Epigenetic regulator UHRF1 inactivates REST and growth suppressor gene expression via DNA methylation to promote axon regeneration.

Injured peripheral sensory neurons switch to a regenerative state after axon injury, which requires transcriptional and epigenetic changes. However, the roles and mechanisms of gene inactivation after injury are poorly understood. Here, we show that DNA methylation, which generally leads to gene silencing, is required for robust axon regeneration after peripheral nerve lesion. Ubiquitin-like containing PHD ring finger 1 (UHRF1), a critical epigenetic regulator involved in DNA methylation, increases upon axon injury and is required for robust axon regeneration. The increased level of UHRF1 results from a decrease in miR-9. The level of another target of miR-9, the transcriptional regulator RE1 silencing transcription factor (REST), transiently increases after injury and is required for axon regeneration. Mechanistically, UHRF1 interacts with DNA methyltransferases (DNMTs) and H3K9me3 at the promoter region to repress the expression of the tumor suppressor gene phosphatase and tensin homolog (PTEN) and REST. Our study reveals an epigenetic mechanism that silences tumor suppressor genes and restricts REST expression in time after injury to promote axon regeneration.

Differential suppression of intracranial self-stimulation, food-maintained operant responding, and open field activity by paw incision and spinal nerve ligation in rats.

BACKGROUND: Detection of ongoing spontaneous pain behaviors in laboratory animals remains a research challenge. Most preclinical pain studies measure elicited behavioral responses to an external noxious stimulus; however, ongoing spontaneous pain in humans and animals may be unrelated to hypersensitivity, and likely diminishes many behaviors, particularly motivated behaviors, that we hypothesize will decrease after induction of acute and chronic pain. METHODS: In this study, 201 male rats were subjected to paw incision (INC), L5/L6 spinal nerve ligation (SNL), or INC in SNL rats, and the effects on paw withdrawal threshold (PWT) were assessed. For comparison, the behavioral-decreasing effects on nonevoked measures, including lever pressing for rewarding electrical stimulation of the ventral tegmental area intracranial self-stimulation (VTA ICSS) or food reinforcement (FR), and open field activity (OFA), were also assessed in these same rats. RESULTS: INC decreased PWT for 4 days, decreased VTA ICSS for 2 days, and FR for 1 day but did not alter OFA. SNL decreased PWT similarly to INC but did not decrease VTA ICSS or FR; SNL did however decrease OFA. INC in SNL rats reduced PWT, VTA ICSS, and FR similarly to INC alone and did not decrease OFA compared with SNL alone. CONCLUSIONS: The acute effects of INC on decreasing lever pressing for VTA ICSS and FR (1-2 days after incision) correspond to the timeframe in which ongoing spontaneous pain is expected to occur after INC. Therefore, these decreases are likely mediated by ongoing spontaneous pain, which may be unrelated to mechanical hypersensitivity that persists for up to 4 days after INC. PWT is decreased similarly by SNL, yet operant behavior (lever pressing for VTA ICSS and FR) was not decreased by SNL. SNL, but not INC, decreased rearing behavior but not total distance traveled during OFA. This further indicates that the presence and the extent of hypersensitivity are not predictive of many behavioral changes in rats thought to be mediated by the presence of ongoing pain. Surprisingly, the behavioral effects of INC are not exacerbated in SNL rats. These data support the growing belief that acute pain models produce short-lived spontaneous pain behaviors that are often less pronounced or absent in neuropathic pain models, and highlight the need for assessment of both evoked and nonevoked pain behaviors in developing future therapies for acute and chronic pain.

Intracranial self-stimulation of the paraventricular nucleus of the hypothalamus: increased faciliation by morphine compared to cocaine.

