Characteristics of auditory steady-state responses to different click frequencies in awake intact macaques.

BACKGROUND: Auditory steady-state responses (ASSRs) are periodic evoked responses to constant periodic auditory stimuli, such as click trains, and are suggested to be associated with higher cognitive functions in humans. Since ASSRs are disturbed in human psychiatric disorders, recording ASSRs from awake intact macaques would be beneficial to translational research as well as an understanding of human brain function and its pathology. However, ASSR has not been reported in awake macaques. RESULTS: Electroencephalograms (EEGs) were recorded from awake intact macaques, while click trains at 20-83.3 Hz were binaurally presented. EEGs were quantified based on event-related spectral perturbation (ERSP) and inter-trial coherence (ITC), and ASSRs were significantly demonstrated in terms of ERSP and ITC in awake intact macaques. A comparison of ASSRs among different click train frequencies indicated that ASSRs were maximal at 83.3 Hz. Furthermore, analyses of laterality indices of ASSRs showed that no laterality dominance of ASSRs was observed. CONCLUSIONS: The present results demonstrated ASSRs, comparable to those in humans, in awake intact macaques. However, there were some differences in ASSRs between macaques and humans: macaques showed maximal ASSR responses to click frequencies higher than 40 Hz that has been reported to elicit maximal responses in humans, and showed no dominant laterality of ASSRs under the electrode montage in this study compared with humans with right hemisphere dominance. The future ASSR studies using awake intact macaques should be aware of these differences, and possible factors, to which these differences were ascribed, are discussed.

Early-life experiences altered the maturation of the lateral habenula in mouse models, resulting in behavioural disorders in adulthood.

Background: Abnormally high activity in the lateral habenula causes anxiety- or depression-like behaviours in animal experimental models. It has also been reported in humans that excessive stress in early life is correlated with the onset of psychiatric disorders in adults. These findings raise the question of whether maturation of the lateral habenula is affected under the influence of early-life experiences, which could govern behaviours throughout life. Methods: We examined the maturation of the lateral habenula in mice based on neuronal activity markers and plastic components: Zif268/Egr1, parvalbumin and perineuronal nets. We examined the effect of early-life stress using repeated maternal deprivation. Results: First, we found a transient highly sensitive period of the lateral habenula under stress. The lateral habenula matured through 4 stages: postnatal days 1-9 (P1-9), P10-20, around P35 and after P35. At P10-20, the lateral habenula was highly sensitive to stress. We also observed experience-dependent maturation of the lateral habenula. Only mice exposed to chronic stress from P10-20 exhibited changes specific to the lateral habenula at P60: abnormally high stress reactivity shown by Zif268/Egr1 and fewer parvalbumin neurons. These mice showed anxiety- or depression-like behaviours in the light-dark box test and forced swim test. Limitations: The effect of parvalbumin neurons in the lateral habenula on behavioural alterations remains unknown. It will be important to understand the "sensitive period" of the neuronal circuits in the lateral habenula and how the period P10-20 is different from P9 or earlier, or P35 or later. Conclusion: In mice, early-life stress in the period P10-20 led to late effects in adulthood: hyperactivity in the lateral habenula and anxiety or depression, indicating differences in neuronal plasticity between stages of lateral habenula maturation.

Stress-Related Neuronal Clusters in Sublenticular Extended Amygdala of Basal Forebrain Show Individual Differences of Positions.

