L. I. Khozhai

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.

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.

Training in the Morris Water Maze of Female and Male Rats Exposed to Hypoxia at Various Periods of Prenatal Development

Capability for learning was studied in the offspring of rats exposed to hyporbaric hypoxia on the days 11–13, 14–16 or 18–20 of pregnancy. Training in the Morris water maze has been shown to lead to consequences of effect of prenatal hypoxia evident in males, but not in females. The most pronounced changes are found at training in the male rats whose mothers were exposed to hypoxia on the days 14–16 of pregnancy. The revealed differences in the character of learning depend on experimental conditions. Under “severe” stress conditions (at the water temperature of 16–17°C), prenatal hypoxia leads to an improvement of learning parameters as compared with control, while under more favorable conditions (at the water temperature of 23–24°C), to their deterioration.

Reactions of neural elements of neocortex to action of hypoxia at the early neonatal period in rats

We studied reactions of neural elements from different neocortex regions (sensorimotor, visual, auditory) to acute normobaric hypoxia on the model of human incomplete pregnancy (the 2-day-old rat pups) and revealed similar and unidirectional reorganization in all these regions. The chosen parameters of hypoxia induced the earliest detectable changes as fast as in a day since exposure: a decrease in cell body size and cytoplasmic volume, intensification of apoptotic cell death. By the end of the neonatal period (day 5), several ultrastructural changes were observed by indicating deceleration of processes of nerve cell differentiation: arrest of complication of smooth and rough endoplasmic reticulum and Golgi apparatus, the reduced number of free ribosomes and polysomes in cytoplasm as well as of axonal and dendritic growth cones in neuropil, delayed and disordered myelination of nerve fibers. All these morphofunctional abnormalities may be the structural basis for development of neonatal encephalopathies.

Long-Term Consequences of Hypoxia During the Perinatal Period of Development on the Structural-Functional Characteristics of the Brain in Rats

The experiments reported here showed that acute hypoxia during the perinatal period of development leads to structural changes in the motor and visual areas of the neocortex, detectable by postnatal day 20 as impairments to the structural organization of the layers of the neocortex. We report the first data showing that different hippocampal fields respond differently to hypoxia during the postnatal period and present evidence for the existence of long-term sequelae of perinatal hypoxia in the structure of the hippocampus. Acute hypoxia was followed by cell death in all fields and thinning of the pyramidal neuron layers. The greatest levels of cell death occurred in fields CA4 and CA3. As postnatal age increased, cell death remained significant in field CA4, decreased in field CA3, and was not seen in field CA1, though granule neuron death in the dentate fascia increased with increasing age. In addition, there were decreases in the sizes of pyramidal neuron cell bodies in all hippocampal fields. All hippocampal fields showed activation of astrocyte reactions, more marked in field CA4, where gliosis persisted to prepubertal age. Hypoxia in the early postnatal period could have effects on synaptogenesis, particularly on the formation of giant synapses in the dentate fascia. Studies of the functional characteristics of the nervous system in such animals showed that hypoxia could induce significant impairments in behavioral reactions. Rats of the experimental group showed impairments to the inhibitory functions of the cerebral cortex, increased anxiety, and disturbances to spatial learning and working memory.

Effect of hypoxia in early perinatal ontogenesis on behavior and structural characteristics of the rat brain

The study has shown that acute hypoxia in newborn rat pups can lead to disturbances in processes of formation of brain structures, behavior reactions, and learning in the subsequent ontogenesis. The single normobaric hypoxia at the 2nd day of life causes retardation of such integrative parameter or general development and growth as body mass at the period of feeding. In such animals, essential disturbances of the sensorimotor development were revealed in forms of delay of reflex reactions of turning on a plane, negative geotaxis, and avoidance of edge. Also detected was action of hypoxia on hanging on a rope by using front legs (a symptom of muscle weakness). Morphological study has shown all studied functional zones of neocortex (motor, sensomotor, auditory, visual) to response stereotypically to the early postnatal action of hypoxia. The death of nerve cells is predominant in the II–III associative layers, the sizes and number of pyramidal neurons are sharply decreased. Different hippocampus fields maturing after death in mammals show a characteristic response to hypoxia. In individual hippocampus fields there was detected a different degree of death of neurons, with predominance in the CA3 and CA4 fields. A possibility of modeling the perinatal encephalopathy with minimal brain dysfunctions in children is discussed.

Distribution of GABAergic Neurons in the Neocortex in Rats during the Postnatal Period after Perinatal Hypoxia

The distribution of GABAergic neurons in different areas of the neocortex (frontal, sensorimotor, and visual) was studied in Wistar rats at different time points in the postnatal period of development after exposure to perinatal hypoxia. These neurons were detected using antibodies to GAD-67, a marker for GABAergic neurons. Perinatal hypoxia was found to lead to significant decreases in the numbers of GAD-67-expressing neurons in both the upper and the deep layers of the cortex at juvenile age (at day 20 of postnatal development), persisting to the pubertal period (day 40). The number of GAD-67-synthesizing neurons in each of the neocortical layers in experimental animals on day 40 were half those in controls. The sharp decrease in the number of GABAergic neurons in the neocortex is probably due to the harmful action of acute perinatal hypoxia on precursor cell migration from the subventricular zone or on the synthesis of the factors controlling these migration processes and the maturation of GABAergic neurons, and thus decreasing the late expression of GAD-67.

