Serotonin: a regulator of neuronal morphology and circuitry.

Serotonin is an important neuromodulator associated with a wide range of physiological effects in the central nervous system. The exact mechanisms whereby serotonin influences brain development are not well understood, although studies in invertebrate and vertebrate model organisms are beginning to unravel a regulatory role for serotonin in neuronal morphology and circuit formation. Recent data suggest a developmental window during which altered serotonin levels permanently influence neuronal circuitry, however, the temporal constraints and molecular mechanisms responsible are still under investigation. Growing evidence suggests that alterations in early serotonin signaling contribute to a number of neurodevelopmental and neuropsychiatric disorders. Thus, understanding how altered serotonin signaling affects neuronal morphology and plasticity, and ultimately animal physiology and pathophysiology, will be of great significance.

Serotonergic dystrophy induced by excess serotonin.

Administration of certain serotonin-releasing amphetamine derivatives (fenfluramine and/or 3,4-methylenedioxymethamphetamine, MDMA, 'ecstasy') results in dystrophic serotonergic morphology in the mammalian brain. In addition to drug administration, dystrophic serotonergic neurites are also associated with neurodegenerative disorders. We demonstrate here that endogenously elevated serotonin in the Drosophila CNS induces aberrant enlarged varicosities, or spheroids, that are morphologically similar to dystrophic mammalian serotonergic fibers. In Drosophila these spheroids are specific to serotonergic neurons, distinct from typical varicosities, and form only after prolonged increases in cytoplasmic serotonin. Our results also suggest that serotonin levels during early development determine later sensitivity of spheroid formation to manipulations of the serotonin transporter (SERT). Elevated serotonin also interacts with canonical protein aggregation and autophagic pathways to form spheroids. The data presented here support a model in which excess cytoplasmic neurotransmitter triggers a cell-specific pathway inducing aberrant morphology in fly serotonergic neurons that may be shared in certain mammalian pathologies.

Drosophila serotonergic varicosities are not distributed in a regular manner.

Neurons of the brain form complex tree-like structures that are critical for function. Here we examine the spatial pattern of serotonergic varicosities, the synaptic sites of serotonin release in the central nervous system (CNS). These varicosities are thought to form largely nonjunctional-type connections that partition in a grid-like manner in order to distribute evenly the neuromodulatory neurotransmitter serotonin. We describe the neuropil distribution of serotonergic varicosities in the brain and ventral nerve cord (VNC) of the larval Drosophila CNS. In the brain, we find evidence for avoidance between varicosities at distances lower than 1.75 microm. However, in the VNC, we find a clustered distribution. A similar clustered pattern is found in the Xenopus brain. This pattern produces many varicosities that are clustered together but also includes some varicosities that are very isolated. These isolated varicosities are not found along particular topological sections of the neurite tree or in particular locations in the CNS. In addition, the pattern breaks down when serotonergic branches of adjacent segments invade each other's territory. The pattern is similar to those described by a power law.

Branch architecture of the fly larval abdominal serotonergic neurons.

In the metazoan central nervous system (CNS), serotonergic neurons send projections throughout the synaptic neuropil. Little is known about the rules that govern these widespread neuromodulatory branching patterns. In this study, we utilize the Drosophila as a model to examine serotonergic branching. Using single cell GFP labeling we show that within each segment of the Drosophila ventral nerve cord (VNC), each of two serotonergic neurons tiles distinct innervation patterns in the contralateral neuropil. In addition, branches extend only a short distance from the target segment. Through ablation-mediated isolation of serotonergic cells, we demonstrate that the distinct areas of innervation are not maintained through competition between neighboring like-serotonergic neurites. Furthermore, the basic branching pattern of serotonergic neurons within the neuropil remains unchanged despite alterations of initial axonal trajectories.

A freeware java tool for spatial point analysis of neuronal structures.

Spatial point analysis is an analytical approach towards understanding patterns in the distribution of single points, such as synapses. To aid in this type of analysis of neuronal structures, a freeware tool, called PAJ, has been developed. This Java-based tool takes 3D Cartesian coordinates as input and performs a range of analyses to test for underlying patterns. In addition, Monte Carlo analysis is performed to compare experimental input with randomized input. This tool should be especially useful in determining whether neuronal structures are spatially patterned such that individual units interact with each other.

Developmental effects of SSRIs: lessons learned from animal studies.

Selective serotonin reuptake inhibitors (SSRIs) are utilized in the treatment of depression in pregnant and lactating women. SSRIs may be passed to the fetus through the placenta and the neonate through breastfeeding, potentially exposing them to SSRIs during peri- and postnatal development. However, the long-term effects of this SSRI exposure are still largely unknown. The simplicity and genetic amenability of model organisms provides a critical experimental advantage compared to studies with humans. This review will assess the current research done in animals that sheds light on the role of serotonin during development and the possible effects of SSRIs. Experimental studies in rodents show that administration of SSRIs during a key developmental window creates changes in brain circuitry and maladaptive behaviors that persist into adulthood. Similar changes result from the inhibition of the serotonin transporter or monoamine oxidase, implicating these two regulators of serotonin signaling in developmental changes. Understanding the role of serotonin in brain development is critical to identifying the possible effects of SSRI exposure.

A behavioral stress assay in Drosophila larvae.

