Daily singing of adult songbirds functions to maintain song performance independently of auditory feedback and age.

Many songbirds learn to produce songs through vocal practice in early life and continue to sing daily throughout their lifetime. While it is well-known that adult songbirds sing as part of their mating rituals, the functions of singing behavior outside of reproductive contexts remain unclear. Here, we investigated this issue in adult male zebra finches by suppressing their daily singing for two weeks and examining the effects on song performance. We found that singing suppression decreased the pitch, amplitude, and duration of songs, and that those song features substantially recovered through subsequent free singing. These reversible song changes were not dependent on auditory feedback or the age of the birds, contrasting with the adult song plasticity that has been reported previously. These results demonstrate that adult song structure is not stable without daily singing, and suggest that adult songbirds maintain song performance by preventing song changes through physical act of daily singing throughout their life. Such daily singing likely functions as vocal training to maintain the song production system in optimal conditions for song performance in reproductive contexts, similar to how human singers and athletes practice daily to maintain their performance.

Molecular biology of serotonergic systems in avian brains.

Serotonin (5-hydroxytryptamine, 5-HT) is a phylogenetically conserved neurotransmitter and modulator. Neurons utilizing serotonin have been identified in the central nervous systems of all vertebrates. In the central serotonergic system of vertebrate species examined so far, serotonergic neurons have been confirmed to exist in clusters in the brainstem. Although many serotonin-regulated cognitive, behavioral, and emotional functions have been elucidated in mammals, equivalents remain poorly understood in non-mammalian vertebrates. The purpose of this review is to summarize current knowledge of the anatomical organization and molecular features of the avian central serotonergic system. In addition, selected key functions of serotonin are briefly reviewed. Gene association studies between serotonergic system related genes and behaviors in birds have elucidated that the serotonergic system is involved in the regulation of behavior in birds similar to that observed in mammals. The widespread distribution of serotonergic modulation in the central nervous system and the evolutionary conservation of the serotonergic system provide a strong foundation for understanding and comparing the evolutionary continuity of neural circuits controlling corresponding brain functions within vertebrates. The main focus of this review is the chicken brain, with this type of poultry used as a model bird. The chicken is widely used not only as a model for answering questions in developmental biology and as a model for agriculturally useful breeding, but also in research relating to cognitive, behavioral, and emotional processes. In addition to a wealth of prior research on the projection relationships of avian brain regions, detailed subdivision similarities between avian and mammalian brains have recently been identified. Therefore, identifying the neural circuits modulated by the serotonergic system in avian brains may provide an interesting opportunity for detailed comparative studies of the function of serotonergic systems in mammals.

Temporal hampering of thyroid hormone synthesis just before hatching impeded the filial imprinting in domestic chicks.

Thyroid hormones play a critical role in the initiation of the sensitive period of filial imprinting. The amount of thyroid hormones in the brains of chicks increases intrinsically during the late embryonic stages and peaks immediately before hatching. After hatching, a rapid imprinting-dependent inflow of circulating thyroid hormones into the brain occurs via vascular endothelial cells during imprinting training. In our previous study, inhibition of hormonal inflow impeded imprinting, indicating that the learning-dependent inflow of thyroid hormones after hatching is critical for the acquisition of imprinting. However, it remained unclear whether the intrinsic thyroid hormone level just before hatching affects imprinting. Here, we examined the effect of temporal thyroid hormone decrease on embryonic day 20 on approach behavior during imprinting training and preference for the imprinting object. To this end, methimazole (MMI; a thyroid hormone biosynthesis inhibitor) was administered to the embryos once a day on days 18-20. Serum thyroxine (T4) was measured to evaluate the effect of MMI. In the MMI-administered embryos, the T4 concentration was transiently reduced on embryonic day 20 but recovered to the control level on post-hatch day 0. At the beginning of imprinting training on post-hatch day 1, control chicks approached the imprinting object only when the object was moving. In the late phase of training, control chicks subsequently approached towards the static imprinting object. On the other hand, in the MMI-administered chicks, the approach behavior decreased during the repeated trials in the training, and the behavioral responses to the imprinting object were significantly lower than those of control chicks. This indicates that their persistent responses to the imprinting object were impeded by a temporal thyroid hormone decrease just before hatching. Consequently, the preference scores of MMI-administered chicks were significantly lower than those of control chicks. Furthermore, the preference score on the test was significantly correlated with the behavioral responses to the static imprinting object in the training. These results indicate that the intrinsic thyroid hormone level immediately before hatching is crucial for the learning process of imprinting.

