This involves understanding the interplay between activity-dependent phenomena and regenerative outgrowth and investigating the mechanisms that link neuronal excitability and structural plasticity. Adopting such an approach could lead to more effective treatments for chronic pain syndromes. Emerging from current paradigms that tend to view heightened neuronal excitability following injury as detrimental due to the risk of excitotoxicity( 68 , 69 ), the current study supports an alternative perspective: the necessity of preserving an early phase of excitability to facilitate proper neural regeneration. While efforts to curb excessive excitability post-injury are well-founded, these interventions might inadvertently hinder the inherent pro-regenerative processes associated with the initial surge in neuronal excitability and activity. By excessively dampening this early phase, there could be an unintended loss of the favorable conditions that drive regenerative mechanisms ( 70 ). Thus, striking a balance between mitigating excitotoxicity and allowing for the temporally distinct, early phase excitability, could hold the key to unlocking more effective neural regeneration strategies. 4.3 DRG neuronal plasticity involves alterations in excitability Recent work has explored a related line of inquiry and demonstrated that in vitro , DRG neurons undergo transcriptional de-maturation by downregulating genes critical for synaptic transmission ( 28 ). Genetic deletion of core components of the synapse essential for neurotransmission – such as RIM1/2 or Munc13 – significantly enhances axon growth and regeneration while reducing branching ( 28 ). Together with the findings presented here, these results suggest that inflammation-induced molecular cascades that promote neurite outgrowth are inexorably linked with changes in neural excitability and activity. Activity-dependent phenomena are well-documented in regenerative contexts. Although it is rare for a neuron to lose all input in vivo , as it does in a dissociative culture system, evidence indicates that peripheral neurons may atrophy or even die following long-term sensory deprivation ( 71 – 73 ). In this context, it is possible that pain after injury plays a crucial role in promoting the proper re-maturation of healthy neuronal circuitry. Therefore, the excessive use of analgesics in clinical settings may potentially hinder regenerative outgrowth ( 74 , 75 ). The experience of pain may be a necessary part of the healing process; acknowledging and pursuing this hypothesis may lead to better outcomes. While the precise mechanism linking excitability and structural plasticity remains unclear, several plausible hypotheses can be tested. One such hypothesis is that ionic gradients along neuronal membranes allow for calcium influx during action potentials. Calcium is a well-known second messenger and cofactor for many different molecular cascades that promote both the growth of axons but also neuronal sensitization and pain hypersensitivity( 76 , 77 ). As a driver of calcium currents, inflammation, a central player in injury responses, is well-characterized in driving calcium currents( 78 – 80 ). The early increases in excitability may support increased calcium flux and drive the increased growth of female DRG neurons in response to MCM stimulated with TNFα (T-MCM). Furthermore, male DRG neurons stimulated with T-MCM exhibited a slight but not statistically significant changes in neurite outgrowth, suggesting that the interplay of inflammatory modulation of ionic gradients influencing outgrowth may be sex specific. This interplay suggests potential avenues for pain management, centered around the crosstalk between inflammation and neuronal plastic responses. 4.4 DRG neuronal plasticity: the relationship between excitability and pain Plasticity, excitability, and pain are interconnected processes in the nervous system. The maturation and maintenance of neuronal circuits involve signaling cascades that regulate the functional component and structural generation and pruning of synapses, membrane potentials, and sensory receptor insertion. These processes involve different cell types, including glia( 81 ) and immune cells( 82 ), and are activity driven ( 83 , 84 ). The consequences of these plastic processes can be adaptive or maladaptive, such as sickness behavior and chronic pain. Pain is one of the most common outcomes after injury and inflammation, and has been widely reported in the animal model of neuroinflammatory disease, EAE( 85 – 88 ). Interestingly, pain in the EAE model is also associated with significant changes occurring at the level of DRG including increased inflammation and increased neuronal activity/hyperexcitability ( 54 , 55 ). Our lab has also recently reported that common markers associated with increased neural plasticity and growth are increased in DRG neurons of EAE mice in a sex specific manner, with females exhibiting higher levels of ATF3 and pCREB ( 89 ). Taken together with the results of the experiments described here, we can speculate that DRG neurons from female mice may use the inflammatory stimulation as a priming signal to trigger a change in their growth status. While our results with RTG in the EAE model suggest that inhibiting this increase in neural activity in response to inflammation may be beneficial for pain in the short term, how this treatment affects the long-term plasticity of these neurons remains to be determined. 5. Conclusions The relationship between neuronal excitability and structural plasticity is complex and dynamic, with evidence suggesting that they may be inextricably linked. Both in vitro and in vivo studies have demonstrated the importance of activity-dependent phenomena in promoting regenerative processes and maintaining nervous system function. The role of pain in this process is an intriguing area of investigation, with some evidence suggesting that it may be necessary for proper re-maturation of healthy circuitry. The mechanisms underlying the link between excitability and plasticity remain unclear, but the role of Ca 2+ as a second messenger and cofactor for molecular cascades presents a plausible hypothesis that warrants further investigation. Overall, a deeper understanding of the relationship between neuronal excitability and structural plasticity has important implications for the development of new strategies to promote nerve regeneration and functional recovery after injury or disease. Declarations Ethics declaration All animal experiments were performed according to Canadian Council on Animal Care’s Guidelines and Policies with approval from University Of Alberta Health Sciences Animal Care and Use Committee. Author Contribution TNF, SML, ADM and TH conducted experiments and analyzed data. TNF wrote the manuscript. BJH, JRP, HTK and BJK designed experiments and edited the manuscript. Acknowledgements: Funding for this project was provided by operating grants from the MS Society of Canada (EGID-MSSC-3761), a Project grant from the Canadian Institutes for Health Research (CIHR, FRN162434), the Alberta MS Collaboration and the University Hospital Fund (University of Alberta). TNF was supported by studentships from the MS Society of Canada and by the Alexander Graham Bell Canada Graduate Scholarship from NSERC. The authors wish to thank G. Tenorio for assistance in figure preparation. Availability of data and materials The datasets supporting the conclusions of this manuscript are available upon request. References Solaro C, Trabucco E, Messmer Uccelli M. Pain and Multiple Sclerosis: Pathophysiology and Treatment. Curr Neurol Neurosci Rep. 2013;13(1). Stephen GW. Acquired channelopathies in nerve injury and MS. Neurology. 2001;56(12):1621. Urits I, Adamian L, Fiocchi J, Hoyt D, Ernst C, Kaye AD, Viswanath O. 