(231) Transcranial Direct Current Stimulation of the Prefrontal Cortex Modulates both Pain and Nociceptive Flexion Reflex (NFR) Activity

S32 The Journal of Pain

Abstracts

examined pain and functional related behavioral changes in doxycycline inducible HIV-1 Tat transgenic (iTat Tg) mice following the induction of Tat expression. Two induction regimen were compared: 7-day daily injection vs. 21-day daily injection of doxycycline (i.p. at 100mg/kg). Sensory sensitivities (mechanical, heat and cold) were assessed to evaluate pain-like behaviors. Sensory (pin prick assay) and motor (hind limb grip strength, toe spread reflex and toe spacing score) functional tests were used to evaluate hind paw functions associated with Tat expression. Both regimens induced significant mechanical hypersensitivity and significant reduction of grip strength from day 3 post the initiation of Tat induction to the end of the experiment (day 35) in iTat Tg mice. Tat induction for 21 days also significantly induced cold hypersensitivity in iTat Tg mice up to day 14 post-the first doxycycline injection. Neither regimen induced significant heat hypersensitivity or affected other functional measurements. Together, our results suggest the utility of iTat Tg mice in studying the role of Tat in HIV SN. Sex-depended differences and Tat-induced molecular pathways will be further investigated in future studies. (Grant support: R21 DA044886 [PI Cao])

preference were used to assess changes in evoked pain thresholds (hyperalgesia) and impaired reward, respectively. After 96 hours of morphine treatment, fecal samples were collected and processed for 16s rDNA sequencing. Manipulation of the gut microbiome was used to assess the causal relationship between the gut microbiome and opioid dependent behaviors. While both intermittent and sustained morphine treatment led to alterations in the gut microbiota composition, intermittent morphine treatment led to exacerbated inflammation, hyperalgesia, and negative affect. Depletion of the gut microbiota via antibiotic treatment led to a similar phenotypic outcome to opioid dependence, including central inflammation, morphine tolerance, and decreased negative affect. Recolonizing antibiotic-depleted animals with a control microbiota restored microglia to a resting phenotype, improved reward behavior, and blocked hyperalgesia. Recolonization with an opioid microbiota (isolated from mice treated with intermittent morphine), maintained the opioid-dependent phenotype. Our findings suggest that differing opioid regimens uniquely influence the gut microbiome, and that this process is causally related to the development of opioid dependent behaviors.

(228) Pain Hypersensitivity in Chronic Traumatic Neuropathy is Mediated by Peripheral Macrophages and AT2R Signaling

(230) The Role of nNOS Positive Interneurons in Spinal Somatosensory Circuits

A. Shepherd and D. Mohapatra; Washington University School of Medicine, St. Louis, MO Traumatic modes of experimental neuropathy in rodents have been extensively utilized in pre-clinical studies to understand mechanisms underlying chronic neuropathic pain, and to test the in vivo efficacy of experimental and known analgesic drugs. The vast majority of studies probe anatomical, transcriptomic and tissue/cell functional analysis, as well as pain hypersensitivity-related behavioral outcome measures within 1 to 3 weeks of the induction of trauma/surgical procedures. This models the acute/sub-chronic phases of neuropathic pain in humans, and thus represents a disconnect from chronic neuropathy conditions/diseases and the associated pain in humans. With this in mind, we investigated whether the mechanisms elucidated at 1 to 3 weeks post-injury also drive pain hypersensitivity at more chronic stages (>100 days post-injury). We used evoked and voluntary behavioral measures of mechanical and cold hypersensitivity along with immunohisochemical analysis in the spared nerve injury (SNI) model of traumatic neuropathy in mice. Elevated macrophage density was detectable in the injured nerve at this late stage, and mechanical and cold hypersensitivity also persist in these animals. We recently reported a role for peripheral macrophages and the function of the type II angiotensin II receptor (known as AT2R) therein, at the site of nerve injury in the development of neuropathic pain. In the present study, we found that the AT2R antagonist EMA401, which is currently in phase II clinical trials for post-herpetic neuralgia and diabetic neuropathic pain, attenuated mechanical and cold pain hypersensitivity in SNI mice. Collectively, our observations suggest that in addition to being a crucial driver of neuropathic pain hypersensitivity in the more acute stages, peripheral macrophage infiltration and AT2R signaling therein continues to drive sustained pain hypersensitivity associated with neuropathy in chronic pathological states of the disease.

