I. P. Butkevich

Prenatal stress alters time characteristics and intensity of formalin-induced pain responses in juvenile rats.

Previous studies found that stressful events during pregnancy can alter the offsprings pain sensitivity to the phasic nociceptive stimuli. The present data constitute the first demonstration of the consequences of prenatal stress to formalin-induced pain in juvenile rats. Injection of formalin into a hind paw of a 25-day-old rat that had not been stressed prenatally produced the typical biphasic specific nociceptive behavioral response consisting of an early short phase lasting 1-4 min followed by a second prolonged phase (12-24 min). Between them there was an interphase that lasted 6-9 min during which the specific behaviors were not shown. This period is generally considered to be a period of inactivity. Prenatally stressed rat pups showed significant increase in flexing+shaking behaviors and in the duration of the second phase of formalin-induced pain in flexing+shaking and licking behaviors and decrease of the duration of the interphase. Disinhibition of the pain behaviors during the interphase was greatly more pronounced in female than in male rats. Sex differences indicate increased vulnerability of inhibitory processes to prenatal stress in females compared with males. These data also underline the importance of understanding the nature of the interphase and provide data on the mechanisms that underlie that component of the formalin test.

Maternal stress differently alters nociceptive behaviors in the formalin test in adult female and male rats.

The long-term effects of restraint stress in Wistar rats during the last week of gestation were investigated on the acute and tonic phases of the specific biphasic nociceptive behavioral response in the formalin test in offspring, females and males, at 90 days. Prenatal stress produced significant changes in formalin-induced pain, which was more pronounced in females as compared to males. The distorted response in females was more at the supraspinal level with an increased intensity of the licking response in both phases as well as with an increased their duration. Results concerning changes of the interphase length indicate the impairments of inhibitory mechanisms in the central nervous system. Furthermore, profound difference in the effects of prenatal stress on the first phase but similarity in these effects on the second phase in females and males are indirect strong support of the view that the second phase in the formalin test can not be mediated by central sensitization alone but greatly depends on signals ongoing from nociceptive primary afferents. Finally, the results obtained in males are important argument in favor of assumption about different mechanisms of acute and tonic pain. Taken together, these studies show that prenatal stress alters nociceptive behaviors in the formalin test in rats at 90 days in a sex-specific manner.

Decreased serotonin level during pregnancy alters morphological and functional characteristics of tonic nociceptive system in juvenile offspring of the rat

Serotonin (5-HT) contributes to the prenatal development of the central nervous system, acting as a morphogen in the young embryo and later as a neurotransmitter. This biologically active agent influences both morphological and biochemical differentiation of raphe neurons, which give rise to the descending serotonergic paths that regulate the processing of acutely evoked nociceptive inputs. The involvement of 5-HT in the prenatal development of tonic nociceptive system has not been studied. In the present study we evaluated the effects of a single injection (400 mg/kg, 2 ml, i.p.) of the 5-HT synthesis inhibitor, para-chlorophenylalanine (pCPA), given to pregnant rats during the critical period fetal serotonin development. The functional integrity of the tonic nociceptive response was investigated in 25 day old rats using the classic formalin test. Morphological analysis of brain structures involved in formalin-induced pain and 5-HT levels in the heads of 12-day embryos were also evaluated. Embryonic levels of 5-HT were significantly lowered by the treatment. The juvenile rats from pCPA-treated females showed altered brain morphology and cell differentiation in the developing cortex, hippocampus, raphe nuclei, and substantia nigra. In the formalin test, there were significant decreases in the intensity and duration of the second phase of the formalin-induced response, characterizing persistent, tonic pain. The extent of impairments in the brain structures correlated positively with the level of decrease in the behavioral responses. The data demonstrate the involvement of 5-HT in the prenatal development of the tonic nociceptive system. The decreased tonic component of the behavioral response can be explained by lower activity of the descending excitatory serotonergic system originating in the raphe nuclei, resulting in decreased tonic pain processing organized at the level of the dorsal horn of the spinal cord.