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Sex differences in the inflammatory response of the mouse DRG and its connection to pain in experimental autoimmune encephalomyelitis. Sci Rep. 2022;12(1). Additional Declarations No competing interests reported. Supplementary Files Friedmanetal.SupplementaryTable11.pdf Supplementary Table 1. Summary of antibodies and reagents used in immuno-histo/cyto-chemistry experiments Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4094312","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":279484805,"identity":"e91d65d5-8f43-4dc3-a054-4e9cfa8ec75c","order_by":0,"name":"Timothy N. 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(A) Schematic of DRG neuron cultures and CM stimulation. (B-I) Representative images of neurites stained with βIII tubulin at experimental endpoint. Quantification of male (J) and female (K) neurites with sex-matched condition media treatments, normalized to the extent of outgrowth measured in the control (blank) conditions. Scale bar = 50μm; * p \u0026lt; 0.05. Data is analyzed by one-way ANOVA with Dunnett’s multiple comparison test. Bar graphs represent mean ± SEM.\u003c/p\u003e","description":"","filename":"OnlineFigure1..png","url":"https://assets-eu.researchsquare.com/files/rs-4094312/v1/f9f5852d7cb50b248c74713b.png"},{"id":52791836,"identity":"c69bb54e-26c8-4587-85fd-5d3d189779c3","added_by":"auto","created_at":"2024-03-15 20:10:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":479213,"visible":true,"origin":"","legend":"\u003cp\u003eWhole-cell patch clamp recordings of female DRG neurons incubated in BMDM-conditioned media (CM) for 6 hours reveals distinct firing patterns and electrophysiological profiles. (A) Schematic of experimental workflow; neurons were recorded 6 hours post-plating and CM stimulation. (B) Example traces of the three characteristic firing patterns: Quiescent, Evoked, and Spontaneous. (C) Quantification of categorical labels for stereotypic firing patterns across CM treatments. Quantification of rheobase (D), resting membrane potential (RMP) (E) across CM treatments. * p \u0026lt; 0.05. Data is analyzed by unpaired two-tailed t-test. Bar graphs represent mean ± SEM. Categorical data is represented by parts-of-whole transformation and analyzed by Fisher’s exact test on untransformed values.\u003c/p\u003e","description":"","filename":"OnlineFigure2..png","url":"https://assets-eu.researchsquare.com/files/rs-4094312/v1/2767fbae66ec08c7e16b7ac8.png"},{"id":52791627,"identity":"9a79d7c1-8a4c-4d9c-92b9-7398e95d204c","added_by":"auto","created_at":"2024-03-15 20:02:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":468969,"visible":true,"origin":"","legend":"\u003cp\u003eWhole-cell patch clamp recordings of established female DRG neurons incubated in BMDM-conditioned media (CM) reveals distinct firing patterns and electrophysiological profiles. (A) Schematic of experimental workflow; neurons were recorded 48 hours post-plating and CM stimulation. (B) Example traces of the three characteristic firing patterns: Quiescent, Evoked, and Spontaneous. (C) Quantification of categorical labels for stereotypic firing patterns across CM treatments. Quantification of rheobase (D) and resting membrane potential (RMP) (E) across CM treatments. * p \u0026lt; 0.05. Data is analyzed by unpaired two-tailed t-test. Bar graphs represent mean ± SEM. Categorical data is represented by parts-of-whole transformation.\u003c/p\u003e","description":"","filename":"OnlineFigure3..png","url":"https://assets-eu.researchsquare.com/files/rs-4094312/v1/52e1d148153777f2ad0c8b26.png"},{"id":52791629,"identity":"4ec42da5-4dcf-4a38-895f-3ebef4a06dfa","added_by":"auto","created_at":"2024-03-15 20:02:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4228050,"visible":true,"origin":"","legend":"\u003cp\u003eWhole-cell patch clamp recordings of established female DRG neurons with distinct structural phenotypes have distinct firing patterns and electrophysiological profiles. (A-C) Representative brightfield images of the distinct neuronal phenotypes (no outgrowth, arborizing, elongating) observed at 48 hours post-plating and CM stimulation. (Ai-Ci) Quantification of categorical labels for stereotypic firing patterns across CM treatments. Categorical data is represented by parts-of-whole transformation.\u003c/p\u003e","description":"","filename":"OnlineFigure4..png","url":"https://assets-eu.researchsquare.com/files/rs-4094312/v1/b8393bcd63567b15bf67c714.png"},{"id":52791632,"identity":"b98185bd-adee-4489-892b-28a22bf8c217","added_by":"auto","created_at":"2024-03-15 20:02:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1772730,"visible":true,"origin":"","legend":"\u003cp\u003eNeurite outgrowth of DRG neurons is diminished by retigabine (RTG) treatment. (A) Example traces of spontaneously firing DRG neurons which become quiescent under RTG treatment. (B) Quantification of action potential (AP) frequency in binned time domains centred around the period of RTG stimulation. (C) Individual traces of unstimulated and RTG stimulated APs. Note the deeper AHP curve and slower return to RMP in the RTG trace. (D-G) Representative images of neurites stained with βIII tubulin at experimental endpoint after treatment with blank media (D and F) or RTG (E and G). (H) Quantification of male and female neurites after treatment with RTG or control (blank media). \u0026nbsp;Scale bar = 50μm; * p \u0026lt; 0.05. Data is analyzed by unpaired two-tailed t-test. Bar graphs represent mean ± SEM. Categorical data is represented by parts-of-whole transformation.\u003c/p\u003e","description":"","filename":"OnlineFigure5..png","url":"https://assets-eu.researchsquare.com/files/rs-4094312/v1/821fe7377d8ad5a1450f4f5a.png"},{"id":52791630,"identity":"9b4bc14c-7aeb-409d-9106-72b454089999","added_by":"auto","created_at":"2024-03-15 20:02:30","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":100816,"visible":true,"origin":"","legend":"\u003cp\u003eRetigabine prevents the growth promoting action of inflammatory conditioned media. (A-C) Representative images of neurites\u0026nbsp;from mouse DRG neurons after culture for 48h with\u0026nbsp;either no treatment, BMDM-conditioned media treatment, or BMDM-conditioned media + 100 µM RTG treatment. (D) Quantification of mean area of neurite outgrowth. Scale bar = 100µm, and dashed line represents mean of untreated control; ****P\u0026lt;0.0001. Data is analyzed by one-way ANOVA. Bar graphs represent mean ± SEM normalized to untreated controls, and dashed line represents mean of untreated control bar.\u0026nbsp;\u003c/p\u003e","description":"","filename":"OnlineFigure6..png","url":"https://assets-eu.researchsquare.com/files/rs-4094312/v1/efe44a8333c5c02bccc5dab6.png"},{"id":52791633,"identity":"397fe63e-00c5-40c1-b412-a90c40121e97","added_by":"auto","created_at":"2024-03-15 20:02:30","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":52994,"visible":true,"origin":"","legend":"\u003cp\u003eRetigabine prevents pain hypersensitivity in the EAE model of neuroinflammatory disease. (A) Withdrawal thresholds to von Frey hair stimulation in mice immunized for EAE and treated with vehicle control (left) or RTG (10mg/kg) (right). Vehicle treated mice exhibit stereotypical reductions in withdrawal thresholds indicative of pain hypersensitivity. Mice that begin treatment with RTG on day 7 post-immunization exhibit a complete reversal of these behaviours. *P\u0026lt;0.05, two-way ANOVA, Tukey host hoc test. (B) Number of action potentials from DRGNs of EAE immunized mice, treated with retigabine (RTG, 100 μM) vs. vehicle control. Frequency of action potentials (Hz) were recorded from 3s current injections in 10 pA increments. (C) Resting membrane potential (RMP, mV) from DRGNs of EAE immunized mice treated with retigabine (RTG, 100 μM) vs. vehicle control. **** P\u0026lt;0.001, two-tailed unpaired Student’s t-test, DRGNs treated with RTG (100 μM) vs. vehicle control. (D) Action potential injection threshold (rheobase, pA) from DRGNs of EAE immunized mice treated with retigabine (RTG, 100 μM) vs. vehicle control. *** P\u0026lt;0.001, two-tailed unpaired Student’s t-test, DRGNs treated with RTG (100 μM) vs. vehicle control.