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Behavioral Alterations in Rats Prenatally Exposed to Valproic Acid: Animal Model of Autism

Autism is a severe behavioral disorder characterized by pervasive impairments in social interactions, deficits in verbal and nonverbal communication, and stereotyped, repetitive patterns of behaviors and interests. Recently, a new rodent model of autism was created by exposure of rat fetuses to valproic acid (VPA) on the 12.5th day of gestation (VPA rats). The model has striking anatomical, pathological, and etiological similarities to human data; however, it has not been characterized behaviorally. In order to determine if VPA rats present behavioral aberrations observed in autism, their behavior was extensively evaluated in a battery of tests. The results of the present experiments demonstrate that VPA rats exhibit: (1) lower sensitivity to pain and higher sensitivity to nonpainful stimuli, (2) diminished acoustic prepulse inhibition, (3) locomotor and repetitive/stereotypic-like hyperactivity combined with lower exploratory activity, and (4) decreased number of social behaviors and increased latency to social behaviors. In addition, VPA rats showed delayed maturation, lower body weight, delayed motor development, and attenuated integration of a coordinated series of reflexes, delayed nest-seeking response mediated by olfactory system, and normal negative geotaxis. Interestingly, all behavioral aberrations described in this paper appear before puberty, which could distinguish the VPA rat model of autism from other animal models of neurodevelopmental disorders, especially rodent models of schizophrenia. Our results bring further support to validity of the proposed VPA animal model of autism, suggesting similarities between the observed pattern of behavioral alterations in VPA rats and features of disturbed behavior in autistic patients.

Opioids in chronic pain

The advance in our understanding of the biogenesis of various endogenous opioid peptides, their anatomical distribution, and the characteristics of the multiple receptors with which they interact open a new avenue for understanding the role of opioid peptide systems in chronic pain. The main groups of opioid peptides: enkephalins, dynorphins and beta-endorphin derive from proenkephalin, prodynorphin and proopiomelanocortin, respectively. Recently, a novel group of peptides has been discovered in the brain and named endomorphins, endomorphin-1 and -2. They are unique in comparison with other opioid peptides by atypical structure and high selectivity towards the mu-opioid receptor. Another group, which joined the endogenous opioid peptide family in the last few years is the pronociceptin system comprising the peptides derived from this prohormone, acting at ORL1 receptors. Three members of the opioid receptor family were cloned in the early 1990s, beginning with the mouse delta-opioid receptor (DOR1) and followed by cloning of mu-opioid receptor (MOR1) and kappa-opioid receptor (KOR1). These three receptors belong to the family of seven transmembrane G-protein coupled receptors, and share extensive structural homologies. These opioid receptor and peptide systems are significantly implicated in antinociceptive processes. They were found to be represented in the regions involved in nociception and pain. The effects of opioids in animal models of inflammatory pain have been studied in great detail. Inflammation in the periphery influences the central sites and changes the opioid action. Inflammation increased spinal potency of various opioid receptor agonists. In general, the antinociceptive potency of opioids is greater against various noxious stimuli in animals with peripheral inflammation than in control animals. Inflammation-induced enhancement of opioid antinociceptive potency is characteristic predominantly for mu opioid receptors, since morphine elicits a greater increase in spinal potency of mu- than of delta- and kappa-opioid receptor agonists. Enhancement of the potency of mu-opioid receptor agonists during inflammation could arise from the changes occurring in opioid receptors, predominantly in affinity or number of the mu-opioid receptors. Inflammation has been shown to alter the expression of several genes in the spinal cord dorsal horn. Several studies have demonstrated profound alterations in the spinal PDYN system when there is peripheral inflammation or chronic arthritis. Endogenous dynorphin biosynthesis also increases under various conditions associated with neuropathic pain following damage to the spinal cord and injury of peripheral nerves. Interestingly, morphine lacks potent analgesic efficacy in neuropathic pain. A vast body of clinical evidence suggests that neuropathic pain is not opioid-resistant but only that reduced sensitivity to systemic opioids is observed in this condition, and an increase in their dose is necessary in order to obtain adequate analgesia. Reduction of morphine antinociceptive potency was postulated to be due to the fact that nerve injury reduced the activity of spinal opioid receptors or opioid signal transduction. Our recent study with endogenous ligands of the mu-opioid receptor, endomorphins, further complicates the issue, since endomorphins appear to be effective in neuropathic pain. Identification of the involved differences may be of importance to the understanding of the molecular mechanism of opioid action in neuropathic pain, as well as to the development of better and more effective drugs for the treatment of neuropathic pain in humans.

