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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.

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.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.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.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.

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).

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.

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.

The broad use and misuse of prescription opioids during pregnancy has resulted in a surge of infants diagnosed with Neonatal Opioid Withdrawal Syndrome (NOWS). Short-term irritability and neurological complications are hallmarks of NOWS, but the long-term consequences are unknown. Our newly-developed preclinical model of oxycodone self-administration enables adult female rats to readily drink oxycodone (0.06-0.12 mg/ml, ∼10/mg/kg/day) continuously before and during pregnancy and after delivery, to achieve similar liquid intake in oxycodone moms relative to water-only controls. Oxycodone levels were detected in the serum of mothers and pups. Growth parameters in dams and pups, and litter mass and size were similar to controls. Maternal behavior at postnatal day 1 (PN1) was unchanged by perinatal oxycodone consumption. Regarding the plantar thermal response, there were no differences in paw retraction latency between oxycodone and control pups at PN2 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 controls. Finally, during naltrexone precipitated withdrawal at PN9, oxycodone males vocalized more than control pups, consistent with a previously-published withdrawal phenotype. Thus, our rat model of continuous oral oxycodone self-administration in pregnancy shows exacerbated affect/social communication in pups in a sex-dependent manner but spared cognition and locomotion. Our preclinical, high face validity NOWS model reproduces key aspects of human opioid use during pregnancy, enabling longitudinal analysis of how maternal oxycodone changes emotional behavior in the offspring.HIGHLIGHTSFemale rats self-administered oxycodone at clinically relevant doses before and during pregnancy and for the first two weeks after parturition.Both dams and pups, for the14 day postnatal experimental period, had detectable levels of oxycodone in their bloodDams drinking oxycodone only or water only did not differ in weight gain, water intake, or the number of pups born and their pups did not differ in weight throughout.Sensory and motor function in the pups was not altered, nor was hippocampal dependent spatial learning.Oxycodone exposed pups were physically dependent and displayed increased withdrawal behaviors with or without the opioid antagonist naltrexone.Pups expressed more negative affect, expressed by increased ultrasonic vocalizations, following naltrexone precipitated withdrawal or when separated from their mother.

The increased abuse of opioids - such as oxycodone - poses major challenges for health and socioeconomic systems. Human prescription opioid abuse is marked by continuous, voluntary, oral intake, and sex differences. Therefore the field would benefit from a preclinical in-depth characterization of sex differences in a chronic oral voluntary, free choice, and continuous access paradigm. Here we show in an oral oxycodone continuous access two-bottle choice paradigm sex-dependent voluntary drug intake, dependence, and motivation to take the drug. Adult female and male Long-Evans rats were given unlimited, continuous home cage access to two bottles containing water (Control) or one bottle of water and one bottle of oxycodone dissolved in water (Experimental). Most experimental rats voluntarily drank oxycodone (∼10 mg/kg/day) and escalated their intake over 22 weeks. Females self-administered twice as much oxycodone as males, leading to greater blood levels of oxycodone, and engaged in more gnawing behavior. Precipitated withdrawal revealed high levels of dependence in both sexes. Reflecting motivation to drink oxycodone, ascending concentration 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. Thus, female rats consumed and preferred oxycodone more than males in this chronic two-bottle oral choice paradigm. Both sexes displayed many features of human oxycodone abuse, and behavioral pre-screening predicted parameters of intake and withdrawal. This model provides an additional paradigm for understanding mechanisms that mediate long-term voluntary drug use and for exploring potential treatment options.HIGHLIGHTSAdult rats offered continuous choice of oral oxycodone vs. water preferred oxycodoneRats self-titrated oxycodone, yet females preferred and escalated more than malesBoth sexes were motivated to drink oxycodone, as shown by a citric acid aversion testBoth sexes became dependent on oxycodone, as shown by precipitated withdrawalBehavioral prescreening predicted later aspects of oxycodone intake and dependence

