Inducible and reversible silencing of selected neurons in vivo is critical to understanding the structure and dynamics of brain circuits. We have developed Molecules for Inactivation of Synaptic Transmission (MISTs) that can be genetically targeted to allow the reversible inactivation of neurotransmitter release. MISTs consist of modified presynaptic proteins that interfere with the synaptic vesicle cycle when crosslinked by small molecule "dimerizers." MISTs based on the vesicle proteins VAMP2/Synaptobrevin and Synaptophysin induced rapid ( approximately 10 min) and reversible block of synaptic transmission in cultured neurons and brain slices. In transgenic mice expressing MISTs selectively in Purkinje neurons, administration of dimerizer reduced learning and performance of the rotarod behavior. MISTs allow for specific, inducible, and reversible lesions in neuronal circuits and may provide treatment of disorders associated with neuronal hyperactivity.

Connectomics-the study of how neurons wire together in the brain-is at the forefront of modern neuroscience research. However, many connectomics studies are limited by the time and precision needed to correctly segment large volumes of electron microscopy (EM) image data. We present here a semi-automated segmentation pipeline using freely available software that can significantly decrease segmentation time for extracting both nuclei and cell bodies from EM image volumes.

Two methods for rapid characterization of molecular shape are presented. Both techniques are based on the density of atoms near the molecular surface. The Fast Atomic Density Evaluation (FADE) algorithm uses fast Fourier transforms to quickly estimate densities. The Pairwise Atomic Density Reverse Engineering (PADRE) method derives modified density measures from the relationship between atomic density and total potentials. While many shape-characterization techniques define shape relative to a surface, the descriptors returned by FADE and PADRE can measure local geometry from points within the three-dimensional space surrounding a molecule. The methods can be used to find crevices and protrusions near the surface of a molecule and to test shape complementarity at the interface between docking molecules.

A wide range of organisms use sex pheromones to communicate with each other and to identify appropriate mating partners. While the evolution of chemical communication has been suggested to cause sexual isolation and speciation, the mechanisms that govern evolutionary transitions in sex pheromone production are poorly understood. Here, we decipher the molecular mechanisms underlying the rapid evolution in the expression of a gene involved in sex pheromone production in Drosophilid flies. Long-chain cuticular hydrocarbons (e.g., dienes) are produced female-specifically, notably via the activity of the desaturase DESAT-F, and are potent pheromones for male courtship behavior in Drosophila melanogaster. We show that across the genus Drosophila, the expression of this enzyme is correlated with long-chain diene production and has undergone an extraordinary number of evolutionary transitions, including six independent gene inactivations, three losses of expression without gene loss, and two transitions in sex-specificity. Furthermore, we show that evolutionary transitions from monomorphism to dimorphism (and its reversion) in desatF expression involved the gain (and the inactivation) of a binding-site for the sex-determination transcription factor, DOUBLESEX. In addition, we documented a surprising example of the gain of particular cis-regulatory motifs of the desatF locus via a set of small deletions. Together, our results suggest that frequent changes in the expression of pheromone-producing enzymes underlie evolutionary transitions in chemical communication, and reflect changing regimes of sexual selection, which may have contributed to speciation among Drosophila.

Spine growth and retraction with synapse formation and elimination plays an important role in shaping brain circuits during development and in the adult brain, yet the temporal relationship between spine morphogenesis and the formation of functional synapses remains poorly defined. We imaged hippocampal pyramidal neurons to identify spines of different ages. We then used two-photon glutamate uncaging, whole-cell recording, and Ca(2+) imaging to analyze the properties of nascent spines and their older neighbors. New spines expressed glutamate-sensitive currents that were indistinguishable from mature spines of comparable volumes. Some spines exhibited negligible AMPA receptor-mediated responses, but the occurrence of these "silent" spines was uncorrelated with spine age. In contrast, NMDA receptor-mediated Ca(2+) accumulations were significantly lower in new spines. New spines reconstructed using electron microscopy made synapses. Our data support a model in which outgrowth and enlargement of nascent spines is tightly coupled to formation and maturation of glutamatergic synapses.

Imaging neurons and neural circuits over large volumes at high speed and subcellular resolution is a difficult task. Incorporating a Bessel focus module into a two-photon fluorescence mesoscope, we achieved rapid volumetric imaging of neural activity over the mesoscale with synaptic resolution. We applied the technology to calcium imaging of entire dendritic spans of neurons as well as neural ensembles within multiple cortical regions over two hemispheres of the awake mouse brain.

The Drosophila cuticle carries a rich array of morphological details. Thus, cuticle examination has had a central role in the history of genetics. This protocol describes a procedure for mounting adult cuticles in Hoyer's medium, a useful mountant for both larval and adult cuticles. The medium digests soft tissues rapidly, leaving the cuticle cleared for observation. In addition, samples can be transferred directly from water to Hoyer's medium. However, specimens mounted in Hoyer's medium degrade over time. For example, the fine denticles on the larval dorsum are best observed soon after mounting; they begin to fade after 1 week, and can disappear completely after several months. More robust features, such as the ventral denticle belts, will persist for a longer period of time. Because adults cannot profitably be mounted whole in Hoyer's medium, some dissection is necessary.

Electron microscopy (EM) allows for the reconstruction of dense neuronal connectomes but suffers from low throughput, limiting its application to small numbers of reference specimens. We developed a protocol and analysis pipeline using tissue expansion and lattice light-sheet microscopy (ExLLSM) to rapidly reconstruct selected circuits across many samples with single synapse resolution and molecular contrast. We validate this approach in Drosophila, demonstrating that it yields synaptic counts similar to those obtained by EM, can be used to compare counts across sex and experience, and to correlate structural connectivity with functional connectivity. This approach fills a critical methodological gap in studying variability in the structure and function of neural circuits across individuals within and between species.

To successfully perform goal-directed navigation, animals must know where they are and what they are doing—e.g., looking for water, bringing food back to the nest, or escaping from a predator. Hippocampal neurons code for these critical variables conjunctively, but little is known about how this where/what code is formed or flexibly routed to other brain regions. To address these questions, we performed intracellular whole-cell recordings in mouse CA1 during a cued, two-choice virtual navigation task. We demonstrate that plateau potentials in CA1 pyramidal neurons rapidly strengthen synaptic inputs carrying conjunctive information about position and choice. Plasticity-induced response fields were modulated by cues only in animals previously trained to collect rewards based on these cues. Thus, we reveal that gradual learning is required for the formation of a conjunctive population code, upstream of CA1, while plateau-potential-induced synaptic plasticity in CA1 enables flexible routing of the code to downstream brain regions.