[Sex-dependent differences in parameters of a long-term pain caused by inflammatory focus in prenatally stressed newborn rats].

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

Mapping of pain circuitry in early post-natal development using manganese-enhanced MRI in rats.

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

Opiate withdrawal during development: are NMDA receptors indispensable?

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.

[EFFECT OF PRENATAL STRESS ON SEROTONERGIC NEURONS IN DORSAL RAPHE NUCLEUS AND ON PAIN BEHAVIOR DURING NEONATAL PERIOD].

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

Antinociceptive effects of locally administered morphine in infant rats.

Opiates injected into a site of injury are analgesic in adult animals, but there are no data on the effectiveness of this route of administration in immature organisms. Since the biological processes that regulate the effects of locally administered opiates are in flux during the early postnatal life of the rat, it is not clear whether or not opiates given directly into local tissue would be effective as analgesics. To test this we injected morphine (0.12, 0.60, 3.0 microg/injection) directly into the hindpaw (intraplantar) of infant rats at 3, 10 and 21 days of age, and assessed the behavioral response and the induction of Fos like immunocytochemistry in the dorsal horn of the spinal cord in the formalin test. Controls included saline injections to the paw, or comparable doses of morphine injected subcutaneously. At 3 days of age, the two higher doses were behaviorally analgesic when given into the paw, but there was limited selectivity over the subcutaneous route. At both 10 and 21 days of age, intraplantar injections were effective analgesics, whereas subcutaneous injections were not. The number of Fos stained cells in the dorsal horn of the spinal cord, induced by the formalin treatment, was decreased significantly by the 3.0 mg dose of morphine at all three ages. The results demonstrate that local treatment with morphine is an effective and selective analgesic in the infant rat.

The induction of Fos-like immunoreactivity by noxious thermal, mechanical and chemical stimuli in the lumbar spinal cord of infant rats.

The present study examined the maturation of nociceptive primary afferents using expression of Fos-like immunoreactivity in the second-order spinal cord neurons as an anatomical and functional marker. Pinch, immersion in hot water, or formalin injection applied to the hindpaw was used as the peripheral noxious stimulus in awake 0-, 1-, 2-, 3-, and 14-day-old rat pups. On the day of birth, all 3 stimuli elicited expression of the Fos protein in dorsal horn cells indicating that nociceptive primary afferents are functional at this age. The expression of the Fos protein was related to the intensity of stimulation since greater injection volumes of formalin or prolonged application of the thermal stimulus increased the number of stained nuclei. The number of stained nuclei was age dependent and older pups exhibited a greater number of stained nuclei. The results of this study are consistent with electrophysiological studies that have demonstrated that the primary nociceptive afferents continue to mature during the rats' postnatal life. Furthermore, the number of Fos immunoreactive neurons in immature rats are age and stimulus-intensity dependent.

Effects of chronic antenatal and postnatal administration of narcotics on naloxone-induced anorexia in preweanling rats.

Previous studies have reported age-related changes in opiate receptors and in their response to narcotics during the process of normal growth of the brain. By inducing alterations in this developmental sequence, the present study attempted to provide correlates of the opiate receptor system with naloxone-induced anorexia. Offspring of mothers treated with morphine (7.5 mg/kg twice daily, s.c.) or saline during pregnancy, and infants from untreated mothers given morphine (5 mg/kg), naltrexone (10 mg/kg) or saline subcutaneously on postnatal days 1-5, were tested at days 10, 12 and 14 for deprivation-induced milk consumption following an acute dose of naloxone (1 or 5 mg/kg, i.p.). Naloxone reduced the food intake of 10- and 12-day old infants chronically treated with morphine postnatally. At age 14, naloxone reduced the food consumed by all the pretreatment groups, and pretreatment with morphine altered the dose-response curves for feeding modulation induced by naloxone. Naloxone had no effect on the food consumed by 10- or 12-day old offspring of mothers treated with morphine or saline, or on those age-groups that received naltrexone chronically or saline pretreatments postnatally. The observed changes occurred in the absence of gross malformations, drug-withdrawal symptoms and differences in activity. These results demonstrate that opiate receptors may participate in feeding.

