Rigorous science demands support of transgender scientists.

To build a just, equitable, and diverse academy, scientists and institutions must address systemic barriers that sex and gender minorities face. This Commentary summarizes (1) critical context informing the contemporary oppression of transgender people, (2) how this shapes extant research on sex and gender, and (3) actions to build an inclusive and rigorous academy for all.

Anipose: A toolkit for robust markerless 3D pose estimation.

Quantifying movement is critical for understanding animal behavior. Advances in computer vision now enable markerless tracking from 2D video, but most animals move in 3D. Here, we introduce Anipose, an open-source toolkit for robust markerless 3D pose estimation. Anipose is built on the 2D tracking method DeepLabCut, so users can expand their existing experimental setups to obtain accurate 3D tracking. It consists of four components: (1) a 3D calibration module, (2) filters to resolve 2D tracking errors, (3) a triangulation module that integrates temporal and spatial regularization, and (4) a pipeline to structure processing of large numbers of videos. We evaluate Anipose on a calibration board as well as mice, flies, and humans. By analyzing 3D leg kinematics tracked with Anipose, we identify a key role for joint rotation in motor control of fly walking. To help users get started with 3D tracking, we provide tutorials and documentation at http://anipose.org/.

Central processing of leg proprioception in Drosophila.

Proprioception, the sense of self-movement and position, is mediated by mechanosensory neurons that detect diverse features of body kinematics. Although proprioceptive feedback is crucial for accurate motor control, little is known about how downstream circuits transform limb sensory information to guide motor output. Here we investigate neural circuits in Drosophila that process proprioceptive information from the fly leg. We identify three cell types from distinct developmental lineages that are positioned to receive input from proprioceptor subtypes encoding tibia position, movement, and vibration. 13Bα neurons encode femur-tibia joint angle and mediate postural changes in tibia position. 9Aα neurons also drive changes in leg posture, but encode a combination of directional movement, high frequency vibration, and joint angle. Activating 10Bα neurons, which encode tibia vibration at specific joint angles, elicits pausing in walking flies. Altogether, our results reveal that central circuits integrate information across proprioceptor subtypes to construct complex sensorimotor representations that mediate diverse behaviors, including reflexive control of limb posture and detection of leg vibration.

A size principle for recruitment of Drosophila leg motor neurons.

To move the body, the brain must precisely coordinate patterns of activity among diverse populations of motor neurons. Here, we use in vivo calcium imaging, electrophysiology, and behavior to understand how genetically-identified motor neurons control flexion of the fruit fly tibia. We find that leg motor neurons exhibit a coordinated gradient of anatomical, physiological, and functional properties. Large, fast motor neurons control high force, ballistic movements while small, slow motor neurons control low force, postural movements. Intermediate neurons fall between these two extremes. This hierarchical organization resembles the size principle, first proposed as a mechanism for establishing recruitment order among vertebrate motor neurons. Recordings in behaving flies confirmed that motor neurons are typically recruited in order from slow to fast. However, we also find that fast, intermediate, and slow motor neurons receive distinct proprioceptive feedback signals, suggesting that the size principle is not the only mechanism that dictates motor neuron recruitment. Overall, this work reveals the functional organization of the fly leg motor system and establishes Drosophila as a tractable system for investigating neural mechanisms of limb motor control.

In the body, spindly nerve cells called motor neurons connect the brain to the muscles. Their role is to control movement, as they translate the electrical signals from the brain into instructions to the muscles. In humans, it takes over 150,000 motor neurons to control the movement of one leg; in contrast, fruit flies only need 50 neurons to operate a leg, despite also executing a variety of movements. Fruit flies are commonly used in laboratories to study an array of biological processes, yet little is known about how their motor neurons direct movements. In particular, it was unclear whether the same principles that control how muscles contract in mammals also applied in the tiny fruit fly. To begin investigating, Azevedo et al. mapped out the arrangement of motor neurons that control muscles in the fruit fly leg. As the leg moved, the activity of both the neurons and the muscles they controlled was recorded, as well as the force that had been generated. The experiments showed that each motor neuron controls a certain range of leg force and speed: some produced small, slow motion important for posture and dexterity, while others created large, fast movements essential to running or escape. In addition, the neurons activate in a particular order ­ cells that control slow movements fire first, and those that direct fast maneuvers later. These processes are also found in other organisms, but the difference is that flies have so few neurons, allowing scientists to reliably identify each motor neuron. Future experiments will therefore be able to test how flies recruit the right neurons to create specific movement sequences. Fruit flies are often used to research human illnesses that affect movement, such as motor neuron disease. A better understanding of the way their neural circuits coordinate the body could help reveal how these conditions emerge.

AMGSEFLamide, a member of a broadly conserved peptide family, modulates multiple neural networks in Homarus americanus.

