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Abstract Background Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is frequently over-expressed in simple epithelia, progenitors, embryonic and tissue stem cells, carcinoma and cancer-initiating cells. Besides functioning as a homophilic adhesion protein, EpCAM is an oncogenic receptor that requires regulated intramembrane proteolysis for activation of its signal transduction capacity. Upon cleavage, the extracellular domain EpEX is released as a soluble ligand while the intracellular domain EpICD translocates into the cytoplasm and eventually into the nucleus in combination with four-and-a-half LIM domains protein 2 (FHL2) and β-catenin, and drives cell proliferation. Methods EpCAM cleavage, induction of the target genes, and transmission of proliferation signals were investigated under varying density conditions using confocal laser scanning microscopy, immunoblotting, cell counting, and conditional cell systems. Results EpCAM cleavage, induction of the target genes, and transmission of proliferation signals were dependent on adequate cell-to-cell contact. If cell-to-cell contact was prohibited EpCAM did not provide growth advantages. If cells were allowed to undergo contact to each other, EpCAM transmitted proliferation signals based on signal transduction-related cleavage processes. Accordingly, the pre-cleaved version EpICD was not dependent on cell-to-cell contact in order to induce c-myc and cell proliferation, but necessitated nuclear translocation. For the case of contact-inhibited cells, although cleavage of EpCAM occurred, nuclear translocation of EpICD was reduced, as were EpCAM effects. Conclusion Activation of EpCAM's cleavage and oncogenic capacity is dependent on cellular interaction (juxtacrine) to provide for initial signals of regulated intramembrane proteolysis, which then support signalling via soluble EpEX (paracrine).

Aims: Opioid use disorder (OUD) remains a serious public health problem. Opioid maintenance treatment is effective but under-utilized, hard to access under existing federal regulations, and, once patients achieve OUD stability, challenging to discontinue. Fewer than 2% of persons with OUD stop using opioids completely. There have been calls from public advocacy groups, governmental agencies, and public health officials for new treatments for OUD. Dezocine, a non-scheduled opioid previously used in the United States and currently widely prescribed in China for pain management, could be a candidate for a novel OUD treatment medication in the U.S. Nonetheless, to date, there have been no reviews of the clinical and preclinical literature detailing dezocine's abuse potential, a key consideration in assessing its clinical utility., Discussion: There are no English language reports of human abuse, dependence, or overdose of dezocine, despite years of extensive clinical use. There are a few case reports of dezocine abuse in the Chinese literature, but there are no reports of overdose deaths. Dezocine is perceived as an opioid and is "liked" by opioid-experienced human and non-human primates, properties that are not dose-dependent and are mitigated by ceiling effects-higher doses do not result in more "liking." There is little withdrawal, spontaneous or precipitated, in humans, monkeys, rats, or mice treated chronically with dezocine alone. However, at some doses, dezocine can precipitate withdrawal in humans and monkeys dependent on other opioids. In rodents, dezocine reduces the severity of morphine withdrawal and the rewarding properties of other opioids., Conclusions: Although dezocine is reinforcing in humans and monkeys with prior or concurrent opioid use within a restricted dose range, there are only a few anecdotal reports of dezocine abuse despite of the long history of use in humans. Given the evidence of dezocine's limited abuse potential, it could be useful both as a treatment for OUD. However, in-depth studies would be required for dezocine to be re-considered for clinical use., (© 2024 The Author(s). CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

Background: In the short term, parental presence while a human infant is in pain buffers the immediate pain responses, although emerging evidence suggests repeated social buffering of pain may have untoward long-term effects., Methods/finding: To explore the short- and long-term impacts of social buffering of pain, we first measured the infant rat pup's [postnatal day (PN) 8, or 12] response to mild tail shock with the mother present compared to shock alone or no shock. Shock with the mother reduced pain-related behavioral activation and USVs of pups at both ages and reduced Fos expression in the periaqueductal gray, hypothalamic paraventricular nucleus, and the amygdala at PN12 only. At PN12, shock with the mother compared to shock alone differentially regulated expression of several hundred genes related to G-protein-coupled receptors (GPCRs) and neural development, whereas PN8 pups showed a less robust and less coherent expression pattern. In a second set of experiments, pups were exposed to daily repeated Shock-mother pairings (or controls) at PN5-9 or PN10-14 (during and after pain sensitive period, respectively) and long-term outcome assessed in adults. Shock+mother pairing at PN5-9 reduced adult carrageenan-induced thermal hyperalgesia and reduced Fos expression, but PN10-14 pairings had minimal impact. The effect of infant treatment on adult affective behavior showed a complex treatment by age dependent effect. Adult social behavior was decreased following Shock+mother pairings at both PN5-9 and PN10-14, whereas shock alone had no effect. Adult fear responses to a predator odor were decreased only by PN10-14 treatment and the infant Shock alone and Shock+mother did not differ., Conclusions/significance: Overall, integrating these results into our understanding of long-term programming by repeated infant pain experiences, the data suggest that pain experienced within a social context impacts infant neurobehavioral responses and initiates an altered developmental trajectory of pain and affect processing that diverges from experiencing pain alone., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Barr et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Dezocine is a partial mu opioid receptor agonist previously used as an analgesic for perioperative acute pain in the US and is now the most used perioperative analgesic in China. In general, dezocine is well-tolerated, with relatively minimal risk of fatal respiratory depression. To our knowledge, there are no reports of dezocine addiction, which suggests that the abuse liability of dezocine is low. The overarching goal of this study was to determine the efficacy of a novel formulation of dezocine (Cyc-dezocine), developed for intraperitoneal or intranasal administration, to reduce voluntary opioid taking in rats. One cohort of male rats self-administered intravenous oxycodone on a fixed-ratio 5 schedule of reinforcement. Once oxycodone taking stabilized, rats were pretreated with systemic injections of vehicle or Cyc-dezocine. Cyc-dezocine dose-dependently reduced intravenous oxycodone self-administration. A second cohort of male and female rats self-administered oral oxycodone from drinking water. Once oxycodone taking stabilized, rats were pretreated with intra-nasal Cyc-dezocine. Consistent with the effects of i.p. Cyc-dezocine in our intravenous oxycodone studies, intra-nasal Cyc-dezocine attenuated oral oxycodone self-administration. Together, these findings support the need for further studies investigating the therapeutic potential of Cyc-dezocine for treating opioid use disorder., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Introduction: Pain assessment is challenging in neonates. Behavioral and physiological pain scales do not assess neocortical nociception, essential to pain encoding and central pain pathway development. Functional near-infrared spectroscopy (fNIRS) can assess neocortical activation to noxious stimuli from changes in oxy-(HbO) and total-hemoglobin concentrations (HbT). This study aims to assess fNIRS nociceptive functional activation in the prefrontal cortex of neonates undergoing circumcision through changes in HbO and HbT, and the correlation between changes in fNIRS and Neonatal Infant Pain Scale (NIPS), a behavioral pain assessment scale., Methods: In healthy term neonates, HbO, HbT, and NIPS were recorded during sequential circumcision events 1-Prep before local anesthetic injection; 2-Local anesthetic injection; 3-Prep before incision; 4-Oral sucrose; 5-Incision; 6-Gomco (hemostatic device) attached; 7-Gomco twisted on; and 8-Gomco removed. fNIRS and NIPS changes after each event were assessed with Wilcoxon signed-rank test and summarized as median and interquartile range (IQR). Changes in fNIRS vs. NIPS were correlated with Spearman coefficient., Results: In 31 neonates fNIRS increased (median [IQR] µmol/L) with noxious events: Local injection (HbO: 1.1 [0.5, 3.1], p < .001; HbT: 2.3 [0.2, 7.6], p < .001), Gomco attached (HbO: 0.7 [0.1, 1.7], p = .002; HbT: 0.7 [-0.2, 2.9], p = .02), and Gomco twisted on (HbO: 0.5 [-0.2, 1.7], p = .03; HbT: 0.8 [-0.1, 3.3], p = .02). fNIRS decreased with non-noxious event: Prep before incision (HbO: -0.6 [-1.2, -0.2] p < .001; HbT: -1 [-1.8, -0.4], p < .001). Local anesthetic attenuated fNIRS increases to subsequent sharp stimuli. NIPS increased with subsequent sharp stimuli despite local anesthetic. Although fNIRS and NIPS changed in the same direction, there was not a strong correlation between them., Conclusions: During neonatal circumcision, changes in fNIRS differed between different types of painful stimuli, which was not the case for NIPS, suggesting that fNIRS may complement NIPS to assess the quality of pain., (© 2021 John Wiley & Sons Ltd.)

