Juvenile social isolation affects the structure of the tanycyte-vascular interface in the hypophyseal portal system of the adult mice.

Accumulating evidence indicates that social stress in the juvenile period affects hypothalamic-pituitary-adrenal (HPA) axis activity in adulthood. The biological mechanisms underlying this phenomenon remain unclear. We aimed to elucidate them by comparing adult mice that had experienced social isolation from postnatal day 21-35 (juvenile social isolation (JSI) group) with those reared normally (control group). JSI group mice showed an attenuated HPA response to acute swim stress, while the control group had a normal response to this stress. Activity levels of the paraventricular nucleus in both groups were comparable, as shown by c-Fos immunoreactivities and mRNA expression of c-Fos, Corticotropin-releasing factor (CRF), Glucocorticoid receptor, and Mineralocorticoid receptor. We found greater vascular coverage by tanycytic endfeet in the median eminence of the JSI group mice than in that of the control group mice under basal condition and after acute swim stress. Moreover, CRF content after acute swim stress was greater in the median eminence of the JSI group mice than in that of the control group mice. The attenuated HPA response to acute swim stress was specific to JSI group mice, but not to control group mice. Although a direct link awaits further experiments, tanycyte morphological changes in the median eminence could be related to the HPA response.

Entrance-sealing behavior in the home cage: a defensive response to potential threats linked to the serotonergic system and manifestation of repetitive/stereotypic behavior in mice.

The security of animal habitats, such as burrows and nests, is vital for their survival and essential activities, including eating, mating, and raising offspring. Animals instinctively exhibit defensive behaviors to protect themselves from imminent and potential threats. In 1963, researchers reported wild rats sealing the entrances to their burrows from the inside using materials such as mud, sand, and vegetation. This behavior, known as "entrance sealing (ES)," involves repetitive movements of their nose/mouth and forepaws and is likely a proactive measure against potential intruders, which enhances burrow security. These observations provide important insights into the animals' ability to anticipate potential threats that have not yet occurred and take proactive actions. However, this behavior lacks comprehensive investigation, and the neural mechanisms underpinning it remain unclear. Hypothalamic perifornical neurons expressing urocortin-3 respond to novel objects/potential threats and modulate defensive responses to the objects in mice, including risk assessment and burying. In this study, we further revealed that chemogenetic activation of these neurons elicited ES-like behavior in the home-cage. Furthermore, behavioral changes caused by activating these neurons, including manifestations of ES-like behavior, marble-burying, and risk assessment/burying of a novel object, were effectively suppressed by selective serotonin-reuptake inhibitors. The c-Fos analysis indicated that ES-like behavior was potentially mediated through GABAergic neurons in the lateral septum. These findings underscore the involvement of hypothalamic neurons in the anticipation of potential threats and proactive defense against them. The links of this security system with the manifestation of repetitive/stereotypic behaviors and the serotonergic system provide valuable insights into the mechanisms underlying the symptoms of obsessive-compulsive disorder.

Protocol for behavioral tests using chemogenetically manipulated mice.

Behavioral analyses using mice chemogenetically manipulated by designer receptors exclusively activated by designer drugs (DREADDs) are powerful tools to elucidate neural functions. Here, we describe the detailed protocols for stereotaxic surgery, adeno-associated virus (AAV)-mediated introduction to Gq-DREADDs in mice, and for behavioral testing and analyses related to anxiety, risk assessment, and burying behaviors. A series of these tests are useful in evaluating animal anxiety and their defensive response patterns to potential threats. For complete details on the use and execution of this protocol, please refer to Horii-Hayashi et al. (2021).

The effects of maternal separation on behaviours under social-housing environments in adult male C57BL/6 mice.

Adverse experience in early life can affect the formation of neuronal circuits during postnatal development and exert long-lasting influences on neural functions that can lead to the development of a variety of psychiatric disorders including depression, anxiety disorders, and post-traumatic stress disorder. Many studies have demonstrated that daily repeated maternal separation, an animal model of early-life stress, can induce impairments in emotional behaviours and cognitive function during adolescence and adulthood. However, the behavioural phenotypes of maternally separated mice under long-term group-housing conditions are largely unknown. In this study, we applied our newly developed assay system to investigate the effects of maternal separation on behaviours under group-housing conditions during four days of continuous observations. Using our system, we found that repeated maternal separation resulted in inappropriate social distance from cagemates, altered approach preferences to others, and induced a lower rank in the time spent on the running wheel under group-housing conditions in adult male mice. Focussing on these behavioural abnormalities that appear in an environment with a social context will be important insights to understand the pathogenesis of psychiatric disorders.

