Switching by cuttlefish of preying tactics targeted at moving prey.

Previous studies have demonstrated that the size of the prey relative to the cuttlefish is important to the choice between tentacular strike and jump-on tactics. In the present study, we investigated the decision-making in the cuttlefish's tactical switch when preying on the same size prey. A servomotor system controlling the movement of a shrimp was used to elicit the cuttlefish's preying behavior. The success rate of prey capture and the kinematics of visual attack were examined systematically. The results showed that the jump-on behavior appeared mostly after a miss attack by previous tentacular strike on a moving shrimp. Compared with a visual attack with tentacles, the jump-on tactic has over a shorter attacking distance and wider attacking angles. Thus, these two different preying tactics have different operating ranges relative to the prey. More importantly, the cuttlefish can adjust their preying tactics adaptively depending on their prior preying experience.

Impact of Lidocaine on Pain-Related Grooming in Cuttlefish.

Nociception is the neural process of encoding noxious stimuli and is typically accompanied by a reflex withdrawal response away from the potentially injurious stimulus. Studies on nociception in cephalopods have so far focused on octopus and squid, with no investigations to our knowledge on cuttlefish. Yet, these are an important species both in scientific and commercial use. Therefore, the present study demonstrated that a standard pain stimulus, acetic acid, induced grooming behaviour directed towards the injection site in cuttlefish and that the injection of lidocaine reduces grooming behaviours in acetic-acid-injected cuttlefish. Wound-directed behaviour demonstrates that the animal is aware of the damage; thus, when subjecting these animals to any painful treatments in the laboratory, researchers should consider alleviating pain by the administration of pain-relieving drugs.

The effect of unexpected rewards on decision making in cuttlefish.

Despite numerous studies demonstrating the cognitive ability of cephalopods, there is currently no study showing an emotion-like behavior in this group of animals. To examine whether cuttlefish have different internal states, we developed a behavioral paradigm to assess if prior surprised events are able to alter the choice made by cuttlefish. By presenting unexpected food rewards to cuttlefish before the test, we investigated whether the reaction time of choosing between two shrimps, an intuitive response toward the prey without previous learning, at three different levels of discriminative tests (easy, difficult, and ambiguous), are different compared to the one without an unexpected reward. This behavioral paradigm serves to demonstrate whether cuttlefish are aware of ambiguous situations, and their choice outcome and reaction time are dependent of their internal states. The results show that the response latency was significantly shortened in the difficult and ambiguous tests when choosing from two shrimps that are either moderately different in size or similar sizes, respectively, when cuttlefish have received unexpected rewards before the test. These results were compared with tests during which the cuttlefish did not receive any reward in advance. Furthermore, this shortening of latency did not result in a difference in choice outcome during the difficult and ambiguous tests. Interestingly, even when cuttlefish have obtained the expected food rewards or simply made tentacular strike without prey capture each time before test, these prior experiences were sufficient to shorten the response latency in the difficult and ambiguous tests. However, different from the result of unexpected rewards, food consumption alone or prey capture failure did affect the choice outcome during the simple and difficult tests. Taken together, our findings suggest that pre-test treatments of unexpected and expected rewards or simply unsuccessful visual attack seem to induce cuttlefish to adopt different foraging behaviors. This context dependent decision making suggests that cuttlefish's foraging strategies are influenced by the previously surprised event and their internal states. It also shows a speed-accuracy tradeoff in difficult and ambiguous situations when foraging for prey. This observation may lead to a future investigation of the presence of emotional state in cephalopods.

Design and Ex Vivo Experimental Validations of the CMOS 256-Pixel Photovoltaic-Powered Subretinal Prosthetic Chip With Auto-Adaptive Pixels for a Wide Image Illuminance Range.

