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.

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.

Suppressive Modulation of the Chick Forebrain Network for Imprinting by Thyroid Hormone: An in Vitro Study.

The thyroid hormone 3,5,3'-triiodothyronine (T3) is considered to act acutely in the chick forebrain because focal infusion of T3 to the intermediate medial mesopallium (IMM) causes 4 to 6-day-old hatchlings to become imprintable approximately 30 min after the infusion. To understand the mechanism of this acute T3 action, we examined synaptic responses of IMM neurons in slice preparations in vitro. Extracellular field potential responses to local electrical stimulation were pharmacologically dissociated to synaptic components mediated by AMPA and NMDA receptors, as well as GABA-A and -B receptors. Bath-applied T3 (20-40 µM) enhanced the positive peak amplitude of the field potential, which represented the GABA-A component. Bicuculline induced spontaneous epileptic bursts by NMDA receptor activation, and subsequent application of T3 suppressed the bursting frequency. Pretreatment of slices with T3 failed to influence the synaptic potentiation caused by tetanic stimulation. Intracellular whole-cell recording using a patch electrode confirmed the T3 actions on the GABA-A and NMDA components. T3 enhanced the GABA-A response and suppressed the NMDA plateau potential without changes in the resting membrane potential or the threshold of action potentials. Contrary to our initial expectation, T3 suppressed the synaptic drives of IMM neurons, and did not influence activity-dependent synaptic potentiation. Imprinting-associated T3 influx may act as an acute suppressor of the IMM network.

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.

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.

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.

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.

The dorsal arcopallium of chicks displays the expression of orthologs of mammalian fear related serotonin receptor subfamily genes.

Fear is an adaptive emotion that elicits defensive behavioural responses against aversive threats in animals. In mammals, serotonin receptors (5-HTRs) have been shown to modulate fear-related neural circuits in the basolateral amygdala complex (BLA). To understand the phylogenetic continuity of the neural basis for fear, it is important to identify the neural circuit that processes fear in other animals. In birds, fear-related behaviours were suggested to be processed in the arcopallium/amygdala complex and modulated by the serotonin (5-HT) system. However, details about the distribution of 5-HTRs in the avian brain are very sparsely reported, and the 5-HTR that is potentially involved in fear-related behaviour has not been elucidated. In this study, we showed that orthologs of mammalian 5-HTR genes that are expressed in the BLA, namely 5-HTR1A, 5-HTR1B, 5-HTR2A, 5-HTR2C, 5-HTR3A, and 5-HTR4, are expressed in a part of the chick arcopallium/amygdala complex called the dorsal arcopallium. This suggests that serotonergic regulation in the dorsal arcopallium may play an important role in regulating fear-related behaviour in birds. Our findings can be used as a basis for comparing the processing of fear and its serotonergic modulation in the mammalian amygdala complex and avian arcopallium/amygdala complex.

Subtypes of dementia with Lewy bodies are associated with α-synuclein and tau distribution.

OBJECTIVE: To determine whether Lewy body disease subgroups have different clinical profiles. METHODS: Participants had dementia, autopsy-confirmed transitional or diffuse Lewy body disease (TLBD or DLBD) (n = 244), or Alzheimer disease (AD) (n = 210), and were seen at least twice (mean follow-up 6.2 ± 3.8 years). TLBD and DLBD groups were partitioned based on the presence or absence of neocortical neurofibrillary tangles using Braak staging. Four Lewy body disease subgroups and AD were compared on clinical features, dementia trajectory, and onset latency of probable dementia with Lewy bodies (DLB) or a DLB syndrome defined as probable DLB or dementia with one core feature of parkinsonism or probable REM sleep behavior disorder. RESULTS: In TLBD and DLBD without neocortical tangles, diagnostic sensitivity was strong for probable DLB (87% TLBD, 96% DLBD) and the DLB syndrome (97% TLBD, 98% DLBD) with median latencies <1 year from cognitive onset, and worse baseline attention-visual processing but better memory-naming scores than AD. In DLBD with neocortical tangles, diagnostic sensitivity was 70% for probable DLB and 77% for the DLB syndrome with respective median latencies of 3.7 years and 2.7 years from cognitive onset, each associated with tangle distribution. This group had worse baseline attention-visual processing than AD, but comparable memory-naming impairment. TLBD with neocortical tangles had 48% diagnostic sensitivity for probable DLB and 52% for the DLB syndrome, with median latencies >6 years from cognitive onset, and were cognitively similar to AD. Dementia trajectory was slowest for TLBD without neocortical tangles, and fastest for DLBD with neocortical tangles. CONCLUSIONS: The phenotypic expression of DLB was associated with the distribution of α-synuclein and tau pathology.

