Annika F. Barber

Traumatic brain injury (TBI) impacts millions of individuals annually causing death, disability, and a heightened risk for long-term neurological and neuropsychiatric disorders. In recent years the fruit fly, Drosophila melanogaster has become a valuable model organism to study the cellular and molecular responses following TBI. AP-1 mediated transcriptional responses to TBI have previously been identified in Drosophila using pan-glial approaches. Fruit flies possess multiple glial subtypes which vary greatly in both cellular morphology and function, including glia of the blood hemolymph barrier, cortex, astrocyte-like, and ensheathing glia. By generating and utilizing a nuclear localized AP-1 transcriptional reporter, we identified glial subtype-specific differences in the extent of AP-1 activation following injury. Our findings identify a strong AP-1 response in the blood hemolymph barrier and ensheathing glia, a moderate response in cortex glia and little to no AP-1 activation in astrocyte-like glia. In addition, we inhibited AP-1 signaling in each glial subtype and tested the effect on acute survival. We found that inhibition of the AP-1 response in neuropil ensheathing glia leads to increased mortality following mild and moderate TBI. These results show that AP-1 activation levels vary across glial subtypes after TBI, with activation in neuropil ensheathing glia having a particularly important role in promoting post-injury survival.

SUMMARY: Both aging and a high-fat diet (HFD) dampen circadian gene transcription rhythms and promote chronic inflammation. How aging and HFD interact to affect peripheral molecular clocks and circadian behavior remains unclear. Using Drosophila melanogaster , we showed that aging and HFD additively dampened circadian behavior, with their adverse effects converging on the fat body (FB), a tissue that regulates systemic metabolism and innate immunity. Applying longitudinal in vivo bioluminescence recording in small, genetically defined cell populations, we found that molecular clocks in the FB were uniquely vulnerable to aging- and HFD-induced dampening of rhythm amplitude, whereas those in the clock neurons declined with age but were resistant to dietary stress. To test the consequences of this FB clock decline, we disrupted individual components of the core molecular clock specifically in the FB. We found that only CLOCK (CLK) disruption shortened lifespan on HFD, whereas disrupting its binding partner CYCLE (CYC), or the repressors PERIOD and TIMELESS, did not. Furthermore, CLK, but not CYC, disruption upregulated antimicrobial peptide expression in the FB, dampened behavioral rhythms, and suppressed locomotor activity, even though both CLK and CYC disruption comparably dampened clock gene oscillation in the FB. Together, these results indicate that FB CLK has a unique role in suppressing pro-inflammatory signals independently of CYC. Our findings provide insight into how stressors such as aging and HFD selectively disrupt the peripheral metabolic clock, and into the distinct roles of individual clock components, with implications for age-related inflammation and metabolic disease.

Central pacemaker neurons use a combination of external stimuli and neuropeptide signaling to synchronize molecular oscillations leading to circadian behaviors. The clock network structure and signaling between these pacemaker neuron groups have been well described, but how these pacemakers communicate with specific brain output regions remains poorly understood. Here, we identified how “core” clock neurons in Drosophila , the ventrolateral neurons (LNvs), signal to the proto-hypothalamic region, the pars intercerebralis (PI). Previously thought to communicate with the PI only indirectly, we provide evidence to show that LNvs functionally modulate insulin-producing cells (IPCs) of the PI in a time-of-day-dependent manner. This functional connectivity relies on neuropeptidergic signaling of two classical clock neuropeptides: pigment dispersing factor (PDF) and short Neuropeptide F (sNPF). Connectomic analysis does not identify any direct synaptic inputs from clock neurons to IPCs. Small ventrolateral clock neurons, which secrete both PDF and sNPF are 15-20 μm away from IPCs, suggesting that volume transmission across these distances may be possible in the fly dorsal protocerebrum. Peptide application with functional imaging of IPCs provides insight into how these two neuropeptides may act synergistically via their receptors to signal to IPCs. Our findings indicate that LNvs can signal directly to IPCs by volume transmission and also form indirect multisynaptic circuits with IPCs, which may model more broadly how circadian clock peptides communicate with other clock output regions. Author Summary: Circadian clocks in the brains of animals from flies to humans allow animals to anticipate daily environmental changes and temporally coordinate internal processes. The central clock in the brain serves as a master pacemaker that sets the pace of circadian clocks in all other tissues. Fruit flies have been an essential model system for discovering the genetic and cellular underpinnings of circadian rhythms, with work in flies being awarded the 2017 Nobel Prize in Physiology or Medicine. Brain clocks in flies and mammals use classical synaptic communication and neuromodulatory peptides to signal between the neurons that make up the brain clock. Our study asks whether the neuropeptides that signal between clock neurons also signal from clock to non-clock neurons. We found that the signature fly clock neuropeptide pigment dispersing factor signals outside the brain clock to insulin producing cells of the brain. There are no synaptic connections from clock neurons to insulin producing cells, instead signaling occurs by diffusion of peptide signals across tens of microns, termed volume transmission. Our findings point to the possibility that neuropeptide volume transmission may be a general feature not only of intra-clock signaling but also of brain clock output signaling to non-clock neurons.

