The central complex (CX) plays a key role in many higher-order functions of the insect brain including navigation and activity regulation. Genetic tools for manipulating individual cell types, and knowledge of what neurotransmitters and neuromodulators they express, will be required to gain mechanistic understanding of how these functions are implemented. We generated and characterized split-GAL4 driver lines that express in individual or small subsets of about half of CX cell types. We surveyed neuropeptide and neuropeptide receptor expression in the central brain using fluorescent in situ hybridization. About half of the neuropeptides we examined were expressed in only a few cells, while the rest were expressed in dozens to hundreds of cells. Neuropeptide receptors were expressed more broadly and at lower levels. Using our GAL4 drivers to mark individual cell types, we found that 51 of the 85 CX cell types we examined expressed at least one neuropeptide and 21 expressed multiple neuropeptides. Surprisingly, all co-expressed a small molecule neurotransmitter. Finally, we used our driver lines to identify CX cell types whose activation affects sleep, and identified other central brain cell types that link the circadian clock to the CX. The well-characterized genetic tools and information on neuropeptide and neurotransmitter expression we provide should enhance studies of the CX.

Astrocytes perform multifarious roles in the formation, regulation, and function of synapses in the brain, but the mechanisms involved are incompletely understood. Interestingly, astrocytes abundantly express neuroligins, postsynaptic adhesion molecules that function as synaptic organizers by binding to presynaptic neurexins. Here we examined the function of neuroligins in astrocytes with a rigorous genetic approach that uses the conditional deletion of all major neuroligins ( Nlgn1-3 ) in astrocytes in vivo and complemented this approach by a genetic deletion of neuroligins in glia cells that are co-cultured with human neurons. Our results show that early postnatal deletion of neuroligins from astrocytes in vivo has no detectable effect on cortical or hippocampal synapses and does not alter the cytoarchitecture of astrocytes when evaluated in young adult mice. Moreover, deletion of astrocytic neuroligins in co-cultures of human neurons produced no detectable consequences for the formation and function of synapses. Thus, astrocytic neuroligins are unlikely to fundamentally shape synapse formation or astrocyte morphogenesis but likely perform other important roles that remain to be discovered.

Astrocytes perform multifarious roles in the formation, regulation, and function of synapses in the brain, but the mechanisms involved are incompletely understood. Interestingly, astrocytes abundantly express neuroligins, postsynaptic adhesion molecules that function as synaptic organizers by binding to presynaptic neurexins. Here we examined the function of neuroligins in astrocytes with a rigorous genetic approach that uses the conditional deletion of all major neuroligins ( Nlgn1-3 ) in astrocytes in vivo and complemented this approach by a genetic deletion of neuroligins in glia cells that are co-cultured with human neurons. Our results show that early postnatal deletion of neuroligins from astrocytes in vivo has no detectable effect on cortical or hippocampal synapses and does not alter the cytoarchitecture of astrocytes when evaluated in young adult mice. Moreover, deletion of astrocytic neuroligins in co-cultures of human neurons produced no detectable consequences for the formation and function of synapses. Thus, astrocytic neuroligins are unlikely to fundamentally shape synapse formation or astrocyte morphogenesis but likely perform other important roles that remain to be discovered.

Lactate, once considered an inert byproduct of glycolysis, is now increasingly appreciated as a metabolite with diverse roles in cellular metabolism and physiologic regulation. Our understanding of the role of lactate in biological systems can be considered in two categories: metabolic substrate and metabolic signal. These shared roles of lactate can be reconciled through the lens of a metabolite that is suited to regulate cellular function to license adaptation in line with the bioenergetic state of the cell. The mechanisms through which lactate production, transport, and consumption occur within cells and organisms are an area of longstanding and still active research. Here, we focus on how lactate production and utilization as a metabolic substrate feed into its role as a metabolic signal and the emerging mechanisms through which it regulates biological processes.

Deep Mutational Scanning (DMS) is an emerging method to systematically test the functional consequences of thousands of sequence changes to a protein target in a single experiment. Because of its utility in interpreting both human variant effects and protein structure-function relationships, it holds substantial promise to improve drug discovery and clinical development. However, applications in this domain require improved experimental and analytical methods. To address this need, we report novel DMS methods to precisely and quantitatively interrogate disease-relevant mechanisms, protein-ligand interactions, and assess predicted response to drug treatment. Using these methods, we performed a DMS of the melanocortin-4 receptor (MC4R), a G-protein-coupled receptor (GPCR) implicated in obesity and an active target of drug development efforts. We assessed the effects of >6600 single amino acid substitutions on MC4R's function across 18 distinct experimental conditions, resulting in >20 million unique measurements. From this, we identified variants that have unique effects on MC4R-mediated Gαs- and Gαq-signaling pathways, which could be used to design drugs that selectively bias MC4R's activity. We also identified pathogenic variants that are likely amenable to a corrector therapy. Finally, we functionally characterized structural relationships that distinguish the binding of peptide versus small molecule ligands, which could guide compound optimization. Collectively, these results demonstrate that DMS is a powerful method to empower drug discovery and development.

