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.

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.

Coupling Neuropeptide Levels to Structural Plasticity in Drosophila Clock Neurons.

We have previously reported that pigment dispersing factor (PDF) neurons, which are essential in the control of rest-activity cycles in Drosophila, undergo circadian remodeling of their axonal projections, a phenomenon called circadian structural plasticity. Axonal arborizations display higher complexity during the day and become simpler at night, and this remodeling involves changes in the degree of connectivity. This phenomenon depends on the clock present within the ventrolateral neurons (LNvs) as well as in glia. In this work, we characterize in detail the contribution of the PDF neuropeptide to structural plasticity at different times across the day. Using diverse genetic strategies to temporally restrict its downregulation, we demonstrate that even subtle alterations to PDF cycling at the dorsal protocerebrum correlate with impaired remodeling, underscoring its relevance for the characteristic morning spread; PDF released from the small LNvs (sLNvs) and the large LNvs (lLNvs) contribute to the process. Moreover, forced depolarization recruits activity-dependent mechanisms to mediate growth only at night, overcoming the restriction imposed by the clock on membrane excitability. Interestingly, the active process of terminal remodeling requires PDF receptor (PDFR) signaling acting locally through the cyclic-nucleotide-gated channel ion channel subunit A (CNGA). Thus, clock-dependent PDF signaling shapes the connectivity of these essential clock neurons on daily basis.

Organization of Circadian Behavior Relies on Glycinergic Transmission.

The small ventral lateral neurons (sLNvs) constitute a central circadian pacemaker in the Drosophila brain. They organize daily locomotor activity, partly through the release of the neuropeptide pigment-dispersing factor (PDF), coordinating the action of the remaining clusters required for network synchronization. Despite extensive efforts, the basic principles underlying communication among circadian clusters remain obscure. We identified classical neurotransmitters released by sLNvs through disruption of specific transporters. Adult-specific RNAi-mediated downregulation of the glycine transporter or impairment of glycine synthesis in LNv neurons increased period length by nearly an hour without affecting rhythmicity of locomotor activity. Electrophysiological recordings showed that glycine reduces spiking frequency in circadian neurons. Interestingly, downregulation of glycine receptor subunits in specific sLNv targets impaired rhythmicity, revealing involvement of glycine in information processing within the network. These data identify glycinergic inhibition of specific targets as a cue that contributes to the synchronization of the circadian network.

Acetylcholine from Visual Circuits Modulates the Activity of Arousal Neurons in Drosophila.

Drosophila melanogaster's large lateral ventral neurons (lLNvs) are part of both the circadian and sleep-arousal neuronal circuits. In the past, electrophysiological analysis revealed that lLNvs fire action potentials (APs) in bursting or tonic modes and that the proportion of neurons firing in those specific patterns varies circadianly. Here, we provide evidence that lLNvs fire in bursts both during the day and at night and that the frequency of bursting is what is modulated in a circadian fashion. Moreover, we show that lLNvs AP firing is not only under cell autonomous control, but is also modulated by the network, and in the process we develop a novel preparation to assess this. We demonstrate that lLNv bursting mode relies on a cholinergic input because application of nicotinic acetylcholine receptor antagonists impairs this firing pattern. Finally, we found that bursting of lLNvs depends on an input from visual circuits that includes the cholinergic L2 monopolar neurons from the lamina. Our work sheds light on the physiological properties of lLNvs and on a neuronal circuit that may provide visual information to these important arousal neurons. SIGNIFICANCE STATEMENT: Circadian rhythms are important for organisms to be able to anticipate daily changes in environmental conditions to adjust physiology and behavior accordingly. These rhythms depend on an endogenous mechanism that operates in dedicated neurons. In the fruit fly, the large lateral ventral neurons (lLNvs) are part of both the circadian and sleep-arousal neuronal circuits. Here, we provide new details about the firing properties of these neurons and demonstrate that they depend, not only on cell-autonomous mechanisms, but also on a specific neurotransmitter derived from visual circuits. Our work sheds light on the physiological properties of lLNvs and on a neuronal circuit that may provide visual information to these important arousal neurons.

Pumilio-2 regulates translation of Nav1.6 to mediate homeostasis of membrane excitability.

