Brain-derived neurotrophic factor from microglia regulates neuronal development in the medial prefrontal cortex and its associated social behavior.

Microglia and brain-derived neurotrophic factor (BDNF) are essential for the neuroplasticity that characterizes critical developmental periods. The experience-dependent development of social behaviors-associated with the medial prefrontal cortex (mPFC)-has a critical period during the juvenile period in mice. However, whether microglia and BDNF affect social development remains unclear. Herein, we aimed to elucidate the effects of microglia-derived BDNF on social behaviors and mPFC development. Mice that underwent social isolation during p21-p35 had increased Bdnf in the microglia accompanied by reduced adulthood sociability. Additionally, transgenic mice overexpressing microglial Bdnf-regulated using doxycycline at different time points-underwent behavioral, electrophysiological, and gene expression analyses. In these mice, long-term overexpression of microglial BDNF impaired sociability and excessive mPFC inhibitory neuronal circuit activity. However, administering doxycycline to normalize BDNF from p21 normalized sociability and electrophysiological function in the mPFC, whereas normalizing BDNF from later ages (p45-p50) did not normalize electrophysiological abnormalities in the mPFC, despite the improved sociability. To evaluate the possible role of BDNF in human sociability, we analyzed the relationship between adverse childhood experiences and BDNF expression in human macrophages, a possible proxy for microglia. Results show that adverse childhood experiences positively correlated with BDNF expression in M2 but not M1 macrophages. In summary, our study demonstrated the influence of microglial BDNF on the development of experience-dependent social behaviors in mice, emphasizing its specific impact on the maturation of mPFC function, particularly during the juvenile period. Furthermore, our results propose a translational implication by suggesting a potential link between BDNF secretion from macrophages and childhood experiences in humans.

Efficacy of modified thoracoabdominal nerves block through perichondrial approach in open gynecological surgery: a prospective observational pilot study and a cadaveric evaluation.

BACKGROUND: Modified thoracoabdominal nerves block through perichondrial approach (M-TAPA) was first described as a peripheral nerve block by Tulgar in 2019. This technique provides an analgesic effective range from Th7-11 with a single puncture per side. Although the efficacy and effective duration of M-TAPA have been reported, further examination is required. Therefore, this study aimed to evaluate the analgesic range and effective duration of M-TAPA in open gynecologic surgery. METHODS: Following approval, 10 adult female patients scheduled for open radical hysterectomy via a vertical incision or laparotomy using a midline incision from under the xiphoid process to the symphysis pubis were enrolled. The primary outcome was the number of anesthetized dermatomes at 2 and 24 h postoperatively. Secondary outcomes included numerical rating scale scores and the total amount of fentanyl used. Cadaveric evaluation was performed to assess the spread of the dye. RESULTS: The median numbers (interquartile range) of anesthetized dermatomes at 2 and 24 h postoperatively were 6 (5-7) and 6.5 (5-7) in the anterior cutaneous branch area and 5 (4-7) and 7 (5-7) in the lateral cutaneous branch area, respectively. There was an 85% chance of simultaneously acquiring analgesia in areas innervated by Th8-11, including complete block in areas innervated by the anterior cutaneous branches of Th9-10. Cadaveric evaluation showed the spread of the dye in Th8-11. CONCLUSIONS: M-TAPA may have analgesic effects in the areas supplied by the anterior cutaneous branches of Th8-11. TRAIL REGISTRATION: IRB approval (No.2700; registered on July 10, 2020) and registration (UMIN Clinical Trials Registry: UMIN000041137 ; registered on July 17, 2020).

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).

Scaffold attachment factor B: distribution and interaction with ERα in the rat brain.

Scaffold attachment factor (SAFB) 1 and its homologue SAFB2 are multifunctional proteins that are involved in various cellular mechanisms, including chromatin organization and transcriptional regulation, and are also corepressors of estrogen receptor alpha (ERα). Both SAFBs are expressed at high levels in the brain. However, the distributions of SAFB1 and SAFB2 have yet to be characterized in detail and it is unclear whether both proteins interact with ERα in the brain. In this study, we investigated the expression and distribution of both SAFBs and their interaction with ERα in adult male rat brain. Immunohistochemical staining showed that SAFB1 and SAFB2 have a similar distribution pattern and are widely expressed throughout the brain. Double-fluorescence immunohistochemical and immunocytochemical analyses in primary cultures showed that the two SAFB proteins are localized in nuclei of neurons, astrocytes, and oligodendrocytes. Of note, SAFB2 was also found in cytoplasmic regions in these cell lineages. Both SAFB proteins were also expressed in ERα-positive cells in the medial preoptic area (MPOA) and arcuate and ventromedial hypothalamic nuclei. Co-immunoprecipitation experiments revealed that both SAFB proteins from the MPOA reciprocally interact with endogenous ERα. These results indicate that, in addition to a role in basal cellular function in the brain, the SAFB proteins may serve as ERα corepressors in hormone-sensitive regions.