Deep Mutational Scanning (DMS) is an emerging method to systematically test the functional consequences of thousands of sequence changes to a protein target in a single experiment. Because of its utility in interpreting both human variant effects and protein structure-function relationships, it holds substantial promise to improve drug discovery and clinical development. However, applications in this domain require improved experimental and analytical methods. To address this need, we report novel DMS methods to precisely and quantitatively interrogate disease-relevant mechanisms, protein-ligand interactions, and assess predicted response to drug treatment. Using these methods, we performed a DMS of the melanocortin-4 receptor (MC4R), a G-protein-coupled receptor (GPCR) implicated in obesity and an active target of drug development efforts. We assessed the effects of >6600 single amino acid substitutions on MC4R’s function across 18 distinct experimental conditions, resulting in >20 million unique measurements. From this, we identified variants that have unique effects on MC4R-mediated Gαs- and Gαq-signaling pathways, which could be used to design drugs that selectively bias MC4R’s activity. We also identified pathogenic variants that are likely amenable to a corrector therapy. Finally, we functionally characterized structural relationships that distinguish the binding of peptide versus small molecule ligands, which could guide compound optimization. Collectively, these results demonstrate that DMS is a powerful method to empower drug discovery and development.

Affiliative social connections facilitate well-being and survival in numerous species. Engaging in social interactions requires positive or negative motivational drive, elicited through coordinated activity across neural circuits. However, the identity, interconnectivity, and functional encoding of social information within these circuits remains poorly understood. Here, we focus on downstream projections of dorsal raphe nucleus (DRN) dopamine neurons (DRN DAT ), which we previously implicated in social motivation alongside an aversive affective state. We show that three prominent DRN DAT projections – to the bed nucleus of the stria terminalis (BNST), central amygdala (CeA), and posterior basolateral amygdala (BLP) – play separable roles in behavior, despite substantial collateralization. Photoactivation of the DRN DAT -CeA projection promoted social behavior and photostimulation of the DRN DAT -BNST projection promoted exploratory behavior, while the DRN DAT -BLP projection supported place avoidance, suggesting a negative affective state. Downstream regions showed diverse receptor expression, poising DRN DAT neurons to act through dopamine, neuropeptide, and glutamate transmission. Furthermore, we show ex vivo that the effect of DRN DAT photostimulation on downstream neuron excitability depended on region and baseline cell properties, resulting in excitatory responses in BNST cells and diverse responses in CeA and BLP. Finally, in vivo microendoscopic cellular-resolution recordings in the CeA with DRN DAT photostimulation revealed a correlation between social behavior and neurons excited by social stimuli– suggesting that increased dopamine tone may recruit different CeA neurons to social ensembles. Collectively, these circuit features may facilitate a coordinated, but flexible, response in the presence of social stimuli that can be flexibly guided based on the internal social homeostatic need state of the individual.

Affiliative social connections facilitate well-being and survival in numerous species. Engaging in social interactions requires positive or negative motivational drive, elicited through coordinated activity across neural circuits. However, the identity, interconnectivity, and functional encoding of social information within these circuits remains poorly understood. Here, we focus on downstream projections of dorsal raphe nucleus (DRN) dopamine neurons (DRN DAT ), which we previously implicated in social motivation alongside an aversive affective state. We show that three prominent DRN DAT projections – to the bed nucleus of the stria terminalis (BNST), central amygdala (CeA), and posterior basolateral amygdala (BLP) – play separable roles in behavior, despite substantial collateralization. Photoactivation of the DRN DAT -CeA projection promoted social behavior and photostimulation of the DRN DAT -BNST projection promoted exploratory behavior, while the DRN DAT -BLP projection supported place avoidance, suggesting a negative affective state. Downstream regions showed diverse receptor expression, poising DRN DAT neurons to act through dopamine, neuropeptide, and glutamate transmission. Furthermore, we show ex vivo that the effect of DRN DAT photostimulation on downstream neuron excitability depended on region and baseline cell properties, resulting in excitatory responses in BNST cells and diverse responses in CeA and BLP. Finally, in vivo microendoscopic cellular-resolution recordings in the CeA with DRN DAT photostimulation revealed a correlation between social behavior and neurons excited by social stimuli– suggesting that increased dopamine tone may recruit different CeA neurons to social ensembles. Collectively, these circuit features may facilitate a coordinated, but flexible, response in the presence of social stimuli that can be flexibly guided based on the internal social homeostatic need state of the individual.