The second‐generation Drosophila traumatic brain injury (TBI) device dTBI2 improves Drosophila TBI administration by providing a moderate‐throughput, tunable, head‐specific injury. Our updated device design improves user‐friendliness, eliminates inconsistencies in injury timing, and has an updated circuit design to extend the longevity of delicate electronic components. dTBI2 improves reproducibility across users and runs, and results in more consistent post‐injury phenotypes. This protocol describes the construction, calibration, and use of the dTBI2 device, which uses an Arduino‐controlled piezoelectric actuator to deliver a force that compresses a fly head against a metal collar. The duration and depth of head compression is tunable, allowing calibration of injury severity. All device components are commercially available, and the entire device can be constructed in under a week for less than $1000. The dTBI2 design will enable any lab to build a highly reliable, low‐cost device for Drosophila TBI, facilitating increased adoption and ease of exploration of closed‐head TBI in Drosophila for forward genetic screens. We describe below the three protocols necessary for constructing a dTBI2 device. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Construction of the dTBI2 control deviceBasic Protocol 2: Construction of the piezoelectric actuator housingBasic Protocol 3: Administration of dTBI2 injuries

Circadian cycles of sleep:wake and gene expression change with age in all organisms examined. Metabolism is also under robust circadian regulation, but little is known about how metabolic cycles change with age and whether these contribute to the regulation of behavioral cycles. To address this gap, we compared cycling of metabolites in young and old Drosophila and found major age‐related variations. A significant model separated the young metabolic profiles by circadian timepoint, but could not be defined for the old metabolic profiles due to the greater variation in this dataset. Of the 159 metabolites measured in fly heads, we found 17 that cycle by JTK analysis in young flies and 17 in aged. Only four metabolites overlapped in the two groups, suggesting that cycling metabolites are distinct in young and old animals. Among our top cyclers exclusive to young flies were components of the pentose phosphate pathway (PPP). As the PPP is important for buffering reactive oxygen species, and overexpression of glucose‐6‐phosphate dehydrogenase (G6PD), a key component of the PPP, was previously shown to extend lifespan in Drosophila , we asked if this manipulation also affects sleep:wake cycles. We found that overexpression in circadian clock neurons decreases sleep in association with an increase in cellular calcium and mitochondrial oxidation, suggesting that altering PPP activity affects neuronal activity. Our findings elucidate the importance of metabolic regulation in maintaining patterns of neural activity, and thereby sleep:wake cycles.

The gut microbiome is well known to impact host physiology and health. Given widespread control of physiology by circadian clocks, we asked how the microbiome interacts with circadian rhythms in the Drosophila gut. The microbiome did not cycle in flies fed ad libitum, and timed feeding (TF) drove limited cycling only in clockless per ⁰¹ flies. However, TF and loss of the microbiome influenced the composition of the gut cycling transcriptome, independently and together. Moreover, both interventions increased the amplitude of rhythmic gene expression, with effects of TF at least partly due to changes in histone acetylation. Contrary to expectations, timed feeding rendered animals more sensitive to stress. Analysis of microbiome function in circadian physiology revealed that germ-free flies reset more rapidly with shifts in the light:dark cycle. We propose that the microbiome stabilizes cycling in the host gut to prevent rapid fluctuations with changing environmental conditions.