Song-related brain auditory activity in Bengalese finches as examined by immediate early gene expressions: Comparison of arousal states and the correlational analyses between brain regions.

Songbirds use auditory feedback to memorize a tutor song in juveniles and to maintain it in adults. In Bengalese finches, electrophysiological studies showed the auditory responses in the premotor area HVC remained active regardless of asleep/awake status, in contrast to auditory gating phenomenon identified in zebra finches. We investigated the correlations in auditory activity between the brain regions and differences in the activity during wakefulness and sleeping in Bengalese finches. We used the immediate early gene egr-1 as a marker of neural activity that can detect regions responding to auditory stimuli in the whole brain. Results showed that auditory response, as measured by egr-1 expression to the bird's own song while sleeping and awake, was similar in HVC and NCM. Higher activity during awake than sleep was found only in the lower auditory area MLd. Analyses showed egr-1 expressions between brain regions induced by the bird's own song playback in awake/sleep conditions, suggesting that auditory information correlated with the inter part, not the outer part, of MLd with the higher song-related regions. Furthermore, the sleep condition suppressed the spontaneous activity, but not the song-induced activity in Area X. Altogether, this study presents a new attempt to explore the auditory-motor network using a molecular tool to map neurons of the nearly whole brain.

Comparisons of Hepatobiliary Phase Imaging Using Combinations of Parallel Imaging and Variable Degrees of Compressed Sensing With Use of Parallel Imaging Alone.

OBJECTIVE: This study aimed to compare the image quality in the hepatobiliary phase images of gadoxetic acid-enhanced liver magnetic resonance imaging using parallel imaging (PI) and compressed sensing (CS) reconstruction, using variable CS factors with the standard method using the PI technique. METHODS: In this study, 64 patients who underwent gadoxetic acid-enhanced liver magnetic resonance imaging at 3.0 T were enrolled. Hepatobiliary phase images were acquired 6 times using liver acquisition with volume acceleration (LAVA) and CS reconstruction with 5 CS factors 1.4, 1.6, 1.8, 2.0, and 2.5 (LAVA-CS 1.4, 1.6, 1.8, 2.0, and 2.5) and standard LAVA (LAVA-noCS). For objective analysis, the signal intensity ratios (SIRs) of the liver-to-spleen (SIR liver/spleen ), liver-to-portal vein (SIR liver/portal vein ), and liver-to-fat (SIR liver/fat ) were estimated. For subjective analysis, 2 radiologists independently evaluated the quality of all the images. RESULTS: The objective analysis demonstrated no significant difference in all evaluation parameters of all the images. Subjective analysis revealed that the scores of all evaluation items were higher for LAVA-noCS images than for LAVA-CS images, and only LAVA-CS 1.4 did not significantly differ from LAVA-noCS in all evaluation items ( P = 1.00 in 2 readers). CONCLUSIONS: A CS factor of 1.4 in the hepatobiliary phase image with combined PI and CS can reduce the scan time without degrading the image quality compared with the standard method.

Dopaminergic nuclei in the chick midbrain express serotonin receptor subfamily genes.