The effects of prenatal stress on the immunocytochemical reaction for serotonin (5-HT) in the dorsal raphe nucleus (DRN) were studied in seven-day-old male rat pups subjected to pain in the formalin test (inflammatory pain; controls received injections of physiological saline. Prenatal stress enhanced the animals’ pain behavior in the formalin test. Prenatal stress decreased the numbers of 5-HT-immunoreactive cells as compared to those in prenatally unstressed animals, both in rat pups with inflammatory pain and in controls. The formalin treatment did not alter the number of 5-HT-immunoreactive cells in the DRN in rat pups in either prenatally stressed or unstressed animals. Thus, we demonstrate that 5-HT-ergic neurons in the brainstem DRN in seven-day-old rat pups are the targets of prenatal stress. We suggest that stress due to separation from the mother during the experiment masked differences in the immunocytochemical parameters in rat pups with inflammatory pain and in controls.

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.

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-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), 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 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 maternal deprivation-isolation 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 maternal deprivation-isolation 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.

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.

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.

Neuropathic pain is an example of 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 day 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 one day after compression injury when performed at PN14, 21 or 28. Thermal withdrawal latencies return to near baseline by 7 days post-surgery (PS7) when the injuries were at PN14, and lasted up to 14 days when imposed at PN28. There was mechanical allodynia following nerve injury at 7 or 14 days after injury at PN14. Measurements of mRNA from spinal cord at 1, 7 and 14 days post-injury 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 PN21/28. This may be due to the use of a transient, and not sustained, compression compared to that of ligation models.

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 well-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.

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.

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 gray of the midbrain (PAG) or medial rostral ventral medulla (RVM) matures late, towards the end or past the preweaning period. Descending facilitation takes precedence over inhibition. Yet opiates injected ICV or directly into the PAG induce analgesia relatively early in development. Our goal was to reexamine the role of opiates specific to individual receptor types in analgesia at several supraspinal sites.

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. J. Comp. Neurol. 519:3433–3455, 2011. © 2011 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.

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.

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.

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.

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. 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. 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. 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. 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 the flexion-shaking patterns organized at the spinal level in response to injection of formalin (10%, 10 μl) 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 flexion-shakings 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 one-week 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.

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.

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.

Microarrays are one of several technologies that allow for measurement the expression of thousands of genes simultaneously. This technological advance provides a challenge for the analysis of these data. In this review we discuss these analytical issues from the initial quality control to normalization, differential expression, clustering and finally functional pathway analysis. We focus on Affymetrix data but many of the issues are the same for other array platforms.

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.

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 a-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 ineffectivness 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. # 2004 ISDN. Published by Elsevier Ltd. All rights reserved.

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.

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.

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.

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.

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.

During early ontogeny, stimuli that pose a threat to an animal change. Unrelated adult male rats may kill young rats, but infanticide ends around weaning. Predation, on the other hand, may increase during early ontogeny when rats begin to extend their activity range. We investigated the developmental course of two defensive responses, immobility and analgesia, in young rats exposed to an adult male rat or to predator cues. Preweaning 14-day-old rats became immobile and analgesic when exposed to the male and showed immobility but not analgesia when exposed to cat odor. On Day 26, around weaning, the presence of the male rat no longer induced immobility and analgesia whereas cat odor produced higher levels of immobility and analgesia compared to control and male-exposed animals. This developmental change in responsivity may reflect the differences in the risk of being harmed by a male or a cat during different periods of ontogeny.

Opiate withdrawal behaviors in the infant differ from those of the adult. The neural circuitry underlying opioid withdrawal in the adult rat is well defined and includes the locus coeruleus (LC) and periaqueductal gray (PAG), with a minor role of the amygdala. Because the different behaviors that constitute the infant syndrome may be mediated by different neural circuits, we tested the hypothesis that these three sites are involved in opiate withdrawal. Pups were injected with morphine from day 1- 6 after birth (b.i.d.) and on the morning of the seventh day. Withdrawal was then elicited by local injection of the opioid antagonist methylnaloxonium into the LC, PAG, or amyg- dala. Withdrawal signs were precipitated in a dose-dependent manner following injec- tion into the LC or PAG, but not the amygdala. The withdrawal behaviors elicited from the LC and PAG included both the same and different behaviors. The results support the hypothesis that the neural circuitry mediating opiate withdrawal behaviors is similar in infant and adult animals, but the behaviors expressed are age-specific. Synapse 39: 139 -151, 2001. © 2001 Wiley-Liss, Inc.