Dezocine is an opioid that was used in clinical practice for acute pain management in the US (1986 to 2011) and is currently in use in China. It is not listed as a controlled substance in the US due to no reported cases of addiction. Dezocine is a partial agonist at the mu opioid receptor (MOR); however, it is unclear whether dezocine can activate both the G protein pathway and the beta-arrestin pathway. In this study we hypothesized that dezocine does not activate the beta-arrestin pathway, which could be the potential molecular mechanism by which dezocine is not addictive or at least less addictive than other classic opioids. Both morphine, a MOR full agonist and buprenorphine, a partial MOR agonist similar to dezocine, were used for comparison purposes. The major side effects of dezocine in clinical usage are its gastrointestinal side effects and first pass effects; therefore, we explored the possibility of administering dezocine intranasally in rodents to demonstrate the feasibility of intranasal administration for new clinical usage purposes. With proper formulation it is possible to administer dezocine intranasally to achieve a high concentration in the brain in the rodent model. The results indicate that dezocine does not activate the beta-arrestin pathway in MOR. Intranasal delivery of dezocine achieves a much higher medication concentration in the blood and brain as compared to intraperitoneal injection. It also persists a longer time before it falls below detection in the blood. This study provides a possible explanation of why dezocine is not addictive or at least less addictive than other commonly used opioids. This study also demonstrates that intranasal administration offers an alternative strategy for its potential clinical applications.

Traumatic brain injury (TBI) is a serious public health problem associated with numerous physical and neuropsychiatric comorbidities. Chronic pain is prevalent and interferes with post-injury functioning and quality of life, whereas substance use disorder (SUD) is the third most common neuropsychiatric diagnosis after TBI. Neither of these conditions has a clear mechanistic explanation based on the known pathophysiology of TBI. Dynorphin is an endogenous opioid neuropeptide that is significantly dysregulated after TBI. Both dynorphin and its primary receptor, the ĸ-opioid receptor (KOR), are implicated in the neuropathology of chronic pain and SUD. Here, we review the known roles of dynorphin and KORs in chronic pain and SUDs. We synthesize this information with our current understanding of TBI and highlight potential mechanistic parallels between and across conditions that suggest a role for dynorphin in long-term sequelae after TBI. In pain studies, dynorphin/KOR activation has either antinociceptive or pro-nociceptive effects, and there are similarities between the signaling pathways influenced by dynorphin and those underlying development of chronic pain. Moreover, the dynorphin/KOR system is considered a key regulator of the negative affective state that characterizes drug withdrawal and protracted abstinence in SUD, and molecular and neurochemical changes observed during the development of SUD are mirrored by the pathophysiology of TBI. We conclude by proposing hypotheses and directions for future research aimed at elucidating the potential role of dynorphin/KOR in chronic pain and/or SUD after TBI.

The world is in the midst of an opioid crisis. Nearly 92,000 persons in the U.S. alone died from illicit drugs and prescription opioids in 2020 [1]. This number does not include the countless other individuals who die as a result of the violent crime that accompanies the illicit drug trade. To address this crisis, we need to appreciate aspects of drug addiction. The goal of this brief review is to highlight some major facets of addiction neurobiology, focused on opioids, to provide a basic understanding of the research and terminology encountered in more detailed in-depth articles and discussions on addiction.

Exposure to stress at an early age programs the HPA axis which can lead to cognitive deficits in adults. However, it is not known whether these deficits emerge in adulthood or are expressed earlier in life. The aims of the study were to investigate (1) the immediate effects of early injury-induced stress in one-day-old (P1) and repeated stress on at P1 and P2 rat pups on plasma corticosterone levels; and (2) examine the subsequent long-term effects of this early stress on spatial learning and memory, and stress reactivity in early P26-34 and late P45-53 adolescent male and female rats. Intra-plantar injection of formalin induced prolonged and elevated levels of corticosterone in pups and impaired spatial learning and short- and long-term memory in late adolescent males and long-term memory in early adolescent females. There were sex differences in late adolescence in both learning and short-term memory. Performance on the long-term memory task was better than that on the short-term memory task for all early adolescent male and female control and stressed animals. Short-term memory was better in the late age control rats of both sexes and for formalin treated females as compared with the early age rats. These results are consistent with an impaired function of structures involved in memory (the hippocampus, amygdala, prefrontal cortex) after newborn pain. However, activation of the HPA axis by neonatal pain did not directly correlate with spatial learning and memory outcomes and the consequences of neonatal pain remain are likely multi-determined., 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., (Copyright © 2021 Butkevich, Mikhailenko, Vershinina and Barr.)

Background: The broad use/misuse of prescription opioids during pregnancy has resulted in a surge of infants with Neonatal Opioid Withdrawal Syndrome (NOWS). Short-term irritability and neurological complications are its hallmarks, but the long-term consequences are unknown., Methods: A newly-developed preclinical model of oxycodone self-administration enables adult female rats to drink oxycodone (∼10/mg/kg/day) before and during pregnancy, and after delivery, and to maintain normal liquid intake, titrate dosing, and avoid withdrawal., Results: Oxycodone was detected in the serum of mothers and pups. Growth parameters in dams and pups and litter mass and size were similar to controls. There were no differences in paw retraction latency to a thermal stimulus between Oxycodone and Control pups at postnatal (PN) 2 or PN14. Oxycodone and Control pups had similar motor coordination, cliff avoidance, righting time, pivoting, and olfactory spatial learning from PN3 through PN13. Separation-induced ultrasonic vocalizations at PN8 revealed higher call frequency in Oxycodone pups relative to Control pups (p<0.031; Cohen's d=1.026). Finally, Oxycodone pups displayed withdrawal behaviors (p's<0.029; Cohen's d's>0.806), and Oxycodone males only vocalized more than Control pups in the first minute of testing (p's<0.050; Cohen's d's>.866). Significant effects were corroborated by estimation plots., Conclusions: Our rat model of oral oxycodone self-administration in pregnancy shows exacerbated affect/social communication in pups in a sex-dependent manner but spared cognition and sensory-motor behaviors. This preclinical model reproduces selective aspects of human opioid use during pregnancy, enabling longitudinal analysis of how maternal oxycodone changes emotional behavior in the offspring., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Newborns in intensive care are regularly exposed to minor painful procedures at developmental time points when noxious stimulation would be normally absent. Pain from these interventions is inconsistently treated and often exists concurrently with systemic infection, a common comorbidity of prematurity. Our understanding of the independent and combined effects of early painful experiences and infection on pain response is incomplete. The main goals of this research therefore were to understand how pain and infection experienced early in life influence future nociceptive and affective responses to painful stimuli. Rat pups were infected with E-coli on postnatal day 2 (PN2) and had left hind paw injury with carrageenan on PN3. Standard thermal tests for acute pain, formalin tests for inflammatory pain, and conditioned place aversion testing were performed at different ages to assess the nociceptive and affective components of the pain response. Early E-coli infection and early inflammatory injury with carrageenan both independently increased pain scores following hind paw reinjury with formalin on PN8, with effects persisting into adulthood in the carrageenan exposed group. When experienced concurrently, early E-coli infection and carrageenan exposure also increased conditioned aversion to pain in adults. Effect of sex was significant only in formalin testing, with males showing higher pain scores in infancy and females showing higher pain scores as adults. These findings demonstrate that infection experienced early in life can alter both the nociceptive and affective components of the pain response and that there is a cumulative effect of local and systemic pro-inflammatory processes on the aversive component of pain., Competing Interests: The authors have no conflicts of interest to disclose., (© 2020 The Authors.)

