mEMbrain: an interactive deep learning MATLAB tool for connectomic segmentation on commodity desktops.

Connectomics is fundamental in propelling our understanding of the nervous system's organization, unearthing cells and wiring diagrams reconstructed from volume electron microscopy (EM) datasets. Such reconstructions, on the one hand, have benefited from ever more precise automatic segmentation methods, which leverage sophisticated deep learning architectures and advanced machine learning algorithms. On the other hand, the field of neuroscience at large, and of image processing in particular, has manifested a need for user-friendly and open source tools which enable the community to carry out advanced analyses. In line with this second vein, here we propose mEMbrain, an interactive MATLAB-based software which wraps algorithms and functions that enable labeling and segmentation of electron microscopy datasets in a user-friendly user interface compatible with Linux and Windows. Through its integration as an API to the volume annotation and segmentation tool VAST, mEMbrain encompasses functions for ground truth generation, image preprocessing, training of deep neural networks, and on-the-fly predictions for proofreading and evaluation. The final goals of our tool are to expedite manual labeling efforts and to harness MATLAB users with an array of semi-automatic approaches for instance segmentation. We tested our tool on a variety of datasets that span different species at various scales, regions of the nervous system and developmental stages. To further expedite research in connectomics, we provide an EM resource of ground truth annotation from four different animals and five datasets, amounting to around 180 h of expert annotations, yielding more than 1.2 GB of annotated EM images. In addition, we provide a set of four pre-trained networks for said datasets. All tools are available from https://lichtman.rc.fas.harvard.edu/mEMbrain/. With our software, our hope is to provide a solution for lab-based neural reconstructions which does not require coding by the user, thus paving the way to affordable connectomics.

Connectomics of the Octopus vulgaris vertical lobe provides insight into conserved and novel principles of a memory acquisition network.

Here, we present the first analysis of the connectome of a small volume of the Octopus vulgaris vertical lobe (VL), a brain structure mediating the acquisition of long-term memory in this behaviorally advanced mollusk. Serial section electron microscopy revealed new types of interneurons, cellular components of extensive modulatory systems, and multiple synaptic motifs. The sensory input to the VL is conveyed via~1.8 × 106 axons that sparsely innervate two parallel and interconnected feedforward networks formed by the two types of amacrine interneurons (AM), simple AMs (SAMs) and complex AMs (CAMs). SAMs make up 89.3% of the~25 × 106VL cells, each receiving a synaptic input from only a single input neuron on its non-bifurcating primary neurite, suggesting that each input neuron is represented in only~12 ± 3.4SAMs. This synaptic site is likely a 'memory site' as it is endowed with LTP. The CAMs, a newly described AM type, comprise 1.6% of the VL cells. Their bifurcating neurites integrate multiple inputs from the input axons and SAMs. While the SAM network appears to feedforward sparse 'memorizable' sensory representations to the VL output layer, the CAMs appear to monitor global activity and feedforward a balancing inhibition for 'sharpening' the stimulus-specific VL output. While sharing morphological and wiring features with circuits supporting associative learning in other animals, the VL has evolved a unique circuit that enables associative learning based on feedforward information flow.

mEMbrain: an interactive deep learning MATLAB tool for connectomic segmentation on commodity desktops.

Connectomics is fundamental in propelling our understanding of the nervous system’s organization, unearthing cells and wiring diagrams reconstructed from volume electron microscopy (EM) datasets. Such reconstructions, on the one hand, have benefited from ever more precise automatic segmentation methods, which leverage sophisticated deep learning architectures and advanced machine learning algorithms. On the other hand, the field of neuroscience at large, and of image processing in particular, has manifested a need for user-friendly and open source tools which enable the community to carry out advanced analyses. In line with this second vein, here we propose mEMbrain, an interactive MATLAB-based software which wraps algorithms and functions that enable labeling and segmentation of electron microscopy datasets in a user-friendly user interface compatible with Linux and Windows. Through its integration as an API to the volume annotation and segmentation tool VAST, mEMbrain encompasses functions for ground truth generation, image preprocessing, training of deep neural networks, and on-the-fly predictions for proofreading and evaluation. The final goals of our tool are to expedite manual labeling efforts and to harness MATLAB users with an array of semi-automatic approaches for instance segmentation. We tested our tool on a variety of datasets that span different species at various scales, regions of the nervous system and developmental stages. To further expedite research in connectomics, we provide an EM resource of ground truth annotation from 4 different animals and 5 datasets, amounting to around 180 hours of expert annotations, yielding more than 1.2 GB of annotated EM images. In addition, we provide a set of 4 pre-trained networks for said datasets. All tools are available from https://lichtman.rc.fas.harvard.edu/mEMbrain/ . With our software, our hope is to provide a solution for lab-based neural reconstructions which does not require coding by the user, thus paving the way to affordable connectomics.