Administration of NK1 antagonists in adult animals attenuates the nociceptive response in the formalin test, indicating that the neurokinins and the NK1 receptor play a role in mediating this pain response. The number and distribution of NK1 receptors change dramatically during development, and the age at which they become involved in pain processing is not known. We examined the role of NK1 receptors in the formalin model in rats ranging in age between 3- and 21-days old. An NK1 antagonist, CP99,994, and its less active enantiomer CP100,263 were administered to the spinal cord (intrathecal), systemically (subcutaneous), or locally (intraplantar). Intrathecal administration of CP99,994, but not CP100,263, attenuated pain behaviors in the second phase of the formalin response in 14-day and 21-day old rats, but did not alter the pain response in 3-day or 10-day old rats. CP99,994 also reduced the expression of the c-fos protein in the superficial dorsal horn of 21-day old rats. Systemic and intraplantar injection of either CP99,994 or CP100,263 reduced the pain response to formalin in 3-day and 21-day old rats, suggesting a non-NK1 mediated mechanism of action. These results indicate that, within the spinal cord, NK1 receptors start to play a role in the pain response to formalin between 10 and 21 days. Moreover, analgesia induced by systemic or local injection of NK1 antagonists involves mechanisms other than, or in addition to, the NK1 receptor.

Offspring of women exposed to opiate drugs such as heroin and methadone during pregnancy have a high incidence of morbidity and mortality. Infants also show opiate withdrawal. In this study, we examined the behavioral effects of precipitated withdrawal in morphine-dependent fetal rats at gestational day (GD) 20. The dam was implanted on GD 14 with a pellet containing 75.0 mg of morphine. On GD 20, the dam underwent chemoyelotomy at L1/L2. The uterine horns were externalized and four subject fetuses were selected for behavioral observation, two from each uterine horn. The fetus was then injected subcutaneously with either saline or naltrexone (1.0 mg/kg) and the behaviors of the fetus recorded every 15 sec for 20 min. The results show that naltrexone injected fetuses that had been chronically exposed to morphine demonstrated increased limb and mouth movements, face wiping, and body curls, and spent less time quiet as compared with control fetuses. These results indicate that a morphine withdrawal-like syndrome occurs in the fetal rat.

During early ontogeny infant rats show specific responses to a variety of age-dependent threatening situations. When isolated from nest and dam, they emit ultrasonic vocalizations and show decreased reactivity to noxious stimulation, or analgesia. When exposed to an unfamiliar adult male, they become immobile and analgesic. The midbrain periaqueductal gray (PAG) is an important area within the circuitry that controls responses to threatening stimuli in the adult. Little is known about the functions of the PAG in early life. It was hypothesized that the PAG mediates the responses to the age-specific threats social isolation and male exposure in the infant rat. Rat pups were lesioned electrolytically either in the lateral or the ventrolateral PAG on postnatal day 7, tested in social isolation on day 10, and exposed to a male on day 14. On day 10 during isolation, ultrasonic vocalizations and isolation-induced analgesia were decreased in both lesion groups. On day 14, male-induced immobility and analgesia were decreased in ventrally lesioned animals. In conclusion, the PAG seems to play a developmentally continuous role in age-specific responses to threat such as ultrasonic vocalization, analgesia, and immobility.

An important yet unanswered question is how neonates respond to painful stimuli, given the immaturity of their neural pathways. We examined the development of the neurokinin system using a novel approach, examining changes of this system by observing the pain responses of mice lacking the NK1 receptor at different stages of development We show that the NK1 receptor is not involved in nociception to heat, mechanical or chemical stimuli, at 3 days. In contrast, the NK1 receptor is involved in nociceptive responses to high intensity heat and mechanical stimuli, and mediates the second phase of the formalin response in 21-day-old mice. This indicates that nociception in neonates does not require the NK1 receptor and that the functional maturation of the NK1 receptor allows diversity in both the type of stimuli that activate the pain system and the types of responses elicited by nociceptive stimuli.