Hypothalamic perifornical Urocortin-3 neurons modulate defensive responses to a potential threat stimulus.

Defensive behaviors are evolved responses to threat stimuli, and a potential threat elicits risk assessment (RA) behavior. However, neural mechanisms underlying RA behavior are hardly understood. Urocortin-3 (Ucn3) is a member of corticotropin-releasing factor peptide family and here, we report that Ucn3 neurons in the hypothalamic perifornical area (PeFA) are involved in RA of a novel object, a potential threat stimulus, in mice. Histological and in vivo fiber photometry studies revealed that the activity of PeFA Ucn3 neurons was associated with novel object investigation involving the stretch-attend posture, a behavioral marker for RA. Chemogenetic activation of these neurons increased RA and burying behaviors toward a novel object without affecting anxiety and corticosterone levels. Ablation of these neurons caused the abnormal behaviors of gnawing and direct contacts with novel objects, especially in a home-cage. These results suggest that PeFA Ucn3 neurons modulate defensive responses to a potential threat stimulus.

A Newly Defined Area of the Mouse Anterior Hypothalamus Involved in Septohypothalamic Circuit: Perifornical Area of the Anterior Hypothalamus, PeFAH.

Although the hypothalamus is classified into more than 10 compartments, it still contains uncharacterized areas. In this study, we identified a new triangular-shaped area between the paraventricular hypothalamic nucleus (PVN) and the fornix area in the mouse anterior hypothalamus, which is enriched in chondroitin sulfate proteoglycans (CSPGs). We designated this region as the perifornical area of the anterior hypothalamus (PeFAH) based on its anatomical location. As evidenced by Nissl staining, the PeFAH was distinguishable as an area of relatively low density. Immunohistochemical and DNA microarray analyses indicated that PeFAH contains sparsely distributed calretinin-positive neurons and densely clustered enkephalin-positive neurons. Furthermore, the PeFAH was shown to have bidirectional neural connections with the lateral septum. Indeed, we confirmed enkephalinergic projections from PeFAH neurons to the lateral septum, and inversely, calbindin-positive lateral septum neurons as afferents to the PeFAH. Finally, c-Fos expression analysis revealed that the activity of certain PeFAH neuronal populations tended to be increased by psychological stressors, but not that of enkephalinergic neurons. We proposed PeFAH as a new region in the AH.

Long-term effects of maternal separation coupled with social isolation on reward seeking and changes in dopamine D1 receptor expression in the nucleus accumbens via DNA methylation in mice.

Early-life stress has long-lasting effects on the stress response, emotions, and behavior throughout an individual's life. Clinical reports have demonstrated that child abuse victims exhibit impairments in reward-associated behavior; yet, the mechanism for this effect remains unclear. Maternal separation (MS) or MS coupled with social isolation (SI) (MS+SI) is widely used as a model for early-life stress in rodent studies. We employed mice subjected to MS+SI to clarify the long-term effect of early-life stress on reward-seeking involving palatable foods by a conditioned place-preference (CPP) paradigm. Prior MS+SI experience decreased exploration time in a chocolate-paired compartment in adult female mice, but not in male mice. We then focused on the mesolimbic dopamine pathway associated with reward-seeking behavior and measured both mRNA and protein levels of tyrosine hydroxylase (TH) in the ventral tegmental area (VTA) and dopamine D1 and D2 receptors in the nucleus accumbens (NAc). MS+SI female mice had significantly lower D1 receptor mRNA and protein levels than controls, whereas the expression of TH and the D2 receptor was similar in the 2 groups. All mRNA and protein levels were unchanged in MS+SI male mice. When attempting to elucidate the mechanism underlying downregulation of the D1 receptor in the NAc of MS+SI females, we found hypermethylation of the Drd1a promoter region. These results suggest that early-life stress affects reward-seeking behavior in female mice, which may be associated with the downregulation of D1 receptor in the NAc via epigenetic modification of its promoter region.

