Serotonin signaling plays a key role in the regulation of development, mood and behavior. Drosophila is well suited for the study of the basic mechanisms of serotonergic signaling, but the small size of its nervous system has previously precluded the direct measurements of neurotransmitters. This study demonstrates the first real-time measurements of changes in extracellular monoamine concentrations in a single larval Drosophila ventral nerve cord. Channelrhodopsin2-mediated, neuronal type-specific stimulation is used to elicit endogenous serotonin release, which is detected using fast-scan cyclic voltammetry at an implanted microelectrode. Release is decreased when serotonin synthesis or packaging are pharmacologically inhibited, confirming that the detected substance is serotonin. Similar to tetanus-evoked serotonin release in mammals, evoked serotonin concentrations are 280 -640 nM in the fly, depending on the stimulation length. Extracellular serotonin signaling is prolonged after administering cocaine or fluoxetine, showing that transport regulates the clearance of serotonin from the extracellular space. When ChR2 is targeted to dopaminergic neurons, dopamine release is measured demonstrating that this method is broadly applicable to other neurotransmitter systems. This study shows that the dynamics of serotonin release and reuptake in Drosophila are analogous to those in mammals, making this simple organism more useful for the study of the basic physiological mechanisms of serotonergic signaling.

Synthesis and reuptake regulate 5-HT release | 189 25-30 min prior to the initiation of experiments, a time that is sufficient for drugs to diffuse into the nerve cord (Borue et al. 2009). An electrode was implanted in the neuropil using a micromanipulator and allowed to equilibrate for 5 min before data were collected. After the collection of at least 30 s of baseline data, VNCs were exposed to 10 s of intense blue light to stimulate release. This stimulation length produces close to maximal serotonin release (Borue et al. 2009). The light source was a 10 W halogen microscope bulb with a standard fluorescein excitation filter (450-490 nm) that was manually switched. Data analysisElectrochemical data were analyzed using Tar Heel CV software (gift of Mark Wightman). For a detailed description of serotonin detection in the fly using fast-scan cyclic voltammetry, see Borue et al. (2009). Cyclic voltammograms (CVs) were used to verify serotonin was detected. The current at the maximum serotonin voltage was converted to serotonin concentration using postcalibration data for each electrode and concentration changes plotted versus time. The initial stimulated peak height was used to normalize data for multiple stimulation assays, where stimulations were performed with the start of the 10 s stimulation occurring every 1, 2, 5 or 10 min. A series of in vitro experiments performed in a flow cell showed that electrode sensitivity to serotonin decreased slightly over the course of multiple exposures (Fig. S1). A linear correction coefficient was applied to all data from multiple stimulation experiments to correct for changes in electrode sensitivity. This correction does not change the interpretation of data because it does not change the relationships between categories of VNCs. Statistical analyses of pooled data including two-tailed Student's t-tests were conducted using Excel. One-and two-way ANOVAs was performed using GraphPad Prism software (San Diego, CA, USA). Mean values are given as ± SEM. ResultsNeuronal serotonin content after pharmacological manipulations We explored the effects of pharmacological manipulations on neuronal serotonin content as measured by immunohistochemistry, the most common method for studying neurotransmitters in Drosophila. While immunohistochemistry provides only an estimate of neuronal serotonin tissue levels, because of antibody-mediated amplification of fixed serotonin, these rough numbers can be used to show the relative importance of each of the drugs on tissue content (Mize et al. 1988). A control VNC, incubated in buffer, shows normal serotonergic morphology and serotonin tissue content (Fig. 1a). The VNC is a segmented structure that contains four serotonergic neurons per segment, two on each side of the midline (Chen and Condron 2008). The serotonergic cell bodies are visible as pairs of brightly stained circles on either side of the neuropil while the projections appear as two strips of brightly stained material overlying the cell bodies. The imaging was optimized to show residua...

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.

Serotonin signaling plays a key role in the regulation of development, mood and behavior. Selective serotonin reuptake inhibitors (SSRIs) have been the standard of treatment for several mental disorders, including depression. However, our understanding of their effects is incomplete and the serotonergic system in mammals is complex, making it difficult to study. Drosophila is well suited for the study of the basic mechanisms of serotonergic signaling, but the small size of its nervous system has previously precluded the direct measurements of neurotransmitters. We have developed a novel combination of methods to study serotonergic signaling in the larval Drosophila central nervous system. Fast-scan cyclic voltammetry at inserted microelectrodes is used to detect serotonin elicited by channelrhodopsin-2 (ChR2) mediated depolarization. This dissertation demonstrates the first real-time measurements of serotonin dynamics in a single larval Drosophila nerve cord. A characterization of serotonin release and clearance in the fly, including the estimation of Michaelis-Menten constants, shows that they are analogous to those in mammals, making this simple organism more useful for the study of the basic physiological mechanisms of serotonergic signaling. The effects of pharmacologically inhibiting serotonin synthesis or reuptake on the releasable pool of serotonin are probed with multiple stimulation experiments. Reuptake is shown to be important for the clearance of serotonin from the extracellular space as well as the rapid replenishment of the releasable pool. Synthesis is critical to the longerterm replenishment of the releasable pool, especially when reuptake is concurrently inhibited. Decreases in serotonin are rescued by inhibiting action potential propagation with tetrodotoxin, implicating endogenous activity in depletion of neuronal serotonin. These results give insight into the possible effects of SSRIs on the serotonergic system and the important role that synthesis may play in this phenomenon as well as in overall serotonergic neuron function. The have also paved the way for future use of Drosophila for large-scale genetic analysis of neurotransmitter dynamics. This dissertation was completed under the direction of Jill Venton, PhD, in the Department of Chemistry and Neuroscience Graduate Program.