OBJECTIVE: To design and verify a CMOS 256-pixel photovoltaic-powered subretinal prosthetic chip with key advances over the state-of-the-art. The three key advances are: 1) automatic adaptation to changing background illuminance levels; 2) increase of injection charges with reduced crosstalk leakage charges, enhanced charge balance, and low process variations; 3) stable stimulation voltage to keep the safety of water window. METHODS: The novel auto-adaptive pixel circuit is designed to realize the Michealis - Menten equation (MME) so that the automatic adaptation to changing background illuminance can be achieved. Both improved biphasic constant current stimulator (CCS) via bi-directional shared electrodes (BDSEs) with optimized stimulation pattern and improved constant current generator/ring oscillator are designed to achieve the above second advance on injection charges. The closed-loop charge pump is designed to achieve the third advance. RESULTS: The measured dynamic range of image illuminance is increased to 54.7 dB. The maximum stimulation charge is 8.89nC. The measured stimulation current mismatch is 1.7% and the measured residual charge is 0.150 nC. The measured variations of stimulation frequencies are from 26 Hz to 29.7 Hz. The results of ex vivo tests have shown that the proposed subretinal chip can evoke spiking responses of RGCs. The function of adaptation process to background illuminance has also been verified. CONCLUSION AND SIGNIFICANCE: Through both electrical measurement and ex vivo tests, the functions of designed subretinal chip have been validated successfully. It is shown that the proposed subretinal chip is a promising solution for subretinal prostheses.

Mild stress culture conditions promote neurite outgrowth of retinal explants from postnatal mice.

The axons of retinal ganglion cells (RGCs) in adult mammals fail to regenerate after injury. It has been suggested that some extrinsic factors, such as neural activity, may promote the regeneration process. The present study tested the hypothesis that environmental stress such as slightly elevated osmolarity and temperature can enhance neural activity and thus promote axon regeneration of RGCs in postnatal mice. Retinal explants from P9-11 mice were cultured for 5 days to study the capacity of RGC neurite outgrowth. The neural activity of retinal explants in these two stress conditions was examined using the multi-electrode array. We found that RGC neurite outgrowth from P9-P11 mouse explants was significantly enhanced when the concentration of the culture medium was increased by 1.25 fold, but not when increased by 1.5 fold. Similarly, retinal explants from P9-P11 mice grew longer neurites when the overall temperature was increased from 35 °C to 38 °C, 40 °C or 42 °C for one hour each day, but not when they were kept at 40 °C or 42 °C constantly for five days. We further showed that there was increased neural activity during these two mild stress conditions. It was found that short-term 42 °C heat stress increased the expression of heat shock proteins 27 and 70 in postnatal retinas and they were RGC neural activity dependent. The present study thus provides insights into the cellular mechanism of retinal axon regeneration under the mild stress conditions.

Visual Attack on the Moving Prey by Cuttlefish.

Visual attack for prey capture in cuttlefish involves three well characterized sequential stages: attention, positioning, and seizure. This visually guided behavior requires accurate sensorimotor integration of information on the target's direction and tentacular strike control. While the behavior of cuttlefish visual attack on a stationary prey has been described qualitatively, the kinematics of visual attack on a moving target has not been analyzed quantitatively. A servomotor system controlling the movement of a shrimp prey and a high resolution imaging system recording the behavior of the cuttlefish predator, together with the newly developed DeepLabCut image processing system, were used to examine the tactics used by cuttlefish during a visual attack on moving prey. The results showed that cuttlefish visually tracked a moving prey target using mainly body movement, and that they maintained a similar speed to that of the moving prey right before making their tentacular strike. When cuttlefish shot out their tentacles for prey capture, they were able to either predict the target location based on the prey's speed and compensate for the inherent sensorimotor delay or adjust the trajectory of their tentacular strike according to the prey's direction of movement in order to account for any changes in prey position. These observations suggest that cuttlefish use the various visual tactics available to them flexibly in order to capture moving prey, and that they are able to extract direction and speed information from moving prey in order to allow an accurate visual attack.

Awareness of danger inside the egg: Evidence of innate and learned predator recognition in cuttlefish embryos.