Insights from extracellular matrix studies in the hypothalamus: structural variations of perineuronal nets and discovering a new perifornical area of the anterior hypothalamus.

The hypothalamus controls metabolism, stress responses, and instinctive behaviors for individual survival and species preservation. Recent studies suggest that hypothalamic neurons retain plasticity throughout adulthood, which enables these neurons to respond to various kinds of changes in environment, nutrients, and fluctuating hormones. One of the mechanisms underlying the regulation of neural plasticity is the formation of a stable extracellular matrix (ECM) structure called perineuronal nets (PNNs). PNNs are large aggregates of heterogeneous ECM molecules such as chondroitin sulfate proteoglycans (CSPGs), hyaluronan, their link proteins, and tenascin-R. PNNs surround the cell body and proximal dendrites of a subset of neurons and limit adult neural plasticity. This review describes the CSPG-based ECM, including the PNNs, with a special focus on the hypothalamus of mice. We first provide an overview of PNNs in terms of their structure, molecular components, and functions, most of which have been demonstrated by extrahypothalamic studies. Second, we show the presence or absence of PNNs within individual hypothalamic regions and then describe non-PNN-formed ECM containing CSPGs that can be observed in particular hypothalamic regions. Finally, we will introduce a newly identified mouse hypothalamic area that we named the perifornical area of the anterior hypothalamus (PeFAH), which contains a cluster of PNN-positive neurons. PeFAH neurons express enkephalin and have bidirectional connections with the lateral septum. The anterior hypothalamus and lateral septum are thought to regulate defensive behaviors; therefore, the PeFAH neurons and PNNs around them could be involved in the regulation of defensive behaviors.

Bone marrow stromal cell sheets may promote axonal regeneration and functional recovery with suppression of glial scar formation after spinal cord transection injury in rats.

OBJECTIVE Transplantation of bone marrow stromal cells (BMSCs) is a theoretical potential as a therapeutic strategy in the treatment of spinal cord injury (SCI). Although a scaffold is sometimes used for retaining transplanted cells in damaged tissue, it is also known to induce redundant immunoreactions during the degradation processes. In this study, the authors prepared cell sheets made of BMSCs, which are transplantable without a scaffold, and investigated their effects on axonal regeneration, glial scar formation, and functional recovery in a completely transected SCI model in rats. METHODS BMSC sheets were prepared from the bone marrow of female Fischer 344 rats using ascorbic acid and were cryopreserved until the day of transplantation. A gelatin sponge (GS), as a control, or BMSC sheet was transplanted into a 2-mm-sized defect of the spinal cord at the T-8 level. Axonal regeneration and glial scar formation were assessed 2 and 8 weeks after transplantation by immunohistochemical analyses using anti-Tuj1 and glial fibrillary acidic protein (GFAP) antibodies, respectively. Locomotor function was evaluated using the Basso, Beattie, and Bresnahan scale. RESULTS The BMSC sheets promoted axonal regeneration at 2 weeks after transplantation, but there was no significant difference in the number of Tuj1-positive axons between the sheet- and GS-transplanted groups. At 8 weeks after transplantation, Tuj1-positive axons elongated across the sheet, and their numbers were significantly greater in the sheet group than in the GS group. The areas of GFAP-positive glial scars in the sheet group were significantly reduced compared with those of the GS group at both time points. Finally, hindlimb locomotor function was ameliorated in the sheet group at 4 and 8 weeks after transplantation. CONCLUSIONS The results of the present study indicate that an ascorbic acid-induced BMSC sheet is effective in the treatment of SCI and enables autologous transplantation without requiring a scaffold.

Bioinspired design of a polymer gel sensor for the realization of extracellular Ca(2+) imaging.

Although the role of extracellular Ca(2+) draws increasing attention as a messenger in intercellular communications, there is currently no tool available for imaging Ca(2+) dynamics in extracellular regions. Here we report the first solid-state fluorescent Ca(2+) sensor that fulfills the essential requirements for realizing extracellular Ca(2+) imaging. Inspired by natural extracellular Ca(2+)-sensing receptors, we designed a particular type of chemically-crosslinked polyacrylic acid gel, which can undergo single-chain aggregation in the presence of Ca(2+). By attaching aggregation-induced emission luminogen to the polyacrylic acid as a pendant, the conformational state of the main chain at a given Ca(2+) concentration is successfully translated into fluorescence property. The Ca(2+) sensor has a millimolar-order apparent dissociation constant compatible with extracellular Ca(2+) concentrations, and exhibits sufficient dynamic range and excellent selectivity in the presence of physiological concentrations of biologically relevant ions, thus enabling monitoring of submillimolar fluctuations of Ca(2+) in flowing analytes containing millimolar Ca(2+) concentrations.