\u003c/p\u003e","description":"","filename":"OnlineFigure7..png","url":"https://assets-eu.researchsquare.com/files/rs-4094312/v1/5fb8ae53de264417c675dccd.png"},{"id":52792456,"identity":"ce065182-3607-4aa1-9281-022dfd776dca","added_by":"auto","created_at":"2024-03-15 20:18:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2655968,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4094312/v1/cd83a38a-dd34-4a4e-b886-e76a5e760229.pdf"},{"id":52791628,"identity":"1378fcb9-481a-4c76-8e9b-00acb746cad4","added_by":"auto","created_at":"2024-03-15 20:02:30","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":23245,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 1. Summary of antibodies and reagents used in immuno-histo/cyto-chemistry experiments\u003c/p\u003e","description":"","filename":"Friedmanetal.SupplementaryTable11.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4094312/v1/e0d54542c293e908a46eb1f5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Plasticity of mouse dorsal root ganglion neurons by innate immune activation is influenced by electrophysiological activity","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePain is a prevalent symptom in autoimmune diseases such as multiple sclerosis and rheumatoid arthritis, and its management remains a challenge. Based on the current understanding of their pathophysiological mechanisms, these diseases involve the dysregulated activation of the innate and adaptive immune system, leading to high levels of inflammation that damage tissue in the periphery and central nervous system along with the sensitization of primary sensory neurons, culminating in pain and disability (\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInflammation is not always detrimental, however. For instance, after injury, immune-mediated inflammation can trigger regenerative mechanisms, assisting in debris clearance or promoting regeneration (\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Inflammation is a dynamic process that can be initiated in response to various stimuli and then resolve when no longer required (\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). In individuals with multiple sclerosis (MS), an inflammatory autoimmune disease characterized by periods of attack and remission, these inflammatory processes can coincide with periods of disability. Although inflammation can resolve in MS, it is not always the case, and persistent low-grade inflammation may lead to neurodegeneration, emphasizing the importance of the resolution process (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Chronic inflammation can lead to disability and may also play a role in the induction of chronic pain. As inflammation is adaptive and designed to resolve, its persistence may indicate an impairment in the resolution process.\u003c/p\u003e \u003cp\u003eDorsal root ganglion (DRG) neurons have an innate capacity for growth after injury to their distal axons(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). This plasticity can be demonstrated in cell culture. Dissociated DRG neurons can readily establish and send out axons in short periods of time, a process that can be encouraged by the priming of these cells with \u0026lsquo;conditioning lesions\u0026rsquo;(\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e) or discouraged by inhibition of retrograde transport along the injured axon(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Furthermore, the structural plasticity of neurons can be modulated by pharmacological manipulation of ion channels. For example, inhibition of Ca\u003csup\u003e2+\u003c/sup\u003e channel trafficking to the membrane(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) with gabapentin can modulate synaptogenesis and neural circuitry rewiring(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). This effect is also observed \u003cem\u003ein vivo\u003c/em\u003e as gabapentinoids can be used to increase regeneration of axons after spinal cord injury (\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Similarly, Baclofen reduces the amplitude of voltage-gated Ca\u003csup\u003e2+\u003c/sup\u003e-currents in DRG neurons (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) and promotes their regeneration after spinal cord injury. The growth state of sensory neurons can also be modulated by inflammation (\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Not only can the inflammatory response to injury, even potentially sterile injury, have effects on outgrowth, but inflammation triggered by adaptive or innate immunity can also affect outgrowth. Therefore, inflammation can be seen as a critical driver of structural plasticity and the growth status of sensory neurons (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt is also widely recognized that inflammation can alter the excitability of neurons, and traditional mechanisms of inflammation can result in increased action potential frequencies and lowered thresholds for action potential generation (i.e., sensitization). Inflammatory mediators like TNFα and IL-1β can regulate neuronal expression of voltage-gated sodium and potassium channels, increasing neuronal excitability (\u003cspan additionalcitationids=\"CR35 CR36 CR37\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). This phenomenon has been observed in both electrophysiological recordings of single cells (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e) and nerve conduction studies (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Heightened excitability and sensitization of peripheral sensory neurons results in an increased perception of pain, although this phenomenon may resolve as inflammation subsides.\u003c/p\u003e \u003cp\u003eWhile inflammation can independently affect both the excitability and growth status of neurons, increasing excitability through electrical stimulation can promote axonal growth, indicating that there may be a connection between the two processes (\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Additionally, some researchers have proposed that the inflammation-induced changes in excitability may be directly involved in changes to neuronal growth status (\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Increasing neuronal excitability using optogenetics enhances axonal growth in the presence of an inflammatory stimulus (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). This suggests that the increase in neuronal excitability may be one of the mechanisms by which inflammation promotes structural plasticity.\u003c/p\u003e \u003cp\u003eOverall, the relationship between inflammation, excitability, and the capacity for structural plasticity of DRG neurons is complex and likely involves multiple signaling cascades. To investigate this relationship, we conducted a series of experiments examining the effect of inflammation on neuronal excitability and neurite outgrowth. Our findings indicate that an early phase of increased neuronal excitability results in an increased growth status of primary sensory neurons, and that the neuron\u0026rsquo;s excitability is intricately linked to its structural phenotype.