A brainstem substrate for analgesia elicited by intraoral sucrose.

Previous studies demonstrated that nursing or intraoral infusion of certain components of mother's milk (e.g. sugars and fats) produces calming and opiate receptor-dependent analgesia in newborn rats and humans. However, the neural circuitry underlying such analgesia is unknown. The aim of the present study was to specify the central pathways by which taste stimuli engage neural antinociceptive mechanisms. For this purpose, midcollicular transactions were used to investigate the role of the forebrain in analgesia elicited by intraoral infusion of 0.2 M sucrose in neonatal rats. Sucrose-induced analgesia persisted, and was enhanced, following midcollicular transection, indicating that it did not require neural circuits confined to the forebrain. Fos immunohistochemistry was used to identify brainstem neurons activated by a brief (90 s) intraoral infusion of a small volume (90 microl, 0.2M) of sucrose or a salt solution (0.1 M ammonium chloride) in 10-day-old rat pups. Compared with control groups (intact, cannula, distilled water), both sucrose and ammonium chloride induced Fos expression in the rostral nucleus tractus solitarius, the first relay in the ascending gustatory pathway. Sucrose also elicited Fos expression in several brainstem areas associated with centrally mediated analgesia, including the periaqueductal gray and the nucleus raphe magnus. Taken together, these findings demonstrate that analgesia elicited by intraoral sucrose does not require involvement of the forebrain. Intraoral sucrose activates neurons in the periaqueductal gray and nucleus raphe magnus, two key brainstem sites critically involved in descending pain modulation.

Alterations in stress-associated behaviors and neurochemical markers in adult rats after neonatal short-lasting local inflammatory insult.

Recently, there has been a growing interest in long-term consequences of neonatal pain because modern neonatal intensive care units routinely employ procedures that cause considerable pain and may be followed by local inflammation and hyperalgesia lasting for several hours or even days. To address this question, we developed a rat model of short lasting (<2 days) early local inflammatory insult produced by a single injection of 0.25% carrageenan (CAR) into the plantar surface of a hindpaw. Previously, we demonstrated that rats receiving this treatment within the first week after birth grow into adults with a global reduction in responsiveness to acute pain. Here, we report that these animals also manifest a low anxiety trait associated with reduced emotional responsiveness to stress. This conclusion is based in the following observations: (a) rats in our model display reduced anxiety on an elevated plus-maze; (b) in the forced swim test, these rats exhibit behavioral characteristics associated with stronger ability for stress coping; and (c) these animals have reduced basal and stress-induced plasma levels of such stress-related neuroendocrine markers as corticotropin-releasing factor, vasopressin, and adrenocorticotrophic hormone. In addition, we used DNA microarray and real-time reverse-transcriptase polymerase chain reaction to profile long-term changes in gene expression in the midbrain periaqueductal gray (PAG; a region involved in both stress and pain modulation) in our animal model. Among the affected genes, serotonergic receptors were particularly well represented. Specifically, we detected increase in the expression of 5-HT1A, 5-HT1D, 5-HT2A, 5-HT2C and 5-HT4 receptors. Several of these receptors are known to be involved in the anxiolytic and analgesic activity of the PAG. Finally, to determine whether neonatal inflammatory insult induces elevation in maternal care, which may play a role in generating long-term behavioral alterations seen in our model, we examined maternal behavior for 3 days following CAR injection. Indeed, we observed a substantial increase in maternal attention to the pups at the time of inflammation, but this increase was not without its cost: a period of significant maternal neglect afterward.

Characterization of basal and re-inflammation-associated long-term alteration in pain responsivity following short-lasting neonatal local inflammatory insult.

Recently, several studies have suggested that neonatal noxious insult could alter future responses to painful stimuli. However, the manifestations, mechanisms, and even developmental nature of these alterations remain a matter of controversy. In part, this is due to the lack of detailed information on the neonatal sensitive period(s) during which noxious stimulation influences future nociception, and the time-course and distribution of the resultant abnormalities. The present paper describes these parameters in a rat model of short-lasting ( approximately 24 h) neonatal local inflammation of a hindpaw produced by injection of 0.25% carrageenan (1 microl/g). Examinations of paw withdrawal responses to thermal and mechanical stimulations in adult animals, which as neonates were subjected to this insult, showed that the previously-reported long-term hypoalgesia and hyperalgesia are not mutually exclusive outcomes of early noxious experience. Long-term hypoalgesia was apparent at the basal conditions and was equally strong in the previously injured and uninjured paws, which suggests a globally-driven deficit. In contrast, long-term excessive hyperalgesia had the strongest manifestation in the neonatally-injured paw after re-inflammation, indicating significant segmental involvement in its generation. The differences between mechanisms underlying the observed hypoalgesia and hyperalgesia are further underscored by the finding that, while the former is detectable only after animals reach the second month of life, the latter is elicitable immediately upon cessation of the initial neonatal inflammation. Nevertheless, we detected a significant overlap in the neonatal sensitive periods for generation of these effects (both occurring within the first postnatal week). Also, neither the basal hypoalgesia nor excessive re-inflammation-associated hyperalgesia subsided with age and were detectable in 120-125-day-old rats. These finding provide a framework within which the entire complex of long-term effects of early noxious experience can be understood and examined.