The increasing abuse of opioids - such as oxycodone - poses major challenges for health and socioeconomic systems. Human prescription opioid abuse is marked by chronic, voluntary, oral intake and sex differences. To develop interventions, the field would benefit from a preclinical paradigm that similarly provides rodents with chronic, continuous, oral, voluntary and free-choice access to oxycodone. Here we show female and male rats voluntarily ingest and choose oxycodone over water and show both dependence and motivation to take oxycodone during a chronic oral voluntary, two-bottle choice, continuous access paradigm. Adult female and male Long-Evans rats were given unlimited, continuous homecage access to two bottles containing water (Control) or one bottle of water and one bottle of oxycodone dissolved in water (Experimental). Virtually all experimental rats voluntarily drank oxycodone (~10 mg/kg/day) and escalated their intake over 22 weeks. Females self-administered twice as much oxycodone by body weight (leading to higher blood levels of oxycodone) and engaged in more gnawing behavior of wooden blocks relative to males. Precipitated withdrawal revealed high levels of dependence in both sexes. Reflecting motivation to drink oxycodone, ascending concentrations of citric acid suppressed the intake of oxycodone (Experimental) and the intake of water (Control); however, Experimental rats returned to pre-citric acid preference levels whereas Controls rats did not. Pre-screening behaviors of rats on open field exploration predicted oxycodone intake. Thus, rats consumed and preferred oxycodone over time in this chronic two-bottle oral choice paradigm and both sexes displayed many features of human oxycodone abuse., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: Premature and ill neonates undergo painful but medically necessary procedures while hospitalized. Although opiate drugs are administered as analgesics, problems associated with their side effects, tolerance, and potential dependence necessitate research into alternative pain-relieving medications. Here we test two plant-derived compounds in infant rats: sinomenine, which targets the Mas-related G-protein-coupled receptor member X2 opioid receptor; and salvinorin A, which is a κ opioid receptor agonist. In adult animals both sinomenine and salvinorin A are analgesic, but neither has been tested in infants., Methods: We used the formalin and thermal plantar tests in rats 7 and 21 days of age (PN7 and PN21) for behavioral signs of pain. In addition, brain sections were stained using Fos immunohistochemistry to examine patterns of brain activation in the midbrain periaqueductal gray and the paraventricular nucleus of the hypothalamus., Results: Sinomenine was analgesic in both the formalin and thermal tests on animals 21 days of age. At PN7 only the highest dose elevated response latencies in the thermal test and there were no effects of sinomenine in the formalin test. Analysis of Fos expression in the sinomenine-treated animals showed no drug effect, in contrast to the behavioral results. Salvinorin A was analgesic in the formalin test only at the highest dose at 21 days of age but not in the thermal test at either age., Conclusion: The increased modest effectiveness of sinomenine in older animals and the minimum salvinorin A drug effect suggest that the compounds act on sites that develop during the preweaning period (sinomenine) or after weaning (salvinorin A)., (Copyright © 2020 Shanghai Changhai Hospital. Published by Elsevier B.V. All rights reserved.)

How obesity exacerbates migraine and other pain disorders remains unknown. Trigeminal nociceptive processing, crucial in migraine pathophysiology, is abnormal in mice with diet induced obesity. However, it is not known if this is also true in genetic models of obesity. We hypothesized that obese mice, regardless of the model, have trigeminal hyperalgesia. To test this, we first evaluated trigeminal thermal nociception in leptin deficient (ob/ob) and control mice using an operant thermal assay. Unexpectedly, we found significant hypoalgesia in ob/ob mice. Because thermal hypoalgesia also occurs in mice lacking the transient receptor potential vanilloid 1 channel (TRPV1), we tested capsaicin-evoked trigeminal nociception. Ob/ob and control mice had similar capsaicin-evoked nocifensive behaviors, but ob/ob mice were significantly less active after a facial injection of capsaicin than were diet-induced obese mice or lean controls. Conditioned place aversion in response to trigeminal stimulation with capsaicin was similar in both genotypes, indicating normal negative affect and pain avoidance. Supporting this, we found no difference in TRPV1 expression in the trigeminal ganglia of ob/ob and control mice. Finally, we assessed the possible contribution of hyperphagia, a hallmark of leptin deficiency, to the behavior observed in the operant assay. Ob/ob and lean control mice had similar reduction of intake when quinine or capsaicin was added to the sweetened milk, excluding a significant contribution of hyperphagia. In summary, ob/ob mice, unlike mice with diet-induced obesity, have trigeminal thermal hypoalgesia but normal responses to capsaicin, suggesting specificity in the mechanisms by which leptin acts in pain processing., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

The selective serotonin reuptake inhibitor fluoxetine and the 5-HT1A receptor agonist buspirone are used to treat depression and anxiety. Previously we demonstrated that chronic stress during pregnancy (prenatal stress) in rats, used as a model of maternal depression risk, increased inflammatory pain and depressive-like behavior in the offspring; buspirone injected to pregnant dams was protective. Clinically, the addition of buspirone to fluoxetine increases the latter's efficacy in treating depression in patients. Here, we investigated the influence of repeated prenatal injections of fluoxetine, buspirone or their combination on pain- and depressive-like behaviors in prenatally stressed young male and female rats. Prenatal stress augmented depressive-like behavior and both thermal and inflammatory pain (formalin test), replicating our prior findings, and increased basal levels of corticosterone in the blood plasma. Both drugs and their combination reduced the effects of prenatal stress on thermal pain and depressive-like behavior independently of sex. The combination of fluoxetine and buspirone, compared with fluoxetine, was more antinociceptive in the hot plate test in both sexes, and when compared with buspirone, was more antinociceptive only in males. A detailed study of the time-course of formalin-induced pain showed a nuanced effect of these drugs that was sex-dependent. The combination of the two drugs was less effective in females than males during the initial acute phase of nociceptive behavior in flexing + shaking behaviors, whereas that combination was more effective than fluoxetine alone in the first acute phase of licking behavior in females. The antinociceptive effect of buspirone dominated that of the drug combination and of fluoxetine alone, especially during the interphase of the formalin test in both sexes for both flexing + shaking and licking, suggesting a more effective prenatal action of buspirone on the development of a descending serotonergic inhibitory system modulating pain in the spinal cord dorsal horn neurons. Our results indicate that inflammatory pain-like responses integrated at the spinal level in males were more vulnerable to prenatal stress than females. In licking, the antinociceptive effect of fluoxetine and drug combination in the interphase was more in males than females. The data underscore the importance of considering sexual dimorphism when using drug therapy.

Premature or ill full-term infants are subject to a number of noxious procedures as part of their necessary medical care. Although we know that human infants show neural changes in response to such procedures, we know little of the sensory or affective brain circuitry activated by pain. In rodent models, the focus has been on spinal cord and, more recently, midbrain and medulla. The present study assesses activation of brain circuits using manganese-enhanced magnetic resonance imaging (MEMRI). Uptake of manganese, a paramagnetic contrast agent that is transported across active synapses and along axons, was measured in response to a hindpaw injection of dilute formalin in 12-day-old rat pups, the age at which rats begin to show aversion learning and which is roughly the equivalent of full-term human infants. Formalin induced the oft-reported biphasic response at this age and induced a conditioned aversion to cues associated with its injection, thus demonstrating the aversiveness of the stimulation. Morphometric analyses, structural equation modeling and co-expression analysis showed that limbic and sensory paths were activated, the most prominent of which were the prefrontal and anterior cingulate cortices, nucleus accumbens, amygdala, hypothalamus, several brainstem structures, and the cerebellum. Therefore, both sensory and affective circuits, which are activated by pain in the adult, can also be activated by noxious stimulation in 12-day-old rat pups., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

