Tatsuhiro Hisatsune

GABAergic Excitation Promotes Neuronal Differentiation in Adult Hippocampal Progenitor Cells

Hippocampal activity influences neurogenesis in the adult dentate gyrus; however, little is known about the involvement of the hippocampal circuitry in this process. In the subgranular zone of the adult dentate gyrus, neurogenesis involves a series of differentiation steps from radial glia-like stem/progenitor (type-1) cells, to transiently amplifying neuronal progenitor (type-2) cells, to postmitotic neurons. In this study, we conducted GFP-targeted recordings of progenitor cells in fresh hippocampal slices from nestin-GFP mice and found that neuronal progenitor (type-2) cells receive active direct neural inputs from the hippocampal circuitry. This input was GABAergic but not glutamatergic. The GABAergic inputs depolarized type-2 cells because of their elevated [Cl(-)](i). This excitation initiated an increase of [Ca(2+)](i) and the expression of NeuroD. A BrdU-pulse labeling study with GABA(A)-R agonists demonstrated the promotion of neuronal differentiation via this GABAergic excitation. Thus, it appears that GABAergic inputs to hippocampal progenitor cells promote activity-dependent neuronal differentiation.

BMP2-mediated alteration in the developmental pathway of fetal mouse brain cells from neurogenesis to astrocytogenesis

We show that when telencephalic neural progenitors are briefly exposed to bone morphogenetic protein 2 (BMP2) in culture, their developmental fate is changed from neuronal cells to astrocytic cells. BMP2 significantly reduced the number of cells expressing microtubule-associated protein 2, a neuronal marker, and cells expressing nestin, a marker for undifferentiated neural precursors, but BMP2 increased the number of cells expressing S100-β, an astrocytic marker. In telencephalic neuroepithelial cells, BMP2 up-regulated the expression of negative helix–loop–helix (HLH) factors Id1, Id3, and Hes-5 (where Hes is homologue of hairy and Enhancer of Split) that inhibited the transcriptional activity of neurogenic HLH transcription factors Mash1 and neurogenin. Ectopic expression of either Id1 or Id3 (where Id is inhibitor of differentiation) inhibited neurogenesis of neuroepithelial cells, suggesting an important role for these HLH proteins in the BMP2-mediated changes in the neurogenic fate of these cells. Because gliogenesis in the brain and spinal cord, derived from implanted neural stem cells or induced by injury, is responsible for much of the failure of neuronal regeneration, this work may lead to a therapeutic strategy to minimize this problem.

Developmental Requirement of gp130 Signaling in Neuronal Survival and Astrocyte Differentiation

gp130 is a signal-transducing receptor component used in common by the interleukin-6 (IL-6) family of hematopoietic and neurotrophic cytokines, including IL-6, IL-11, leukemia-inhibitory factor, ciliary neurotrophic factor, oncostatin-M, and cardiotrophin-1. We have examined in this study a role of gp130 in the nervous system by analyzing developmental cell death of several neuronal populations and the differentiation of astrocytes in gp130-deficient mice. A significant reduction was observed in the number of sensory neurons in L5 dorsal root ganglia and motoneurons in the facial nucleus, the nucleus ambiguus, and the lumbar spinal cord in gp130 −/− mice on embryonic day 18.5. On the other hand, no significant neuronal loss was detectable on day 14.5, suggesting a physiological role of gp130 in supporting newly generated neurons during the late phase of development when naturally occurring cell death takes place. Moreover, expression of an astrocyte marker, GFAP, was severely reduced in the brain of gp130 −/− mice. Our data demonstrate that gp130 expression is essential for survival of subgroups of differentiated motor and sensory neurons and for the differentiation of major populations of astrocytesin vivo.

Cholinergic activation of hippocampal neural stem cells in aged dentate gyrus.