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 \u003cem\u003eAnimals\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eUnless otherwise stated, all mice are wild-type C57/BL6, purchased from Charles River and included in experiments at 8 to 10 weeks of age (C57BL/6NCrl, Strain Code: 027, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.criver.com/products-services/find-model/c57bl6-mouse?region=24#\u003c/span\u003e\u003cspan address=\"https://www.criver.com/products-services/find-model/c57bl6-mouse?region=24#\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). All mice were given access to food and water ad libitum and maintained in the University of Alberta facility under 12 hour light/dark cycles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 \u003cem\u003eBone Marrow Derived Macrophage (BMDM) Cultures and Conditioned Media\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eBone marrow derived macrophage cultures were generated under a previously described protocol(\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Briefly, female and male 8 to 10 week old C57BL/6 mice were euthanized by Euthanyl\u0026reg; (sodium pentobarbital) injected intraperitoneally. After euthanasia, bone marrow cells were isolated from femurs as single-cell suspensions and filtered to remove excess debris. Bone marrow cells were allowed to differentiate into macrophages (BMDMs) for 8 to 10 days in L929 media. Upon completed differentiation, bone marrow derived macrophages were harvested and resuspended into freshly prepared DMEM containing 1% each of sodium pyruvate, Glutamax, Pen/Strep, and FBS. This media is hereon referred to as \u0026lsquo;low-serum media\u0026rsquo;. Cells were cultured in T75 flasks and were allowed to rest for 48 hours after which a full change of media was performed. A small volume of TNFα (final concentration: 1ng/ml) or vehicle (low-serum media) was added and allowed to incubate for 24 hours. After incubation, the stimulation media was fully replaced with a final volume of low-serum media and the stimulated BMDMs were allowed to rest for 6 hours. This \u0026lsquo;6-hour CM\u0026rsquo; was captured at the experimental endpoint, aliquoted, and stored at -80\u0026deg;C until further usage avoiding freeze-thaw cycles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 \u003cem\u003eDorsal Root Ganglia Neuron Cultures\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eDorsal root ganglion (DRG) neurons were acquired from male and female mice, using a modified protocol from previously described work(\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Briefly, animals were euthanized by Euthanyl\u0026reg; (sodium pentobarbital) injected intraperitoneally. After injection, animals were monitored for level of consciousness and dissections did not proceed until no response to toe pinch or corneal contact was observed. Cardiac punctures were performed to confirm euthanization and animals were perfused with 10mL of ice-cold saline. Perfused animals underwent spinal laminectomies and gross dissection of the spinal cord to expose the DRG. DRG were micro-dissected from the spinal column, taking care to remove as much residual nerve as possible while avoiding damage to the DRG. Isolated DRG were placed in ice-cold HBSS -/- until dissections were completed. To acquire single-cell suspensions of DRG neurons, the HBSS -/- was replaced with a warmed dilution of Stemxyme I (Worthington, LS004106) and DNase (Worthington, LS002007) in HBSS -/- and incubated in a 37\u0026deg;C water bath for approximately 45 minutes. Following digestion, enzyme activity was quenched with equal volumes of low ovomucoid (Worthington, LS003086) and mechanically titrated with a P1000 pipette until tissue was fully dissociated. The cell suspension was filtered through a 70 um mesh filter (Biologix, 15-1070) and gently layered on top of a 20% BSA solution. This layered gradient was spun at 300G for 10 minutes at room temperature to pellet neuronal cells and remove cellular debris (mainly myelin). Debris was gently removed, and the cell pellet was resuspended in a small volume of 0.5% BSA in HBSS -/- and quantified for neuronal yield with a hemocytometer. The cell suspension was adjusted to 1000 cells / 100 uL and 100 uL of this suspension was added to equilibrated media in a 24 well (CellVis, P24-1.5H-N), poly-D-lysine coated plate (Sigma, P6407). For electrophysiological recordings, cells were plated onto glass coverslips (Fisher, 1254583) identically coated in poly-D-lysine and transferred into the electrophysiological recording setup at experimental timepoints. Unless otherwise stated, plated neurons were incubated at 37\u0026deg;C with 10% CO\u003csub\u003e2\u003c/sub\u003e for 48 hours. A full list of reagents used in cell culture experiments can be found in \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 \u003cem\u003ePharmacology\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eFor experiments involving retigabine (RTG), a master stock of 50mM was prepared by reconstituting 10ng of lyophilized RTG (Tocris, 6233) in DMSO, aliquoted, and stored at -20\u0026deg;C. On experimental timepoints, a 10x concentration (200 uM) was prepared by dilutions of thawed aliquots in DMEM. The final concentration of RTG was adjusted by direct dilution into plate wells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 \u003cem\u003eImmunocytochemistry\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eAt experimental endpoints, an equivalent volume of 8% PFA was added to the media of culture plates containing the adherent cells. Samples were incubated at room temperature in the diluted fixative for 15 minutes and then washed 3 times in DPBS. Permeabilization and non-specific IgG binding was blocked by one hour incubation with 10% NDS in DPBSTx 0.2% at room temperature, followed by an overnight incubation with rabbit anti-βIII tubulin (Sigma, T2200) diluted in DPBS. On the following day, cells were washed 3 times in DPS, incubated with DAPI (Invitrogen, D1306) and 594-conjugated anti-rabbit secondary antibodies (Jackson ImmunoResearch, 711-586-152) for one hour at room temperature before a final three washes. Imaging was performed on an ImageXpress Micro system and analyzed using MetaXpress 6.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.6\u003c/b\u003e \u003cb\u003eNeurite Extension Well Average Analysis\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eImaging was performed on an ImageXpress Micro system, using a single protocol for all experiments. Fluorescence images were collected with a 10x objective, corresponding to a field-of-view of 1406um2 per image, tiled in a 6-by-6 grid with 10% overlap. Basic neurite extension as performed using the \u0026lsquo;Neurite Extension\u0026rsquo; plugin in the MetaXpress 6 software. Data was aggregated by well, averaging outgrowth of Total # of Neuronal Cell Bodies / Total # of Neurites. Experiments were replicated with internal controls for normalizing, where one replicate corresponds to a single well\u0026rsquo;s average for that condition. Additional metrics were retrieved from the \u0026lsquo;Neurite Extension\u0026rsquo; analysis including: # of Neuronal Cells, Total Outgrowth, # of Branches per Cell, # of Processes per Cell, and % Significant Outgrowth (data not shown).