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Chemobehavioural phenotyping presents unique opportunities for analyzing neurotoxicants and discovering behavior-modifying neuroceuticals in small aquatic model organisms such as zebrafish (Danio rerio). A recently popularized approach in this field involves the utilization of zebrafish embryos for a photo-motor response (PMR) bioassay. The PMR bioassay entails stimulating zebrafish embryos between 24 and 36 h post fertilization (hpf) with a high-intensity light stimulus, inducing a transient increase in the frequency of photo-induced embryo body flexions. These flexions can be computationally analyzed to derive behavioral signatures, enabling the categorization of neuromodulating chemicals. Despite the significant advantages of the PMR bioassay, its widespread implementation is hindered by lack of well described and straightforward high-throughput bioinformatic analysis of behavioral data. In this methods article, we present an easily implementable bioinformatics protocol specifically designed for rapid behavioral analysis of large cohorts of zebrafish specimens in PMR bioassays. We also address common pitfalls encountered during PMR analysis, discuss its limitations, and propose future directions for developing next-generation biometric analysis techniques in chemobehavioural assays utilizing zebrafish embryos., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Assessment of animals' sensory-motor functions requires precise and electronically controlled stimuli to induce and quantify specific behavioral phenotypes. However, accessible and inexpensive tools for conducting diverse sensory-motor biotests with fish are lacking. In this work, we present an open-source software and hardware interface that enables automated delivery of three independent and fully programmable stimuli for behavioral bioassays. We demonstrate the proof-of-concept application of this low-cost technology in establishing reproducible fear responses using a mechanical tap-startle stimulus in larval zebrafish. This response is characterized by a sudden burst of motion in response to a nondirectional mechanical stimulus delivered to the fish chamber. We propose that the simplicity and flexibility of this interface offer innovative opportunities for studying sensory-motor functions in various fields, including neurobiology, neuropharmacology, neurotoxicology, and aquatic ecotoxicology.

Due to technical limitations, there have been minimal studies performed on thermal preferences and thermotactic behaviors of aquatic ectotherm species commonly used in ecotoxicity testing. In this work, we demonstrate an innovative, purpose-built and miniaturized electrothermal array for rapid thermal preference behavioral tests. We applied the novel platform to define thermal preferences in multiple invertebrate and vertebrate species. Specifically, Dugesia notogaea (freshwater planarians), Chironomus tepperi (nonbiting midge larvae), Ostracoda (seed shrimp), Artemia franciscana (brine shrimp), Daphnia carinata (water flea), Austrochiltonia subtenuis (freshwater amphipod), Physa acuta (freshwater snail), Potamopyrgus antipodarum (New Zealand mud snail) and larval stage of Danio rerio (zebrafish) were tested. The Australian freshwater water fleas, amphipods, snail Physa acuta as well as zebrafish exhibited the most consistent preference to cool zones and clear avoidance of zones >27 °C out of nine species tested. Our results indicate the larval stage of zebrafish as the most responsive species highly suitable for prospective development of multidimensional behavioral test batteries. We also showcase preliminary data that environmentally relevant concentrations of pharmaceutical pollutants such as non-steroidal anti-inflammatory drug (NSAID) ibuprofen (9800 ng/L) and insecticide imidacloprid (4600 ng/L) but not anti-depressant venlafaxine (2200 ng/L) and (iv) anticonvulsant medications gabapentin (400 ng/L) can perturb thermal preference behavior of larval zebrafish. Collectively our results demonstrate the utility of simple and inexpensive thermoelectric technology in rapid exploration of thermal preference in diverse species of aquatic animals. We postulate that more broadly such technologies can also have added value in ecotoxicity testing of emerging contaminants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Many targeted natural product isolation approaches rely on the use of pre-existing bioactivity information to inform the strategy used for the isolation of new bioactive compounds. Bioactivity information can be available either in the form of prior assay data or via Structure Activity Relationship (SAR) information which can indicate a potential chemotype that exhibits a desired bioactivity. The work described herein utilizes a unique method of targeted isolation using structure-based virtual screening to identify potential antibacterial compounds active against MRSA within the marine sponge order Verongiida. This is coupled with molecular networking-guided, targeted isolation to provide a novel drug discovery procedure. A total of 12 previously reported bromotyrosine-derived alkaloids were isolated from the marine sponge species Pseudoceratina durissima, and the compound, (+)-aeroplysinin-1 (1) displayed activity against the MRSA pathogen (MIC: <32 µg/mL). The compounds (1−3, 6 and 9) were assessed for their central nervous system (CNS) interaction and behavioral toxicity to zebrafish (Danio rerio) larvae, whereby several of the compounds were shown to induce significant hyperactivity. Anthelmintic activity against the parasitic nematode Haemonchus contorutus was also evaluated (2−4, 6−8)., Competing Interests: The authors declare no conflict of interest.

