Paul Szyszka

In turbulent environments, odorants from different sources arrive at different times, potentially providing cues for odor source segregation. In several invertebrate species, short differences in odorant onset enable freely moving animals to discriminate odorant mixtures. In vertebrates, however, studies of sensitivity to odorant onset asynchrony have been conducted under highly constrained sampling conditions, such as with odor delivery tightly coupled to respiration. In this study, we investigated whether domestic dogs could detect odorant onset asynchrony in odorant mixtures under conditions that preserve key features of natural odor sampling. Dogs performed a discrimination task in which odor stimuli were presented as ongoing pulse trains that began independently of animal behavior, avoiding artificial synchronization of odor delivery with sniff cycles. Dogs were trained to discriminate between mixtures of two odorants with synchronous onsets and mixtures with asynchronous onsets. Of the dogs trained, one was able to discriminate odorant onset asynchronies as short as 633 ms. Dogs also displayed sensitivity to auditory stimulus onset asynchrony, discriminating auditory asynchronies as short as 30 ms. These results provide the first demonstration of temporal sensitivity in canine olfaction and the first evidence that vertebrates can use odorant onset asynchrony under conditions that permit free odor sampling.

Loss-of-function variants in AP3B2, a neuronal adaptor protein required for synaptic vesicle formation, cause a severe early-onset neurodevelopmental epilepsy known as Developmental and Epileptic Encephalopathy 48 (DEE48). To investigate how AP3B2 loss alters brain development, leading to increased seizure susceptibility, we generated a Xenopus laevis model by targeting the orthologous gene using CRISPR/Cas9. Ap3b2.S-/- (mosaic) F0 tadpoles displayed increased locomotor activity with frequent seizure-like episodes when compared to sibling controls. Visualisation of forebrain and midbrain activity using the genetically encoded Ca2+ sensor GCaMP6s detected spontaneous, large amplitude, prolonged and widespread neural activity, alongside increased interhemispheric synchrony of both regions. Comparison of whole-brain transcriptomes from ap3b2 CRISPants and unedited sibling controls detected mainly downregulation of brain expressed genes, with significant over-representation of pathways involved in ion transport, axon formation and guidance, inhibitory (GABA) neurotransmission, and transport across the blood-brain barrier (BBB). In a simple assay for BBB integrity, CRISPant tadpoles were confirmed to have faster leakage of sodium fluorescein. Acute exposure to the angiotensin receptor blocker losartan significantly reduced locomotor hyperactivity, and CRISPant cohorts treated with losartan tended to have lower neural activity, indicating incomplete rescue of the ap3b2.S CRISPant phenotype. These findings demonstrate how AP3B2 loss of function alters brain development and the establishment of the BBB, with the resulting alterations in neurotransmitter pathways predisposing the brain to spontaneous seizures. Our results suggest that traditional anti-seizure medications designed to alter ion transport and GABA metabolism could be augmented with drugs targeting neuroinflammation, as adjunct seizure control options in infants with DEE48.

In insects, olfactory receptor neurons (ORNs) are localized in sensilla. Within a sensillum, different ORN types are typically co-localized and exhibit nonsynaptic reciprocal inhibition through ephaptic coupling. This inhibition is hypothesized to aid odor source discrimination in environments where odor molecules (odorants) are dispersed by wind, resulting in turbulent plumes. Under these conditions, odorants from a single source arrive at the ORNs synchronously, while those from separate sources arrive asynchronously. Ephaptic inhibition is expected to be weaker for asynchronous arriving odorants from separate sources, thereby enhancing their discrimination. Previous studies have focused on ephaptic inhibition of sustained ORN responses to constant odor stimuli. This begs the question of whether ephaptic inhibition also affects transient ORN responses and if this inhibition is modulated by the temporal arrival patterns of different odorants. To address this, we recorded co-localized ORNs in the fruit fly Drosophila melanogaster and exposed them to dynamic odorant mixtures. We found reciprocal inhibition, strongly suggesting the presence of ephaptic coupling. This reciprocal inhibition does indeed modulate transient ORN responses and is sensitive to the relative timing of odor stimuli. Notably, the strength of inhibition decreases as the synchrony and correlation between arriving odorants decrease. These results support the hypothesis that ephaptic inhibition aids odor source discrimination.

Insect olfactory receptor neurons (ORNs) are often co-localized within sensilla and exhibit non-synaptic reciprocal inhibition through ephaptic coupling. It has been postulated that this inhibition aids odour source discrimination, as synchronous arrival of different odour molecules (odorants) from a single source should increase ephaptic inhibition, whereas asynchronous arrival of odorants from different sources should decrease ephaptic inhibition. However, it was as yet unknown whether temporal arrival patterns of different odorants indeed modulate ephaptic inhibition, since past studies have focused on ephaptic inhibition of sustained ORN responses to prolonged and constant odour stimuli. However, most natural odour stimuli are not constant but rather transient and fluctuate as a result of dispersion in turbulent plumes in the air. To investigate the role of ephaptic inhibition in olfaction within turbulent environments, we recorded co-localized ORNs in the fruit fly Drosophila melanogaster exposed to dynamic odorant mixtures. We found that ephaptic inhibition does modulate transient ORN responses, and the strength of ephaptic inhibition decreases as the synchrony between arriving odorants decreases. These results support the hypothesis that ephaptic inhibition aids odour source discrimination.

