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We have recently reported a sex difference in morphine-induced analgesia in a visceral pain model. To test the hypothesis that estrogen plays a role in mediating this sex difference, the effect of morphine on the visceromotor response (vmr) to colorectal distention was compared between ovariectomized (OVx) and OVx with estrogen replacement (E2) rats. After demonstrating that estrogen attenuates the potency of systemically administered morphine, we tested peripheral, spinal, and supraspinal sites for estrogen modulation of micro-opioid receptor (MOR) activity. The peripheral MOR antagonist naloxone methiodide reversed the effect of systemic morphine. The peripheral MOR agonist loperamide also attenuated the vmr and in addition was more potent in OVx rats than E2 rats, demonstrating estrogen modulation of peripheral micro-opioid analgesia. Intrathecally injected morphine attenuated the vmr, with no difference in potency noted between the 2 groups. Morphine given by intracerebroventricular injection was more potent in OVx rats than in E2 rats, suggesting estrogen modulation of supraspinal micro-opioid receptors. Results from all administration routes revealed that the potency of morphine in OVx and E2 rats was similar to male and intact female rats, respectively, suggesting that estrogen is one of the key factors contributing to the sex difference in micro-opioid analgesia. PERSPECTIVE: Female rats are less sensitive to morphine analgesia of visceral pain than male rats. This study demonstrates that estrogen decreases the analgesic potency of peripheral and supraspinal but not spinal morphine in a model of visceral pain and may be a key factor contributing to the sex difference in micro-opioid analgesia. [ABSTRACT FROM AUTHOR]

Specific Aim: Provide an overview of the literature addressing major areas pertinent to pain in transgender persons and to identify areas of primary relevance for future research., Methods: A team of scholars that have previously published on different areas of related research met periodically though zoom conferencing between April 2021 and February 2023 to discuss relevant literature with the goal of providing an overview on the incidence, phenotype, and mechanisms of pain in transgender patients. Review sections were written after gathering information from systematic literature searches of published or publicly available electronic literature to be compiled for publication as part of a topical series on gender and pain in the Frontiers in Pain Research., Results: While transgender individuals represent a significant and increasingly visible component of the population, many researchers and clinicians are not well informed about the diversity in gender identity, physiology, hormonal status, and gender-affirming medical procedures utilized by transgender and other gender diverse patients. Transgender and cisgender people present with many of the same medical concerns, but research and treatment of these medical needs must reflect an appreciation of how differences in sex, gender, gender-affirming medical procedures, and minoritized status impact pain., Conclusions: While significant advances have occurred in our appreciation of pain, the review indicates the need to support more targeted research on treatment and prevention of pain in transgender individuals. This is particularly relevant both for gender-affirming medical interventions and related medical care. Of particular importance is the need for large long-term follow-up studies to ascertain best practices for such procedures. A multi-disciplinary approach with personalized interventions is of particular importance to move forward., Competing Interests: ETS and UCSD has received funding and/or product for research from Epimed International (Dallas, TX) SPR Therapeutics (Cleveland, OH), Infutronix (Natick, MA), and Avanos (Irvine, CA). LC. Associate Editor for Frontiers in Pain Research. LC did not participate in the journal review process leading to the acceptance of this manuscript. MV. Chief Specialty Editor in Frontiers in Medical Technology. UW serves on the External Consultant Board for the “NIH Preclinical Screening Platform for Pain” (NIH/NINDS). In her capacity as a special government employee of the US Food and Drug Administration (FDA), she has served as a voting member of the FDA Anesthetic and Analgesic Drug Products Advisory Committee. In the past 3 years she has received compensation for serving on advisory boards or for consulting activities for Aphrodite Health Inc., Wilmington, DE, Avenue Therapeutics Inc., New York, NY, Bayer Aktiengesellschaft, Leverkusen, Germany, Biohaven Pharmaceuticals, New Haven, CT, and Syneos Health, Morrisville, NC, all unrelated to the submitted work. Reports research grants from the US National Institutes of Health. TY reports being a founder of Raft Pharmaceutical, SAB Navega Pharma all unrelated to the submitted work and is the Editor in Chief for Frontiers in Pain Research. TY did not participate in the journal review process leading to the acceptance of this manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 Anger, Case, Baranowski, Berger, Craft, Damitz, Gabriel, Harrison, Kaptein, Lee, Murphy, Said, Smith, Thomas, Valdés Hernández, Trasvina, Wesselmann and Yaksh.)

Gordon Holmes syndrome (GHS) is a neurological disorder associated with neuroendocrine, cognitive, and motor impairments with corresponding neurodegeneration. Mutations in the E3 ubiquitin ligase RNF216 are strongly linked to GHS. Previous studies show that deletion of Rnf216 in mice led to sex-specific neuroendocrine dysfunction due to disruptions in the hypothalamic-pituitary-gonadal axis. To address RNF216 action in cognitive and motor functions, we tested Rnf216 knock-out (KO) mice in a battery of motor and learning tasks for a duration of 1 year. Although male and female KO mice did not demonstrate prominent motor phenotypes, KO females displayed abnormal limb clasping. KO mice also showed age-dependent strategy and associative learning impairments with sex-dependent alterations of microglia in the hippocampus and cortex. Additionally, KO males but not females had more negative resting membrane potentials in the CA1 hippocampus without any changes in miniature excitatory postsynaptic current (mEPSC) frequencies or amplitudes. Our findings show that constitutive deletion of Rnf216 alters microglia and neuronal excitability, which may provide insights into the etiology of sex-specific impairments in GHS., (Copyright © 2024 George et al.)