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.

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.

Differences in elements between intact and disrupted human ligamenta capitum femorum.

To compare the element compositions between intact (i.e., intact throughout its length) and disrupted (i.e., ligament no longer attached to the attachment) ligaments, the contents of elements in the human ligamenta capitum femorum (LCF) were analyzed by inductively coupled plasma-atomic emission spectrometry. Histological and immunohistological assessments were also performed in both groups. The subjects were 8 men and 32 women. Trace element analyses showed that the sulfur and iron contents were significantly greater in the intact group than in the disrupted group, while the phosphorus and magnesium contents were significantly smaller in the intact group than in the disrupted group. The calcium and zinc contents were smaller in the intact group than in the disrupted group, with no significant differences. Histologically, there were fibrocartilage cells and extracellular matrix metachromasia in ligaments of the intact group. In contrast, fibrocartilage cells disappeared, and fat cells appeared instead of collagen fibrils in ligaments of the disrupted group. The LCFs of the intact group were immunohistologically positive for all components examined including collagens, glycosaminoglycans, and proteoglycans. The increase in sulfur suggested the presence of high glycosaminoglycan levels associated with fibrocartilaginous metaplasia in the ligament by compressive force. The reduction in iron may show a decreased number of blood vessels in the synovium after ligament disruption. The increases in phosphorus, magnesium, and calcium are indicative of degenerative changes including calcification and ossification. We conclude that differences in the contents of elements between intact and disrupted LCFs indicate degenerative alterations to the ligament structure after disruption.

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.

Intranuclear dynamics of corticosteroid receptors and effects of proteasomal activity in cultured hippocampal neural cells.

Adrenal corticosteroids readily enter the brain and exert markedly diverse effects, such as stress responses in the target neural cells. These effects are regulated by two receptor systems via the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR), which are both ligand-dependent transcription factors. Several steroid hormone receptors including GR, estrogen receptor, and androgen receptor, have been shown to move rapidly in the nucleus even after ligand treatment, supposedly corresponding to transcriptional "fine-tuning". We applied fluorescence recovery after photobleaching (FRAP) to assess the mobility of green fluorescent protein (GFP)-tagged GR and -MR in the nucleus of transiently transfected cultured hippocampal neurons. FRAP results showed high mobility of GR and MR in the nucleus. Half-recovery time of GR was longer than that of MR in the presence of 10(-6)M corticosterone (CORT), but shorter in the presence of 10(-9)M CORT. Proteasome inhibition reduced the subnuclear mobility of GR and MR, and increased the transcriptional activity at both concentrations of CORT. We also investigated the differential effects of CORT concentration and proteasome inhibition on the nuclear retention level of these receptors. Our findings may provide intriguing new insights into the dynamics of corticosteroid receptors in neural cells and the molecular basis of stress regulation by these receptors in the hippocampus.

Expression of G protein-coupled receptor 30 in the spinal somatosensory system.

Estrogens were originally identified as the primary sex steroid hormones in females and regulators of reproductive function and sexual behavior, but it has long been suggested that estrogens also have local effects on the somatosensory system at the spinal cord level. It is well known that the effects of estrogens are mediated by nuclear estrogen receptors (ERs) through genomic action, but recently a membrane-bound G protein-coupled receptor, GPR30, was identified as a non-genomic estrogen receptor. In this study we investigated the presence and localization of GPR30 in the rat spinal cord and dorsal root ganglion (DRG) in comparison with ERalpha. Using immunohistochemistry and in situ hybridization, we showed the expression of GPR30 in DRG neurons in male and female rats at mRNA and protein levels without specific sexual difference. A dense accumulation of GPR30 immunoreactivity was observed in the outer layer of the spinal dorsal horn, and selective spinal dorsal rhizotomy revealed that GPR30 was transported from the DRG to terminals located in the spinal dorsal horn. GPR30 expression was downregulated in DRG neurons of ovariectomized female rats. The spinal somatosensory system might be modulated by estradiol via putative membrane ER, GPR30-mediated mechanism.

Expression and intracellular distribution of the G protein-coupled receptor 30 in rat hippocampal formation.

