The olfactory system remains plastic throughout life because of continuous neurogenesis of sensory neurons in the nose and inhibitory interneurons in the olfactory bulb. Here, we reveal that ...transgenic expression of an odorant receptor has non–cell autonomous effects on axons expressing this receptor from the endogenous gene. Perinatal expression of transgenic odorant receptor causes rerouting of like axons to new glomeruli, whereas expression after the sensory map is established does not lead to rerouting. Further, chemical ablation of the map after rerouting does not restore the normal map, even when the transgenic receptor is no longer expressed. Our results reveal that glomeruli are designated as targets for sensory neurons expressing specific odorant receptors during a critical period in the formation of the olfactory sensory map.
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Mapping neural circuits across defined synapses is essential for understanding brain function. Here we describe trans-Tango, a technique for anterograde transsynaptic circuit tracing and ...manipulation. At the core of trans-Tango is a synthetic signaling pathway that is introduced into all neurons in the animal. This pathway converts receptor activation at the cell surface into reporter expression through site-specific proteolysis. Specific labeling is achieved by presenting a tethered ligand at the synapses of genetically defined neurons, thereby activating the pathway in their postsynaptic partners and providing genetic access to these neurons. We first validated trans-Tango in the Drosophila olfactory system and then implemented it in the gustatory system, where projections beyond the first-order receptor neurons are not fully characterized. We identified putative second-order neurons within the sweet circuit that include projection neurons targeting known neuromodulation centers in the brain. These experiments establish trans-Tango as a flexible platform for transsynaptic circuit analysis.
•A genetic approach for transsynaptic tracing and manipulation of neural circuits•Genetic access to neurons based on their connectivity•Glomerulus-specific patterns of second-order neurons in the fly olfactory system•Identifying second-order gustatory neurons and their target areas in the fly brain
Talay and Richman et al. develop a genetic method for transsynaptic labeling of neural circuits in Drosophila. They validate it in the olfactory system and implement it in the gustatory system to reveal second-order projections of sweet tastant-responsive neurons.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The modified DNA base 5-hydroxymethylcytosine (5hmC) is enriched in neurons where it may contribute to gene regulation and cellular identity. To determine how 5hmC influences gene expression in an in ...vivo neuronal population, we assessed the patterning and function of the base along the developmental lineage of the main olfactory epithelium—from multipotent stem cells through neuronal progenitors to mature olfactory sensory neurons (mOSNs). We find that 5hmC increases over gene bodies during mOSN development with substantial patterning occuring between the progenitor and mOSN stages. Although gene-body 5hmC levels correlate with gene expression in all three developmental cell types, this association is particularly pronounced within mOSNs. Overexpression of Tet3 in mOSNs markedly alters gene-body 5hmC levels and gene expression in a manner consistent with a positive role for 5hmC in transcription. Moreover, Tet3 overexpression disrupts olfactory receptor expression and the targeting of axons to the olfactory bulb, key molecular and anatomical features of the olfactory system. Our results suggest a physiologically significant role for gene-body 5hmC in transcriptional facilitation and the maintenance of cellular identity independent of its function as an intermediate to demethylation.
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The mushroom body (MB) is a well-characterized associative memory structure within the
brain. Analyzing MB connectivity using multiple approaches is critical for understanding the functional ...implications of this structure. Using the genetic anterograde transsynaptic tracing tool,
Tango, we identified divergent projections across the brain and convergent downstream targets of the MB output neurons (MBONs). Our analysis revealed at least three separate targets that receive convergent input from MBONs: other MBONs, the fan-shaped body (FSB), and the lateral accessory lobe (LAL). We describe, both anatomically and functionally, a multilayer circuit in which inhibitory and excitatory MBONs converge on the same genetic subset of FSB and LAL neurons. This circuit architecture enables the brain to update and integrate information with previous experience before executing appropriate behavioral responses. Our use of
-Tango provides a genetically accessible anatomical framework for investigating the functional relevance of components within these complex and interconnected circuits.
We have developed an experimental strategy to monitor protein interactions in a cell with a high degree of selectivity and sensitivity. A transcription factor is tethered to a membrane-bound receptor ...with a linker that contains a cleavage site for a specific protease. Activation of the receptor recruits a signaling protein fused to the protease that then cleaves and releases the transcription factor to activate reporter genes in the nucleus. This strategy converts a transient interaction into a stable and amplifiable reporter gene signal to record the activation of a receptor without interference from endogenous signaling pathways. We have developed this assay for three classes of receptors: G protein-coupled receptors, receptor tyrosine kinases, and steroid hormone receptors. Finally, we use the assay to identify a ligand for the orphan receptor GPR1, suggesting a role for this receptor in the regulation of inflammation.
