Down-regulation of 3H-imipramine binding sites in rat cerebral cortex after prenatal exposure to antidepressants

Several antidepressant drugs were given to pregnant rats in the last 15 days of gestation and 3H-imipramine binding (3H-IMI) was subsequently measured in the cerebral cortex of the offspring. The selective serotonin (5-HT) uptake blockers chlorimipramine and fluoxetine as well as the selective monoamine oxidase (MAO) inhibitors clorgyline and deprenyl induced, after prenatal exposure, a down-regulation of 3H-IMI binding sites at postnatal day 25. The density of these binding sites was still reduced at postnatal day 90 in rats exposed in utero to the MAO inhibitors. The antidepressants desipramine and nomifensine were ineffective in this respect. After chronic treatment of adult animals, only chlorimipramine was able to down-regulate the 3H-IMI binding sites. Consequently, prenatal exposure of rats to different antidepressant drugs affecting predominantly the 5-HT systems induces more marked and long-lasting effects on cortical 3H-IMI binding sites. The results suggest that the developing brain is more susceptible to the actions of antidepressants.     

Activity of Acetylcholinesterase in the Motor-Sensory Cortex in Early Ontogenesis of Rats Exposed to Prenatal Hypoxia

Effects of acute prenatal hypoxia (13–14 days of gestation, 3 h, O₂ = 7%) on acetylcholinesterase (AChE, EC 3.1.1.7) activity in homogenates, synaptosomes, and cytosol of the motor-sensory cortex of Wistar rats were studied on the days 1, 5, 10, 19 and 30 after birth. In homogenates of normally developing cortex, the AChE activity did not significantly change with age. Activity of AChE in synaptosomes increased 4 times throughout the entire period of observation, while in the cytosol, 4.3 times to reach maximum at the 19th day. Maximum rise of the AChE activity in synaptosomes was observed at the period from the 5th to the 10th day. Activity of AChE in homogenate and synaptosomes of rats submitted to prenatal hypoxia decreased during the first five days after birth (p < 0.001) but later, starting from the day 10, it increased in all fractions. A statistically significantly higher activity of AChE than in controls was revealed in homogenate of the motor-sensory cortex on day 19 (p < 0.01), while in synaptosomes, on the days 19 and 26 (p < 0.001 and p < 0.05, respectively), and in cytosol, on the days 10, 26, and 30 (p < 0.05, p < 0.05, and p < 0.001). Maximum change in the ratio of AChE activities in cytosol and synaptosomes was found on the day 19 (p < 0.01). At the same period of development, changes in the ratio of AChE activity in synaptosomes and homogenate of the control and hypoxic animals were also observed. Thus, prenatal hypoxia leads to in changes in the activity both of the cytosol and synaptosomal membrane-bound forms of AChE in the motor-sensory cortex of rats, which agrees with our own and literature data on disorder of neuro- and neuritogenesis in the process of formation of CNS and of behavioral reactions in early postnatal ontogenesis under the effect of pathogenic factors at certain days of prenatal ontogenesis.     

The neonatal glucocorticoid treatment-produced long-term changes of the pituitary-adrenal function and brain corticosteroid receptors in rats.

Two distinct periods of sensitivity to elevated glucocorticoid hormone levels during postnatal development of the pituitary-adrenal axis were studied. Wistar rats were injected subcutaneously (s.c.) with cortisol (1 mg/kg) on postnatal days 1-5 or 14-18. The steroid treatment during the first postnatal week resulted in a decrease of the morning basal and stress-induced plasma corticosterone levels in 30 day-old male rats, as well as in rats that were injected with cortisol on the third postnatal week. Stress-induced corticosterone levels in 90-day old cortisol-treated rats were determined in blood samples drawn from the tail vein before the restraint stress, immediately after the 20-min long stress, then 60 and 180 min afterwards. Only the rats treated with cortisol during the third week showed a prolonged stress-induced corticosterone secretion, with the highest corticosterone level in 180 min after the restraint stress. The early neonatal cortisol treatment had no effect on (3)H-corticosterone binding in all studied brain areas of the 90-day old rats. The rats treated with cortisol at the 14-17th postnatal days showed a significantly lower (3)H-corticosterone binding in the frontal cortex, hippocampus, and hypothalamus. These findings suggest that the third week of life in rats is more sensitive to elevated levels of corticosterone than the first one. The high level of glucocorticoids at this period has long-term effects on the efficiency of the negative feedback mechanisms provided by hypothalamus-pituitary-adrenal axis.     

