Wings of Change: aPKC/FoxP-dependent plasticity in steering motor neurons underlies operant self-learning in Drosophila.

Background: Motor learning is central to human existence, such as learning to speak or walk, sports moves, or rehabilitation after injury. Evidence suggests that all forms of motor learning share an evolutionarily conserved molecular plasticity pathway. Here, we present novel insights into the neural processes underlying operant self-learning, a form of motor learning in the fruit fly Drosophila. Methods: We operantly trained wild type and transgenic Drosophila fruit flies, tethered at the torque meter, in a motor learning task that required them to initiate and maintain turning maneuvers around their vertical body axis (yaw torque). We combined this behavioral experiment with transgenic peptide expression, CRISPR/Cas9-mediated, spatio-temporally controlled gene knock-out and confocal microscopy. Results: We find that expression of atypical protein kinase C (aPKC) in direct wing steering motoneurons co-expressing the transcription factor FoxP is necessary for this type of motor learning and that aPKC likely acts via non-canonical pathways. We also found that it takes more than a week for CRISPR/Cas9-mediated knockout of FoxP in adult animals to impair motor learning, suggesting that adult FoxP expression is required for operant self-learning. Conclusions: Our experiments suggest that, for operant self-learning, a type of motor learning in Drosophila, co-expression of atypical protein kinase C (aPKC) and the transcription factor FoxP is necessary in direct wing steering motoneurons. Some of these neurons control the wing beat amplitude when generating optomotor responses, and we have discovered modulation of optomotor behavior after operant self-learning. We also discovered that aPKC likely acts via non-canonical pathways and that FoxP expression is also required in adult flies.

Replacing academic journals.

Replacing traditional journals with a more modern solution is not a new idea. Here, we propose ways to overcome the social dilemma underlying the decades of inaction. Any solution needs to not only resolve the current problems but also be capable of preventing takeover by corporations: it needs to replace traditional journals with a decentralized, resilient, evolvable network that is interconnected by open standards and open-source norms under the governance of the scholarly community. It needs to replace the monopolies connected to journals with a genuine, functioning and well-regulated market. In this new market, substitutable service providers compete and innovate according to the conditions of the scholarly community, avoiding sustained vendor lock-in. Therefore, a standards body needs to form under the governance of the scholarly community to allow the development of open scholarly infrastructures servicing the entire research workflow. We propose a redirection of money from legacy publishers to the new network by funding bodies broadening their minimal infrastructure requirements at recipient institutions to include modern infrastructure components replacing and complementing journal functionalities. Such updated eligibility criteria by funding agencies would help realign the financial incentives for recipient institutions with public and scholarly interest.

Mastodon over Mammon: towards publicly owned scholarly knowledge.

Twitter is in turmoil and the scholarly community on the platform is once again starting to migrate. As with the early internet, scholarly organizations are at the forefront of developing and implementing a decentralized alternative to Twitter, Mastodon. Both historically and conceptually, this is not a new situation for the scholarly community. Historically, scholars were forced to leave social media platform FriendFeed after it was bought by Facebook in 2006. Conceptually, the problems associated with public scholarly discourse subjected to the whims of corporate owners are not unlike those of scholarly journals owned by monopolistic corporations: in both cases the perils associated with a public good in private hands are palpable. For both short form (Twitter/Mastodon) and longer form (journals) scholarly discourse, decentralized solutions exist, some of which are already enjoying some institutional support. Here we argue that scholarly organizations, in particular learned societies, are now facing a golden opportunity to rethink their hesitations towards such alternatives and support the migration of the scholarly community from Twitter to Mastodon by hosting Mastodon instances. Demonstrating that the scholarly community is capable of creating a truly public square for scholarly discourse, impervious to private takeover, might renew confidence and inspire the community to focus on analogous solutions for the remaining scholarly record-encompassing text, data and code-to safeguard all publicly owned scholarly knowledge.

Collective action or individual choice: Spontaneity and individuality contribute to decision-making in Drosophila.

Our own unique character traits make our behavior consistent and define our individuality. Yet, this consistency does not entail that we behave repetitively like machines. Like humans, animals also combine personality traits with spontaneity to produce adaptive behavior: consistent, but not fully predictable. Here, we study an iconically rigid behavioral trait, insect phototaxis, that nevertheless also contains both components of individuality and spontaneity. In a light/dark T-maze, approximately 70% of a group of Drosophila fruit flies choose the bright arm of the T-Maze, while the remaining 30% walk into the dark. Taking the photopositive and the photonegative subgroups and re-testing them reveals the spontaneous component: a similar 70-30 distribution emerges in each of the two subgroups. Increasing the number of choices to ten choices, reveals the individuality component: flies with an extremely negative series of first choices were more likely to show photonegative behavior in subsequent choices and vice versa. General behavioral traits, independent of light/dark preference, contributed to the development of this individuality. The interaction of individuality and spontaneity together explains why group averages, even for such seemingly stereotypical behaviors, are poor predictors of individual choices.