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A powerful feature of adaptive memory is its inherent flexibility. Alcohol and other addictive substances can remold neural circuits important for memory to reduce this flexibility. However, the ...mechanism through which pertinent circuits are selected and shaped remains unclear. We show that circuits required for alcohol-associated preference shift from population level dopaminergic activation to select dopamine neurons that predict behavioral choice in
. During memory expression, subsets of dopamine neurons directly and indirectly modulate the activity of interconnected glutamatergic and cholinergic mushroom body output neurons (MBON). Transsynaptic tracing of neurons important for memory expression revealed a convergent center of memory consolidation within the mushroom body (MB) implicated in arousal, and a structure outside the MB implicated in integration of naïve and learned responses. These findings provide a circuit framework through which dopamine neuronal activation shifts from reward delivery to cue onset, and provide insight into the maladaptive nature of memory.
Olfactory receptor (OR) choice represents an example of genetically hardwired stochasticity, where every olfactory neuron expresses one out of ~2000 OR alleles in the mouse genome in a probabilistic, ...yet stereotypic fashion. Here, we propose that topographic restrictions in OR expression are established in neuronal progenitors by two opposing forces: polygenic transcription and genomic silencing, both of which are influenced by dorsoventral gradients of transcription factors NFIA, B, and X. Polygenic transcription of OR genes may define spatially constrained OR repertoires, among which one OR allele is selected for singular expression later in development. Heterochromatin assembly and genomic compartmentalization of OR alleles also vary across the axes of the olfactory epithelium and may preferentially eliminate ectopically expressed ORs with more dorsal expression destinations from this 'privileged' repertoire. Our experiments identify early transcription as a potential 'epigenetic' contributor to future developmental patterning and reveal how two spatially responsive probabilistic processes may act in concert to establish deterministic, precise, and reproducible territories of stochastic gene expression.
Behavior cannot be predicted from a “connectome” because the brain contains a chemical “map” of neuromodulation superimposed upon its synaptic connectivity map. Neuromodulation changes how neural ...circuits process information in different states, such as hunger or arousal. Here we describe a genetically based method to map, in an unbiased and brain-wide manner, sites of neuromodulation under different conditions in the Drosophila brain. This method, and genetic perturbations, reveal that the well-known effect of hunger to enhance behavioral sensitivity to sugar is mediated, at least in part, by the release of dopamine onto primary gustatory sensory neurons, which enhances sugar-evoked calcium influx. These data reinforce the concept that sensory neurons constitute an important locus for state-dependent gain control of behavior and introduce a methodology that can be extended to other neuromodulators and model organisms.
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► Tango-map method visualizes dopaminergic modulation in the Drosophila brain ► Upon starvation, dopamine is released onto sugar-sensing neurons ► Starvation enhances feeding behavior via dopamine modulation of sugar-sensing neurons ► Dopamine modulation of sweet-sensing neurons enhances sucrose-evoked calcium influx
Visualizing levels of dopamine signaling across the whole brain reveals how starved flies become more keenly receptive to sugar. Hunger promotes dopamine release onto sugar-sensing neurons, enhancing calcium influx upon encountering food.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The expression of a single odorant receptor (OR) gene from a large gene family in individual sensory neurons is an essential feature of the organization and function of the olfactory system. We have ...used chromosome conformation capture to demonstrate the specific association of an enhancer element,
H, on chromosome 14 with multiple OR gene promoters on different chromosomes. DNA and RNA fluorescence in situ hybridization (FISH) experiments allow us to visualize the colocalization of the
H enhancer with the single OR allele that is transcribed in a sensory neuron. In transgenic mice bearing additional
H elements, sensory neurons that express OR pseudogenes also express a second functional receptor. These data suggest a model of receptor choice in which a single
trans-acting enhancer element may allow the stochastic activation of only one OR allele in an olfactory sensory neuron.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Olfactory sensory neurons (OSNs) are functionally defined by their expression of a unique odorant receptor (OR). Mechanisms underlying singular OR expression are well studied, and involve a massive ...cross-chromosomal enhancer interaction network. Trace amine-associated receptors (TAARs) form a distinct family of olfactory receptors, and here we find that mechanisms regulating Taar gene choice display many unique features. The epigenetic signature of Taar genes in TAAR OSNs is different from that in OR OSNs. We further identify that two TAAR enhancers conserved across placental mammals are absolutely required for expression of the entire Taar gene repertoire. Deletion of either enhancer dramatically decreases the expression probabilities of different Taar genes, while deletion of both enhancers completely eliminates the TAAR OSN populations. In addition, both of the enhancers are sufficient to drive transgene expression in the partially overlapped TAAR OSNs. We also show that the TAAR enhancers operate in cis to regulate Taar gene expression. Our findings reveal a coordinated control of Taar gene choice in OSNs by two remote enhancers, and provide an excellent model to study molecular mechanisms underlying formation of an olfactory subsystem.
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