Synthesis of 1,3-Dibromopyrene as Precursor of 1-, 3-, 6-, and 8-Substituted Long-Axially Symmetric Pyrene Derivatives.

Pyrene derivatives bearing substituents at positions 1, 3, 6, and 8 find numerous applications, as exemplified by their use in lasers, sensors, and bioimaging probes. However, these derivatives typically have point-symmetric or short-axially symmetric structures, whereas long-axially symmetric derivatives remain underexplored because of the difficulty in obtaining their precursor, 1,3-dibromopyrene. To address this problem, we herein synthesized 1,3-dibromopyrene from 1-methoxypyrene in an overall yield (71 % over four steps) considerably exceeding those of existing methods. 1,3-Dibromopyrene was converted into 13OPA, a long-axially symmetric pyrene dye with electron-donor (alkoxy) groups at positions 1 and 3 and electron-acceptor (formyl) groups at positions 6 and 8. 13OPA exhibited photophysical properties distinct from those of its point-symmetric and short-axially symmetric isomers, featuring a broad and strongly redshifted absorption, strong fluorescence with reduced sensitivity to protic solvents, and small dipole moment change upon photoexcitation. The derivatization of 13OPA into a Schiff base and its functionalization via Lewis acid-base pairing were also demonstrated. Thus, our work expands the design scope of pyrene-based molecules, particularly those used as emitters.

Analysis of visual discrimination in Japanese eel (Anguilla japonica).

Japanese eels were trained to discriminate between a checkerboard panel and a plain gray panel in a circular pool with three pipes. One of the pipes was open, whereas the others were closed. The correct choice of discriminative stimulus was reinforced by entering the pipe. When the panels were displayed vertically (on the wall), the eels successfully learned discrimination, but they were unable to acquire the task when the panels were presented horizontally (on the floor). Enucleation of the retina impaired discrimination, whereas ablation of the olfactory plates did not. In the second experiment, the eels underwent three tests after discriminative training with vertical stimuli displayed. When plain black or white panels were presented instead of a checkerboard panel, the eels could not discriminate. Thus, the discriminative stimulus must have both black and white components. The eels exhibited a generalization gradient along the fines of the checkerboard. Finally, the pallium was damaged by coagulation, and the eels did not maintain the discrimination after the lesions. The behavioral deficits were classified into successful relearning and no relearning. Damage to the dorso-lateral (DL) or dorso-central (DC) pallium was associated with severe impairment (no relearning), although it was not possible to isolate the particular brain area or combination of brain areas which was required. The DL damage probably causes memory deficits, but the deficits caused by the DC damage might be motor or motivational deficits.

Spontaneous tempo production in cockatiels (Nymphicus hollandicus) and jungle crows (Corvus macrorhynchos).

Musical and rhythmical abilities are poorly documented in non-human animals. Most of the existing studies focused on synchronisation performances to external rhythms. In humans, studies demonstrated that rhythmical processing (e. g. rhythm discrimination or synchronisation to external rhythm) is dependent of an individual measure: the individual tempo. It is assessed by asking participants to produce an endogenous isochronous rhythm (known as spontaneous motor tempo) without any specific instructions nor temporal cue. In non-human animal literature, studies describing spontaneous and endogenous production of motor tempo without any temporal clue are rare. This exploratory study aims to describe and compare the spontaneous motor tempo of cockatiels and jungle crows. Data were collected on spontaneous beak drumming behaviours of birds housed in laboratory. Inter beak strokes intervals were calculated from sound tracks of videos. The analyses revealed that inter beak strokes intervals are non-randomly distributed intervals and are isochronous. Recorded spontaneous motor tempos are significantly different among some cockatiels. Since we could only conduct statistical analysis with one corvid, we cannot conclude about this species. Our results suggest that cockatiels and jungle crows have individual tempos, thus encouraging further investigations.

Infrared thermography for non-invasive measurement of social inequality aversion in rodents and potential usefulness for future animal-friendly studies.

