Neurophysiological measurements of planarian brain activity: a unique model for neuroscience research.

Planarians are well-known model organisms for regeneration and developmental biology research due to their remarkable regenerative capacity. Here we aim to advocate for the use of planaria as a valuable model for neurobiology, as well. Planarians have most of the major qualities of more developed organisms, including a primal brain. These traits combined with their exceptional regeneration capabilities, allow neurobiological experiments not possible in any other model organism, as we demonstrate by electrophysiological recording from planaria with two heads that controlling a shared body. To facilitate planarian neuroscience research, we developed an extracellular multi-unit recording procedure for the planarians fragile brain (Dugesia japonica). We created a semi-intact preparation restrained with fine dissection pins, enabling hours of reliable recording, via a suction electrode. Here we demonstrate the feasibility and potential of planarian neurophysiological research by characterizing the neuronal activity during simple learning processes and responses to various stimuli. In addition, we examined the use of linalool as anesthetic agent to allows recordings from an intact, large worm and for fine electrophysiological approaches such as intracellular recording. The demonstrated ability for neurophysiological measurements, along with the inherent advantages of planarians, promotes this exceptional model organism for neuroscience research.

Embodied mechanisms of motor control in the octopus.

Achieving complex behavior in soft-bodied animals is a hard task, because their body morphology is not constrained by a fixed number of jointed elements, as in skeletal animals, and thus the control system has to deal with practically an infinite number of control variables (degrees of freedom). Almost 30 years of research on Octopus vulgaris motor control has revealed that octopuses efficiently control their body with strategies that emerged during the adaptive coevolution of their nervous system and body morphology. In this minireview, we highlight principles of embodied organization that were revealed by studying octopus motor control, and that are used as inspiration for soft robotics. We describe the evolved solutions to the problem, implemented from the lowest level, the muscular system, to the network organization in higher motor control centers of the octopus brain. We show how the higher motor control centers, where the sensory-motor interface lies, can control and coordinate limbs with large degrees of freedom without using body-part maps to represent sensory and motor information, as they do in vertebrates. We demonstrate how this unique control mechanism, which allows efficient control of the body in a large variety of behaviors, is embodied within the animal's body morphology.

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.

Feel the light: sight-independent negative phototactic response in octopus arms.

Controlling the octopus's flexible hyper-redundant body is a challenging task. It is assumed that the octopus has poor proprioception which has driven the development of unique mechanisms for efficient body control. Here we report on such a mechanism: a phototactic response of extraocular photoreception. Extraocular photoreception has been observed in many and diverse species. Previous research on cephalopods revealed that increased illumination on their skin evokes chromatophore expansion. Recently, the mechanism was investigated and has been termed 'light-activated chromatophore expansion' (LACE). In this work we show that in response to illumination, the arm tip reacts in a reflex-like manner, folding in and moving away from the light beam. We performed a set of behavioral experiments and surgical manipulations to elucidate and characterize this phototactic response. We found that in contrast to the local activation and control of LACE, the phototactic response is mediated by the brain, although it is expressed in a reflex-like pattern. Our research results and observations led us to propose that the phototaxis is a means for protecting the arms in an instinctive manner from potential daytime predators such as fish and crabs, that could identify the worm-like tips as food. Indeed, observations of the octopuses revealed that their arm tips are folded in during the daytime, whereas at night they are extended. Thus, the phototactic response might compensate for the octopus's poor proprioception by keeping their arms folded in illuminated areas, without the need to be aware of their state.

Ocular anatomy and correlation with histopathologic findings in two common octopuses (Octopus vulgaris) and one giant Pacific octopus (Enteroctopus dofleini) diagnosed with inflammatory phakitis and retinitis.

