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The Serotonin Transporter (SERT) regulates extracellular serotonin levels and is the target of most current drugs used to treat depression. The mechanisms by which inhibition of SERT activity influences behavior are poorly understood. To address this question in the model organism Drosophila melanogaster , we developed new loss of function mutations in Drosophila SERT ( dSERT) . Previous studies in both flies and mammals have implicated serotonin as an important neuromodulator of sleep, and our newly generated dSERT mutants show an increase in total sleep and altered sleep architecture that is mimicked by feeding the SSRI citalopram. Differences in daytime versus nighttime sleep architecture as well as genetic rescue experiments unexpectedly suggest that distinct serotonergic circuits may modulate daytime versus nighttime sleep. dSERT mutants also show defects in copulation and food intake, akin to the clinical side effects of SSRIs and consistent with the pleomorphic influence of serotonin on the behavior of D . melanogaster . Starvation did not overcome the sleep drive in the mutants and in male dSERT mutants, the drive to mate also failed to overcome sleep drive. dSERT may be used to further explore the mechanisms by which serotonin regulates sleep and its interplay with other complex behaviors.

The transcriptional effects of SSRIs and other serotonergic drugs remain unclear, in part due to the heterogeneity of postsynaptic cells, which may respond differently to changes in serotonergic signaling. Relatively simple model systems such as Drosophila afford more tractable microcircuits in which to investigate these changes in specific cell types. Here, we focus on the mushroom body, an insect brain structure heavily innervated by serotonin and comprised of multiple different but related subtypes of Kenyon cells. We use fluorescence-activated cell sorting of Kenyon cells, followed by either bulk or single-cell RNA sequencing to explore the transcriptomic response of these cells to SERT inhibition. We compared the effects of two different Drosophila Serotonin Transporter (dSERT) mutant alleles as well as feeding the SSRI citalopram to adult flies. We find that the genetic architecture associated with one of the mutants contributed to significant artefactual changes in expression. Comparison of differential expression caused by loss of SERT during development versus aged, adult flies, suggests that changes in serotonergic signaling may have relatively stronger effects during development, consistent with behavioral studies in mice. Overall, our experiments revealed limited transcriptomic changes in Kenyon cells, but suggest that different subtypes may respond differently to SERT loss-of-function. Further work exploring the effects of SERT loss-of-function in other circuits may be used help to elucidate how SSRIs differentially affect a variety of different neuronal subtypes both during development and in adults.

Sensory systems rely on neuromodulators, such as serotonin, to provide flexibility for information processing as stimuli vary, such as light intensity throughout the day. Serotonergic neurons broadly innervate the optic ganglia of Drosophila melanogaster, a widely used model for studying vision. It remains unclear whether serotonin modulates the physiology of interneurons in the optic ganglia. To address this question, we first mapped the expression patterns of serotonin receptors in the visual system, focusing on a subset of cells with processes in the first optic ganglion, the lamina. Serotonin receptor expression was found in several types of columnar cells in the lamina including 5-HT2B in lamina monopolar cell L2, required for spatiotemporal luminance contrast, and both 5-HT1A and 5-HT1B in T1 cells, whose function is unknown. Subcellular mapping with GFP-tagged 5-HT2B and 5-HT1A constructs indicated that these receptors localize to layer M2 of the medulla, proximal to serotonergic boutons, suggesting that the medulla neuropil is the primary site of serotonergic regulation for these neurons. Exogenous serotonin increased basal intracellular calcium in L2 terminals in layer M2 and modestly decreased the duration of visually induced calcium transients in L2 neurons following repeated dark flashes, but otherwise did not alter the calcium transients. Flies without functional 5-HT2B failed to show an increase in basal calcium in response to serotonin. 5-HT2B mutants also failed to show a change in amplitude in their response to repeated light flashes but other calcium transient parameters were relatively unaffected. While we did not detect serotonin receptor expression in L1 neurons, they, like L2, underwent serotonin-induced changes in basal calcium, presumably via interactions with other cells. These data demonstrate that serotonin modulates the physiology of interneurons involved in early visual processing in Drosophila.