Over the last two decades, the number of infants exposed to opioids in utero has quadrupled in the United States, with some states reporting rates as high as 55 infants per 1000 births. Clinical studies report that children previously exposed to opioids during gestation show significant deficits in social behavior, including an inability to form friendships or other social relationships. To date, the neural mechanisms whereby developmental opioid exposure disrupts social behavior remain unknown. Using a novel paradigm of perinatal opioid administration, we tested the hypothesis that chronic opioid exposure during critical developmental periods would disrupt juvenile play. As oxytocin is a major regulator of sociability, the impact of perinatal morphine exposure on oxytocin peptide expression was also examined. Juvenile play was assessed in vehicle- or morphine-exposed male and female rats at P25, P35, and P45. Classical features of juvenile play were measured, including time spent engaged in social play, time not in contact, number of pins, and number of nape attacks. We report that morphine-exposed males and females spend less time engaged in play behavior than control males and females, with a corresponding increase in time spent alone. Morphine-exposed males and females also initiated fewer pins and nape attacks. Together, these data suggest that male and female rats exposed to morphine during critical developmental periods are less motivated to participate in social play, potentially due to alterations in oxytocin-mediated reward signaling., Competing Interests: Declaration of competing interest This work was supported by the National Institutes of Health (1RO1DA041529). The funding source was not involved in study design; data collection, analysis, or interpretation; manuscript writing; or the decision to publish this article. Morphine sulfate was provided by the NIDA Drug Supply Program. The authors declare no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Preclinical and clinical studies have reported sex differences in pain and analgesia. These differences may be linked to anatomical structures of the central nervous system pain modulatory circuitry, and/or hormonal milieu. The midbrain periaqueductal gray (PAG) is a critical brain region for descending inhibition of pain. The PAG projects to the rostral ventromedial medulla (RVM), which projects bilaterally to the spinal cord to inhibit pain. In addition to pain, this descending circuit (or pathway) can be engaged by endogenous opioids (i.e., endorphins) or exogenous opioids (i.e., morphine), and we have previously reported sex differences in the activation of this circuit during pain and analgesia. Forebrain structures, including the amygdala, project to and engage the PAG-RVM circuit during persistent inflammatory pain. However, there are limited studies in females detailing this amygdalar-PAG pathway and its involvement during persistent inflammatory pain. The objective of the present study was to delineate the neural projections from the amygdala to the PAG in male and female rats to determine if they are sexually distinct in their anatomical organization. We also examined the activation of this pathway by inflammatory pain and the co-localization of receptors for estrogen. Injection of the retrograde tracer fluorogold (FG) into the ventrolateral PAG (vlPAG) resulted in dense retrograde labeling in both the central amygdala (CeA) and medial amygdala (MeA). While the number of CeA-vlPAG neurons were comparable between the sexes, there were more MeA-vlPAG neurons in females. Inflammatory pain resulted in greater activation of the amygdala in males; however, females displayed higher Fos expression within CeA-vlPAG projection neurons. Females expressed higher ERα in the MeA and CeA and the same was true of the projection neurons. Together, these data indicate that although the MeA-vlPAG projections are denser in females, inflammatory pain does not significantly activate these projections. In contrast, inflammatory pain resulted in a greater activation of the CeA-vlPAG pathway in females. As females experience a greater number of chronic pain syndromes, the CeA-vlPAG pathway may play a facilitatory (and not inhibitory) role in pain modulation., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Opioids have decreased analgesic potency (but not efficacy) in aged rodents compared with adults; however, the neural mechanisms underlying this attenuated response are not yet known. The present study investigated the impact of advanced age and biological sex on opioid signaling in the ventrolateral periaqueductal gray (vlPAG) in the presence of chronic inflammatory pain. Assays measuring µ-opioid receptor (MOR) radioligand binding, GTPγS binding, receptor phosphorylation, cAMP inhibition, and regulator of G-protein signaling (RGS) protein expression were performed on vlPAG tissue from adult (2-3 months) and aged (16-18 months) male and female rats. Persistent inflammatory pain was induced by intraplantar injection of complete Freund's adjuvant (CFA). Adult males exhibited the highest MOR binding potential (BP) and highest G-protein activation (activation efficiency ratio) in comparison to aged males and females (adult and aged). No impact of advanced age or sex on MOR phosphorylation state was observed. DAMGO-induced cAMP inhibition was highest in the vlPAG of adult males compared with aged males and females (adult and aged). vlPAG levels of RGS4 and RGS9-2, critical for terminating G-protein signaling, were assessed using RNAscope. Adult rats (both males and females) exhibited lower levels of vlPAG RGS4 and RGS9-2 mRNA expression compared with aged males and females. The observed age-related reductions in vlPAG MOR BP, G-protein activation efficiency, and cAMP inhibition, along with the observed age-related increases in RGS4 and RGS9-2 vlPAG expression, provide potential mechanisms whereby the potency of opioids is decreased in the aged population. SIGNIFICANCE STATEMENT Opioids have decreased analgesic potency (but not efficacy) in aged rodents compared with adults; however, the neural mechanisms underlying this attenuated response are not yet known. In the present study, we observed age-related reductions in ventrolateral periaqueductal gray (vlPAG) µ-opioid receptor (MOR) binding potential (BP), G-protein activation efficiency, and cAMP inhibition, along with the observed age-related increases in regulator of G-protein signaling (RGS)4 and RGS9-2 vlPAG expression, providing potential mechanisms whereby the potency of opioids is decreased in the aged population. These coordinated decreases in opioid receptor signaling may explain the previously reported reduced potency of opioids to produce pain relief in females and aged rats., (Copyright © 2022 the authors.)

Oxytocinergic actions within the hippocampal CA2 are important for neuromodulation, memory processing and social recognition. However, the source of the OTergic innervation, the cellular targets expressing the OT receptors (OTRs) and whether the PVN-to-CA2 OTergic system is altered during heart failure (HF), a condition recently associated with cognitive and mood decline, remains unknown. Using immunohistochemistry along with retrograde monosynaptic tracing, RNAscope and a novel OTR-Cre rat line, we show that the PVN (but not the supraoptic nucleus) is an important source of OTergic innervation to the CA2. These OTergic fibers were found in many instances in close apposition to OTR expressing cells within the CA2. Interestingly, while only a small proportion of neurons were found to express OTRs (~15%), this expression was much more abundant in CA2 astrocytes (~40%), an even higher proportion that was recently reported for astrocytes in the central amygdala. Using an established ischemic rat heart failure (HF) model, we found that HF resulted in robust changes in the PVN-to-CA2 OTergic system, both at the source and target levels. Within the PVN, we found an increased OT immunoreactivity, along with a diminished OTR expression in PVN neurons. Within the CA2 of HF rats, we observed a blunted OTergic innervation, along with a diminished OTR expression, which appeared to be restricted to CA2 astrocytes. Taken together, our studies highlight astrocytes as key cellular targets mediating OTergic PVN inputs to the CA2 hippocampal region. Moreover, they provide the first evidence for an altered PVN-to-CA2 OTergic system in HF rats, which could potentially contribute to previously reported cognitive and mood impairments in this animal model., (© 2022 British Society for Neuroendocrinology.)

Recessive mutations in RNF216/TRIAD3 cause Gordon Holmes syndrome (GHS), in which dysfunction of the hypothalamic-pituitary-gonadal (HPG) axis and neurodegeneration are thought to be core phenotypes. We knocked out Rnf216/Triad3 in a gonadotropin-releasing hormone (GnRH) hypothalamic cell line. Rnf216/Triad3 knockout (KO) cells had decreased steady-state GnRH and calcium transients. Rnf216/Triad3 KO adult mice had reductions in GnRH neuron soma size and GnRH production without changes in neuron densities. In addition, KO male mice had smaller testicular volumes that were accompanied by an abnormal release of inhibin B and follicle-stimulating hormone, whereas KO females exhibited irregular estrous cycling. KO males, but not females, had reactive microglia in the hypothalamus. Conditional deletion of Rnf216/Triad3 in neural stem cells caused abnormal microglia expression in males, but reproductive function remained unaffected. Our findings show that dysfunction of RNF216/TRIAD3 affects the HPG axis and microglia in a region- and sex-dependent manner, implicating sex-specific therapeutic interventions for GHS., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Nociceptor sensitization following nerve injury or inflammation leads to chronic pain. An increase in the nociceptor hyperpolarization-activated current, I h , is observed in many models of pathological pain. Pharmacological blockade of I h prevents the mechanical and thermal hypersensitivity that occurs during pathological pain. Alterations in the Hyperpolarization-activated Cyclic Nucleotide-gated ion channel 2 (HCN2) mediate I h -dependent thermal and mechanical hyperalgesia. Limited knowledge exists regarding the nature of these changes during chronic inflammatory pain. Modifications in HCN2 expression and post-translational SUMOylation have been observed in the Complete Freund's Adjuvant (CFA) model of chronic inflammatory pain. Intra-plantar injection of CFA into the rat hindpaw induces unilateral hyperalgesia that is sustained for up to 14 days following injection. The hindpaw is innervated by primary afferents in lumbar DRG, L4-6. Adjustments in HCN2 expression and SUMOylation have been well-documented for L5 DRG during the first 7 days of CFA-induced inflammation. Here, we examine bilateral L4 and L6 DRG at day 1 and day 3 post-CFA. Using L4 and L6 DRG cryosections, HCN2 expression and SUMOylation were measured with immunohistochemistry and proximity ligation assays, respectively. Our findings indicate that intra-plantar injection of CFA elicited a bilateral increase in HCN2 expression in L4 and L6 DRG at day 1, but not day 3, and enhanced HCN2 SUMOylation in ipsilateral L6 DRG at day 1 and day 3. Changes in HCN2 expression and SUMOylation were transient over this time course. Our study suggests that HCN2 is regulated by multiple mechanisms during CFA-induced inflammation.