Octopus bimaculoides' arm recruitment and use during visually evoked prey capture.

Octopus' limb hyper-redundancy complicates traditional motor control system theory due to its extensive sensory inputs, subsequent decision-making, and arm coordination. Octopuses are thought to reduce flexibility control complexity by relying on highly stereotypical motor primitives (e.g., reaching1,2,3,4 and crawling5) and multi-level processes to coordinate movement,6,7 utilizing extensive peripheral nervous system (PNS) processing.2,8,9 Division of labor along the anterior-posterior axis10 and limb specialization of the four anterior arms in T-maze food retrieval11 further simplify control. However, specific arm recruitment and coordination during visually guided reaching behavior remains poorly understood. Here, we investigated visually evoked Octopus bimaculoides' prey capture capabilities12,13 by eliciting and examining prey-specific arm recruitment. When striking crabs, octopuses preferred synchronous arm recruitment, while sequential arm recruitment with a characteristic swaying movement is employed for shrimp. Such behavioral selection aligns with specific prey escape strategies and the octopus' flexible arm biomechanical constraints. Although side bias existed, we found significant bilateral symmetry, with one side being functionally a mirror of the other rather than anterior arm use being functionally equal and differing to posterior arm use. Among arms, the second limb is unequivocally dominant for goal-directed monocularly driven prey capture. Although the eight arms share gross anatomy and are considered equipotential,10,14 such arm use for specific actions could reflect subtle evolutionary adaptations. Finally, we quantitatively show, corroborating earlier observations,10,15 that octopuses employ a dimension reduction strategy by actively deciding to recruit adjacent arms over other available arms during either sequential or synchronous visually evoked prey attack.

Alteration of predatory behaviour and growth in juvenile cuttlefish by fluoxetine and venlafaxine.

Antidepressants in coastal waters may affect ontogeny of predatory behaviour in cuttlefish, which may, as a result, affect growth of newly-hatched cuttlefish. We investigated the effects of two of the most prescribed antidepressants, fluoxetine (FLX) and venlafaxine (VEN) in environmentally realistic concentrations on the predatory behaviour of hatchlings of Sepia officinalis. Newly-hatched cuttlefish were exposed from 1 h (i.e., day 1) to 5 days after hatching to either FLX alone (5 ng·L-1) or combined with VEN (2.5 ng·L-1 or 5 ng·L-1 each) to simulate an environmentally realistic exposure scenario. Their predatory behaviour was analysed through several parameters: prey detection, feeding motivation and success in catching the prey. All parameters improved in control animals over the first five days. The combination of FLX and VEN at 5 ng·L-1 each altered the predatory behaviour of the hatchlings by increasing the latency before attacking the prey, i.e., reducing feeding motivation, as well as by reducing the number of successful attacks. The changes in predatory behaviour tended to reduce food intake and affected growth significantly at 28 days post-hatching. Exposures to either FLX at 5 ng·L-1 or FLX and VEN in mixture at 2.5 ng·L-1 each tended to produce similar effects, even though they were not statistically significant. It is likely that the antidepressants affect maturation of the predatory behaviour and/or learning processes associated with the development of this behaviour. The slightest delay in maturation processes may have detrimental consequences for growth and population fitness.

The antidepressant venlafaxine may act as a neurodevelopmental toxicant in cuttlefish (Sepia officinalis).

The Serotonin/Norepinephrine Reuptake Inhibitor (SNRI) antidepressant venlafaxine (VEN, Effexor(®)) has become one of the most common antidepressants detected in North American and European streams. Mammalian research has established that VEN exposure is associated with a range of structural, neurochemical, and functional alterations of the brain in adults and newborns. However, the neurodevelopmental effects of VEN on non-target organisms have never been investigated. The aim of our research was to decrease this gap in knowledge by characterizing the effects of VEN exposure on a cephalopod mollusk, the common cuttlefish Sepia officinalis. This species inhabits VEN-contaminated waters and possesses an unusually sophisticated brain. These characteristics render it a unique invertebrate species for studying the neurodevelopmental effects of VEN. Cuttlefish were exposed to environmentally-relevant concentrations of VEN (Measured concentrations ≈5 and 100ngL(-)(1)) or to filtered natural seawater (control) in a closed-loop system with regular water changes during the first 20days after hatching. We evaluated brain maturation as well as neurochemical changes and behavioral performances during this critical period of development. Our results show that both VEN-exposed groups exhibited a decrease in norepinephrine levels, along with a reduction in the relative number of glutamate NMDA-like receptors binding sites in the group exposed to 5ngL(-1) of VEN after 20days of exposure. Brain regional changes in cellular proliferation were observed in VEN-exposed groups in the vertical lobe (i.e. a key structure involved in cognitive processes) and in the optic lobes (i.e. main visual processing centers) in the absence of significant change in their volume. Along with these neurodevelopmental changes, 20days of exposure to 100ngL(-1) of VEN was associated with a decrease in camouflage ability. Overall, our study suggests that VEN is a neurodevelopmental toxicant in non-target aquatic organisms at environmentally-relevant concentrations.

