Mating and egg laying are tightly cooordinated events in the reproductive life of all oviparous females. Oviposition is typically rare in virgin females but is initiated after copulation. Here we identify the neural circuitry that links egg laying to mating status in Drosophila melanogaster. Activation of female-specific oviposition descending neurons (oviDNs) is necessary and sufficient for egg laying, and is equally potent in virgin and mated females. After mating, sex peptide-a protein from the male seminal fluid-triggers many behavioural and physiological changes in the female, including the onset of egg laying. Sex peptide is detected by sensory neurons in the uterus, and silences these neurons and their postsynaptic ascending neurons in the abdominal ganglion. We show that these abdominal ganglion neurons directly activate the female-specific pC1 neurons. GABAergic (γ-aminobutyric-acid-releasing) oviposition inhibitory neurons (oviINs) mediate feed-forward inhibition from pC1 neurons to both oviDNs and their major excitatory input, the oviposition excitatory neurons (oviENs). By attenuating the abdominal ganglion inputs to pC1 neurons and oviINs, sex peptide disinhibits oviDNs to enable egg laying after mating. This circuitry thus coordinates the two key events in female reproduction: mating and egg laying.

Chemogenetics enables non-invasive chemical control over cell populations in behaving animals. However, existing small molecule agonists show insufficient potency or selectivity. There is also need for chemogenetic systems compatible with both research and human therapeutic applications. We developed a new ion channel-based platform for cell activation and silencing that is controlled by low doses of the anti-smoking drug varenicline. We then synthesized novel sub-nanomolar potency agonists, called uPSEMs, with high selectivity for the chemogenetic receptors. uPSEMs and their receptors were characterized in brains of mice and a rhesus monkey by in vivo electrophysiology, calcium imaging, positron emission tomography, behavioral efficacy testing, and receptor counterscreening. This platform of receptors and selective ultrapotent agonists enables potential research and clinical applications of chemogenetics.

Neuronal diversity is essential for mammalian brain function but poses a challenge to molecular profiling. To address the need for tools that facilitate cell-type-specific epigenomic studies, we developed the first affinity purification approach to isolate nuclei from genetically defined cell types in a mammal. We combine this technique with next-generation sequencing to show that three subtypes of neocortical neurons have highly distinctive epigenomic landscapes. Over 200,000 regions differ in chromatin accessibility and DNA methylation signatures characteristic of gene regulatory regions. By footprinting and motif analyses, these regions are predicted to bind distinct cohorts of neuron subtype-specific transcription factors. Neuronal epigenomes reflect both past and present gene expression, with DNA hyper-methylation at developmentally critical genes appearing as a novel epigenomic signature in mature neurons. Taken together, our findings link the functional and transcriptional complexity of neurons to their underlying epigenomic diversity.

Fluorescent protein-based sensors for detecting neuronal activity have been developed largely based on non-neuronal screening systems. However, the dynamics of neuronal state variables (e.g., voltage, calcium, etc.) are typically very rapid compared to those of non-excitable cells. We developed an electrical stimulation and fluorescence imaging platform based on dissociated rat primary neuronal cultures. We describe its use in testing genetically-encoded calcium indicators (GECIs). Efficient neuronal GECI expression was achieved using lentiviruses containing a neuronal-selective gene promoter. Action potentials (APs) and thus neuronal calcium levels were quantitatively controlled by electrical field stimulation, and fluorescence images were recorded. Images were segmented to extract fluorescence signals corresponding to individual GECI-expressing neurons, which improved sensitivity over full-field measurements. We demonstrate the superiority of screening GECIs in neurons compared with solution measurements. Neuronal screening was useful for efficient identification of variants with both improved response kinetics and high signal amplitudes. This platform can be used to screen many types of sensors with cellular resolution under realistic conditions where neuronal state variables are in relevant ranges with respect to timing and amplitude.