Background Repetitive Transcranial Magnetic Stimulation (rTMS) is commonly administered to Major Depressive Disorder (MDD) patients taking psychotropic medications, yet the effects on treatment outcomes remain unknown. We explored how concomitant medication use relates to clinical response to a standard course of rTMS. Methods Medications were tabulated for 181 MDD patients who underwent a six‐week rTMS treatment course. All patients received 10 Hz rTMS administered to left dorsolateral prefrontal cortex (DLPFC), with 1 Hz administered to right DLPFC in patients with inadequate response to and/or intolerance of left‐sided stimulation. Primary outcomes were change in Inventory of Depressive Symptomatology Self Report (IDS‐SR30) total score after 2, 4, and 6 weeks. Results Use of benzodiazepines was associated with less improvement at week 2, whereas use of psychostimulants was associated with greater improvement at week 2 and across 6 weeks. These effects were significant controlling for baseline variables including age, overall symptom severity, and severity of anxiety symptoms. Response rates at week 6 were lower in benzodiazepine users versus non‐users (16.4% vs. 35.5%, p = 0.008), and higher in psychostimulant users versus non‐users (39.2% vs. 22.0%, p = 0.02). Conclusions Concomitant medication use may impact rTMS treatment outcome. While the differences reported here could be considered clinically significant, results were not corrected for multiple comparisons and findings should be replicated before clinicians incorporate the evidence into clinical practice. Prospective, hypothesis‐based treatment studies will aid in determining causal relationships between medication treatments and outcome. Repetitive Transcranial Magnetic Stimulation (rTMS) is commonly used to treat Major Depressive Disorder (MDD) patients who are taking psychotropic medications, yet the effects on treatment outcome remains unknown. Exploratory hypothesis‐generating analyses of concomitant medication use in a clinical sample (n = 181) suggested that specific categories of medication use (psychostimulants and benzodiazepines, respectively) may be associated with better or worse outcome to an acute course of rTMS. Prospective, hypothesis‐based treatment studies are needed to determine potential causal relationships between medication treatments and outcome.

Amphetamines elevate extracellular dopamine, but the underlying mechanisms remain uncertain. Here we show in rodents that acute pharmacological inhibition of the vesicular monoamine transporter (VMAT) blocks amphetamine-induced locomotion and self-administration without impacting cocaine-induced behaviours. To study VMAT’s role in mediating amphetamine action in dopamine neurons, we have used novel genetic, pharmacological and optical approaches in Drosophila melanogaster . In an ex vivo whole-brain preparation, fluorescent reporters of vesicular cargo and of vesicular pH reveal that amphetamine redistributes vesicle contents and diminishes the vesicle pH-gradient responsible for dopamine uptake and retention. This amphetamine-induced deacidification requires VMAT function and results from net H + antiport by VMAT out of the vesicle lumen coupled to inward amphetamine transport. Amphetamine-induced vesicle deacidification also requires functional dopamine transporter (DAT) at the plasma membrane. Thus, we find that at pharmacologically relevant concentrations, amphetamines must be actively transported by DAT and VMAT in tandem to produce psychostimulant effects. Amphetamines are known to enhance extracellular dopamine levels, but the underlying mechanisms are unclear. Utilising a new pH biosensor for synaptic vesicles, the authors show that amphetamines diminish vesicle pH gradients, disrupting dopamine packaging and leading to increased neurotransmitter release.

Aminergic nuclei in mammals are generally composed of relatively small numbers of cells with broad projection patterns. Despite the gross similarity of many individual neurons, recent transcriptomic, anatomic and behavioral studies suggest previously unsuspected diversity. Smaller clusters of aminergic neurons in the model organism Drosophila melanogaster provide an opportunity to explore the ramifications of neuronal diversity at the level of individual cells. A group of approximately 10 tyraminergic/octopaminergic neurons innervates the female reproductive tract in flies and has been proposed to regulate multiple activities required for fertility. The projection patterns of individual neurons within the cluster are not known and it remains unclear whether they are functionally heterogenous. Using a single cell labeling technique, we show that each region of the reproductive tract is innervated by a distinct subset of tyraminergic/octopaminergic cells. Optogenetic activation of one subset stimulates oviduct contractions, indicating that the cluster as a whole is not required for this activity, and underscoring the potential for functional diversity across individual cells. Using whole cell patch clamp, we show that two adjacent and morphologically similar cells are tonically inhibited, but each responds differently to injection of current or activation of the inhibitory GluCl receptor. GluCl appears to be expressed at relatively low levels in tyraminergic/octopaminergic neurons within the cluster, suggesting that it may regulate their excitability via indirect pathways. Together, our data indicate that specific tyraminergic/octopaminergic cells within a relatively homogenous cluster have heterogenous properties and provide a platform for further studies to determine the function of each cell.