The effects of opiate antagonists on milk intake of preweanling rats.

The observation that acute administration of opiate agonists to rodent adults increased food intake while the reverse was true with a single injection of an opiate antagonist has led to the suggestion that endogenous opioids may in part mediate feeding behaviour in adult rodents. Studies were performed in infant rats to explore further the generality and ontogeny of this function. In three models of food availability, the effects of naloxone and naltrexone were assessed in deprivation-induced feeding tests in 3-, 10-, 12, 14- and 19-day-old preweanling rats. Naloxone or naltrexone attenuated food intake only after 14 days. Functional immaturity of the opioid systems vis-a-vis ingestive behaviour may account for this developmental sequence.

The ontogeny of opioid receptors mediating opiate-induced feeding in rats.

Acute administration of naloxone to preweanling rats does not attenuate independent ingestion of milk until 14 days of age suggesting that the full expression of an endogenous opioid system(s), regulating feeding rats, is not complete prior to this age. The present study was undertaken to examine the functional ontogeny of opioid receptors mediating opiate-induced feeding in rats. Rat pups, satiated with milk, were given intraperitoneal injections of the opiate receptor agonist, morphine, and were allowed free access to milk. Morphine stimulated the intake of milk at 3, 5, 7, 14 and 21 days of age, within 2 hr of injection. A time-course analysis in 7-day-old pups showed greater enhancement of intake between hours 2 and 4, than between hours 0 and 2, for large doses of morphine (0.3 and 1.0 mg/kg) suggesting that morphine-induced behavioral depression, which was observed early in the test session, confounded intake at earlier hours. Administration of the opiate receptor antagonist, naltrexone, produced no effect on intake of its own, but blocked the stimulation of intake by morphine in 5-day-old pups confirming that the effect of morphine on the intake of milk was mediated by opioid receptors. Thus, while a functional endogenous opioid system(s), regulating feeding in rats, is not fully mature until 14 days postpartum, the present results suggest that opioid receptors mediating feeding are functional very early in the postnatal development of the rat.

The appearance of Fos protein-like immunoreactivity in the hypothalamus of developing rats in response to cold ambient temperatures.

This study examined cellular activity in the hypothalamus of developing rats in response to cold environmental temperatures. The appearance of the nuclear protein, Fos, in response to cold ambient temperatures in rats three to 35 days of age was used as a marker of neuronal activation. Fos-positive nuclei were first seen in response to cold ambient temperatures in the ventromedial nucleus at three days of age, the paraventricular nucleus at eight days of age, the preoptic-anterior hypothalamus at 10 days of age and the anterior hypothalamic nucleus at 21 days of age. The rectal temperature of the 10-day-old pups dropped by less than half that measured in the nine-day-old pups after 1 h in the cold. It is possible that the activation of neurons in the preoptic-anterior hypothalamus in the 10-day-old animal may contribute to the decreased hypothermia observed in the 10-day-old after 1 h in the cold. The high density of Fos-like immunoreactive-labeled nuclei in the preoptic-anterior hypothalamus in the 10-day-old rats that were exposed to the cold environment indicates increased neuronal activity at this site. The results suggest that, at least in the preoptic-anterior hypothalamus, the appearance of the cellular response to cold ambient temperatures is coincidental with a thermogenic response.

Stress-induced preproenkephalin mRNA expression in the amygdala changes during early ontogeny in the rat.

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

Screening for cognitive toxicity of anticholinergic drugs.

Impairment of memory by muscarinic anticholinergic drugs has been demonstrated with a variety of agents using both acute administration in normal volunteers and chronic treatment in patients. Cognitive toxicity must be considered in the evaluation of the risks versus the benefits of psychopharmacologic treatment. The relationship between serum levels of anticholinergic drugs measured by radioreceptor assay and performance on the Buschke Selective Reminding Test were investigated to develop strategies for identifying patients with drug-related cognitive impairment. The radioreceptor assay may be of value in screening for patients at high risk for cognitive toxicity. However, there appears to be no alternative to careful longitudinal evaluation of learning and memory in patients after appropriate trial modifications of their medication regimens to identify patients with drug-related impairment.