Predation can be a very strong selective pressure on prey. Many studies have shown the existence of innate anti-predator responses, mostly in the early developmental stages of juvenile vertebrates. Learning to recognize predators is another possible defensive resource, but such a method involves a high death risk. There is evidence that prenatal learning exists in animals but few studies have explicitly tested for embryonic learning. The aim of this study was to test innate and learned predator recognition in cuttlefish embryos. For this, naïve embryos were exposed to chemical and visual cues emanating from predators, non-predators, and ink. Their response was assessed by measuring their ventilation rate (VR). We first show that VR decreased in response to both visual and chemical predatory cues and ink but not to non-predatory cues. Second, we show that when non-predatory cues (visual or chemical) are paired with predatory cues or ink for several days, embryonic VR significantly decreased. Such a response is likely adaptive, especially in a translucent egg, since it results in reduced movement and hence may lower the risk of detection by visual predators. This freezing-like behavior may also reduce the bioelectric field, thus lessening the predation risk by non-visual foragers. Our results report that cuttlefish embryos had an innate capacity to differentiate between harmless and harmful chemical and visual cues. They were also capable of learning to respond to harmless cues when they were paired with danger (predator or ink) based on conditioning. The combination of these behavioral mechanisms is an example of the early adaptability of cephalopods. Such behavioral plasticity may give the newly hatched cuttlefish a selective advantage when dealing with either known or unfamiliar threats. Nevertheless, more experiments are needed to test the efficiency of the embryos' response faced with known or new predators.

Spatiotemporal integration of visual stimuli and its relevance to the use of a divisional power supply scheme for retinal prosthesis.

A wireless photovoltaic retinal prosthesis is currently being studied with the aim of providing prosthetic vision to patients with retinitis pigmentosa (RP) and age-related macular degeneration (AMD). The major challenge of a photovoltaic device is its limited power efficiency. Our retinal prosthetic design implements a unique divisional power supply scheme (DPSS) system that provides the electrical power generated by all of the solar cells to only a subset of electrodes at any moment in time. The aim of the present study was to systematically characterize the spatiotemporal integration performance of the system under various DPSS conditions using human subjects and a psychophysical approach. A 16x16 pixels LED array controlled by Arduino was used to simulate the output signal of the DPSS design, and human performance under different visual stimulations at various update frequencies was then used to assess the spatiotemporal capability of retinal prostheses. The results showed that the contrast polarity of the image, image brightness, and division number influenced the lower limit of the update frequency of the DPSS system, while, on the other hand, visual angle, ambient light level, and stimulation order did not affect performance significantly. Pattern recognition by visual persistence with spatiotemporal integration of multiple frames of sparse dots is a feasible approach in retinal prosthesis design. These findings provide an insight into how to optimize a photovoltaic retinal prosthesis using a DPSS design with an appropriate update frequency for reliable pattern recognition. This will help the development of a wireless device able to restore vision to RP and AMD patients in the future.

Learned valuation during forage decision-making in cuttlefish.

Decision-making, when humans and other animals choose between two options, is not always based on the absolute values of the options but can also depend on their relative values. The present study examines whether decision-making by cuttlefish is dependent on relative values learned from previous experience. Cuttlefish preferred a larger quantity when making a choice between one or two shrimps (1 versus 2) during a two-alternative forced choice. However, after cuttlefish were primed under conditions where they were given a small reward for choosing one shrimp in a no shrimp versus one shrimp test (0 versus 1) six times in a row, they chose one shrimp significantly more frequently in the 1 versus 2 test. This reversed preference for a smaller quantity was not due to satiation at the time of decision-making, as cuttlefish fed a small shrimp six times without any choice test prior to the experiment still preferred two shrimps significantly more often in a subsequent 1 versus 2 test. This suggests that the preference of one shrimp in the quantity comparison test occurs via a process of learned valuation. Foraging preference in cuttlefish thus depends on the relative value of previous prey choices.