Neonatal pain and stress induce long-term changes in pain sensitivity and behavior. Previously we found alterations in pain sensitivity in adolescent rats exposed to early-life adverse events. We tested whether these alterations have long-lasting effects and if those effects can be improved by the 5-hydroxytryptamine 1A (5-HT1A) receptor agonist buspirone injected chronically during the adolescent period. This study investigates: (1) effects of inflammatory pain (the injection of formalin into the pad of a hind paw) or stress (short maternal deprivation-isolation, MI), or their combination in 1-2-day-old rats on the adult basal pain, formalin-induced pain, anxiety and depression; (2) effects of adolescent buspirone in adult rats that experienced similar early-life insults. Changes in nociceptive thresholds were evaluated using the hot plate (HP) and formalin tests; levels of anxiety and depression were assessed with the elevated plus maze and forced swim tests respectively. Both neonatal painful and stressful treatments induced long-term alterations in the forced swim test. Other changes in adult behavioral responses were dependent on the type of neonatal treatment. There was a notable lack of long-term effects of the combination of early inflammatory pain and stress of MI on the pain responses, anxiety levels or on the effects of adolescent buspirone. This study provides the first evidence that chronic injection of buspirone in adolescent rats alters antinociceptive and anxiolytic effects limited to adult rats that showed behavioral alterations induced by early-life adverse treatments. These data highlight the role of 5-HT1A receptors in long-term effects of neonatal inflammatory pain and stress of short MI on adaptive behavior and possibility of correction of the pain and psychoemotional behavior that were altered by adverse pain/stress intervention using buspirone during critical adolescent period.

Neuropathic pain is chronic pain that develops after nerve injury and is less frequent in infants and children than in adults. Likewise, in animal models of neuropathic pain, allodynia and hyperalgesia are non-existent or attenuated in the infant, with a "switch" during development by which acute nerve injury transitions to chronic pain. Concomitant with the delay in neuropathic pain, there is a parallel delay in the ability of nerve injury to activate the immune system. Models of neuropathic pain in the infant have used various ligation methods and find that neuropathic pain does not occur under after postnatal days 21-28 (PN21-PN28), linked to activation of immune processes and developmental regulation of anti-inflammatory cytokines. We applied a model of neuropathic pain in the adult using a transient compression of the cervical nerve or nerve root in infant rats (injured at 10, 14, 21, or 28 days of age) to define transition periods during which injury results in no change in thermal and mechanical pain sensitivity or in short-term changes in pain. There was little to no hyperalgesia when the injury was imposed at PN10, but significant thermal hyperalgesia and mechanical allodynia 1 day after compression injury when performed at PN14, 21, or 28. Thermal withdrawal latencies returned to near baseline by 7 days postsurgery when the injuries were at PN14, and lasted up to 14 days when the injury was imposed at PN28. There was mechanical allodynia following injury at 1 day postinjury and at 14 days after injury at PN14. Measurements of mRNA from spinal cord at 1, 7, and 14 days postinjury at PN14, 21, and 28 showed that both the magnitude and duration of elevated immune markers and chemokines/cytokines were greater in the older animals, corresponding to the development of hyperalgesia. Thus, we confirm the late onset of neuropathic pain but found no evidence of emergent hyperalgesia if the injury was before PN21. This may be due to the use of a transient, and not sustained, compression ligation model.

We examined the neural substrates of fear memory formation and maintenance when repeated recall was used to prevent forgetting in young animals. In contrast to adult rats, juveniles failed to show contextual fear responses at 4 d post-fear conditioning. Reconsolidation sessions 3 and 6 d after conditioning restored contextual fear responses in juveniles 7 d after initial training. In juveniles that received reconsolidation sessions, protein kinase M zeta (PKMζ) increased in the amygdala, but not in the hippocampus. These data suggest that repeated reminders and increased PKMζ maintain fear responses in juvenile animals that otherwise would not exhibit this behavior., (© 2016 Oliver et al.; Published by Cold Spring Harbor Laboratory Press.)

In the adult, immune and neural processes jointly modulate pain. During development, both are in transition and little is known about the role that the immune system plays in pain processing in infants and children. The objective of this study was to determine if inhibition or augmentation of the immune system would alter pain processing in the infant rat, as it does in the adult. In Experiment 1, rat pups aged 3, 10, or 21 (PN3, PN10, and PN21) days of age were pretreated with NS398 (selective cyclooxygenase (COX)-2 inhibitor) or SC560 (selective COX-1 inhibitor) and tested in the intraplantar formalin test to assess effects of COX inhibition on nociception. Neither drug had an effect on the behavioral response at PN3 or PN10 pups but both drugs attenuated nociceptive scores in PN21 pups. cFos expression in the spinal cord likewise was reduced only at PN21. In Experiment 2, pups were injected with lipopolysaccharide (LPS) prior to the formalin test at PN3 or PN21. LPS increased the nociceptive response more robustly at PN21 than at PN3, while increasing cytokine mRNA equally at both ages. The augmentation of pain responding at PN21 was largely during the late stages of the formalin test, as reported in the adult. These data support previous findings demonstrating late maturing immune modulation of nociceptive behaviors., (© 2014 Wiley Periodicals, Inc.)

The effects of prenatal stress on immunocytochemical reaction on serotonin (5-HT) in the dorsal raphe nucleus (DRN) of the brainstem were investigated in 7-day-old male rat pups exposed to impact of pain in the formalin test (a control is an injection of saline). A strengthening effect of prenatal stress on pain behavior was revealed in animals in the formalin test. Prenatal stress decreased the number of 5-HT-immunoreactive neurons as compared with that in prenatally non-stressed animals in both pups with inflammatory pain and the controls. There were no differences in the number of 5-HT-immunoreactive cells between pups with inflammatory pain and saline in both prenatally non-stressed animals and prenatally stressed ones. The data suggest that stress of maternal separation during the experiment hides the difference in immunocytochemical result in the rat pups with inflammatory pain and in the control. Thus, it was demonstrated for the first time that in 7-day-old rat pups the 5-HT-ergic neurons in DRN of the brainstem are a target of the prenatal stress.

Children form a strong attachment to their caregiver--even when that caretaker is abusive. Paradoxically, despite the trauma experienced within this relationship, the child develops a preference for trauma-linked cues--a phenomenon known as trauma bonding. Although infant trauma compromises neurobehavioral development, the mechanisms underlying the interaction between infant trauma bonding (i.e., learned preference for trauma cues) and the long-term effects of trauma (i.e., depressive-like behavior, amygdala dysfunction) are unknown. We modeled infant trauma bonding by using odor-shock conditioning in rat pups, which engages the attachment system and produces a life-long preference for the odor that was paired with shock. In adulthood, this trauma-linked odor rescues depressive-like behavior and amygdala dysfunction, reduces corticosterone (CORT) levels, and exerts repair-related changes at the molecular level. Amygdala microarray after rescue implicates serotonin (5-HT) and glucocorticoids (GCs), and a causal role was verified through microinfusions. Blocking amygdala 5-HT eliminates the rescue effect; increasing amygdala 5-HT and blocking systemic CORT mimics it. Our findings suggest that infant trauma cues share properties with antidepressants and safety signals and provide insight into mechanisms by which infant trauma memories remain powerful throughout life.

Pain is a serious problem for infants and children and treatment options are limited. Moreover, infants born prematurely or hospitalized for illness likely have concurrent infection that activates the immune system. It is now recognized that the immune system in general and glia in particular influence neurotransmission and that the neural bases of pain are intimately connected to immune function. We know that injuries that induce pain activate immune function and suppressing the immune system alleviates pain. Despite this advance in our understanding, virtually nothing is known of the role that the immune system plays in pain processing in infants and children, even though pain is a serious clinical issue in pediatric medicine. This brief review summarizes the existing data on immune-neural interactions in infants, providing evidence for the immaturity of these interactions., (© 2014 Wiley Periodicals, Inc.)