PURPOSE: Review octopus ocular anatomy and describe the histopathologic findings in three octopuses diagnosed with phakitis and retinitis. ANIMALS: Two common octopuses (Octopus vulgaris) and one giant Pacific octopus (Enteroctopus dofleini) with a history of ophthalmic disease. METHODS: A literature search was performed for the ocular anatomy section. Both eyes from all three octopuses, and two control eyes, were submitted for histopathologic evaluation. Hematoxylin and eosin stain was used for standard histopathologic evaluation; GMS stain was used to screen for fungi, gram stain for bacteria; and Fite's acid fast stain for acid fast bacteria. RESULTS: Anatomically, the anterior chamber of the octopus has direct contact with ambient water due to an opening in the dorsal aspect of a pseudocornea. The octopus lens is divided into anterior and posterior segments. The anterior half is exposed to the environment through the opening into the anterior chamber. Neither part of the lens has a lens capsule. The retina is everted, unlike the inverted vertebrate retina, and consists of just two layers. Histopathology revealed inflammatory phakitis and retinitis of varying severity in all six eyes of the study animals. No intraocular infectious organisms were recognized but one common octopus eye had clusters of coccidian parasites, identified as Aggregata sp., in extraocular tissues and blood vessels. CONCLUSION: We describe inflammatory phakitis and retinitis in two species of octopuses. The underlying cause for the severe intraocular response may be direct intraocular infection, water quality, an ocular manifestation of a systemic disease, or natural senescence.

The Diversity of Muscles and Their Regenerative Potential across Animals.

Cells with contractile functions are present in almost all metazoans, and so are the related processes of muscle homeostasis and regeneration. Regeneration itself is a complex process unevenly spread across metazoans that ranges from full-body regeneration to partial reconstruction of damaged organs or body tissues, including muscles. The cellular and molecular mechanisms involved in regenerative processes can be homologous, co-opted, and/or evolved independently. By comparing the mechanisms of muscle homeostasis and regeneration throughout the diversity of animal body-plans and life cycles, it is possible to identify conserved and divergent cellular and molecular mechanisms underlying muscle plasticity. In this review we aim at providing an overview of muscle regeneration studies in metazoans, highlighting the major regenerative strategies and molecular pathways involved. By gathering these findings, we wish to advocate a comparative and evolutionary approach to prompt a wider use of "non-canonical" animal models for molecular and even pharmacological studies in the field of muscle regeneration.

From synaptic input to muscle contraction: arm muscle cells of Octopus vulgaris show unique neuromuscular junction and excitation-contraction coupling properties.

The muscular-hydrostat configuration of octopus arms allows high manoeuvrability together with the efficient motor performance necessary for its multitasking abilities. To control this flexible and hyper-redundant system the octopus has evolved unique strategies at the various levels of its brain-to-body organization. We focus here on the arm neuromuscular junction (NMJ) and excitation-contraction (E-C) properties of the arm muscle cells. We show that muscle cells are cholinergically innervated at single eye-shaped locations where acetylcholine receptors (AChR) are concentrated, resembling the vertebrate neuromuscular endplates. Na+ and K+ contribute nearly equally to the ACh-activated synaptic current mediating membrane depolarization, thereby activating voltage-dependent L-type Ca2+ channels. We show that cell contraction can be mediated directly by the inward Ca2+ current and also indirectly by calcium-induced calcium release (CICR) from internal stores. Indeed, caffeine-induced cell contraction and immunohistochemical staining revealed the presence and close association of dihydropyridine (DHPR) and ryanodine (RyR) receptor complexes, which probably mediate the CICR. We suggest that the dynamics of octopus arm contraction can be controlled in two ways; motoneurons with large synaptic inputs activate vigorous contraction via activation of the two routs of Ca2+ induced contraction, while motoneurons with lower-amplitude inputs may regulate a graded contraction through frequency-dependent summation of EPSP trains that recruit the CICR. Our results thus suggest that these motoneuronal pools are likely to be involved in the activation of different E-C coupling modes, thus enabling a dynamics of muscles activation appropriate for various tasks such as stiffening versus motion generation.

Updated View on the Relation of the Pineal Gland to Autism Spectrum Disorders.

Identification of the biological features of autism is essential for designing an efficient treatment and for prevention of the disorder. Though the subject of extensive research, the neurophysiological features of autism remain unclear. One of the proposed biological causes of autism is malfunction of the pineal gland and deficiency of its principal hormone, melatonin. The main function of melatonin is to link and synchronize the body's homeostasis processes to the circadian and seasonal rhythms, and to regulate the sleep-wake cycle. Therefore, pineal dysfunction has been implicated based on the common observation of low melatonin levels and sleep disorders associated with autism. In this perspective, we highlight several recent findings that support the hypothesis of pineal gland/melatonin involvement in autism. Another common symptom of autism is abnormal neuroplasticity, such as cortical overgrowth and dendritic spine dysgenesis. Here, we synthesize recent information and speculate on the possibility that this abnormal neuroplasticity is caused by hyperactivity of endogenous N,N-dimethyltryptamine (DMT). The pineal gland was proposed as the source of DMT in the brain and therefore, our assumption is that besides melatonin deficiency, pineal dysfunction might also play a part in the development of autism through abnormal metabolism of DMT. We hope that this manuscript will encourage future research of the DMT hypothesis and reexamination of several observations that were previously attributed to other factors, to see if they could be related to pineal gland/melatonin malfunction. Such research could contribute to the development of autism treatment by exogenous melatonin and monitored light exposure.

