Tango-seq: overlaying transcriptomics on connectomics to identify neurons downstream of Drosophila clock neurons.

Knowing how neural circuits change with neuronal plasticity and differ between individuals is important to fully understand behavior. Connectomes are typically assembled using electron microscopy, but this is low throughput and impractical for analyzing plasticity or mutations. Here, we modified the trans-Tango genetic circuit-tracing technique to identify neurons synaptically downstream of Drosophila s-LNv clock neurons, which show 24hr plasticity rhythms. s-LNv target neurons were labeled specifically in adult flies using a nuclear reporter gene, which facilitated their purification and then single cell sequencing. We call this Tango-seq, and it allows transcriptomic data - and thus cell identity - to be overlayed on top of anatomical data. We found that s-LNvs preferentially make synaptic connections with a subset of the CNMa+ DN1p clock neurons, and that these are likely plastic connections. We also identified synaptic connections between s-LNvs and mushroom body Kenyon cells. Tango-seq should be a useful addition to the connectomics toolkit.

Dopaminergic systems create reward seeking despite adverse consequences.

Resource-seeking behaviours are ordinarily constrained by physiological needs and threats of danger, and the loss of these controls is associated with pathological reward seeking1. Although dysfunction of the dopaminergic valuation system of the brain is known to contribute towards unconstrained reward seeking2,3, the underlying reasons for this behaviour are unclear. Here we describe dopaminergic neural mechanisms that produce reward seeking despite adverse consequences in Drosophila melanogaster. Odours paired with optogenetic activation of a defined subset of reward-encoding dopaminergic neurons become cues that starved flies seek while neglecting food and enduring electric shock punishment. Unconstrained seeking of reward is not observed after learning with sugar or synthetic engagement of other dopaminergic neuron populations. Antagonism between reward-encoding and punishment-encoding dopaminergic neurons accounts for the perseverance of reward seeking despite punishment, whereas synthetic engagement of the reward-encoding dopaminergic neurons also impairs the ordinary need-dependent dopaminergic valuation of available food. Connectome analyses reveal that the population of reward-encoding dopaminergic neurons receives highly heterogeneous input, consistent with parallel representation of diverse rewards, and recordings demonstrate state-specific gating and satiety-related signals. We propose that a similar dopaminergic valuation system dysfunction is likely to contribute to maladaptive seeking of rewards by mammals.

Sexually dimorphic mechanisms of VGLUT-mediated protection from dopaminergic neurodegeneration.

Parkinson's disease (PD) targets some dopamine (DA) neurons more than others. Sex differences offer insights, with females more protected from DA neurodegeneration. The mammalian vesicular glutamate transporter VGLUT2 and Drosophila ortholog dVGLUT have been implicated as modulators of DA neuron resilience. However, the mechanisms by which VGLUT2/dVGLUT protects DA neurons remain unknown. We discovered DA neuron dVGLUT knockdown increased mitochondrial reactive oxygen species in a sexually dimorphic manner in response to depolarization or paraquat-induced stress, males being especially affected. DA neuron dVGLUT also reduced ATP biosynthetic burden during depolarization. RNA sequencing of VGLUT+ DA neurons in mice and flies identified candidate genes that we functionally screened to further dissect VGLUT-mediated DA neuron resilience across PD models. We discovered transcription factors modulating dVGLUT-dependent DA neuroprotection and identified dj-1ß as a regulator of sex-specific DA neuron dVGLUT expression. Overall, VGLUT protects DA neurons from PD-associated degeneration by maintaining mitochondrial health.

Gliotransmission of D-serine promotes thirst-directed behaviors in Drosophila.

Thirst emerges from a range of cellular changes that ultimately motivate an animal to consume water. Although thirst-responsive neuronal signals have been reported, the full complement of brain responses is unclear. Here, we identify molecular and cellular adaptations in the brain using single-cell sequencing of water-deprived Drosophila. Water deficiency primarily altered the glial transcriptome. Screening the regulated genes revealed astrocytic expression of the astray-encoded phosphoserine phosphatase to bi-directionally regulate water consumption. Astray synthesizes the gliotransmitter D-serine, and vesicular release from astrocytes is required for drinking. Moreover, dietary D-serine rescues aay-dependent drinking deficits while facilitating water consumption and expression of water-seeking memory. D-serine action requires binding to neuronal NMDA-type glutamate receptors. Fly astrocytes contribute processes to tripartite synapses, and the proportion of astrocytes that are themselves activated by glutamate increases with water deprivation. We propose that thirst elevates astrocytic D-serine release, which awakens quiescent glutamatergic circuits to enhance water procurement.

Transposon expression in the Drosophila brain is driven by neighboring genes and diversifies the neural transcriptome.

