To coordinate cellular physiology, eukaryotic cells rely on the inter-organelle transfer of molecules at specialized organelle-organelle contact sites1,2. Endoplasmic reticulum-mitochondria contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signaling molecules, lipids, and metabolites3. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle4,5. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation6,7, a clear understanding of their nanoscale structure and regulation is still lacking. Here, we combine 3D electron microscopy with high-speed molecular tracking of a model organelle tether, VAPB, to map the structure and diffusion landscape of ERMCSs. From EM reconstructions, we identified subdomains within the contact site where ER membranes dramatically deform to match local mitochondrial curvature. In parallel live cell experiments, we observed that the VAPB tethers that mediate this interface were not immobile, but rather highly dynamic, entering and leaving the site in seconds. These subdomains enlarged during nutrient stress, indicating ERMCSs can readily remodel under different physiological conditions. An ALS-associated mutation in VAPB altered the normal fluidity of contact sites, likely perturbing effective communication across the contact site and preventing remodeling. These results establish high speed single molecule imaging as a new tool for mapping the structure of contact site interfaces and suggest that the diffusion landscape of VAPB is a crucial component of ERMCS homeostasis.

To coordinate cellular physiology, eukaryotic cells rely on the rapid exchange of molecules at specialized organelle-organelle contact sites. Endoplasmic reticulum-mitochondrial contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signalling molecules, lipids and metabolites. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation, a clear understanding of their nanoscale organization and regulation is still lacking. Here we combine three-dimensional electron microscopy with high-speed molecular tracking of a model organelle tether, Vesicle-associated membrane protein (VAMP)-associated protein B (VAPB), to map the structure and diffusion landscape of ERMCSs. We uncovered dynamic subdomains within VAPB contact sites that correlate with ER membrane curvature and undergo rapid remodelling. We show that VAPB molecules enter and leave ERMCSs within seconds, despite the contact site itself remaining stable over much longer time scales. This metastability allows ERMCSs to remodel with changes in the physiological environment to accommodate metabolic needs of the cell. An amyotrophic lateral sclerosis-associated mutation in VAPB perturbs these subdomains, likely impairing their remodelling capacity and resulting in impaired interorganelle communication. These results establish high-speed single-molecule imaging as a new tool for mapping the structure of contact site interfaces and reveal that the diffusion landscape of VAPB at contact sites is a crucial component of ERMCS homeostasis.

Functional imaging in behaving animals is essential to understanding brain function. However, artifacts resulting from animal motion, including locomotion, can severely corrupt functional measurements. To dampen tissue motion, we designed a new optical window with minimal optical aberrations. Using the newly developed high-speed continuous volumetric imaging system based on an optical phase-locked ultrasound lens, we quantified motion of the cerebral cortex and hippocampal surface during two-photon functional imaging in behaving mice. We find that the out-of-plane motion is generally greater than the axial dimension of the point-spread-function during mouse locomotion, which indicates that high-speed continuous volumetric imaging is necessary to minimize motion artifacts.

Many animals utilize acoustic signals-or songs-to attract mates. During courtship, Drosophila melanogaster males vibrate a wing to produce trains of pulses and extended tone, called pulse and sine song, respectively. Courtship songs in the genus Drosophila are exceedingly diverse, and different song features appear to have evolved independently of each other. How the nervous system allows such diversity to evolve is not understood. Here, we identify a wing muscle in D. melanogaster (hg1) that is uniquely male-enlarged. The hg1 motoneuron and the sexually dimorphic development of the hg1 muscle are required specifically for the sine component of the male song. In contrast, the motoneuron innervating a sexually monomorphic wing muscle, ps1, is required specifically for a feature of pulse song. Thus, individual wing motor pathways can control separate aspects of courtship song and may provide a "modular" anatomical substrate for the evolution of diverse songs.

Following considerable progress on the molecular and cellular basis of taste perception in fly sensory neurons, the time is now ripe to explore how taste information, integrated with hunger and satiety, undergo a sensorimotor transformation to lead to the motor actions of feeding behavior. I examine what is known of feeding circuitry in adult flies from more than 250 years of work in larger flies and from newer work in Drosophila. I review the anatomy of the proboscis, its muscles and their functions (where known), its motor neurons, interneurons known to receive taste inputs, interneurons that diverge from taste circuitry to provide information to other circuits, interneurons from other circuits that converge on feeding circuits, proprioceptors that influence the motor control of feeding, and sites of integration of hunger and satiety on feeding circuits. In spite of the several neuron types now known, a connected pathway from taste inputs to feeding motor outputs has yet to be found. We are on the threshold of an era where these individual components will be assembled into circuits, revealing how nervous system architecture leads to the control of behavior.