Due to increasing numbers of anthropogenic chemicals with unknown neurotoxic properties, there is an increasing need for a paradigm shift toward rapid and higher throughput behavioral bioassays. In this work, we demonstrate application of a purpose-built high throughput multidimensional behavioral test battery on larval stages of Danio rerio (zebrafish) at 5 days post fertilization (dpf). The automated battery comprised of the established spontaneous swimming (SS), simulated predator response (SPR), larval photomotor response (LPR) assays as well as a new thermotaxis (TX) assay. We applied the novel system to characterize environmentally relevant concentrations of emerging pharmaceutical micropollutants including anticonvulsants (gabapentin: 400 ng/L; carbamazepine: 3000 ng/L), inflammatory drugs (ibuprofen: 9800 ng/L), and antidepressants (fluoxetine: 300 ng/L; venlafaxine: 2200 ng/L). The successful integration of the thermal preference assay into a multidimensional behavioral test battery provided means to reveal ibuprofen-induced perturbations of thermal preference behaviors upon exposure during embryogenesis. Moreover, we discovered that photomotor responses in larval stages of fish are also altered by the as yet understudied anticonvulsant gabapentin. Collectively our results demonstrate the utility of high-throughput multidimensional behavioral ecotoxicity test batteries in prioritizing emerging risks associated with neuroactive drugs that can perturb neurodevelopment. Moreover, we showcase the added value of thermotaxis bioassays for preliminary screening of emerging contaminants.

Cell adhesion molecules are membrane-bound proteins predominantly expressed in the central nervous system along principal axonal pathways with key roles in nervous system development, neural cell differentiation and migration, axonal growth and guidance, myelination, and synapse formation. Here, we describe ten affected individuals with bi-allelic variants in the neuronal cell adhesion molecule NRCAM that lead to a neurodevelopmental syndrome of varying severity; the individuals are from eight families. This syndrome is characterized by developmental delay/intellectual disability, hypotonia, peripheral neuropathy, and/or spasticity. Computational analyses of NRCAM variants, many of which cluster in the third fibronectin type III (Fn-III) domain, strongly suggest a deleterious effect on NRCAM structure and function, including possible disruption of its interactions with other proteins. These findings are corroborated by previous in vitro studies of murine Nrcam-deficient cells, revealing abnormal neurite outgrowth, synaptogenesis, and formation of nodes of Ranvier on myelinated axons. Our studies on zebrafish nrcama Δ mutants lacking the third Fn-III domain revealed that mutant larvae displayed significantly altered swimming behavior compared to wild-type larvae (p < 0.03). Moreover, nrcama Δ mutants displayed a trend toward increased amounts of α-tubulin fibers in the dorsal telencephalon, demonstrating an alteration in white matter tracts and projections. Taken together, our study provides evidence that NRCAM disruption causes a variable form of a neurodevelopmental disorder and broadens the knowledge on the growing role of the cell adhesion molecule family in the nervous system., Competing Interests: Declaration of interests C.G.-J. is a full-time employee of the Regeneron Genetics Center and receives stock options as part of compensation. All other authors have no conflicts to declare., (Copyright © 2022 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Large-scale chemobehavioral phenotyping with zebrafish embryos is a promising avenue for accelerated neurotoxicity testing and discovery of behavior-modifying neuroceuticals. These strategies are hampered by lack of effective embryo in-test positioning, wide-field imaging, and high-throughput bioinformatic analytics. In this study, we demonstrate advantages of using custom large-density embryo arrays in conjunction with an open-source ultra-high-definition video imaging system. Moreover, we present a high-throughput bioinformatics workflow for rapid behavioral analysis of large cohorts of specimens in photomotor response bioassays. The system validation was showcased in a proof-of-concept neurotoxicity analysis.

