Anne Milasincic Andrews, Ph.D. - US grants

This proposal is concerned with the application of "fast" electrochemical methods for the determination of real-time serotonin reuptake and release in brain tissue derived from serotonin transporter knockout mice. These mice were produced to gain a clearer understanding of the role of the serotonin transporter in normative behavior, and in anxiety and mood disorders. Serotonin transporter knockout mice display elevations in spontaneous anxiety-like behavior and decreases in locomotor activation in response to 3,4- methylenedioxymethamphetamine (MDMA), a popular drug of abuse. It is hypothesized that these alterations in phenotype are directly attributable to long-term decreases in serotonin transporter function, which ultimately result in neuroadaptive changes in the serotonergic system and its postsynaptic targets. Many of the neurochemical and behavioral parameters studied to date show intermediate levels of change in mice bearing one functional copy of the serotonin transporter gene. However, transporter function initially assessed by [3H]serotonin uptake appears unaltered in heterozygote knockout mice. Therefore, we hypothesize that the characterization of serotonin uptake by classical radiochemical methods does not provide the temporal resolution necessary to detect important variations in the kinetics of the uptake process. The research described will: (1) Evaluate the kinetics of serotonin uptake using high-speed chronoamperometry in synaptosomes derived from serotonin transporter knockout mice; and (2) Characterize the dynamics of serotonin reuptake and release in specific brain regions in slice preparations from serotonin transporter knockout mice using fast scan cyclic voltammetry. We aim to answer the question of whether intermediate changes in transporter expression lead to significant modifications in transporter function. This proposal represents the novel application of voltammetric techniques to the characterization of changes in serotonergic neurotransmission in transgenic mouse models.

DESCRIPTION (provided by applicant): An age-dependent decline in serotonergic innervation of the forebrain has been well documented and may contribute to late onset neurodegenerative disorders, increased incidence of depression or loss of cognitive function that occur with age in humans. This proposal will investigate the relationship between long-term elevations in synaptic serotonin and age and their effects on neuronal innervation in mice with a genetic inactivation of the serotonin transporter gene. Our recent studies indicate that decreased serotonin transporter expression results in a reversal or prevention of normal age-related degeneration of serotonergic axons. We hypothesize that increased extracellular serotonin may be functioning as a trophic factor to promote the survival of serotonergic axons in the forebrain. Mice that lack the serotonin transporter display a 5-fold increase in extracellular serotonin compared to wild type mice. In mice with a 50% reduction in serotonin transporter expression, a 5-fold increase in extracellular serotonin has been detected. In fact, 70% of the normal human population expresses approximately 30% less serotonin transporter, so these findings will have direct applicability to aging in humans. We will investigate the effect decreased serotonin transporter expression on the survival and integrity of forebrain serotonergic, catecholaminergic and cholinergic axons by immunocytochemistry. In addition, we will evaluate the age-related effects of reduced serotonin transporter expression on regional serotonin, dopamine, norepinephrine and acetylcholine neurotransmitter levels. These studies have been designed to integrate anatomical and neurochemical data to assess age-related changes in axonal innervation across the lifespan resulting from decreased of the serotonin transporter expression.

[unreadable] DESCRIPTION (provided by applicant): Understanding the modulation of individual BDNF mRNA splice variants by two clinically relevant forms of reduced serotonin reuptake will facilitate our understanding of the role of BDNF in the regulation of mood and anxiety-related behavior. It will also assist us in addressing long-range questions such as why BDNF is subject to complex transcriptional regulation and how this is reflected in different BDNF protein levels in specific subregions or neuronal subtypes in psychiatric and degenerative brain disorders? Ultimately, we anticipate that having the capability to selectively modulate BDNF at the level of transcription in different cell types and brain regions will be a powerful avenue for the future development of novel therapeutics for the treatment of depression, anxiety disorders and Alzheimer's disease. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The serotonin neurotransmitter system is known to regulate emotion, anxiety states and cognition. Moreover, altered serotonin transmission is hypothesized to be involved in the etiology and treatment of mood and anxiety disorders, and neurodegenerative diseases including Alzheimer's disease. This project is designed to take advantage of the high temporal and spatial resolution afforded by carbon fiber microelectrode voltammetry analytical methods to characterize changes in serotonin neurotransmission in three important human and mouse models with direct relevance to psychiatric and degenerative disorders. The proposed research will: (1) Use high-speed chronoamperometry to evaluate differences in the kinetics of serotonin reuptake in human lymphoblast cell cultures derived from individuals with variable serotonin transporter expression driven by two common promoter polymorphisms (5-HTTLPR and rs25531) in combination with an lle425Val coding region substitution mutation found in rare familiar forms of obsessive compulsive disorder;and (2) Employ fast cyclic voltammetry to characterize alterations in the dynamics of serotonin release and reuptake in vivo in serotonin transporter knockout mice and brain-derived neurotrophic factor (BDNF) knockout mice. Our overarching hypothesis states that potentially subtle but biologically important changes in serotonergic neurotransmission occur in mice and humans with altered serotonin transporter expression. Further, we theorize that changes in serotonin transmission underlie age-related degenerative loss of serotonergic innervation in mice with reduced BDNF. We postulate that application of fast electrochemical methods is necessary to detect these changes in brain neurotransmission, which are fundamental to the investigation of basic and disease-related processes. This outcome of these studies will reveal the extent to which voltammetric techniques are able to differentiate altered serotonin neurotransmission in genetically engineered mice and human cells with genetic alterations important for advancing our knowledge of the pathogenesis and treatment of psychiatric and neurodegenerative diseases.