K. Smith, J. Hachisuka, H. Koerber, and S. Ross; University of Pittsburgh, Pittsburgh, PA Somatosensory information, encoding pain, touch, itch, heat and cold all enter the central nervous system at the spinal cord dorsal horn. Here this information is modulated through both descending control and local microcircuitry before being relayed to higher brain centres for perception of the stimulus. In this study, we use a semi intact, ex vivo preparation to investigate the role of a population of dorsal horn interneurons that express neuronal nitric oxide synthase (nNOS) in local spinal microcircuitry. In this preparation we dissect (in continuum) spinal cord, together with L2 and L3 roots, dorsal root ganglia, saphenous nerve and hind limb skin. This allows natural stimulation of the skin while performing whole cell patch clamp recordings from identified dorsal horn neurons. nNOScreER mice were crossed with either Ai9 or Ai32 reporter animals and given tamoxifen (0.4mg/kg, i.p) at day P14. Electrophysiology experiments were performed 3 weeks later. In some cases, DiI was injected to the parabrachial nucleus 4 days before experiments to retrogradely label spinal projection neurons. We found that nNOS positive interneurons receive input from both innocuous and noxious mechanical stimulation, but not a scratch stimulus. Further, optogenetic activation of these interneurons results in action potential discharge in identified projection neurons, suggesting an ability to send sensory information to higher brain areas. Using this ex vivo preparation will allow us to characterise spinal somatosensory circuits both under normal conditions (current study) and both acute and chronic pain states. This will help us understand how spinal microcircuitry is disrupted following injury.

(231) Transcranial Direct Current Stimulation of the Prefrontal Cortex Modulates both Pain and Nociceptive Flexion Reflex (NFR) Activity (229) Revealing a Brain-Gut Microbiome Connection following Chronic Opioid Treatment A. Taylor, K. Lee, H. Vuong, D. Nusbaum, E. Hsiao, and C. Evans; University of California Los Angeles, Los Angeles, CA Chronic opioid use is characterized by several phenotypic markers, including hyperalgesia and negative affect. We and other have shown previously that inflammation in the brain contributes to these behavioral symptoms, although the mechanism by which opioids drive central inflammation remains unclear. Recent reports have shown that neuroinflammation observed in other psychiatric conditions characterized by negative affect can be caused by disruptions to gut microbiota along a poorly understood gut-brain signaling axis. In this study, we explored how opioid use and cessation impacts the mouse gut microbiome. We hypothesized that opioid-induced changes in the gut microbiota influences inflammation-driven hyperalgesia and impaired reward behavior. Male adult C57Bl6/J mice were treated with either intermittent or sustained morphine. A separate group of animals treated with saline served as a control. Tail withdrawal and conditioned place

P. Slepian, J. Rhudy, B. Clark, and C. France; Ohio University, Athens, OH Transcranial direct current stimulation (tDCS) is a form of noninvasive brain stimulation that can either enhance (via anodal stimulation) or depress (via cathodal stimulation) neural excitability in focal areas of the cortex through application of a low-intensity direct current. Although prior studies indicate that cortical application of tDCS is associated with modulation of experimental pain, the present study provides the first test of the simultaneous effect of tDCS on both pain ratings and modulation of nociceptive flexion reflex (NFR). Thirty-two healthy participants (15 female) completed three days of NFR testing, each separated by at least one week. On each testing day, participants received 15 minutes of either anodal, cathodal, or sham tDCS over the dorsolateral prefrontal cortex (order counterbalanced across participants). Active stimulation was delivered at 2mA. tDCS was immediately followed by 24 discrete noxious electrical stimuli delivered over the sural nerve, immediately posterior to the ankle, to elicit NFR. NFR was evoked concurrently with presentation of pleasant, unpleasant, or neutral images. Following each stimulation participants rated pain

Abstracts intensity using a 0-100 rating scale. Mixed linear modeling was used to analyze tDCS effects on pain and NFR magnitude while controlling for image content and within-session habituation. Results indicated that tDCS had a significant effect on both pain, F(2, 2169) = 4.37, p = .01, and NFR, F(2, 2169) = 36.39, p < .001. Pain was higher after cathodal versus sham stimulation, p < 0.001, d = 0.10, but did not differ in any other comparison. NFR magnitude was higher after cathodal versus anodal (p < 0.001, d = 0.43) and sham (p < 0.001, d = 0.48) stimulation. These findings indicate that tDCS stimulation affects both pain reports and spinal nociceptive modulation, and highlights the importance of the dorsolateral prefrontal cortex in descending inhibition of pain and nociception.