Although the expression and distribution of nuclear estrogen receptors in the hippocampus has been described, it has been proposed that the nuclear receptors may not explain all aspects of estrogen function in the hippocampus. Recently, a G protein-coupled receptor for estrogen, GPR30, was identified as a membrane-localized estrogen receptor in several cancer cell lines. In this study, we examined the expression and intracellular distribution of GPR30 in the rat hippocampal formation. We found expression of GPR30 in pyramidal cells of CA1-3 and granule cells of the dentate gyrus at both mRNA and protein levels. Specific markers for intracellular organelles and immunoelectron microscopy revealed that GPR30 was mainly localized to the Golgi apparatus and partially in the endoplasmic reticulum of the neuron but could not detect the protein at the cell surface. Expression levels were not different among male, female in proestrus and female in estrus at the adult stage, but were higher in newborn male than newborn female.

Intranuclear mobility of estrogen receptor alpha and progesterone receptors in association with nuclear matrix dynamics.

We analyzed the intranuclear dynamics of estrogen receptor alpha (ER alpha) and progesterone receptor (PR)-A/B labeled with different spectral variants of green fluorescent protein (GFP) in living cells. The distribution of ER alpha and PR-A/B were changed from a diffuse to discrete pattern after the addition of both ligands, but the extent of discrete cluster formation of PR-A/B was lower than that of ER alpha. The nuclear areas where PR-A/B were accumulated were colocalized with the cluster of ER alpha, suggesting that cross-talk in the transcriptional regulation occurred in the loci. Fluorescence recovery after photobleaching (FRAP) analysis revealed that the mobility of PR-A/B was hastened by the coexistence of ER alpha, while the mobility of ER alpha was not changed by the coexistence of PR-A/B. Cluster formation was correlated with the nuclear matrix binding, because nuclear matrix binding capacity was also lower in PR-A/B than ER alpha. By ATP-depletion from the cells, most of ER alpha and PR-A/B were bound to the nuclear matrix and their mobilities were extinguished both in the absence and presence of ligand. Fluorescent protein (FP) tagged nuclear matrix component protein (NuMA), which was colocalized with ER alpha and PR-A/B, showed ATP-dependent rapid exchange in the nucleus. These results indicate that the mobility of ER alpha and PR-A/B is associated with the dynamics of the nuclear matrix.

Expression of G protein-coupled receptor-30, a G protein-coupled membrane estrogen receptor, in oxytocin neurons of the rat paraventricular and supraoptic nuclei.

The regulatory actions of estrogens on magnocellular oxytocin (OT) neurons of the paraventricular and supraoptic nuclei are well documented. Although the expression and distribution of nuclear estrogen receptor-beta, but not estrogen receptor-alpha, in the OT neuron has been described, the nuclear receptors may not explain all aspects of estrogen function in the hypothalamic OT neuron. Recently a G protein-coupled receptor (GPR) for estrogens, GPR30, has been identified as a membrane-localized estrogen receptor in several cancer cell lines. In this study, we therefore investigated the expression and localization of GPR30 in magnocellular OT neurons to understand the mode of rapid estrogen actions within these neurons. Here we show that, in the paraventricular nucleus and supraoptic nucleus, GPR30 is expressed in magnocellular OT neurons at both mRNA and protein levels but is not expressed in vasopressin neurons. Specific markers for intracellular organelles and immunoelectron microscopy revealed that GPR30 was localized mainly in the Golgi apparatus of the neurons but could not be detected at the cell surface. In addition, the expression of GPR30 is also detected in the neurohypophysis. These results suggest that GPR30 may serve primarily as a nongenomic transducer of estrogen actions in the hypothalamo-neurohypophyseal system.

Direct and rapid cytosolic delivery using cell-penetrating peptides mediated by pyrenebutyrate.

Intracellular delivery of bioactive molecules using arginine-rich peptides, including oligoarginine and HIV-1 Tat peptides, is a recently developed technology. Here, we report a dramatic change in the methods of internalization for these peptides brought about by the presence of pyrenebutyrate, a counteranion bearing an aromatic hydrophobic moiety. In the absence of pyrenebutyrate, endocytosis plays a major role in cellular uptake. However, the addition of pyrenebutyrate results in direct membrane translocation of the peptides yielding diffuse cytosolic peptide distribution within a few minutes. Using this method, rapid and efficient cytosolic delivery of the enhanced green fluorescent protein (EGFP) was achieved in cells including rat hippocampal primary cultured neurons. Enhancement of bioactivity on the administration of anapoptosis-inducing peptide is also demonstrated. Thus, coupling arginine-rich peptides with this hydrophobic anion dramatically improved their ability to translocate cellular membranes, suggesting the great impact of this approach on exploring and controlling cell function.