Low concentrations of hydrogen sulphide alter monoamine levels in the developing rat central nervous system.

The central nervous system is one of the primary target organs for hydrogen sulphide (H2S) toxicity; however, there are limited data on the neurotoxic effects of low-dose chronic exposure on the developing nervous system. Levels of serotonin and norepinephrine in the developing rat cerebellum and frontal cortex were determined following chronic exposure to 20 and 75 ppm H2S during perinatal development. Both monoamines were altered in rats exposed to 75 ppm H2S compared with controls; serotonin levels were significantly increased at days 14 and 21 postnatal in both brain regions, and norepinephrine levels were significantly increased at days 7, 14, and 21 postnatal in cerebellum and at day 21 in the frontal cortex. Exposure to 20 ppm H2S significantly increased the levels of serotonin in the frontal cortex at day 21, whereas levels of norepinephrine were significantly reduced in the frontal cortex at days 14 and 21, and at day 14 in the cerebellum.     

Development of GABAA receptors in the central visual structures of rat brain. Effect of visual pattern deprivation

The postnatal development of high-affinity 3H-muscimol binding to GABAA receptors was studied in the lateral geniculate nucleus, superior colliculus, frontal and visual cortex of the rat brain. In the lateral geniculate nucleus 3H-muscimol binding rises from day 10 through day 37 reaching the highest value during the entire development followed by a slight decrease until adulthood. In the superior colliculus 3H-muscimol binding increased continuously from day 10 through day 37, and then decreased until day 50 reaching the adult value. In the visual and frontal cortex, binding reached the highest levels on days 14 and 25, respectively, persisted until day 37 followed by a slight decrease until adulthood. The ontogeny of 3H-muscimol binding sites in the visual regions does not essentially differ from that in other brain regions, suggesting that the appearance of 3H-muscimol binding sites in the visual system is not correlated with the functional maturation of the visual system. Unilateral eyelid closure from day 11 until day 25 did not affect the development of GABAA receptors in any of the central visual regions examined, indicating the lack of environmentally controlled mechanism.     

Pre- and Postsynaptic Localization of 3H-Imipramine Binding Sites in Rat Cerebral Cortex

Abstract

The subcellular localization of ³H-imipramine binding sites in brain was investigated with the aim of learning about the possible mechanism of action of this antidepressant. The rat cerebral cortex was submitted to a systematic fractionation and both the nuclear and the synaptosomal fractions were purified by gradient centrifugation. Using a centrifugation assay for the binding, we found that the synaptosomal membranes had the highest specific activity and showed two binding sites, one of high affinity with a K_D of 14 nM and a Bmax of 3.1 pmol per mg protein, and another of lower affinity with a K_D of 99 nM and a Bmax of 14.2 pmol per mg protein. Purified nuclei have a lower specific activity than the synaptosomal membrane, specially when expressed per g tissue. On the other hand, myelin and capillaries have few binding sites. Synaptosomal membranes were treated with 0.1, 0.2 and 0.5% Triton X-100 to dissolve the pre- and post-synaptic membrane and submitted to ³H-imipramine binding in the presence of the detergent or after washing of the residue. The results obtained suggest that although most ³H-imipramine binding sites are localized pre-synaptically, a certain proportion are post-synaptic. These findings are discussed in relation to previous studies from this laboratory on the localization of central receptors with reference to the synaptic region and to the antidepressant action of imipramine.     

Imipramine treatment differentially affects platelet 3H-imipramine binding and serotonin uptake in depressed patients

Uptake of serotonin and 3H-imipramine binding in platelets of depressed patients were investigated simultaneously with changes in clinical state. Both Vmax for serotonin uptake and Bmax for 3H-imipramine binding were significantly lower in unmedicated depressed patients with respect to normal subjects. Successful treatment with imipramine led to a significant increase in Bmax for 3H-imipramine binding, without significant change in Vmax for serotonin uptake. Bmax values increased to the normal range following complete, rather than partial clinical improvement. These data indicate that successful antidepressant treatment may increase the density of 3H-imipramine binding sites on platelets by a process which is independent of the uptake of serotonin.     