Development and Structural Variety of the Chondroitin Sulfate Proteoglycans-Contained Extracellular Matrix in the Mouse Brain.

Chondroitin sulfate proteoglycans (CSPGs) are major components of the extracellular matrix (ECM) in the brain. In adult mammals, CSPGs form the specialized ECM structure perineuronal nets (PNNs) that surround somata and dendrites of certain types of neurons. PNNs restrict synaptic plasticity and regulate the closure of critical periods. Although previous studies have examined the starting period of PNN formation, focusing on primary sensory cortices, there are no systematic studies at the whole brain level. Here, we examined the starting period of PNN formation in male mice ranging in age from postnatal day 3 to week 11, mainly focusing on several cortical areas, limbic structures, hypothalamus, and brain stem, using lectin histochemistry with Wisteria floribunda agglutinin (WFA). Results showed that early PNN formation was observed in several reticular formations of the brain stem related to the cranial nerves and primary somatosensory cortices. In the limbic system, PNN formation in the hippocampus started earlier than that of the amygdala. Furthermore, in the medial amygdaloid nucleus and some hypothalamic regions, WFA labeling did not show typical PNN-like forms. The present study suggests spatiotemporal differences at the beginning of PNN formation and a structural variety of CSPG-contained ECM in the brain.

A newly identified mouse hypothalamic area having bidirectional neural connections with the lateral septum: the perifornical area of the anterior hypothalamus rich in chondroitin sulfate proteoglycans.

While previous studies and brain atlases divide the hypothalamus into many nuclei and areas, uncharacterised regions remain. Here, we report a new region in the mouse anterior hypothalamus (AH), a triangular-shaped perifornical area of the anterior hypothalamus (PeFAH) between the paraventricular hypothalamic nucleus and fornix, that abundantly expresses chondroitin sulfate proteoglycans (CSPGs). The PeFAH strongly stained with markers for chondroitin sulfate/CSPGs such as Wisteria floribunda agglutinin and antibodies against aggrecan and chondroitin 6 sulfate. Nissl-stained sections of the PeFAH clearly distinguished it as a region of comparatively low density compared to neighboring regions, the paraventricular nucleus and central division of the anterior hypothalamic area. Immunohistochemical and DNA microarray analyses suggested that PeFAH contains sparsely distributed calretinin-positive neurons and a compact cluster of enkephalinergic neurons. Neuronal tract tracing revealed that both enkephalin- and calretinin-positive neurons project to the lateral septum (LS), while the PeFAH receives input from calbindin-positive LS neurons. These results suggest bidirectional connections between the PeFAH and LS. Considering neuronal subtype and projection, part of PeFAH that includes a cluster of enkephalinergic neurons is similar to the rat perifornical nucleus and guinea pig magnocellular dorsal nucleus. Finally, we examined c-Fos expression after several types of stimuli and found that PeFAH neuronal activity was increased by psychological but not homeostatic stressors. These findings suggest that the PeFAH is a source of enkephalin peptides in the LS and indicate that bidirectional neural connections between these regions may participate in controlling responses to psychological stressors.

Effects of early life adverse experiences on the brain: implications from maternal separation models in rodents.

During postnatal development, adverse early life experiences affect the formation of neuronal networks and exert long-lasting effects on neural function. Many studies have shown that daily repeated maternal separation (MS), an animal model of early life stress, can regulate the hypothalamic-pituitary-adrenal axis (HPA axis) and affect subsequent brain function and behavior during adulthood. However, the molecular basis of the long-lasting effects of early life stress on brain function has not been fully elucidated. In this mini review, we present various cases of MS in rodents and illustrate the alterations in HPA axis activity by focusing on corticosterone (CORT). We then show a characterization of the brain regions affected by various patterns of MS, including repeated MS and single time MS at various stages before weaning, by investigating c-Fos expression. These CORT and c-Fos studies suggest that repeated early life stress may affect neuronal function in region- and temporal-specific manners, indicating a critical period for habituation to early life stress. Next, we introduce how early life stress can impact behavior, namely by inducing depression, anxiety or eating disorders, and alterations in gene expression in adult mice subjected to MS.