Improved Charge Pump Design and Ex Vivo Experimental Validation of CMOS 256-Pixel Photovoltaic-Powered Subretinal Prosthetic Chip.

An improved design of CMOS 256-pixel photovoltaic-powered implantable chip for subretinal prostheses is presented. In the proposed subretinal chip, a high-efficiency fully-integrated 4× charge pump is designed and integrated with on-chip photovoltaic (PV) cells and a 256-pixel array with active pixel sensors (APS) for image light sensing, biphasic constant current stimulators, and electrodes. Thus the PV voltage generated by infrared (IR) light can be boosted to above 1V so that the charge injection is increased. The proposed chip adopts the 32-phase divisional power supply scheme (DPSS) to reduce the required supply current and thus the required area of the PV cells. The proposed chip is designed and fabricated in 180-nm CMOS image sensor (CIS) technology and post-processed with biocompatible IrOx electrodes and silicone packaging. From the electrical measurement results, the measured stimulation frequency is 28.3 Hz under the equivalent electrode impedance load. The measured maximum output stimulation current is 7.1 µA and the amount of injected charges per pixel is 7.36 nC under image light intensity of 3200 lux and IR light intensity of 100 mW/cm2. The function of the proposed chip has been further validated successfully with the ex vivo experimental results by recording the electrophysiological responses of retinal ganglion cells (RGCs) of retinas from retinal degeneration (rd1) mice with a multi-electrode array (MEA). The measured average threshold injected charge is about 3.97 nC which is consistent with that obtained from the patch clamp recording on retinas from wild type (C57BL/6) mice with a single electrode pair.

Multiple Approaches for Enhancing Neural Activity to Promote Neurite Outgrowth of Retinal Explants.

Activity is important for neural development and regeneration. Enhancing neural activity can facilitate axon regrowth of retinal ganglion cells. Here, we describe various methods, including electrical stimulation, pharmacological manipulation, and optogenetics, to elevate neural activity of retinal explants in mice and to analyze their effects on promoting neurite outgrowth in organotypic culture.

Male Alternative Reproductive Tactics and Associated Evolution of Anatomical Characteristics in Loliginid Squid.

Loliginid squids provide a unique model system to explore male alternative reproductive tactics (ARTs) and their linkage to size, behavioral decision making, and possibly age. Large individuals fight one another and the winners form temporary consortships with females, while smaller individuals do not engage in male-male agonistic bouts but use various sneaker tactics to obtain matings, each with varying mating and fertilization success. There is substantial behavioral flexibility in most species, as smaller males can facultatively switch to the alternative consort behaviors as the behavioral context changes. These forms of ARTs can involve different: mating posture; site of spermatophore deposition; fertilization success; and sperm traits. Most of the traits of male dimorphism (both anatomical and behavioral) are consistent with traditional sexual selection theory, while others have unique features that may have evolved in response to the fertilization environment faced by each temporary or permanent male morph.

Blue Light Promotes Neurite Outgrowth of Retinal Explants in Postnatal ChR2 Mice.

Neurons in the adult mammalian CNS fails to regenerate after severe injury. However, it is known that an increase in neural activity occurs in mouse retinal ganglion cells (RGCs) after extrinsic stimulation and this can induce axon growth. In the present study, we applied an optogenetic approach using a mouse model, specifically involving channelrhodopsin-2 (ChR2) expression in RGCs. We investigated whether modulation of RGC neural activity exclusively by blue light stimulation is able to promote neurite outgrowth of postnatal retinal explants. The results showed that activation of RGCs expressing ChR2 by 20 Hz blue light for 1 h is a most effective way of enhancing neurite outgrowth in postnatal retinas. This is achieved via gap junctions that spread neural activity across the whole retina. Moreover, we found that activation of intrinsic photosensitive RGCs (ipRGCs) by blue light also contributes significantly to the promotion of neurite outgrowth in the same postnatal retinal explants. Our findings not only demonstrate that a short-term increase in RGC neural activity is sufficient to facilitate the neurite outgrowth of retinal explants, but also highlight the fact that the temporal pattern of neural activity in RGCs is a critical factor in regulating axon regeneration.