Adult hippocampal neurogenesis contributes to the hippocampal circuits role in cognitive functioning. New neurons are generated from hippocampal neural stem cells (NSCs) throughout life, but their generation is substantially diminished in aged animals due to a decrease in NSC proliferation. Because acetylcholine (ACh) is an important neurotransmitter released in the hippocampus during learning and exercise that is known to decrease with aging, we investigated whether aged NSCs can respond to ACh. In this study, we found that cholinergic stimulation has a positive effect on NSC proliferation in both young adult (8–12 weeks old) and aged mice (>2 years old). In fresh hippocampal slices, we observed a rapid calcium increase in NSCs in the dentate gyrus after muscarinic cholinergic stimulation, in both age groups. Furthermore, we found that the exercise‐induced promotion of aged NSC proliferation was abrogated by the specific lesioning of the septal cholinergic system. In turn, cholinergic activation by either eserine (physostigmine) or donepezil treatment promoted the proliferation of NSCs in aged mice. These results indicate that NSCs respond to cholinergic stimulation by proliferating in aged animals. Physiological and/or pharmacological cholinergic stimulation(s) may ameliorate cognitive decline in aged animals, by supporting adult hippocampal neurogenesis.

Increased number of new neurons in the olfactory bulb and hippocampus of adult non-human primates after focal ischemia

Adult neurogenesis is modulated by growth factors, physical conditions, and other alterations in the physical microenvironment. We studied the effects of focal ischemia on neurogenesis in the subventricular zone (SVZ), olfactory bulb (OB), and hippocampal dentate gyrus (DG) (known to be persistent neurogenic regions) in the adult non-human primate, the cynomolgus monkey. Three monkeys underwent middle cerebral artery occlusion-induced focal ischemia and were given multiple BrdU injections during the first 2 weeks after ischemia. Twenty-eight days later, the animals were perfused. The number of new neurons (3182 +/- 408/mm3) in the ipsilateral DG of ischemic monkeys was 4.7-fold that in the DG of non-operated monkeys. The number of new neurons (9176 +/- 2295/mm3) in the ipsilateral olfactory bulb of ischemic monkeys was 18.0-fold that in normal olfactory bulb. These observations suggest an increase in the number of new OB neurons, as well as new DG neurons, after focal ischemia in a primate. This substantial increase in new neurons after focal ischemia could result from the enhancement of cell proliferation rather than a change in the rate of cell commitment. Of the three monkeys subjected to ischemia, only one animal possessed a unique progenitor cell type at the most anterior aspect of the ipsilateral SVZ. Within this region, a short migration (approximately 500 microm) of doublecortin-expressing immature neuronal progenitor cells was observed.

Rapid integration of young newborn dentate gyrus granule cells in the adult hippocampal circuitry

Newborn dentate gyrus granule cells (DGCs) are integrated into the hippocampal circuitry and contribute to the cognitive functions of learning and memory. The dendritic maturation of newborn DGCs in adult mice occurs by the first 3–4 weeks, but DGCs seem to receive a variety of neural inputs at both their dendrites and soma even shortly after their birth. However, few studies on the axonal maturation of newborn DGCs have focused on synaptic structure. Here, we investigated the potentiality of output and input in newborn DGCs, especially in the early period after terminal mitosis. We labeled nestin‐positive progenitor cells by injecting GFP Cre‐reporter adenovirus into Nestin‐Cre mice, enabling us to trace the development of progenitor cells by their GFP expression. In addition to GABAergic input from interneurons, we observed that the young DGCs received axosomatic input from the medial septum as early as postinfection day 7 (PID 7). To evaluate the axonal maturation of the newborn DGCs compared with mature DCGs, we performed confocal and electron microscopic analyses. We observed that newborn DGCs projected their mossy fibers to the CA3 region, forming small terminals on hilar or CA3 interneurons and large boutons on CA3 pyramidal cells. These terminals expressed vesicular glutamate transporter 1, indicating they were glutamatergic terminals. Intriguingly, the terminals at PID 7 had already formed asymmetric synapses, similar to those of mature DGCs. Together, our findings suggest that newborn DGCs may form excitatory synapses on both interneurons and CA3 pyramidal cells within 7 days of their terminal mitosis.