Environmental Enrichment Reverses Behavioral Alterations in Rats Prenatally Exposed to Valproic Acid: Issues for a Therapeutic Approach in Autism

Environmental enrichment has been repeatedly shown to affect multiple aspects of brain function, and is known to improve cognitive, behavioral, and histopathological outcome after brain injuries. The purpose of the present experiments was to determine the effect of an enriched environment on behavioral aberrations observed in male rats exposed to valproic acid on day 12.5 of gestation (VPA rats), and proposed on the basis of etiological, anatomical, and behavioral data as an animal model of autism. Environmental enrichment reversed almost all behavioral alterations observed in the model. VPA rats after environmental enrichment (VPA-E) compared to VPA rats reared in standard conditions have higher sensitivity to pain and lower sensitivity to nonpainful stimuli; stronger acoustic prepulse inhibition; lower locomotor, repetitive/stereotypic-like activity, and enhanced exploratory activity; decreased anxiety; increased number of social behaviors; and shorter latency to social explorations. In comparison with control animals (Con), VPA-E rats exhibited increased number of pinnings in adolescence and social explorations in adulthood, and were less anxious in the elevated plus maze. Similar differences in social behavior and anxiety were observed between control rats exposed to environmental enrichment (Con-E) and control group reared in standard conditions. These results suggest that postnatal environmental manipulations can counteract the behavioral alterations in VPA rats. We propose environmental enrichment as an important tool for the treatment of autism spectrum disorders.

Gender-specific behavioral and immunological alterations in an animal model of autism induced by prenatal exposure to valproic acid.

Autism is a severe behavioral disorder characterized by pervasive impairments in social interactions, deficits in verbal and non-verbal communication, and stereotyped behaviors, with a four times higher incidence in boys than in girls. The core symptoms are frequently accompanied by a spectrum of neurobehavioral and immunological derangements, including: aberrant sensitivity to sensory stimulation, anxiety, and decreased cellular immune capacity. Recently, a new potential rodent model of autism induced by prenatal exposure to valproic acid (VPA rats) has been proposed. In order to determine if gender has an influence on alterations observed in VPA rats, male and female rats have been evaluated in a battery of behavioral, immunological, and endocrinological tests. A plethora of aberrations has been found in male VPA rats: lower sensitivity to pain, increased repetitive/stereotypic-like activity, higher anxiety, decreased level of social interaction, increased basal level of corticosterone, decreased weight of the thymus, decreased splenocytes proliferative response to concanavaline A, lower IFN-gamma/IL-10 ratio, and increased production of NO by peritoneal macrophages. Female VPA rats exhibited only increased repetitive/stereotypic-like activity and decreased IFN-gamma/IL-10 ratio. Sexual dimorphism characteristics for measured parameters have been observed in both groups of animals, except social interaction in VPA rats. Our results confirm existence of similarities between the observed pattern of aberrations in VPA rats and features of disturbed behavior and immune function in autistic patients, and suggest that they are gender-specific, which is intriguing in light of disproportion in boys to girls ratio in autism.

Inhibition of nitric oxide synthase enhances morphine antinociception in the rat spinal cord

NG-nitro-L-arginine methyl ester (L-NAME, 400-1500 micrograms), administered intrathecally (ith.), elicits a slight but dose-related antinociception in rats, assessed by tail-flick and paw pressure tests. L-NAME (400 micrograms) and morphine (0.5 microgram) coadministered ith. elicit a profound and long-lasting antinociception, which is abolished by ith. administration of 3-morpholino-sydnonimine (SIN-1, 100 micrograms). Hemoglobin (266 micrograms) administered ith. also slightly potentiates morphine antinociception. These results suggest that nitric oxide (NO) is involved in spinal nociceptive events, and that the increased production of NO following the nociceptive input may diminish the efficiency of opioid antinociception in the spinal cord.

Local peripheral opioid effects and expression of opioid genes in the spinal cord and dorsal root ganglia in neuropathic and inflammatory pain.

ABSTRACT We investigated the efficacy of local intraplantar (i.pl.) injection of peptide and non‐peptide μ‐, δ‐ and κ‐opioid receptor agonists in rat models of inflammatory and neuropathic pain. Locally applied agonists dose‐dependently reduced formalin‐induced flinching of the inflamed paw and induced antiallodynic and antihyperalgesic effects in sciatic nerve ligation‐induced neuropathic pain. These effects were mediated by peripheral opioid receptors localized at the side of tissue/nerve injury, as was demonstrated by selective and non‐selective opioid receptors antagonists. The ED50 dose range of μ‐ and κ‐agonists required to induce analgesia in neuropathy was much higher than the ED50 for inflammation; moreover, only δ‐agonists were effective in the same dose range in both pain models. Additionally, effective antinociception was achieved at a lower dose of peptide, compared to non‐peptide, opioids. Such findings support the use of the peripheral administration of opioid peptides, especially δ‐agonists, in treating chronic pain. Furthermore, in order to assess whether adaptations in the expression of opioid genes could underlie the clinical observation of reduced opioid effectiveness in neuropathic pain, we analyzed the abundance of opioid transcripts in the spinal cord and dorsal root ganglia (DRG) during the neuropathy and inflammation. Nerve injury down‐regulated mRNA for all types of opioid receptors in the DRG, which is predicted to decrease in the synthesis of opioid receptors to possibly account for the reduced effectiveness of locally administered opioids in neuropathy. The obtained results differentiate inflammatory and neuropathic pain and provide a novel insight into the peripheral effectiveness of opioids in both types of pain.