To understand functional neuronal circuits for emotion in the basal forebrain, patterns of neuronal activation were examined in mice by immunohistochemistry of immediate-early gene products (Zif268/Egr1 and c-Fos). In all mice examined, clusters of 30-50 neurons expressing Zif268 were found on both sides in the area between the extended amygdala (EA) and globus pallidus (GP), generally designated as sublenticular extended amygdala (SLEA). The clusters consisted of 79.9 ± 3.0% of GABAergic neurons in GAD65-mCherry mice. The expression of the cholinergic marker choline acetyltransferase and the GP markers parvalbumin, proenkephalin, and FoxP2 indicated that these neurons were different from known types of neurons in the EA and GP; therefore, we named them the sublenticular extended amygdalar Zif268/Egr1-expressing neuronal cluster (SLEA-zNC). Sublenticular extended amygdalar Zif268/Egr1-expressing neuronal clusters participated in stress processing because increasing numbers of cells were observed in SLEA-zNCs after exposure to restraint stress (RS), the induction of which was suppressed by diazepam treatment. Mapping SLEA-zNCs showed that their positions and arrangement varied individually; SLEA-zNCs were distributed asymmetrically and tended to be situated mainly in the middle region between the anterior commissure (AC) and posterior end of the GP. However, the total cell number in SLEA-zNCs was compatible between the right and left hemispheres after activation by RS. Therefore, SLEA-zNCs were distributed asymmetrically but were not lateralized. Because time courses of activation differed between the Zif268 and c-Fos, the sequential dual treatment of RSs enabled us to differentiate SLEA-zNCs activated by the first and second RS. The results supported that the same SLEA-zNCs responded to both the first and second RS, and this also applied for all SLEA-zNCs. Thus, we concluded that the cluster positions were invariable under RS in each mouse but were distributed differently between individual mice. We name these newly identified neuronal clusters as stress-related neuronal clusters, SLEA-zNCs, which are considered to be novel functional units of "islands of activation." Moreover, SLEA-zNCs were situated at different positions in all mice examined, showing individual differences in their positions.

Atlas of the telencephalon based on cytoarchitecture, neurochemical markers, and gene expressions in Rhinogobius flumineus [Mizuno, 1960].

Gobiida is a basal subseries of percomorphs in teleost fishes, holding a useful position for comparisons with other orders of Percomorpha as well as other cohort of teleosts. Here, we describe a telencephalic atlas of a Gobiida species Rhinogobius flumineus (Mizuno, Memoirs of the College of Science, University of Kyoto, Series B: Biology, 1960; 27, 3), based on cytoarchitectural observations, combined with analyses of the distribution patterns of neurochemical markers and transcription factors. The telencephalon of R. flumineus shows a number of features distinct from those of other teleosts. Among others, the followings were of special note. (a) The lateral part of dorsal telencephalon (Dl), which is known as a visual center in other teleosts, is composed of as many as seven regions, some of which are conspicuous, circumscribed by cell plates. These subdivisions of the Dl can be differentiated clearly by differential soma size and color with Nissl-staining, and distribution patterns of neural markers. (b) Cell populations continuous with the ventral region of dorsal part of ventral telencephalon (vVd) exhibit extensive dimension. Especially, portion 1 of the central part of ventral telencephalon appears to represent a cell population laterally translocated from the vVd, forming a large cluster of small cells that penetrate deep into the central part of dorsal telencephalon. (c) The magnocellular subdivision of dorsal part of dorsal telencephalon (Ddmg) contains not only large cells but also vglut2a-positive clusters of small cells that cover a wide range of the caudal Ddmg. Such clusters of small cells have not been observed in the Ddmg of other teleosts.

An Evolutionary Hypothesis of Binary Opposition in Functional Incompatibility about Habenular Asymmetry in Vertebrates.