The ability to regulate intrinsic membrane excitability, to maintain consistency of action potential firing, is critical for stable neural circuit activity. Without such mechanisms, Hebbian-based synaptic plasticity could push circuits toward activity saturation or, alternatively, quiescence. Although now well documented, the underlying molecular components of these homeostatic mechanisms remain poorly understood. Recent work in the fruit fly, Drosophila melanogaster, has identified Pumilio (Pum), a translational repressor, as an essential component of one such mechanism. In response to changing synaptic excitation, Pum regulates the translation of the voltage-gated sodium conductance, leading to a concomitant adjustment in action potential firing. Although similar homeostatic mechanisms are operational in mammalian neurons, it is unknown whether Pum is similarly involved. In this study, we report that Pum2 is indeed central to the homeostatic mechanism regulating membrane excitability in rat visual cortical pyramidal neurons. Using RNA interference, we observed that loss of Pum2 leads to increased sodium current (I(Na)) and action potential firing, mimicking the response by these neurons to being deprived of synaptic depolarization. In contrast, increased synaptic depolarization results in increased Pum2 expression and subsequent reduction in INa and membrane excitability. We further show that Pum2 is able to directly bind the predominant voltage-gated sodium channel transcript (NaV1.6) expressed in these neurons and, through doing so, regulates translation of this key determinant of membrane excitability. Together, our results show that Pum2 forms part of a homeostatic mechanism that matches membrane excitability to synaptic depolarization in mammalian neurons.

Adult-specific electrical silencing of pacemaker neurons uncouples molecular clock from circadian outputs.

BACKGROUND: Circadian rhythms regulate physiology and behavior through transcriptional feedback loops of clock genes running within specific pacemaker cells. In Drosophila, molecular oscillations in the small ventral lateral neurons (sLNvs) command rhythmic behavior under free-running conditions releasing the neuropeptide PIGMENT DISPERSING FACTOR (PDF) in a circadian fashion. Electrical activity in the sLNvs is also required for behavioral rhythmicity. Yet, how temporal information is transduced into behavior remains unclear. RESULTS: Here we developed a new tool for temporal control of gene expression to obtain adult-restricted electrical silencing of the PDF circuit, which led to reversible behavioral arrhythmicity. Remarkably, PERIOD (PER) oscillations during the silenced phase remained unaltered, indicating that arrhythmicity is a direct consequence of the silenced activity. Accordingly, circadian axonal remodeling and PDF accumulation were severely affected during the silenced phase. CONCLUSIONS: Although electrical activity of the sLNvs is not a clock component, it coordinates circuit outputs leading to rhythmic behavior.

Drosophila glial glutamate transporter Eaat1 is regulated by fringe-mediated notch signaling and is essential for larval locomotion.

In the mammalian CNS, glial cells expressing excitatory amino acid transporters (EAATs) tightly regulate extracellular glutamate levels to control neurotransmission and protect neurons from excitotoxic damage. Dysregulated EAAT expression is associated with several CNS pathologies in humans, yet mechanisms of EAAT regulation and the importance of glutamate transport for CNS development and function in vivo remain incompletely understood. Drosophila is an advanced genetic model with only a single high-affinity glutamate transporter termed Eaat1. We found that Eaat1 expression in CNS glia is regulated by the glycosyltransferase Fringe, which promotes neuron-to-glia signaling through the Delta-Notch ligand-receptor pair during embryogenesis. We made Eaat1 loss-of-function mutations and found that homozygous larvae could not perform the rhythmic peristaltic contractions required for crawling. We found no evidence for excitotoxic cell death or overt defects in the development of neurons and glia, and the crawling defect could be induced by postembryonic inactivation of Eaat1. Eaat1 fully rescued locomotor activity when expressed in only a limited subpopulation of glial cells situated near potential glutamatergic synapses within the CNS neuropil. Eaat1 mutants had deficits in the frequency, amplitude, and kinetics of synaptic currents in motor neurons whose rhythmic patterns of activity may be regulated by glutamatergic neurotransmission among premotor interneurons; similar results were seen with pharmacological manipulations of glutamate transport. Our findings indicate that Eaat1 expression is promoted by Fringe-mediated neuron-glial communication during development and suggest that Eaat1 plays an essential role in regulating CNS neural circuits that control locomotion in Drosophila.

Alternative splicing in the voltage-gated sodium channel DmNav regulates activation, inactivation, and persistent current.

Diversity in neuronal signaling is a product not only of differential gene expression, but also of alternative splicing. However, although recognized, the precise contribution of alternative splicing in ion channel transcripts to channel kinetics remains poorly understood. Invertebrates, with their smaller genomes, offer attractive models to examine the contribution of splicing to neuronal function. In this study we report the sequencing and biophysical characterization of alternative splice variants of the sole voltage-gated Na+ gene (DmNav, paralytic), in late-stage embryos of Drosophila melanogaster. We identify 27 unique splice variants, based on the presence of 15 alternative exons. Heterologous expression, in Xenopus oocytes, shows that alternative exons j, e, and f primarily influence activation kinetics: when present, exon f confers a hyperpolarizing shift in half-activation voltage (V1/2), whereas j and e result in a depolarizing shift. The presence of exon h is sufficient to produce a depolarizing shift in the V1/2 of steady-state inactivation. The magnitude of the persistent Na+ current, but not the fast-inactivating current, in both oocytes and Drosophila motoneurons in vivo is directly influenced by the presence of either one of a pair of mutually exclusive, membrane-spanning exons, termed k and L. Transcripts containing k have significantly smaller persistent currents compared with those containing L. Finally, we show that transcripts lacking all cytoplasmic alternatively spliced exons still produce functional channels, indicating that splicing may influence channel kinetics not only through change to protein structure, but also by allowing differential modification (i.e., phosphorylation, binding of cofactors, etc.). Our results provide a functional basis for understanding how alternative splicing of a voltage-gated Na+ channel results in diversity in neuronal signaling.