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

For over half a century, male rodents have been the default model organism in preclinical neuroscience research, a convention that has likely contributed to higher rates of misdiagnosis and adverse side effects from drug treatment in women. Studying both sexes could help to rectify these public health problems, but incentive structures in publishing and career advancement deter many researchers from doing so. Moreover, funding agency directives to include male and female animals and human participants in grant proposals lack mechanisms to hold recipients accountable. In this Perspective, we highlight areas of behavioral, cellular and systems neuroscience in which fundamental sex differences have been identified, demonstrating that truly rigorous science must include males and females. We call for a cultural and structural change in how we conduct research and evaluate scientific progress, realigning our professional reward systems and experimental standards to produce a more equitable, representative and therefore translational body of knowledge.

The present study investigated the impact of advanced age on morphine modulation of persistent inflammatory pain in male and female rats. The impact of age, sex, and pain on μ-opioid receptor (MOR) expression and binding in the ventrolateral periaqueductal gray (vlPAG) was also examined using immunohistochemistry and receptor autoradiography. Intraplantar administration of complete Freund's adjuvant induced comparable levels of edema and hyperalgesia in adult (2-3 mos) and aged (16-18 mos) male and female rats. Morphine potency was highest in adult males, with a greater than two-fold increase in morphine EC 50 observed in adult versus aged males (3.83 mg/kg vs. 10.16 mg/kg). Adult and aged female rats also exhibited significantly higher EC 50 values (7.76 mg/kg and 8.74 mg/kg, respectively) than adult males. The upward shift in EC 50 from adult to aged males was paralleled by a reduction in vlPAG MOR expression and binding. The observed age-related reductions in morphine potency and vlPAG MOR expression and binding have significant implications in pain management in the aged population., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Unilateral injection of Complete Freund's Adjuvant (CFA) into the intra-plantar surface of the rodent hindpaw elicits chronic inflammation and hyperalgesia in the ipsilateral hindlimb. Mechanisms contributing to this hyperalgesia may act over multiple time courses and can include changes in ion channel expression and post-translational SUMOylation. Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels mediate the hyperpolarization-activated current, I h . An HCN2-mediated increase in C-nociceptor I h contributes to mechanical hyperalgesia in the CFA model of inflammatory pain. Changes in HCN2 post-translational SUMOylation and protein expression have not been systematically documented for a given dorsal root ganglia (DRG) throughout the time course of inflammation., Methods: This study examined HCN2 protein expression and post-translational SUMOylation in a rat model of CFA-induced hindpaw inflammation. L5 DRG cryosections were used in immunohistochemistry experiments and proximity ligation assays to investigate HCN2 expression and SUMOylation, respectively, on days 1 and 3 post-CFA., Results: Unilateral CFA injection elicited a significant bilateral increase in HCN2 staining intensity in small diameter DRG neurons on day 1 post-CFA, and a significant bilateral increase in the number of small neurons expressing HCN2 but not staining intensity on day 3 post-CFA. HCN2 channels were hyper-SUMOylated in small diameter neurons of ipsilateral relative to contralateral DRG on days 1 and 3 post-CFA., Conclusions: Unilateral CFA injection elicits unilateral mechanical hyperalgesia, a bilateral increase in HCN2 expression and a unilateral increase in post-translational SUMOylation. This suggests that enhanced HCN2 expression in L5 DRG is not sufficient for mechanical hyperalgesia in the early stages of inflammation and that hyper-SUMOylation of HCN2 channels may also be necessary., Significance: Nociceptor HCN2 channels mediate an increase in I h that is necessary for mechanical hyperalgesia in a CFA model of chronic pain, but the mechanisms producing the increase in nociceptor I h have not been resolved. The data presented here suggest that the increase in I h during the early stages of inflammation may be mediated by an increase in HCN2 protein expression and post-translational SUMOylation., (© 2020 The Authors. European Journal of Pain published by John Wiley & Sons Ltd on behalf of European Pain Federation - EFIC®.)

Each year, over 50 million Americans suffer from persistent pain, including debilitating headaches, joint pain, and severe back pain. Although morphine is amongst the most effective analgesics available for the management of severe pain, prolonged morphine treatment results in decreased analgesic efficacy (i.e., tolerance). Despite significant headway in the field, the mechanisms underlying the development of morphine tolerance are not well understood. The midbrain ventrolateral periaqueductal gray (vlPAG) is a primary neural substrate for the analgesic effects of morphine, as well as for the development of morphine tolerance. A growing body of literature indicates that activated glia (i.e., microglia and astrocytes) facilitate pain transmission and oppose morphine analgesia, making these cells important potential targets in the treatment of chronic pain. Morphine affects glia by binding to the innate immune receptor toll-like receptor 4 (TLR4), leading to the release of proinflammatory cytokines and opposition of morphine analgesia. Despite the established role of the vlPAG as an integral locus for the development of morphine tolerance, most studies have examined the role of glia activation within the spinal cord. Additionally, the role of TLR4 in the development of tolerance has not been elucidated. This review attempts to summarize what is known regarding the role of vlPAG glia and TLR4 in the development of morphine tolerance. These data, together, provide information about the mechanism by which central nervous system glia regulate morphine tolerance, and identify a potential therapeutic target for the enhancement of analgesic efficacy in the clinical treatment of chronic pain., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The long-term consequences of perinatal opioid exposure and subsequent development of neonatal opioid withdrawal syndrome is largely unknown and likely dependent on a multitude of factors, including co-morbid drug use, pre- and post-natal care, and individual factors including the maternal-infant relationship and home environment. This review summarizes the current literature from clinical and preclinical studies on perinatal opioid exposure, focusing on the consequences in the offspring. Although a large number of preclinical studies have been conducted examining the impact of prenatal opioid exposure, the models employed are not necessarily representative of clinical use patterns, making it challenging to translate these results to the impacted population. Use of more clinically-relevant models of perinatal opioid exposure are requisite for the development of improved pharmacological and behavioral treatment strategies to improve quality of life for this vulnerable population.

Morphine remains one of the most widely prescribed opioids for alleviation of persistent and/or severe pain; however, multiple preclinical and clinical studies report that morphine is less efficacious in females compared to males. Morphine primarily binds to the mu opioid receptor, a prototypical G-protein coupled receptor densely localized in the midbrain periaqueductal gray. Anatomical and physiological studies conducted in the 1960s identified the periaqueductal gray, and its descending projections to the rostral ventromedial medulla and spinal cord, as an essential descending inhibitory circuit mediating opioid-based analgesia. Remarkably, the majority of studies published over the following 30 years were conducted in males with the implicit assumption that the anatomical and physiological characteristics of this descending inhibitory circuit were comparable in females; not surprisingly, this is not the case. Several factors have since been identified as contributing to the dimorphic effects of opioids, including sex differences in the neuroanatomical and neurophysiological characteristics of the descending inhibitory circuit and its modulation by gonadal steroids. Recent data also implicate sex differences in opioid metabolism and neuroimmune signaling as additional contributing factors. Here we cohesively present these lines of evidence demonstrating a neural basis for sex differences in opioid modulation of pain, with a focus on the PAG as a sexually dimorphic core of descending opioid-induced inhibition and argue for the development of sex-specific pain therapeutics.