Epilepsy, a clinical diagnosis characterised by paroxysmal episodes known as seizures, affects 1% of people worldwide. Safe and patient‐specific treatment is vital and can be achieved by the development of rapid pre‐clinical models of for identified epilepsy genes. Epilepsy can result from either brain injury or gene mutations, and can also be induced chemically. Xenopus laevis tadpoles could be a useful model for confirmation of variants of unknown significance found in epilepsy patients, and for drug re‐purposing screens that could eventually lead to benefits for patients. Here, we characterise and quantify seizure‐related behaviours in X. laevis tadpoles arrayed in 24‐well plates. To provoke acute seizure behaviours, tadpoles were chemically induced with either pentylenetetrazole (PTZ) or 4‐aminopyridine (4‐AP). To test the capacity to adapt this method for drug testing, we also exposed induced tadpoles to the anti‐seizure drug valproate (VPA). Four induced seizure‐like behaviours were described and manually quantified, and two of these (darting, circling) could be accurately detected automatically, using the video analysis software TopScan. Additionally, we recorded swimming trajectories and mean swimming velocity. Automatic detection showed that either PTZ or 4‐AP induced darting behaviour and increased mean swimming velocity compared to untreated controls. Both parameters were significantly reduced in the presence of VPA. In particular, darting behaviour was a shown to be a sensitive measure of epileptic seizure activity. While we could not automatically detect the full range of seizure behaviours, this method shows promise for future studies since X. laevis is a well‐characterised and genetically tractable model organism. image

Developmental and epileptic encephalopathies (DEE) are a genetically diverse group of disorders with similar early clinical presentations. DEE65 is caused by de novo , non-synonymous, gain-of-function mutations in CYFIP2. It presents in early infancy as hypotonia, epileptic spasms and global developmental delay. While modelling loss-of-function mutations can be done using knockdown or knockout techniques to reduce the amount of functional protein, modelling gain-of-function mutations requires different approaches. Here, we show that transient ectopic expression of the Arg87Cys pathogenic variant of cyfip2 mRNA in Xenopus laevis tadpoles resulted in increased seizure-related behaviours such as rapid darting and swimming in circles. In contrast, expression of a second pathological variant, Tyr108Cys, did not alter tadpole behaviour. Expression of either pathogenic variant resulted in spontaneous epileptic activity in the brain. For both variants, neuronal hyperactivity was reduced by treating the tadpole with 5 mM of the anti-seizure drug valproate (VPA). mRNA overexpression of gain-of-function variants in X. laevis tadpoles may be useful both for understanding the aetiology of DEE and for pre-clinical drug testing.

Epilepsy, a clinical diagnosis characterized by paroxysmal episodes known as seizures, affects 1% of people worldwide. Safe and patient-specific treatment is vital and can be achieved by the development of rapid pre-clinical models of for identified epilepsy genes. Epilepsy can result from either brain injury or gene mutations, and can also be induced chemically. Xenopus laevis tadpoles could be a useful model for confirmation of variants of unknown significance found in epilepsy patients, and for drug re-purposing screens that could eventually lead to benefits for patients. Here, we characterise and quantify seizure-related behaviours in X. laevis tadpoles arrayed in 24-well plates. To provoke acute seizure behaviours, tadpoles were chemically induced with either pentylenetetrazole (PTZ) or 4-aminopyridine (4-AP). To test the capacity to adapt this method for drug testing, we also exposed induced tadpoles to the anti-seizure drug valproate (VPA). Four induced seizure-like behaviours were described and manually quantified, and two of these (darting, circling) could be accurately detected automatically, using the video analysis software TopScan. Additionally, we recorded swimming trajectories and mean swimming velocity. Automatic detection showed that either PTZ or 4-AP induced darting behaviour and increased mean swimming velocity compared to untreated controls. Both parameters were significantly reduced in the presence of VPA. In particular, darting behaviour was a shown to be a sensitive measure of epileptic seizure activity. While we could not automatically detect the full range of seizure behaviours, this method shows promise for future studies, since X. laevis is a well-characterised and genetically tractable model organism.

Background: Pest insects are often baited with poisoned feeding stimulants, the most common of which are sugars. However, sugars are attractive for most animal species, which makes it difficult to target only a specific pest insect species. Here, we assessed different sugar alcohols for their potential as more species‐selective feeding stimulants for pest insects.Results: We tested the attractiveness of the sugar alcohols sorbitol, xylitol and erythritol with a capillary feeder assay in wasps (as potential pest insects, because introduced wasps are a pest in many regions) and bees (as non‐target insects). For the common wasp (Vespula vulgaris), sorbitol and xylitol acted as nutritive feeding stimulants, and erythritol acted as a non‐nutritive feeding stimulant. For the buff‐tailed bumble bee (Bombus terrestris), sorbitol acted as a feeding stimulant, while for the honey bee (Apis mellifera), none of the sugar alcohols acted as feeding stimulant.Conclusion: The species‐specific preferences for sugar alcohols suggest their potential as species‐selective insect baits. The wasp‐specific preference for xylitol suggests its potential as a bee‐safe alternative to sugar‐containing bait for controlling the common wasp. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.