INTRODUCTIONThis protocol describes a simple behavioral assay designed to test the response of Drosophila larvae to a homeostatic insult: bright light. Treating normally photophobic larvae with bright light before placing them near a target food source reveals a greater latency to reach the target when compared to controls not receiving light treatment. This effect is reversible given a recovery period after light treatment, and it may reveal a method by which to measure behavioral plasticity and stress responses in fruit fly larvae.

A solid-phase immunostaining protocol for high-resolution imaging of delicate structures in the Drosophila larval central nervous system (CNS).

INTRODUCTIONThis protocol describes a method for mounting and immunostaining Drosophila larval tissue in preparation for high-resolution fluorescent imaging of fine structures in the central nervous system (CNS). Affixing the tissue directly to the coverslip and then moving the coverslip between wash solutions provides a simple solid-phase method of immunostaining that assists in preserving fine structures. This method also easily allows for manipulations and/or viewing of the live sample prior to fixation if desired. Finally, putting the tissue in direct contact with the coverslip places fine structures immediately adjacent to the objective lens. We also briefly describe a method to create three-dimensional (3D) models of confocal Z-stacks in order to better characterize fine structures by measuring their volume and obtaining 3D Cartesian coordinates in space.

Development and sensitivity to serotonin of Drosophila serotonergic varicosities in the central nervous system.

Serotonin is a classical small-molecule neurotransmitter with known effects on developmental processes. Previous studies have shown a developmental role for serotonin in the fly peripheral nervous system. In this study, we show that serotonin can modulate the development of serotonergic varicosities within the fly central nervous system. We have developed a system to examine the development of serotonergic varicosities in the larval CNS. We use this method to describe the normal serotonergic development in the A7 abdominal ganglion. From first to third instar larvae, the volume of the neuropil and number of serotonergic varicosities increase substantially while the varicosity density remains relatively constant. We hypothesize that serotonin is an autoregulator for serotonergic varicosity density. We tested the sensitivity of serotonergic varicosities to serotonin by adding neurotransmitter at various stages to isolated larval ventral nerve cords. Addition of excess exogenous serotonin decreases native varicosity density in older larvae, and these acute effects are reversible. The effects of serotonin appear to be selective for serotonergic varicosities, as dopaminergic and corazonergic varicosities remain qualitatively intact following serotonin application.

An assay of behavioral plasticity in Drosophila larvae.

Stress, or threats to homeostasis, is a universal part of life. Organisms face changing and challenging situations everyday, and the ability to respond to such stress is essential for survival. When subjected to acute stress, the body responds molecularly and behaviorally in order to recover a steady state. We developed a simple and robust assay of behavioral plasticity for Drosophila larvae in which well-defined behavioral responses and recovery can be observed and quantified. After experiencing different control and bright light treatments, populations of photophobic fly larvae were placed a defined distance from a food source to which they crawled. Half-times (t(1/2)), or times at which half the total number of larvae reached the food, were used to compare different treatments and larval populations. Repeated control treatments with a main experimental strain gave tight, reproducible t(1/2) ranges. Control treatments with the wild type strains Oregon R and Canton S, the "rover" and "sitter" alleles of the forager locus, and eyeless mutants gave comparable results to those of the experimental strain. Exposure to bright light for a defined time period resulted in a reproducible slowing of locomotion. However, given a defined recovery period, the larvae recover full, normal locomotion. In addition, bright light treatments with Canton S gave comparable results to those of the experimental strain. Eyeless mutants, which are partially blind, do not show a response to bright light treatment. Thus, our assay measures the behavioral responses to bright light in Drosophila larvae and therefore might be useful as a general assay for studying behavioral plasticity and, potentially, adaptation to a stressful stimulus.

Variation in serotonergic and dopaminergic neuronal survival in the central nervous system of adult Drosophila.

Loss of serotonergic and dopaminergic neurons may have serious implications for normal brain function. Drosophila models of neurodegenerative diseases utilize the short life-span and simple anatomy of the fly to characterize the molecular and genetic processes characteristic of each dysfunctional state. In fly embryonic and larval ventral nerve cords, serotonergic and dopaminergic neurons are positioned in a stereotypic pattern that is reorganized during metamorphosis. In this study, we examine the adult pattern of serotonergic and dopaminergic neurons within the adult fly ventral nerve cord. We find that the number of cells lost following metamorphosis is highly variable. Changes in cell number attributable to age are therefore likely to be highly masked by developmental variation. The source of this variation is probably apoptosis-based cell loss during pupal development.

robo2 and robo3 interact with eagle to regulate serotonergic neuron differentiation.

The function of the central nervous system (CNS) depends crucially upon the correct differentiation of neurons and formation of axonal connections. Some aspects of neuronal differentiation are known to occur as axonal connections are forming. Although serotonin is a highly conserved neurotransmitter that is important for many CNS functions, little is known about the process of serotonergic neuron differentiation. We show that in Drosophila, expression of the serotonin transporter (SerT) is both temporally and physically related to midline crossing. Additionally, we show that the axon guidance molecules roundabout2 and roundabout3 (robo2/3) are necessary for serotonergic neuron differentiation and function independently of their ligand, slit. Loss of robo2 or robo3 causes a loss of SerT expression in about half of neurons, and resembles the phenotype seen in mutants for the transcription factor eagle (eg). Finally, we show a direct relationship between robo2/3 and eg: robo2/3 mutants lose Eg expression in serotonergic neurons, and robo2 and eg interact genetically to regulate SerT expression. We propose that post-midline expression of Robo2/3 is part of a signal that regulates serotonergic neuron differentiation and is transduced by the transcription factor Eg.