Chronic pain is a debilitating condition that impacts tens of millions each year, resulting in lost wages for workers and exacting considerable costs in health care and rehabilitation. A thorough understanding of the neural mechanisms underlying pain and analgesia is critical to facilitate the development of therapeutic strategies and personalized medicine. Clinical and epidemiological studies report that women experience greater levels of pain than men and have higher rates of pain-related disorders. Studies in both rodents and humans report sex differences in the anatomical and physiologic properties of the descending antinociceptive circuit, mu opioid receptor (MOR) expression and binding, morphine metabolism, and immune system activation, all of which likely contribute to the observed sex differences in pain and opioid analgesia. Although more research is needed to elucidate the underlying mechanisms, these sex differences present potential therapeutic targets to optimize pain management strategies for both sexes.

Preclinical studies report that the effective dose for morphine is approximately 2-fold higher in females than males. Following systemic administration, morphine is metabolized via Phase II glucuronidation in the liver and brain into two active metabolites: morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G), each possessing distinct pharmacological profiles. M6G binds to μ opioid receptors and acts as a potent analgesic. In contrast, M3G binds to toll-like receptor 4 (TLR4), initiating a neuroinflammatory response that directly opposes the analgesic effects of morphine and M6G. M3G serum concentrations are 2-fold higher in females than males, however, sex-specific effects of morphine metabolites on analgesia and glial activation in vivo remain unknown. The present studies test the hypothesis that increased M3G, and subsequent TLR4-mediated activation of glia, is a primary mechanism driving the attenuated response to morphine in females. We demonstrate that intra-PAG M6G results in a greater analgesic response in females than morphine alone. M6G analgesia was reversed with co-administration of (-)-naloxone, but not (+)-naloxone, suggesting that this effect is μ opioid receptor mediated. In contrast, intra-PAG administration of M3G significantly attenuated the analgesic effects of systemic morphine in males only, increasing the 50% effective dose of morphine two-fold (5.0 vs 10.3mg/kg) and eliminating the previously observed sex difference. An increase in IL-1β, IL-6 and TNF was observed in females following intra-PAG morphine or M6G. In males, only IL-1β levels increased following morphine. Changes in cytokine levels following M3G were limited to TNF in females. Together, these data implicate sex differences in morphine metabolism, specifically M3G, as a contributing factor in the attenuated response to morphine observed in females., (Copyright © 2017 Elsevier Inc. All rights reserved.)

We hypothesize that dorsal hippocampal (dHC) neurons, which are critical for episodic memory, form a memory of a meal and inhibit the initiation of the next meal and the amount ingested during that meal. In support, we showed previously that (1) consuming a sucrose meal induces expression of the synaptic plasticity marker activity-regulated cytoskeleton-associated protein (Arc) in dHC neurons and (2) reversible inactivation of these neurons immediately following a sucrose meal accelerates the onset of the next meal and increases the size of that meal. These data suggest that hippocampal-dependent memory inhibits intake; therefore, the following experiments were conducted to determine whether hippocampal-dependent memory impairments are associated with increased intake. We reported recently that one episode of early life inflammatory pain impairs dHC-dependent memory in adult rats. The present study determined whether neonatal inflammatory pain also increases sucrose intake and attenuates sucrose-associated Arc expression. Male and female Sprague-Dawley rats were given an intraplantar injection of the inflammatory agent carrageenan (1%) on the day of birth and sucrose intake and sucrose-associated dHC Arc expression were measured in adulthood. Neonatal inflammatory pain increased sucrose intake in adult female and male rats, decreased sucrose-associated dHC Arc expression in female rats, and tended to have a similar effect on Arc expression in male rats. Neonatal inflammatory pain significantly decreased the interval between two sucrose meals in female but not in male rats. Morphine administration at the time of insult attenuated the effects of injury on sucrose intake. Collectively, these findings indicate that one brief episode of inflammatory pain on the day of birth has a long long-lasting, sex-dependent impact on intake of a palatable food in adulthood., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Although morphine remains the primary drug prescribed for alleviation of severe or persistent pain, both preclinical and clinical studies have shown that females require two to three times more morphine than males to produce comparable levels of analgesia. In addition to binding to the neuronal μ-opioid receptor, morphine binds to the innate immune receptor toll-like receptor 4 (TLR4) localized primarily on microglia. Morphine action at TLR4 initiates a neuroinflammatory response that directly opposes the analgesic effects of morphine. Here, we test the hypothesis that the attenuated response to morphine observed in females is the result of increased microglia activation in the periaqueductal gray (PAG), a central locus mediating the antinociceptive effects of morphine. We report that, whereas no overall sex differences in the density of microglia were noted within the PAG of male or female rats, microglia exhibited a more "activated" phenotype in females at baseline, with the degree of activation a significant predictor of morphine half-maximal antinociceptive dose (ED 50 ) values. Priming microglia with LPS induced greater microglia activation in the PAG of females compared with males and was accompanied by increased transcription levels of IL-1β and a significant rightward shift in the morphine dose-response curve. Blockade of morphine binding to PAG TLR4 with (+)-naloxone potentiated morphine antinociception significantly in females such that no sex differences in ED 50 were observed. These results demonstrate that PAG microglia are sexually dimorphic in both basal and LPS-induced activation and contribute to the sexually dimorphic effects of morphine in the rat. SIGNIFICANCE STATEMENT We demonstrate that periaqueductal gray (PAG) microglia contribute to the sexually dimorphic effects of morphine. Specifically, we report that increased activation of microglia in the PAG contributes to the attenuated response to morphine observed in females. Our data further implicate the innate immune receptor toll-like receptor 4 (TLR4) as an underlying mechanism mediating these effects and establish that TLR4 inhibition in the PAG of females reverses the sex differences in morphine responsiveness. These data suggest novel methods to improve current opioid-based pain management via inhibition of glial TLR4 and illustrate the necessity for sex-specific research and individualized treatment strategies for the management of pain in men and women., (Copyright © 2017 the authors 0270-6474/17/373202-13$15.00/0.)

Opioid tolerance and the potential for addiction is a significant burden associated with pain management, yet its precise underlying mechanism and prevention remain elusive. Immune signaling contributes to the decreased efficacy of opioids, and we recently demonstrated that Toll-like receptor 4 (TLR4)-mediated neuroinflammation in the periaqueductal gray (PAG) drives tolerance. Tumor necrosis factor (TNF), a product of TLR4 signaling, promotes inflammation and facilitates glutamatergic signaling, key components of opioid tolerance. Therefore, we hypothesize that TLR4-mediated opioid tolerance requires TNF signaling. By expression of a dominant-negative TNF peptide via lentiviral vector injection in rat PAG to sequester soluble TNF (solTNF), we demonstrate that solTNF mediates morphine tolerance induced by TLR4 signaling, stimulates neuroinflammation (increased IL-1β and TLR4 mRNA), and disrupts glutamate reuptake (decreased GLT-1 and GLAST mRNA). We further demonstrate the efficacy of the brain-permeant PEGylated version of the anti-solTNF peptide, XPro1595, injected systemically, to normalize morphine-induced CNS neuroinflammation and morphine- and endotoxin-induced changes in glutamate transport, effectively preserving the efficacy of morphine analgesia and eliminating tolerance. Our findings provide a novel pharmacological target for the prevention of opioid-induced immune signaling, tolerance, and addiction.

