Biology subjects

Baines, R. A.

Publications and source records attributed to Baines, R. A..

13 recordsLinked to original sources

bioRxiv2025

Neuronal excitability is permanently altered by activity manipulation during an embryonic critical period in Drosophila

Neuronal intrinsic excitability provides the baseline that homeostatic mechanisms act to preserve, yet the processes that establish a baseline remain poorly defined. Developmental critical periods (CPs) are thought to play a central role, but the link between early activity and long-term intrinsic properties is not well characterised. To address this, we used the genetic tractability of the Drosophila larval locomotor circuit to manipulate individual neurons during an embryonic CP. Following optogenetic excitation or inhibition, during the CP, we assessed intrinsic excitability of the same neurons in third-instar larvae (i.e. [~]5 days thereafter). We compared an excitatory premotor interneuron (A27h), an inhibitory premotor interneuron (A31k), and a motor neuron (aCC). Both interneurons exhibited anti-homeostatic responses: excitatory perturbation increased intrinsic excitability, while inhibitory perturbation decreased it, effects that persisted throughout larval development. In contrast, motor neurons showed no significant changes under the same conditions, revealing cell type-specific sensitivity to early activity. These findings build on the general principles of the functional relationships between CP activity and neuronal excitability and how intrinsic excitability is not passively set but actively shaped during these windows, with long-lasting, neuron-specific consequences. More broadly, our results highlight how developmental perturbations can alter the excitatory-inhibitory balance of mature neural circuits that may contribute to the aetiology of neurodevelopmental disorders.

neuroscience↗

bioRxiv2025

How the Drosophila Cryptochrome C-terminus mediates magnetosensitivity

The Earths magnetic field plays an important role in the seasonal migrations of many species of animals. A Cryptochrome (CRY)-based radical pair mechanism (RPM) has been suggested to underlie the mechanistic basis of animal magnetosensitivity and navigation. The quantum spin state of a radical pair involving flavin adenine dinucleotide (FAD) bound to CRY in the canonical pocket is sensitive to external magnetic fields that can alter the signalling concentration of activated CRY1-5. However, several experimental observations challenge this model including the finding that the C-terminal fragment of Drosophila CRY (DmCRY), which lacks any canonical FAD binding pocket, and human CRY2, which lacks affinity for FAD, are sufficient to support magnetosensitivity6-9. Here, we use all-atom molecular dynamic (MD) simulations, alongside in vitro and in vivo analyses to reveal that the C-terminus of Drosophila CRY (DmCRY-CT) binds FAD. FAD binding is required for transduction of a magnetic signal within cells, and, in vitro, initiates formation of high molecular weight DmCRY-CT oligomers, including large insoluble aggregates reminiscent of CRY photobodies observed in plants10-14. These results provide a plausible mechanistic basis for several experimental observations that have reported non-canonical magnetosensitivity in animals.

biochemistry↗

bioRxiv2025

Circadian Rhythms Time Seizure Severity in Drosophila

It is established that epilepsy patients can exhibit 24-hour rhythms in seizure severity and occurrence. While the pathways underlying seizure rhythmicity remain poorly understood, it seems likely that a contribution from the biological clock is involved. A better understanding of any such contribution may translate to better treatments. Here, the influence of the 24 h circadian rhythm on seizure activity in Drosophila melanogaster is investigated. Seizure-susceptible bang-sensitive julius seizure (jus) mutants were subjected to mechanically induced seizure at six different zeitgeber points. A clear sex- dependent phenotype was observed, with seizure severity showing a greater time-of-day effect in females, than males. The temporal pattern of seizure recovery time was bimodal, exhibiting both a morning and an evening peak. Rearing flies in constant light, which renders the molecular clock dysfunctional, abolished the seizure rhythm. Conversely, female seizure mutants reared in constant darkness, allowing free running of the circadian clock, continued to exhibit a bimodal rhythm of seizure severity. These findings support a role for the biological clock in seizure activity, at least in female Drosophila. Thus, this study validates Drosophila as a potential model for the identification of mechanisms modulating seizure rhythmicity, with the potential to aid future treatment of epilepsy.

neuroscience↗

bioRxiv2024

Conserved mitochondrial reactive oxygen species and HIF1-alpha signalling during an embryonic critical period directs subsequent nervous system development