Modulation of Signal-Transducing Function of Neuronal Membrane Na+,K by Endogenous Ouabain and Low-Power Infrared Radiation Leads to Pain Relief

Effects of infrared (IR) radiation generated by a low-power Co2-laser on sensory neurons of chick embryos were investigated by organotypic culture method. Low-power IR radiation firstly results in marked neurite suppressing action, probably induced by activation of Na+,K+-ATPase signal-transducing function. A further increase in energy of radiation leads to stimulation of neurite growth. We suggest that this effect is triggered by activation of Na+,K+-ATPase pumping function. Involvement of Na+,K+-ATPase in the control of the transduction process was proved by results obtained after application of ouabain at very low concentrations. Physiological significance of low-power IR radiation and effects of ouabain at nanomolar level was investigated in behavioral experiments (formalin test). It is shown that inflammatory pain induced by injection of formalin is relieved both due to ouabain action and after IR irradiation.

Sex differences in formalin-induced pain in prenatally stressed infant rats

The aim of this work was to study the effects of prenatal stress on nociceptive responses in the formalin test in female and male infant (7‐day‐old) Long‐Evans hooded rats. Prenatally stressed infant rats displayed biphasic flinching+ shaking behavior whereas non‐stressed animals showed only a weak second phase. Pain sensitivity in prenatally stressed males was significantly greater than that of prenatally non‐stressed males during the second phase only; there were no differences in pain sensitivity between prenatally stressed and non‐stressed females. Moreover prenatally stressed male rats pups demonstrated that the second phase of the response to formalin was enhanced relative to the second phase in stressed females. The current and previous data [Butkevich IP, Barr GA, Mikhailenko VA, Otellin VA. Increased formalin‐induced pain and expression of fos neurons in the lumbar spinal cord of prenatally stressed infants rats. Neurosci Lett 2006a;403:222–226] show increased tonic pain in prenatally stressed infant rats and a large increase in the number of formalin‐induced fos‐like immunoreactivity in the spinal cord dorsal horn. There is a concomitant decrease in serotonin‐like immunoreactivity in the lumbar spinal cord dorsal horn [Butkevich IP, Barr GA, Otellin VA. Effect of prenatal stress on behavioral and neural indices of formalin‐induced pain in infant rats. Abstracts, 35th Annual Meeting of Soc. For Neurosci. 2005a. Program No. 512.4 Washington, DC: Society for Neuroscience]. Given the decreased level of perinatal testosterone in prenatally stressed rats to which infant males are more sensitive than females, we suggest that these hormonal, behavioral and neuronal indices are strongly interrelated in prenatally stressed 7‐day‐old rat pups and that the decreased surge of testosterone may contribute to the increased behavioral response in the second phase in male rat pups. Mechanisms underlying the behavioral pain response induced by inflammation in prenatally stressed rat pups are characterized by sexual dimorphism even prior to the activational effects of sex hormones.

Reduced serotonin synthesis during early embryogeny changes effect of subsequent prenatal stress on persistent pain in the formalin test in adult male and female rats

The considerable evidence supporting a role for serotonin (5-HT) in the embryonic formation of CNS, mediation of prenatal stress, and pain processing is reviewed. Long-term influences of prenatal 5-HT depletion as well as its combination with prenatal stress effects on tonic nociceptive system in 90-day-old Wistar rats were studied in the formalin test. Pregnant dams were injected with para-chlorophenylalanine (pCPA, 400 mg/kg/2 ml, ip), producing 5-HT depletion during the early period of fetal serotonergic system development. The adult offspring from pCPA-treated dams revealed changes in behavioral indices of persistent pain (flexing + shaking and licking) in the formalin test (2.5%, 50 microl) that were accompanied by irreversible morphological alterations in the dorsal raphe nuclei. In the other series of experiments, the role of 5-HT in the mediation of prenatal stress on the behavioral indices of persistent pain was investigated in the adult offspring from dams with 5-HT depletion followed by restraint stress. Stress during the last embryonic week caused much more increase in flexing + shaking and licking in the second tonic phase of the response to formalin in offspring from pCPA- than saline-treated (control) dams. The former was characterized by alterations in the durations of the interphase, the second phase, and the whole behavioral response too. In offspring from pCPA-treated dams, sex dimorphism was revealed in tonic pain evaluated by licking. Together with our previous results in juvenile rats demonstrating the necessity of definite level of prenatal 5-HT for normal development of tonic nociceptive system, the present pioneering findings obtained in adult rats indicate that prenatal 5-HT depletion causes long-term morphological abnormalities in the dorsal raphe nuclei accompanied by alterations in behavioral indices of tonic pain. Early prenatal 5-HT depletion increases vulnerability of tonic nociceptive circuits to the following prenatal stress.