Serotonin (5-hydroxytryptamine, 5-HT) is a phylogenetically conserved modulator of numerous aspects of neural functions. Serotonergic neurons in the dorsal and median raphe nucleus provide ascending innervation to the entire forebrain and midbrain. Another important neural modulatory system exists in the midbrain, the dopaminergic system, which is associated to reward processing and motivation control. Dopaminergic neurons are distributed and clustered in the brain, classically designated as groups A8-A16. Among them, groups A8-A10 associated with reward processing and motivation control are located in the midbrain and projected to the forebrain. Recently, midbrain dopaminergic neurons were shown to be innervated by serotonergic neurons and modulated by 5-HT, with the crosstalk between serotonergic and dopaminergic systems attracting increased attention. In birds, previous studies revealed that midbrain dopaminergic neurons are located in the A8-A10 homologous clusters. However, the detailed distribution of dopaminergic neurons and the crosstalk between serotonergic and dopaminergic systems in the bird are poorly understood. To improve the understanding of the regulation of the dopaminergic by the serotonergic system, we performed in situ hybridization in the chick brainstem. We prepared RNA probes for chick orthologues of dopaminergic neuron-related genes; tyrosine hydroxylase (TH) and dopa decarboxylase (DDC), noradrenaline related genes; noradrenaline transporter (NAT) and dopamine beta-hydroxylase (DBH), and serotonin receptor genes; 5-HTR1A, 5-HTR1B, 5-HTR1D, 5-HTR1E, 5-HTR1F, 5-HTR2A, 5-HTR2B, 5-HTR2C, 5-HTR3A, 5-HTR4, 5-HTR5A, and 5-HTR7. We confirmed that the expression of tyrosine hydroxylase (TH) and NAT was well matched in all chick dopaminergic nuclei examined. This supported that the compensation of the function of dopamine transporter (DAT) by NAT is a general property of avian dopaminergic neurons. Furthermore, we showed that 5-HTR1A and 5-HTR1B were expressed in midbrain dopaminergic nuclei, suggesting the serotonergic regulation of the dopaminergic system via these receptors in chicks. Our findings will help us understand the interactions between the dopaminergic and serotonergic systems in birds at the molecular level.

Effect of Darkness on Intrinsic Motivation for Undirected Singing in Bengalese Finch (Lonchura striata Domestica): A Comparative Study With Zebra Finch (Taeniopygia guttata).

The zebra finch (ZF) and the Bengalese finch (BF) are animal models that have been commonly used for neurobiological studies on vocal learning. Although they largely share the brain structure for vocal learning and production, BFs produce more complex and variable songs than ZFs, providing a great opportunity for comparative studies to understand how animals learn and control complex motor behaviors. Here, we performed a comparative study between the two species by focusing on intrinsic motivation for non-courtship singing ("undirected singing"), which is critical for the development and maintenance of song structure. A previous study has demonstrated that ZFs dramatically increase intrinsic motivation for undirected singing when singing is temporarily suppressed by a dark environment. We found that the same procedure in BFs induced the enhancement of intrinsic singing motivation to much smaller degrees than that in ZFs. Moreover, unlike ZFs that rarely sing in dark conditions, substantial portion of BFs exhibited frequent singing in darkness, implying that such "dark singing" may attenuate the enhancement of intrinsic singing motivation during dark periods. In addition, measurements of blood corticosterone levels in dark and light conditions provided evidence that although BFs have lower stress levels than ZFs in dark conditions, such lower stress levels in BFs are not the major factor responsible for their frequent dark singing. Our findings highlight behavioral and physiological differences in spontaneous singing behaviors of BFs and ZFs and provide new insights into the interactions between singing motivation, ambient light, and environmental stress.

Chick Hippocampal Formation Displays Subdivision- and Layer-Selective Expression Patterns of Serotonin Receptor Subfamily Genes.

Hippocampal formation (HF) plays a key role in cognitive and emotional processing in mammals. In HF neural circuits, serotonin receptors (5-HTRs) modulate functions related to cognition and emotion. To understand the phylogenetic continuity of the neural basis for cognition and emotion, it is important to identify the neural circuits that regulate cognitive and emotional processing in animals. In birds, HF has been shown to be related to cognitive functions and emotion-related behaviors. However, details regarding the distribution of 5-HTRs in the avian brain are very sparse, and 5-HTRs, which are potentially involved in cognitive functions and emotion-related behaviors, are poorly understood. Previously, we showed that 5-HTR1B and 5-HTR3A were expressed in chick HF. To identify additional 5-HTRs that are potentially involved in cognitive and emotional functions in avian HF, we selected the chick orthologs of 5-HTR1D, 5-HTR1E, 5-HTR1F, 5-HTR2B, 5-HTR5A, and 5-HTR7 and performed in situ hybridization in the chick telencephalon. We found that 5-HTR1D, 5-HTR1E, 5-HTR5A, and 5-HTR7 were expressed in the chick HF, especially 5-HTR1D and 5-HTR1E, which showed subdivision- and layer-selective expression patterns, suggesting that the characteristic 5-HT regulation is involved in cognitive functions and emotion-related behaviors in these HF regions. These findings can facilitate the understanding of serotonin regulation in avian HF and the correspondence between the HF subdivisions of birds and mammals.

Imprintability of Newly Hatched Domestic Chicks on an Artificial Object: A Novel High Time-Resolution Apparatus Based on a Running Disc.