As developing networks transition from spontaneous irregular to patterned activity, they undergo plastic tuning phases, termed "critical periods"; "critical" because disturbances during these phases can lead to lasting changes in network development and output. Critical periods are common to developing nervous systems, with analogous features shared from insects to mammals, yet the core signalling mechanisms that underly cellular critical period plasticity have remained elusive. To identify these, we exploited the Drosophila larval locomotor network as an advantageous model system. It has a defined critical period and offers unparalleled access to identified network elements, including the neuromuscular junction as a model synapse. We find that manipulations of a single motoneuron or muscle cell during the critical period lead to predictable, and permanent, cell-specific changes. This demonstrates that critical period adjustments occur at a single cell level. Mechanistically, we identified mitochondrial reactive oxygen species (ROS) as causative. Specifically, we show that ROS produced by complex I of the mitochondrial electron transport chain, generated by the reverse flow of electrons, are necessary and instructive for critical period-regulated plasticity. Downstream of ROS, we identified the Drosophila homologue of hypoxia inducible factor (HIF-1), as required for transducing the mitochondrial ROS signal to the nucleus. This signalling axis is also sufficient to cell autonomously specify changes in neuronal properties and animal behaviour but, again, only when activated during the embryonic critical period. Thus, we have identified specific mitochondrial ROS and HIF-1 as primary signals that mediate critical period plasticity.

developmental biology↗

bioRxiv2024

Circadian control in the timing of critical periods during Drosophila larval neuronal development.

Critical periods (CPs) of development are temporal windows of heightened neural plasticity. Activity perturbation during CPs can produce significant, and permanent, alterations to the development of neural circuits. In this study we report a circadian mechanism underlying the timing of CPs in Drosophila embryonic and larval development. These CPs occur at [~]24 hr intervals and are open to manipulation through blue light (BL)-activation of the circadian regulator Cryptochrome (CRY). This manipulation is sufficient to destabilize the larval CNS, evidenced by an induced seizure phenotype when tested at third instar (L3). In addition to CRY nulls, genetic ablation of the period gene also mitigates the BL exposure seizure phenotype and, moreover, alleles of period that affect circadian timing alter the timing of the CPs. Our analysis shows a clear role for the main clock neuropeptide, pigment dispersing factor (PDF), to transduce the output of these CPs. Targeted PDF receptor knockdown, in either GABAergic or CRY-positive neurons, is sufficient to prevent the CRY-mediated seizure phenotype. This study is a first demonstration of a circadian mechanism in Drosophila larvae, and whilst this alone is of major significance, our results highlight the potential of using Drosophila larvae as a model to investigate the impact of circadian rhythms on early neuronal development in higher organisms, which remains experimentally challenging. Significance StatementWhilst the role of the biological clock is well understood in adult organisms, the same is not true for embryonic development. How the maternal clock impacts the mammalian fetus remains poorly understood. Given that many expectant mothers experience altered circadian rhythms, largely due to nightshift working, it is important to address these concerns. Here we identify clock-mediated periods in neural development of the embryonic Drosophila which can be manipulated by light. These findings provide an experimental opportunity to better understand the role of the circadian clock in early development.

neuroscience↗

bioRxiv2023

Balance of Activity during a Critical Period Tunes a Developing Network

Developing neural circuits are influenced by activity and are especially sensitive to changes in activity during critical periods (CPs) of development. Changes occurring during a CP often become locked-in so that they affect the mature network. Indeed, several neurodevelopmental disorders have been linked to excessive activity during such periods. It is, therefore, important to identify those aspects of neural circuit development that are influenced by neural activity during a CP. In this study, we take advantage of the genetic tractability of Drosophila to show that activity perturbation during an embryonic CP permanently alters properties of the locomotor circuit. Specific changes we identify include increased synchronicity of motoneuron activity, and greater strengthening of excitatory over inhibitory synaptic drive to motoneurons. These changes are sufficient to reduce network robustness, evidenced by increased sensitivity to induced seizure. We also show that we can rescue these changes when increased activity is mitigated by inhibition provided by mechanosensory neurons. Similarly, we demonstrate a dose-dependent relationship between inhibition experienced during the CP, and the extent to which it is possible to rescue the hyperexcitable phenotype characteristic of the parabss mutation. This suggests that developing circuits must be exposed to a properly balanced sum of excitation and inhibition during the CP to achieve normal mature network function. Our results, therefore, provide novel insight into how activity during a CP shapes specific elements of a circuit, and how activity during this period is integrated to tune neural circuits to the environment in which they will likely function.

