Offspring Of Ethanol-fed Rats Research Articles

Effects of ethanol and ipsapirone on the development of midline raphe glial cells and astrocytes

Previously, results of studies from our laboratory have shown that the offspring of ethanol-fed female rats have a significant decrease in serotonin (5-HT) neurons and glia that contain S100B, an essential trophic factor for the development of 5-HT neurons. The deficiency of S100B-immunopositive glia was detected during the vulnerable period in 5-HT neuron development and in brain areas proximal to these neurons. The reductions of both 5-HT neurons and S100B-positive glia were prevented by maternal treatment with a 5-HT 1A agonist (i.e., ipsapirone or buspirone). In the current study, we investigated whether the offspring of ethanol-fed rats had a general decrease in the density of glial cells in the brain areas that contain 5-HT neurons, and we determined whether these changes were prevented by maternal treatment with ipsapirone between gestational days (GDs) 13 and 20. We estimated the density of vimentin-positive glia of the midline raphe glial structure (MRGS) at GD 20 and postnatal day (PND) 5 and of glial fibrillary acidic protein (GFAP)–positive astrocytes proximal to the dorsal and median raphe at PNDs 5 and 19. The results of this study provide evidence that in utero ethanol exposure is associated with a reduced density of GFAP-immunopositive astrocytes proximal to the dorsal and median raphe. Maternal ipsapirone treatment significantly increased astroglial density in the dorsal raphe at PNDs 5 and 19 and in the median raphe at PND 5, such that it either prevented (dorsal raphe, PNDs 5 and 19) or blunted (median raphe, PND 5) the effects of ethanol.

Astrocyte-mediated trophic support of developing serotonin neurons: effects of ethanol, buspirone, and S100B

Previously, this laboratory demonstrated that the development of serotonin (5-HT) neurons and S100B-immunopositive glia proximal to these neurons is impaired in the offspring of ethanol-fed rats. However, maternal treatment with a 5-HT 1A agonist, e.g., buspirone or ipsapirone, between gestational days 13 and 20 prevented most of the ethanol-associated changes to developing 5-HT neurons and S100B-immunopositive glia in offspring. The present in vitro studies examined the hypothesis that the protective effects of a 5-HT 1A agonist on ethanol-exposed, developing 5-HT neurons are mediated in part by astrocyte-produced factors such as S100B. Primary cultures of fetal 5-HT neurons were maintained in conditioned medium (CM) that was obtained from ethanol- and buspirone-treated astrocytes. In order to assess the potential contribution of S100B to the protective effects of buspirone, a mouse monoclonal antibody to S100B was added to the CM to block the biological effects of this protein. These studies demonstrated that CM, obtained from ethanol-treated astrocytes, was unable to support normal development of 5-HT neurons; there was a significant reduction in the number of 5-HT neurons/well. However, CM that was obtained from astrocytes that were co-treated with buspirone and ethanol prevented the ethanol-associated reduction, and the protective effects of buspirone required S100B. We also investigated whether exogenous S100B could protect 5-HT neurons from damage caused by direct exposure to ethanol. Direct exposure of fetal brainstem neurons to ethanol in chemically-defined medium was associated with a significant reduction in the number of 5-HT immunopositive neurons/well. However, exogenous S100B protected 5-HT neurons from the ethanol-associated reduction. Our observations suggest that the protective effects of buspirone on ethanol-exposed, developing 5-HT neurons are mediated in part by the astrocyte-produced factor S100B.

Treatment of pregnant alcohol-consuming rats with buspirone: effects on serotonin and 5-hydroxyindoleacetic acid content in offspring.