Inhibition of nitric oxide synthase attenuates the development of morphine tolerance and dependence in mice

The effect of the nitric oxide (NO) synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME, 5-20 mg/kg i.p.) and NG-nitro-L-arginine (NO2Arg, 5-20 mg/kg i.p.) on morphine-induced analgesia, as well as on morphine induced tolerance and dependence was examined in male albino Swiss mice. Neither acute nor repeated (for 5 days) administration of the nitric oxide synthase inhibitor, L-NAME affected the morphine induced analgesia, as measured by hot plate and tail-flick tests. On the other hand, administration of L-NAME or NO2Arg along with morphine prevented the development of tolerance to the analgesic effect of morphine for at least 7 days, whereas the analgesic effect of morphine alone disappeared after 5 days. L-NAME and NO2Arg also attenuated some signs of morphine dependence, as assessed by naloxone (2 mg/kg)-precipitated withdrawal. These results indicate that NO may play a role in the development of morphine tolerance and dependence.

Mixed opioid/nonopioid effects of dynorphin and dynorphin related peptides after their intrathecal injection in rats

Dynorphin dose-dependently increased the tail flick latency of rats to radiant heat following its intrathecal injection. This effect was accompanied by an alteration in motor function that was characterized by a flaccid extension of the hindlimbs and flaccidity of the tail. Naloxone (10 but not 1 mg/kg) blocked the antinociceptive effect and motor disturbance produced by dynorphin. The non-opioid analogue des-Tyr1-dynorphin(1-13) also increased tail flick latency and produced paralysis. Dynorphin(1-8) significantly elevated tail flick latency without affecting motor function. Furthermore, the effect of dynorphin(1-8) was blocked by 1 mg/kg naloxone. These data suggest a possible physiological role of dynorphin in influencing motor function in the spinal cord and a role of dynorphin(1-8) in modulating pain transmission. Another finding of the present study was that dynorphin was approximately ten times more potent in producing its effects when injected one day after surgery compared to when it when it was injected one week or more after surgery.

Spinal analgesic action of endomorphins in acute, inflammatory and neuropathic pain in rats.

We studied spinal analgesic and antiallodynic effects of endomorphin-1 and endomorphin-2 administered i.t. in comparison with Tyr-D-Ala-Gly-MePhe-Gly-ol (DAMGO) or morphine, during acute, inflammatory and neuropathic pain in rats chronically implanted with intrathecal cannulas. Endomorphin-1 and endomorphin-2 (2.5, 5, 10 microg i.t.) increased the tail-flick latency and, to the lesser extent, the paw pressure latency. The range of potencies in both those models of acute pain was as follows: DAMGO > morphine = endomorphin-1 > endomorphin-2. In a model of inflammatory pain, the number of formalin-induced flinching episodes was decreased by endomorphin-1. The effect of endomorphin-2 was much less pronounced. Both DAMGO and morphine significantly inhibited the pain-related behavior evoked by formalin. In a neuropathic pain model (sciatic nerve crushing in rats), endomorphin-1 and -2 (5 microg i.t.) had a statistically significant effect on the tail-flick latency and on the cold-water tail flick latency. Morphine, 5 microg, was found to be ineffective. Endomorphin-1 and -2 (2.5 and 5 microg i.t.) dose-dependently antagonized allodynia. Those effects of endomorphins were antagonized in acute (30 microg), inflammatory (30 microg) and neuropathic pain models (60 microg) by cyprodime, a selective mu-opioid receptor antagonist. In conclusion, our results show a strong analgesic action of endomorphins at the spinal cord level. The most interesting finding is a strong, stronger than in the case of morphine, antiallodynic effect of endomorphins in rats subjected to sciatic nerve crushing, which suggests a possible use of these compounds in a very difficult therapy of neuropathic pain.

Immunocytochemical localization of somatostatin receptor sst2A in the rat spinal cord and dorsal root ganglia

Intrathecal administration of octreotide, a stable somatostatin analogue, provides pain relief in patients, and locally applied somatostatin inhibits firing of nociceptive dorsal horn neurons. In the present study, we have raised polyclonal antibodies that specifically detect the somatostatin receptor sst2A and used these antisera for immunocytochemical localization of the receptor protein in the rat spinal cord and dorsal root ganglia. In the superficial layers of the dorsal horn, sst2A‐like immunoreactivity (Li) formed a dense network consisting of neuronal perikarya and dendrites which were often closely apposed by, but not co‐contained within, somatostatin‐14‐immunoreactive nerve fibres and terminals. sst2A‐Li was resistant to dorsal rhizotomy and did not colocalize with either substance P or calcitonin gene‐related peptide suggesting that sst2A‐Li was not located to primary afferents, but rather confined to second‐order spinal neurons. The position of sst2A‐Li perikarya and dendrites in the dorsal horn appeared to be similar to those containing μ‐opioid receptor‐Li; however, double labelling experiments revealed no instances of coexistence of these two receptors. sst2A‐Li was also observed in the dorsal root ganglia predominantly targeted to the somatic plasmalemma of medium size neurons distinct from those expressing somatostatin‐14 or δ‐opioid receptors. Thus, the present results not only provide a morphological substrate for spinal octreotide analgesia but also show that somatostatin and opioids are poised to modulate nociceptive transmission by distinct anatomical systems.