BACKGROUND: Neuropathic pain attenuates opioid facilitation of rewarding electrical stimulation of limbic dopaminergic pathways originating from the ventral tegmental area. Whether neuropathic pain alters opioid effects of other brain-reward systems is unknown. METHODS: Control and spinal nerve-ligated (SNL) rats had electrodes implanted into the paraventricular nucleus (PVN) of the hypothalamus or medial forebrain bundle. Control and SNL rats were trained to lever-press for intracranial self-stimulation (ICSS), and modulation by morphine or cocaine was assessed. RESULTS: Control and SNL rats lever-pressed for stimulation of the PVN and medial forebrain bundle. Morphine produced greater reductions in the frequency at which rats emitted 50% of maximal responding for PVN ICSS (maximal effect 24.67 ± 4.60 [mean ± SEM] and 24.11 ± 5.96 in SNL [n = 6] and control [n = 8] rats, respectively, compared with medial forebrain bundle ICSS (12.38 ± 6.77 [n = 8] and 12.69 ± 1.55 [n = 7]). In contrast, cocaine was less efficacious in potentiating PVN ICSS (maximal effect 11.76 ± 2.86 and 12.38 ± 4.01 in SNL [n = 12] and control [n = 8] rats, respectively) compared with medial forebrain bundle ICSS (30.58 ± 3.40 [n = 9] and 27.55 ± 4.51 [n = 7]). CONCLUSIONS: PVN ICSS is facilitated to a greater extent by morphine than cocaine, and the effects of each drug on this behavior are unaltered after spinal nerve ligation. These effects contrast those observed with direct stimulation of limbic dopamine pathways, suggesting that the PVN may have a greater role in the reinforcing effects of opioids than classic limbic regions, particularly in the presence of chronic pain.

Opioid facilitation of rewarding electrical brain stimulation is suppressed in rats with neuropathic pain.

INTRODUCTION: Opioids are powerful analgesics, but are also common drugs of abuse. Few studies have examined how neuropathic pain alters the pharmacology of opioids in modulating limbic pathways that underlie abuse liability. METHODS: Rats with or without spinal nerve ligation (SNL) were implanted with electrodes into the left ventral tegmental area and trained to lever press for electrical stimulation. The effects of morphine, heroin, and cocaine on facilitating electrical stimulation of the ventral tegmental area and mechanical allodynia were assessed in SNL and control subjects. RESULTS: Responding for electrical stimulation of the ventral tegmental area was similar in control and SNL rats. The frequency at which rats emitted 50% of maximal responding was 98.2 ± 5.1 (mean ± SEM) and 93.7 ± 2.8 Hz in control and SNL rats, respectively. Morphine reduced the frequency at which rats emitted 50% of maximal responding in control (maximal shift of 14.8 ± 3.1 Hz), but not SNL (2.3 ± 2.2 Hz) rats. Heroin was less potent in SNL rats, whereas cocaine produced similar shifts in control (42.3 ± 2.0 Hz) and SNL (37.5 ± 4.2 Hz) rats. CONCLUSIONS: Nerve injury suppressed potentiation of electrical stimulation of the ventral tegmental area by opioids, suggesting that the positive reinforcing effects are diminished by chronic pain. Given concerns regarding prescription opioid abuse, developing strategies that assess both analgesia and abuse liability within the context of chronic pain may aid in determining which opioids are most suitable for treating chronic pain when abuse is a concern.

Rewarding electrical brain stimulation in rats after peripheral nerve injury: decreased facilitation by commonly abused prescription opioids.

BACKGROUND: Prescription opioid abuse is a significant concern in treating chronic pain, yet few studies examine how neuropathic pain alters the abuse liability of commonly abused prescription opioids. METHODS: Normal and spinal nerve ligated (SNL) rats were implanted with electrodes into the left ventral tegmental area (VTA). Rats were trained to lever press for intracranial electrical stimulation (VTA ICSS), and the effects of methadone, fentanyl, hydromorphone, and oxycodone on facilitation of VTA ICSS were assessed. A second group of neuropathic rats were implanted with intrathecal catheters, and the effects of intrathecal clonidine, adenosine, and gabapentin on facilitation of VTA ICSS were assessed. The effects of electrical stimulation of the VTA on mechanical allodynia were assessed in SNL rats. RESULTS: Responding for VTA ICSS was similar in control and SNL rats. Methadone, fentanyl, and hydromorphone were less potent in facilitating VTA ICSS in SNL rats. Oxycodone produced a significant facilitation of VTA ICSS in control (maximum shift 24.10 ± 6.19 Hz) but not SNL rats (maximum shift 16.32 ± 7.49 Hz), but also reduced maximal response rates in SNL rats. Intrathecal administration of clonidine, adenosine, and gabapentin failed to facilitate VTA ICSS in SNL rats, and electrical stimulation of the VTA did not alter mechanical allodynia following nerve injury. CONCLUSIONS: The present data suggests that the positive reinforcing effects of commonly abused prescription opioids are diminished following nerve injury. In addition, alleviation of mechanical allodynia with nonopioid analgesics does not appear to stimulate limbic dopamine pathways originating from the VTA in SNL rats.