OASIS regulates chondroitin 6-O-sulfotransferase 1 gene transcription in the injured adult mouse cerebral cortex.

Old astrocyte specifically induced substance (OASIS), a basic leucine zipper transcription factor of the cAMP response element binding/Activating transcription factor family, is induced in reactive astrocytes in vivo and has important roles in quality control of protein synthesis at the endoplasmic reticulum. Reactive astrocytes produce a non-permissive environment for regenerating axons by up-regulating chondroitin sulfate proteoglycans (CSPGs). In this study, we focus on the potential role of OASIS in CSPG production in the adult mouse cerebral cortex. CS-C immunoreactivity, which represents chondroitin sulfate moieties, was significantly attenuated in the stab-injured cortices of OASIS knockout mice compared to those of wild-type mice. We next examined expression of the CSPG-synthesizing enzymes and core proteins of CSPGs in the stab-injured cortices of OASIS knockout and wild-type mice. The levels of chondroitin 6-O-sulfotransferase 1 (C6ST1, one of the major enzymes involved in sulfation of CSPGs) mRNA and protein increased after cortical stab injury of wild-type, but not of OASIS knockout, mice. A C-terminal deletion mutant OASIS over-expressed in rat C6 glioma cells increased C6ST1 transcription by interacting with the first intron region. Neurite outgrowth of cultured hippocampal neurons was inhibited on culture dishes coated with membrane fractions of epidermal growth factor-treated astrocytes derived from wild type but not from OASIS knockout mice. These results suggest that OASIS regulates the transcription of C6ST1 and thereby promotes CSPG sulfation in astrocytes. Through these mechanisms, OASIS may modulate axonal regeneration in the injured cerebral cortex. OASIS, an ER stress-responsive CREB/ATF family member, is up-regulated in the reactive astrocytes of the injured brain. We found that the up-regulated OASIS is involved in the transcriptional regulation of C6ST1 gene, which promotes chondroitin sulfate proteoglycan (CSPG) sulfation. We conclude that OASIS functions as an anti-regenerative transcription factor by establishing a non-permissive microenvironment to regenerating axons.

Distribution of corticosteroid receptors in mature oligodendrocytes and oligodendrocyte progenitors of the adult mouse brain.

The expression of glucocorticoid receptors (GRs) was investigated immunohistochemically in two different lineages of oligodendrocytes, using carbonic anhydrase (CA) II and neuron glial antigen (NG) 2 as markers of mature oligodendrocytes and oligodendrocyte progenitors, respectively. We focused on the gray matter regions, including CA1, CA3 and the dentate gyrus of the hippocampus, the primary somatosensory cortex barrel field and the basolateral amygdala, and the white matter regions, including the corpus callosum, external capsule and fimbria of the hippocampus. More than 80% of CAII-immunoreactive (IR) cells and more than 95% of NG2-IR cells expressed GRs in various regions of the brain. In contrast, neither CAII-IR cells nor NG2-IR cells expressed mineralocorticoid receptors (MRs) in the same regions. The intensity of GR expression was drastically reduced in CA II-IR cells and NG2-IR cells in the same regions in adrenalectomized mice. Finally, steroid receptor co-activator (SRC)-1 and p300, both of which are cofactors for GR, were expressed in the gray and white matter regions in NG2-IR cells, but not in CAII-IR cells. These results suggest that the expression of GRs in oligodendrocytes and their progenitor cells mediates several functions in vivo, including differentiation and myelination, as a major target of glucocorticoids and their cofactors.

Chondroitin sulfate proteoglycan tenascin-R regulates glutamate uptake by adult brain astrocytes.