In filial imprinting, newly hatched chicks repeatedly approach a conspicuous object nearby and memorize it, even though it is an artificial object instead of their mother hen. Imprinting on an artificial object in a laboratory setting has a clear sensitive period from post hatch days 1-3 in the case of domestic chicks. However, the establishment of imprintability are difficult to investigate because of the limitations of the behavioral apparatus. In this study, we developed a novel behavioral apparatus, based on a running disc, to investigate the learning processes of imprinting in newly hatched domestic chicks. In the apparatus, the chick repeatedly approaches the imprinting object on the disc. The apparatus sends a transistor-transistor-logic signal every 1/10 turn of the disc to a personal computer through a data acquisition system following the chick's approach to the imprinting object on the monitor. The imprinting training and tests were designed to define the three learning processes in imprinting. The first process is the one in which chicks spontaneously approach the moving object. The second is an acquired process in which chicks approach an object even when it is static. In the third process, chicks discriminate between the differently colored imprinting object and the control object in the preference test. Using the apparatus, the difference in the chicks' behavior during or after the sensitive period was examined. During the sensitive period, the chicks at post hatch hour 12 and 18 developed the first imprinting training process. The chicks at post hatch hour 24 maintained learning until the second process. The chicks at post hatch hour 30 reached the discrimination process in the test. After the sensitive period, the chicks reared in darkness until post hatch day 4 exhibited poor first learning process in the training. Thus, this apparatus will be useful for the detection of behavioral changes during neuronal development and learning processes.

Mismatch Responses Evoked by Sound Pattern Violation in the Songbird Forebrain Suggest Common Auditory Processing With Human.

Learning sound patterns in the natural auditory scene and detecting deviant patterns are adaptive behaviors that aid animals in predicting future events and behaving accordingly. Mismatch negativity (MMN) is a component of the event-related potential (ERP) that is reported in humans when they are exposed to unexpected or rare stimuli. MMN has been studied in several non-human animals using an oddball task by presenting deviant pure tones that were interspersed within a sequence of standard pure tones and comparing the neural responses. While accumulating evidence suggests the homology of non-human animal MMN-like responses (MMRs) and human MMN, it is still not clear whether the function and neural mechanisms of MMRs and MMN are comparable. The Java sparrow (Lonchura oryzivora) is a songbird that is a vocal learner, is highly social, and maintains communication with flock members using frequently repeated contact calls and song. We expect that the songbird is a potentially useful animal model that will broaden our understanding of the characterization of MMRs. Due to this, we chose this species to explore MMRs to the deviant sounds in the single sound oddball task using both pure tones and natural vocalizations. MMRs were measured in the caudomedial nidopallium (NCM), a higher-order auditory area. We recorded local field potentials under freely moving conditions. Significant differences were observed in the negative component between deviant and standard ERPs, both to pure tones and natural vocalizations in the oddball sequence. However, the subsequent experiments using the randomized standard sequence and regular pattern sequence suggest the possibility that MMR elicited in the oddball paradigm reflects the adaptation to a repeated standard sound but not the genuine deviance detection. Furthermore, we presented contact call triplet sequences and investigated MMR in the NCM in response to sound sequence order. We found a significant negative shift in response to a difference in sequence pattern. This demonstrates MMR elicited by violation of the pattern of the triplet sequence and the ability to extract sound sequence information in the songbird auditory forebrain. Our study sheds light on the electrophysiological properties of auditory sensory memory processing, expanding the scope of characterization of MMN-like responses beyond simple deviance detection, and provides a comparative perspective on syntax processing in human.

Subtype-selective contribution of muscarinic acetylcholine receptors for filial imprinting in newly-hatched domestic chicks.