Involvement of the lateral amygdala in the antiallodynic and reinforcing effects of heroin in rats after peripheral nerve injury.

BACKGROUND: Neuropathic pain alters opioid self-administration in rats. The brain regions altered in the presence of neuropathic pain mediating these differences have not been identified, but likely involve ascending pain pathways interacting with the limbic system. The amygdala is a brain region that integrates noxious stimulation with limbic activity. METHODS: µ-Opioid receptors were blocked in the amygdala using the irreversible antagonist, ß-funaltrexamine, and the antiallodynic and reinforcing effects of heroin were determined in spinal nerve-ligated rats. In addition, the effect of ß-funaltrexamine was determined on heroin self-administration in sham-operated rats. RESULTS: ß-Funaltrexamine decreased functional activity of µ-opioid receptors by 60 ± 5% (mean ± SD). Irreversible inhibition of µ-opioid receptors in the amygdala significantly attenuated the ability of doses of heroin up to 100 µg/kg to reverse hypersensitivity after spinal nerve ligation. Heroin intake by self-administration in spinal nerve-ligated rats was increased from 5.0 ± 0.3 to 9.9 ± 2.1 infusions/h after administration of 2.5 nmol of ß-funaltrexamine in the lateral amygdala, while having no effect in sham-operated animals (5.8 ± 1.6 before, 6.7 ± 0.9 after). The antiallodynic effects of 60 µg/kg heroin were decreased up to 4 days, but self-administration was affected for up to 14 days. CONCLUSIONS: µ-Opioid receptors in the lateral amygdala partially meditate heroin's antiallodynic effects and self-administration after peripheral nerve injury. The lack of effect of ß-funaltrexamine on heroin self-administration in sham-operated subjects suggests that opioids maintain self-administration through a distinct mechanism in the presence of pain.

Ascending dorsal column sensory neurons respond to spinal cord injury and downregulate genes related to lipid metabolism.

Regeneration failure after spinal cord injury (SCI) results in part from the lack of a pro-regenerative response in injured neurons, but the response to SCI has not been examined specifically in injured sensory neurons. Using RNA sequencing of dorsal root ganglion, we determined that thoracic SCI elicits a transcriptional response distinct from sciatic nerve injury (SNI). Both SNI and SCI induced upregulation of ATF3 and Jun, yet this response failed to promote growth in sensory neurons after SCI. RNA sequencing of purified sensory neurons one and three days after injury revealed that unlike SNI, the SCI response is not sustained. Both SCI and SNI elicited the expression of ATF3 target genes, with very little overlap between conditions. Pathway analysis of differentially expressed ATF3 target genes revealed that fatty acid biosynthesis and terpenoid backbone synthesis were downregulated after SCI but not SNI. Pharmacologic inhibition of fatty acid synthase, the enzyme generating palmitic acid, decreased axon growth and regeneration in vitro. These results support the notion that decreased expression of lipid metabolism-related genes after SCI, including fatty acid synthase, may restrict axon regenerative capacity after SCI.

Effects of opioid receptor ligands in rats trained to discriminate 22 from 2 hours of food deprivation suggest a lack of opioid involvement in eating for hunger.