Mu opioid receptors in the ventrolateral periaqueductal gray mediate stress-induced analgesia but not immobility in rat pups.

Rat pups become immobile and analgesic when exposed to an adult male rat. The aim of this study was to determine whether these reactions are under the control of endogenous opioids and to determine the role of the midbrain periaqueductal gray (PAG), which mediates stress-induced immobility and analgesia in adult animals. In Experiment 1, 14-day-old rats were injected systemically with the general opioid receptor antagonist naltrexone (1 mg/kg), which blocked male-induced analgesia to thermal stimulation but did not affect immobility. In Experiment 2, the selective mu opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP; 50 or 100 ng/200 nl) was microinjected into the ventrolateral and lateral PAG. CTOP suppressed male-induced analgesia when injected into the ventrolateral PAG. Male-induced immobility was not affected by CTOP. Male proximity therefore seems to induce analgesia in rat pups by releasing endogenous opioids that bind to mu opioid receptors in the ventrolateral PAG.

Ontogeny of morphine withdrawal in the rat.

The present studies examined behavioral changes during precipitated morphine withdrawal in 7- to 42-day-old rat pups. One group of rats was injected with morphine sulfate (10.0 mg/kg) twice daily for 6.5 days. Another group of 7-day-old rats received a lower dose of morphine (3.0 mg/kg). Controls were saline injected or untreated litters (7-day-old pups only). On Day 7, a target pup was injected with saline or naltrexone (0.3-10.0 mg/kg). Preweaning pups were observed in a warm chamber with the litter. Forty-two-day-old rats were tested individually. Morphine-treated pups tested with naltrexone showed significant alterations in behavior that varied at different ages. For example, rolling, stretching, and head and paw moves were observed at the younger ages, whereas burrowing, diarrhea, jumps, teeth chatter, and wet dog shakes occurred in the older rats. These data indicate that morphine-abstinent rats demonstrate withdrawal signs that are within the developmental repertoire of the rat.

Self-stimulation in 7- and 10-day-old rats.

Recently, it has been shown that the infant rat exhibits learned behaviors characteristic of the adult. With a modified self-stimulation paradigm, this study explored whether 7- and 10-day-old rat pups could learn a discriminated operant to obtain direct electrical stimulation in neural sites that support self-stimulation in adults. By nudging one of two response manipulanda, at two ages (7 and 10 days) and temperatures (22 and 35 degrees C), pups self-stimulated with electrodes implanted in a variety of forebrain sites, including the prefrontal cortex, bed nucleus of the stria terminalis, medial nucleus of the amygdala, and the medial forebrain bundle. The only temperature-sensitive site might be the nucleus accumbens which was positive only at the higher temperature in 10-day-olds. These results indicate that several forebrain sites demonstrate rewarding properties of stimulation in the preweanling rat pup.

Naltrexone-precipitated morphine withdrawal in infant rat is attenuated by acute administration of NOS inhibitors but not NMDA receptor antagonists.

RATIONALE: There is increasing evidence that the N-methyl-D-aspartate (NMDA) receptor and the nitric oxide system are involved in opiate dependence in the adult rat, but whether these results in the adult apply to the infant rat is unknown. OBJECTIVES: Here we examined the effects of NMDA receptor antagonists and nitric oxide synthase (NOS) inhibitors, which reduce the opiate abstinence syndrome in adult animals, on morphine withdrawal in the infant rat. METHODS: Neonatal rats were injected with morphine sulfate (10.0 mg/kg) twice daily for 6.5 days. On the 7th day, pups were injected with NOS inhibitors (L-NAME or 7-NI), NMDA receptor antagonists (MK-801 or AP-5), or vehicle. After 15 min, the pups were injected with naltrexone (1 mg/kg) to precipitate withdrawal. Behavior for each pup was identified and recorded every 15 s for 10 min before naltrexone injection and 15 min after naltrexone injection. RESULTS: Both L-NAME and 7-NI significantly reduced most withdrawal behaviors in the infant rat, a result in line with previous studies in the adult rat. In contrast, AP-5 reduced some withdrawal behaviors but also increased others (e.g., moving paws). MK-801 was likewise ineffective in reducing most withdrawal behaviors and increased certain withdrawal behaviors (walking and wall climbing). CONCLUSIONS: In the infant rat, the production of nitric oxide is involved in opiate withdrawal whereas the NMDA receptor may not yet be functionally active or may play only a minor role.