Morphine has been and continues to be one of the most potent and widely used drugs for the treatment of pain. Clinical and animal models investigating sex differences in pain and analgesia demonstrate that morphine is a more potent analgesic in males than in females. In addition to binding to the neuronal μ-opioid receptor, morphine binds to the innate immune receptor toll-like receptor 4 (TLR4), located on glial cells. Activation of glial TLR4 initiates a neuroinflammatory response that directly opposes morphine analgesia. Females of many species have a more active immune system than males; however, few studies have investigated glial cells as a potential mechanism driving sexually dimorphic responses to morphine. This Mini-Review illustrates the involvement of glial cells in key processes underlying observed sex differences in morphine analgesia and suggests that targeting glia may improve current treatment strategies for pain. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Prairie vole breeder pairs form monogamous pair bonds, which are maintained through the expression of selective aggression toward novel conspecifics. Here, we utilize behavioral and anatomical techniques to extend the current understanding of neural mechanisms that mediate pair bond maintenance. For both sexes, we show that pair bonding up-regulates mRNA expression for genes encoding D1-like dopamine (DA) receptors and dynorphin as well as enhances stimulated DA release within the nucleus accumbens (NAc). We next show that D1-like receptor regulation of selective aggression is mediated through downstream activation of kappa-opioid receptors (KORs) and that activation of these receptors mediates social avoidance. Finally, we also identified sex-specific alterations in KOR binding density within the NAc shell of paired males and demonstrate that this alteration contributes to the neuroprotective effect of pair bonding against drug reward. Together, these findings suggest motivational and valence processing systems interact to mediate the maintenance of social bonds.

From an evolutionary perspective, adaptations of an organism to its early environment are essential for survival. The occurrence of early life perturbation, coincident with increased developmental plasticity, provides a unique opportunity for such adaptations to become programmed and persist throughout life. However, adaptations that are beneficial to maintaining homeostasis in one's early environment may result in extreme response strategies that confer vulnerability or dysfunction later in life. This review summarizes recent findings in human and animal studies demonstrating that early life pain results in a hypo-/hyper-sensitive phenotype in response to acute and persistent pain and stress later in life. Changes in cognition and immune function in response to early life pain have also been observed. Recent data on the neural mechanisms underlying these long-term changes are discussed, as well as potential strategies to minimize the impact of early life pain.

Approximately 1 in 6 infants are born prematurely each year. Typically, these infants spend 25 days in the Neonatal Intensive Care Unit (NICU) where they experience 10-18 painful and inflammatory procedures each day. Remarkably, pre-emptive analgesics and/or anesthesia are administered less than 25% of the time. Unalleviated pain during the perinatal period is associated with permanent decreases in pain sensitivity, blunted cortisol responses and high rates of neuropsychiatric disorders. To date, the mechanism(s) by which these long-term changes in stress and pain behavior occur, and whether such alterations can be prevented by appropriate analgesia at the time of insult, remains unclear. Work in our lab using a rodent model of early life pain suggests that inflammatory pain experienced on the day of birth blunts adult responses to stress- and pain-provoking stimuli, and dysregulates the hypothalamic pituitary adrenal (HPA) axis in part through a permanent upregulation in central endogenous opioid tone. This review focuses on the long-term impact of neonatal inflammatory pain on adult anxiety- and stress-related responses, and underlying neuroanatomical changes in the context of endogenous pain control and the HPA axis. These two systems are in a state of exaggerated developmental plasticity early in postnatal life, and work in concert to respond to noxious or aversive stimuli. We present empirical evidence from animal and clinical studies, and discuss historical perspectives underlying the lack of analgesia/anesthetic use for early life pain in the modern NICU., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The present experiment tested the hypothesis that neonatal injury disrupts adult hippocampal functioning and that normal aging or chronic stress during adulthood, which are known to have a negative impact on hippocampal function, exacerbate these effects. Male and female Sprague-Dawley rats were given an intraplantar injection of the inflammatory agent carrageenan (1%) on the day of birth and their memory was tested in the hippocampal-dependent spatial water maze in adulthood and again in middle age. We found that neonatal injury impaired hippocampal-dependent memory in adulthood, that the effects of injury on memory were more pronounced in middle-aged male rats, and that chronic stress accelerated the onset of these memory deficits. Neonatal injury also decreased glucocorticoid receptor mRNA in the dorsal CA1 area of middle-aged rats, a brain region critical for spatial memory. Morphine administration at the time of injury completely reversed injury-induced memory deficits, but neonatal morphine treatments in the absence of injury produced significant memory impairments in adulthood. Collectively, these findings are consistent with our hypothesis that neonatal injury produces long-lasting disruption in adult hippocampal functioning., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Previous studies in rats have established that inflammatory pain experienced on the day of birth (P0) decreases sensitivity to acute noxious, anxiety- and stress-provoking stimuli. However, to date, the impact of early-life pain on adult responses to chronic stress is not known. Further, the ability of morphine, administered at the time of injury, to mitigate changes in adult behavioral and hormonal responses to acute or chronic stressors has not been examined. P0 male and female Sprague-Dawley rat pups were given an intraplantar injection of 1% carrageenan or handled in an identical manner in the presence or absence of morphine. As adults, rats that experienced early-life pain displayed decreased sensitivity to acute stressors, as indicated by increased time in the inner area of the Open Field, and increased latency to immobility and decreased time immobile in the Forced Swim Test (FST). An accelerated return of corticosterone to baseline was also observed. Morphine administration at the time of injury completely reversed this 'hyporesponsive' phenotype. By contrast, following 7 days of chronic variable stress, injured animals displayed a 'hyperresponsive' phenotype in that they initiated immobility and spent significantly more time immobile in the FST than controls. Responses to chronic stress were also rescued in animals that received morphine at the time of injury. These data suggest that analgesia for early-life pain prevents adult hyposensitivity to acute anxiety- and stress-provoking stimuli and increased vulnerability to chronic stress, and have important clinical implications for the management of pain in infants., (© 2014 S. Karger AG, Basel.)

The last decade has witnessed profound growth in studies examining the role of fundamental neuroimmune processes as key mechanisms that might form a natural bridge between normal physiology and pathological outcomes. Rooted in core concepts from psychoneuroimmunology, this review utilizes a succinct, exemplar-driven approach of several model systems that contribute significantly to our knowledge of the mechanisms by which neuroimmune processes interact with stress physiology. Specifically, we review recent evidence showing that (i) stress challenges produce time-dependent and stressor-specific patterns of cytokine/chemokine expression in the CNS; (ii) inflammation-related genes exhibit unique expression profiles in males and females depending upon individual, cooperative or antagonistic interactions between steroid hormone receptors (estrogen and glucocorticoid receptors); (iii) adverse social experiences incurred through repeated social defeat engage a dynamic process of immune cell migration from the bone marrow to brain and prime neuroimmune function and (iv) early developmental exposure to an inflammatory stimulus (carageenin injection into the hindpaw) has a lasting influence on stress reactivity across the lifespan. As such, the present review provides a theoretical framework for understanding the role that neuroimmune mechanisms might play in stress plasticity and pathological outcomes, while at the same time pointing toward features of the individual (sex, developmental experience, stress history) that might ultimately be used for the development of personalized strategies for therapeutic intervention in stress-related pathologies.