It is well accepted that opioids promote feeding for reward. Some studies suggest a potential involvement in hunger-driven intake, but they suffer from the scarcity of methodologies differentiating between factors that intersect eating for pleasure versus energy. Here, we used a unique food deprivation discrimination paradigm to test a hypothesis that, since opioids appear to control feeding reward, injection of opioid agonists would not produce effects akin to 22 h of food deprivation. We trained rats to discriminate between 22 h and 2 h food deprivation in a two-lever, operant discrimination procedure. We tested whether opioid agonists at orexigenic doses produce discriminative stimulus effects similar to 22 h deprivation. We injected DAMGO, DSLET, or orphanin FQ in the paraventricular hypothalamic nucleus (PVN), a site regulating hunger/satiety, and butorphanol subcutaneously (to produce maximum consumption). We assessed the ability of the opioid antagonist, naltrexone, to reduce the discriminative stimulus effects of 22 h deprivation and of the 22 h deprivation-like discriminative stimulus effects of PVN-injected hunger mediator, neuropeptide Y (NPY). In contrast to PVN NPY, centrally or peripherally injected opioid agonists failed to induce discriminative stimuli similar to those of 22 h deprivation. In line with that, naltrexone did not reduce the hunger discriminative stimuli induced by either 22 h deprivation or NPY administration in 2 h food-restricted subjects, even though doses used therein were sufficient to decrease deprivation-induced feeding in a non-operant setting in animals familiar with consequences of 2 h and 22 h deprivation. We conclude that opioids promote feeding for reward rather than in order to replenish lacking energy.

Nociceptor Deletion of Tsc2 Enhances Axon Regeneration by Inducing a Conditioning Injury Response in Dorsal Root Ganglia.

Neurons of the PNS are able to regenerate injured axons, a process requiring significant cellular resources to establish and maintain long-distance growth. Genetic activation of mTORC1, a potent regulator of cellular metabolism and protein translation, improves axon regeneration of peripheral neurons by an unresolved mechanism. To gain insight into this process, we activated mTORC1 signaling in mouse nociceptors via genetic deletion of its negative regulator Tsc2. Perinatal deletion of Tsc2 in nociceptors enhanced initial axon growth after sciatic nerve crush, however by 3 d post-injury axon elongation rate became similar to controls. mTORC1 inhibition prior to nerve injury was required to suppress the enhanced axon growth. Gene expression analysis in purified nociceptors revealed that Tsc2-deficient nociceptors had increased activity of regeneration-associated transcription factors (RATFs), including cJun and Atf3, in the absence of injury. Additionally, nociceptor deletion of Tsc2 activated satellite glial cells and macrophages in the dorsal root ganglia (DRG) in a similar manner to nerve injury. Surprisingly, these changes improved axon length but not percentage of initiating axons in dissociated cultures. The pro-regenerative environment in naïve DRG was recapitulated by AAV8-mediated deletion of Tsc2 in adult mice, suggesting that this phenotype does not result from a developmental effect. Consistently, AAV8-mediated Tsc2 deletion did not improve behavioral recovery after a sciatic nerve crush injury despite initially enhanced axon growth. Together, these data show that neuronal mTORC1 activation induces an incomplete pro-regenerative environment in the DRG that improves initial but not later axon growth after nerve injury.

Tests of behavioral-economic assessments of relative reinforcer efficacy: economic substitutes.

This experiment was conducted to test predictions of two behavioral-economic approaches to quantifying relative reinforcer efficacy. According to the first of these approaches, characteristics of averaged normalized demand curves may be used to predict progressive-ratio breakpoints and peak responding. The second approach, the demand analysis, rejects the concept of reinforcer efficacy, arguing instead that traditional measures of relative reinforcer efficacy (breakpoint, peak response rate, and choice) correspond to specific characteristics of non-normalized demand curves. The accuracy of these predictions was evaluated in rats' responding for food or fat: two reinforcers known to function as partial substitutes. Consistent with the first approach, predicted peak normalized response output values (Omax) obtained under single-schedule conditions ordinally predicted progressive-ratio breakpoints and peak responding. Predictions of the demand analysis had mixed success. Pmax and Omax were significantly correlated with PR breakpoints and peak responding (respectively) when fat, but not when food, was the reinforcer. Relative consumption of food and fat under single schedules of reinforcement did not predict preference better than chance. The normalized demand analysis is supplemented with the economic concept of diminishing marginal utility, to predict preference shifts across the range of food and fat prices examined.

Tests of behavioral-economic assessments of relative reinforcer efficacy II: economic complements.