Responses of Rat Brain Interneuronal Synapses to Hypoxia in the Early Neonatal Period

The responses of forming synapses in the rat neocortex to the actions of hypoxia in the early period of neonatal life (day 2) were studied. Immunocytochemical studies were used to detect synaptophysin and these results, along with electron microscopic studies, addressed the sensorimotor cortex in rat pups at 3, 5, and 10 days of postnatal development (using groups of 6–10 individuals) in an experimental group and a control group (intact animals). Immunocytochemical studies of control animal showed significant differences in the quantitative distribution of synaptophysin-positive structures in different layers of the neocortex during the early neonatal period of development (day 5). Perinatal hypoxia decreased the optical density of the immunocytochemical reaction product more than twofold, and this was accompanied by reductions in the density of synaptophysin-positive granules in all layers of the neocortex. In addition, electron-dense terminals, providing evidence of degenerative processes, were seen. The neuropil of the neocortex showed a sharp decrease in the number of growth cones, small processes, and forming synapses, along with a significant increase in the electron density of synaptic elements, especially postsynaptic membranes and densities. In experimental animals, increases in the numbers of growth cones and forming synaptic structures were seen only by postnatal day 10. Thus, the consequences of hypoxia during the early neonatal period, inducing impairments to synaptogenesis, persisted throughout the study period.

Prenatal serotonin depletion changes the behavioral response in the nociceptive formalin test in rats.

In the past years, the attention of researchers is attracted to studying the mechanisms of the development of the central nervous system during prenatal ontogeny, when its major structural and functional characteristics are formed. A substantiated hypothesis assumes that serotonin (5-HT) is involved in these processes. It is known that, at early stages of embryonic development, 5-HT serves as an inducer of cellular and tissue processes and later acquires the properties of a neurotransmitter [1]. Change in the 5-HT content in the prenatal period induces disturbances in neuroembryogenesis leading to abnormalities in the brain development, which may be retained in postnatal ontogeny [1, 2]. The role of 5-HT in the prenatal development of the nociceptive system, in which it acts first as a morphogen and later as a key neurotransmitter, remains to be understood. In this study, we attempted to estimate, using the standard nociceptive formalin test [3, 4], the effect of prenatal 5-HT depletion on the parameters of pain sensitivity of Wistar rats after birth. Formalin test is an adequate model of a prolonged tonic pain, whose modulation in CNS, unlike the modulation of acute phasic [5] pain, is determined by other, unknown, mechanisms and which allows objective quantitative estimation of pain sensitivity. Earlier, in experiments with 25-day-old rats, we described in detail the behavioral response to formalin injection [6] (which already at this age was represented by two phases characteristic of mature rats [7]). This study was performed using young rats of the same age. The 5-HT depletion was performed by injecting an aqueous solution of an inhibitor of the 5-HT synthesis, parachlorphenylalanine (pCPA; ICN, United States) at a dose of 400 mg/kg (2 ml) intraperitoneally to the rat on day 9 of pregnancy. The term of injection was selected taking into account the published data that the maximum effect of pCPA (the greatest decrease in the 5-HT content not only in the mother, but also in the embryo) is expressed with in two to three days, i.e., in the crucial period (day 11‐12) of the formation of the serotonergic system [1, 8]. In the study, we used the fo lowing groups of female and male rats of offspring: (1) intact females; (2) control females that were injected with saline during pregnancy; (3) females that were subjected to 5-HT depletion during pregnancy. At the age of 25 days, the rat was placed into a chamber (25 〈 25 〈 25 cm) with transparent walls, and its twophase behavioral response (TPBR) to the formalin injection (2.5%, 10 μ l) into the plantar pad of the left hind foot was studied. We recorded the number of flexions and shakes patterns (the spinal level) and the duration (in second) of the licking (the supraspinal level) of the injected foot during each minute, until the response was finished (for 60 min). We analyzed the intensity and duration (in minutes) of the first and second phases and the duration of the period of rest (an interphase interval during which specific responses are absent). The formalin injection caused the above specific

Dynamics of Changes in the Expression of the GLUR2 Subunit of the Ionotropic Glutamate Receptor in the Ventrolateral Part of the Solitary Tract Nucleus during the Early Postnatal Period in Health and Prenatal Serotonin Deficiency

Experiments on Wistar rats (n = 18) were performed to study changes in the expression of ionotropic glutamate receptor subunits (GluR2) in the respiratory subnuclei (ventral and lateral) of the solitary tract nucleus (STN). Observations were made during the early postnatal period (days 5, 10, and 20, 5–6 animals per group) in normal conditions and with prenatal decreases in serotonin levels due to inhibition of tryptophan hydroxylase with parachlorophenylalanine. Immunocytochemical detection of GluR2 revealed significant increases in GluR2 expression in the respiratory subnuclei of the STN in the early postnatal period (a 2-fold increase in the lateral subnucleus, 2.6-fold in the ventral). Prenatal serotonin deficiency altered GluR2 expression in the respiratory subnuclei of the STN. There was a significant delay in GluR2 expression in the early period, which increased to control levels by two weeks after birth, though GluR2 expression by juvenile age was lower (twofold) than in controls. Impairments to the glutamatergic receptor networks in the respiratory nuclei may be the basis of respiratory dysfunction.