Neurons in multiple brain regions fire trains of action potentials anticipating specific movements, but this 'preparatory activity' has not been systematically compared across behavioral tasks. We compared preparatory activity in auditory and tactile delayed-response tasks in male mice. Skilled, directional licking was the motor output. The anterior lateral motor cortex (ALM) is necessary for motor planning in both tasks. Multiple features of ALM preparatory activity during the delay epoch were similar across tasks. First, majority of neurons showed direction-selective activity and spatially intermingled neurons were selective for either movement direction. Second, many cells showed mixed coding of sensory stimulus and licking direction, with a bias toward licking direction. Third, delay activity was monotonic and low-dimensional. Fourth, pairs of neurons with similar direction selectivity showed high spike-count correlations. Our study forms the foundation to analyze the neural circuit mechanisms underlying preparatory activity in a genetically tractable model organism.Short-term memories link events separated in time. Neurons in frontal cortex fire trains of action potentials anticipating specific movements, often seconds before the movement. This 'preparatory activity' has been observed in multiple brain regions, but has rarely been compared systematically across behavioral tasks in the same brain region. To identify common features of preparatory activity, we developed and compared preparatory activity in auditory and tactile delayed-response tasks in mice. The same cortical area is necessary for both tasks. Multiple features of preparatory activity, measured with high-density silicon probes, were similar across tasks. We find that preparatory activity is low-dimensional and monotonic. Our study forms the foundation to analyze the circuit mechanisms underlying preparatory activity in a genetically tractable model organism.

Pathogen-mediated activation of macrophages arms innate immune responses that include enhanced surface ruffling and macropinocytosis for environmental sampling and receptor internalization and signaling. Activation of macrophages with bacterial lipopolysaccharide (LPS) generates prominent dorsal ruffles, which are precursors for macropinosomes. Very rapid, high-resolution imaging of live macrophages with lattice light sheet microscopy (LLSM) reveals new features and actions of dorsal ruffles, which redefine the process of macropinosome formation and closure. We offer a new model in which ruffles are erected and supported by F-actin tent poles that cross over and twist to constrict the forming macropinosomes. This process allows for formation of large macropinosomes induced by LPS. We further describe the enrichment of active Rab13 on tent pole ruffles and show that CRISPR deletion of Rab13 results in aberrant tent pole ruffles and blocks the formation of large LPS-induced macropinosomes. Based on the exquisite temporal and spatial resolution of LLSM, we can redefine the ruffling and macropinosome processes that underpin innate immune responses.

PURPOSE: To develop switchable and tunable labels with high contrast ratio for MRI using magnetocaloric materials that have sharp first-order magnetic phase transitions at physiological temperatures and typical MRI magnetic field strengths.

METHODS: A prototypical magnetocaloric material iron-rhodium (FeRh) was prepared by melt mixing, high-temperature annealing, and ice-water quenching. Temperature- and magnetic field-dependent magnetization measurements of wire-cut FeRh samples were performed on a vibrating sample magnetometer. Temperature-dependent MRI of FeRh samples was performed on a 4.7T MRI.

RESULTS: Temperature-dependent MRI clearly demonstrated image contrast changes due to the sharp magnetic state transition of the FeRh samples in the MRI magnetic field (4.7T) and at a physiologically relevant temperature (~37°C).

CONCLUSION: A magnetocaloric material, FeRh, was demonstrated to act as a high contrast ratio switchable MRI contrast agent due to its sharp first-order magnetic phase transition in the DC magnetic field of MRI and at physiologically relevant temperatures. A wide range of magnetocaloric materials are available that can be tuned by materials science techniques to optimize their response under MRI-appropriate conditions and be controllably switched in situ with temperature, magnetic field, or a combination of both.

Identifying the neurotransmitters used by specific neurons is a critical step in understanding the function of neural circuits. However, methods for the consistent and efficient detection of neurotransmitter markers remain limited. Fluorescence hybridization (FISH) enables direct labeling of type-specific mRNA in neurons. Recent advances in FISH allow this technique to be carried out in intact tissue samples such as whole-mount brains. Here, we present a FISH platform for high-throughput detection of eight common neurotransmitter phenotypes in brains. We greatly increase FISH throughput by processing samples mounted on coverslips and optimizing fluorophore choice for each probe to facilitate multiplexing. As application examples, we demonstrate cases of neurotransmitter co-expression, reveal neurotransmitter phenotypes of specific cell types and explore the onset of neurotransmitter expression in the developing optic lobe. Beyond neurotransmitter markers, our protocols can in principle be used for large scale FISH detection of any mRNA in whole-mount fly brains.

