Both synthesis and reuptake are critical for replenishing the releasable serotonin pool in Drosophila

Abstract

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...

Sex- and SERT-Mediated Differences in Stimulated Serotonin Revealed by Fast Microdialysis

“…31,48 Increased synthesis could contribute to an increase in the readily releasable pool of serotonin; this pool is known to be sensitive to changes in uptake and synthesis. 49 Although the current study does not provide direct evidence for differences in serotonin release per se, we hypothesize that presynaptic serotonin 1A autoreceptor desensitization/downregulation, 47,50 in conjunction with increased synthesis, 48 Mice genetically lacking SERT were previously discovered to show a loss of frequency-dependent serotonin release when brain slices containing the substantia nigra were investigated by FSCV. 51 The authors concluded that abolishing SERT leads to a large summation of extracellular serotonin regardless of stimulus strength.…”

Functional groups modulate the sensitivity and electron transfer kinetics of neurochemicals at carbon nanotube modified microelectrodes

“…Homozygous 3-day-old larvae expressing a Tph-GAL4;UAS-ChR2 genotype were fed all-trans retinal for two days 25,26 . The central nervous system (CNS) of a wandering, 3 rd instar (5-day-old) larvae was dissected and incubated as described previously 27 .…”

Serotonin: a regulator of neuronal morphology and circuitry

“…The fruit fly also offers considerable advantages, as locomotor circuits of the larva are well studied [90, 91] and genetic amenability not only provides access to specific gene function but also allows cell-labeling techniques crucial for circuit observation. Electrochemical techniques such as fast scan cyclic voltammetry have been modified to measurereal-time serotonin release in Drosophila larval CNS [92,93], meaning that serotonergic input and locomotor output [90] could be quantified simultaneously in addition to monitoring morphology of the serotonergic neurons and the locomotor circuit.…”