Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus

Fluoxetine-induced dematuration of hippocampal neurons and adult cortical neurogenesis in the common marmoset

The selective serotonin reuptake inhibitor fluoxetine (FLX) is widely used to treat depression and anxiety disorders. Chronic FLX treatment reportedly induces cellular responses in the brain, including increased adult hippocampal and cortical neurogenesis and reversal of neuron maturation in the hippocampus, amygdala, and cortex. However, because most previous studies have used rodent models, it remains unclear whether these FLX-induced changes occur in the primate brain. To evaluate the effects of FLX in the primate brain, we used immunohistological methods to assess neurogenesis and the expression of neuronal maturity markers following chronic FLX treatment (3 mg/kg/day for 4 weeks) in adult marmosets (n = 3 per group). We found increased expression of doublecortin and calretinin, markers of immature neurons, in the hippocampal dentate gyrus of FLX-treated marmosets. Further, FLX treatment reduced parvalbumin expression and the number of neurons with perineuronal nets, which indicate mature fast-spiking interneurons, in the hippocampus, but not in the amygdala or cerebral cortex. We also found that FLX treatment increased the generation of cortical interneurons; however, significant up-regulation of adult hippocampal neurogenesis was not observed in FLX-treated marmosets. These results suggest that dematuration of hippocampal neurons and increased cortical neurogenesis may play roles in FLX-induced effects and/or side effects. Our results are consistent with those of previous studies showing hippocampal dematuration and increased cortical neurogenesis in FLX-treated rodents. In contrast, FLX did not affect hippocampal neurogenesis or dematuration of interneurons in the amygdala and cerebral cortex

Immature-like molecular expression patterns in the hippocampus of a mouse model of dementia with Lewy body-linked mutant β-synuclein

Abstract Aim Maturation abnormalities of the brain cells have been suggested in several neuropsychiatric disorders, including schizophrenia, bipolar disorder, autism spectrum disorders, and epilepsy. In this study, we examined the expression patterns of neuronal maturation markers in the brain of a mouse model of dementia with Lewy body-linked mutant β-synuclein (βS), especially in the hippocampus, to explore whether such brain abnormalities occur in neurodegenerative disorders as well. Methods Quantitative PCR (qPCR) and immunohistochemical analyses were performed using the hippocampus of 14-month-old P123H βS transgenic (Tg) mice to evaluate the expression of molecular markers for maturation of dentate granule cells. Results Based on qPCR results, expression of Tdo2 and Dsp (markers of mature granule cells) was decreased and that of Drd1a (a marker of immature granule cells) was increased in the hippocampus of P123H βS Tg mice compared to that in wild-type controls. Immunohistochemical analysis revealed decreased expression of mature granule cell markers Calb1 and Gria1, along with increased expression of the microglial marker Iba1, in the hippocampal dentate gyrus region of P123H βS Tg mice. P123H βS Tg mice exhibited immature-like neuronal molecular expression patterns and microgliosis in the hippocampus. Pseudo-immaturity of dentate granule cells, associated with neuroinflammation, may be a shared endophenotype in the brains of at least a subgroup of patients with neuropsychiatric disorders and neurodegenerative diseases

Expression of progenitor cell/immature neuron markers does not present definitive evidence for adult neurogenesis

It is agreed upon that adult hippocampal neurogenesis (AHN) occurs in the dentate gyrus (DG) in rodents. However, the existence of AHN in humans, particularly in elderly individuals, remains to be determined. Recently, several studies reported that neural progenitor cells, neuroblasts, and immature neurons were detected in the hippocampus of elderly humans, based on the expressions of putative markers for these cells, claiming that this provides evidence of the persistence of AHN in humans. Herein, we briefly overview the phenomenon that we call “dematuration, ” in which mature neurons dedifferentiate to a pseudo-immature status and re-express the molecular markers of neural progenitor cells and immature neurons. Various conditions can easily induce dematuration, such as inflammation and hyper-excitation of neurons, and therefore, the markers for neural progenitor cells and immature neurons may not necessarily serve as markers for AHN. Thus, the aforementioned studies have not presented definitive evidence for the persistence of hippocampal neurogenesis throughout adult life in humans, and we would like to emphasize that those markers should be used cautiously when presented as evidence for AHN. Increasing AHN has been considered as a therapeutic target for Alzheimer’s disease (AD); however, given that immature neuronal markers can be re-expressed in mature adult neurons, independent of AHN, in various disease conditions including AD, strategies to increase the expression of these markers in the DG may be ineffective or may worsen the symptoms of such diseases

Circadian Gene Circuitry Predicts Hyperactive Behavior in a Mood Disorder Mouse Model

SummaryBipolar disorder, also known as manic-depressive illness, causes swings in mood and activity levels at irregular intervals. Such changes are difficult to predict, and their molecular basis remains unknown. Here, we use infradian (longer than a day) cyclic activity levels in αCaMKII (Camk2a) mutant mice as a proxy for such mood-associated changes. We report that gene-expression patterns in the hippocampal dentate gyrus could retrospectively predict whether the mice were in a state of high or low locomotor activity (LA). Expression of a subset of circadian genes, as well as levels of cAMP and pCREB, possible upstream regulators of circadian genes, were correlated with LA states, suggesting that the intrinsic molecular circuitry changes concomitant with infradian oscillatory LA. Taken together, these findings shed light onto the molecular basis of how irregular biological rhythms and behavior are controlled by the brain

Expression of tryptophan 2,3-dioxygenase in mature granule cells of the adult mouse dentate gyrus