Astrocyte Differentiation of Fetal Neuroepithelial Cells by Interleukin-11 via Activation of a Common Cytokine Signal Transducer, gp130, and a Transcription Factor, STAT3

Abstract: The interleukin (IL)‐6 family cytokines utilize membrane glycoprotein gp130 in common as a critical signal‐transducing receptor component. IL‐11, a cytokine initially identified as a plasmacytoma growth factor, belongs to this family. We show here that IL‐11 and its cognate receptor components are expressed in fetal mouse neuroepithelial cells. We also show that after 4 days of culture with IL‐11, cells with typical astrocytic morphologies expressing glial fibrillary acidic protein (GFAP; a marker for astrocytes) come out. This differentiation process is totally dependent on the gp130‐mediated signal‐transduction pathway involving activation of a latent cytoplasmic transcription factor, STAT3 (for signal transducer and activator of transcription 3), because (a) IL‐11‐induced astrocyte differentiation is not observed when neuroepithelial cells prepared from gp130‐deficient mice were used, (b) stimulation of neuroepithelial cells by IL‐11 rapidly induces tyrosine‐phosphorylation of STAT3, and (c) transfection of neuroepithelial cells with a dominant‐negative form of STAT3 inhibits IL‐11‐induced activation of the GFAP gene promoter. We have further identified, in the GFAP promoter region, a STAT3 site at which nucleotide substitutions almost completely abolished the IL‐11‐induced GFAP promoter activation. Taken together, it is suggested that IL‐11 contributes to astrocytogenesis in fetal brain via activation of gp130 and STAT3.

Excitatory GABAergic Activation of Cortical Dividing Glial Cells

Adult neocortex contains dividing satellite glia population even though their characteristics and functions have still remained unknown. Nestin(+)/NG2(+) cells as major fraction of dividing glial cells express bicuculline-sensitive gamma-aminobutyric acid A (GABA(A)) receptors and receive GABAergic inputs. Due to their high [Cl(-)](i), GABAergic activation depolarized the cells and then induced Ca(2+) influx into them. To assess an effect of this GABAergic excitation, we looked for the expression of neurotrophic factors. Among them, we detected the expression of brain-derived neurotrophic factor (BDNF) on the cells. The level of BDNF expression was elevated after cortical ischemia, and this elevation was blocked by bumetanide, an inhibitor for NKCC1 that blocks the GABAergic depolarization. Furthermore, performing a modified adhesive removal test, we observed that the treatment of bumetanide significantly attenuated the recovery in somatosensory dysfunction. Our results may shed a light on satellite glia population in the cortex and imply their roles in the functional recovery after ischemic injuries.

β-Alanyl-L-Histidine Rescues Cognitive Deficits Caused by Feeding a High Fat Diet in a Transgenic Mouse Model of Alzheimer's Disease

Our goal in this study was to determine whether or not feeding young (4 months old) Alzheimers disease model transgenic mice with a high fat diet (HFD), consisting of 32% fat, is capable of causing cognitive decline and whether treatment with β-alanyl-L-histidine (carnosine) is capable of reducing these effects. Carnosine is an endogenous antioxidant and antiglycating agent that is abundantly present in the brain and muscle tissues of vertebrates. After 8 weeks of feeding with HFD, we observed a significant decline in the contextual memory in transgenic mice fed with HFD as compared to transgenic mice fed with a normal diet as well as to normal diet-wild type mice. Treatment with carnosine at a dose of 5 mg/day for 6 weeks was effective in preventing cognitive decline, as the transgenic group fed with HFD and treated with carnosine displayed a level of cognition comparable to controls. No differences in senile plaque load were observed between all groups. However, we observed an increase in the expression of RAGE in blood vessels as well as increased microglial activation in the hippocampus of animals fed with HFD, effects that were reversed when treated with carnosine. Given these results, there is a possibility that inflammation and cerebrovascular abnormalities might be the cause of cognitive decline in this model.

Primate-specific alterations in neural stem/progenitor cells in the aged hippocampus

In the dentate gyrus of the hippocampus, new neurons are generated from neural stem/progenitor cells (NPCs) throughout life. As aging progresses, the rate of neurogenesis decreases exponentially, which might be responsible, in part, for age-dependent cognitive decline in animals and humans. However, few studies have analyzed the alterations in NPCs during aging, especially in primates. Here, we labeled NPCs by triple immunostaining for FABP7, Sox2, and GFAP and found that their numbers decreased in aged macaque monkeys (>20 years old), but not in aged mice. Importantly, we observed marked morphological alterations of the NPCs in only the aged monkeys. In the aged monkey hippocampus, the processes of the NPCs were short and ran horizontally rather than vertically. Despite these alterations, the proliferation rate of the NPCs in aged monkeys was similar to that in young monkeys. Thus, morphological alterations do not affect the proliferation rate of NPCs, but may be involved in the maintenance of NPCs in aged primates, including elderly humans.