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.7\u003c/b\u003e \u003cb\u003eWhole-cell Current Clamp Electrophysiology\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eAction potentials recordings from mouse DRG neurons were acquired in current-clamp mode using an Axopatch 200B amplifier (Molecular Devices), Digidata 1440 digitizer and Clampex10 software (Molecular Devices). Whole-cell configuration was obtained in voltage-clamp mode before manually switching to current-clamp recording mode. Recordings were filtered at 5 kHz and sampled at 10 kHz. Patch pipettes were manufactured from soda lime capillary glass (Thermo Fisher Scientific) using a Sutter P-97 (Sutter Instrument) puller. Electrodes had a tip resistance of 2\u0026ndash;4 MΩ when filled with an internal (pipette) solution. Pipette solution was comprised of; 130 mM K-gluconate, 4 mM Mg-ATP, 0.3 mM Na-GTP, 10 mM EGTA, 2 mM CaCl2, and 10 mM HEPES (adjusted to pH 7.2 with KOH). The bath was perfused with an external solution containing: 135 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2 and 10 mM HEPES (adjusted to pH 7.3 with NaOH). Patch clamp experiments were performed at room temperature (22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C). DRG neuron dimensions (cell size) were estimated using a microscope eyepiece reticle (27 mm, 10 mm scale). For identification of IB4\u0026thinsp;+\u0026thinsp;cells, DRG neurons were labelled with isolectin GS-B4 Alexa Fluor\u0026trade; 488-conjugated antibody (Thermo Fisher, I21411) at least 15 minutes prior to transferring cells to the recording chamber. Images of DRG neurons in the recording chamber were acquired using a high resolution USB2.0 CMOS, 1280 x 1024, Camera (Thorlabs, DCC1645C) and ThorCam\u0026trade; software. The resting membrane potential was determined immediately following whole-cell break-in at I\u0026thinsp;=\u0026thinsp;0 pA. Threshold (Rheobase) was established by the first action potential to be elicited by a series of 3s stepwise current injections that increased from 0 pA in 10 pA increments. Action potential frequencies were calculated by the number of spikes during the 3 second stepwise current injections from 0 pA in 10 pA increments. Frequency of action potentials during acute retigabine application (RTG) was analyzed using the event detection, Threshold search feature of Clampfit 10.7. The frequency of action potentials was calculated by the number of spikes over the time exposed to a specific condition (control, RTG application, washout). The baseline was set at the resting membrane potential at the beginning of the recording. The threshold level for the inclusion criteria of an action potential was set at 0 mV. Patch clamp recordings and analysis were performed independent from DRG extraction and culture; as such, the electrophysiologist was blinded to all experimental groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 \u003cem\u003eEAE induction and behavioural assessment\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eExperimental autoimmune encephalomyelitis (EAE) was induced in female C57BL/6 mice (n\u0026thinsp;=\u0026thinsp;12; 8\u0026ndash;10 weeks old; Charles River) by subcutaneously injecting 50 ug myelin oligodendrocyte glycoprotein (MOG\u003csub\u003e35\u0026thinsp;\u0026minus;\u0026thinsp;55\u003c/sub\u003e) emulsified in complete Freund\u0026rsquo;s adjuvant (CFA) (Hooke Laboratories, Hook Kits\u0026trade;, EK-2110). Mice were given 100 ng pertussis toxin, \u003cem\u003eBordatella pertussis\u003c/em\u003e, (Hooke Laboratories, BT-0105) via IP injection on the day of induction and 24 hours later. Mice were monitored daily until the end of the experiment.\u003c/p\u003e \u003cp\u003eMice were assessed for pain hypersensitivity by measuring withdrawal thresholds to punctate mechanical stimulation using calibrated von Frey Hair monofilaments. Mechanical withdrawal thresholds were measured by the von Frey assay. Animals were repeatedly habituated to a 7.5x10x7.5 cm plexiglass box on a suspended mesh platform and allowed to explore for 30 minutes. On test days, the up-down method was used to determine the 50% withdrawal threshold (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Briefly, filaments of various force (0.04-4 g) were applied to both hind paws and positive and negative responses were recorded. The 50% withdrawal threshold was calculated, as previously described (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Prior to immunization, baseline withdrawal thresholds were recorded. Mice were then tested on day 7, 11 and 15 after immunization for EAE. Mice were treated with either RTG (10 mg/kg, IP, n\u0026thinsp;=\u0026thinsp;6) or vehicle (DMSO is saline, 10%, n\u0026thinsp;=\u0026thinsp;6) daily beginning on day 7 (after von Frey testing had been completed). DRGs from these mice were harvested on day 16 and subjected to electrophysiological analysis using the same parameters described above. All animal experiments were performed according to Canadian Council on Animal Care\u0026rsquo;s Guidelines and Policies with approval from University of Alberta Health Sciences Animal Care and Use Committee.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 DRG neuronal plasticity after stimulation with BMDM conditioned media\u003c/h2\u003e \u003cp\u003ePrevious experiments from our group have shown that the conditioned media from male and female macrophages after pro-inflammatory stimulation elicit different pain phenotypes when injected into the hind paw of adult mice(\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Male mice were relatively resistant to change in their tactile thresholds after injection of macrophage conditioned media while females exhibited heightened pain sensitivity (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). In the current study, we sought to establish the relationship between the growth status of sensory neurons and the conditioned media that elicited these different pain phenotypes. We first cultured DRG neurons in the base media used in macrophage conditioned media experiments to confirm if neurons would readily survive and extend neurites in a non-traditional media. Optimization experiments confirmed that DRG neurons attached and begin neurite outgrowth within 24 hours post-plating in base, \u0026lsquo;low-serum\u0026rsquo; media and survive at least 96 hours in culture (data not shown). We next investigated the effect on outgrowth of neurons in the presence of conditioned media from unstimulated male or female bone marrow derived macrophages (macrophage conditioned media, MCM), or with the conditioned media from sex-matched macrophages after undergoing stimulation with tumor necrosis factor alpha (TNFα macrophage conditioned media, T-MCM). Neurons were cultured for 48 hours in the presence of these different conditioned media and structural plasticity was assessed by the pan-neuronal and cytoskeletal βIII tubulin staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-G). Mean outgrowth of neurites from neurons was quantified at the experimental endpoint for both male (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH) and female (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI) treated neurons. Both male and female neurons incubated with the standard, unstimulated macrophage condition media (MCM) had no change in outgrowth compared to regular media treated controls. However, female DRG neurons specifically demonstrated an increase in neurite outgrowth after incubation with female T-MCM (1-way ANOVA, F\u003csub\u003e2,35\u003c/sub\u003e = 7.635, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0018, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI\u003cb\u003e)\u003c/b\u003e. Male neurons incubated with the male equivalent T-MCM did not exhibit a statistically significant increase in neurite outgrowth (1-way ANOVA, F\u003csub\u003e2,35\u003c/sub\u003e = 3.035, p\u0026thinsp;=\u0026thinsp;0.0609, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Electrophysiological assessment of DRG neurons early after culturing\u003c/h2\u003e \u003cp\u003eThe function of neurons is dictated by their structure and excitability. We next assessed the excitability profiles of neurons treated with the different macrophage-conditioned medias using whole-cell current clamp recordings, incubated under identical conditions to previous experiments (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). We decided to focus on female DRG neurons due to previous work indicating a specific mechanism of