Spermatogenesis-associated 5 like 1 (SPATA5L1) represents an orphan gene encoding a protein of unknown function. We report 28 bi-allelic variants in SPATA5L1 associated with sensorineural hearing loss in 47 individuals from 28 (26 unrelated) families. In addition, 25/47 affected individuals (53%) presented with microcephaly, developmental delay/intellectual disability, cerebral palsy, and/or epilepsy. Modeling indicated damaging effect of variants on the protein, largely via destabilizing effects on protein domains. Brain imaging revealed diminished cerebral volume, thin corpus callosum, and periventricular leukomalacia, and quantitative volumetry demonstrated significantly diminished white matter volumes in several individuals. Immunofluorescent imaging in rat hippocampal neurons revealed localization of Spata5l1 in neuronal and glial cell nuclei and more prominent expression in neurons. In the rodent inner ear, Spata5l1 is expressed in the neurosensory hair cells and inner ear supporting cells. Transcriptomic analysis performed with fibroblasts from affected individuals was able to distinguish affected from controls by principal components. Analysis of differentially expressed genes and networks suggested a role for SPATA5L1 in cell surface adhesion receptor function, intracellular focal adhesions, and DNA replication and mitosis. Collectively, our results indicate that bi-allelic SPATA5L1 variants lead to a human disease characterized by sensorineural hearing loss (SNHL) with or without a nonprogressive mixed neurodevelopmental phenotype., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

Mucopolysaccharidosis IIIA (MPS IIIA, Sanfilippo syndrome type A), a paediatric neurological lysosomal storage disease, is caused by impaired function of the enzyme N -sulfoglucosamine sulfohydrolase (SGSH) resulting in impaired catabolism of heparan sulfate glycosaminoglycan (HS GAG) and its accumulation in tissues. MPS IIIA represents a significant proportion of childhood dementias. This condition generally leads to patient death in the teenage years, yet no effective therapy exists for MPS IIIA and a complete understanding of the mechanisms of MPS IIIA pathogenesis is lacking. Here, we employ targeted CRISPR/Cas9 mutagenesis to generate a model of MPS IIIA in the zebrafish, a model organism with strong genetic tractability and amenity for high-throughput screening. The sgsh Δex5-6 zebrafish mutant exhibits a complete absence of Sgsh enzymatic activity, leading to progressive accumulation of HS degradation products with age. sgsh Δex5-6 zebrafish faithfully recapitulate diverse CNS-specific features of MPS IIIA, including neuronal lysosomal overabundance, complex behavioural phenotypes, and profound, lifelong neuroinflammation. We further demonstrate that neuroinflammation in sgsh Δex5-6 zebrafish is largely dependent on interleukin-1β and can be attenuated via the pharmacological inhibition of Caspase-1, which partially rescues behavioural abnormalities in sgsh Δex5-6 mutant larvae in a context-dependent manner. We expect the sgsh Δex5-6 zebrafish mutant to be a valuable resource in gaining a better understanding of MPS IIIA pathobiology towards the development of timely and effective therapeutic interventions.

An early step in pancreas development is marked by the expression of the transcription factor Pdx1 within the pancreatic endoderm, where it is required for the specification of all endocrine cell types. Subsequently, Pdx1 expression becomes restricted to the β-cell lineage, where it plays a central role in β-cell function. This pivotal role of Pdx1 at various stages of pancreas development makes it an attractive target to enhance pancreatic β-cell differentiation and increase β-cell function. In this study, we used a newly generated zebrafish reporter to screen over 8000 small molecules for modulators of pdx1 expression. We found four hit compounds and validated their efficacy at different stages of pancreas development. Notably, valproic acid treatment increased pancreatic endoderm formation, while inhibition of TGFβ signaling led to α-cell to β-cell transdifferentiation. HC toxin, another HDAC inhibitor, enhances β-cell function in primary mouse and human islets. Thus, using a whole organism screening strategy, this study identified new pdx1 expression modulators that can be used to influence different steps in pancreas and β-cell development., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The midcingulate cortex and therefore the underlying midcingulum bundle (MCB) as well play a major role in attention. Although a specific structure's function does strongly depend on its neuroanatomical characteristics, research assessing the morphological variability of the midcingulate region is rather sparse. The present study examined the micro- and macrostructure of the MCB in both hemispheres by means of diffusion-tensor imaging. Besides, effects of gender (Female = 40, Male = 39) and handedness (Lefthanders = 45, Righthanders = 34) were assessed as well. Measures of fractional anisotropy, mean diffusion, as well as the white matter volumes of the MCBs were assessed. By integration of multi-modal images, the MCB was isolated and confounding with callosal fibers was avoided. Evidence was found indicating differences between hemispheres and gender regarding both volume and microstructural characteristics of the MCB. Interestingly, gender-related effects seem to be substantially associated with variations in individual brain volumes. Handedness did not emerge as relevant factor in the analyses. These findings might indicate a higher functional connectivity of the left MCB and in males as compared to females.