Pre-hatching fluoxetine-induced neurochemical, neurodevelopmental, and immunological changes in newly hatched cuttlefish.

Embryonic and early postembryonic development of the cuttlefish Sepia officinalis (a cephalopod mollusk) occurs in coastal waters, an environment subject to considerable pressure from xenobiotic pollutants such as pharmaceutical residues. Given the role of serotonin in brain development and its interaction with neurodevelopmental functions, this study focused on fluoxetine (FLX), a selective serotonin reuptake inhibitor (SSRI, antidepressant). The goal was to determine the effects of subchronic waterborne FLX exposure (1 and 10 µg L(-1)) during the last 15 days of embryonic development on neurochemical, neurodevelopmental, behavioral, and immunological endpoints at hatching. Our results showed for the first time that organic contaminants, such as FLX, could pass through the eggshell during embryonic development, leading to a substantial accumulation of this molecule in hatchlings. We also found that FLX embryonic exposure (1 and 10 µg L(-1)) (1) modulated dopaminergic but not serotonergic neurotransmission, (2) decreased cell proliferation in key brain structures for cognitive and visual processing, (3) did not induce a conspicuous change in camouflage quality, and (4) decreased lysozyme activity. In the long term, these alterations observed during a critical period of development may impair complex behaviors of the juvenile cuttlefish and thus lead to a decrease in their survival. Finally, we suggest a different mode of action by FLX between vertebrate and non-vertebrate species and raise questions regarding the vulnerability of early life stages of cuttlefish to the pharmaceutical contamination found in coastal waters.

An HPLC-ECD method for monoamines and metabolites quantification in cuttlefish (cephalopod) brain tissue.

The cuttlefish belongs to the mollusk class Cephalopoda, considered as the most advanced marine invertebrates and thus widely used as models to study the biology of complex behaviors and cognition, as well as their related neurochemical mechanisms. Surprisingly, methods to quantify the biogenic monoamines and their metabolites in cuttlefish brain remain sparse and measure a limited number of analytes. This work aims to validate an HPLC-ECD method for the simultaneous quantification of dopamine, serotonin, norepinephrine and their main metabolites in cuttlefish brain. In comparison and in order to develop a method suitable to answer both ecological and biomedical questions, the validation was also carried out on a phylogenetically remote species: mouse (mammals). The method was shown to be accurate, precise, selective, repeatable and sensitive over a wide range of concentrations for 5-hydroxyindole-3-acetic acid, serotonin, dopamine, 3,4-dihydroxyphenylacetic acid and norepinephrine in the both extracts of cuttlefish and mouse brain, though with low precision and recovery for 4-hydroxy-3-methoxyphenylethylene glycol. Homovanillic acid, accurately studied in rodents, was not detectable in the brain of cuttlefish. Overall, we described here the first fully validated HPLC method for the routine measurement of both monoamines and metabolites in cuttlefish brain. Copyright © 2016 John Wiley & Sons, Ltd.

Cryptic and biochemical responses of young cuttlefish Sepia officinalis exposed to environmentally relevant concentrations of fluoxetine.

Antidepressants released in the environment have the potential to generate neural disrupting effects in non-target organisms, yet their putative effects on behaviors have never been studied in cephalopod molluscs. This study assessed the impact of the antidepressant fluoxetine (FLX) on the efficiency of cryptic behaviors (body patterns on uniform, checkerboard and sandy substrates), locomotor activity, and brain chemistry in young cuttlefish exposed to environmental concentrations (1 and 100ngL(-1) of FLX) during the perinatal period. Behavioral responses of cuttlefish were monitored at hatching and two weeks later, and brain monoamine contents were quantified at one month of age. FLX significantly altered the camouflage efficiencies on uniform and sandy backgrounds only at the lowest concentration, but not at 100ngL(-1). Hatchlings exposed to 1ngL(-1) of FLX exhibited a duration exposure-dependent decrease in the uniform camouflage. They also showed a significant increase of the frequency of sand digging behaviors which might make them highly visible to predators in nature. When tested again two weeks later, cuttlefish seemed to have recovered and no more behavioral alterations were observed showing a transitory effect of the antidepressant. FLX did not affect the levels of serotonin, norepinephrine and their metabolites; however, it seemed to influence dopaminergic activity between the two FLX-exposed groups. The results show for the time that environmentally realistic concentrations of a single SSRI significantly impair the cryptic performances of newly hatched cuttlefish, and may ultimately reduce their chance for survival.