pain in our autoimmune inflammatory mouse model (\u003cspan additionalcitationids=\"CR56\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e) as well as the female bias of autoimmunity and chronic pain in the human population (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). We analyzed general metrics of neuronal excitability including the minimum electric current required to elicit an action potential (rheobase) and the resting membrane potential (RMP) after 6 hours of incubation in the different conditioned medias. We found that all conditions displayed varying degrees of spontaneous activity and quiescence, with the T-MCM treated neurons exhibiting the most spontaneous activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). While neurons treated with MCM had more negative RMP compared to media only control (two-tailed t-test, MCM RMP: t\u0026thinsp;=\u0026thinsp;2.156, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e, the rheobase of these MCM treated neurons was unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Neurons treated with T-MCM however, exhibited a lower rheobase and had less negative resting membrane potential (RMP) relative to media only treated neurons (two-tailed t-test, T-MCM Rheobase: t\u0026thinsp;=\u0026thinsp;2.019, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; T-MCM RMP: t\u0026thinsp;=\u0026thinsp;2.115, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, E). Interestingly, the T-MCM treated neurons that had a higher excitability profile overall (i.e. most spontaneous activity, lowered rheobase and less negative resting membrane potential) at 6 hours, correlated with the neurons exhibiting the greatest structural outgrowth at 48 hours described in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. These findings suggest a potential positive relationship between increased early excitability of the sensory neuron and later increased structural plasticity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Electrophysiological assessment of DRG neurons in established cultures\u003c/h2\u003e \u003cp\u003eTo evaluate neurons at later time points in culture, we conducted an identical electrophysiological assessment 48-hours after plating. Notably, we observed an overall shift in the excitability profiles in all treatment conditions. Specifically, the MCM condition exhibited no spontaneous activity at this later timepoint, also exhibiting an elevated rheobase and the most negative resting membrane potential (two-tailed t-test, MCM Rheobase: t\u0026thinsp;=\u0026thinsp;2.410, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; MCM RMP: t\u0026thinsp;=\u0026thinsp;0.639, p\u0026thinsp;=\u0026thinsp;0.53, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D, E\u003cb\u003e)\u003c/b\u003e. In contrast, the T-MCM treated neurons still displayed some spontaneous activity but were identical in rheobase and resting membrane potential compared to their media only controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D, E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Growth status and electrophysiological characteristics of DRG neurons\u003c/h2\u003e \u003cp\u003eAs we were interested in relating electrical excitability to structural plasticity, we noted that neurons adopt distinct morphologies 48-hours after plating consistent with observations from Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. At this time point, neurons were consistently observed to fall into three categories: a population of neurons yet to extend any neurites (\u003cem\u003eno outgrowth\u003c/em\u003e); a population that display a highly complex, \u0026lsquo;arborizing\u0026rsquo; phenotype characterized by numerous branches from the primary outgrowth (\u003cem\u003earborizing\u003c/em\u003e); and a population that extend \u0026lsquo;elongating\u0026rsquo; neurites characterized by low amounts of branching and a high displacement of primary growth (\u003cem\u003eelongating\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). Identifying and subdividing these morphological populations by visual inspection at the time of patch clamp recording, we found that the spontaneous activity seen in the T-MCM condition was restricted to neurons with no outgrowth (Fig.\u0026nbsp;4\u003cb\u003eAi)\u003c/b\u003e. Furthermore, neurons in the \u0026lsquo;arborizing\u0026rsquo; and \u0026lsquo;elongating\u0026rsquo; categories were primarily quiescent regardless of treatment condition (Fig.\u0026nbsp;4\u003cb\u003eBi, Ci\u003c/b\u003e). Overall, these data suggest that incubation of neurons with different types of inflammatory-conditioned media convey unique excitability profiles in neurons that have yet to display outgrowth and that this can inform the extent of outgrowth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Structural plasticity is modulated by Kv7 channel activity\u003c/h2\u003e \u003cp\u003eTo assess the direct impact that electrical activity/excitability has on structural plasticity, we turned to pharmacological manipulation of these neurons in our \u003cem\u003ein vitro\u003c/em\u003e system. Retigabine (RTG) is an anticonvulsant drug that was initially developed as a treatment for epilepsy. It acts as a positive allosteric modulator of voltage-gated potassium channels (Kv7), which stabilizes the resting membrane potential and reduces neuronal excitability (\u003cspan additionalcitationids=\"CR60\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). We repurposed this drug to act as a general inhibitor of neuronal activity to mimic the quiescent phenotype we identified \u003cem\u003ein vitro\u003c/em\u003e. The effect of RTG is near instantaneous as repetitively evocable neurons exhibit a complete loss of electrical activity upon wash-in of RTG during recording (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Repetitive firing of neurons is then restored when RTG is washed off (1-way ANOVA, F\u003csub\u003e2,14\u003c/sub\u003e = 4.40, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Control vs. RTG, p\u0026thinsp;=\u0026thinsp;0.0584, RTG vs. Washout, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Basal neurite outgrowth of both male and female DRG neurons cultured in the presence of retigabine is also significantly reduced after 48 hours in culture (2-way ANOVA, treatment effect F\u003csub\u003e1,92\u003c/sub\u003e = 15.54, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Although the quiescent phenotype was primarily shown in the later stages of outgrowth, we additionally tested the addition of RTG at a later timepoint (24 hours post plating), but this had an identical effect to the early addition (data not shown). These data indicate that neurons are dependent on some level of spontaneous activity for axonal outgrowth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.6 Plasticity driven by BMDM conditioned media is dependent on excitability.