Ontogeny of analgesia elicited by non-nutritive suckling in acute and persistent neonatal rat pain models.

Significant analgesic and calming effects in human infants and neonatal rodents are produced by orogustatory and orotactile stimuli associated with nursing. These naturally occurring analgesic stimuli may help to protect the vulnerable developing nervous system from the long-term effects of neonatal tissue injury. However, the efficacy of orotactile-induced analgesia across the pre-weaning period, as well as its effects on persistent inflammatory pain, is unknown. Here, we investigated the developmental profile of analgesia produced by orotactile stimulation during non-nutritive suckling in rats. The effects of suckling, as compared to non-suckling littermates, on nocifensive withdrawal responses to thermal and mechanical stimuli were examined at postnatal (P) days P0, P3, P10, P17 and P21. In some rats, Complete Freund's adjuvant (CFA) was injected in a fore- or hindpaw to produce inflammation. For thermal stimuli, suckling significantly increased forepaw withdrawal latencies at P3, P10 and P17, while hindpaw responses were increased at P3 and P10, but not at P17. In inflamed pups, suckling increased fore- and hindpaw response latencies at P10 and P17, but not at P0 or P21. Suckling-induced analgesia was naloxone-insensitive. For mechanical stimuli, suckling-induced analgesia was present at P3, P10 and P17, but not at P21, for both fore- and hindpaws in naïve and inflamed animals. Additionally, suckling had a small but significant effect at P0 for the forepaw in inflamed pups. In nearly all experiments, the peak effect of suckling for thermal and mechanical stimuli occurred at P10. These results indicate that orotactile analgesia, like orogustatory analgesia, is absent or minimal at P0, appears consistently at approximately P3 and is maximal at P10. Unlike gustatory analgesia in rats however, orotactile analgesia persists at least to P17. Orotactile stimulation during suckling effectively reduces transient pain elicited by thermal and mechanical stimuli, as well as persistent hyperalgesia and allodynia caused by inflammation.

A comparative study of the effects of morphine in the dorsal periaqueductal gray and nucleus accumbens of rats submitted to the elevated plus-maze test.

We studied the effects of morphine injected either systemically or into the dorsal periaqueductal gray (DPAG) or nucleus accumbens (NA) using conventional and ethological analyses of behavior of rats submitted to the elevated plus-maze test with transparent walls. Intraperitoneal morphine (0.1 mg/kg and 0.3 mg/kg) increased both standard and ethological measures, expressing general exploratory activity such as total arm entries, end-exploration, scanning, head-dipping, and rearing. Morphine 10 (7.6 microg/microl) and 30 nmol (23 microg/microl) injected into nucleus accumbens produced similar effects, which were blocked by i.p. naltrexone (2.0 mg/kg), an opioid antagonist with good affinity for mu-opioid receptors. Morphine injected into the DPAG produced either antiaversive (10 nmol) or aversive effects (30 nmol), which respectively reduced and increased entries and time spent in the open arms and behaviors associated with risk assessment (peeping out, stretched attend postures, and flat back approach). The proaversive effects were inhibited by i.p. norbinaltorphimine (2.0 mg/kg), a selective inhibitor for kappa-opioid receptors. These findings support the contention that at least some of the motivational effects of morphine may be due to activation of opioid mechanisms in nucleus accumbens, and DPAG has neural substrates for antiaversive and aversive effects of morphine. Moreover, on the basis of previous and present data obtained in this laboratory, it is suggested that stimulation of mu-opioid receptors inhibits and stimulation of kappa-receptors activates the neural substrate of aversion in the DPAG. On the other hand, the increase in exploratory behavior due to interaction of morphine with mu-opioid receptors in the nucleus accumbens may be due to the stimulation of the interface between neural substrates of motivation and motor output in this structure.

Neurochemical mechanisms of the defensive behavior in the dorsal midbrain.

Some regions in the mesencephalon, such as dorsal periaqueductal gray, inferior colliculus and deep layers of superior colliculus have been grouped together as a continuous strip of midbrain structures involved in the integration of the different components of aversive states in the brain. In fact, escape behavior and defensive, or fear-like behavior often result when these sites are electrically or chemically stimulated. Moreover, the behavioral responses induced by stimulation of these structures are, in general, accompanied by increases in mean arterial blood pressure, heart rate and respiration, and by analgesia. Both the behavioral and autonomic consequences of electrical stimulation of the mesencephalic tectum was shown to be attenuated by minor tranquilizers, probably through enhancement of GABAergic neurotransmission. Besides GABAergic interneurons which exert a tonic inhibitory control on neural circuits responsible for the behavioral correlates of the aversion in the above-mentioned structures, several other mechanisms such as opioid, neuropeptides, serotonergic and excitatory amino acids have also been implicated in the regulation of these processes. As to the analgesia that accompanies these aversive states it is mediated by non-opioid mechanisms, particularly by serotonergic ones through 5-HT2 receptors. Now, efforts have been made to characterize the mode of action of these neurotransmitters on their multiple receptors and how they interact with each other to produce or regulate the neural substrates of aversion in the midbrain.