Increased formalin-induced pain and expression of fos neurons in the lumbar spinal cord of prenatally stressed infant rats

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.

Buspirone before prenatal stress protects against adverse effects of stress on emotional and inflammatory pain-related behaviors in infant rats: age and sex differences.

Prenatal stress strengthens tonic pain and provokes depression. The serotoninergic system is involved in these processes. We recently showed that maternal buspirone, a 5-HT1A receptor agonist, protects against the adverse effects of in utero stress on depression and pain in adult rat offspring. Using a similar maternal treatment with buspirone, we focus here on the infant stage, which is important for the correction of prenatal abnormalities. Maternal buspirone before restraint stress during the last week of pregnancy decreased the time of immobility in the forced swim test in the infant offspring. Prenatal stress increased formalin-induced pain in the second part of the time-course of the response to formalin in males of middle infancy but in the first part of the response in males of late infancy. The effect was reversed by maternal buspirone. Pain dominated in males of both middle and late infancy but the time-course of formalin pain in infant females revealed a slower development of the processes. The results show that the time-course of formalin-induced pain in infant rats reacts to prenatal stress in an age-dependent and sexually dimorphic manner. Our finding of opposite influences of prenatal stress and buspirone before prenatal stress on formalin-induced pain during the interphase indicates that functional maturity of the descending serotonergic inhibitory system occurs in late infancy males (11-day-olds), and 5-HT1A receptors participate in this process. The data provide evidence that maternal treatment with buspirone prior to stress during pregnancy alleviates depression-like and tonic pain-related behaviors in the infant offspring.

Gonadal ERα/β, AR and TRPV1 gene expression: Modulation by pain and morphine treatment in male and female rats

The results of several studies strongly indicate a bidirectional relationship among gonadal hormones and pain. While gonadal hormones play a key role in pain modulation, they have been found to be affected by pain therapies in different experimental and clinical conditions. However, the effects of pain and pain therapy on the gonads are still not clear. In this study, we determined the long-lasting (72 h) effects of inflammatory pain (formalin test) and/or morphine on estrogen receptor (ER), androgen receptor (AR) and TRPV1 gene expression in the rat testis and ovary. The animals were divided into groups: animals receiving no treatment, animals exposed only to the experimental procedure (control group), animals receiving no pain but morphine (sham/morphine), animals receiving pain and morphine (formalin/morphine), and animals receiving only formalin (formalin/saline). Testosterone (T) and estradiol (E) were determined in the plasma at the end of the testing. In the sham/morphine rats, there were increases of ERα, ERβ, AR and TRPV1 mRNA expression in the ovary; in the testis, ERα and ERβ mRNA expression were reduced while AR and TRPV1 expression were unaffected by treatment. T and E plasma levels were increased in morphine-treated female rats, while T levels were greatly reduced in morphine-treated and formalin-treated males. In conclusion, both testicular and ovarian ER (ERα and ERβ) and ovarian AR and TRPV1 gene expression appear to be affected by morphine treatment, suggesting long-lasting interactions among opioids and gonads.

Maternal buspirone protects against the adverse effects of in utero stress on emotional and pain-related behaviors in offspring

Previous investigations from our laboratory demonstrated that prenatal stress exacerbates inflammatory pain-related behavior in adult rats and that fetal serotonin (5-HT) is involved in this phenomenon. In the present study we test the hypothesis that injections of buspirone, a 5-HT1A agonist, to rat dams before restraint stress during the last week of pregnancy (between pregnant days 15 and 20) can improve the characteristics of emotional and inflammatory pain-related behaviors in the adult offspring. Buspirone was injected to dams between the 9 and 20 days of pregnancy, during restraint stress, five min before it. The depression-like behavior in the forced swim test, formalininduced pain and body weight were investigated in the adult offspring. Prenatal stress exacerbated the licking behavior, the index of formalin-induced pain, and increased the time of immobility, the index of depression-like behavior. Buspirone normalized the licking behavior and profoundly reduced the time of immobility, which indicates differences in the mechanisms of antinociceptive and antidepressant effects of buspirone. The present new findings demonstrate that adverse influences of prenatal stress on emotional and inflammatory pain-related behaviors can be prevented by using prenatal buspirone, which shows long-term anxiolytic, antidepressant and antinociceptive effects. The new fact of body weight decrease in buspirone+stress males is worth noting in the context of the important problem of body weight gain as a common side effect of treatment with antidepressant drugs.