Ontogeny of the hypothalamo-neurohypophysial system in rats--an immunohistochemical study.

In fetal rats neurophysin has been visualized immunohistochemically first at the 16th gestation day in perikaryons of the supraoptic nucleus, followed by the median eminence and the neurohypophysis at the 17th day, and the paraventricular neurons at the 19th day. The external zone of the median eminence contains abundantly immunoreactive fibres at the first days post partum. In the perikaryons of the suprachiasmatic nucleus immunoreactive material appears after the 3rd day of postnatal development.     

State of the Na+, K+-ATPase system of the female rat brain cerebral cortex in the postnatal period during ethanol consumption in pregnancy and lactation

The ontogenetic development of the rat brain cortex Na+, K(+)-ATPase and Mg(2+)-ATPase activities under female ethanol (20% v/v) consumption in the third trimester of gestation or in postpartum period was studied. The weight characteristics (body, whole brain and cortex weight) of viable rats on the first day after birth were not affected critically by prenatal alcohol exposure. It is revealed that the delay of postnatal rat growth 10 days after birth under translactational ethanol consumption is accompanied by reliable decrease of plasma membrane Na+, K(+)-ATPase activity in comparison with control animals. The comparable decrease in activities was observed for the ouabain-sensitive and ouabain-resistant Na+, K(+)-ATPase components (isoform species). From the 20th day the differences in enzyme activity were not revealed. Mg(2+)-ATPase increases in postnatal period independent of Na+, K(+)-ATPase activity and it remains insensitive to postnatal maternal alcohol intake. It is suggested, the first ten day period of lactation is critical for ethanol effect on the developmental control of the brain Na+, K(+)-ATPase functional expression and the course of adaptive processes in the rat organism.     

Disturbances in Formation of the New and Old Cortex at Changes of Conditions of Embryonic Development

Using a model of acute hypoxia during pregnancy of rats, changes in the development of old (hippocampus) and new (sensorimotor) cortex associated with disturbance of neuronogenesis have been revealed in the studied brain structures at the period of action of a pathological factor. It was found that in rats submitted to hypoxia at the 13–14th days of embryogenesis, the number of degenerating neurons (including the pyramidal ones) at various levels of chromatolysis increased since the 5th day after birth; the increase was present for the entire first month of postnatal development. In the cortex of rat pups submitted to prenatal hypoxia there were observed deformation of neuronal bodies, vacuoles in the cytoplasm, shrinkage of apical dendrites of pyramidal neurons and delayed development of the structure (time of the appearance of spikes, formation of structural elements and the size of the cells) of the nervous tissue of the brain of the rat pups exposed to prenatal hypoxia. The columnar structure of the cortex was disturbed. In hippocampus, the process of degeneration of neurons started by 2–3 days later than in the cortex; by two weeks of postnatal development a massive degeneration and death of a part of neurons were also revealed. The morphometrical analysis showed a decrease in the number of neurons and their total area in the sensorimotor cortex (the layer V) and an increase in the number of glial elements at the 10–17th days after birth. In the hippocampus a decrease in the area occupied by neurons and in their size was detected in adult animals. The adult rats submitted to prenatal hypoxia were found to have disturbances of memory and learning. A correlation was shown between the disturbances of the conditions of embryonic development and the changes in the ability of learning and storage of new skills in the offspring.     

Expression of thrombospondin-1 and CD36 and CD47 receptors in the rat brain after exposure to damaging factors in the early postnatal period

The expression of thrombospondin-1 (TS-1) and its receptors CD47 and CD36 in the cerebral cortex and hippocampus of rats under damaging factors in the early postnatal period was studied. After hypoxia on the 7th day of postnatal development, an increase in the number of CD47-expressing cerebral endothelial cells (days of postnatal development: P28–P70) and reduction in the number of TS-1-expressing astrocytes in the cortex at P28 were observed. In animals subjected to early postnatal stress at the age of P2–P15, a decrease in TS-1-expressing astrocytes in the cortex and hippocampus was registered (predominantly at the age of P28). It was noted that these changes characterize the period of long-term effects (P28–P70) of early stress that is relevant to the processes of reparative angiogenesis and arresting of neurological deficits.     