Muscarinic acetylcholine receptors (mAChRs) play an important role in many brain functions. Our previous study revealed that the injection of mAChRs antagonist scopolamine into the intermediate medial mesopallium (IMM) region, which is critical for filial imprinting, impairs memory formation. In avian brains, four mAChR subtypes have been identified (M2, M3, M4 and M5). M3 and M5 receptors increase the excitability of neurons, whereas M2 and M4 receptors reduce the excitability. Because the scopolamine blocks all subtypes, the previous study did not identify which subtype contributes to the memory formation. By injecting several types of mAChR antagonists into the IMM, in this study we determined which mAChR subtype plays a critical role in imprinting. First, the effects of antagonists on the excitatory receptor subtypes M3 and M5 were examined. Injection of the M3 antagonist (DAU5884) at 20 mM or the M5 antagonist (ML381) at 2 mM impaired imprinting. Considering the pKi value of DAU5884, the impairment seems to be caused by DAU5884 binding to M3 and/or M4 receptors. Second, the effect of antagonists on the inhibitory receptor subtype M2 was examined. The results showed that the M2 antagonist (AQ-RA741) impaired imprinting at a concentration of 20 mM. Considering the pKi value of AQ-RA741, the impairment seems to be caused by AQ-RA741 binding to M2 and/or M4. The findings of this study suggests that the excitatory receptor subtypes M3 and M5 and the inhibitory receptor subtype M2 and/or M4 cooperate to achieve the appropriate balance of acetylcholine signaling to execute imprinting.

Oxytocin variation and brain region-specific gene expression in a domesticated avian species.

The Bengalese finch was domesticated more than 250 years ago from the wild white-rumped munia (WRM). Similar to other domesticated species, Bengalese finches show a reduced fear response and have lower corticosterone levels, compared to WRMs. Bengalese finches and munias also have different song types. Since oxytocin (OT) has been found to be involved in stress coping and auditory processing, we tested whether the OT sequence and brain expression pattern and content differ in wild munias and domesticated Bengalese finches. We sequenced the OT from 10 wild munias and 11 Bengalese finches and identified intra-strain variability in both the untranslated and protein-coding regions of the sequence, with all the latter giving rise to synonymous mutations. Several of these changes fall in specific transcription factor-binding sites, and show either a conserved or a relaxed evolutionary trend in the avian lineage, and in vertebrates in general. Although in situ hybridization in several hypothalamic nuclei did not reveal significant differences in the number of cells expressing OT between the two strains, real-time quantitative PCR showed a significantly higher OT mRNA expression in the cerebrum of the Bengalese finches relative to munias, but a significantly lower expression in their diencephalon. Our study thus points to a brain region-specific pattern of neurochemical expression in domesticated and wild avian strains, which could be linked to domestication and the behavioral changes associated with it.

Gene expression profiles of the muscarinic acetylcholine receptors in brain regions relating to filial imprinting of newly-hatched domestic chicks.

Muscarinic acetylcholine receptors (mAChRs) in the central nervous system play an important role in regulating complex functions such as learning, memory, and selective attention. Five subtypes of the mAChRs (M1-M5) have been identified in mammals, and are classified into two subfamilies: excitatory (M1, M3, and M5) and inhibitory (M2 and M4) subfamilies. Filial imprinting of domestic chicks is a useful model in the laboratory to investigate the mechanisms of memory formation in early learning. We recently found that mAChRs in the intermediate medial mesopallium (IMM) are involved in the memory formation of imprinting. However, expression profiles of each mAChR subtype in the brain regions including the IMM remain unexplored. Here we show the unique gene expression of each mAChR subtype in the pallial regions involved in imprinting. In terms of the excitatory mAChRs, M5 was expressed in the IMM region and other parts of the pallium, whereas M3 was less expressed in the IMM but highly expressed in the hyperpallium and nidopallium. Regarding the inhibitory mAChRs, M2 was sparsely distributed but clearly in some cells throughout the pallial regions. M4 was highly expressed in the IMM region and other parts of the pallium. These expression profiles can be used as a basis for understanding cholinergic modulation in the memory formation of imprinting and other learning processes in birds, and compared to those of mammals.

Neural correlates of vocal initiation in the VTA/SNc of juvenile male zebra finches.

Initiation and execution of complex learned vocalizations such as human speech and birdsong depend on multiple brain circuits. In songbirds, neurons in the motor cortices and basal ganglia circuitry exhibit preparatory activity before initiation of song, and that activity is thought to play an important role in successful song performance. However, it remains unknown where a start signal for song is represented in the brain and how such a signal would lead to appropriate vocal initiation. To test whether neurons in the midbrain ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) show activity related to song initiation, we carried out extracellular recordings of VTA/SNc single units in singing juvenile male zebra finches. We found that a subset of VTA/SNc units exhibit phasic activity precisely time-locked to the onset of the song bout, and that the activity occurred specifically at the beginning of song. These findings suggest that phasic activity in the VTA/SNc represents a start signal that triggers song vocalization.

