Recommendations for empowering early career researchers to improve research culture and practice.

Early career researchers (ECRs) are important stakeholders leading efforts to catalyze systemic change in research culture and practice. Here, we summarize the outputs from a virtual unconventional conference (unconference), which brought together 54 invited experts from 20 countries with extensive experience in ECR initiatives designed to improve the culture and practice of science. Together, we drafted 2 sets of recommendations for (1) ECRs directly involved in initiatives or activities to change research culture and practice; and (2) stakeholders who wish to support ECRs in these efforts. Importantly, these points apply to ECRs working to promote change on a systemic level, not only those improving aspects of their own work. In both sets of recommendations, we underline the importance of incentivizing and providing time and resources for systems-level science improvement activities, including ECRs in organizational decision-making processes, and working to dismantle structural barriers to participation for marginalized groups. We further highlight obstacles that ECRs face when working to promote reform, as well as proposed solutions and examples of current best practices. The abstract and recommendations for stakeholders are available in Dutch, German, Greek (abstract only), Italian, Japanese, Polish, Portuguese, Spanish, and Serbian.

High-Frequency Neuronal Bursting is Essential for Circadian and Sleep Behaviors in Drosophila.

Circadian rhythms have been extensively studied in Drosophila; however, still little is known about how the electrical properties of clock neurons are specified. We have performed a behavioral genetic screen through the downregulation of candidate ion channels in the lateral ventral neurons (LNvs) and show that the hyperpolarization-activated cation current Ih is important for the behaviors that the LNvs influence: temporal organization of locomotor activity, analyzed in males, and sleep, analyzed in females. Using whole-cell patch clamp electrophysiology we demonstrate that small LNvs (sLNvs) are bursting neurons, and that Ih is necessary to achieve the high-frequency bursting firing pattern characteristic of both types of LNvs in females. Since firing in bursts has been associated to neuropeptide release, we hypothesized that Ih would be important for LNvs communication. Indeed, herein we demonstrate that Ih is fundamental for the recruitment of pigment dispersing factor (PDF) filled dense core vesicles (DCVs) to the terminals at the dorsal protocerebrum and for their timed release, and hence for the temporal coordination of circadian behaviors.SIGNIFICANCE STATEMENT Ion channels are transmembrane proteins with selective permeability to specific charged particles. The rich repertoire of parameters that may gate their opening state, such as voltage-sensitivity, modulation by second messengers and specific kinetics, make this protein family a determinant of neuronal identity. Ion channel structure is evolutionary conserved between vertebrates and invertebrates, making any discovery easily translatable. Through a screen to uncover ion channels with roles in circadian rhythms, we have identified the Ih channel as an important player in a subset of clock neurons of the fruit fly. We show that lateral ventral neurons (LNvs) need Ih to fire action potentials in a high-frequency bursting mode and that this is important for peptide transport and the control of behavior.

Dopamine Signaling in Wake-Promoting Clock Neurons Is Not Required for the Normal Regulation of Sleep in Drosophila.

Dopamine is a wake-promoting neuromodulator in mammals and fruit flies. In Drosophila melanogaster, the network of clock neurons that drives sleep/activity cycles comprises both wake-promoting and sleep-promoting cell types. The large ventrolateral neurons (l-LNvs) and small ventrolateral neurons (s-LNvs) have been identified as wake-promoting neurons within the clock neuron network. The l-LNvs are innervated by dopaminergic neurons, and earlier work proposed that dopamine signaling raises cAMP levels in the l-LNvs and thus induces excitatory electrical activity (action potential firing), which results in wakefulness and inhibits sleep. Here, we test this hypothesis by combining cAMP imaging and patch-clamp recordings in isolated brains. We find that dopamine application indeed increases cAMP levels and depolarizes the l-LNvs, but, surprisingly, it does not result in increased firing rates. Downregulation of the excitatory D1-like dopamine receptor (Dop1R1) in the l-LNvs and s-LNvs, but not of Dop1R2, abolished the depolarization of l-LNvs in response to dopamine. This indicates that dopamine signals via Dop1R1 to the l-LNvs. Downregulation of Dop1R1 or Dop1R2 in the l-LNvs and s-LNvs does not affect sleep in males. Unexpectedly, we find a moderate decrease of daytime sleep with downregulation of Dop1R1 and of nighttime sleep with downregulation of Dop1R2. Since the l-LNvs do not use Dop1R2 receptors and the s-LNvs also respond to dopamine, we conclude that the s-LNvs are responsible for the observed decrease in nighttime sleep. In summary, dopamine signaling in the wake-promoting LNvs is not required for daytime arousal, but likely promotes nighttime sleep via the s-LNvs.SIGNIFICANCE STATEMENT In insect and mammalian brains, sleep-promoting networks are intimately linked to the circadian clock, and the mechanisms underlying sleep and circadian timekeeping are evolutionarily ancient and highly conserved. Here we show that dopamine, one important sleep modulator in flies and mammals, plays surprisingly complex roles in the regulation of sleep by clock-containing neurons. Dopamine inhibits neurons in a central brain sleep center to promote sleep and excites wake-promoting circadian clock neurons. It is therefore predicted to promote wakefulness through both of these networks. Nevertheless, our results reveal that dopamine acting on wake-promoting clock neurons promotes sleep, revealing a previously unappreciated complexity in the dopaminergic control of sleep.