\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTo assess whether the neurite growth promoting effects of T-MCM are on dependent on changes to the excitability profile of the neurons, we treated DRG neurons for 48 hours with T-MCM in the presence or absence of RTG. As seen previously (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), compared to neurons treated with media only, T-MCM promoted a significant increase in mean outgrowth (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B, D). This effect was abrogated by co-incubation of T-MCM with RTG (1-way ANOVA, treatment effect F\u003csub\u003e2,33\u003c/sub\u003e = 194, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Attenuating DRG excitability prevents pain in mice with EAE\u003c/h2\u003e \u003cp\u003eGiven the robust effects on neural activity and outgrowth from DRG neurons treated with RTG in the presence of inflammatory macrophage conditioned media, we next wanted to determine how this might impact pain in the context of neuroinflammatory disease. We have previously described the emergence of pain hypersensitivity to tactile stimulation in the hind paws of mice with experimental autoimmune encephalomyelitis (EAE) that is accompanied by significant increases in DRG neuron excitability and activity (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). Mice were immunized for EAE and seven days after immunization we began treatment with either RTG (10 mg/kg, IP) or vehicle-control. Vehicle treated mice exhibited well characterized, significant reductions in paw withdrawal thresholds indicative of pain hypersensitivity that became statistically different from baseline values on day 11 post immunization. In contrast, mice that began treatment with RTG on day 7 displayed a complete reversal of this behavioural hypersensitivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-way RM ANOVA, treatment effect F\u003csub\u003e1,10\u003c/sub\u003e = 6.035, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). RTG treatment effectively supressed action potential firing from DRG nociceptors compared to vehicle treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) and altered the overall excitability of these neurons by lowering the resting membrane potential and significantly elevating the rheobase \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, D\u003cb\u003e)\u003c/b\u003e. Collectively, reducing DRG excitability with RTG limits DRG axon outgrowth \u003cem\u003ein vitro\u003c/em\u003e and pain hypersensitivity following EAE \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.1 BMDM Conditioned Media conveys plastic potential to DRG neurons\u003c/h2\u003e \u003cp\u003eBuilding upon previous research indicating sex differences in innate immune inflammatory activity, we have investigated the sex-specific changes in DRG sensory neuron structural plasticity and excitability when exposed to inflammatory mediators from innate immune cells (BMDMs). Here, we demonstrate that incubating peripheral neurons with sex-matched inflammatory conditioned media from innate immune cells can impact both the structural plasticity and the excitability profile of these neurons. We find that excitability parameters of neurons that exhibit high amounts of structural outgrowth correspond to the greatest shift in excitability, becoming quiescent after an initial period of high spontaneous activity. Furthermore, we demonstrate that a pharmacological intervention limiting neuronal activation can prevent this capacity for structural plasticity in the inflammatory conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.2 DRG neurons are highly plastic cells\u003c/h2\u003e \u003cp\u003eIn early neuronal development, the maturation of neurons involves complex signaling cascades that are crucial for the transcriptional profile and development of the functional component of these cells (\u003cspan additionalcitationids=\"CR63\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e). Maintenance of neuronal circuitry in adult organisms is tightly regulated and these plastic processes can result in adaptive or maladaptive consequences depending on the context. For instance, heightened pain sensitivity during inflammation may trigger acute plasticity that is adaptive for preventing further injury, but intense or prolonged inflammation also has the capacity to promote signalling within sensory neurons that leads to long term changes and chronic pain syndromes (\u003cspan additionalcitationids=\"CR66\" citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePain management is a critical aspect of first-line treatments for nervous system injury. However, strategies that directly address pain without considering the structural consequences have proven to be ineffective in preventing chronic pain. Thus, it may be worthwhile to consider both the functional and structural aspects of neural plasticity when developing pain management strategies. This involves understanding the interplay between activity-dependent phenomena and regenerative outgrowth and investigating the mechanisms that link neuronal excitability and structural plasticity. Adopting such an approach could lead to more effective treatments for chronic pain syndromes. Emerging from current paradigms that tend to view heightened neuronal excitability following injury as detrimental due to the risk of excitotoxicity(\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e), the current study supports an alternative perspective: the necessity of preserving an early phase of excitability to facilitate proper neural regeneration. While efforts to curb excessive excitability post-injury are well-founded, these interventions might inadvertently hinder the inherent pro-regenerative processes associated with the initial surge in neuronal excitability and activity. By excessively dampening this early phase, there could be an unintended loss of the favorable conditions that drive regenerative mechanisms (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e). Thus, striking a balance between mitigating excitotoxicity and allowing for the temporally distinct, early phase excitability, could hold the key to unlocking more effective neural regeneration strategies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e4.3 DRG neuronal plasticity involves alterations in excitability\u003c/h2\u003e \u003cp\u003eRecent work has explored a related line of inquiry and demonstrated that \u003cem\u003ein vitro\u003c/em\u003e, DRG neurons undergo transcriptional de-maturation by downregulating genes critical for synaptic transmission (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Genetic deletion of core components of the synapse essential for neurotransmission \u0026ndash; such as RIM1/2 or Munc13 \u0026ndash; significantly enhances axon growth and regeneration while reducing branching (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Together with the findings presented here, these results suggest that inflammation-induced molecular cascades that promote neurite outgrowth are inexorably linked with changes in neural excitability and activity.\u003c/p\u003e \u003cp\u003eActivity-dependent phenomena are well-documented in regenerative contexts. Although it is rare for a neuron to lose all input \u003cem\u003ein vivo\u003c/em\u003e, as it does in a dissociative culture system, evidence indicates that peripheral neurons may atrophy or even die following long-term sensory deprivation (\u003cspan additionalcitationids=\"CR72\" citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e). In this context, it is possible that pain after injury plays a crucial role in promoting the proper re-maturation of healthy neuronal circuitry. Therefore, the excessive use of analgesics in clinical settings may potentially hinder regenerative outgrowth (\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e). The experience of pain may be a necessary part of the healing process; acknowledging and pursuing this hypothesis may lead to better outcomes.