Pumilio binds para mRNA and requires Nanos and Brat to regulate sodium current in Drosophila motoneurons.

Homeostatic regulation of ionic currents is of paramount importance during periods of synaptic growth or remodeling. Our previous work has identified the translational repressor Pumilio (Pum) as a regulator of sodium current (I(Na)) and excitability in Drosophila motoneurons. In this current study, we show that Pum is able to bind directly the mRNA encoding the Drosophila voltage-gated sodium channel paralytic (para). We identify a putative binding site for Pum in the 3' end of the para open reading frame (ORF). Characterization of the mechanism of action of Pum, using whole-cell patch clamp and real-time reverse transcription-PCR, reveals that the full-length protein is required for translational repression of para mRNA. Additionally, the cofactor Nanos is essential for Pum-dependent para repression, whereas the requirement for Brain Tumor (Brat) is cell type specific. Thus, Pum-dependent regulation of I(Na) in motoneurons requires both Nanos and Brat, whereas regulation in other neuronal types seemingly requires only Nanos but not Brat. We also show that Pum is able to reduce the level of nanos mRNA and as such identify a potential negative-feedback mechanism to protect neurons from overactivity of Pum. Finally, we show coupling between I(Na) (para) and I(K) (Shal) such that Pum-mediated change in para results in a compensatory change in Shal. The identification of para as a direct target of Pum represents the first ion channel to be translationally regulated by this repressor and the location of the binding motif is the first example in an ORF rather than in the canonical 3'-untranslated region of target transcripts.

Down-regulation of torp4a, encoding the Drosophila homologue of torsinA, results in increased neuronal degeneration.

Early-onset torsion dystonia is a dominant motor disorder linked to mutations in torsinA. TorsinA is weakly related to a superfamily of chaperone-like proteins. The function of the torsin group remains largely unknown. Here we use RNAi and over-expression to analyze the function of torp4a, the only Drosophila torsin. Targeted down-regulation in the eye causes progressive degeneration of the retina. Conversely, over-expression of torp4a protects from age-related degeneration. In the retinas of young animals, a correlation with the lysosome-related organelle, the pigment granule, is also observed. Lowering torp4a causes an increase in pigment granules, while over-expression causes loss of granules. We have performed a screen for genetic interactors of torp4a identifying a number mutants, including two members of the AP-3 complex. Other genetic interactors found included genes related to actin and myosin function. Our findings implicate the Drosophila torsin, torp4a, to function with molecules consistent with already predicted roles in the endoplasmic reticulum/nuclear envelope compartment, and have identified potential new interactions with AP-3 like components.

Reversible demyelination, blood-brain barrier breakdown, and pronounced neutrophil recruitment induced by chronic IL-1 expression in the brain.

Interleukin-1beta (IL-1) expression is associated with a spectrum of neuroinflammatory processes related to chronic neurodegenerative diseases. The single-bolus microinjection of IL-1 into the central nervous system (CNS) parenchyma gives rise to delayed and localized neutrophil recruitment, transient blood-brain barrier (BBB) breakdown, but no overt damage to CNS integrity. However, acute microinjections of IL-1 do not mimic the chronic IL-1 expression, which is a feature of many CNS diseases. To investigate the response of the CNS to chronic IL-1 expression, we injected a recombinant adenovirus expressing IL-1 into the striatum. At the peak of IL-1 expression (days 8 and 14 post-injection), there was a marked recruitment of neutrophils, vasodilatation, and breakdown of the BBB. Microglia and astrocyte activation was evident during the first 14 days post-injection. At days 8 and 14, extensive demyelination was observed but the number of neurons was not affected by any treatment. Finally, at 30 days, signs of inflammation were no longer present, there was evidence of tissue reorganization, the BBB was intact, and the process of remyelination was noticeable. In summary, our data show that chronic expression of IL-1, in contrast to its acute delivery, can reversibly damage CNS integrity and implicates this cytokine or downstream components as major mediators of demyelination in chronic inflammatory and demyelinating diseases.