Infrared thermography is a method that detects thermal radiation energy and can measure the body surface temperature of animals from a distance. While rectal temperature has traditionally been used to measure animals' core temperature, thermal imaging can avoid the stress and potential rise of body temperature deriving from handling of the animals. Additionally, being non-invasive and contactless, thermal imaging allows free movement of the animals. The validity of this technique as a psychophysiological method has been proven in a series of stress-induced hyperthermia (SIH) studies of mice under social inequality conditions. Restraint in a holder elicits SIH in mice. A restrained mouse surrounded by freely moving cage mates displays increased SIH suggesting that social inequality enhances the stress. Social inequality can be examined also in unrestrained mice, in particular through unequal distribution of food. In this protocol, a food-deprived mouse is given a small piece of cheese, while its cage mate is given a large piece of cheese. This inequity causes SIH, suggesting social inequality aversion in mice. Thus, social inequality in different situations similarly increased SIH. Importantly, in future studies infrared thermography could also be used to evaluate emotional arousal states different from stress (for example to assess reactivity to rewards or in social and sexual preference tests). Moreover, the technique could be used to investigate also cognitive arousal induced by novelty. Indeed, infrared thermography could be a particularly useful tool for animal-friendly studies of cognition and emotion in rodents.

A versatile and fast-sampling rate wearable analog data logger.

We propose a wearable, versatile, and open-source data logger that harvests the capacities of a low-cost microcontroller and enables fast-sampling recording of Analog signals into a microSD card. We describe here the circuit design and an exhaustive list of instructions to build a small, lightweight, and fast sampling rate data logger (up to 5 kHz for simultaneous recording of 3 channels and up to 40 kHz when using a single channel). We provide data analysis instructions, including publicly available scripts to facilitate its replication and customization. As a straightforward proof-of-concept, we tested our device embedded with a three-axial Analog accelerometer and were able to record triple axis acceleration of body movements in high resolution. A Fourier transform followed by a principal component analysis discriminated accurately between body motions of two participants and two types of movement recorded (walking VS running). Our wearable and fast-sampling rate data logger overcomes limits that we identified in previous studies, by being low-cost, capable of fast sampling rate, and easily replicated. Moreover, it can be customized to fit with a wide variety of applications in biomedical research by substituting the three-axial Analog accelerometer with virtually any type of Analog sensors or devices that output Analog signals. •We present a method to build and use a low-cost, fast-sampling rate and wearable Analog data logger, where having an engineering background is not required.•The data logger we present can collect Analog signals from 3 channels simultaneously at 5kHz and up to 40 kHz when using a single channel.•We demonstrate that our data logger can record data from a triple axis Analog accelerometer at 5 kHz, however, signals from virtually any Analog sensor or device that outputs Analog signals can be collected.

NIR-II-Excitable Dye-Loaded Nanoemulsions for Two-Photon Microscopy Imaging of Capillary Blood Vessels in the Entire Hippocampal CA1 Region of Living Mice.

For in vivo two-photon fluorescence microscopy (2PM) imaging, the development of techniques that can improve the observable depth and temporal resolution is an important challenge to address biological and biomedical concerns such as vascular dynamics in the deep brain (typically the hippocampal region) of living animals. Improvements have been achieved through two approaches: an optical approach using a highly tissue-penetrating excitation laser oscillating in the second near-infrared wavelength region (NIR-II, 1100-1350 nm) and a chemical approach employing fluorescent probes with high two-photon brightness (characterized by the product of the two-photon absorption cross section, σ2, and the fluorescence quantum yield, Φ). To integrate these two approaches, we developed a fluorescent dye exhibiting a sufficiently high σ2Φ value of 68 Goeppert-Mayer units at 1100 nm. When a nanoemulsion encapsulating >1000 dye molecules per particle and a 1100 nm laser were employed for 2PM imaging, capillary blood vessels in almost the entire hippocampal CA1 region of the mouse brain (approximately 1.1-1.5 mm below the surface) were clearly visualized at a frame rate of 30 frames s-1 (averaged over eight frames, practically 3.75 frames s-1). This observable depth and frame rate are much higher than those in previous reports on 2PM imaging. Furthermore, this nanoemulsion allowed for the visualization of blood vessels at a depth of 1.8 mm, corresponding to the hippocampal dentate gyrus. These results highlight the advantage of combining bright probes with NIR-II lasers. Our probe is a promising tool for studying the vascular dynamics of living animals and related diseases.

Philadelphia Chromosome-positive Acute Lymphoblastic Leukemia Incidentally Detected by Fluorodeoxyglucose-positron Emission Tomography/Computed Tomography at a Health Checkup.