The fractal dimension (FD) was used to reveal brain structure irregularities in patients with schizophrenia. FD provides a unique way of quantifying the shape complexity of cortical folding of the human brain. MR images were obtained from seven patients with schizophrenia that were compared with six healthy control subjects. The MR images were first segmented, and the FD was calculated for the grey/white matter boundary for the whole brain and the hemispheres separately, using the box-counting and Minkowski-Bouligand methods. The results showed that the patients had larger FD values than the controls, for the whole brain volume and right hemisphere.

The functional integrity of the corticospinal tract (CST) is of crucial importance for fine and independent finger movement. Therefore, it has been frequently speculated that interhemispheric asymmetries in this tract might be associated with handedness. The objective of the present study was to examine this proposed relationship, by studying the effects of handedness on macro- and microstructural properties of the CST. For this purpose, 30 consistently left and 30 consistently right-handed participants of either sex were examined applying a combination of anatomical T1-weighted and diffusion-tensor (DTI) MR imaging. In an automated procedure and based on white matter probability maps as well as on DTI fibre direction information, the left and right hemispheric CST was segmented in the individual brain. Focussing the analysis on the posterior limb of the internal capsule, the size as well as two DTI parameters (mean diffusion and fractional anisotropy) was determined. Although the statistical analysis revealed substantial CST asymmetries regarding size, mean diffusion, and anisotropy, neither of these metrics showed a significant hemisphere-by-handedness interaction. From this it was concluded that handedness might be related to motorcortical asymmetries which seem not to be directly reflected in the corticospinal fibre tract characteristics.

The anterior cingulate cortex (ACC) is thought to play a major role in executive processes. Studies assessing neuroanatomical attributes of this region report a high degree of morphological variability. Recent theories consider the fissurization of the cortex to be a product of gross mechanical processes related to cortical growth and local cytoarchitectural characteristics. Hence, local sulcal patterning and gray matter volume are supposed to be associated. ACC fissurization was quantified in left- and right-handers of both sexes by recording the presence and extension of the paracingulate sulcus (PCS). Differences between groups regarding local gray matter volume were assessed by means of optimized voxel-based morphometry (oVBM) including additional modulation. Overall, the PCS occurred more often and was more pronounced in the left as compared to the right anterior cingulate region, although hemispheric differences were less pronounced in male left- and female right-handers. These discrepancies between groups seem to stem from variations of cingulate morphology in the left rather than the right hemisphere. The pattern of relevant comparisons in the oVBM analysis indicated a similar interaction. Therefore, evidence was found for discrepancies between groups and hemispheres on the macrostructural level.

The present study examined how interhemispheric transfer time (IHTT) is affected by interindividual differences in corpus callosum (CC) architecture. For this purpose the CC of 42 healthy male subjects was assessed by applying a combination of morphological and diffusion-tensor magnetic resonance imaging to characterize the CC on macro- (midsagittal area) and microstructural level (mean diffusion, fractional anisotropy). Following the so-called Poffenberger paradigm, IHTT was determined with both reaction time measures and event-related potentials recorded in response to stimuli briefly presented to either left or right visual hemifield. Statistical analysis revealed significant negative correlations between mean diffusion and IHTT estimates derived from the P100 component (at O1/O2 electrode pair), particularly in the posterior CC subregion. Interpreting mean diffusion as an index of microstructural tissue properties, IHTT appears to be directly related to the structural integrity of the posterior CC.