Isolation of substances inhibiting the specific binding of imipramine and serotonin uptake from the brain of the bull

Several fractions that inhibit specific binding of 3H-imipramine and reverse uptake of 3H-serotonin in rat brain synaptosomes were obtained by gel chromatography on Sephadex G-10 from an acidic extract of brain homogenates. The profiles of inhibition of 3H-imipramine specific binding and reverse uptake of 3H-serotonin were found to be in a good agreement. This finding suggests that substances identified in the brain extract may have both types of activities. Further investigations relative to the purification and identification of the substances isolated from bovine brain should be aimed at the search for endogenous ligands for the "imipramine receptor".     

Effect of deafferentation of the forelimb on the postnatal development of the synaptic plasticity in the hippocampus

The postnatal development of LTP in CA1 area of hippocampus was studied in hippocampal slices from 13-20-day-old intact rats, after unilateral resection of n. medianus on the 13th day, and sham-operated animals. In slices from the intact rats prepared on the 15th-16th-day of postnatal development, the LTP magnitude and duration were significantly larger than in adult animals. Partial deafferentation eliminated this overshoot. However, a less pronounced increase in synaptic plasticity was observed in operated animals on the 17th day. The LTP suppression in the experimental animals may be explained by a decrease in the NMDA receptor activity due to enhanced synaptic activity in the hippocampus. We think that the limited sensory inflow from the partially deafferented forelimb to the hippocampus via the entorhinal cortex may be compensated by activation of other inputs from specific or/and nonspecific pathways. In contrast, the LTP magnitude and duration were significantly increased in slices from the sham-operated rats. This increase may be explained by a decline of synaptic activation of the hippocampus under anesthesia.     

The acute and the chronic effects of imipramine on the spontaneous motility in chick embryos

The effect of imipramine on the spontaneous motility and development of chick embryos was studied from the 4th to the 19th day of incubation. On acute administration (a single dose of 12.5 of 25 mg/kg egg weight), imipramine already induced significant depression of spontaneous motility in 11-day embryos--an effect which increased significantly after the 15th day of incubation. The similar effect of imipramine in spinal embryos testifies to its direct action on the spinal cord and draws attention to certain details of the role of supraspinal structures of the CNS in the acute effect of imipramine. The chronic administration of imipramine showed that it had an almost 100% lethal effect from 4th to the 7th day of incubation. Between the 8th and the 10th day it caused longlasting depression of spontaneous motility. When it was administered between the 11th and 16th day of incubation, no significant effect on the development of spontaneous motor activity was found in chick embryos.     

Prenatal Diazepam Exposure Affects β-Adrenergic Receptors in Brain Regions of Adult Rat Offspring

The postnatal development of [3H]dihydroalprenolol binding to beta-adrenergic receptors has been studied in frontal cortex, cerebellum, striatum, and hypothalamus of the rat after prenatal and perinatal exposure to diazepam. Dams were injected subcutaneously with single daily doses of 1 mg of diazepam/kg from day 7 to 20 of gestation or from day 15 of gestation to day 6 after birth. Prenatal exposure had no effect on litter size or length of gestation or on the postnatal development of body and brain weights of the progeny. However, a reduced mortality of the pups was observed in relation to vehicle-treated controls until postnatal day 10. Prenatal diazepam administration decreased [3H]dihydroalprenolol binding in frontal cortex, striatum, and hypothalamus but not in cerebellum. This decrease in beta-adrenergic receptor binding was due to a decrease in receptor density rather than in receptor affinity. In contrast, perinatal diazepam exposure led to a transient decrease in [3H]dihydroalprenolol binding limited to the frontal cortex. The permanent reduction in number of beta-adrenergic receptors, which depends on the scaling and duration of the drug application period, points to the necessity of a prolonged evaluation of effects of exposure to psychotropic drugs in early stages of brain development.     