We herein report a case of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-ALL) that was incidentally detected by fluorodeoxyglucose-positron emission tomography (18F-FDG PET)/computed tomography (CT) at a health checkup. At that time, the findings of a physical examination and blood tests were all normal, except for the diffuse bone marrow uptake (maximum standardized uptake value: 6.3). One month later, when the blood counts remained in the normal ranges, a bone marrow examination confirmed the diagnosis of Ph-ALL. Although a diffuse bone marrow uptake of 18F-FDG is observed in some benign conditions, physicians should also consider the possibility of hematological malignancies, including acute leukemia, even when that is the only abnormal finding.

Synthesis and photophysical properties of a new push-pull pyrene dye with green-to-far-red emission and its application to human cellular and skin tissue imaging.

Herein, we discuss a new pyrene-based push-pull dye (PC) and our investigation of its photophysical properties and applicability to biological studies. The newly synthesized dye exhibits highly polarity-sensitive fluorescence over a significantly wide range (i.e., the green to far-red region), accompanied by high fluorescence quantum yields (ΦFL > 0.70 in most organic solvents) and superior photostability to that of the commonly used Nile Red (NR) dye, which also fluoresces in the green to red region. When human prostate cancer cells stained with PC were imaged using a confocal laser scanning fluorescence microscope, PC was found to selectively stain the lipid droplets. Under the cell conditions where the formation of droplets was inhibited, PC could be distributed to both the remaining droplets and the intercellular membranes, which could be distinguished based on the fluorescence solvatochromic function of PC. Furthermore, PC efficiently stained normal human skin tissue blocks treated with a transparency-enhancing agent and enabled clear visualization of individual cells in each tissue architecture by means of two-photon fluorescence microscopy (2PM). Interestingly, PC provides bright 2PM images under tissue-penetrative 960 nm excitation, realizing much clearer and deeper tissue imaging than conventional pyrene dyes and NR. These results suggest that PC could replace several commonly used dyes in various biological applications, particularly the rapid and accurate diagnosis of tissue diseases, typified by biopsy.

Visual snake aversion in Octodon degus and C57BL/6 mice.

Phobia against spiders or snakes is common in humans, and similar phobia-like behaviors have been observed in non-human animals. Visual images of snakes elicit phobia in humans, but sensory modalities that cause snake aversion in non-human animals are not well examined. In this study, we examined visually induced snake aversion in two rodent species. Using a three-compartment experimental chamber, reactions to images of snakes were compared between the diurnal precocious rodent Octodon degus and nocturnal laboratory mice. The snakes whose images were presented do not live in the original habitats of degus or mice. Snake aversion was assessed by presenting snake vs. no-image, snake vs. flower, snake vs. degu, and snake vs. mouse images. The time spent in a compartment with the snake image and with the non-snake images were measured. Degus avoided images of snakes in every tests. In contrast, mice did not display snake aversion. Degus are diurnal animals, i.e., visual information is important for their survival. Since mice are nocturnal, visual information is less important for survival. Such behavioral differences in the two species may explain the difference in visually induced aversion to snakes. A principal component analysis of the stimulus images suggests that elementary cues, such as color, do not explain the differences in the species' aversion to snakes. Finally, snake aversion in degus suggests that aversion is innate, since the animals were born and raised in a laboratory.

Impairments in spatial learning by telencephalic lesions in Japanese eels (Anguilla japonica).

This study aimed to use Japanese eels (Anguilla japonica) as subjects to examine the effects of telencephalic lesions on spatial learning. Ten Japanese eels were trained on a Morris-type spatial learning task. Four pipes were placed in a pool; however, the eels could hide in only one of these pipes. The learning task ensured that the eels learned about the position of the open pipe. Subsequently, their telencephalons were damaged. The lesioned eels could not maintain their learning and demonstrated deficits in re-learning as some of them were unable to relearn the task. An analysis of the lesion sizes revealed that while damage to the dorsolateral pallium correlates with maintenance of learning, damage to the dorsomedial pallium correlates with re-learning.

Preference and discrimination of facial expressions of humans, rats, and mice by C57 mice.