Ethopharmacological analysis of behaviour of rats using variations of the elevated plus-maze.

Besides allowing better observation and recording of the behaviour of rodents, we have shown that the elevated plus-maze with transparent walls is as sensitive to anxiolytic and anxiogenic drug effects as a traditional apparatus with opaque walls. In this study we extend these observations with an ethopharmacological analysis of the behaviour of rats in the elevated plus-maze with transparent walls. A factor analysis of the behaviour of control (saline-treated) rats on the modified maze and on a standard maze revealed a characteristic distribution of the behavioural categories in six factors. For the modified maze, the first three factors, representing standard indices of anxiety, locomotion and risk assessment, were similar to the factor distribution reported by this and other studies using the standard elevated plus-maze test. Distinct features appeared on Factor 4, with the loading of rears in the modified maze together with the occurrence of the percentage of centre time and flat-back approach common to both mazes. Scanning and grooming, which are generally grouped on Factor 5 in the standard maze, appeared as isolated behavioural elements loading on Factors 5 and 6, respectively, in the modified maze. An ethopharmacological analysis of behaviour in the modified maze showed that midazolam (1 and 2 mg/kg) produced an anxioselective profile, whereas pentylenetetrazol (5 and 10 mg/kg) produced an anxiogenic-like profile. These results point to the distinct features of the elevated plus-maze with transparent walls, which may be beneficial in the study of the different facets of anxiety and the mode of action of anxiolytic drugs in rats.

Behavioral and pharmacological validation of the elevated plus maze constructed with transparent walls.

In this study we compared the performance of male Wistar rats, weighing 250-300 g, submitted to the standard plus maze (vertical surfaces of the closed arms with opaque walls) to their performance in a modified maze with raised Plexiglas edges in the closed arms (transparent walls). The animals (N = 12 for each group) continued to show a clear preference for the closed arms with transparent walls of the modified elevated plus maze. In addition, exploratory activity was higher in the open arms of the modified plus maze (4.25 +/- 0.42 entries and 53.50 +/- 5.10 s) as compared to that of the standard plus maze (2.10 +/- 0.25 entries and 24.00 +/- 4.91 s). Intraperitoneal injection of midazolam produced an increase in the number of entries (6.40 +/- 1.21 and 8.50 +/- 1.15 for 1.0 and 2.0 mg/kg, respectively) and in the time spend in the open arms (85.32 +/- 14.56 and 125.50 +/- 22.16 s for 1.0 and 2.0 mg/kg, respectively) while pentylenetetrazole caused a decrease in the number of entries (3.68 +/- 0.54 and 2.33 +/- 0.62 for 5.0 and 10 mg/kg, respectively) and in the time spent in the open arms of the modified maze (39.60 +/- 6.67 and 23.60 +/- 6.40 s for 5.0 and 10 mg/kg, respectively). The anxiolytic effect of midazolam and the anxiogenic effect of pentylenetetrazole were similar to those usually reported in the literature by authors using the standard test. These results behaviorally and pharmacologically validate the elevated plus maze with transparent walls and suggest that this test could be a useful tool for the study of anxiolytic drugs and the neurobiology of anxiety.

Behavioral and pharmacological validation of the elevated plus maze contructed with transparent walls

In this study we compared the performance of male Wistar rats, weighing 250-300g, submitted to the standard plus maze (vertical surfaces of the closed arms with opaque walls) to their performance in a modified maze with raised Plexiglas edges in the closed arms (transparent walls). The animals (N = 12 for each group) continued to show a clear preference for the closed arms with transparent walls of the modified elevated plus maze. In addition, exploratory activity was higher in the open arms of the modified plus maze (4.25 ñ 0.42 entries and 53.50 ñ 5.10s) as compared to that of the standard plus maze (2.10 ñ 0.25 entries and 24.00 ñ 4.91 s). Intraperitoneal injection of midazolam produced an increase in the number of entries (6.40 ñ 1.21 and 8.50 ñ 1.15 for 1.0 and 2.0 mg/Kg, rspectively) and in the time spent in the open arms (85.32 ñ 14.56 and 125.50 ñ 22.16 s for 1.0 and 2.0 mg/Kg, respectively) while pentylenetetrazole caused a decrease in the number of entries (3.68 ñ 0.54 and 2.33 ñ 0.62 for 5.0 and 10 mg/Kg, respectively) and in the time spent in the open arms of the modified maze (39.60 ñ 6.67 and 23.60 ñ 6.40 s for 5.0 and 10 mg/Kg, respectively). The anxiolytic effect of midazolam and the anxiogenic effect of pentylenetetrazole were similar to those usually reported in the literature by authors using the standard test. The4se results behaviorally and pharmacologically validate the elevated plus maze with transparebnt walls and suggest that this test could be a useful tool for the study of anxiolytic drugs and the neurobiology of anxiety