Effect of gangliosides on adenylyl cyclase activity in the cortex and striatum of rats

By studying the effects of gangliosides (G) on learning and memory we have found that i.p. administration of G led to a decrease in AC activity in the cortex (Cx) and striatum (Str) as well as in the threshold of the sensitivity of striatal neurons to the effect of cholinergic agonists. G also modified the sensitivity of AC from the Cx and Str to such agents as Gpp[NH]p and forskolin. The aim of this work was to analyze the correlation between the changes in the activity of AC in the Cx and Str, concentration of G in these brain structures as well as formation of motor reactions of newborn rats during the first month of postnatal ontogenesis. It was found that new born rats develop normal body rotation by sixth day, locomotion by fifteenth day and stabilization of locomotor activity and supporting body balance by 20–22nd day. As shown previously, the concentration of gangliosides in the Cx and Str is gradually increasing during the first month of animal development. The activity of AC (pmol cAMP/min/mg of protein) in the Cx was found to decrease from 34.75 to 4.09 and in the Str from 46.00 to 11.67 during the first week after birth. However in the periods of formation of general behavioural reactions we observed a statistically significant increase in AC activity: in the Str on 10th and 26th days (p < 0.01) and in the Cx from 10th to 19th days (p < 0.05) compared to the AC activity on 5th and 30th days. Thus, formation of locomotor activity and posture‐tonic reactions during development of rats in early postnatal ontogenesis correlates with increasing concentration of G and basal activity of AC. Supported by RFBR (99‐04‐49751) and RAS (99‐06‐287).     

Differential ontogeny of type 1 and type 2 benzodiazepine receptors

The postnatal development of Type 1 and Type 2 benzodiazepine receptors in rat cerebral cortex was studied using CL 218,872, a novel triazolopyridazine. On postnatal day 1 most ³H-flunitrazepam binding sites appeared to be Type 2 receptors, which increased rapidly during the first week of life and reached adult levels by 3–4 weeks of age. Type 1 receptors, on the other hand, represented only a small percentage of the binding sites on postnatal day 1 and did not begin to increase in number until approximately 7–16 days of age. These results demonstrate a differential postnatal development of two sub-populations of benzodiazepine receptors.     

Steroid 5α-Reductase in Adult Rat Brain After Neonatal Dihydrotestosterone Administration

Testosterone (T) is known to play an important masculinizing role in the developing brain of rat, including the regulation of 5α-reductase (5α-R) isozymes. However, the effects of dihydrotesterone (DHT), a more potent androgen than T, have not been elucidated. In this study, DHT was administered from day 5 through day 20 of postnatal life (period of postnatal sexual differentiation of the central nervous system) at doses of: 12 mg/kg/d on days 5, 6, 7, 8, 19, and 20; 15 mg/kg/d on days 9, 10, 11, 12, 16, 17, and 18; and 18 mg/kg/d on days 13, 14, and 15. In adulthood, quantitative RT-PCR was used to measure mRNA levels of 5α-R1 and 5α-R2 isozymes in the prefrontal cortex (PFC) of male and female rats with varied androgenic status. Under our study conditions, neonatal DHT administration influenced on adult PFC 5α-R isozymes levels and their regulation pattern by androgens, and this pattern was the inverse of that reported in adult neonatally T-treated rats.     

Pre- and Postsynaptic Localization of 3H-Imipramine Binding Sites: Action of 5–7 Dihydroxytryptamine and Triton X-100 on Subcellular Fractions of Rat Brain

Abstract

In rats injected intraventricularly with 5–7 dihydroxytryptamine there was a considerable reduction of 5-hydroxytryptamine and 5-hydroxyindol acetic acid content in cerebral cortex and hippocampus. After cell fractionation of these structures, a 37% reduction of ³H-imipramine binding was observed in the crude mitochondrial fraction of the treated rats, that contains the synaptosomes. In purified synaptosomal membranes the reduction was about 20%. Dissolution of the presynaptic membrane with 0.1 and 0.2% Triton X-100 on the treated membranes further reduced ³H-imipramine binding respectively by 25% and 40%, values similar to those obtained on control synaptosomal membranes. These findings were further substantiated using saturation experiments for the high affinity site of ³H-imipramine. The results obtained are discussed in relation to the possible mechanism of action of antidepressant drugs at the synaptic region, and the possible postsynaptic effect is emphasized.