The influence of sex has been neglected in clinical studies on pain and analgesia, with the vast majority of research conducted exclusively in males. However, both preclinical and clinical studies indicate that males and females differ in both the anatomical and physiological composition of central nervous system circuits that are involved in pain processing and analgesia. These differences influence not only the response to noxious stimuli, but also the ability of pharmacological agents to modify this response. Morphine is the most widely prescribed opiate for the alleviation of persistent pain in the clinic; however, it is becoming increasingly clear that morphine is less potent in women compared to men. This review highlights recent research identifying neuroanatomical and physiological dimorphisms underlying sex differences in pain and opioid analgesia, focusing on the endogenous descending pain modulatory circuit., (Published by Elsevier Inc.)

Less than 60% of infants undergoing invasive procedures in the neonatal intensive care unit receive analgesic therapy. These infants show long-term decreases in pain sensitivity and cortisol reactivity. In rats, we have previously shown that inflammatory pain experienced on the day of birth significantly decreases adult somatosensory thresholds and responses to anxiety- and stress-provoking stimuli. These long-term changes in pain and stress responsiveness are accompanied by two-fold increases in central met-enkephalin and β-endorphin expression. However, the time course over which these changes in central opioid peptide expression occur, relative to the time of injury, are not known. The present studies were conducted to determine whether the observed changes in adult opioid peptide expression were present within the first postnatal week following injury. The impact of neonatal inflammation on plasma corticosterone, a marker for stress reactivity, was also determined. Brain, spinal cord, and trunk blood were harvested at 24 h, 48 h, and 7 d following intraplantar administration of the inflammatory agent carrageenan on the day of birth. Radioimmunoassay was used to determine plasma corticosterone and met-enkephalin and β-endorphin levels within the forebrain, cortex, midbrain, and spinal cord. Within 24 h of injury, met-enkephalin levels were significantly increased in the midbrain, but decreased in the spinal cord and cortex; forebrain β-endorphin levels were significantly increased as a result of early life pain. Corticosterone levels were also significantly increased. At 7 d post-injury, opioid peptides remained elevated relative to controls, suggesting a time point by which injury-induced changes become programmed and permanent., (Copyright © 2013 Wiley Periodicals, Inc.)

Inflammatory pain experienced on the day of birth (postnatal day 0: PD0) significantly dampens behavioral responses to stress- and anxiety-provoking stimuli in adult rats. However, to date, the mechanisms by which early life pain permanently alters adult stress responses remain unknown. The present studies examined the impact of inflammatory pain, experienced on the day of birth, on adult expression of receptors or proteins implicated in the activation and termination of the stress response, including corticotrophin releasing factor receptors (CRFR1 and CRFR2) and glucocorticoid receptor (GR). Using competitive receptor autoradiography, we show that Sprague Dawley male and female rat pups administered 1% carrageenan into the intraplantar surface of the hindpaw on the day of birth have significantly decreased CRFR1 binding in the basolateral amygdala and midbrain periaqueductal gray in adulthood. In contrast, CRFR2 binding, which is associated with stress termination, was significantly increased in the lateral septum and cortical amygdala. GR expression, measured with in situ hybridization and immunohistochemistry, was significantly increased in the paraventricular nucleus of the hypothalamus and significantly decreased in the hippocampus of neonatally injured adults. In parallel, acute stress-induced corticosterone release was significantly attenuated and returned to baseline more rapidly in adults injured on PD0 in comparison to controls. Collectively, these data show that early life pain alters neural circuits that regulate responses to and neuroendocrine recovery from stress, and suggest that pain experienced by infants in the Neonatal Intensive Care Unit may permanently alter future responses to anxiety- and stress-provoking stimuli., (Published by Elsevier Ltd.)

The ventrolateral periaqueductal gray (vlPAG) is an integral locus for morphine action. Although it is clear that glia contribute to the development of morphine tolerance, to date, the investigation of their role has been limited to spinal and medullary loci. Opioids induce a neuroinflammatory response that opposes acute and long-term analgesia, thereby limiting their efficacy as therapeutic agents. Recent data suggest that the innate immune receptor Toll-like receptor 4 (TLR4), along with its coreceptor myeloid differentiation factor-2 (MD-2), mediates these effects. To date, the brain loci through which TLR4 modulates morphine tolerance have not been identified. We have previously demonstrated that chronic subcutaneous morphine results in tolerance that is accompanied by increases in vlPAG glial cell activity. Using in vivo pharmacological manipulations of vlPAG glia and TLR4 in the adult male rat, we show that intra-vlPAG administration of the general glial cell metabolic inhibitor propentofylline or the astrocyte activity inhibitor fluorocitrate attenuate tolerance to morphine. Characterization of MD-2 expression within the PAG revealed dense MD-2 expression throughout the vlPAG. Further, antagonizing vlPAG TLR4 dose dependently prevented the development of morphine tolerance, and vlPAG microinjections of TLR4 agonists dose dependently produced a "naive" tolerance to subsequent challenge doses of morphine. Finally, using a model of persistent inflammatory pain and pharmacological manipulation of TLR4 we demonstrate that systemic antagonism of TLR4 potentiated acute morphine antihyperalgesia. These results, together, indicate that vlPAG glia regulate morphine tolerance development via TLR4 signaling, and implicate TLR4 as a potential therapeutic target for the treatment of pain.

Unlabelled: Morphine is among the most prevalent analgesics prescribed for chronic pain. However, prolonged morphine treatment results in the development of analgesic tolerance. An abundance of evidence has accumulated indicating that central nervous system glial cell activity facilitates pain transmission and opposes morphine analgesia. While the midbrain ventrolateral periaqueductal gray (vlPAG) is an important neural substrate mediating pain modulation and the development of morphine tolerance, no studies have directly assessed the role of PAG glia. Here we test the hypothesis that morphine-induced increases in vlPAG glial cell activity contribute to the development of morphine tolerance. As morphine is primarily consumed for the alleviation of severe pain, the influence of persistent inflammatory pain was also assessed. Administration of morphine, in the absence of persistent inflammatory pain, resulted in the rapid development of morphine tolerance and was accompanied by a significant increase in vlPAG glial activation. In contrast, persistent inflammatory hyperalgesia, induced by intraplantar administration of complete Freund's adjuvant (CFA), significantly attenuated the development of morphine tolerance. No significant differences were noted in vlPAG glial cell activation for CFA-treated animals versus controls. These results indicate that vlPAG glia are modulated by a persistent pain state, and implicate vlPAG glial cells as possible regulators of morphine tolerance., Perspective: The development of morphine tolerance represents a significant impediment to its use in the management of chronic pain. We report that morphine tolerance is accompanied by increased glial cell activation within the vlPAG, and that the presence of a persistent pain state prevented vlPAG glial activation and attenuated morphine tolerance., (Published by Elsevier Inc.)