\u003c/p\u003e \u003cp\u003eWhile the precise mechanism linking excitability and structural plasticity remains unclear, several plausible hypotheses can be tested. One such hypothesis is that ionic gradients along neuronal membranes allow for calcium influx during action potentials. Calcium is a well-known second messenger and cofactor for many different molecular cascades that promote both the growth of axons but also neuronal sensitization and pain hypersensitivity(\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e). As a driver of calcium currents, inflammation, a central player in injury responses, is well-characterized in driving calcium currents(\u003cspan additionalcitationids=\"CR79\" citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e). The early increases in excitability may support increased calcium flux and drive the increased growth of female DRG neurons in response to MCM stimulated with TNFα (T-MCM). Furthermore, male DRG neurons stimulated with T-MCM exhibited a slight but not statistically significant changes in neurite outgrowth, suggesting that the interplay of inflammatory modulation of ionic gradients influencing outgrowth may be sex specific. This interplay suggests potential avenues for pain management, centered around the crosstalk between inflammation and neuronal plastic responses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e4.4 DRG neuronal plasticity: the relationship between excitability and pain\u003c/h2\u003e \u003cp\u003ePlasticity, excitability, and pain are interconnected processes in the nervous system. The maturation and maintenance of neuronal circuits involve signaling cascades that regulate the functional component and structural generation and pruning of synapses, membrane potentials, and sensory receptor insertion. These processes involve different cell types, including glia(\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e) and immune cells(\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e), and are activity driven (\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e). The consequences of these plastic processes can be adaptive or maladaptive, such as sickness behavior and chronic pain. Pain is one of the most common outcomes after injury and inflammation, and has been widely reported in the animal model of neuroinflammatory disease, EAE(\u003cspan additionalcitationids=\"CR86 CR87\" citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e). Interestingly, pain in the EAE model is also associated with significant changes occurring at the level of DRG including increased inflammation and increased neuronal activity/hyperexcitability (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). Our lab has also recently reported that common markers associated with increased neural plasticity and growth are increased in DRG neurons of EAE mice in a sex specific manner, with females exhibiting higher levels of ATF3 and pCREB (\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e). Taken together with the results of the experiments described here, we can speculate that DRG neurons from female mice may use the inflammatory stimulation as a priming signal to trigger a change in their growth status. While our results with RTG in the EAE model suggest that inhibiting this increase in neural activity in response to inflammation may be beneficial for pain in the short term, how this treatment affects the long-term plasticity of these neurons remains to be determined.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThe relationship between neuronal excitability and structural plasticity is complex and dynamic, with evidence suggesting that they may be inextricably linked. Both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies have demonstrated the importance of activity-dependent phenomena in promoting regenerative processes and maintaining nervous system function. The role of pain in this process is an intriguing area of investigation, with some evidence suggesting that it may be necessary for proper re-maturation of healthy circuitry. The mechanisms underlying the link between excitability and plasticity remain unclear, but the role of Ca\u003csup\u003e2+\u003c/sup\u003e as a second messenger and cofactor for molecular cascades presents a plausible hypothesis that warrants further investigation. Overall, a deeper understanding of the relationship between neuronal excitability and structural plasticity has important implications for the development of new strategies to promote nerve regeneration and functional recovery after injury or disease.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics declaration\u003c/h2\u003e \u003cp\u003eAll animal experiments were performed according to Canadian Council on Animal Care\u0026rsquo;s Guidelines and Policies with approval from University Of Alberta Health Sciences Animal Care and Use Committee. \u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eTNF, SML, ADM and TH conducted experiments and analyzed data. TNF wrote the manuscript. BJH, JRP, HTK and BJK designed experiments and edited the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e \u003cp\u003eFunding for this project was provided by operating grants from the MS Society of Canada (EGID-MSSC-3761), a Project grant from the Canadian Institutes for Health Research (CIHR, FRN162434), the Alberta MS Collaboration and the University Hospital Fund (University of Alberta). TNF was supported by studentships from the MS Society of Canada and by the Alexander Graham Bell Canada Graduate Scholarship from NSERC. The authors wish to thank G. Tenorio for assistance in figure preparation.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eThe datasets supporting the conclusions of this manuscript are available upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSolaro C, Trabucco E, Messmer Uccelli M. 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Sci Rep. 2022;12(1).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"DRG, pain, inflammation, neurite extension, TNFα, plasticity, electrophysiology, Kv7 channels","lastPublishedDoi":"10.21203/rs.3.rs-4094312/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4094312/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground.\u003c/h2\u003e \u003cp\u003eThe intricate interplay between inflammation, its effects on neuronal excitability, and the ensuing plasticity of dorsal root ganglion (DRG) sensory neurons remains to be fully explored. In this study, we have investigated the impact of inflammatory conditioned media on the excitability and activity of DRG neurons and how this relates to subsequent growth responses of these cells.\u003c/p\u003e\u003ch2\u003eFindings.\u003c/h2\u003e \u003cp\u003eWe show here that an early phase of increased neuronal activity in response to inflammatory conditioned media is critical for the engagement of plastic processes, and that neuronal excitability profiles are linked through time to the structural phenotype of individual neurons. Pharmacological blockade of neuronal activity was able to abolish the growth promoting effects of inflammatory media. Our findings also demonstrate a sex specificity of these responses.\u003c/p\u003e\u003ch2\u003eConclusions.\u003c/h2\u003e \u003cp\u003eOur results suggest that targeting the activity of DRG neurons may provide a novel therapeutic avenue to manipulate their growth status and potential for plasticity in response to inflammation. While further studies are needed to fully elucidate the underlying mechanisms of the relationship between neural activity and growth status, a more complete understanding of this relationship may ultimately lead to the development of new treatments for neuropathic pain in disorders associated with heightened immune responses such as rheumatoid arthritis and MS.\u003c/p\u003e","manuscriptTitle":"Plasticity of mouse dorsal root ganglion neurons by innate immune activation is influenced by electrophysiological activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-15 20:02:25","doi":"10.21203/rs.3.rs-4094312/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d5278bb6-0f23-4e9c-803b-709fbba3f7bb","owner":[],"postedDate":"March 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-15T20:02:27+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-15 20:02:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4094312","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4094312","identity":"rs-4094312","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}