(232) Assessing Chronic Pain Onset in Native Americans: Follow-Up Results from the Oklahoma Study of Native American Pain Risk (OK-SNAP) E. Ross, F. Huber, B. Kuhn, E. Lannon, C. Sturycz, M. Payne, N. Hellman, T. Toledo, Y. Gu€ereca, S. Palit, M. Demuth, J. Shadlow, and J. Rhudy; The University of Tulsa, Tulsa, OK Chronic pain leads to considerable suffering and disability. Epidemiological evidence suggests that Native Americans (NA) suffer from chronic pain at higher rates than any other U.S. racial/ethnic group. However, to our knowledge, no study has examined this issue prospectively to examine whether healthy, pain-free NAs are more likely to develop chronic pain after controlling for other health variables. For the present study, healthy, pain-free participants were enrolled in a study (OK-SNAP) that assessed a variety of variables associated with pain processing and pain risk. Enrollment began in March 2014. Following enrollment, follow-up surveys were administered every 6-months to assess whether chronic pain developed. Of the 139 NA and 147 non-Hispanic white (NHW) participants enrolled, 208 (73%) responded to at least 1 follow-up. Participants were deemed to have chronic pain if they experienced persistent pain for >3 months that did not remit at subsequent follow-ups (N=34; 16%). On average these individuals reported several locations with persistent pain (M=3.71, SD=3.64), including lower (56%) and upper (35%) back, neck (29%), shoulder (29%), feet (29%), knees (26%), legs (21%), and hips (21%). This chronic pain group was compared to those that did not develop chronic pain (N=174). A logistic regression was conducted with race (NHW vs. NA) as a predictor, after controlling for age, BMI, income, and sex. Race emerged as the only significant predictor (OR=3.015, 95% CI: 1.309, 6.945) indicating that NAs were 3x more likely to develop chronic pain than NHWs, an effect that explained 10.4% of the variance in pain onset (Nagelkerke R2=.104). These prospective findings are consistent with prior epidemiological reports that there is a pain disparity within the NA community. Future studies are needed to examine the mechanisms for this disparity.

(233) Conditional Knockout of Prdm12 from Sensory Neurons in Mice Recapitulates Aspects of Human Congenital Insensitivity to Pain M. Landy and H. Lai; UT Southwestern, Dallas, TX Painful conditions are a huge medical burden with an estimated prevalence of 32% of the population, and an economic cost of $635 billion, or 4% of the GDP. Currently, the best available medical analgesics are opioids, but due to their vast side effect profile and risk of overdose, these are far from ideal drugs. Recently, the study of genetic mutations leading to congenital insensitivity to pain (CIP) has resulted in submission of novel drugs to the FDA for clinical trial. Here, we show our work on one such mutation of the gene Prdm12. We first characterized the expression pattern of Prdm12 within different sensory neurons present in the dorsal root ganglia (DRG) by performing in-situ hybridization paired with immunohistochemistry, and found it to be expressed in both myelinated and unmyelinated small-diameter axons of the thoracic and lumbar DRGs. We then generated a new strain of mice in which Prdm12 has been conditionally knocked out of neurons in DRG and cranial ganglia, and subjected these mice to a variety of behavioral assays alongside littermate controls. We have shown that the Prdm12cKO mice have reduced sensitivity to nociceptive thermal and mechanical stimuli, and correlated this with a decrease in the number of pain-sensitive IB4+ and CGRP+ Ad and C fibers in the DRG. We believe that further study of this mouse model, along with investigation of the downstream effectors and genomic targets of PRDM12, will provide a new avenue for the pursuit of analgesic drug discovery.

