Patricia M. Whitaker-azmitia - US grants

Serotonin (5-Hydroxytryptamine, 5-HT) can , in many ways, be considered one of the most pervasive and potent regulatory neurotransmitter systems in the mammalian brain. The diversity and selectivity of the receptors which respond to serotonin are evidence of the fine controlling role which this neurotransmitter plays in normal brain function. In our work, we have localized a high affinity serotonin receptor (5-HT1) to astroglial cells. These cells surround neurons and play a vital role in their maintenance as well as in modulating their transmitting activity. We now wish to extend our original studies into several important new areas, including: 1) identification of the receptor subtype, i.e., 5-HT1A, 5-HT1B or 5-HT1C receptor 2) identification of the second messenger linkage, and 3) role of these receptors in development of serotonergic neurons. As a source of astroglial cells, we use primary cultures derived from newborn rat brain. Astrocytes derived from brainstem and cortex will be examined. First, we will establish the sub-type of serotonin receptor localized to astrocytes by using the specific receptor ligands 3H-PAT, 125I-iodo-LSD and 125I- iodocyanopindolol. Secondly, we will establish second messenger linkages by examining the production (or inhibition) of cAMP and the hydrolysis of phospotidylinostitol. Finally, in assessing the role in development, we will further characterize an inhibitory growth factor, which is produced by our cultures in response to stimulation of their membrane serotonin receptors. We will also begin studies of co-cultures of astrocytes and serotonin neurons, to further elucidate this role.

It is now clearly established that the neurotransmitter serotonin acts as a developmental signal in the maturing brain prior to assuming its role in neurotransmission. Our work suggests that this action is mediated through the presence of two high affinity serotonin receptors in the fetal brain. We have hypothesized that these receptors are the 5-HT1a and 5-HT1b receptors which have been characterized in the adult brain. Our work in now concerned with more clearly determining the role each receptor plays and the biochemical, morphological and behavioral consequences of that role. Our study will begin by using radioligand binding assays to characterize the receptors which occur in various regions of the developing rat brain. We will use the radioligands 3H-PAT (for 5- HT1a) and 125I-CYP (for 5-HT1b) in saturation analysis studies in tissues taken from 14 day embryos up to 30 days post-natal. On the basis of these studies, we will test the abilities of selective agonists (PAT for 5-HT1a and TFMPP for 5-HT1b) to alter neuronal development in a tissue culture model of serotenergic neurons. This model will be assessed biochemically (by specific high affinity uptake of 3H-serotonin) and morphologically (by immunocytochemistry, using an antibody raised against serotonin, and morphometric analysis). Our work will then return to the whole animal, where we will study the distribution and pharmacokinetics of the selective agonists in rat fetuses, after maternal administration. This information will enable us to accurately deliver to the fetus the concentrations of agonist which have been shown in tissue culture to alter neuronal development. The offspring will be assessed biochemically, morphologically and behaviorally for developmental abnormalities. As a result of our work, we will gain insights into human developmental disorders such as Down's syndromes and autism, where serotonergic abnormalities are known to occur.

DESCRIPTION (adapted from applicant's abstract): A number of different genes can be localized to the region of the chromosome 21 which is considered to be obligate for Down's Syndrome (DS) and one or more could be responsible for various aspects of the syndrome. Using animal models, it may be possible to determine the role of different genes in the phenotype. In particular, the studies included in this proposal will look at the contribution which S-100beta overexpression could make in the phenotype using an S-100beta transgenic mouse. The findings will be compared with the results in another model of DS, the TS65 Dn mouse, a segmental trisomic mouse for the distal end of chromosome 16, which contains most of the genes found on human chromosomes 21, and specifically overexpresses APP and SOD. However the, the TS65Dn mouse does not include S-100beta, which is found on chromosome 10. Since S-100beta acts as a serotoninergic growth factor and stabilizer of the neuronal cytoskeleton, and these are disrupted in DS, these are the components on the phenotype which will be examined. The investigators propose that although many genes may contribute to the phenotype of Downs syndrome, the morphologic and serotonergic changes, may be due to the over expression of S-100beta. They further propose that these changes may have behavioral consequences. This hypothesis will be tested in the S-100 beta and Ts65Dn mice in the following Specific Aims: 1.Study the morphologic development and degeneration of neurons in the hippocampus. 2 Study the development and degeneration of the serotonergic system. 3 Study any behavioral consequences in the animals, by using age appropriate behaviors. The results of these studies may lead to a clearer understanding of the role of S-100beta in DS.

[unreadable] DESCRIPTION (provided by applicant): Organophosphate (OP) toxicity has acute and chronic components. Although the acute toxicity is through the known mechanism of action of OP's, the inhibition of cholinesterases, the chronic apparently is not. Recent research has suggested that the chronic toxicity is through the production of free radicals, and that this may be particularly damaging during fetal brain development. One way in which the brain responds to an increase in free radicals is through an astrogliotic reaction, which includes release of S100B. S100B is a trophic factor promoting both serotonin terminal outgrowth and synaptogenesis. Thus, in fetal OP toxicity, there will be an increase in serotonin terminals and synaptic density at a young age, but as the animal ages, both will be prematurely lost. In tests of learning and memory, therefore, the young animal may show no deficits, but the older animals, with loss of synapses, will show deficits. The proposal uses a model of OP toxicity previously found to increase serotonin and synaptic markers at a young age- injection of 1 mg/kg of chlorpyrifos (CPF) to rats on gestational days 17-20. The hypothesis will be tested as follows: Specific Aim 1: Using immunochemistry, gestational day (GD) 20 rat pups or mice will be examined for S100B content. Heat shock protein (HSP) -27 and clusterin will be used as markers of oxidative stress and microglial activation as a marker of neuroinflammation. Findings will be compared with litters given a Vitamin E-enriched diet, as an antioxidant Specific Aim 2: Using immunocytochemistry, the hippocampus will be examined for serotonin terminal density, S100B content and changes in astroglial and microglial morphology. Animals will be tested at three ages: postnatal days (PND), PND 35 (adolescence), PND 60 (adult) and PND 365 (aged). Specific Aim 3: Synaptic density will be determined at the same ages, first using immunocytochemistry of microtubule-associated protein (MAP)-2 and synaptophysin. Any changes observed will be confirmed with electronmicroscopy. Specific Aim 4: Animals will be tested for changes in learning and memory at PND 35, 60 and 365. [unreadable] [unreadable] [unreadable]