This laboratory previously demonstrated that in utero ethanol exposure markedly impairs the development of the serotonergic system in rat brain. Developmental abnormalities could be detected as early as G15 in the brainstem and G19 in the cortex. Because of the importance of fetal serotonin (5-HT) and 5-HT1A receptors for the normal development of 5-HT containing neurons, we initiated studies to determine whether administration of a 5-HT1A agonist, buspirone, to pregnant rats could overcome the adverse effects of in utero ethanol exposure on the developing serotonergic system in offspring. Female, Sprague-Dawley rats were given daily subcutaneous injections of buspirone (4.5 mg/kg) from gestational day 13 (G13) to G20. 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) content were determined in the cortex and cortical regions. These experiments demonstrated that the ethanol-associated abnormalities in the development of the serotonergic system can be partially overcome by in utero exposure to buspirone. Specifically, whereas untreated ethanol rats had a deficiency of 5-HT and/or 5-HIAA in whole cortex on PN5, and in the motor cortex on PN19 and 35, no significant differences were detected in these regions of the age-matched offspring of buspirone-treated, ethanol-fed rats. In contrast, the 5-HT and 5-HIAA deficiency in the somatosensory cortex of 19-day-old offspring of ethanol-fed rats was not corrected by in utero buspirone treatment. These results suggest that the abnormal development of cortical projections of serotonergic neurons may be due in part to the low fetal 5-HT content in ethanol-exposed rats and may potentially be overcome by in utero treatment with a 5-HT1A agonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of in Utero Ethanol Exposure on the Developing Serotonergic System

Previous work in this laboratory demonstrated that the 19- and 35-day-old offspring of ethanol-fed rats have a significant deficiency of cortical serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA), as well as a decrease in the number of total 5-HT1 receptors in the motor and somatosensory cortex. The present studies extend our previous reports by demonstrating that there is also a deficit of 5-HT and 5-HIAA in the motor cortex but not in the somatosensory cortex. In addition, we have shown that a deficit of 5-HT1A receptors in the motor and somatosensory cortices contributes to the deficit of total 5-HT1 receptors. In contrast, we did not observe any changes in the binding to 5-HT1B receptors in these cortical regions from the 19-day-old offspring of ethanol-fed rats. The present studies also examined the effects of in utero ethanol exposure on the early development of the serotonergic system. The results of these studies demonstrated a deficit of 5-HT and/or 5-HIAA in the brain stem as early as the 15th day of gestation (G15) and in the cortex as early as G19. In addition, we demonstrated a delay in both the normal developmental decline of 5-HT1A receptors in the brain stem and in the acquisition of cortical 5-HT1A receptors. No changes were found in the binding of [125I]cyanopindolol to 5-HT1B receptors in either region of fetal or neonatal rats exposed to ethanol in utero.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of in utero ethanol exposure on serotonin uptake in cortical regions

Previously, this laboratory found that the 19- and 35- to 37-day-old offspring of rats that consumed ethanol on a chronic basis prior to parturition had a decreased cortical content of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) as well as a decreased number of cortical 5-HT 1 binding sites. These results emphasized the sensitivity of the developing cortical serotonergic nerves to the effects of in utero ethanol exposure. In the present study, we examined the effects of in utero ethanol exposure on an additional component of the developing cortical serotonergic systems. Specifically, we examined the uptake of [ 3H]-5-HT by synaptosomes which were isolated from the motor or somatosensory regions of the cerebral cortex. The results demonstrated that the V max for serotonin uptake was significantly decreased ( p<0.025) by approximately 15–20% in the motor cortices of the 19- and 35-day-old offspring of rats that consumed ethanol on a chronic basis prior to parturition. In addition, there was a significant ( p<0.025)∼30% decrease in the K m for serotonin uptake in the motor cortex of 35-day-old offspring of ethanol-fed rats. In contrast, neither the (K m) nor the V max for serotonin uptake were significantly altered ( p>0.05) in the somatosensory cortices in 19- or 35-day-old offspring of ethanol-fed rats. These results emphasize the selective sensitivity of developing cortical projections of the serotonergic system.