Intrinsic motivation for singing in songbirds is enhanced by temporary singing suppression and regulated by dopamine.

Behaviors driven by intrinsic motivation are critical for development and optimization of physical and brain functions, but their underlying mechanisms are not well studied due to the complexity and autonomy of the behavior. Songbirds, such as zebra finches, offer a unique opportunity to study neural substrates of intrinsic motivation because they spontaneously produce many renditions of songs with highly-quantifiable structure for vocal practice, even in the absence of apparent recipients ("undirected singing"). Neural substrates underlying intrinsic motivation for undirected singing are still poorly understood partly because singing motivation cannot be easily manipulated due to its autonomy. Also, undirected singing itself acts as an internal reward, which could increase singing motivation, leading to difficulty in measuring singing motivation independent of singing-associated reward. Here, we report a simple procedure to easily manipulate and quantify intrinsic motivation for undirected singing independent of singing-associated reward. We demonstrate that intrinsic motivation for undirected singing is dramatically enhanced by temporary suppression of singing behavior and the degree of enhancement depends on the duration of suppression. Moreover, by examining latencies to the first song following singing suppression as a measure of singing motivation independent of singing-associated reward, we demonstrate that intrinsic singing motivation is critically regulated by dopamine through D2 receptors. These results provide a simple experimental tool to manipulate and measure the intrinsic motivation for undirected singing and illustrate the importance of zebra finches as a model system to study the neural basis of intrinsically-motivated behaviors.

Seasonal regulation of singing-driven gene expression associated with song plasticity in the canary, an open-ended vocal learner.

Songbirds are one of the few animal taxa that possess vocal learning abilities. Different species of songbirds exhibit species-specific learning programs during song acquisition. Songbirds with open-ended vocal learning capacity, such as the canary, modify their songs during adulthood. Nevertheless, the neural molecular mechanisms underlying open-ended vocal learning are not fully understood. We investigated the singing-driven expression of neural activity-dependent genes (Arc, Egr1, c-fos, Nr4a1, Sik1, Dusp6, and Gadd45ß) in the canary to examine a potential relationship between the gene expression level and the degree of seasonal vocal plasticity at different ages. The expression of these genes was differently regulated throughout the critical period of vocal learning in the zebra finch, a closed-ended song learner. In the canary, the neural activity-dependent genes were induced by singing in the song nuclei throughout the year. However, in the vocal motor nucleus, the robust nucleus of the arcopallium (RA), all genes were regulated with a higher induction rate by singing in the fall than in the spring. The singing-driven expression of these genes showed a similar induction rate in the fall between the first year juvenile and the second year adult canaries, suggesting a seasonal, not age-dependent, regulation of the neural activity-dependent genes. By measuring seasonal vocal plasticity and singing-driven gene expression, we found that in RA, the induction intensity of the neural activity-dependent genes was correlated with the state of vocal plasticity. These results demonstrate a correlation between vocal plasticity and the singing-driven expression of neural activity-dependent genes in RA through song development, regardless of whether a songbird species possesses an open- or closed-ended vocal learning capacity.

Generation of Tetrafluoroethylene-Propylene Elastomer-Based Microfluidic Devices for Drug Toxicity and Metabolism Studies.

Polydimethylsiloxane (PDMS) is widely used to fabricate microfluidic organs-on-chips. Using these devices (PDMS-based devices), the mechanical microenvironment of living tissues, such as pulmonary respiration and intestinal peristalsis, can be reproduced in vitro. However, the use of PDMS-based devices in drug discovery research is limited because of their extensive absorption of drugs. In this study, we investigated the feasibility of the tetrafluoroethylene-propylene (FEPM) elastomer to fabricate a hepatocyte-on-a-chip (FEPM-based hepatocyte chip) with lower drug absorption. The FEPM-based hepatocyte chip expressed drug-metabolizing enzymes, drug-conjugating enzymes, and drug transporters. Also, it could produce human albumin. Although the metabolites of midazolam and bufuralol were hardly detected in the PDMS-based hepatocyte chip, they were detected abundantly in the FEPM-based hepatocyte chip. Finally, coumarin-induced hepatocyte cytotoxicity was less severe in the PDMS-based hepatocyte chip than in the FEPM-based hepatocyte chip, reflecting the different drug absorptions of the two chips. In conclusion, the FEPM-based hepatocyte chip could be a useful tool in drug discovery research, including drug metabolism and toxicity studies.