To survive, all mammalian species must recognize and respond appropriately to threatening stimuli. In adults, the prelimbic medial prefrontal cortex (mPFC) appears to be involved in fear expression, whereas the infralimbic mPFC mediates fear extinction. In juvenile rats (PN26), the mPFC receives information on potential predators but does not act on it. To test whether the prefrontal cortex is capable of fear regulation in the young organism, we exposed juvenile rats to a threatening or nonthreatening stimulus and assessed fear and brain Fos activation of the mPFC subdivisions, amygdala and periaqueductal gray (PAG). In response to the threat, juveniles froze more, spent more time far from the threat, and had elevated numbers of Fos-positive cells in the prelimbic mPFC, the medial amygdala, and ventral PAG. To test the hypothesis that the mPFC has a dual role in modulating the amygdala and PAG in juveniles, we pharmacologically disinhibited each of the two subdivisions of the mPFC and assessed freezing and downstream activation to the threat. Juvenile rats infused with picrotoxin into the prelimbic mPFC and exposed to a threatening stimulus froze less, spent less time far from the threat, and increased Fos expression. Infusion of picrotoxin into the infralimbic mPFC also reduced fear responding to the threatening stimulus but had no effect on Fos expression. In sum, it appears that the mPFC can process threatening stimuli in juveniles at this age, even though it is normally not involved in the fear responses.

Background: Stimulation of a variety of brain sites electrically or by opiates activates descending inhibitory pathways to attenuate noxious input to the spinal cord dorsal horn and produce analgesia. Analgesia induced by electrical stimulation of the periaqueductal grey (PAG) of the midbrain or medial rostral ventral medulla (RVM) matures late, towards the end or past the pre-weaning period. Descending facilitation takes precedence over inhibition. Yet opiates injected intracerebroventricularly or directly into the PAG induce analgesia relatively early in development. Our goal was to re-examine the role of opiates specific to individual receptor types in analgesia at several supraspinal sites., Methods: Antinociception was tested following microinjection of DAMGO (μ-opiate agonist), DPDPE (∂-opiate agonist) or U50,488 (κ-opiate agonist) into the PAG, RVM or dorsal lateral pons (DLP) in 3-, 10- and 14-day-old rats., Results: DAMGO produced analgesia at 3 days of age at each brain area; the RVM was the most effective and the dorsal PAG was the least effective site. DPDPE produced modest analgesia at 10 and 14 days of age at the ventral PAG, RVM or DLP, but not the dorsal PAG. U50,488H was ineffective at all sites and all ages., Conclusions: Antinociception could be elicited at all three sites by DAMGO as early as 3 days of age and DPDPE at 10 and 14 days of age. The degree of analgesia increased gradually during the first 2 weeks of life, and likely reflects the maturation of connections within the brain and of descending inhibitory paths from these sites., (© 2012 European Federation of International Association for the Study of Pain Chapters.)

Adult central nervous system (CNS) neurons do not regenerate severed axons unaided but may regenerate axons into apposed predegenerated peripheral nerve grafts (PNGs). We examined gene expression by using microarray technology in laser-dissected lateral vestibular (LV) neurons whose axons were severed by a lateral hemisection at C3 (HX) and in lateral vestibular nucleus (LVN) neurons that were hemisected at C3 and that received immunosuppression with cyclosporine A (CsA) and a predegenerated PNG (termed I-PNG) into the lesion site. The results provide an expression analysis of temporal changes that occur in LVN neurons in nonregenerative and potentially regenerative states and over a period of 42 days. Axotomy alone resulted in a prolonged change in regulation of probe sets, with more being upregulated than downregulated. Apposition of a PNG with immunosuppression muted gene expression overall. Axotomized neurons (HX) upregulated genes commonly associated with axonal growth, whereas axotomized neurons whose axons were apposed to the PNG showed diminished expression of many of these genes but greater expression of genes related to energy production. The results suggest that axotomized LVN neurons express many genes thought to be associated with regeneration to a greater extent than LVN neurons that are apposed to a PNG. Thus the LVN neurons remain in a regenerative state following axotomy but the conditions provided by the I-PNG appear to be neuroprotective, preserving or enhancing mitochondrial activity, which may provide required energy for regeneration. We speculate that the graft also enables sufficient axonal synthesis of cytoskeletal components to allow axonal growth without marked increase in expression of genes normally associated with regeneration., (Copyright © 2011 Wiley-Liss, Inc.)

Estrogen modulates pain perception but how it does so is not fully understood. The aim of this study was to determine if estradiol reduces nociceptive responses in part via hypothalamic-pituitary-adrenal (HPA) axis regulation of cyclooxygenase (COX)-1/COX-2 activity. The first study examined the effects of estradiol (20%) or vehicle with concurrent injection nonsteroidal antiinflammatory drugs (NSAIDs) on formalin-induced nociceptive responding (flinching) in ovariectomized (OVX) rats. The drugs were ibuprofen (COX-1 and COX-2 inhibitor), SC560 (COX-1 inhibitor), or NS398 (COX-2 inhibitor). In a second study, estradiol's effects on formalin-induced nociception were tested in adrenalectomized (ADX), OVX, and ADX+OVX rats. Serum levels of prostaglandins (PG) PGE(2) and corticosterone were measured. Estradiol significantly decreased nociceptive responses in OVX rats with effects during both the first and the second phase of the formalin test. The nonsteroidal antiinflammatory drugs (NSAIDs) did not alter nociception at the doses used here. Adrenalectomy neither altered flinching responses in female rats nor reversed estradiol-induced antinociceptive responses. Estradiol alone had no effect on corticosterone (CORT) or prostaglandin levels after the formalin test, dissociating the effects of estradiol on behavior and these serum markers. Ibuprofen and NS398 significantly reduced PGE2 levels. CORT was not decreased by OVX surgery or by estradiol below that of ADX. Only IBU significantly increased corticosterone levels. Taken together, our results suggest that estradiol-induced antinociception in female rats is independent of COX activity and HPA axis activation., (Copyright © 2010 Wiley-Liss, Inc.)

In adult animals, the medial prefrontal cortex (mPFC) plays a significant role in regulating emotions and projects to the amygdala and periaqueductal gray (PAG) to modulate emotional responses. However, little is known about the development of this neural circuit and its relevance to unlearned fear in pre-adulthood. To address these issues, we examined the mPFC of 14-d-old (infants), 26-d-old (juveniles), and 38- to 42-d-old (adolescents) rats to represent different developmental and social milestones. The expression patterns of the neuronal marker FOS were used to assess neurological activity. Muscimol, a GABA agonist, was used to inactivate the prelimbic and infralimbic mPFC subdivisions (400 ng in 200 nl). Animals were exposed to either a threatening or nonthreatening stimulus that was ecologically relevant and age specific. Freezing was measured as an indicator of innate fear behavior. The data indicated that the mPFC is neither active nor responsive to innate fear in infant rats. In juveniles, the prelimbic mPFC became responsive in processing aversive sensory stimulation but did not regulate freezing behavior. Finally, during adolescence, inactivation of the prelimbic mPFC significantly attenuated freezing and decreased FOS expression in the ventral PAG. Surprisingly, across all ages, there were no significant differences in FOS levels in the medial and basolateral/lateral amygdala when either mPFC subdivision was inactivated. Together, unlearned fear has a unique developmental course with different brain areas involved in unlearned fear in the immature animal than the adult. In particular, the mPFC neural circuitry is different in young animals and progressively develops more capacities as the animal matures.

How exogenous estrogen affects inflammatory responses is poorly understood despite the large numbers of women receiving estrogen-alone hormone therapy. The aim of this study was to determine if estradiol alters injury- or inflammation-induced nociceptive responses after carrageenan administration in females and whether its effects are mediated through cyclo-oxygenase (COX) and prostaglandins (PG). To this end, paw withdrawal latencies and serum levels of PGE2 and PGD2 were measured in rats treated with estradiol (0, 10, 20, and 30%) and/or SC560 (COX-1 inhibitor) or NS398 (COX-2 inhibitor) after intraplantar carrageenan administration. Estradiol significantly increased withdrawal latencies before (baseline condition) and after carrageenan administration to one hindpaw. NS398 was anti-nociceptive only in carrageenan treated animals. SC560 increased withdrawal latencies in both paws at 1 and 5hours after carrageenan administration. Co-administration of estradiol and NS398, but not SC560, was additive except for a prolonged anti-nociceptive effects of estradiol combined with NS398. The anti-nociceptive effect extended beyond that observed with either drug or estradiol alone at the 5-hour time point. Estradiol had no significant effect on PGE2 serum levels, but both COX antagonists decreased them. Although neither estradiol nor the COX inhibitors alone had an effect on PGD2 serum levels, co-administration of NS398 and estradiol significantly elevated PGD2 levels. Taken together, our results suggest that estradiol is anti-nociceptive in the thermal test and reduces carrageenan-induced hyperalgesia. These effects are minimally altered through PG-mediated mechanisms., (Published by Elsevier B.V.)