The stability of memories during brain remodeling: A perspective.

One of the most important features of the nervous system is memory: the ability to represent and store experiences, in a manner that alters behavior and cognition at future times when the original stimulus is no longer present. However, the brain is not always an anatomically stable structure: many animal species regenerate all or part of the brain after severe injury, or remodel their CNS toward a new configuration as part of their life cycle. This raises a fascinating question: what are the dynamics of memories during brain regeneration? Can stable memories remain intact when cellular turnover and spatial rearrangement modify the biological hardware within which experiences are stored? What can we learn from model species that exhibit both, regeneration and memory, with respect to robustness and stability requirements for long-term memories encoded in living tissues? In this Perspective, we discuss relevant data in regenerating planaria, metamorphosing insects, and hibernating ground squirrels. While much remains to be done to understand this remarkable process, molecular-level insight will have important implications for cognitive science, regenerative medicine of the brain, and the development of non-traditional computational media in synthetic bioengineering.

An automated training paradigm reveals long-term memory in planarians and its persistence through head regeneration.

Planarian flatworms are a popular system for research into the molecular mechanisms that enable these complex organisms to regenerate their entire body, including the brain. Classical data suggest that they may also be capable of long-term memory. Thus, the planarian system may offer the unique opportunity to study brain regeneration and memory in the same animal. To establish a system for the investigation of the dynamics of memory in a regenerating brain, we developed a computerized training and testing paradigm that avoided the many issues that confounded previous, manual attempts to train planarians. We then used this new system to train flatworms in an environmental familiarization protocol. We show that worms exhibit environmental familiarization, and that this memory persists for at least 14 days - long enough for the brain to regenerate. We further show that trained, decapitated planarians exhibit evidence of memory retrieval in a savings paradigm after regenerating a new head. Our work establishes a foundation for objective, high-throughput assays in this molecularly tractable model system that will shed light on the fundamental interface between body patterning and stored memories. We propose planarians as key emerging model species for mechanistic investigations of the encoding of specific memories in biological tissues. Moreover, this system is lik ely to have important implications for the biomedicine of stem-cell-derived treatments of degenerative brain disorders in human adults.

Transmembrane voltage potential controls embryonic eye patterning in Xenopus laevis.

Uncovering the molecular mechanisms of eye development is crucial for understanding the embryonic morphogenesis of complex structures, as well as for the establishment of novel biomedical approaches to address birth defects and injuries of the visual system. Here, we characterize change in transmembrane voltage potential (V(mem)) as a novel biophysical signal for eye induction in Xenopus laevis. During normal embryogenesis, a striking hyperpolarization demarcates a specific cluster of cells in the anterior neural field. Depolarizing the dorsal lineages in which these cells reside results in malformed eyes. Manipulating V(mem) of non-eye cells induces well-formed ectopic eyes that are morphologically and histologically similar to endogenous eyes. Remarkably, such ectopic eyes can be induced far outside the anterior neural field. A Ca(2+) channel-dependent pathway transduces the V(mem) signal and regulates patterning of eye field transcription factors. These data reveal a new, instructive role for membrane voltage during embryogenesis and demonstrate that V(mem) is a crucial upstream signal in eye development. Learning to control bioelectric initiators of organogenesis offers significant insight into birth defects that affect the eye and might have significant implications for regenerative approaches to ocular diseases.

Alternative sites of synaptic plasticity in two homologous "fan-out fan-in" learning and memory networks.

BACKGROUND: To what extent are the properties of neuronal networks constrained by computational considerations? Comparative analysis of the vertical lobe (VL) system, a brain structure involved in learning and memory, in two phylogenetically close cephalopod mollusks, Octopus vulgaris and the cuttlefish Sepia officinalis, provides a surprising answer to this question. RESULTS: We show that in both the octopus and the cuttlefish the VL is characterized by the same simple fan-out fan-in connectivity architecture, composed of the same three neuron types. Yet, the sites of short- and long-term synaptic plasticity and neuromodulation are different. In the octopus, synaptic plasticity occurs at the fan-out glutamatergic synaptic layer, whereas in the cuttlefish plasticity is found at the fan-in cholinergic synaptic layer. CONCLUSIONS: Does this dramatic difference in physiology imply a difference in function? Not necessarily. We show that the physiological properties of the VL neurons, particularly the linear input-output relations of the intermediate layer neurons, allow the two different networks to perform the same computation. The convergence of different networks to the same computational capacity indicates that it is the computation, not the specific properties of the network, that is self-organized or selected for by evolutionary pressure.