New granule cells are continuously generated in the dentate gyrus of the adult hippocampus. During granule cell maturation, the mechanisms that differentiate new cells not only describe the degree of cell differentiation, but also crucially regulate the progression of cell differentiation. Here, we describe a gene, tryptophan 2,3-dioxygenase (TDO), whose expression distinguishes stem cells from more differentiated cells among the granule cells of the adult mouse dentate gyrus. The use of markers for proliferation, neural progenitors, and immature and mature granule cells indicated that TDO was expressed in mature cells and in some immature cells. In mice heterozygous for the alpha-isoform of calcium/calmodulin-dependent protein kinase II, in which dentate gyrus granule cells fail to mature normally, TDO immunoreactivity was substantially downregulated in the dentate gyrus granule cells. Moreover, a 5-bromo-2'-deoxyuridine labeling experiment revealed that new neurons began to express TDO between 2 and 4 wk after the neurons were generated, when the axons and dendrites of the granule cells developed and synaptogenesis occurred. These findings indicate that TDO might be required at a late-stage of granule cell development, such as during axonal and dendritic growth, synaptogenesis and its maturation

Oral Clostridium butyricum on mice endometritis through uterine microbiome and metabolic alternations

Endometritis occurs frequently in humans and animals, which can negatively affect fertility and cause preterm parturition syndrome. Orally administered Clostridium butyricum, a butyrate-producing gram-positive anaerobe, exhibits anti-inflammatory effects. However, the precise mechanism by which Clostridium butyricum attenuates endometritis remains unclear. This in vivo study evaluated the anti-inflammatory effects of orally administered Clostridium butyricum on uterine tissues. In addition, we conducted uterine microbiome and lipid metabolome analyses to determine the underlying mechanisms. Female Balb/c mice were divided into the following four groups (n = 5–20): (1) mock group, (2) only operation group (mice only underwent operation to exposed uterine horns from the side), (3) control group (mice underwent the same operation with the operation group + perfusion of lipopolysaccharide solution from uterine horns), and (4) Clostridium butyricum administration group (mice underwent the same operation with the control group + oral Clostridium butyricum administration from days 0 to 9). Clostridium butyricum was administered via oral gavage. On day 10, we investigated protein expression, uterine microbiome, and lipid metabolism in uterine tissues. Consequently, orally administered Clostridium butyricum altered the uterine microbiome and induced proliferation of Lactobacillus and Limosilactobacillus species. The effects can contribute to show the anti-inflammatory effect through the interferon-β upregulation in uterine tissues. Additionally, oral Clostridium butyricum administration resulted in the upregulations of some lipid metabolites, such as ω-3 polyunsaturated fatty acid resolvin D5, in uterine tissues, and resolvin D5 showed anti-inflammatory effects. However, the orally administered Clostridium butyricum induced anti-inflammatory effect was attenuated with the deletion of G protein-coupled receptor 120 and 15-lipooxgenase inhibition. In conclusion, Clostridium butyricum in the gut has anti-inflammatory effects on uterine tissues through alterations in the uterine microbiome and lipid metabolism. This study revealed a gut-uterus axis mechanism and provided insights into the treatment and prophylaxis of endometritis

Factors that contribute to long-term survival in patients with leukemia not in remission at allogeneic hematopoietic cell transplantation

<p>Abstract</p> <p>Background</p> <p>There has been insufficient examination of the factors affecting long-term survival of more than 5 years in patients with leukemia that is not in remission at transplantation.</p> <p>Method</p> <p>We retrospectively analyzed leukemia not in remission at allogeneic hematopoietic cell transplantation (allo-HCT) performed at our institution between January 1999 and July 2009. Forty-two patients with a median age of 39 years received intensified conditioning (n = 9), standard (n = 12) or reduced-intensity conditioning (n = 21) for allo-HCT. Fourteen patients received individual chemotherapy for cytoreduction during the three weeks prior to reduced-intensity conditioning. Diagnoses comprised acute leukemia (n = 29), chronic myeloid leukemia-accelerated phase (n = 2), myelodysplastic syndrome/acute myeloid leukemia (MDS/AML) (n = 10) and plasma cell leukemia (n = 1). In those with acute leukemia, cytogenetic abnormalities were intermediate (44%) or poor (56%). The median number of blast cells in bone marrow (BM) was 26.0% (range; 0.2-100) before the start of chemotherapy for allo-HCT. Six patients had leukemic involvement of the central nervous system. Stem cell sources were related BM (7%), related peripheral blood (31%), unrelated BM (48%) and unrelated cord blood (CB) (14%).</p> <p>Results</p> <p>Engraftment was achieved in 33 (79%) of 42 patients. Median time to engraftment was 17 days (range: 9-32). At five years, the cumulative probabilities of acute graft-versus-host disease (GVHD) and chronic GVHD were 63% and 37%, respectively. With a median follow-up of 85 months for surviving patients, the five-year Kaplan-Meier estimates of leukemia-free survival rate and overall survival (OS) were 17% and 19%, respectively. At five years, the cumulative probability of non-relapse mortality was 38%. In the univariable analyses of the influence of pre-transplant variables on OS, poor-risk cytogenetics, number of BM blasts (>26%), MDS overt AML and CB as stem cell source were significantly associated with worse prognosis (p = .03, p = .01, p = .02 and p < .001, respectively). In addition, based on a landmark analysis at 6 months post-transplant, the five-year Kaplan-Meier estimates of OS in patients with and without prior history of chronic GVHD were 64% and 17% (p = .022), respectively.</p> <p>Conclusion</p> <p>Graft-versus-leukemia effects possibly mediated by chronic GVHD may have played a crucial role in long-term survival in, or cure of active leukemia.</p