Unlabelled: In the fetal, infant, and adult rat, injury induces a well-defined behavioral response and induces c-fos expression in the spinal cord dorsal horn. There is more limited information about the processing of noxious stimulation in the infant brain. We describe here the appearance of the Fos protein in the brain of fetal and infant rats following formalin-induced injury. Regions were chosen for analysis with a special focus on brain loci that express c-fos in the adult. No Fos positive cells were found in the brains of fetuses; newborns did not show increased Fos expression after formalin injection in any structure examined. At 3 and 14 days of age, there was a significant increase in Fos staining induced by formalin in the ventral lateral medulla. In contrast, paraventricular and medial dorsal nuclei of the thalamus, the paraventricular nucleus of the hypothalamus, and periaqueductal gray of the midbrain showed increased levels of Fos protein only at 14 days of age. We hypothesize that this developmental pattern is related not only to the maturation of pain perception but also to development of autonomic and defensive reactions to pain in the infant., Perspective: Because the infant processes pain differently than the adult, knowledge of those differences informs pediatric clinical practice. Using Fos expression as a marker of neural activity in the rat, we show that the pattern of brain activation is immature at birth but is in place by 14 days of age., (Copyright © 2011 American Pain Society. Published by Elsevier Inc. All rights reserved.)

Human infants may be exposed to opiates through placental transfer from an opiate-using mother or through the direct administration of such drugs to relieve pain (e.g., due to illness or neonatal surgery). Infants of many species show physical dependence and tolerance to opiates. The magnitude of tolerance and the nature of withdrawal differ from those of the adult. Moreover, the mechanisms that contribute to the chronic effects of opiates are not well understood in the infant but include biological processes that are both common to and distinct from those of the adult. We review the animal research literature on the effects of chronic and acute opiate exposure in infants and identify mechanisms of withdrawal and tolerance that are similar to and different from those understood in adults. These mechanisms include opioid pharmacology, underlying neural substrates, and the involvement of other neurotransmitter systems. It appears that brain circuitry and opioid receptor types are similar but that NMDA receptor function is immature in the infant. Intracellular signaling cascades may differ but data are complicated by differences between the effects of chronic versus acute morphine treatment. Given the limited treatment options for the dependent infant patient, further study of the biological functions that are altered by chronic opiate treatment is necessary to guide evidenced-based treatment modalities.

Behavioral transitions characterize development. Young infant rats paradoxically prefer odors that are paired with shock, but older pups learn aversions. This transition is amygdala and corticosterone dependent. Using microarrays and microdialysis, we found downregulated dopaminergic presynaptic function in the amygdala with preference learning. Corticosterone-injected 8-d-old pups and untreated 12-d-old pups learned aversions and had dopaminergic upregulation in the amygdala. Dopamine injection into the amygdala changed preferences to aversions, whereas dopamine antagonism reinstated preference learning.

Human infants are often exposed to opiates chronically but the mechanisms by which opiates induce dependence in the infant are not well studied. In the adult the brain regions involved in the physical signs of opiate withdrawal include the periaqueductal gray area, the locus coeruleus, amygdala, ventral tegmental area, nucleus accumbens, hypothalamus, and spinal cord. Microinjection studies show that many of these brain regions are involved in opiate withdrawal in the infant rat. Our goal here was to determine if these regions become metabolically active during physical withdrawal from morphine in the infant rat as they do in the adult. Following chronic morphine or saline treatment, withdrawal was precipitated in 7-day-old pups with the opiate antagonist naltrexone. Cells positive for Fos-like immunoreactivity were quantified within select brain regions. Increased Fos-like labeled cells were found in the periaqueductal gray, nucleus accumbens, locus coeruleus, and spinal cord. These are consistent with other studies showing that the neural circuits underlying the physical signs of opiate withdrawal are similar in the infant and adult.

The aim of this work was to study the effects of prenatal stress on nociceptive responses in the formalin test in female and male infant (7-day-old) Long-Evans hooded rats. Prenatally stressed infant rats displayed biphasic flinching+ shaking behavior whereas non-stressed animals showed only a weak second phase. Pain sensitivity in prenatally stressed males was significantly greater than that of prenatally non-stressed males during the second phase only; there were no differences in pain sensitivity between prenatally stressed and non-stressed females. Moreover prenatally stressed male rats pups demonstrated that the second phase of the response to formalin was enhanced relative to the second phase in stressed females. The current and previous data [Butkevich IP, Barr GA, Mikhailenko VA, Otellin VA. Increased formalin-induced pain and expression of fos neurons in the lumbar spinal cord of prenatally stressed infants rats. Neurosci Lett 2006a;403:222-226] show increased tonic pain in prenatally stressed infant rats and a large increase in the number of formalin-induced fos-like immunoreactivity in the spinal cord dorsal horn. There is a concomitant decrease in serotonin-like immunoreactivity in the lumbar spinal cord dorsal horn [Butkevich IP, Barr GA, Otellin VA. Effect of prenatal stress on behavioral and neural indices of formalin-induced pain in infant rats. Abstracts, 35th Annual Meeting of Soc. For Neurosci. 2005a. Program No. 512.4 Washington, DC: Society for Neuroscience]. Given the decreased level of perinatal testosterone in prenatally stressed rats to which infant males are more sensitive than females, we suggest that these hormonal, behavioral and neuronal indices are strongly interrelated in prenatally stressed 7-day-old rat pups and that the decreased surge of testosterone may contribute to the increased behavioral response in the second phase in male rat pups. Mechanisms underlying the behavioral pain response induced by inflammation in prenatally stressed rat pups are characterized by sexual dimorphism even prior to the activational effects of sex hormones.

Rationale: Neonates respond to noxious stimuli at or before birth, but the organization of nociceptive systems changes well into postnatal life. It is unknown how nociceptive information is processed in the immature animal and, specifically, whether noxious stimulation is transmitted by glutamatergic circuits, known to play an important role in nociception in the adult. Both N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are found within the neonatal spinal cord, but in immature form, and when they become involved in pain processing in vivo is not known., Objectives: The objective was to determine the age-related changes in the involvement of spinal NMDA and AMPA receptors in formalin-induced nociception during early life. Because the formalin test provides a measure of immediate nociceptive responding (first phase) and of peripheral and central sensitization (second phase), a second aim was to determine if there is specificity of the effects to either phase., Materials and Methods: NMDA antagonists (MK801, AP5) or an AMPA antagonist (YM872) was administered intrathecally, and pups were assessed in the formalin test behaviorally and by Fos expression within the spinal cords of 3-, 10-, and 21-day-old rats., Results: The NMDA antagonists attenuated formalin-induced behavioral responses at the youngest age tested with some selectivity for the second phase of responding. MK-801 did not induce motor impairment at any age. YM872 also attenuated formalin-induced nociceptive responses at all ages throughout the test session, although there was some motor impairment in the 3-day-old subjects. Spinal administration of either YM872 or MK-801 reduced Fos expression in the spinal cord at all ages., Conclusions: These data suggest that spinal NMDA and AMPA receptor are functional and involved in formalin-induced nociception throughout development.