A second-generation device for automated training and quantitative behavior analyses of molecularly-tractable model organisms.

A deep understanding of cognitive processes requires functional, quantitative analyses of the steps leading from genetics and the development of nervous system structure to behavior. Molecularly-tractable model systems such as Xenopus laevis and planaria offer an unprecedented opportunity to dissect the mechanisms determining the complex structure of the brain and CNS. A standardized platform that facilitated quantitative analysis of behavior would make a significant impact on evolutionary ethology, neuropharmacology, and cognitive science. While some animal tracking systems exist, the available systems do not allow automated training (feedback to individual subjects in real time, which is necessary for operant conditioning assays). The lack of standardization in the field, and the numerous technical challenges that face the development of a versatile system with the necessary capabilities, comprise a significant barrier keeping molecular developmental biology labs from integrating behavior analysis endpoints into their pharmacological and genetic perturbations. Here we report the development of a second-generation system that is a highly flexible, powerful machine vision and environmental control platform. In order to enable multidisciplinary studies aimed at understanding the roles of genes in brain function and behavior, and aid other laboratories that do not have the facilities to undergo complex engineering development, we describe the device and the problems that it overcomes. We also present sample data using frog tadpoles and flatworms to illustrate its use. Having solved significant engineering challenges in its construction, the resulting design is a relatively inexpensive instrument of wide relevance for several fields, and will accelerate interdisciplinary discovery in pharmacology, neurobiology, regenerative medicine, and cognitive science.

The octopus vertical lobe modulates short-term learning rate and uses LTP to acquire long-term memory.

Analyzing the processes and neuronal circuitry involved in complex behaviors in phylogenetically remote species can help us understand the evolution and function of these systems. Cephalopods, with their vertebrate-like behaviors but much simpler brains, are ideal for such an analysis. The vertical lobe (VL) of Octopus vulgaris is a pivotal brain station in its learning and memory system. To examine the organization of the learning and memory circuitry and to test whether the LTP that we discovered in the VL is involved in behavioral learning, we tetanized the VL to induce a global synaptic enhancement of the VL pathway. The effects of tetanization on learning and memory of a passive avoidance task were compared to those of transecting the same pathway. Tetanization accelerated and transection slowed short-term learning to avoid attacking a negatively reinforced object. However, both treatments impaired long-term recall the next day. Our results suggest that the learning and memory system in the octopus, as in mammals [9], is separated into short- and long-term memory sites. In the octopus, the two memory sites are not independent; the VL, which mediates long-term memory acquisition through LTP, also modulates the circuitry controlling behavior and short-term learning.

The octopus: a model for a comparative analysis of the evolution of learning and memory mechanisms.

Comparative analysis of brain function in invertebrates with sophisticated behaviors, such as the octopus, may advance our understanding of the evolution of the neural processes that mediate complex behaviors. Until the last few years, this approach was infeasible due to the lack of neurophysiological tools for testing the neural circuits mediating learning and memory in the brains of octopus and other cephalopods. Now, for the first time, the adaptation of modern neurophysiological methods to the study of the central nervous system of the octopus allows this avenue of research. The emerging results suggest that a convergent evolutionary process has led to the selection of vertebrate-like neural organization and activity-dependent long-term synaptic plasticity. As octopuses and vertebrates are very remote phylogenetically, this convergence suggests the importance of the shared properties for the mediation of learning and memory.

A learning and memory area in the octopus brain manifests a vertebrate-like long-term potentiation.

Cellular mechanisms underlying learning and memory were investigated in the octopus using a brain slice preparation of the vertical lobe, an area of the octopus brain involved in learning and memory. Field potential recordings revealed long-term potentiation (LTP) of glutamatergic synaptic field potentials similar to that in vertebrates. These findings suggest that convergent evolution has led to the selection of similar activity-dependent synaptic processes that mediate complex forms of learning and memory in vertebrates and invertebrates.