C. elegans’ major food source is bacteria, and worms are naturally attracted to many bacterial species, including pathogenic Pseudomonas ; in fact, worms prefer PA14 as well as wild bacteria over the lab E. coli strain (OP50) standardly used in the laboratory setting. Many labs have shown that despite this natural attraction to PA14, prior exposure to PA14 causes the worms to instead avoid PA14. This behavioral switch can happen on a relatively fast time scale, even within the duration of the choice assay. Here we show that accurate assessment of the animals’ true first choice requires the use of a paralytic (azide) to trap the worms at their initial choice, and to prevent the switch from attraction to avoidance of PA14 within the assay period. We previously discovered that exposure of C. elegans to 25°C plate-grown PA14 at 20°C for 24hrs not only leads to these animals switching from attraction to avoidance of PA14, but also to their progeny avoiding PA14 in the naïve state, and this avoidance persists through the F4 generation. Other types of PA14 training can also cause P0 and/or F1 avoidance, but do not induce transgenerational (F2 and beyond) inheritance. We also previously showed that the transgenerational (P0-F4) learned avoidance is mediated by P11, a small RNA produced by PA14. P11 is both necessary and sufficient for transgenerational epigenetic inheritance of avoidance behavior. P11 is highly expressed in our standard growth conditions (25°C on surfaces), but not in other conditions, suggesting that reported failure to observe F2-F4 avoidance is most likely due to the absence of P11 expression in PA14 in the experimenters’ growth conditions. Through mutant analyses, we have tested many genes – including germline regulators, small RNA uptake, RNA interference/processing, chromatin modifiers, and neuronal genes - for their involvement in transgenerational inheritance of learned pathogen avoidance, allowing us to better understand the molecular requirements for this process. We previously found that wild C. elegans strains also show TEI of learned pathogen avoidance, and that at least two other wild bacteria, P. vranovensis and P. fluorescens 15, induce this transgenerational avoidance. The avoidance induced by each Pseudomonas species functions through a specific, distinct small RNA (Pv1 in P. vranovensis and Pfs1 in P. fluorescens 15 , respectively) that either directly or indirectly reduce the levels of the gene maco-1 , which in turn regulates daf-7 expression in the ASI neuron and subsequent avoidance behavior. The conservation of multiple components of this small RNA TEI mechanism across C. elegans strains and in multiple Pseudomonas species suggests that this transgenerational learned avoidance behavior is likely to be functional and physiologically important in wild conditions.

C. elegans’ major food source is bacteria, and worms are naturally attracted to many bacterial species, including pathogenic Pseudomonas ; in fact, worms prefer PA14 as well as wild bacteria over the lab E. coli strain (OP50) standardly used in the laboratory setting. Many labs have shown that despite this natural attraction to PA14, prior exposure to PA14 causes the worms to instead avoid PA14. This behavioral switch can happen on a relatively fast time scale, even within the duration of the choice assay. Here we show that accurate assessment of the animals’ true first choice requires the use of a paralytic (azide) to trap the worms at their initial choice, and to prevent the switch from attraction to avoidance of PA14 within the assay period. We previously discovered that exposure of C. elegans to 25°C plate-grown PA14 at 20°C for 24hrs not only leads to these animals switching from attraction to avoidance of PA14, but also to their progeny avoiding PA14 in the naïve state, and this avoidance persists through the F4 generation. Other types of PA14 training can also cause P0 and/or F1 avoidance, but do not induce transgenerational (F2 and beyond) inheritance. We also previously showed that the transgenerational (P0-F4) learned avoidance is mediated by P11, a small RNA produced by PA14. P11 is both necessary and sufficient for transgenerational epigenetic inheritance of avoidance behavior. P11 is highly expressed in our standard growth conditions (25°C on surfaces), but not in other conditions, suggesting that reported failure to observe F2-F4 avoidance is most likely due to the absence of P11 expression in PA14 in the experimenters’ growth conditions. Through mutant analyses, we have tested many genes – including germline regulators, small RNA uptake, RNA interference/processing, chromatin modifiers, and neuronal genes - for their involvement in transgenerational inheritance of learned pathogen avoidance, allowing us to better understand the molecular requirements for this process. We previously found that wild C. elegans strains also show TEI of learned pathogen avoidance, and that at least two other wild bacteria, P. vranovensis and P. fluorescens 15, induce this transgenerational avoidance. The avoidance induced by each Pseudomonas species functions through a specific, distinct small RNA (Pv1 in P. vranovensis and Pfs1 in P. fluorescens 15 , respectively) that either directly or indirectly reduce the levels of the gene maco-1 , which in turn regulates daf-7 expression in the ASI neuron and subsequent avoidance behavior. The conservation of multiple components of this small RNA TEI mechanism across C. elegans strains and in multiple Pseudomonas species suggests that this transgenerational learned avoidance behavior is likely to be functional and physiologically important in wild conditions.