Social animals likely recognize emotional expressions in other animals. Recent studies suggest that mice can visually perceive emotional expressions of other mice. In the first experiment, we measured the preference of mice for two different facial expressions (a normal facial expression and an expression of negative emotion such as pain) of rats, mice, and humans. Results revealed that mice showed a slight preference for the normal expression over the face expressing pain in the case of rats, but no preference in the case of others. In the second experiment, we trained mice to discriminate between the two facial expressions in an operant chamber with a touch screen. They could discriminate facial expressions of mice and rats, but they did not show discrimination of human facial expressions. Principal component analysis of the images of stimuli reveals negative correlation between pixel-based dissimilarity of training stimuli and the number of sessions to criterion. The mice showed generalization to novel images of the mouse faces with and without pain but did not maintain their discriminative behavior when new rat faces were shown. These results suggest that mice display category discrimination of conspecific facial expressions but not of other species.

Higher-Order Conditioning in the Spatial Domain.

Spatial learning and memory, the processes through which a wide range of living organisms encode, compute, and retrieve information from their environment to perform goal-directed navigation, has been systematically investigated since the early twentieth century to unravel behavioral and neural mechanisms of learning and memory. Early theories about learning to navigate space considered that animals learn through trial and error and develop responses to stimuli that guide them to a goal place. According to a trial-and error learning view, organisms can learn a sequence of motor actions that lead to a goal place, a strategy referred to as response learning, which contrasts with place learning where animals learn locations with respect to an allocentric framework. Place learning has been proposed to produce a mental representation of the environment and the cartesian relations between stimuli within it-which Tolman coined the cognitive map. We propose to revisit some of the best empirical evidence of spatial inference in animals, and then discuss recent attempts to account for spatial inferences within an associative framework as opposed to the traditional cognitive map framework. We will first show how higher-order conditioning can successfully account for inferential goal-directed navigation in a variety of situations and then how vectors derived from path integration can be integrated via higher-order conditioning, resulting in the generation of higher-order vectors that explain novel route taking. Finally, implications to cognitive map theories will be discussed.

Spatial inference without a cognitive map: the role of higher-order path integration.

The cognitive map has been taken as the standard model for how agents infer the most efficient route to a goal location. Alternatively, path integration - maintaining a homing vector during navigation - constitutes a primitive and presumably less-flexible strategy than cognitive mapping because path integration relies primarily on vestibular stimuli and pace counting. The historical debate as to whether complex spatial navigation is ruled by associative learning or cognitive map mechanisms has been challenged by experimental difficulties in successfully neutralizing path integration. To our knowledge, there are only three studies that have succeeded in resolving this issue, all showing clear evidence of novel route taking, a behaviour outside the scope of traditional associative learning accounts. Nevertheless, there is no mechanistic explanation as to how animals perform novel route taking. We propose here a new model of spatial learning that combines path integration with higher-order associative learning, and demonstrate how it can account for novel route taking without a cognitive map, thus resolving this long-standing debate. We show how our higher-order path integration (HOPI) model can explain spatial inferences, such as novel detours and shortcuts. Our analysis suggests that a phylogenetically ancient, vector-based navigational strategy utilizing associative processes is powerful enough to support complex spatial inferences.

Spatial Learning in Japanese Eels Using Extra- and Intra-Maze Cues.

Japanese eels (Anguilla japonica) were trained on a spatial-learning paradigm in a pool placed in an experimental room where several extra-maze cues were present. Four tubes were placed in the pool, of which one was open and could be entered by the eels. The open tube was placed at a fixed position in the pool and contained a triangular block that served as an intra-maze cue. The eels learned to identify the open tube, and their performance was maintained when the pool was rotated. However, they were unable to maintain their performance in a dark room, which suggests that spatial learning is based on visual cues. To determine the influence of the extra- and intra-maze cues, the tube with the triangle was moved to a new position and another open tube was kept in its place. The eels chose either the tube at the original position or the tube with the triangle at its new position, suggesting that spatial discrimination may be based on either extra- or intra-maze cues. We thus conclude that the eels employed an adjunctive strategy of multiple cues. In the next experiment, the eels were trained to visually discriminate the position of the stimulus (triangle), which changed in every trial. After the training, the eels were submitted to a test in which, in addition to the triangular pattern, a rectangular pattern was introduced. The eels discriminated between the tubes with the triangular and rectangular patterns, suggesting that they had the ability to discriminate visual patterns.

Discrimination of artificial starry sky by pigeons.