Somatic transposon expression in neural tissue is commonly considered as a measure of mobilization and has therefore been linked to neuropathology and organismal individuality. We combined genome sequencing data with single-cell mRNA sequencing of the same inbred fly strain to map transposon expression in the Drosophila midbrain and found that transposon expression patterns are highly stereotyped. Every detected transposon is resident in at least one cellular gene with a matching expression pattern. Bulk RNA sequencing from fly heads of the same strain revealed that coexpression is a physical link in the form of abundant chimeric transposon-gene mRNAs. We identified 264 genes where transposons introduce cryptic splice sites into the nascent transcript and thereby significantly expand the neural transcript repertoire. Some genes exclusively produce chimeric mRNAs with transposon sequence; on average, 11.6% of the mRNAs produced from a given gene are chimeric. Conversely, most transposon-containing transcripts are chimeric, which suggests that somatic expression of these transposons is largely driven by cellular genes. We propose that chimeric mRNAs produced by alternative splicing into polymorphic transposons, rather than transposon mobilization, may contribute to functional differences between individual cells and animals.

No evidence for a magnetite-based magnetoreceptor in the lagena of pigeons.

It is well established that an array of avian species sense the Earth's magnetic field and use this information for orientation and navigation. While the existence of a magnetic sense can no longer be disputed, the underlying cellular and biophysical basis remains unknown. It has been proposed that pigeons exploit a magnetoreceptor based on magnetite crystals (Fe3O4) that are located within the lagena [1], a sensory epithelium of the inner ear. It has been hypothesised that these magnetic crystals form a bed of otoconia that stimulate hair cells transducing magnetic information into a neuronal impulse. We performed a systematic high-sensitivity screen for iron in the pigeon lagena using synchrotron X-ray fluorescence microscopy coupled with the analysis of serial sections by transmission electron microscopy. We find no evidence for extracellular magnetic otoconia or intracellular magnetite crystals, suggesting that if an inner ear magnetic sensor does exist it relies on a different biophysical mechanism.

Cellular diversity in the Drosophila midbrain revealed by single-cell transcriptomics.

To understand the brain, molecular details need to be overlaid onto neural wiring diagrams so that synaptic mode, neuromodulation and critical signaling operations can be considered. Single-cell transcriptomics provide a unique opportunity to collect this information. Here we present an initial analysis of thousands of individual cells from Drosophila midbrain, that were acquired using Drop-Seq. A number of approaches permitted the assignment of transcriptional profiles to several major brain regions and cell-types. Expression of biosynthetic enzymes and reuptake mechanisms allows all the neurons to be typed according to the neurotransmitter or neuromodulator that they produce and presumably release. Some neuropeptides are preferentially co-expressed in neurons using a particular fast-acting transmitter, or monoamine. Neuromodulatory and neurotransmitter receptor subunit expression illustrates the potential of these molecules in generating complexity in neural circuit function. This cell atlas dataset provides an important resource to link molecular operations to brain regions and complex neural processes.

Resolving the prevalence of somatic transposition in Drosophila.

Somatic transposition in mammals and insects could increase cellular diversity and neural mobilization has been implicated in age-dependent decline. To understand the impact of transposition in somatic cells it is essential to reliably measure the frequency and map locations of new insertions. Here we identified thousands of putative somatic transposon insertions in neurons from individual Drosophila melanogaster using whole-genome sequencing. However, the number of de novo insertions did not correlate with transposon expression or fly age. Analysing our data with exons as 'immobile genetic elements' revealed a similar frequency of unexpected exon translocations. A new sequencing strategy that recovers transposon: chromosome junction information revealed most putative de novo transposon and exon insertions likely result from unavoidable chimeric artefacts. Reanalysis of other published data suggests similar artefacts are often mistaken for genuine somatic transposition. We conclude that somatic transposition is less prevalent in Drosophila than previously envisaged.

The role of Tuba1a in adult hippocampal neurogenesis and the formation of the dentate gyrus.

The multitubulin hypothesis holds that each tubulin isotype serves a unique role with respect to microtubule function. Here we investigate the role of the α-tubulin subunit Tuba1a in adult hippocampal neurogenesis and the formation of the dentate gyrus. Employing birth date labelling and immunohistological markers, we show that mice harbouring an S140G mutation in Tuba1a present with normal neurogenic potential, but that this neurogenesis is often ectopic. Morphological analysis of the dentate gyrus in adulthood revealed a disorganised subgranular zone and a dispersed granule cell layer. We have shown that these anatomical abnormalities are due to defective migration of prospero-homeobox-1-positive neurons and T-box-brain-2-positive progenitors during development. Such migratory defects may also be responsible for the cytoarchitectural defects observed in the dentate gyrus of patients with mutations in TUBA1A.