A difference in timing for the onset of visual and chemosensory systems during embryonic development in two closely related cuttlefish species.

Embryos perceive environmental stimuli, thanks to their almost mature sensory systems. In cuttlefish, the embryonic development of Sepia officinalis and Sepia pharaonis is similar but the egg capsule transparency is different. S. officinalis' eggs are black (ink), which provide protection from predators. Conversely, those of S. pharaonis are translucent. The aim of this study was to test the visual and chemosensory perception abilities of these two cuttlefish embryos by observation of the ventilation rate (VR) before and after stimulation. Our results show that S. pharaonis responds to light at stage 22 and S. officinalis at stage 24. Conversely, S. pharaonis responds to predator odor at stage 23 and S. officinalis at stage 22. Both species are able to respond to these stimuli before hatching but do not have the same developmental schedule. Neither are the responses of the two cuttlefish exactly the same. In S. officinalis, VR increases after stimulations. In S. pharaonis, VR increases after light stimulation and decreases following the odor stimulation after stage 25. This result could reveal an ability to recognize stimuli at stage 25. The decrease could be identified as freezing-like behavior which would be more adaptive than an increase, since the embryos are visible.

Do cuttlefish have fraction number sense?

Number sense is a key cognitive function in animals. The biological functions of number discrimination have a wide range, including the selection of prey and social interaction. In a previous study, we have shown that cuttlefish are able to distinguish numerical differences among various integers, including 1 vs. 2, 2 vs. 3, 3 vs. 4, and 4 vs. 5. However, it is not known whether cuttlefish are able to discriminate various fractions, that is, various non-integer numbers. In addition, no study on invertebrates has examined fraction number sense. Using the active preying behavior of cuttlefish (Sepia pharaonis), we investigated the spontaneous preference for larger quantity by presenting two-alternative choice between 1 vs. 1.5, 1.5 vs. 2, 2 vs. 2.5, and 2.5 vs. 3. In this context, the quantity1.5 is composed of one large shrimp and one small shrimp, in which the size of the small shrimp is one-half of that of the large shrimp. The result shows that the cuttlefish chose larger quantity in the first three pairs, but they could not distinguish the pair 2.5 vs. 3. Despite that the absolute differences in these pairs are the same (0.5), the relative differences in these pairs decrease (0.5, 0.33, 0.25, and 0.2, respectively). This implies that the perceived difference in quantity is proportional to the initial quantity (Weber's law). Although the present study does not truly differentiate the number difference from the quantity difference, this result does raise the possibility that cuttlefish may be equipped with the primitive concept of fractions, and if so, the perceived just noticeable difference is similar for both integer and fraction number discrimination.

Morphological changes of the optic lobe from late embryonic to adult stages in oval squids Sepioteuthis lessoniana.

The optic lobe is the largest brain area within the central nervous system of cephalopods and it plays important roles in the processing of visual information, the regulation of body patterning, and locomotive behavior. The oval squid Sepioteuthis lessoniana has relatively large optic lobes that are responsible for visual communication via dynamic body patterning. It has been observed that the visual behaviors of oval squids change as the animals mature, yet little is known about how the structure of the optic lobes changes during development. The aim of the present study was to characterize the ontogenetic changes in neural organization of the optic lobes of S. lessoniana from late embryonic stage to adulthood. Magnetic resonance imaging and micro-CT scans were acquired to reconstruct the 3D-structure of the optic lobes and examine the external morphology at different developmental stages. In addition, optic lobe slices with nuclear staining were used to reveal changes in the internal morphology throughout development. As oval squids mature, the proportion of the brain making up the optic lobes increases continuously, and the optic lobes appear to have a prominent dent on the ventrolateral side. Inside the optic lobe, the cortex and the medulla expand steadily from the late embryonic stage to adulthood, but the cell islands in the tangential zone of the optic lobe decrease continuously in parallel. Interestingly, the size of the nuclei of cells within the medulla of the optic lobe increases throughout development. These findings suggest that the optic lobe undergoes continuous external morphological change and internal neural reorganization throughout the oval squid's development. These morphological changes in the optic lobe are likely to be responsible for changes in the visuomotor behavior of oval squids from hatching to adulthood.