neuroscience↗

bioRxiv2022

Targeting neuronal homeostasis to prevent seizures

Manipulating neuronal homeostasis, which enables neurons to regulate their intrinsic excitability, offers an attractive opportunity to prevent seizures. However, no anticonvulsant compounds have yet been reported that directly manipulate neuronal homeostasis. Here, we describe a novel class of anticonvulsant compounds, based on 4-tert-butyl-benzaldehyde (4-TBB), with a mode-of-action that includes increased expression of the homeostatic regulator Pumilio (Pum). In Drosophila and mouse we use a pentylenetetrazole (PTZ) induced seizure model, and an electrically induced seizure model for refractory seizures to evaluate anticonvulsant efficacy. The pyrazole analogue (RAB216) demonstrates best efficacy, protecting 50% of mice from PTZ-induced seizure. Knock-down of Pum, in Drosophila, blocks anticonvulsive effects, whilst analysis of validated Pum targets show significant reductions following exposure of mouse brain to 4-TBB. This study provides proof-of-principle that anticonvulsant effects can be achieved through regulation of neuronal homeostasis and identifies a chemical lead compound for future development.

neuroscience↗

bioRxiv2021

Characterization of induction methods for Drosophila seizure mutations

Epilepsy is one of the most common neurological disorders. Around one third of patients do not respond to current medications. This lack of treatment indicates a need for better understanding of the underlying mechanisms and, importantly, the identification of novel targets for drug manipulation. The fruitfly Drosophila melanogaster has a fast reproduction time, powerful genetics, and facilitates large sample sizes, making it a strong model of seizure mechanisms. However, there has not yet been a systematic analysis of the wide range of behavioral and physiological phenotypes observed across major fly seizure genotypes. To understand this, we systematically measured seizure severity and secondary behavioral phenotypes at both the larval and adult stage. Comparison of several seizure-induction methods; specifically electrical, mechanical and heat-induction, show that larval electroshock is the most effective at inducing seizures across a wide range of seizure-prone mutants. Locomotion in adults and larvae was found to be non-predictive of seizure susceptibility. Recording activity in identified larval motor neurons revealed variations in action potential patterns, across different genotypes, but these patterns did not correlate with seizure susceptibility. To conclude, while there is wide variation in mechanical induction, heat induction, and secondary phenotypes, electroshock is the most consistent method of seizure induction across known major seizure genotypes in Drosophila. Significance StatementEpilepsy is a neurological disorder affecting 1 in 130 people globally, with a significant impact on patients, families, and society. Approximately one third of epileptics do not respond to currently available medication. Thus, better insights into underlying disease mechanisms and identification of new drugs are needed. Fruit flies (Drosophila melanogaster) are a powerful genetic model: a number of single gene mutant flies exhibit seizures, phenotypes that have been shown to respond to established antiepileptic drugs. We compare methods of seizure induction and their utility, to establish which induction method is the most consistent across a range of different seizure-inducing genetic backgrounds. Adopting a common method for seizure analysis in this model will, we predict, speed identification of novel anti-convulsive treatments.

neuroscience↗

bioRxiv2020

Electrophysiological validation of premotor interneurons monosynaptically connected to the aCC motoneuron in the Drosophila larval CNS.