Approximately 500,000 infants are born prematurely each year in the United States. These infants typically require an extensive stay in the neonatal intensive care unit (NICU), where they experience on average 14 painful and invasive procedures each day. These procedures, including repeated heel lance, insertion of intravenous lines, and respiratory and gastric suctioning, typically result in an inflammatory response, inducing pain and stress in the newborn. Remarkably, the majority of these procedures are performed in the complete absence of pre- or post-emptive analgesics. Recent clinical studies report that former NICU patients have increased thresholds for pain and stress later in life as compared with term-born infants. However, to date, the mechanisms whereby early-life inflammation alters later-life response to stress and pain are not known. The present studies were conducted to determine if neonatal injury impairs adult responses to anxiety- and stress-provoking stimuli. As we have previously reported that early-life pain results in a significant increase in opioid peptide expression within the midbrain periaqueductal gray, the role of endogenous opioids in our behavioral studies was also examined. Male and female rats received an intraplantar injection of the inflammatory agent carrageenan (1%) on the day of birth. In adulthood, animals were assessed for changes in response to anxiety- and stress-provoking stimuli using the open field and forced swim tests, respectively. Injury-induced changes in sucrose preference and stress-induced analgesia were also assessed. As adults, neonatally injured animals displayed a blunted response to both anxiety- and stress-provoking stimuli, as indicated by significantly more time spent in the inner area of the open field and a 2-fold increase in latency to immobility in the forced swim test as compared to controls. No change in sucrose preference was observed. Using in situ hybridization and immunohistochemistry, we observed a 2-fold increase in enkephalin mRNA and protein expression, respectively, in stress-related brain regions including the central amygdala and lateral septum. Administration of the opioid receptor antagonist naloxone reversed the attenuated responses to forced swim stress and stress-induced analgesia, suggesting the changes in stress-related behavior were opioid-dependent. Together, these data contribute to mounting evidence that neonatal injury in the absence of analgesics has adverse effects that are both long-term and polysystemic., (Copyright © 2013 S. Karger AG, Basel.)

Despite significant evidence that opioids are involved in attachment by mediating social reward and motivation, the role of opioids in the formation of adult social attachments has not been explored. We used the socially monogamous prairie vole (Microtus ochrogaster) to explore the role of endogenous opioids in social bonding by examining partner preference formation in female prairie voles. We hypothesized that μ-opioid receptors (MORs) in the striatum have a critical role in partner preference formation. We therefore predicted that peripheral administration of an opioid receptor antagonist would inhibit partner preference formation, and more specifically, that μ-opioid selective receptor blockade within the striatum would inhibit partner preference formation. To test our hypotheses, we first administered the non-selective opioid antagonist naltrexone peripherally to females during an 18-h cohabitation with a male and later tested the female with a partner preference test (PPT). Females showed a dose schedule-dependent decrease in partner preference in the PPT, with females in the continuous dose group displaying stranger preferences. Next, we administered microinjections of the MOR selective antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) into either the nucleus accumbens shell (NAS) or the caudate-putamen (CP) immediately before a 24-h cohabitation with a male, and later tested the female with a PPT. Females receiving CTAP into the CP, but not the NAS, showed no preference in the PPT, indicating an inhibition of partner preference formation. We show here for the first time that MORs modulate partner preference formation in female prairie voles by acting in the CP.

While selective serotonin reuptake inhibitors (SSRIs) are widely used to treat anxiety and depression, they also produce profound disruptions of sexual function including delayed orgasm/ejaculation. The nucleus paragigantocellularis (nPGi), a primary source of inhibition of ejaculation in male rats, contains receptors for serotonin (5-HT). The ventrolateral periaqueductal gray (vlPAG) provides serotonin to this region, thus providing an anatomical and neurochemical basis for serotonergic regulation of the nPGi. We hypothesize that 5-HT acting at the nPGi could underlie the SSRI-induced inhibition of ejaculation in rodents. To this end, we produced 5-HT lesions of the source of 5-HT to the nPGi (the vlPAG) and examined sexual behavior. Removing the source of 5-HT to the nPGi facilitated genital reflexes, but not other aspects of sexual behavior, consistent with our hypothesis. Namely, 5-HT lesions produced a significant increase in the mean number of ejaculations and a significant decrease in ejaculation latency as compared to sham lesioned animals, while latency to mating and the post-ejaculatory interval did not differ. These data suggest that the serotonergic vlPAG-nPGi pathway is an important regulatory mechanism for the inhibition of ejaculation in rats and supports the hypothesis that this circuit contributes to SSRI-induced inhibition of ejaculation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Somatic genital reflexes such as ejaculation and vaginocervical contractions are produced through the striated muscles associated with the genitalia. The coordination of these reflexes is surprisingly complex and involves a number of lumbosacral spinal and supraspinal systems. The rat model has been proven to be an excellent source of information regarding these mechanisms, and many parallels to research in humans can be drawn. An understanding of the spinal systems involving the lumbosacral spinal cord, both efferent and afferent, has been generated through decades of research. Spinal and supraspinal mechanisms of descending excitation, through a spinal ejaculation generator in the lumbar spinal cord and thalamus, and descending inhibition, through the ventrolateral medulla, have been identified and characterized both anatomically and physiologically. In addition, delineation of the neural circuits whereby ascending genitosensory information regarding the regulation of somatic genital reflexes is relayed supraspinally has also been the topic of recent investigation. Lastly, the importance of the "social neuropeptides" oxytocin and vasopressin in the regulation of somatic genital reflexes, and associated sociosexual behaviors, is emerging. This work not only has implications for understanding how nervous systems generate sexual behavior but also provides treatment targets for sexual dysfunction in people., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Little is known regarding the descending inhibitory control of genital reflexes such as ejaculation and vaginal contractions. The brainstem nucleus paragigantocellularis (nPGi) projects bilaterally to the lumbosacral motoneuron pools that innervate the genital musculature of both male and female rats. Electrolytic nPGi lesions facilitate ejaculation in males, leading to the hypothesis that the nPGi is the source of descending inhibition to genital reflexes. However, the function of the nPGi in female sexual behavior remains to be elucidated. To this end, male and female rats received bilateral excitotoxic fiber-sparing lesions of the nPGi, and sexual behavior and sexual behavior-induced Fos expression were examined. In males, nPGi lesions facilitated copulation, supporting the hypothesis that the nPGi, and not fibers-of-passage, is the source of descending inhibition of genital reflexes in male rats. nPGi lesions in males did not alter sexual behavior-induced Fos expression in any brain region examined. nPGi-lesioned females spent significantly less time mating with stimulus males and had significantly longer ejaculation-return latencies compared to baseline. These results did not significantly differ from control females, but this trend warranted further analysis of the reinforcing value of sexual behavior. Both lesioned and non-lesioned females formed a conditioned place preference (CPP) for artificial vaginocervical stimulation (aVCS). However, post-reinforcement, nPGi-lesioned females did not differ in the percentage of time spent in the non-reinforced chamber versus the reinforced chamber, suggesting a weakened CPP for aVCS. nPGi lesions in females reduced sexual behavior-induced Fos expression throughout the hypothalamus and amygdala. Taken together, these results suggest that while nPGi lesions in males facilitate copulation, such lesions in females attenuate several aspects of sexual behavior resulting in a reduction in the rewarding value of copulation that may be mediated by nPGi control of genital reflexes. This work has important implications for the understanding and treatment of sexual dysfunction in people including delayed/premature ejaculation, involuntary vaginal spasms, and pain during intercourse., (Published by Elsevier Ltd.)