Current market rates for scholarly publishing services.

For decades, the supra-inflation increase of subscription prices for scholarly journals has concerned scholarly institutions. After years of fruitless efforts to solve this "serials crisis", open access has been proposed as the latest potential solution. However, the prices for open access publishing are also high and are rising well beyond inflation. What has been missing from the public discussion so far is a quantitative approach to determine the actual costs of efficiently publishing a scholarly article using state-of-the-art technologies, such that informed decisions can be made as to appropriate price levels. Here we provide a granular, step-by-step calculation of the costs associated with publishing primary research articles, from submission, through peer-review, to publication, indexing and archiving. We find that these costs range from less than US$200 per article in modern, large-scale publishing platforms using post-publication peer-review, to about US$1,000 per article in prestigious journals with rejection rates exceeding 90%. The publication costs for a representative scholarly article today come to lie at around US$400. We discuss the additional non-publication items that make up the difference between publication costs and final price.

Sensitivity to expression levels underlies differential dominance of a putative null allele of the Drosophila tßh gene in behavioral phenotypes.

The biogenic amine octopamine (OA) and its precursor tyramine (TA) are involved in controlling a plethora of different physiological and behavioral processes. The tyramine-ß-hydroxylase (tßh) gene encodes the enzyme catalyzing the last synthesis step from TA to OA. Here, we report differential dominance (from recessive to overdominant) of the putative null tßhnM18 allele in 2 behavioral measures in Buridan's paradigm (walking speed and stripe deviation) and in proboscis extension (sugar sensitivity) in the fruit fly Drosophila melanogaster. The behavioral analysis of transgenic tßh expression experiments in mutant and wild-type flies as well as of OA and TA receptor mutants revealed a complex interaction of both aminergic systems. Our analysis suggests that the different neuronal networks responsible for the 3 phenotypes show differential sensitivity to tßh gene expression levels. The evidence suggests that this sensitivity is brought about by a TA/OA opponent system modulating the involved neuronal circuits. This conclusion has important implications for standard transgenic techniques commonly used in functional genetics.

The brain as a dynamically active organ.

Nervous systems are typically described as static networks passively responding to external stimuli (i.e., the 'sensorimotor hypothesis'). However, for more than a century now, evidence has been accumulating that this passive-static perspective is wrong. Instead, evidence suggests that nervous systems dynamically change their connectivity and actively generate behavior so their owners can achieve goals in the world, some of which involve controlling their sensory feedback. This review provides a brief overview of the different historical perspectives on general brain function and details some select modern examples falsifying the sensorimotor hypothesis.

Identification of FoxP circuits involved in locomotion and object fixation in Drosophila.

The FoxP family of transcription factors is necessary for operant self-learning, an evolutionary conserved form of motor learning. The expression pattern, molecular function and mechanisms of action of the Drosophila FoxP orthologue remain to be elucidated. By editing the genomic locus of FoxP with CRISPR/Cas9, we find that the three different FoxP isoforms are expressed in neurons, but not in glia and that not all neurons express all isoforms. Furthermore, we detect FoxP expression in, e.g. the protocerebral bridge, the fan-shaped body and in motor neurons, but not in the mushroom bodies. Finally, we discover that FoxP expression during development, but not adulthood, is required for normal locomotion and landmark fixation in walking flies. While FoxP expression in the protocerebral bridge and motor neurons is involved in locomotion and landmark fixation, the FoxP gene can be excised from dorsal cluster neurons and mushroom-body Kenyon cells without affecting these behaviours.

Memory, anticipation, action - working with Troy D. Zars.

We present here our reflections on the scientific work of the late Troy D. Zars (1967 - 2018), on what it was like to work with him, and what it means to us. A common theme running through his work is that memory systems are not for replaying the past. Rather, they are forward-looking systems, providing whatever guidance past experience has to offer for anticipating the outcome of future actions. And in situations where no such guidance is available trying things out is the best option. Working with Troy was inspiring precisely because of the optimism inherent in this concept and that he himself embodied. Our reflections highlight what this means to us as his former mentors, colleagues, and mentees, respectively, and what it might mean for the future of neurogenetics.

MARGO (Massively Automated Real-time GUI for Object-tracking), a platform for high-throughput ethology.