Blockade of muscarinic acetylcholine receptor by scopolamine impairs the memory formation of filial imprinting in domestic chicks (Gallus Gallus domesticus).

Filial imprinting in precocial birds is a useful model for studying memory formation in early learning. The intermediate medial mesopallium (IMM) in the dorsal telencephalon is one of the critical brain regions where the releases of several neurotransmitters increase after the start of imprinting training. Among the increased neurotransmitters, the role of acetylcholine in imprinting has remained unclear. Acetylcholine in the mammalian brain plays an important role in encoding new memories. The muscarinic acetylcholine receptor subtype 1 (M1 receptor) and subtype 3 (M3 receptor) in the hippocampus and cortex of mammalian brain have been shown to be necessary for memory encoding. In this study, we examined whether the imprinting acquisition in chick can be impaired by injecting muscarinic acetylcholine receptor (mAChR) antagonist scopolamine into the bilateral IMM. We show that the injection of scopolamine decreased the preference for the imprinting object in the test, but did not affect the number of approaches to the imprinting object during training. Immunoblotting and immunohistochemistry revealed that M3 receptors were expressed in the IMM. Our data suggest that acetylcholine is involved in the memory formation of imprinting through M3 receptors in the IMM. The scopolamine-injected chicks may be useful as an animal model for dementia such as Alzheimer's disease.

The chick pallium displays divergent expression patterns of chick orthologues of mammalian neocortical deep layer-specific genes.

The avian pallium is organised into clusters of neurons and does not have layered structures such as those seen in the mammalian neocortex. The evolutionary relationship between sub-regions of avian pallium and layers of mammalian neocortex remains unclear. One hypothesis, based on the similarities in neural connections of the motor output neurons that project to sub-pallial targets, proposed the cell-type homology between brainstem projection neurons in neocortex layers 5 or 6 (L5/6) and those in the avian arcopallium. Recent studies have suggested that gene expression patterns are associated with neural connection patterns, which supports the cell-type homology hypothesis. However, a limited number of genes were used in these studies. Here, we showed that chick orthologues of mammalian L5/6-specific genes, nuclear receptor subfamily 4 group A member 2 and connective tissue growth factor, were strongly expressed in the arcopallium. However, other chick orthologues of L5/6-specific genes were primarily expressed in regions other than the arcopallium. Our results do not fully support the cell-type homology hypothesis. This suggests that the cell types of brainstem projection neurons are not conserved between the avian arcopallium and the mammalian neocortex L5/6. Our findings may help understand the evolution of pallium between birds and mammals.

Brain networks underlying conscious tactile perception of textures as revealed using the velvet hand illusion.

Humans are adept at perceiving textures through touch. Previous neuroimaging studies have identified a distributed network of brain regions involved in the tactile perception of texture. However, it remains unclear how nodes in this network contribute to the tactile awareness of texture. To examine the hypothesis that such awareness involves the interaction of the primary somatosensory cortex with higher order cortices, we conducted a functional magnetic resonance imaging (fMRI) study utilizing the velvet hand illusion, in which an illusory velvet-like surface is perceived between the hands. Healthy participants were subjected to a strong illusion, a weak illusion, and tactile perception of real velvet. The strong illusion induced greater activation in the primary somatosensory cortex (S1) than the weak illusion, and increases in such activation were positively correlated with the strength of the illusion. Furthermore, both actual and illusory perception of velvet induced common activation in S1. Psychophysiological interaction (PPI) analysis revealed that the strength of the illusion modulated the functional connectivity of S1 with each of the following regions: the parietal operculum, superior parietal lobule, precentral gyrus, insula, and cerebellum. The present results indicate that S1 is associated with the conscious tactile perception of textures, which may be achieved via interactions with higher order somatosensory areas.

Wnt-2b in the intermediate hyperpallium apicale of the telencephalon is critical for the thyroid hormone-mediated opening of the sensitive period for filial imprinting in domestic chicks (Gallus gallus domesticus).

Filial imprinting is the behavior observed in chicks during the sensitive or critical period of the first 2-3 days after hatching; however, after this period they cannot be imprinted when raised in darkness. Our previous study showed that temporal augmentation of the endogenous thyroid hormone 3,5,3'-triiodothyronine (T3) in the telencephalon, by imprinting training, starts the sensitive period just after hatching. Intravenous injection of T3 enables imprinting of chicks on days 4 or 6 post-hatching, even when the sensitive period has ended. However, the molecular mechanism of how T3 acts as a determinant of the sensitive period is unknown. Here, we show that Wnt-2b mRNA level is increased in the T3-injected telencephalon of 4-day old chicks. Pharmacological inhibition of Wnt signaling in the intermediate hyperpallium apicale (IMHA), which is the caudal area of the telencephalon, blocked the recovery of the sensitive period following T3 injection. In addition, injection of recombinant Wnt-2b protein into the IMHA helped chicks recover the sensitive period without the injection of T3. Lastly, we showed Wnt signaling to be involved in imprinting via the IMHA region on day 1 during the sensitive period. These results indicate that Wnt signaling plays a critical role in the opening of the sensitive period downstream of T3.