Night migratory birds use the star compass for their navigation. Here, I examined discrimination of a starry sky by a nonmigratory bird, the pigeon. Four pigeons were trained in an operant chamber to discriminate between an artificial starry sky created using software (Stella Theater Pro) and a black sky without stars. After they learned the task, they received three generalization tests. Test 1 presented a starry sky with different classes of stars (i.e., the number of stars was changed). Test 2 presented a starry sky at different times (i.e., a horizontal shift of the sky). Test 3 presented a starry sky at different latitudes (i.e., a vertical shift of the sky). The pigeons displayed a clear generalization gradient with a peak shift to the sky with more stars in Test 1 and a peak close to the original sky in Test 3, but almost a flat gradient in Test 2. Therefore, pigeons demonstrated stimulus control using the pattern of the stars, and they were more sensitive to the vertical shift than to the horizontal shift.

The whole is equal to the sum of its parts: Pigeons (Columba livia) and crows (Corvus macrorhynchos) do not perceive emergent configurations.

We previously demonstrated that chimpanzees, like humans, showed better accuracy and faster response time in discriminating visual patterns when the patterns were presented in redundant and uninformative contexts than when they were presented alone. In the present study, we examined the effect of redundant context on pattern discrimination in pigeons (Columba livia) and large-billed crows (Corvus macrorhynchos) using the same task and stimuli as those used in our previous study on chimpanzees. Birds were trained to search for an odd target among homogenous distractors. Each stimulus was presented in one of three ways: (1) alone, (2) with identical context that resulted in emergent configuration to chimpanzees (congruent context), or (3) with identical context that did not result in emergent configuration to chimpanzees (incongruent context). In contrast to the facilitative effect of congruent contexts we previously reported in chimpanzees, the same contexts disrupted target localization performance in both pigeons and crows. These results imply that birds, unlike chimpanzees, do not perceive emergent configurations.

Spatial learning in Japanese eels (Anguilla japonica).

Japanese eels (Anguilla japonica) were trained on a Morris-type spatial learning task. There were four tubes in a pool, but the eels could hide in only one of these. The eels learned the position of the open tube, and maintained their performance when the pool was rotated to remove possible intra-maze cues. The eels could not maintain their performance in a dark room, suggesting that spatial learning involved extra-maze visual cues. When the position of the open tube was randomly changed every day, the performance of the eels in finding the open tube did not improve.

Dark-Field Microscopic Detection of Bacteria using Bacteriophage-Immobilized SiO2@AuNP Core-Shell Nanoparticles.

To replace molecular biological and immunological methods, biosensors have recently been developed for the rapid and sensitive detection of bacteria. Among a wide variety of biological materials, bacteriophages have received increasing attention as promising alternatives to antibodies in biosensor applications. Thus, we herein present a rapid and highly selective detection method for pathogenic bacteria, which combines dark-field light scattering imaging with a plasmonic biosensor system. The plasmonic biosensor system employs bacteriophages as the biorecognition element and the aggregation-induced light scattering signal of gold nanoparticle-assembled silica nanospheres as a signal transducer. Using Staphylococcus aureus strain SA27 as a model analyte, we demonstrated that the plasmonic biosensor system detects S. aureus in the presence of excess Escherichia coli in a highly selective manner. After the sample and the S. aureus phage S13'-conjugated plasmon scattering probe were mixed, S. aureus detection was completed within 15-20 min with a detection limit of 8 × 104 colony forming units per milliliter.

Rapid assessment of the dose-response relationship of methamphetamine using the progressive-dosing procedure.

This paper describes a new method to rapidly obtain dose-response curves for a drug with rewarding properties using the conditioned place preference protocol. In the usual single-dosing procedure, different animals receive single, varying doses. Thus, a large number of animals are required to generate a curve. A new procedure, known as progressive dosing, alternates increasing drug doses with saline. In this way, the same animal can receive multiple tests. The dose-response curves of the rewarding effect of methamphetamine in mice were obtained using both single-dosing and progressive-dosing procedures. Although the progressive-dosing curves were not identical to the single-dosing curves, they showed a similar pattern. The progressive-dosing procedure was replicated with a new set of mice to confirm the reliability of the method and a dose-response curve similar to the previous one was obtained. This new method can reduce the number of animals required and shorten the duration of the experiment.