Fast object tracking in real time allows convenient tracking of very large numbers of animals and closed-loop experiments that control stimuli for many animals in parallel. We developed MARGO, a MATLAB-based, real-time animal tracking suite for custom behavioral experiments. We demonstrated that MARGO can rapidly and accurately track large numbers of animals in parallel over very long timescales, typically when spatially separated such as in multiwell plates. We incorporated control of peripheral hardware, and implemented a flexible software architecture for defining new experimental routines. These features enable closed-loop delivery of stimuli to many individuals simultaneously. We highlight MARGO's ability to coordinate tracking and hardware control with two custom behavioral assays (measuring phototaxis and optomotor response) and one optogenetic operant conditioning assay. There are currently several open source animal trackers. MARGO's strengths are 1) fast and accurate tracking, 2) high throughput, 3) an accessible interface and data output and 4) real-time closed-loop hardware control for for sensory and optogenetic stimuli, all of which are optimized for large-scale experiments.

Reliable novelty: New should not trump true.

Although a case can be made for rewarding scientists for risky, novel science rather than for incremental, reliable science, novelty without reliability ceases to be science. The currently available evidence suggests that the most prestigious journals are no better at detecting unreliable science than other journals. In fact, some of the most convincing studies show a negative correlation, with the most prestigious journals publishing the least reliable science. With the credibility of science increasingly under siege, how much longer can we afford to reward novelty at the expense of reliability? Here, I argue for replacing the legacy journals with a modern information infrastructure that is governed by scholars. This infrastructure would allow renewed focus on scientific reliability, with improved sort, filter, and discovery functionalities, at massive cost savings. If these savings were invested in additional infrastructure for research data and scientific code and/or software, scientific reliability would receive additional support, and funding woes-for, e.g., biological databases-would be a concern of the past.

Corrigendum: Prestigious Science Journals Struggle to Reach Even Average Reliability.

[This corrects the article DOI: 10.3389/fnhum.2018.00037.].

Prestigious Science Journals Struggle to Reach Even Average Reliability.

In which journal a scientist publishes is considered one of the most crucial factors determining their career. The underlying common assumption is that only the best scientists manage to publish in a highly selective tier of the most prestigious journals. However, data from several lines of evidence suggest that the methodological quality of scientific experiments does not increase with increasing rank of the journal. On the contrary, an accumulating body of evidence suggests the inverse: methodological quality and, consequently, reliability of published research works in several fields may be decreasing with increasing journal rank. The data supporting these conclusions circumvent confounding factors such as increased readership and scrutiny for these journals, focusing instead on quantifiable indicators of methodological soundness in the published literature, relying on, in part, semi-automated data extraction from often thousands of publications at a time. With the accumulating evidence over the last decade grew the realization that the very existence of scholarly journals, due to their inherent hierarchy, constitutes one of the major threats to publicly funded science: hiring, promoting and funding scientists who publish unreliable science eventually erodes public trust in science.

Octopamine and Tyramine Contribute Separately to the Counter-Regulatory Response to Sugar Deficit in Drosophila.

All animals constantly negotiate external with internal demands before and during action selection. Energy homeostasis is a major internal factor biasing action selection. For instance, in addition to physiologically regulating carbohydrate mobilization, starvation-induced sugar shortage also biases action selection toward food-seeking and food consumption behaviors (the counter-regulatory response). Biogenic amines are often involved when such widespread behavioral biases need to be orchestrated. In mammals, norepinephrine (noradrenalin) is involved in the counterregulatory response to starvation-induced drops in glucose levels. The invertebrate homolog of noradrenalin, octopamine (OA) and its precursor tyramine (TA) are neuromodulators operating in many different neuronal and physiological processes. Tyrosine-ß-hydroxylase (tßh) mutants are unable to convert TA into OA. We hypothesized that tßh mutant flies may be aberrant in some or all of the counter-regulatory responses to starvation and that techniques restoring gene function or amine signaling may elucidate potential mechanisms and sites of action. Corroborating our hypothesis, starved mutants show a reduced sugar response and their hemolymph sugar concentration is elevated compared to control flies. When starved, they survive longer. Temporally controlled rescue experiments revealed an action of the OA/TA-system during the sugar response, while spatially controlled rescue experiments suggest actions also outside of the nervous system. Additionally, the analysis of two OA- and four TA-receptor mutants suggests an involvement of both receptor types in the animals' physiological and neuronal response to starvation. These results complement the investigations in Apis mellifera described in our companion paper (Buckemüller et al., 2017).

A decision underlies phototaxis in an insect.