Aversive properties of the kappa opioid agonist U50,488 in the week-old rat pup.

mu Opioids have been shown to produce analgesia and to be reinforcing during the first week of life in the rat. kappa Opioids also have analgesic actions in both the infant and adult, but can be aversive in the mature animal. We examined the aversive effects of the kappa opioid agonist U50,488 during the first postnatal week in the rat pup in three ways. In the first experiment, U50,488, injected peripherally (1.0-30.0 mg/kg), was paired with an odor and pups were tested 8 h later for positional preference for avoidance of that odor. This task is similar to conditioned preference/aversion tests used with adult animals. Both 3- and 7-day-old pups learned to avoid the odor adulterated side at the two higher doses. When exposed to odors previously associated with U50,488, pups at both ages decreased locomotor activity. In a second experiment, acute treatment with U50,488 increased ultrasonic distress vocalizations (USV) equally at 3 and 7 days of age, increased locomotor activity, and decreased rectal temperature. Neither of the latter two effects was correlated with the increase in USV production. The third experiment showed that conditioned odor cues increased USV 8 h later in 3- and 7-day-old pups at 1.0-10.0 mg/kg without changes in activity or rectal temperature. The results from these studies suggest that U50,488 can produce aversions in the neonatal rat pup as it does in the adult.

Inescapable shock alters mescaline's disruption of active avoidance acquisition.

Rats were tested on a two-way avoidance acquisition with or without inescapable shock given 24 h prior to training. Mescaline given to nonshock rats disrupted acquisition in a dose-dependent fashion and tolerance developed to this disruption. Mescaline given to shock rats had no effect on acquisition even though levels of acquisition were the same for both shock and nonshock rats without drug. Moreover, subchronic treatment (5 days facilitated acquisition. These experiments demonstrate an interaction between shock, which presumably is a stressor, and mescaline. The data are consistent with the observation that when animals are exposed to presumptive stressors (e.g., shock, handling) hallucinogens can facilitate behavior, while in other situations, hallucinogens disrupt behavior.

The ontogeny of endomorphin-1- and endomorphin-2-like immunoreactivity in rat brain and spinal cord.

Endomorphin-1 and endomorphin-2 are recently described peptides with high affinity and specificity for the mu opioid receptor. They are believed to be the endogenous ligands for that receptor. We describe the maturation of the endomorphins in brain and spinal cord using recently characterized antibodies to each. Endomorphin-1-like immunoreactivity was examined in brain, focusing on the periaqueductal gray of the midbrain and the diagonal band of Broca; endomorphin-2-like immunoreactivity is reported for the medulla and spinal cord. In these regions, and in all other regions studied but not described in this paper, the endomorphins were not seen at birth or at 3 days of age. Staining was present in 7-day-old and older animals. At these early ages, the pattern and density of staining are not fully developed, but appear complete by 21 days of age. The results suggest that both endomorphin-1 and endomorphin-2 develop relatively late compared to other opioid peptides.

Maturation of endomorphin-2 in the dorsal horn of the medulla and spinal cord of the rat.

The endomorphins are potent and selective endogenous agonists at the mu opioid receptor. We describe here the postnatal ontogeny of endomorphin-2 like immunoreactivity (EM2-LI) in the dorsal horn of the rat medulla and spinal cord. EM2-LI is dense in the superficial lamina of the dorsal horn of the adolescent and adult rat, with fibers also present in skin and dorsal root ganglia. No staining was noted at 3 days of age or younger. Faint and limited staining was noted by 7 days of age. The density and distribution of the immunoreactivity increased with age, reaching an adult-like distribution by 21 days of age. Stress-induced responses mediated by endogenous opioids occur late in development and may be related to the late appearance of endomorphin-2.