Preparation of Pipettes and Pipette-Filling Devices for Patch-Clamping Drosophila Neurons.

An essential requirement of every laboratory procedure is to have all materials ready when they are needed, so that the experimental flow is not disrupted. This is particularly true for patch clamping; therefore, effort must be devoted in advance to produce materials such as patch pipettes. This can be a fiddly business; hence, this protocol provides step-by-step advice on how to pull and polish patch-clamp pipettes. It also includes a brief description on how to prepare homemade filling devices to deliver saline efficiently and inexpensively into the pipettes. The protocol ends with guidelines on how to change the filament of a Sutter horizontal puller, a dreaded yet necessary activity that should be learned by anyone who wishes to become an expert patch clamper.

Patch-Clamping Drosophila Brain Neurons.

Drosophila melanogaster is widely used as a model organism in all fields of biomedical research. In neuroscience, vast amounts of information have been gained using this little fly including the identification of neuronal circuits that regulate behaviors, the unraveling of their genetic underpinnings, and the molecular mechanisms involved. With plenty of genetic tools available to manipulate and infer neuronal activity, the direct measurement of electrical properties of fly neurons has lagged behind. This is due to the intricacies of performing electrical recordings in small cells such as fly central neurons. The patch-clamp technique offers the unique possibility of directly measuring the electrical properties of Drosophila neurons. This step-by-step protocol provides detailed advice for mastering this technique.

Dissection of Drosophila Adult Brains for Patch-Clamping Neurons.

The brain of adult flies (Drosophila melanogaster) has been studied in detail from several perspectives, including the anatomical and molecular characterization of hundreds of neuronal types. However, information regarding the electrophysiological properties of most neuronal types is lacking. This protocol provides detailed information on how to dissect the brain of adult flies to produce an ex vivo preparation in which central neurons can be patch-clamped. Immobilizing fresh and tiny tissues, such as fly brains, to perform successful patch-clamp recordings is a critical step; here, we explain how this can be achieved using cyanoacrylate glue.

Dissection of Drosophila Wandering Larval Brains for Patch-Clamping Neurons.

An enormous amount of neuroscientific knowledge has been gained from studying the larval stage of Drosophila From an electrophysiological point of view, the larval neuromuscular junction has played an important role in this quest for knowledge, as it presents practical advantages such as accessibility and a stereotypic pattern. The physiological properties of larval central neurons have been less explored, with information regarding mainly a few identified motoneurons available to date. This protocol describes a quick and easy dissection of the brain of wandering third-instar Drosophila larvae to produce an ex vivo preparation in which central neurons can be patch-clamped. Immobilizing fresh and tiny tissues, such as larval brains, to perform successful patch-clamp recordings is a crucial step; here we explain in detail how this can be achieved using cyanoacrylate glue.

Patch-Clamping Fly Brain Neurons.

The membrane potential of excitable cells, such as neurons and muscle cells, experiences a rich repertoire of dynamic changes mediated by an array of ligand- and voltage-gated ion channels. Central neurons, in particular, are fantastic computators of information, sensing, and integrating multiple subthreshold currents mediated by synaptic inputs and translating them into action potential patterns. Electrophysiology comprises a group of techniques that allow the direct measurement of electrical signals. There are many different electrophysiological approaches, but, because Drosophila neurons are small, the whole-cell patch-clamp technique is the only applicable method for recording electrical signals from individual central neurons. Here, we provide background on patch-clamp electrophysiology in Drosophila and introduce protocols for dissecting larval and adult brains, as well as for achieving whole-cell patch-clamp recordings of identified neuronal types. Patch clamping is a labor-intensive technique that requires a great deal of practice to become an expert; therefore, a steep learning curve should be anticipated. However, the instant gratification of neuronal spiking is an experience that we wish to share and disseminate, as many more Drosophila patch clampers are needed to study the electrical features of so many fly neuronal types unknown to date.

eLife's new model and its impact on science communication.

The eLife Early-Career Advisory Group discusses eLife's new peer review and publishing model, and how the whole process of scientific communication could be improved for the benefit of early-career researchers and the entire scientific community.

Mitigating the impact of conference and travel cancellations on researchers' futures.

The need to protect public health during the current COVID-19 pandemic has necessitated conference cancellations on an unprecedented scale. As the scientific community adapts to new working conditions, it is important to recognize that some of our actions may disproportionately affect early-career researchers and scientists from countries with limited research funding. We encourage all conference organizers, funders and institutions who are able to do so to consider how they can mitigate the unintended consequences of conference and travel cancellations and we provide seven recommendations for how this could be achieved. The proposed solutions may also offer long-term benefits for those who normally cannot attend conferences, and thus lead to a more equitable future for generations of researchers.

Ways to increase equity, diversity and inclusion.

The eLife Early-Career Advisory Group (ECAG), an international group of early-career researchers committed to improving research culture, calls for radical changes at eLife and other journals to address racism in the scientific community and to make science more diverse and inclusive.