DESCRIPTION (provided by applicant): Treatment of mice or rats with serotonin reuptake inhibiting antidepressants (SRIs) during a specific period shortly after birth results in increased anxiety- and depressive-like behavior in adulthood. Here, we will extend our studies on the use of voltammetry methods to investigate the role of the serotonin transporter (SERT) in anxiety and depression to include this important model of postnatal inhibition of serotonin reuptake. We will use chronoamperometry to determine changes in serotonin uptake rates during periadolescence and adulthood arising from postnatal treatment with an SRI. We have demonstrated previously that the use of high-speed chronoamperometry to measure serotonin uptake in brain synaptosomes is superior to traditional radiochemical methods. We will also utilize quantitative autoradiography to evaluate changes in serotonin transporter binding sites to determine whether alterations in SERT function and expression are correlated. There is recent evidence that hippocampal SERT function and expression increase during early and middle adult periods in normal animals. The design of this study will allow us to investigate whether this developmental trajectory begins prior to adulthood. We will also determine whether it is present in brain regions other than hippocampus that are innervated by the serotonergic system and are important for modulating anxiety and mood. Moreover, we will test the hypothesis that postnatal administration of SRIs disrupts normal periadolescent and adult development of SERT function and expression. The results of this study will allow us to interpret whether alterations in normal SERT development occurring during adolescent and/or adult time frames are key elements contributing to the altered phenotype induced by postnatal SRI exposure. This has ramifications for our understanding of normal development of the serotonergic system and for altered trajectories related to early life exposure to SRIs. PUBLIC HEALTH RELEVANCE: Treatment of mice or rats with serotonin reuptake inhibiting antidepressants for a short period after birth leads to elevated anxiety and depressive behavior in adulthood. We will use a state-of-the-art electrochemical method to determine whether reductions in serotonin reuptake persist long after the cessation of drug treatment. We will explore the idea that early life exposure to antidepressants alters the normal development of serotonin transporter function and expression. The results of this study will increase our understanding of normal brain development and altered developmental trajectories related to early life exposure to antidepressants.

Parent Grant Project Summary Current methods to measure neurochemicals in the extracellular space are limited by poor chemical, spatial, and temporal resolution. Researchers are therefore unable to investigate brain chemistries dynamically, particularly at the level of neural circuits and across broad arrays of signaling molecules. To understand cell signaling at the time scales pertinent to intrinsically encoded information, truly transformative sensors are needed that will provide highly multiplexed readouts of changes in extracellular neurochemical concentrations with sub-second response times. The objective of this proposal is to design, develop, test, and optimize neurochemical sensors that approach these critical attributes. Molecular recognition will occur via DNA sequences (aptamers) linked to field-effect transistor (FET) sensor arrays for electronic transduction of reversible binding events via conductance changes. Microscale FETs will be employed initially, followed by the development and implementation of multiplexed nanowire FETs. Lithographically fabricated FETs on silicon microprobes, and the aptamers they are functionalized with will be validated in vitro, ex vivo, and implanted for performance evaluation in vivo. By carrying out the proposed research, we will integrate and extend the unique and diverse capabilities of the members of our team to make critical advances in neurochemical sensing technologies that will enable unprecedented insight into how information is encoded in cell signaling. The impact will be towards understanding the function of the healthy brain in relation to complex behaviors, and corresponding dysfunction in psychiatric and neurodegenerative disorders to ultimately identify new therapeutic targets for these diseases.