Many vertebrates have asymmetrical circuits in the nervous system. There are two types of circuit asymmetry. Asymmetrical circuits in sensory and/or motor systems are usually related to lateralized behaviors. It has been hypothesized that spatial asymmetry in the environment and/or social interactions has led to the evolution of asymmetrical circuits by natural selection. There are also asymmetrical circuits that are not related to lateralized behaviors. These circuits lie outside of the sensory and motor systems. A typical example is found in the habenula (Hb), which has long been known to be asymmetrical in many vertebrates, but has no remarkable relationship to lateralized behaviors. Instead, the Hb is a hub wherein information conveyed to the unilateral Hb is relayed to diverging bilateral nuclei, which is unlikely to lead to lateralized behavior. Until now, there has been no hypothesis regarding the evolution of Hb asymmetry. Here, we propose a new hypothesis that binary opposition in functional incompatibility applies selection pressure on the habenular circuit and leads to asymmetry. Segregation of the incompatible functions on either side of the habenula is likely to enhance information processing ability via creating shorter circuits and reducing the cost of circuit duplication, resulting in benefits for survival. In zebrafish and mice, different evolutionary strategies are thought to be involved in Hb asymmetry. In zebrafish, which use a strategy of structurally fixed asymmetry, the asymmetrical dorsal Hb leads to constant behavioral choices in binary opposition. In contrast, in mice, which use a strategy of functionally flexible lateralization, the symmetrical lateral Hb is functionally lateralized. This makes it possible to process complicated information and to come to variable behavioral choices, depending on the specific situation. These strategies are thought to be selected for and preserved by evolution under selection pressures of rigidity and flexibility of sociability in zebrafish and mice, respectively, as they are beneficial for survival. This hypothesis is highly valuable because it explains how the Hb evolved differently in terms of asymmetry and lateralization among different species. In addition, one can propose possible experiments for the verification of this hypothesis in future research.

Lateralization, maturation, and anteroposterior topography in the lateral habenula revealed by ZIF268/EGR1 immunoreactivity and labeling history of neuronal activity.

We report habenular lateralization in a simple transgenic mouse model used for labeling a facet of neuronal activity history. A transgenic construct comprised of a zif268/egr1 immediate-early gene promoter and a gene for normal Venus fluorescent protein with a membrane tag converted promoter activity into long-life fluorescent proteins, which was thought to describe a facet of neuronal activity history by summing neuronal activity. In addition to mapping the immediate-early gene-immunopositive cells, this method helped demonstrate the functionality of the lateral habenular nucleus (LHb). During postnatal development, the LHb was activated between postnatal days 10 and 16. The water-immersion restraint stress also activated the LHb over a similar period. LHb activation was functionally lateralized, but had no directional bias at the population level. Moreover, the posterior LHb was activated in the early stage after the stress, while the anterior LHb was activated in the later stage. Our results indicate lateralization, maturation, and anteroposterior topography of the LHb during postnatal development and the stress response.

Axons from the medial habenular nucleus are topographically sorted in the fasciculus retroflexus.

We generated transgenic mice lines with a construct consisting of the zif268/egr1 promoter and the gene for the normal long-life yellow fluorescent protein (Venus) with a membrane localization sequence. One of the lines exhibited topographic labeling in the medial habenular nucleus (MHb) during postnatal development, which confirmed the previous findings that the medial, lateral, and dorsal areas of MHb project to the ventral, dorsal, and lateral parts of the interpeduncular nucleus, respectively. In addition, the membranous localization of the labeling allowed us to observe spacial arrangement of the labeled axons in the fasciculus retroflexus (FR) in the transgenic mice. Here, we report topographic sorting of the MHb axons in the FR. At postnatal day (P) 5 and P10, the labeled axons from the medial MHb were fasciculated and ran through the narrow path in the core of the FR. At P24, the labeled axons from the medial and dorsal MHb were fasciculated and ran through the broad path in the FR core. No labeling occurred in the lateral MHb throughout development; correspondingly, parts of the FR core remained unlabeled. The results indicated that the axons from the medial and dorsal areas of the MHb are grouped together in the FR of this transgenic line and are sorted out from the axons from the lateral MHb.

Moderate repulsive effects of E-unit-containing chondroitin sulfate (CSE) on behavior of retinal growth cones.