Gene expression of Dio2 (thyroid hormone converting enzyme) in telencephalon is linked with predisposed biological motion preference in domestic chicks.

Filial imprinting leads to the formation of social attachment if training is performed during a brief sensitive period after hatching. We found that thyroid hormone (3,5,3'-triiodothyronine, T3) acts as a critical determining factor of the sensitive period in domestic chicks. Imprinting upregulates gene expression of the converting enzyme (Dio2, type 2 iodothyronine deiodinase) in the telencephalon, leading to increased brain T3 content. If systemically applied, T3 facilitates imprinting in aged chicks even after the sensitive period is over. Imprinting is also associated with the rapid development of visual perception. Exposure to motion pictures induces a predisposed preference to Johansson's biological motion (BM), and those individuals with higher BM preference are more easily imprinted. Here, we examined whether Dio2 expression is also linked with BM predisposition. Chicks were trained by a rotating red block, and tested for imprinting (experiment 1) and BM preference (experiment 2). To examine the time courses of behavioural and physiological processes, Dio2 expression in telencephalon was compared among three groups: naïve control chicks, and chicks trained for a short (0.5 h) or long period (2 h). In experiment 1, higher Dio2 expression appeared in the 2-h group than in the 0.5-h/control groups, but it was not correlated with the individual imprinting score. In experiment 2, a significant positive correlation appeared between Dio2 expression and BM preference in 2-h-trained chicks. Memory priming by T3 is therefore functionally linked to BM preference induction, leading to successful imprinting to natural objects even when they are initially exposed to artificial objects.

Thyroid Hormone Sensitizes the Imprinting-Associated Induction of Biological Motion Preference in Domestic Chicks.

Filial imprinting is associated with induction of predisposed preference to animations that bear visual features of Johansson's biological motion (BM), and the induction is limited to a few days after hatching. As thyroid hormone (3,5,3'-triiodothyronine, T3) plays a critical role in determining the sensitive period of imprinting, we examined if exogenously applied T3 (or iopanoic acid, IOP; a selective inhibitor for converting enzymes) could also sensitize (or desensitize) the BM induction. Chicks were trained by using a non-BM stimulus (rotating red toy) according to a conventional imprinting procedure. Trained chicks were tested for preference to a point-light BM animation (walking chick) over a non-BM animation (linear motion), and for the preference for the familiarized stimulus (red toy) over an unfamiliar one (yellow toy). In 1-day chicks, those injected with IOP showed significantly lower scores than controls on both BM and imprinting tests. In 4-days chicks, those injected with T3 showed higher scores than control, but the difference in BM score was not significant. Imprinting and the accompanying T3 surge may be necessary for the predisposed BM preference to appear in 1-day chicks. Even after the conventional sensitive period is over, exogenous T3 can partly re-sensitize the BM preference as it does imprinting.

GABA-A and GABA-B Receptors in Filial Imprinting Linked With Opening and Closing of the Sensitive Period in Domestic Chicks (Gallus gallus domesticus).

Filial imprinting of domestic chicks has a well-defined sensitive (critical) period lasting in the laboratory from hatching to day 3. It is a typical model to investigate the molecular mechanisms underlying memory formation in early learning. We recently found that thyroid hormone 3,5,3'-triiodothyronine (T3) is a determinant of the sensitive period. Rapid increases in cerebral T3 levels are induced by imprinting training, rendering chicks imprintable. Furthermore, the administration of exogenous T3 makes chicks imprintable on days 4 or 6 even after the sensitive period has ended. However, how T3 affects neural transmission to enable imprinting remains mostly unknown. In this study, we demonstrate opposing roles for gamma-aminobutyric acid (GABA)-A and GABA-B receptors in imprinting downstream of T3. Quantitative reverse transcription polymerase chain reaction and immunoblotting showed that the GABA-A receptor expression increases gradually from days 1 to 5, whereas the GABA-B receptor expression gradually decreases. We examined whether neurons in the intermediate medial mesopallium (IMM), the brain region responsible for imprinting, express both types of GABA receptors. Immunostaining showed that morphologically identified putative projection neurons express both GABA-A and GABA-B receptors, suggesting that those GABA receptors interact with each other in these cells to modulate the IMM outputs. The roles of GABA-A and GABA-B receptors were investigated using various agonists and antagonists. Our results show that GABA-B receptor antagonists suppressed imprinting on day 1, while its agonists made day 4 chicks imprintable without administration of exogenous T3. By contrast, GABA-A receptor agonists suppressed imprinting on day 1, while its antagonists induced imprintability on day 4 without exogenous T3. Furthermore, both GABA-A receptor agonists and GABA-B receptor antagonists suppressed T3-induced imprintability on day 4 after the sensitive period has ended. Our data from these pharmacological experiments indicate that GABA-B receptors facilitate imprinting downstream of T3 by initiating the sensitive period, while the GABA-A receptor contributes to the termination of the sensitive period. In conclusion, we propose that opposing roles of GABA-A and GABA-B receptors in the brain during development determine the induction and termination of the sensitive period.

