Research reportEffects of in utero ethanol exposure and maternal treatment with a 5-HT1A agonist on S100B-containing glial cells
Introduction
Previous studies from this and other laboratories found that chronic in utero ethanol exposure severely impairs the development of the cell bodies and projections of serotonergic neurons (reviewed in Ref. [12]). In fetal and neonatal rats, ethanol-associated damage included a marked reduction in the concentration of 5-HT [44], [49] and a decrease in 5-HT immunopositive neurons in the brain area containing the raphe nuclei [50], [65] Ethanol-exposed offspring also exhibited an aberrant development of serotonergic projections, resulting in a significant decrease in 5-HT [44], [49] and 5-HT reuptake sites in multiple brain regions innervated by the dorsal and median raphe; 5-HT reuptake sites were lower in the frontal cortex, parietal cortex, lateral hypothalamus, substantia nigra, medial septal area, and striatum [13], [28].
The ethanol-associated deficit of 5-HT occurs during a vulnerable period when fetal 5-HT normally exerts neurotrophic effects, promoting the development of serotonergic neurons. Endogenous 5-HT promotes the development of serotonergic neurons and stimulates the release of S100B, an essential trophic factor, from glia. Both autocrine and paracrine effects of 5-HT are mediated by 5-HT1A receptors through somatodendritic receptors on raphe nuclei [22] and glial 5-HT1A receptors [6], [59], [60]. The autocrine effects include autoamplification of 5-HT synthesis [9], as well as modulation of growth cone elongation and neurite outgrowth [24], [25], [40], and augmented formation of collateral connections between serotonergic neurons [33], [40], [57]. The activation of 5-HT1A receptors by 5-HT causes the release of S100B [58] from glia.
S100B is a trophic factor that is essential for the normal development of serotonergic neurons [4], [31], [35]. Once secreted, S100B acts on serotonergic neurons to promote differentiation and neurite outgrowth [4], [46]. A marked reduction or absence of S100B production, as found in astroglia from S100B-deficient mice, impairs the development of serotonergic neurons [51]. The importance of S100B to the development of 5-HT neurons and the involvement of 5-HT1A receptors in maintaining normal S100B levels has been clearly demonstrated in several studies which manipulated 5-HT levels. When fetal 5-HT levels were reduced either by parachloramphetamine (PCA) treatment [5], [23] or by prenatal cocaine exposure [1], [2], S100 was markedly reduced in young postnatal rats. The reductions in both 5-HT and S100B are prevented by stimulating 5-HT1A receptors with a 5-HT1A agonist [1], [2] or by using a 5-HT reuptake inhibitor [23] to raise the concentration of 5-HT within the synaptic cleft.
During the development of the 5-HT system, S100B is found in proximity with developing raphe nuclei and throughout the brain stem in association with the MRGS. The MRGS is a large, transient structure in the brainstem that contains substantial numbers of S100B-positive cells and which spans multiple serotonergic nuclei, including the dorsal raphe, median raphe, and B9 complex. During the vulnerable period in the development of serotonergic neurons, S100B immunoreactivity in the MRGS can be detected as early as G15 and disappears by PN5 to PN8 [54]. In addition to potentially providing trophic support for developing 5-HT neurons, this structure is believed to guide neuronal migration and serve a function similar to that of radial glia [54].
It is likely that interactions between serotonergic neurons and S100B immunopositive cells are not limited to the MRGS and the area proximal to the dorsal raphe. There is also a high concentration of S100B in the ependyma of the third and fourth ventricles and the aqueduct [45], [54]. S100B immunopositive ependymal cells may influence the development of the serotonergic neurons which form the supra- and subenpendymal plexuses [10], [38] and which innervate ventricles [11], [37], because guidance of axonal growth cones is facilitated by proteins secreted by the ependymal cells [45]. The serotonergic neurons exert a trophic effect on ependymal glial cells [55].
Although the primary site at which ethanol exerts its deleterious effects on the development of the serotonergic system remains unknown, we hypothesized that the fetal deficit in 5-HT potentiates the adverse effects of ethanol on serotonergic neurons by reducing the neurotrophic effects of 5-HT on both neuronal and glial 5-HT1A receptors. In support of this hypothesis, prior studies from this laboratory found that maternal treatment with 5-HT1A agonists buspirone and ipsapirone prevented the ethanol-associated reductions in 5-HT and 5-HT reuptake sites in rats [30], [49]. This treatment also prevented the decreased density of 5-HT immunopositive neurons in the dorsal raphe, median raphe, and B9 complex [50].
In the present study, we investigated whether in utero ethanol exposure impaired production of the essential trophic factor S100B at two ages during the vulnerable period in the development of 5-HT neurons. Specifically, we evaluated the effects of in utero ethanol exposure on the number and density of S100B immunopositive glial cells and associated mRNA levels in rats. Using the optical fractionator method [56], we counted the total number of S100B cells in the MRGS at G20 in a section of brainstem that accommodates the dorsal and median raphe, and quantified the density of S100B immunopositive cells in an area proximal to the dorsal raphe. Both sites of analysis were chosen because of the close proximity of the S100B-producing glial cells to the developing 5-HT neurons. We also determined whether ethanol-associated abnormalities in S100B could be prevented by maternal treatment with 5-HT1A agonists. The effects of both buspirone and ipsapirone were evaluated for comparison with our earlier studies and to determine the specificity of effects.
Section snippets
Animals, diet and drug treatment
The details regarding this laboratory’s animal model of in utero ethanol exposure and maternal treatment with the 5-HT1A agonists ipsapirone and buspirone have been described in detail elsewhere [28], [30], [49], [50]. Briefly, nulliparous female Sprague–Dawley rats were pair-fed using a control or 6.6% ethanol-containing liquid diet for 6 weeks before breeding and during gestation [50]. G=0 is equivalent to the day of conception and P=1 is equivalent to the day of birth. Each day between G13
Results
We used in situ hybridization experiments to visualize cells that express the gene encoding S100B in brain regions of G20 and PN2 offspring. In agreement with prior experiments [35], sagittal sections of PN2 rats exhibited intense labeling of S100B mRNA in the ependymal cells of the third and fourth ventricles; significant message was also found in the lateral ventricle and motor trigeminal nucleus (Fig. 1). The intense expression of the ependymal cells surrounding the aqueduct is emphasized in
Discussion
This investigation clearly demonstrates that in utero ethanol exposure causes a marked reduction in the number of S100B immunopositive glial cells in brain areas containing the cell bodies of serotonergic neurons. It is likely that the deficiency of S100B immunopositive glia and associated deficiency of S100B contribute to the impaired development of serotonergic neurons [13], [14], [28], [50]. S100B is a trophic factor that is essential for the normal development of serotonergic neurons [4],
Acknowledgements
We would like to acknowledge Jamie Firkus and Fred Wang for their assistance in the care and feeding of the animals. We would also like to acknowledge NIAAA/NIH for their support of this research: USPHS AA03490. Jason Eriksen is the recipient of a NRSA predoctoral fellowship: USHPS AA05501.
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