Transcriptional changes in specific subsets of Drosophila neurons following inhibition of the serotonin transporter

Abstract 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

Transcriptional changes in specific subsets of Drosophila neurons following inhibition of the serotonin transporter.

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

An Optical Neuron-Astrocyte Proximity Assay at Synaptic Distance Scales

Astrocytes are complex bushy cells that serve important functions through close contacts between their processes and synapses. However, the spatial interactions and dynamics of astrocyte processes relative to synapses have proven problematic to study in adult living brain tissue. Here, we report a genetically targeted neuron-astrocyte proximity assay (NAPA) to measure astrocyte-synapse spatial interactions within intact brain preparations and at synaptic distance scales. The method exploits resonance energy transfer between extracellularly displayed fluorescent proteins targeted to synapses and astrocyte processes. We validated the method in the striatal microcircuitry following in vivo expression. We determined the proximity of striatal astrocyte processes to distinct neuronal input pathways, to D1 and D2 medium spiny neuron synapses, and we evaluated how astrocyte-to-excitatory synapse proximity changed following cortical afferent stimulation, during ischemia and in a model of Huntington's disease. NAPA provides a simple approach to measure astrocyte-synapse spatial interactions in a variety of experimental scenarios. VIDEO ABSTRACT

Precise CRISPR-Cas9-mediated mutation of a membrane trafficking domain in the Drosophila vesicular monoamine transporter gene

Monoamine neurotransmitters such as noradrenalin are released from both synaptic vesicles (SVs) and large dense-core vesicles (LDCVs), the latter mediating extrasynaptic signaling. The contribution of synaptic versus extrasynaptic signaling to circuit function and behavior remains poorly understood. To address this question, we have previously used transgenes encoding a mutation in the Drosophila Vesicular Monoamine Transporter (dVMAT) that shifts amine release from SVs to LDCVs. To circumvent the use of transgenes with non-endogenous patterns of expression, we have now used CRISPR-Cas9 to generate a trafficking mutant in the endogenous dVMAT gene. To minimize disruption of the dVMAT coding sequence and a nearby RNA splice site, we precisely introduced a point mutation using single-stranded oligonucleotide repair. A predicted decrease in fertility was used as a phenotypic screen to identify founders in lieu of a visible marker. Phenotypic analysis revealed a defect in the ovulation of mature follicles and egg retention in the ovaries. We did not detect defects in the contraction of lateral oviducts following optogenetic stimulation of octopaminergic neurons. Our findings suggest that release of mature eggs from the ovary is disrupted by changing the balance of VMAT trafficking between SVs and LDCVs. Further experiments using this model will help determine the mechanisms that sensitize specific circuits to changes in synaptic versus extrasynaptic signaling

Mutation of the Drosophila melanogaster serotonin transporter dSERT impacts sleep, courtship, and feeding behaviors.

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

Serotonergic modulation of visual neurons in Drosophila melanogaster.

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

Transgenic expression of <i>dSERT</i> with both “<i>TPH”-Gal4</i> and <i>TRH-Gal4</i> is sufficient to rescue increased sleep in <i>dSERT</i>^<i>16</i> mutants.

(A-C) Quantification of total sleep (A), daytime sleep (B) and nighttime sleep (C) in control w1118;TPH-Gal4 (grey), w1118;TPH-Gal4, dSERT16 (red), and w1118;TPH-Gal4, dSERT16; UAS-dSERT (green) flies. (D-F) Quantification of total sleep (D), daytime sleep (E), and nighttime sleep (F) in w1118;TRH-Gal4 (grey), w1118;dSERT16; TRH-Gal4 (red), and w1118;dSERT16; TRH-Gal4/UAS-dSERT (green) flies. (G-I) Quantification of total sleep (G), daytime sleep (H), and nighttime sleep (I) in w1118; TPH-Gal4; TRH-Gal4 (grey), w1118;TPH-Gal4, dSERT16; TRH-Gal4 (red), and w1118;TPH-Gal4, dSERT16; TRH-Gal4/ UAS-dSERT (green) flies. Graphs show individual datapoints and group means ± SEM. one-way ANOVA, with Tukey post-hoc test (p≤0.0332*, p≤0.0021**, p≤0.0002***, p≤0.0001****).</p

<i>dSERT</i> imprecise excision alleles.

(A) Schematic of dSERT gene: grey and blue boxes indicate non-coding exons and coding exons respectively. Deletions of the dSERT10 and dSERT16 flies are indicated with red dotted lines. (B) qRT-PCR was performed on cDNA synthesized from whole, male flies with the ubiquitously expressed ribosomal RNA RplP0 used as a reference. Gene expression of dSERT in the mutants was normalized to controls with +1 representing expression in w1118. dSERT transcript levels were significantly downregulated in dSERT10 (0.01±0.02) and dSERT16 (0.001±0.003) compared to controls. Error bars represent SD. P***≤0.001, Student’s t-test. (C) Western blot analysis shows a band representing dSERT at ~65kD that is reduced in intensity in both mutants. Actin was used as a loading control and shows no difference across genotypes (not shown). (D) Hourly sleep traces in w1118 (black) dSERT10 (orange) and dSERT16 (red) homozygotes. (Sleep trace shows mean ± SEM, n = 31–32 flies per group).</p

Raw data.

(ZIP)</p

<i>dSERT</i>^<i>16</i> mutant sleep phenotype is not an artifact of additional amine-linked behaviors.

(A) dSERT16 mutants (purple) show no change in grooming behavior compared to w1118 controls (grey). The average number of grooming events per minute for three separate 2 min periods with three experimental replicates is shown. In each experimental replicate n = 5 flies for each genotype. Mean± SEM, unpaired Student’s t-test. (B) Male and female dSERT16 mutants behave indistinguishably from control flies in negative geotaxis assays. Mean ± SEM, one-way ANOVA. (PDF)</p