Recent genomic/transcriptomic studies have identified a novel peptide family whose members share the carboxyl terminal sequence -GSEFLamide. However, the presence/identity of the predicted isoforms of this peptide group have yet to be confirmed biochemically, and no physiological function has yet been ascribed to any member of this peptide family. To determine the extent to which GSEFLamides are conserved within the Arthropoda, we searched publicly accessible databases for genomic/transcriptomic evidence of their presence. GSEFLamides appear to be highly conserved within the Arthropoda, with the possible exception of the Insecta, in which sequence evidence was limited to the more basal orders. One crustacean in which GSEFLamides have been predicted using transcriptomics is the lobster, Homarus americanus Expression of the previously published transcriptome-derived sequences was confirmed by reverse transcription (RT)-PCR of brain and eyestalk ganglia cDNAs; mass spectral analyses confirmed the presence of all six of the predicted GSEFLamide isoforms - IGSEFLamide, MGSEFLamide, AMGSEFLamide, VMGSEFLamide, ALGSEFLamide and AVGSEFLamide - in H. americanus brain extracts. AMGSEFLamide, of which there are multiple copies in the cloned transcripts, was the most abundant isoform detected in the brain. Because the GSEFLamides are present in the lobster nervous system, we hypothesized that they might function as neuromodulators, as is common for neuropeptides. We thus asked whether AMGSEFLamide modulates the rhythmic outputs of the cardiac ganglion and the stomatogastric ganglion. Physiological recordings showed that AMGSEFLamide potently modulates the motor patterns produced by both ganglia, suggesting that the GSEFLamides may serve as important and conserved modulators of rhythmic motor activity in arthropods.

Three members of a peptide family are differentially distributed and elicit differential state-dependent responses in a pattern generator-effector system.

C-type allatostatins (AST-Cs) are pleiotropic neuropeptides that are broadly conserved within arthropods; the presence of three AST-C isoforms, encoded by paralog genes, is common. However, these peptides are hypothesized to act through a single receptor, thereby exerting similar bioactivities within each species. We investigated this hypothesis in the American lobster, Homarus americanus, mapping the distributions of AST-C isoforms within relevant regions of the nervous system and digestive tract, and comparing their modulatory influences on the cardiac neuromuscular system. Immunohistochemistry showed that in the pericardial organ, a neuroendocrine release site, AST-C I and/or III and AST-C II are contained within distinct populations of release terminals. Moreover, AST-C I/III-like immunoreactivity was seen in midgut epithelial endocrine cells and the cardiac ganglion (CG), whereas AST-C II-like immunoreactivity was not seen in these tissues. These data suggest that AST-C I and/or III can modulate the CG both locally and hormonally; AST-C II likely acts on the CG solely as a hormonal modulator. Physiological studies demonstrated that all three AST-C isoforms can exert differential effects, including both increases and decreases, on contraction amplitude and frequency when perfused through the heart. However, in contrast to many state-dependent modulatory changes, the changes in contraction amplitude and frequency elicited by the AST-Cs were not functions of the baseline parameters. The responses to AST-C I and III, neither of which is COOH-terminally amidated, are more similar to one another than they are to the responses elicited by AST-C II, which is COOH-terminally amidated. These results suggest that the three AST-C isoforms are differentially distributed in the lobster nervous system/midgut and can elicit distinct behaviors from the cardiac neuromuscular system, with particular structural features, e.g., COOH-terminal amidation, likely important in determining the effects of the peptides. NEW & NOTEWORTHY Multiple isoforms of many peptides exert similar effects on neural circuits. In this study we show that each of the three isoforms of C-type allatostatin (AST-C) can exert differential effects, including both increases and decreases in contraction amplitude and frequency, on the lobster cardiac neuromuscular system. The distribution of effects elicited by the nonamidated isoforms AST-C I and III are more similar to one another than to the effects of the amidated AST-C II.

Non-amidated and amidated members of the C-type allatostatin (AST-C) family are differentially distributed in the stomatogastric nervous system of the American lobster, Homarus americanus.

The crustacean stomatogastric nervous system (STNS) is a well-known model for investigating neuropeptidergic control of rhythmic behavior. Among the peptides known to modulate the STNS are the C-type allatostatins (AST-Cs). In the lobster, Homarus americanus, three AST-Cs are known. Two of these, pQIRYHQCYFNPISCF (AST-C I) and GNGDGRLYWRCYFNAVSCF (AST-C III), have non-amidated C-termini, while the third, SYWKQCAFNAVSCFamide (AST-C II), is C-terminally amidated. Here, antibodies were generated against one of the non-amidated peptides (AST-C I) and against the amidated isoform (AST-C II). Specificity tests show that the AST-C I antibody cross-reacts with both AST-C I and AST-C III, but not AST-C II; the AST-C II antibody does not cross-react with either non-amidated peptide. Wholemount immunohistochemistry shows that both subclasses (non-amidated and amidated) of AST-C are distributed throughout the lobster STNS. Specifically, the antibody that cross-reacts with the two non-amidated peptides labels neuropil in the CoGs and the stomatogastric ganglion (STG), axons in the superior esophageal (son) and stomatogastric (stn) nerves, and ~ 14 somata in each commissural ganglion (CoG). The AST-C II-specific antibody labels neuropil in the CoGs, STG and at the junction of the sons and stn, axons in the sons and stn, ~ 42 somata in each CoG, and two somata in the STG. Double immunolabeling shows that, except for one soma in each CoG, the non-amidated and amidated peptides are present in distinct sets of neuronal profiles. The differential distributions of the two AST-C subclasses suggest that the two peptide groups are likely to serve different modulatory roles in the lobster STNS.