Corrigendum: A Novel Retinal Oscillation Mechanism in an Autosomal Dominant Photoreceptor Degeneration Mouse Model.

[This corrects the article on p. 513 in vol. 9, PMID: 26793064.].

Mismatch between the eye and the optic lobe in the giant squid.

Giant squids (Architeuthis) are a legendary species among the cephalopods. They live in the deep sea and are well known for their enormous body and giant eyes. It has been suggested that their giant eyes are not adapted for the detection of either mates or prey at distance, but rather are best suited for monitoring very large predators, such as sperm whales, at distances exceeding 120 m and at a depth below 600 m (Nilsson et al. 2012 Curr. Biol.22, 683-688. (doi:10.1016/j.cub.2012.02.031)). However, it is not clear how the brain of giant squids processes visual information. In this study, the optic lobe of a giant squid (Architeuthis dux, male, mantle length 89 cm), which was caught by local fishermen off the northeastern coast of Taiwan, was scanned using high-resolution magnetic resonance imaging in order to examine its internal structure. It was evident that the volume ratio of the optic lobe to the eye in the giant squid is much smaller than that in the oval squid (Sepioteuthis lessoniana) and the cuttlefish (Sepia pharaonis). Furthermore, the cell density in the cortex of the optic lobe is significantly higher in the giant squid than in oval squids and cuttlefish, with the relative thickness of the cortex being much larger in Architeuthis optic lobe than in cuttlefish. This indicates that the relative size of the medulla of the optic lobe in the giant squid is disproportionally smaller compared with these two cephalopod species. This morphological study of the giant squid brain, though limited only to the optic lobe, provides the first evidence to support that the optic lobe cortex, the visual information processing area in cephalopods, is well developed in the giant squid. In comparison, the optic lobe medulla, the visuomotor integration centre in cephalopods, is much less developed in the giant squid than other species. This finding suggests that, despite the giant eye and a full-fledged cortex within the optic lobe, the brain of giant squids has not evolved proportionally in terms of performing complex tasks compared with shallow-water cephalopod species.

Neural Organization of the Optic Lobe Changes Steadily from Late Embryonic Stage to Adulthood in Cuttlefish Sepia pharaonis.

The optic lobe is the largest structure in the cuttlefish brain. While the general morphology of the optic lobe in adult cuttlefish has been well described, the 3D structure and ontogenetic development of its neural organization have not been characterized. To correlate observed behavioral changes within the brain structure along the development of this animal, optic lobes from the late embryonic stage to adulthood were examined systematically in the present study. The MRI scan revealed that the so called "cell islands" in the medulla of the cephalopod's optic lobe (Young, 1962, 1974) are in fact a contiguous tree-like structure. Quantification of the neural organizational development of optic lobes showed that structural features of the cortex and radial column zone were established earlier than those of the tangential zone during embryonic and post-hatching stages. Within the cell islands, the density of nuclei was decreased while the size of nuclei was increased during the development. Furthermore, the visual processing area in the optic lobe showed a significant variation in lateralization during embryonic and juvenile stages. Our observation of a continuous increase in neural fibers and nucleus size in the tangential zone of the optic lobe from late embryonic stage to adulthood indicates that the neural organization of the optic lobe is modified along the development of cuttlefish. These findings thus support that the ontogenetic change of the optic lobe is responsible for their continuously increased complexity in body patterning and visuomotor behaviors.