Like a moth into the flame-phototaxis is an iconic example for innate preferences. Such preferences probably reflect evolutionary adaptations to predictable situations and have traditionally been conceptualized as hard-wired stimulus-response links. Perhaps for that reason, the century-old discovery of flexibility in Drosophila phototaxis has received little attention. Here, we report that across several different behavioural tests, light/dark preference tested in walking is dependent on various aspects of flight. If we temporarily compromise flying ability, walking photopreference reverses concomitantly. Neuronal activity in circuits expressing dopamine and octopamine, respectively, plays a differential role in photopreference, suggesting a potential involvement of these biogenic amines in this case of behavioural flexibility. We conclude that flies monitor their ability to fly, and that flying ability exerts a fundamental effect on action selection in Drosophila This work suggests that even behaviours which appear simple and hard-wired comprise a value-driven decision-making stage, negotiating the external situation with the animal's internal state, before an action is selected.

PKC in motorneurons underlies self-learning, a form of motor learning in Drosophila.

Tethering a fly for stationary flight allows for exquisite control of its sensory input, such as visual or olfactory stimuli or a punishing infrared laser beam. A torque meter measures the turning attempts of the tethered fly around its vertical body axis. By punishing, say, left turning attempts (in a homogeneous environment), one can train a fly to restrict its behaviour to right turning attempts. It was recently discovered that this form of operant conditioning (called operant self-learning), may constitute a form of motor learning in Drosophila. Previous work had shown that Protein Kinase C (PKC) and the transcription factor dFoxP were specifically involved in self-learning, but not in other forms of learning. These molecules are specifically involved in various forms of motor learning in other animals, such as compulsive biting in Aplysia, song-learning in birds, procedural learning in mice or language acquisition in humans. Here we describe our efforts to decipher which PKC gene is involved in self-learning in Drosophila. We also provide evidence that motorneurons may be one part of the neuronal network modified during self-learning experiments. The collected evidence is reminiscent of one of the simplest, clinically relevant forms of motor learning in humans, operant reflex conditioning, which also relies on motorneuron plasticity.

Unique transposon landscapes are pervasive across Drosophila melanogaster genomes.

To understand how transposon landscapes (TLs) vary across animal genomes, we describe a new method called the Transposon Insertion and Depletion AnaLyzer (TIDAL) and a database of >300 TLs in Drosophila melanogaster (TIDAL-Fly). Our analysis reveals pervasive TL diversity across cell lines and fly strains, even for identically named sub-strains from different laboratories such as the ISO1 strain used for the reference genome sequence. On average, >500 novel insertions exist in every lab strain, inbred strains of the Drosophila Genetic Reference Panel (DGRP), and fly isolates in the Drosophila Genome Nexus (DGN). A minority (<25%) of transposon families comprise the majority (>70%) of TL diversity across fly strains. A sharp contrast between insertion and depletion patterns indicates that many transposons are unique to the ISO1 reference genome sequence. Although TL diversity from fly strains reaches asymptotic limits with increasing sequencing depth, rampant TL diversity causes unsaturated detection of TLs in pools of flies. Finally, we show novel transposon insertions negatively correlate with Piwi-interacting RNA (piRNA) levels for most transposon families, except for the highly-abundant roo retrotransposon. Our study provides a useful resource for Drosophila geneticists to understand how transposons create extensive genomic diversity in fly cell lines and strains.

Drosophila FoxP mutants are deficient in operant self-learning.

Intact function of the Forkhead Box P2 (FOXP2) gene is necessary for normal development of speech and language. This important role has recently been extended, first to other forms of vocal learning in animals and then also to other forms of motor learning. The homology in structure and in function among the FoxP gene members raises the possibility that the ancestral FoxP gene may have evolved as a crucial component of the neural circuitry mediating motor learning. Here we report that genetic manipulations of the single Drosophila orthologue, dFoxP, disrupt operant self-learning, a form of motor learning sharing several conceptually analogous features with language acquisition. Structural alterations of the dFoxP locus uncovered the role of dFoxP in operant self-learning and habit formation, as well as the dispensability of dFoxP for operant world-learning, in which no motor learning occurs. These manipulations also led to subtle alterations in the brain anatomy, including a reduced volume of the optic glomeruli. RNAi-mediated interference with dFoxP expression levels copied the behavioral phenotype of the mutant flies, even in the absence of mRNA degradation. Our results provide evidence that motor learning and language acquisition share a common ancestral trait still present in extant invertebrates, manifest in operant self-learning. This 'deep' homology probably traces back to before the split between vertebrate and invertebrate animals.