Mapping the wired connectivity of a nervous system is a prerequisite for full understanding of function. In this respect, such endeavours can be likened to genome sequencing projects. These projects similarly produce impressive amounts of data which, whilst a technical tour-de-force, remain under-utilised without validation. Validation of neuron synaptic connectivity requires electrophysiology which has the necessary temporal and spatial resolution to map synaptic connectivity. However, this technique is not common and requires extensive equipment and training to master, particularly when applied to the small CNS of the Drosophila larva. Thus, validation of connectivity in this CNS has been more reliant on behavioural analyses and, in particular, activity imaging using the calcium-sensor GCaMP. Whilst both techniques are powerful, they each have significant limitations for this purpose. Here we use electrophysiology to validate an array of driver lines reported to label specific premotor interneurons that the Drosophila connectome project suggests are monosynaptically connected to an identified motoneuron termed the anterior corner cell (aCC). Our results validate this proposition for four selected lines. Thus, in addition to validating the connectome with respect to these four premotor interneurons, our study highlights the need to functionally validate driver lines prior to use.

neuroscience↗

bioRxiv2019

Motor circuit function is stabilized during postembryonic growth by anterograde trans-synaptic Jelly Belly - Anaplastic Lymphoma Kinase signaling

The brain adapts to a changing environment or growing body size by structural growth and synaptic plasticity. Mechanisms studied to date promote synaptic growth between partner neurons, while negative counterparts that inhibit such interactions have so far remained elusive. Here, we investigate the role of Jeb-Alk signaling in coordinating motor circuit growth during larval stages of Drosophila. We quantify neuronal growth dynamics by intra-vital imaging, and synaptogenesis at nanometer resolution using endogenously labeled synaptic proteins, conditionally tagged with a fluorophore, and link changes in circuit anatomy with altered synaptic physiology and behavior. We find that loss of Jeb-Alk signaling leads to increased strengthening of synaptic excitation by developmental addition of additional postsynaptic but not pre-synaptic specializations. These changes ultimately lead to an epilepsy-like seizure behavior. We thus demonstrate that trans-synaptic anterograde Jeb-Alk signaling acts to stabilize developmental plasticity and circuit function, and that it does so specifically during postembryonic growth.

neuroscience↗

bioRxiv2019

Activity manipulation of an excitatory interneuron, during an embryonic critical period, alters network tuning of the Drosophila larval locomotor circuit

As nervous systems develop, activity perturbations during critical periods can lead to permanently altered network function. However, how activity perturbation influences individual synapses, the network response and the underlying signalling mechanisms are not well understood. Here, we exploit a recently identified critical period in the development of the Drosophila larval locomotor circuit to show that activity perturbation differentially affects individual and identified synaptic pairings. Remarkably, we further show that activity-manipulation of a selective excitatory interneuron is sufficient to fully recapitulate the effects induced by network-wide activity disturbance; indicative that some neurons make a greater contribution to network tuning. We identify nitric oxide (NO)-signalling as a potential mediator of activity-dependent network tuning during the critical period. Significantly, the effect of NO-signalling to network tuning is dictated by the prior activity state of the network. Thus, this study provides mechanistic insight that is currently lacking into how activity during a critical period tunes a developing network.

neuroscience↗

bioRxiv2019

Regulation of neuron-specific gene transcription by stress hormone signalling requires synaptic activity in zebrafish

The Glucocorticoid Receptor (GR) co-ordinates metabolic and behavioural responses to stressors. We hypothesised that GR influences behaviour by modulating specific epigenetic and transcriptional processes in the brain. Using the zebrafish as a model organism, the brain methylomes of wild-type and grs357 mutant adults were analysed and GR-sensitive, differentially methylated regions (GR-DMRs) were identified. Two genes with GR-DMRs exhibited distinct methylation and transcriptional sensitivities to GR: the widely expressed direct GR target fkbp5 and neuron-specific aplp1. In larvae, neural activity is required for GR-mediated transcription of aplp1, but not for that of fkbp5. GR regulates metabotropic glutamate receptor gene expression, the activities of which also modulated aplp1 expression, implicating synaptic neurotransmission as an effector of GR function upstream of aplp1. Our results identify two distinct routes of GR-regulated transcription in the brain, including a pathway through which GR couples endocrine signalling to synaptic activity-regulated transcription by modulating metabotropic glutamate receptor expression.

developmental biology↗

bioRxiv2018

Split-QF system for fine-tuned transgene expression in Drosophila

The Q-system is a binary expression system that works well across species. Here we report the development of a split-QF system that drives strong expression, is repressible by QS and inducible by quinic acid. The split-QF system is fully compatible with existing split-GAL4 and split-LexA lines for advanced intersectional experiments, thus greatly expanding the range of possible anatomical, physiological and behavioural assays in Drosophila.

neuroscience↗