Chronic maternal ethanol consumption results in decreased serotonergic 5-HT 1 sites in cerebral cortical regions from offspring

This laboratory previously demonstrated that chronic maternal ethanol consumption results in a marked deficiency of cortical serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) and of 5-HT uptake sites in the 19- and 35-day-old offspring. In order to determine whether in utero exposure to ethanol similarly affects other components of the serotonergic system we examined the influence of chronic maternal ethanol consumption on cortical, serotonergic 5-HT 1 binding sites in developing offspring. Female Sprague-Dawley rats were pair-fed, using control or 6.6% (v/v) ethanol liquid diets on a chronic basis prior to parturition. Serotonergic 5-HT 1 sites were measured in synaptosomal membranes from whole cortex and cortical regions from developing offspring. Serotonergic 5-HT 1 sites were assessed by measuring the binding of [ 3H]-5-HT to synaptosomal membranes in the presence and absence of nonradioactive 5-HT. Serotonergic 5-HT 2 sites were blocked by including 100 nM spiperone in the assay buffer. The results demonstrated that the 19- and 37-day-old offspring of ethanol-fed rats had a significant (∼10–40%) reduction in the B max for serotonergic 5-HT 1 binding sites on synaptosomal membranes from whole cortex ( p<0.025), motor cortex ( p<0.01), and somatosensory cortex ( p<0.025). However, the binding affinity (K d) for serotonin was not significantly altered ( p>0.05). These results emphasize the sensitivity of the developing cortical serotonergic system to prenatal ethanol exposure.

Maternal ethanol consumption: Binding of l-glutamate to synaptic membranes from whole brain, cortices, and cerebella of offspring

We examined the influence of chronic maternal ethanol consumption on the Na +- and Ca 2+-independent binding of l-glutamate to synaptic plasma membranes from whole brain as well as from cortices and cerebella of developing offspring. The maximum specific binding ( B max) of l-glutamate to the Na +- and Ca 2+-independent binding sites in synaptic plasma membranes of brain peaked at 17 days of age in the offspring of both control and ethanol-fed rats, although at that age there were significantly fewer binding sites in the brains of the offspring of ethanol-fed rats. The regional localization of this deficit is not now known. However, it appears that one major glutamatergic region (the cortex) does not reflect the transient deficiency of l-glutamate sites in brain. In fact, the concentration of l-glutamate binding sites in cortical synaptic plasma membranes was significantly increased in the 20-day-old offspring of ethanol-fed rats. In contrast to the cortex, binding to cerebellar synaptic plasma membranes was comparable in 20-day-old offspring of control and ethanol-fed rats. Despite transient alterations in the concentrations of l-glutamate binding sites in brain and synaptic plasma membranes, the affinity of the sites for l-glutamate ( K d) was consistently normal in the 14- to 26-day-old offspring of ethanol-fed rats.

Maternal ethanol consumption: Effect on (Na +-K +)-ATPase in rat offspring

The activity of Mg 2+-activated, ouabain-sensitive adenosine triphosphatase, (Na +-K +-ATPase), was determined in homogenates of hypothalamus, cortex, cerebellum, and brain stem from the 19-day-old offspring of rats that were pair-fed control or (6.6%, v/v) ethanol liquid diets on a chronic basis prior to parturition. In the offspring of both control and ethanol-fed rats the specific activity of (Na +-K +)-ATPase was significantly ( p<0.01) greater in the cortex than it was in the hypothalamus, brain stem or cerebellum (hypothalamus ∼ brain stem ∼ cerebellum). When the offspring of ethanol-fed and control rats were compared we observed no significant ( p>0.05) differences in the activity of (Na +-K +)-ATPase in any of the four brain regions examined. In addition, the results of kinetic analyses of cortical (Na +-K +)-ATPase were similar in the 19-day-old offspring of ethanol-fed rats and those whose mothers consumed either the control liquid diet or standard laboratory chow. The results of these studies suggest that the activity of the plasma membrane enzyme, (Na +-K +)-ATPase, was not affected in the 19-day-old offspring of ethanol-fed rats.