This experiment was conducted to test the predictions of two behavioral-economic approaches to quantifying relative reinforcer efficacy. The normalized demand analysis suggests that characteristics of averaged normalized demand curves may be used to predict progressive-ratio breakpoints and peak responding. By contrast, the demand analysis holds that traditional measures of relative reinforcer efficacy (breakpoint, peak response rate, and choice) correspond to specific characteristics of non-normalized demand curves. The accuracy of these predictions was evaluated in rats' responding for food or water: two reinforcers known to function as complements. Consistent with the first approach, predicted peak normalized response output values obtained under single-schedule conditions ordinally predicted progressive-ratio breakpoints and peak response rates obtained in a separate condition. Combining the minimum-needs hypothesis with the normalized demand analysis helped to interpret prior findings, but was less useful in predicting choice between food and water--two strongly complementary reinforcers. Predictions of the demand analysis had mixed success. Peak response outputs predicted from the non-normalized water demand curves were significantly correlated with obtained peak responding for water in a separate condition, but none of the remaining three predicted correlations was statistically significant. The demand analysis fared better in predicting choice--relative consumption of food and water under single schedules of reinforcement predicted preference under concurrent schedules significantly better than chance.

Toward an animal model of gambling: delay discounting and the allure of unpredictable outcomes.

Laboratory investigations of gambling are sometimes criticized as lacking ecological validity because the stakes wagered by human subjects are not real or no real monetary losses are experienced. These problems may be partially addressed by studying gambling in laboratory animals. Toward this end, data are summarized which demonstrate that laboratory animals will work substantially harder and prefer to work under gambling-like schedules of reinforcement in which the number of responses per win is unpredictable. These findings are consistent with a delay discounting model of gambling which holds that rewards obtained following unpredictable delays are more valuable than rewards obtained following predictable delays. According to the delay discounting model, individuals that discount delayed rewards at a high rate (like pathological gamblers) perceive unpredictably delayed rewards to be of substantially greater value than predictable rewards. The reviewed findings and empirical model support the utility of studying animal behavior as an ecologically valid first-approximation of human gambling.

Intrathecal Acetyl-L-Carnitine Protects Tissue and Improves Function after a Mild Contusive Spinal Cord Injury in Rats.

Primary and secondary ischemia after spinal cord injury (SCI) contributes to tissue and axon degeneration, which may result from decreased energy substrate availability for cellular and axonal mitochondrial adenosine triphosphate (ATP) production. Therefore, providing spinal tissue with an alternative energy substrate during ischemia may be neuroprotective after SCI. To assess this, rats received a mild contusive SCI (120 kdyn, Infinite Horizons impactor) at thoracic level 9 (T9), which causes loss of ∼ 80% of the ascending sensory dorsal column axonal projections to the gracile nucleus. Immediately afterwards, the energy substrate acetyl-L-carnitine (ALC; 1 mg/day) or phosphate-buffered saline (PBS) was infused intrathecally (sub-arachnoid) for 6 days via an L5/6 catheter attached to a subcutaneous Alzet pump. ALC treatment improved overground locomotor function (Basso-Beattie-Breshnahan [BBB] score 18 vs. 13) at 6 days, total spared epicenter (71% vs. 57%) and penumbra white matter (90% vs. 85%), ventral penumbra microvessels (108% vs. 79%), and penumbra motor neurons (42% vs. 15%) at 15 days post-SCI, compared with PBS treatment. However, the ascending sensory projections (anterogradely traced with cholera toxin B from the sciatic nerves) and dorsal column white matter and perfused blood vessels were not protected. Furthermore, grid walking, a task we have shown to be dependent on dorsal column function, was not improved. Thus, mitochondrial substrate replacement may only be efficacious in areas of lesser or temporary ischemia, such as the ventral spinal cord and injury penumbra in this study. The current data also support our previous evidence that microvessel loss is central to secondary tissue degeneration.

Analgesics as reinforcers with chronic pain: Evidence from operant studies.

Previously preclinical pain research has focused on simple behavioral endpoints to assess the efficacy of analgesics in acute and chronic pain models, primarily reflexive withdrawal from an applied mechanical or thermal stimulus. However recent research has been aimed at investigating other behavioral states in the presence of pain, including spontaneous, non-elicited pain. One approach is to investigate the reinforcing effects of analgesics in animals with experimental pain, which should serve as reinforcers by virtue of their ability to alleviate the relevant subjective states induced by pain. The gold standard for assessing drug reinforcement is generally accepted to be drug self-administration, and this review highlights the ability of drugs to serve as reinforcers in animals with experimental neuropathic pain, and the extent to which this behavior is altered in chronic pain states. Additionally, intracranial self-stimulation is an operant procedure that has been used extensively to study drug reinforcement mechanisms and the manner in which neuropathic pain alters the ability of drugs to serve as reinforcers in this paradigm will also be discussed. Drug self-administration and intracranial self-stimulation have promise as tools to investigate behavioral effects of analgesics in animals with chronic pain, particularly regarding the mechanisms through which these drugs motivate consumption in a chronic pain state.