In experiments on the 7-day-old female and male Long-Evans rat pups, for the first time, there was studied effect of prenatal (immobilization) stress on dynamics of nociceptive behavioral response caused by an inflammatory focus. The nociceptive sensitivity was evaluated for 1 h by the number of 7-day-old organized at the spinal level in response to injection of formalin (10%, 10 microl) to the posterior leg sole. Control rat pups were not submitted to any prenatal stress; in these animals the response in the formalin test was found to be represented by one phase. It the prenatally stressed rat pups the studied patterns were organized into two phases characteristic of the definitive type of response. At the period between them (during interphase), the nociceptive behavior was absent. At the second, tonic phase the number of flexes+shakes in the prenatally stressed males was statistically significantly higher than in the prenatally stressed females, which indicates a sensitization of the neurons involved in the tonic pain chains in male individuals. Thus, the data obtained on prenatally stressed animals confirm the previous data about immaturity of the mechanisms mediating the second phase of response in the formalin test in the 7-day-old rat pups. An important fact is revealed which indicates that in the prenatally stressed rat pups of the same age the second phase of response is already obvious. Mechanisms underlying the behavioral response caused by the inflammatory focus in the formalin test in the number flexes + shakes old stressed rat pups are characterized by sexual dimorphism: the pain sensitivity in males at the second phase of response is statistically significantly higher than in females.

When pregnant dams are stressed, there is a resultant alteration in brain development and behavior in their offspring. Prior work has shown increased nociceptive responses in adolescent or adult rats born of stressed dams. However, the age at which those changes first occur is not known. The aim of the present study was to evaluate the effect of prenatal stress on pain sensitivity in the formalin test in 7-day-old rats, behaviorally and by fos-like immunoreactivity (Fos-LI) in the lumbar spinal cord dorsal horn. The behavioral response to intraplantar injection of formalin is represented by two nociceptive phases separated by an interphase during which nociceptive responses decrease; the interphase is not seen until the start of the third postnatal week and appears as descending inhibitory monoaminergic systems develop. Prenatally stressed infants showed increased nociceptive responses in the second, tonic phase and a large increase in the number of formalin-induced Fos-LI neurons in the lumbar dorsal horn, a result consistent with the behavioral data. The increased nociception in prenatally stressed 7-day-old pups may be associated with the decrease in the intensity of serotonin-like immunoreactivity and density of serotonergic cells in the lumbar spinal cord dorsal horn and the dorsal raphe nucleus reported earlier.

Clinical and preclinical studies have found sex-specific differences in the discrimination and perception of inflammatory stimuli. The emerging picture suggests that the biological basis of these differences resides in the regulatory activity of gonadal hormones in the central nervous system. This study describes the effects of ovarian hormones in inflammatory pain processes. Ovariectomized rats received estradiol and/or progesterone, and the number of paw flinches was measured after 1, 2.5 or 5% formalin administration. Both estradiol and progesterone altered the number of flinches only after 1% formalin administration. Estradiol significantly reduced the overall number of flinches during Phase II of the formalin nociceptive response while progesterone attenuated Phase I of the response. After co-administration of estradiol and progesterone, progesterone reversed estradiol's analgesic effect in Phase II, however, estradiol did not reverse progesterone's analgesic activity in Phase I. To determine if estradiol effects are receptor-mediated, tamoxifen (selective estrogen receptor mediator, 15 mg/kg) or alpha-estradiol (an inactive isomer of estradiol, 20 microg) were utilized. Tamoxifen decreased the number of formalin-induced flinches during Phase II while alpha-estradiol did not affect any formalin-induced responses. When co-administered with estradiol, tamoxifen failed to reverse estradiol's effect, suggesting both tamoxifen and estradiol activate similar intracellular mechanisms. Although Western blot analysis detected the presence of estradiol alpha and beta and progesterone B receptors in the spinal cord, hormone replacement treatments had no effects on the levels of these receptors. We postulate that the mechanisms by which estradiol and progesterone induce analgesia occur through the activation of their receptor at the spinal cord level.

Injury and pain experienced by the infant results in immediate changes in pain sensitivity that last into adulthood. These long-term changes are likely initiated by altered gene expression. Here we measured how injury alters gene expression in the lumbar spinal cord early and late in the preweaning period of the rat. The expression of large numbers of genes was altered significantly at 3 days of age, when injury has long-term consequences. The functional classes of altered genes included transcription factors, cell death related and metal ion genes. The intensity of the stimulus in the 3-day-old pups induced changes in different classes of genes. Fewer changes were noted at 21 days of age. The increased expression of transcription factors and decreased expression of genes whose products are protective against cell death are hypothesized to underlie the long-term changes that are seen after injury in the neonate.

Young animals respond to threatening stimuli in an age-specific way. Their endocrine and behavioral responses reflect the potential threat of the situation at a given age. The aim of the present study was to determine whether corticotropin-releasing factor (CRF) is involved in the endocrine and behavioral responses to threat and their developmental changes in young rats. Preweaning 14-day-old and postweaning 26-day-old rats were exposed to two age-specific threats, cat odor and an adult male rat. The acute behavioral response was determined during exposure. After exposure, the time courses of the corticosterone response and of CRF expression in the paraventricular nucleus of the hypothalamus (PVN) and in extrahypothalamic areas were assessed. Preweaning rats became immobile when exposed to cat odor or the male rat, whereas postweaning rats became immobile to cat odor only. Male exposure increased serum corticosterone levels in 14-day-old rats, but cat odor failed to increase levels at either age. Exposure induced elevation of CRF mRNA levels in the PVN that paralleled changes in corticosterone levels. CRF may thus play a role in endocrine regulation and its developmental changes during early life. Neither cat odor nor the adult male altered CRF mRNA levels in the bed nucleus of the stria terminalis (BNST) or the amygdala, but both stimuli increased levels in the hippocampus. Hippocampal CRF mRNA expression levels did not parallel cat odor or male-induced immobility, indicating that CRF is not involved in this response in young rats but may be involved in aspects of learning and memory.

Kappa opioid receptors stimulation with U50,488 is known to modulate behaviors during the early postnatal period, but the specific neuroanatomical locus of many of these effects is unexplored. In the present study, we infused U50,488 into the midbrain periaqueductal gray (PAG) and investigated the effects of this drug on behavior and heart rate of 1-, 2-, and 3-week-old rats. U50,488 increased activity most potently in 1- and 2-week-old subjects. Ultrasonic vocalization (USV) production was increased in 1-week-old subjects, but not in 2- or 3-week-old pups. Heart rate changes were similarly seen in younger aged subjects. At 1 week, U50,488 decreased heart rate, but at 2 weeks it increased heart rate. There was no effect of this drug on heart rate at 3 weeks. At 1 week, USVs were more potently elicited from dorsal than lateral PAG infusion sites. No other site-specific effects within the PAG were seen. The age-related decline in behavioral effects elicited by U50,488 is consistent with other published reports, and to the extent that kappa receptor activity mediates infant separation responses, implicates the PAG as a modulator of those responses., (2004 Wiley Periodicals, Inc)

Glutamate receptors, especially N-methyl-d-aspartate (NMDA) receptors, are hypothesized to play key roles in opiate tolerance and withdrawal. There is also accumulating evidence that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists and group II metabotropic glutamate receptor (mGluR) agonists attenuate opiate withdrawal. However, most existing data are derived from adult animal models. Glutamate receptor types undergo dramatic developmental changes during early life. Thus, the pharmacological effects on opiate withdrawal of NMDA receptor, AMPA receptor, and mGluR antagonists in the developing organism may not be comparable to those in the adult. Indeed, NMDA receptor antagonists do not block morphine tolerance or withdrawal in the 7-day-old rat, but are partially effective in the 14-day-old, and fully effective in the 21-day-old. Thus, there is a transition period around the second post-natal week for NMDA receptor antagonists to suppress opiate tolerance and withdrawal. A combination of in vivo and in vitro assays was used in the present studies to test the effect of drugs acting on AMPA and group II mGlu receptors on morphine withdrawal in rats at 7, 14, and 21 days of age. These ages represent the critical periods when various glutamate receptor subunits undergo differential change. In contrast to NMDA receptor antagonists' early ineffectiveness in suppressing morphine withdrawal, the AMPA receptor antagonist and the group II mGluR agonists were effective at all ages tested. Thus, for the human infant patient, pharmacotherapies to reduce opiate tolerance and withdrawal should focus on non-NMDA ionotropic and metabotropic receptors.