Phylogeny and mechanisms of shared hierarchical patterns in birdsong.

Organizational patterns can be shared across biological systems, and revealing the factors shaping common patterns can provide insight into fundamental biological mechanisms. The behavioral pattern that elements with more constituents tend to consist of shorter constituents (Menzerath's law [ML]) was described first in speech and language (e.g., words with more syllables consist of shorter syllables) and subsequently in music and animal communication. Menzerath's law is hypothesized to reflect efficiency in information transfer, but biases and constraints in motor production can also lead to this pattern. We investigated the evolutionary breadth of ML and the contribution of production mechanisms to ML in the songs of 15 songbird species. Negative relationships between the number and duration of constituents (e.g., syllables in phrases) were observed in all 15 species. However, negative relationships were also observed in null models in which constituents were randomly allocated into observed element durations, and the observed negative relationship for numerous species did not differ from the null model; consequently, ML in these species could simply reflect production constraints and not communicative efficiency. By contrast, ML was significantly different from the null model for more than half the cases, suggesting additional organizational rules are imposed onto birdsongs. Production mechanisms are also underscored by the finding that canaries and zebra finches reared without auditory experiences that guide vocal development produced songs with nearly identical ML patterning as typically reared birds. These analyses highlight the breadth with which production mechanisms contribute to this prevalent organizational pattern in behavior.

Serotonergic Neurons in the Chick Brainstem Express Various Serotonin Receptor Subfamily Genes.

Serotonin (5-hydroxytryptamine, 5-HT) is a phylogenetically conserved modulatory neurotransmitter. In mammals, 5-HT plays an important role in the regulation of many mental states and the processing of emotions in the central nervous system. Serotonergic neurons in the central nervous system, including the dorsal raphe (DR) and median raphe (MR) nuclei, are spatially clustered in the brainstem and provide ascending innervation to the entire forebrain and midbrain. Both between and within the DR and MR, these serotonergic neurons have different cellular characteristics, developmental origin, connectivity, physiology, and related behavioral functions. Recently, an understanding of the heterogeneity of the DR and MR serotonergic neurons has been developed at the molecular level. In birds, emotion-related behavior is suggested to be modulated by the 5-HT system. However, correspondence between the raphe nuclei of birds and mammals, as well as the cellular heterogeneity in the serotonergic neurons of birds are poorly understood. To further understand the heterogeneity of serotonergic neurons in birds, we performed a molecular dissection of the chick brainstem using in situ hybridization. In this study, we prepared RNA probes for chick orthologs of the following serotonin receptor genes: 5-HTR1A, 5-HTR1B, 5-HTR1D, 5-HTR1E, 5-HTR1F, 5-HTR2A, 5-HTR2B, 5-HTR2C, 5-HTR3A, 5-HTR4, 5-HTR5A, and 5-HTR7. We showed that the expression pattern of 5-HT receptors in the serotonin neurons of chick DR and MR may vary, suggesting heterogeneity among and within the serotonin neurons of the DR and MR in the chick brainstem. Our findings regarding the molecular properties of serotonergic neurons in the bird raphe system will facilitate a good understanding of the correspondence between bird and mammalian raphes.

Structure of the dopamine D2 receptor in complex with the antipsychotic drug spiperone.

In addition to the serotonin 5-HT2A receptor (5-HT2AR), the dopamine D2 receptor (D2R) is a key therapeutic target of antipsychotics for the treatment of schizophrenia. The inactive state structures of D2R have been described in complex with the inverse agonists risperidone (D2Rris) and haloperidol (D2Rhal). Here we describe the structure of human D2R in complex with spiperone (D2Rspi). In D2Rspi, the conformation of the extracellular loop (ECL) 2, which composes the ligand-binding pocket, was substantially different from those in D2Rris and D2Rhal, demonstrating that ECL2 in D2R is highly dynamic. Moreover, D2Rspi exhibited an extended binding pocket to accommodate spiperone's phenyl ring, which probably contributes to the selectivity of spiperone to D2R and 5-HT2AR. Together with D2Rris and D2Rhal, the structural information of D2Rspi should be of value for designing novel antipsychotics with improved safety and efficacy.