Many forms of human and animal behavior involve head movements. A new study reveals the neural code for three-dimensional head movements in the midbrain of freely moving mice.

Skillful control of movement is central to our ability to sense and manipulate the world. A large body of work in nonhuman primates has demonstrated that motor cortex provides flexible, time-varying activity patterns that control the arm during reaching and grasping. Previous studies have suggested that these patterns are generated by strong local recurrent dynamics operating autonomously from inputs during movement execution. An alternative possibility is that motor cortex requires coordination with upstream brain regions throughout the entire movement in order to yield these patterns. Here, we developed an experimental preparation in the mouse to directly test these possibilities using optogenetics and electrophysiology during a skilled reach-to-grab-to-eat task. To validate this preparation, we first established that a specific, time-varying pattern of motor cortical activity was required to produce coordinated movement. Next, in order to disentangle the contribution of local recurrent motor cortical dynamics from external input, we optogenetically held the recurrent contribution constant, then observed how motor cortical activity recovered following the end of this perturbation. Both the neural responses and hand trajectory varied from trial to trial, and this variability reflected variability in external inputs. To directly probe the role of these inputs, we used optogenetics to perturb activity in the thalamus. Thalamic perturbation at the start of the trial prevented movement initiation, and perturbation at any stage of the movement prevented progression of the hand to the target; this demonstrates that input is required throughout the movement. By comparing motor cortical activity with and without thalamic perturbation, we were able to estimate the effects of external inputs on motor cortical population activity. Thus, unlike pattern-generating circuits that are local and autonomous, such as those in the spinal cord that generate left-right alternation during locomotion, the pattern generator for reaching and grasping is distributed across multiple, strongly-interacting brain regions.

The interaction of descending neocortical outputs and subcortical premotor circuits is critical for shaping skilled movements. Two broad classes of motor cortical output projection neurons provide input to many subcortical motor areas: pyramidal tract (PT) neurons, which project throughout the neuraxis, and intratelencephalic (IT) neurons, which project within the cortex and subcortical striatum. It is unclear whether these classes are functionally in series or whether each class carries distinct components of descending motor control signals. Here, we combine large-scale neural recordings across all layers of motor cortex with cell type-specific perturbations to study cortically dependent mouse motor behaviors: kinematically variable manipulation of a joystick and a kinematically precise reach-to-grasp. We find that striatum-projecting IT neuron activity preferentially represents amplitude, whereas pons-projecting PT neurons preferentially represent the variable direction of forelimb movements. Thus, separable components of descending motor cortical commands are distributed across motor cortical projection cell classes.

After committing to an action, a decision-maker can change their mind to revise the action. Such changes of mind can even occur when the stream of information that led to the action is curtailed at movement onset. This is explained by the time delays in sensory processing and motor planning which lead to a component at the end of the sensory stream that can only be processed after initiation. Such post-initiation processing can explain the pattern of changes of mind by asserting an accumulation of additional evidence to a criterion level, termed change-of-mind bound. Here we test the hypothesis that physical effort associated with the movement required to change one's mind affects the level of the change-of-mind bound and the time for post-initiation deliberation. We varied the effort required to change from one choice target to another in a reaching movement by varying the geometry of the choice targets or by applying a force field between the targets. We show that there is a reduction in the frequency of change of mind when the separation of the choice targets would require a larger excursion of the hand from the initial to the opposite choice. The reduction is best explained by an increase in the evidence required for changes of mind and a reduced time period of integration after the initial decision. Thus the criteria to revise an initial choice is sensitive to energetic costs.

Motor neurons are the final common pathway through which the brain controls movement of the body, forming the basic elements from which all movement is composed. Yet how a single motor neuron contributes to control during natural movement remains unclear. Here we anatomically and functionally characterize the individual roles of the motor neurons that control head movement in the fly, Drosophila melanogaster. Counterintuitively, we find that activity in a single motor neuron rotates the head in different directions, depending on the starting posture of the head, such that the head converges towards a pose determined by the identity of the stimulated motor neuron. A feedback model predicts that this convergent behaviour results from motor neuron drive interacting with proprioceptive feedback. We identify and genetically suppress a single class of proprioceptive neuron that changes the motor neuron-induced convergence as predicted by the feedback model. These data suggest a framework for how the brain controls movements: instead of directly generating movement in a given direction by activating a fixed set of motor neurons, the brain controls movements by adding bias to a continuing proprioceptive-motor loop.