Command-like descending neurons can induce many behaviors, such as backward locomotion, escape, feeding, courtship, egg-laying, or grooming (we define 'command-like neuron' as a neuron whose activation elicits or 'commands' a specific behavior). In most animals it remains unknown how neural circuits switch between antagonistic behaviors: via top-down activation/inhibition of antagonistic circuits or via reciprocal inhibition between antagonistic circuits. Here we use genetic screens, intersectional genetics, circuit reconstruction by electron microscopy, and functional optogenetics to identify a bilateral pair of larval 'mooncrawler descending neurons' (MDNs) with command-like ability to coordinately induce backward locomotion and block forward locomotion; the former by stimulating a backward-active premotor neuron, and the latter by disynaptic inhibition of a forward-specific premotor neuron. In contrast, direct monosynaptic reciprocal inhibition between forward and backward circuits was not observed. Thus, MDNs coordinate a transition between antagonistic larval locomotor behaviors. Interestingly, larval MDNs persist into adulthood, where they can trigger backward walking. Thus, MDNs induce backward locomotion in both limbless and limbed animals.

We use super-resolution interferometric photoactivation and localization microscopy (iPALM) and a constrained photoactivatable fluorescent protein integrin fusion to measure the displacement of the head of integrin lymphocyte function-associated 1 (LFA-1) resulting from integrin conformational change on the cell surface. We demonstrate that the distance of the LFA-1 head increases substantially between basal and ligand-engaged conformations, which can only be explained at the molecular level by integrin extension. We further demonstrate that one class of integrin antagonist maintains the bent conformation, while another antagonist class induces extension. Our molecular scale measurements on cell-surface LFA-1 are in excellent agreement with distances derived from crystallographic and electron microscopy structures of bent and extended integrins. Our distance measurements are also in excellent agreement with a previous model of LFA-1 bound to ICAM-1 derived from the orientation of LFA-1 on the cell surface measured using fluorescence polarization microscopy.

Among the proteins required for lipid metabolism in Mycobacterium tuberculosis are a significant number of uncharacterized serine hydrolases, especially lipases and esterases. Using a streamlined synthetic method, a library of immolative fluorogenic ester substrates was expanded to better represent the natural lipidomic diversity of Mycobacterium. This expanded fluorogenic library was then used to rapidly characterize the global structure activity relationship (SAR) of mycobacterial serine hydrolases in M. smegmatis under different growth conditions. Confirmation of fluorogenic substrate activation by mycobacterial serine hydrolases was performed using nonspecific serine hydrolase inhibitors and reinforced the biological significance of the SAR. The hydrolases responsible for the global SAR were then assigned using gel-resolved activity measurements, and these assignments were used to rapidly identify the relative substrate specificity of previously uncharacterized mycobacterial hydrolases. These measurements provide a global SAR of mycobacterial hydrolase activity, a picture of cycling hydrolase activity, and a detailed substrate specificity profile for previously uncharacterized hydrolases.

A number of highly curved membranes in vivo, such as epithelial cell microvilli, have the relatively high sphingolipid content associated with "raft-like" composition. Given the much lower bending energy measured for bilayers with "nonraft" low sphingomyelin and low cholesterol content, observing high curvature for presumably more rigid compositions seems counterintuitive. To understand this behavior, we measured membrane rigidity by fluctuation analysis of giant unilamellar vesicles. We found that including a transmembrane helical GWALP peptide increases the membrane bending modulus of the liquid-disordered (Ld) phase. We observed this increase at both low-cholesterol fraction and higher, more physiological cholesterol fraction. We find that simplified, commonly used Ld and liquid-ordered (Lo) phases are not representative of those that coexist. When Ld and Lo phases coexist, GWALP peptide favors the Ld phase with a partition coefficient of 3-10 depending on mixture composition. In model membranes at high cholesterol fractions, Ld phases with GWALP have greater bending moduli than the Lo phase that would coexist.

Sodium (Na) is a ubiquitous and important inorganic salt mediating many critical biological processes such as neuronal excitation, signaling, and facilitation of various transporters. The hydration states of Na are proposed to play critical roles in determining the conductance and the selectivity of Na channels, yet they are rarely captured by conventional structural biology means. Here we use the emerging cryo-electron microscopy (cryoEM) method micro-electron diffraction (MicroED) to study the structure of a prototypical tetrameric Na-conducting channel, NaK, to 2.5 Å resolution from nano-crystals. Two new conformations at the external site of NaK are identified, allowing us to visualize a partially hydrated Na ion at the entrance of the channel pore. A process of dilation coupled with Na movement is identified leading to valuable insights into the mechanism of ion conduction and gating. This study lays the ground work for future studies using MicroED in membrane protein biophysics.

HIV-1 protease (PR) cleavage of the Gag polyprotein triggers the assembly of mature, infectious particles. Final cleavage of Gag occurs at the junction helix between the capsid protein CA and the SP1 spacer peptide. Here we used MicroED to delineate the binding interactions of the maturation inhibitor bevirimat (BVM) using very thin frozen-hydrated, 3D microcrystals of a CTD-SP1 Gag construct with and without bound BVM. The 2.9-Å MicroED structure revealed that a single BVM molecule stabilizes the six-helix bundle via both electrostatic interactions with the dimethylsuccinyl moiety and hydrophobic interactions with the pentacyclic triterpenoid ring. These results provide insight into the mechanism of action of BVM and related maturation inhibitors that will inform further drug discovery efforts. This study also demonstrates the capabilities of MicroED for structure-based drug design.