It is unclear how neonates respond to noxious stimuli. This study examined the role of neurokinin peptides in 3- and 21-day-old rat pups using the preprotachykinin-A (PPTA) knockout mouse, lacking neurokinin A and substance P. We assessed pain behaviors of these mice before the neurokinin system is putatively active, 3 days after birth, and after this system is active, 21 days after birth. The lack of these peptides failed to alter behavioral responses to nociceptive stimulation in the 3-day-old mice. The 21-day-old mice lacking these peptides were less responsive to 5 microl 2% formalin and to high intensity thermal and mechanical stimuli. Thus, the neurokinins appear not to be an important mechanism in the processing of nociceptive information in the infant.

N-methyl-D-aspartate (NMDA) receptor antagonists are effective in inhibiting the development of morphine tolerance in adult rats. But NMDA receptors undergo dramatic change during the first few weeks of the postnatal life in the rat, and it is unknown whether NMDA receptor antagonists can inhibit the acquisition of opiate tolerance in the developing organism. Here, we investigated the effects of two NMDA receptor antagonists MK-801 and dextromethorphan on the development of morphine tolerance in 7-, 14-, and 21-day-old rats. NMDA receptor antagonists are not effective in attenuating morphine tolerance in the neonatal rat whereas they were partially effected in the 14-day-old and fully effective in rats as old or older than 21 days of age. These data suggest that there exists a transition age, around the second postnatal week in the rat, for the NMDA receptor to play a role in the development of morphine tolerance.

We examined how the experience of a threatening stimulus alters subsequent behavior in a situation where the immediate threat is absent. A small huddle of 12-day-old rats was exposed to a potentially infanticidal adult male rat for 5 min. During male exposure, pups were significantly more immobile than control pups. Thirty, 60, and 180 min after male exposure, the pups were isolated for 5 min from litter and dam in an unfamiliar environment. When isolated, pups that had been previously exposed to the male emitted significantly fewer ultrasonic vocalizations than controls, but did not differ in immobility. Low levels of vocalization were apparent 30 and 60 min after male exposure and were not evident at 180 min. The pups seemed to have adjusted their behavior to a potential male threat in a different context for a limited period of time., (Copyright 2003 Wiley Periodicals, Inc. Dev Psychobiol 42: 386-391, 2003.)

Morphine injected locally to the paw of an adult or an infant rat is analgesic. Opiates specific to micro and kappa opioid receptors, and less consistently to delta opioid receptors, given locally to the site of injury in adult animals are also analgesic in a variety of models of inflammatory pain. To determine which opioid receptor(s) are involved in local analgesia in the immature animal, agonists specific for micro, kappa, and delta opioid receptors were injected into the intraplantar pad in infant rats and the resultant nociceptive behavior and Fos expression assayed in the formalin test. The kappa opioid receptor agonist U50,488 reduced nociceptive behavior in both phases of the formalin test and reduced Fos expression in the dorsal horn of the lumbar spinal cord, at 3 and 21 days of age. Morphiceptin (micro opioid agonist) was analgesic in the 21-day-old pups, but not the 3-day-old pups, measured behaviorally or by Fos expression. DPDPE (delta opioid agonist) was not analgesic at either age. We also tested the effects of opioid receptor antagonists on morphine's local analgesic action. Naltrexone, and to a lesser extent the micro opioid antagonist CTOP, antagonized morphine's analgesic effect. Kappa and delta opioid receptor blockers were inactive. The results demonstrate the ability of the kappa opioid system to mediate analgesia in the neonate at the site of injury in acute and chronic pain models, that the micro opioid agonists are active later in development, but that morphine is analgesic in part through micro opioid receptors., (Copyright 2003 Wiley Periodicals, Inc. Dev Psychobiol 42: 111-122, 2003)

The present study examined the ability of LY235959, a competitive N-methyl-D-aspartate (NMDA) receptor antagonist, to attenuate behaviors and c-fos mRNA expression associated with acute morphine withdrawal in the infant rat. Rat pups were given a single dose of morphine (10.0 mg/kg, s.c.) or saline. Two hours later, pups were removed from the dam and injected with either LY235959 (10.0 mg/kg, s.c.) or saline. Fifteen minutes later acute morphine withdrawal was precipitated with naltrexone (10.0 mg/kg, s.c.) and behaviors were recorded every 15 s for the next 60 min. Immediately after behavioral testing, brain and spinal cord were assayed for c-fos mRNA analysis by solution hybridization. The intensity of the morphine withdrawal syndrome was reduced in pups pre-treated with LY235959. Withdrawal behaviors such as head moves, moving paws, rolling, and walking were decreased, and vocalizations were completely eliminated in pups pre-treated with LY2359559. Acute morphine withdrawal increased c-fos mRNA expression in the brain and the spinal cord, which was attenuated by pre-treatment of LY235959. Thus, in the 7-day-old rat, as in the adult, NMDA receptors play a role in the behavioral and molecular manifestations of acute morphine withdrawal.

Stress activates endogenous opioids that modulate nociceptive transmission. Exposure to a potentially infanticidal adult male rat suppresses pain-related behaviors in pre-weaning but not in older rats. This male-induced analgesia is mediated by l opioid receptors in the periaqueductal gray, a midbrain structure that is innervated by amygdala projections. To determine whether enkephalin, a l and d opioid receptor agonist, is activated by male exposure, mRNA levels of its precursor, preproenkephalin, were measured in subdivisions of the amygdala and the periaqueductal gray. In 14-day-old but not in 21-day-old rats, 5 min of male exposure induced analgesia to heat and increased preproenkephalin mRNA levels in the central nucleus of the amygdala but not in the periaqueductal gray. The change in the activation of enkephalinergic neurons in the central amygdala may contribute to the change in stress-induced analgesia during early ontogeny., (Copyright 2002 IBRO)

Young rats become immobile when exposed to a potentially infanticidal adult male rat. Male-induced immobility declines during the preweaning period, paralleling the decrease in infanticidal threat. To investigate the neural substrates underlying the developmental change in immobility, male-induced expression of the immediate-early gene c-fos was assessed on postnatal days 7, 14 and 21. A huddle of three young rats was exposed to an adult male behind a screen. As control, three littermates were put in the testing chamber but not exposed to the male. On day 7, male exposed and control pups were immobile most of the time and c-fos expression did not differ between conditions. On day 14, rats in the presence of the male stopped ongoing behaviors and became immobile. They had significantly higher c-fos expression in the paraventricular nucleus of the hypothalamus, the amygdala, the periaqueductal gray, and the locus ceruleus. On day 21, the male-exposed rats that were immobile had elevated c-fos expression in a similar pattern as on day 14, however, different nuclei of the amygdala were activated. In contrast, male-exposed 21-day-old rats that showed control levels of immobility did not have elevated c-fos expression in these areas. These results demonstrate that male exposure induced c-fos expression in brain areas of young rats in an age-specific pattern. Some of the activated brain areas seem to have contributed to immobility. Differential activation of neuronal populations may underlie developmental changes in defensive immobility during early ontogeny.

Despite decades of research, the mechanisms that underlie opiate tolerance, dependence and withdrawal remain elusive. Evidence accumulated over the past ten years suggests that the NMDA receptor plays a central role in mediating the neuroplasticity induced by chronic opiate administration in adult animals. Yet, during ontogeny, the NMDA receptor complex undergoes qualitative developmental changes, which renders some of the basic assumptions for a role of the NMDA receptor in opiate withdrawal invalid in infants. Recent data indicate that NMDA receptor antagonists are not effective in blocking morphine tolerance, dependence and withdrawal in the neonatal rat. Roles for other glutamate receptor types (e.g. metabotropic glutamate receptors) have also been proposed recently. In this article, the latest evidence that characterizes the dynamic roles of glutamate receptors in these phenomena during ontogeny will be discussed.