Estrogen reduces the neurite growth of serotonergic cells expressing estrogen receptors.

Serotonergic innervation of the central nervous system has a sexual dimorphism. The serotonin level in the hypothalamus was modulated by estrogen, and the formation of sexual dimorphism of serotonergic fiber innervation in the hypothalamus has been shown by the effect of sexual hormone during the critical perinatal period. In this study, we examined the direct effect of estrogen on the neurite growth of serotonergic neurons in primary culture from embryonic day 14 (E14) of rat mesencephalon. The total neurite length of serotonin-immunoreactive (IR) cells was significantly decreased by estradiol benzoate (E2, 10(-8)M) treatment for 7 days, compared with the case of no treatment. Moreover, the presence of estrogen receptor (ER) alpha and ERbeta mRNA in the E14 mesencephalon with reverse transcription-polymerase chain reaction (RT-PCR), and the ERalpha or ERbeta protein in the cultured serotonin-IR cells with double fluorescence immunohistochemistry were also demonstrated. Our results suggest that the inhibitory effects of E2 on the neurite growth of serotonergic cells expressing ERalpha or ERbeta might be involved in the formation of the sexual dimorphic distribution of serotonergic innervation.

Shuttling components of nuclear import machinery involved in nuclear translocation of steroid receptors exit nucleus via exportin-1/CRM-1* independent pathway.

The nucleocytoplasmic transport processes are mediated by soluble transport factors constantly navigating between nuclear and cytoplasmic compartments. Our understanding about nuclear export of general 'nuclear import factors' that deliver the cargo to the nucleus is still fragmentary. Utilizing green fluorescent protein tagged glucocorticoid receptor (GR) and relA as our working model and with judicious use of LMB, we show in living cells that all the soluble components of the nuclear import machinery exit nucleus via exportin1/CRM1 independent pathway(s).

Imaging analysis of subcellular correlation of androgen receptor and estrogen receptor alpha in single living cells using green fluorescent protein color variants.

Androgen and estrogen act not only in a sex-specific manner but also interactively and synergistically. In the present study, to examine the possible interaction between androgen receptor (AR) and estrogen receptor-alpha (ERalpha), we investigated the subcellular dynamics of AR and ERalpha fused with green fluorescent protein color variants in single living cells using time-lapse microscopy and the technique of fluorescence recovery after photobleaching. AR and ERalpha showed punctate colocalization in the nucleus with estrogen, but not androgen. N-terminal AR deletion mutant did not form a nuclear punctate pattern with either androgen or estrogen. In the presence of AR, but not ERalpha, N-terminal AR deletion mutant formed a punctate nuclear pattern with androgen. AR had different mobility depending on the ligand and the presence of ERalpha. On the other hand, AR had little effect on the stability of ERalpha. ERalpha mutant that does not bind coactivators did not alter the mobility of AR. Taken together, using an imaging technique, we clarified that possible homo/hetero dimerization between AR and ERalpha could be attributed to androgen-estrogen interaction in living cells.

Colocalization and ligand-dependent discrete distribution of the estrogen receptor (ER)alpha and ERbeta.

To investigate the relationships between the loci expressing functions of estrogen receptor (ER)alpha and that of ERbeta, we analyzed the subnuclear distribution of ERalpha and ERbeta in response to ligand in single living cells using fusion proteins labeled with different spectral variants of green fluorescent protein. Upon activation with ligand treatment, fluorescent protein-tagged (FP)-ERbeta redistributed from a diffuse to discrete pattern within the nucleus, showing a similar time course as FP-ERalpha, and colocalized with FP-ERalpha in the same discrete cluster. Analysis using deletion mutants of ERalpha suggested that the ligand-dependent redistribution of ERalpha might occur through a large part of the receptor including at least the latter part of activation function (AF)-1, the DNA binding domain, nuclear matrix binding domain, and AF-2/ligand binding domain. In addition, a single AF-1 region within ERalpha homodimer, or a single DNA binding domain as well as AF-1 region within the ERalpha/ERbeta heterodimer, could be sufficient for the cluster formation. More than half of the discrete clusters of FP-ERalpha and FP-ERbeta were colocalized with hyperacetylated histone H4 and a component of the chromatin remodeling complex, Brg-1, indicating that ERs clusters might be involved in structural changes of chromatin.