In our previous study, the CS-56 antibody, which recognizes a chondroitin sulfate moiety, labeled a subset of adult brain astrocytes, yielding a patchy extracellular matrix pattern. To explore the molecular nature of CS-56-labeled glycoproteins, we purified glycoproteins of the adult mouse cerebral cortex using a combination of anion-exchange, charge-transfer, and size-exclusion chromatographies. One of the purified proteins was identified as tenascin-R (TNR) by mass spectrometric analysis. When we compared TNR mRNA expression patterns with the distribution patterns of CS-56-positive cells, TNR mRNA was detected in CS-56-positive astrocytes. To examine the functions of TNR in astrocytes, we first confirmed that cultured astrocytes also expressed TNR protein. TNR knockdown by siRNA expression significantly reduced glutamate uptake in cultured astrocytes. Furthermore, expression of mRNA and protein of excitatory amino acid transporter 1 (GLAST), which is a major component of astrocytic glutamate transporters, was reduced by TNR knockdown. Our results suggest that TNR is expressed in a subset of astrocytes and contributes to glutamate homeostasis by regulating astrocytic GLAST expression.

Effects of early life stress on brain activity: implications from maternal separation model in rodents.

Adverse experiences in early life can affect the formation of neuronal circuits during postnatal development and exert long-lasting influences on neural function. Many studies have shown that daily repeated maternal separation (RMS), an animal model of early life stress, can modulate the hypothalamic-pituitary-adrenal axis (HPA-axis) and can affect subsequent brain function and emotional behavior during adulthood. However, the molecular basis of the long-lasting effects of early life stress on brain function has not been completely elucidated. In this mini-review, we introduce various cases of maternal separation in rodents and illustrate the alterations in HPA-axis activity by focusing on corticosterone (CORT), an end-product of the HPA-axis in rodents. We then present the characterization of the brain regions affected by various patterns of MS, including RMS and single time maternal separation (SMS) at various stages before weaning, by investigating c-Fos expression, a biological marker of neuronal activity. These CORT and c-Fos studies suggest that repeated early life stress may affect neuronal function in region- and temporal-specific manners, indicating a critical period for habituation to early life stress. Furthermore, we introduce changes in behavioral aspects and gene expression in adult mice exposed to RMS.

Environmental stimulation changes tissue-type plasminogen activator activity in the adult mouse hippocampus.

Tissue-type plasminogen activator (tPA) plays an important role in synaptic plasticity and contributes to several brain functions such as memory, learning, and behaviours. Although a number of studies have demonstrated that various kinds of stimuli such as electrophysiological stimulation, excitotoxic injury, and stress change tPA activity in the brain, no studies have ever examined whether environmental stimulation affect tPA activity in the brain. The aim of this study is to clarify the effect of environmental enrichment on tPA activity in the hippocampus and cerebral cortex of adult mouse brain. Zymographic analysis revealed that hippocampal tPA activity was higher in enriched mice than in standard-caged mice, whereas no significant changes in the cerebral cortex were detected between enriched and control mice. Enrichment-enhanced tPA activity reverted to the pre-enrichment level after mice were returned to a standard housing condition, suggesting that tPA upregulation is stimulation-dependent and reversible. These results suggest that tPA activity is regulated reversibly in an experience-dependent manner in the adult hippocampus.

Chondroitin sulfate demarcates astrocytic territories in the mammalian cerebral cortex.

Adjacent astrocytes in the grey matter of the mammalian cerebral cortex are organized in a tile-like manner and separated from one another, forming discrete domains named "non-overlapping territories". We have previously reported that an anti-chondroitin sulfate (CS) antibody, CS-56, marks a subpopulation of cortical astrocytes which we named the dandelion clock-like structure (DACS) based on its morphological characteristics. In the present study, we found that another anti-CS antibody (anti-CS-C) was also able to detect the DACS and the morphological analysis revealed that a single DACS enwrapped five to six neuronal somata on average, which indicated that DACS coincided with a single astrocyte territory. Double labeling of CS-C and glial fibrillary acidic protein (GFAP) showed a slight overlap between the two territories in the adult cerebral cortex of mice. The neuron number enwrapped by a single DACS was unchanged between 3- and 7-week-old mice, while more extensive processes of DACSs were found in 7-week-old mice compared with those in 3-week-old ones. Moreover, the measurement of a single DACS area was significantly increased by 45% between 3- and 7-week-old mice. In addition, DACSs were found in human, monkey, and domestic pig brains, but not mallard ones, indicating that DACS was conserved in mammalian species. Taken together, CS demarcates territories of a certain population of cortical astrocytes and the cerebral cortex is composed of CS-rich astrocytes and -poor astrocytes in a mosaic fashion.