The objective of the present study was to examine the role of the corpus callosum (CC) in dichotic listening. For this purpose, 40 right-handed healthy male participants were assessed with morphological and diffusion tensor imaging, which yielded macrostructural (midsagittal area) and microstructural (mean diffusion, fractional anisotropy) measures of the total CC and predefined subregions. Applying the standard consonant-vowel Bergen Dichotic Listening Test (including 3 different attentional instructions), the authors found dichotic listening performance to be substantially related to the integrity of the CC at both macro- and microstructural levels. Results indicate a dual role of the CC; it is relevant not only for the stimulus-driven (bottom-up) transfer of left-ear input to the left hemisphere but also for its attentional (top-down) modulation., (Copyright (c) 2006 APA, all rights reserved.)

The present study aimed to examine how differences in functional lateralisation of language are related to interindividual variations in interhemispheric connectivity. Utilising an fMRI silent word-generation paradigm, 89 left- and right-handed subjects were subdivided into four lateralisation subgroups. Applying morphological and diffusion-tensor MRI, midsagittal cross-sectional area as well as quantitative measures of molecular diffusion (anisotropy, mean diffusion) of the corpus callosum were determined to assess interhemispheric connectivity. Statistical analyses revealed group differences in molecular diffusion but not in callosal size, which may be interpreted to reflect a stronger and/or faster interhemispheric connection in strongly left-lateralised subjects as compared to moderately left-lateralised, bilateral, or moderately right-lateralised subjects.

Previous research has often examined whether the asymmetrical structure of the planum temporale (PT) represents an anatomical correlate of lateralized language-processing functions, gathering diverging empirical evidence by comparing PT asymmetry in subjects with differing handedness, gender, or speech lateralization. Apart from other methodological problems, direct comparisons between studies are hampered by insufficient assessment and consideration of all three potential determinants of structural cerebral asymmetry. Based on volumetric assessment of structural Magnetic Resonance Imaging (MRI) scans of 104 healthy subjects, the present study replicated earlier observations of an overall leftward PT asymmetry, which was found to prevail irrespective of handedness, gender, or dichotic-listening ear advantage. However, the mean magnitude of this leftward asymmetry was not determined by either one of these factors in itself, but varied depending on their specific combination. A clear correspondence between structural and functional asymmetry was only observed among right-handed males. In this particular subgroup, more pronounced structural asymmetry was associated with an enlarged PT on the left side, while the enhanced leftward asymmetry of female sinistrals resulted from smaller adjusted volumes of their right PT. The existence of such complex interactions suggests that future research in this area can only be expected to overcome past inconsistencies by adequately considering handedness, gender, and speech lateralization.

The corpus callosum (CC) represents the major commissural tract connecting the two cerebral hemispheres and is supposed to play crucial integrative role in functional hemispheric specialization. The present study examined whether interindividual variations in macro- and microstructure of the human CC are associated with handedness and gender. Therefore, a combined diffusion-tensor (DTI) and high-resolution morphological MRI study was performed on 34 right- and 33 left-handed subjects of both sexes. The mid-sagittal surface areas and quantitative measures of molecular diffusion (relative anisotropy, mean diffusion) of the total CC and its subregions (genu, truncus, posterior third) were determined. Analysis revealed a larger total callosal area in right- as compared to left-handed subjects and in males as compared to females. Throughout all callosal subregions, anisotropy was found to be increased in left-handed as well as in male subjects, while the mean diffusion was diminished only in left-handers. For the posterior third of the CC, a significant negative correlation (r=-0.34) between anisotropy and area was detected in right-handed subjects. Summarized, significant alterations in the molecular diffusion and in the size of the CC with respect to gender and handedness were revealed in the present study. These findings can be interpreted as handedness- and gender-related differences in macro- and microstructure of the callosal pathways. It was demonstrated that the inspection of the callosal microstructure using DTI yields empirical evidence on interhemispheric connectivity that goes well beyond the information revealed by anatomical measurements alone. Thus, DTI has proven to be a useful additional method in cognitive neuroscience.

The aim of the present study was to examine whether handedness and gender are associated with microstructural differences in human corpus callosum (CC). For this purpose, diffusion-tensor magnetic resonance imaging (DTI) was performed on 34 right- and 33 left-handed subjects of both sexes. Four quantitative variables (Mean Diffusion, Relative Anisotropy, parallel and orthogonal diffusion) were computed within the CC. A significantly increased anisotropy was found in left- as compared to right-handed subjects, and in men as compared to women. Additionally, both overall and orthogonal diffusion were significantly lower in left- than in right-handed subjects. Possible interpretations of these findings are discussed. The novel DTI technique promises to further advance current understanding of morphological structure in the living brain.