Chondroitin sulfate (CS), the carbohydrate chain of chondroitin sulfate proteoglycans, is involved in neuronal circuit formation during development. CS shows great structural diversity with combination of disaccharide units of different structure (A-, C-, D-, or E-unit). However, whether its structural diversity contributes to pathway formation remains unclear. We chemically coupled the reducing end of various types of CS to the amino group of phosphatidylethanolamine (lipid-derivatized CS, CS-PE) and established an in vitro time-lapse assay to observe the behaviors of growth cones of retinal ganglion cells from embryonic day 6 chick retina on exposure to beads coated with lipid-derivatized CS (CS-PE beads). Among CS-PEs with different content of the structural units, the beads coated with E-unit-containing CS-PE [E-unit: GlcAß1-3GalNAc(4,6-O-disulfate)] (CSE-PE beads) significantly caused the growth cones to retract and to turn away from the beads, but the beads coated with CSA-, CSC- or CSD-PE beads did not. Importantly, not all the growth cones retracted equally from the CSE-PE beads, but they showed continuum of the repulsive behaviors; some behaved moderately and others remarkably. The growth cones distinguished different samples of CS: CSE and the others. Moreover, the continuum of the repulsive behaviors suggests that CS might be involved with the fine regulation of growth cones' behavior through its characteristic structure.

Influences of retinal axons on the cultural substrate containing biotin-conjugated chondroitin sulfate in vitro.

Although chondroitin sulfate (CS) is known to act as an inhibitory axon guidance cue, retinal axons show substantial growth on a culture substrate containing CS. Thus, the question arises as to how retinal axons elongate on CS-containing culture substrates. To elucidate the effects of retinal axons on a substrate containing CS, we synthesized biotinylated CS (biotin-CS) and developed a culture substrate with streptavidin-conjugated biotin-CS (complex between streptavidin and biotin-CS) to culture retinal axons. The effects of retinal axons on the streptavidin-biotin-CS complex were analyzed immunocytochemically using antibodies against CS and streptavidin, which recognize the carbohydrate and protein portions of the complex, respectively. After the axons were cultured on the substrate, areas that were CS-immunonegative but streptavidin-immunopositive were observed on the surface, corresponding to areas with or without axons, respectively. Absence of CS immunostaining was considered to be caused by structural alterations in the carbohydrate chains of the CS under the influence of the axons.

Restricted distribution of D-unit-rich chondroitin sulfate carbohydrate chains in the neuropil encircling the optic tract and on a subset of retinal axons in chick embryos.

To obtain basic information about the structural diversity and functional specificity of chondroitin sulfates (CSs) in the formation of the retinotectal pathway in chick embryos, the distribution of CSs around the optic tract was investigated by using anti-CS monoclonal antibodies with different specificities. The CSs are unbranched polymers composed of repeating disaccharide units of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc). The disaccharide units are classified into O-, A-, C-, D-, and E-units based on the position(s) of the added sulfate group(s). The MO-225 monoclonal antibody recognizes CSs that are rich in the D-unit [GlcA(2S)beta1-3GalNAc(6S)]; the MO-225 epitopes were distributed in the diencephalotelencephalic boundary and the neuropil encircling the optic tract. In addition, they were distributed on membrane surfaces of the retinal axons running in an interface layer in contact with the neuropil encircling the optic tract. The results suggest that D-unit-rich CSs are involved in delimiting the border of the optic tract and in the chronological sorting of the retinal axons.

Proteoglycans as cues for axonal guidance in formation of retinotectal or retinocollicular projections.

Understanding the formation of neuronal circuits has long been one of the basic problems in developmental neurobiology. Projections from the retina to their higher center, the optic tectum in nonmammalian vertebrates and the superior colliculus in mammals, are most amenable to experimental approaches; thus, much information has been accumulated about the mechanisms of axonal guidance. The retinal axons navigate along the appropriate pathway with the help of a series of guidance cues. Although much of the work has focused on proteinaceous factors, proteoglycans have been identified as playing important roles in retinal axon guidance. Chondroitin sulfate proteoglycans and heparan sulfate proteoglycans are involved in essential decisions of axon steering along the pathway. However, it has not been determined whether diversity of the carbohydrate chains results in differential effects and how their diversity is recognized by growth cones, which represent an important area of future research.