The limbic and neocortical contribution of α-synuclein, tau, and amyloid ß to disease duration in dementia with Lewy bodies.

INTRODUCTION: We sought to assess the individual and combined contribution of limbic and neocortical α-synuclein, tau, and amyloid ß (Aß) to duration of illness in dementia with Lewy bodies (DLB). METHODS: Quantitative digital pathology of limbic and neocortical α-synuclein, tau, and Aß was assessed in 49 patients with clinically probable DLB. Regression modeling examined the unique and shared contribution of each pathology to the variance of illness duration. RESULTS: Patients with diffuse Lewy body disease had more severe pathology of each type and a shorter duration of illness than individuals with transitional Lewy body disease. The three pathologies accounted for 25% of the total variance of duration of illness, with 19% accounted for by α-synuclein alone or in combination with tau and Aß. When the diffuse Lewy body disease group was examined separately, α-synuclein deposition significantly exceeded that of tau and Aß. In this model, 20% of 24% total variance in the model for duration of illness was accounted for independently by α-synuclein. DISCUSSION: In DLB, α-synuclein is an important predictor of disease duration, both independently and synergistically with tau and Aß.

Distinct spatiotemporal accumulation of N-truncated and full-length amyloid-ß42 in Alzheimer's disease.

Accumulation of amyloid-ß peptides is a dominant feature in the pathogenesis of Alzheimer's disease; however, it is not clear how individual amyloid-ß species accumulate and affect other neuropathological and clinical features in the disease. Thus, we compared the accumulation of N-terminally truncated amyloid-ß and full-length amyloid-ß, depending on disease stage as well as brain area, and determined how these amyloid-ß species respectively correlate with clinicopathological features of Alzheimer's disease. To this end, the amounts of amyloid-ß species and other proteins related to amyloid-ß metabolism or Alzheimer's disease were quantified by enzyme-linked immunosorbent assays (ELISA) or theoretically calculated in 12 brain regions, including neocortical, limbic and subcortical areas from Alzheimer's disease cases (n = 19), neurologically normal elderly without amyloid-ß accumulation (normal ageing, n = 13), and neurologically normal elderly with cortical amyloid-ß accumulation (pathological ageing, n = 15). We observed that N-terminally truncated amyloid-ß42 and full-length amyloid-ß42 accumulations distributed differently across disease stages and brain areas, while N-terminally truncated amyloid-ß40 and full-length amyloid-ß40 accumulation showed an almost identical distribution pattern. Cortical N-terminally truncated amyloid-ß42 accumulation was increased in Alzheimer's disease compared to pathological ageing, whereas cortical full-length amyloid-ß42 accumulation was comparable between Alzheimer's disease and pathological ageing. Moreover, N-terminally truncated amyloid-ß42 were more likely to accumulate more in specific brain areas, especially some limbic areas, while full-length amyloid-ß42 tended to accumulate more in several neocortical areas, including frontal cortices. Immunoprecipitation followed by mass spectrometry analysis showed that several N-terminally truncated amyloid-ß42 species, represented by pyroglutamylated amyloid-ß11-42, were enriched in these areas, consistent with ELISA results. N-terminally truncated amyloid-ß42 accumulation showed significant regional association with BACE1 and neprilysin, but not PSD95 that regionally associated with full-length amyloid-ß42 accumulation. Interestingly, accumulations of tau and to a greater extent apolipoprotein E (apoE, encoded by APOE) were more strongly correlated with N-terminally truncated amyloid-ß42 accumulation than those of other amyloid-ß species across brain areas and disease stages. Consistently, immunohistochemical staining and in vitro binding assays showed that apoE co-localized and bound more strongly with pyroglutamylated amyloid-ß11-x fibrils than full-length amyloid-ß fibrils. Retrospective review of clinical records showed that accumulation of N-terminally truncated amyloid-ß42 in cortical areas was associated with disease onset, duration and cognitive scores. Collectively, N-terminally truncated amyloid-ß42 species have spatiotemporal accumulation patterns distinct from full-length amyloid-ß42, likely due to different mechanisms governing their accumulations in the brain. These truncated amyloid-ß species could play critical roles in the disease by linking other clinicopathological features of Alzheimer's disease.