The Journal of Pain

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(234) Cortical Hemodynamic Response to Contact Thermal Stimuli in Older Adults with Knee Osteoarthritis: A Functional Near Infrared Spectroscopy Pilot Study S. Sorkpor, H. Ahn, L. Pollonini, and J. Do; University of Texas Health Science Center, Houston, TX Since the brain is the organ that is principally responsible for processing the experiences of pain, the emergence of neuroimaging for quantifying pain-related responses is promising. Functional nearinfrared spectroscopy (fNIRS) is particularly favorable for studying pain since this optical method is noninvasive, portable, and costeffective. However, no studies have directly evaluated the cortical hemodynamic response to pain in older adults with knee osteoarthritis (OA) using fNIRS. In this pilot study, we sought to investigate the cortical response of older adults with knee OA to contact thermal stimuli. Fourteen community-dwelling adults with knee OA pain aged 50 − 85 years underwent testing. Using a blocked design paradigm consisting of 6 blocks of 20 seconds of stimuli followed by 30 seconds of rest, we applied contact thermal stimuli at 45 degree Celsius to the ventral part of the ipsilateral forearm to the most affected knee using a computer-controlled Medoc TSA-II Neurosensory Analyzer. We used a continuous wave, multichannel fNIRS imaging system (LIGHTNIRS, Shimadzu, Kyoto, Japan) incorporating three semiconductor lasers at 780, 805, and 830 nm and an avalanche photodetector in each optical channel to measure cerebral hemodynamic activity. The instrument encompasses an array of 8 sources and 8 detectors connected to the subject’s scalp in a geometrical layout that covered the prefrontal and somatosensory cortex regions bilaterally. Results indicate substantial brain activation patterns in contralateral prefrontal and somatosensory regions, with differing intensity between subjects in response to the stimulus. Our findings extend the use of fNIRS imaging to older adults with knee OA, and adds to the growing literature regarding neuroimaging measures of pain using fNIRS.

(235) Homeostatic Regulation of Intrinsic Plasticity in Peripheral Nociceptors L. McIlvried, M. Pullen, and R. Gereau; Washington University in St. Louis, St. Louis, MO Stabilization of neuronal activity is crucial for normal nervous system regulation. When changes in the environment chronically alter neuronal activity, homeostatic plasticity creates compensatory shifts (i.e., regulation of intrinsic excitability) to return the system to a physiological level. This plasticity helps prevent circuits from becoming either hyper- or hypoexcitable. Despite this protective mechanism, evidence suggests that the development and maintenance of many chronic pain conditions depend on chronically altered sensory afferent activity. This led us to consider the possibility that malfunction of homeostatic mechanisms may contribute to chronic pain. Since homeostatic plasticity has been primarily studied in the central nervous system, we first tested whether this plasticity also occurs in peripheral nociceptors [i.e., small diameter dorsal root ganglia (DRG) sensory afferent neurons]. We hypothesized that mouse and human primary sensory afferents undergo homeostatic regulation of intrinsic plasticity, evidenced by compensatory changes in neuronal excitability in response to chronic stimuli. Using a combination of pharmacologic and optogenetic approaches with whole-cell electrophysiology, we previously presented that sustained, 24-hour KCl depolarization of dissociated mouse and human DRG neurons led to a compensatory decrease in excitability of small diameter cells. This suggested that putative nociceptors undergo homeostatic regulation of intrinsic excitability. In an effort to demonstrate bidirectionality of this homeostatic plasticity, in this study we used a 24hour incubation with lidocaine and TTX. We found no change in excitability, likely due to the already quiescent nature of DRG neurons in vitro. Ongoing experiments are being performed to determine whether mechanisms of this plasticity are altered in vivo and during chronic pain. This work was supported by grants R01 NS042595 (RG) and T32DA007261 (LM).

(236) Functional and Genetic Dissection of Central Amygdala Neurons Underlying Sensory and Aversive Components of Itch V. Samineni, J. Grajales-Reyes, B. Copits, R. Gereau, and A. Mickle; Washington University St. Louis, St. Louis, MO Itch is defined as an unpleasant sensation that evokes a desire to scratch. The supraspinal mechanisms that are engaged in itch