Unlike other monoamine neurotransmitters, the mechanism by which the brain's histamine content is regulated remains unclear. In mammals, vesicular monoamine transporters (VMATs) are expressed exclusively in neurons and mediate the storage of histamine and other monoamines. We have studied the visual system of Drosophila melanogaster in which histamine is the primary neurotransmitter released from photoreceptor cells. We report here that a novel mRNA splice variant of Drosophila VMAT (DVMAT-B) is expressed not in neurons but rather in a small subset of glia in the lamina of the fly's optic lobe. Histamine contents are reduced by mutation of dVMAT, but can be partially restored by specifically expressing DVMAT-B in glia. Our results suggest a novel role for a monoamine transporter in glia that may be relevant to histamine homeostasis in other systems.

Physiologic and pathogenic changes in amine release induce dramatic behavioral changes, but the underlying cellular mechanisms remain unclear. To investigate these adaptive processes, we have characterized mutations in the Drosophila vesicular monoamine transporter (dVMAT), which is required for the vesicular storage of dopamine, serotonin, and octopamine. dVMAT mutant larvae show reduced locomotion and decreased electrical activity in motoneurons innervating the neuromuscular junction (NMJ) implicating central amines in the regulation of these activities. A parallel increase in evoked glutamate release by the motoneuron is consistent with a homeostatic adaptation at the NMJ. Despite the importance of aminergic signaling for regulating locomotion and other behaviors, adult dVMAT homozygous null mutants survive under conditions of low population density, thus allowing a phenotypic characterization of adult behavior. Homozygous mutant females are sterile and show defects in both egg retention and development; males also show reduced fertility. Homozygotes show an increased attraction to light but are mildly impaired in geotaxis and escape behaviors. In contrast, heterozygous mutants show an exaggerated escape response. Both hetero- and homozygous mutants demonstrate an altered behavioral response to cocaine. dVMAT mutants define potentially adaptive responses to reduced or eliminated aminergic signaling and will be useful to identify the underlying molecular mechanisms.

Aminergic signaling is known to play a critical role in regulating female reproductive processes in both mammals and insects. In Drosophila, the ortholog of noradrenaline, octopamine, is required for ovulation as well as several other female reproductive processes. Two octopamine receptors have already been shown to be expressed in the Drosophila reproductive tract and to be required for egg-laying: OAMB and Octβ2R. The Drosophila genome contains 4 additional octopamine receptors—Octα2R, Octβ1R, Octβ3R, and Oct-TyrR—but their cellular patterns of expression in the reproductive tract and potential contribution(s) to egg-laying are not known. In addition, the mechanisms by which OAMB and Octβ2R regulate reproduction are incompletely understood. Using a panel of MiMIC Gal4 lines, we show that Octα2R, Octβ1R, Octβ3R, and Oct-TyrR receptors are not detectable in either epithelium or muscle but are clearly expressed in neurons within the female fly reproductive tract. Optogenetic activation of neurons that express at least 3 types of octopamine receptors stimulates contractions in the lateral oviduct. We also find that octopamine stimulates calcium transients in the sperm storage organs and that its effects in spermathecal, secretory cells, can be blocked by knock-down of OAMB. These data extend our understanding of the pathways by which octopamine regulates egg-laying in Drosophila and raise the possibility that multiple octopamine receptor subtypes could play a role in this process.

Clinical outcomes of repetitive transcranial magnetic stimulation (rTMS) for treatment of treatment-resistant depression (TRD) vary widely and there is no mood rating scale that is standard for assessing rTMS outcome. It remains unclear whether TMS is as efficacious in older adults with late-life depression (LLD) compared to younger adults with major depressive disorder (MDD). This study examined the effect of age on outcomes of rTMS treatment of adults with TRD. Self-report and observer mood ratings were measured weekly in 687 subjects ages 16–100 years undergoing rTMS treatment using the Inventory of Depressive Symptomatology 30-item Self-Report (IDS-SR), Patient Health Questionnaire 9-item (PHQ), Profile of Mood States 30-item, and Hamilton Depression Rating Scale 17-item (HDRS). All rating scales detected significant improvement with treatment; response and remission rates varied by scale but not by age (response/remission ≥ 60: 38%–57%/25%–33%; <60: 32%–49%/18%–25%). Proportional hazards models showed early improvement predicted later improvement across ages, though early improvements in PHQ and HDRS were more predictive of remission in those < 60 years (relative to those ≥ 60) and greater baseline IDS burden was more predictive of non-remission in those ≥ 60 years (relative to those < 60). These results indicate there is no significant effect of age on treatment outcomes in rTMS for TRD, though rating instruments may differ in assessment of symptom burden between younger and older adults during treatment.