Dorsal column sensory axons degenerate due to impaired microvascular perfusion after spinal cord injury in rats.

The mechanisms contributing to axon loss after spinal cord injury (SCI) are largely unknown but may involve microvascular loss as we have previously suggested. Here, we used a mild contusive injury (120 kdyn IH impactor) at T9 in rats focusing on ascending primary sensory dorsal column axons, anterogradely traced from the sciatic nerves. The injury caused a rapid and progressive loss of dorsal column microvasculature and oligodendrocytes at the injury site and penumbra and an ~70% loss of the sensory axons by 24 h. To model the microvascular loss, focal ischemia of the T9 dorsal columns was achieved via phototoxic activation of intravenously injected rose bengal. This caused an ~53% loss of sensory axons and an ~80% loss of dorsal column oligodendrocytes by 24 h. Axon loss correlated with the extent and axial length of microvessel and oligodendrocyte loss along the dorsal column. To determine if oligodendrocyte loss contributes to axon loss, the glial toxin ethidium bromide (EB; 0.3 µg/µl) was microinjected into the T9 dorsal columns, and resulted in an ~88% loss of dorsal column oligodendrocytes and an ~56% loss of sensory axons after 72 h. EB also caused an ~75% loss of microvessels. Lower concentrations of EB resulted in less axon, oligodendrocyte and microvessel loss, which were highly correlated (R(2) = 0.81). These data suggest that focal spinal cord ischemia causes both oligodendrocyte and axon degeneration, which are perhaps linked. Importantly, they highlight the need of limiting the penumbral spread of ischemia and oligodendrocyte loss after SCI in order to protect axons.

Lack of analgesic efficacy of spinal ondansetron on thermal and mechanical hypersensitivity following spinal nerve ligation in the rat.

The balance between descending inhibition and facilitation is thought to be disturbed in chronic pain states. Increased facilitation by spinally released serotonin has been suggested by demonstration that mechanically evoked neuronal responses of wide dynamic range neurons are inhibited by 5-HT3 receptor antagonists in rats following spinal nerve ligation (SNL) but not sham operation. Despite these physiologic data, the effects of spinal 5-HT3 receptor blockade on behavioral hypersensitivity and neurochemical alterations in spinal serotonergic system have not been thoroughly investigated following spinal nerve ligation in the rat. To test this, we acutely injected intrathecal ondansetron in rats between 14 and 30 days after SNL and assessed effects on thermal and mechanical hypersensitivity. We also determined the density of serotonergic nerve fibers, serotonin content and the levels of 5-HT3 receptors within the spinal cord at this time point. Intrathecal ondansetron (1, 3, 10, 30, and 100microg) produced no effect on behavioral measures of thermal or mechanical hypersensitivity whereas intrathecal morphine (1microg) and gabapentin (200microg) partially reversed thermal and mechanical hypersensitivity following SNL. In addition, SNL did not alter the density of serotonergic fibers or 5-HT3 receptor immunoreactivity or spinal tissue content of 5-HT within the dorsal horn. These results do not support anatomic plasticity of descending serotonergic pathways or tonic 5-HT3 receptor activity in maintaining hypersensitivity after nerve injury and in contrast to previous studies fail to demonstrate an anti-hypersensitivity effect of intrathecal injection of the 5-HT3 receptor antagonist ondansetron. Importantly, behavioral measures of mechanical hypersensitivity assess threshold responses whereas physiological studies of mechanically evoked neuronal responses involve application of suprathreshold stimuli. Thus, suprathreshold or more intense stimuli may be necessary to recruit descending serotonergic facilitatory drive required to observe the inhibitory effects of ondansetron on spinal neuronal excitability and behavioral hypersensitivity.