Over the last several decades, the relative contribution of early life events to individual disease susceptibility has been explored extensively. Only fairly recently, however, has it become evident that abnormal or excessive nociceptive activity experienced during the perinatal period may permanently alter the normal development of the CNS and influence future responses to somatosensory input. Given the significant rise in the number of premature infants receiving high-technology intensive care over the last 20 years, ex-preterm neonates may be exceedingly vulnerable to the long-term effects of repeated invasive interventions. The present review summarizes available clinical and laboratory findings on the lasting impact of exposure to noxious stimulation during early development, with a focus on the structural and functional alterations in nociceptive circuits, and its sexually dimorphic impact.

Studies in both rodents and humans have shown that acute inflammatory pain experienced during the perinatal period produces long-term decreases in pain sensitivity (hypoalgesia) (Grunau et al., 1994a, 2001; Ren et al., 2004; LaPrairie and Murphy, 2007). To date, the mechanisms underlying these long-term adaptations, however, have yet to be elucidated. The present studies tested the hypothesis that neonatal inflammatory pain induces an upregulation in endogenous opioid tone that is maintained into adulthood, and that this increase in opioid tone provides the underlying mechanism for the observed hypoalgesia. On the day of birth (P0), inflammatory pain was induced in male and female Sprague-Dawley rats by intraplantar administration of carrageenan (CGN; 1%). In adulthood (P60), these animals displayed significantly increased paw withdrawal latencies in response to a noxious thermal stimulus in comparison to controls. Systemic administration of the brain-penetrant opioid receptor antagonist naloxone HCl, but not the peripherally restricted naloxone methiodide, significantly attenuated the injury-induced hypoalgesia. Direct administration of naloxone HCl or antagonists directed at the mu or delta opioid receptors into the midbrain periaqueductal gray (PAG) also significantly reversed the injury-induced hypoalgesia in adult rats. Parallel anatomical studies revealed that inflammatory pain experienced on the day of birth significantly increased beta-endorphin and met/leu-enkephalin protein levels and decreased opioid receptor expression in the PAG of the adult rat. Thus, early noxious insult produces long-lasting alterations in endogenous opioid tone, thereby profoundly impacting nociceptive responsiveness in adulthood.

Oxytocin regulates partner preference formation and alloparental behavior in the socially monogamous prairie vole (Microtus ochrogaster) by activating oxytocin receptors in the nucleus accumbens of females. Mating facilitates partner preference formation, and oxytocin-immunoreactive fibers in the nucleus accumbens have been described in prairie voles. However, there has been no direct evidence of oxytocin release in the nucleus accumbens during sociosexual interactions, and the origin of the oxytocin fibers is unknown. Here we show for the first time that extracellular concentrations of oxytocin are increased in the nucleus accumbens of female prairie vole during unrestricted interactions with a male. We further show that the distribution of oxytocin-immunoreactive fibers in the nucleus accumbens is conserved in voles, mice and rats, despite remarkable species differences in oxytocin receptor binding in the region. Using a combination of site-specific and peripheral infusions of the retrograde tracer Fluorogold, we demonstrate that the nucleus accumbens oxytocin-immunoreactive fibers likely originate from paraventricular and supraoptic hypothalamic neurons. This distribution of retrogradely labeled neurons is consistent with the hypothesis that striatal oxytocin fibers arise from collaterals of magnocellular neurons of the neurohypophysial system. If correct, this may serve to coordinate peripheral and central release of oxytocin with appropriate behavioral responses associated with reproduction, including pair bonding after mating, and maternal responsiveness following parturition and during lactation.

Women are more sensitive to most noxious visceral stimuli, both in terms of intensity and frequency. The spinoparabrachial (spino-PBn) pathway is an essential neural circuit for the central relay of viscerosensitive information, but studies characterizing the anatomical and physiological characteristics of this pathway have only been conducted in males. Sex differences in the anatomical and/or physiological organization of the spino-PBn may contribute to the sexually dimorphic incidence rate for visceral pain syndromes. Retrograde labeling and colorectal distention (CRD) induced Fos expression was used to delineate the spino-PBn circuit in male and cycling female Sprague-Dawley rats. The ability of morphine to suppress CRD evoked responses was also examined. Neurons retrogradely labeled from the PBn were localized primarily within the superficial dorsal horn and sacral parasympathetic nucleus of the L5-S1 spinal cord. While no sex differences were noted in either the distribution of spino-PBn neurons or in CRD-induced Fos expression, significantly greater Fos expression was noted specifically in spino-PBn neurons in males compared to females. Morphine selectively attenuated Fos expression in spino-PBn neurons in males, but not females. Subsequent anatomical studies showed significantly reduced mu opioid receptor protein levels and radioligand binding within the PBn of females in comparison to males. Together, these data indicate that there are profound sex differences in how a noxious visceral stimulus and opiates engage the spino-PBn pathway, which may account for the observed clinical differences in visceral pain sensitivity and morphine antinociception.

Persistent inflammatory nociception increases levels of endogenous opioids with affinity for delta opioid receptors in the ventromedial medulla and enhances the antinociceptive effects of the mu opioid receptor (MOPr) agonist [D-Ala2, N-Me-Phe4, Gly5-ol]enkephalin (DAMGO) [Hurley, R.W., Hammond, D.L., 2001. Contribution of endogenous enkephalins to the enhanced analgesic effects of supraspinal mu opioid receptor agonists after inflammatory injury. J. Neurosci. 21, 2536-2545]. It also increases levels of endogenous opioids that act at MOPr elsewhere in the CNS [Zangen, A., Herzberg, U., Vogel, Z., Yadid, G., 1998. Nociceptive stimulus induces release of endogenous beta-endorphin in the rat brain. Neuroscience 85, 659-662]. This study tested the hypothesis that a sustained release of endogenous opioids leads to a downregulation of MOPr in the locus coeruleus (LC) and induces a state of endogenous opioid tolerance. Four days after injection of complete Freund's adjuvant (CFA) in the left hindpaw of the rat, both the magnitude and duration of the antinociception produced by microinjection of DAMGO in the right LC were reduced. Saturation isotherms demonstrated a 50% decrease in MOPr B(max) in homogenates of the LC from CFA-treated rats; K(d) was unchanged. Receptor autoradiography revealed that this decrease was bilateral. The decreased efficacy of DAMGO in CFA-treated rats most likely results from a decreased number of MOPr in the LC. Microinjection of the MOPr antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) in the LC did not exacerbate hyperalgesia in the ipsilateral hindpaw or produce hyperalgesia in the contralateral hindpaw of CFA-treated rats. The downregulation in MOPr is therefore unlikely to result from the induction of endogenous opioid tolerance in the LC. These results indicate that persistent inflammatory nociception alters the antinociceptive actions of MOPr agonists in the CNS by diverse mechanisms that are nucleus specific and likely to have different physiological implications.

Anatomical and physiological studies conducted in the 1960s identified the periaqueductal gray (PAG) and its descending projections to the rostral ventromedial medulla (RVM) and spinal cord dorsal horn, as a primary anatomical pathway mediating opioid-based analgesia. Since these initial studies, the PAG-RVM-spinal cord pathway has been characterized anatomically and physiologically in a wide range of vertebrate species. Remarkably, the majority of these studies were conducted exclusively in males with the implicit assumption that the anatomy and physiology of this circuit were the same in females; however, this is not the case. It is well established that morphine administration produces greater antinociception in males compared to females. Recent studies indicate that the PAG-RVM pathway contributes to the sexually dimorphic actions of morphine. This manuscript will review our anatomical, physiological, and behavioral data identifying sex differences in the PAG-RVM pathway, focusing on its role in pain modulation and morphine analgesia.