Distribution of nestin immunoreactivity in the normal adult human forebrain

The distribution of nestin immunoreactivity was studied in the whole normal adult human forebrains using new anti-human nestin mouse monoclonal and rabbit polyclonal antiserum. The nestin immunoreactive cells could be divided into three types according to their morphological characteristics. The first type contained neuron-like nestin immunoreactive cells, distributed in CA1–3 of hippocampus, septum, the nucleus of diagonal band, amygdala and basal nucleus of Meynert. The second type contained astrocyte-like cells, distributed in the subependymal zone and subgranular layer of dentate gyrus. The third type of cells had smaller cell bodies and fewer processes, also distributed in the subependymal zone and subgranular layer of dentate gyrus. Double immunohistochemical staining showed that the nestin positive, neuron-like cells in the nucleus of diagonal band and hippocampus also expressed NSE. However, the astrocyte-like nestin immunoreactive cells of the subependymal zone and subgranular layer of dentate gyrus were not double labeled with GFAP. Although some nestin immunoreactive fibers were distributed in the infundibulum, no nestin-immunoreactive cells were detected in the cortex. These data indicate that nestin exist in the adult human brain outside of the subependymal zone and dentate gyrus and also implies that nestin-immunoreactive cells may play a role in the modulation of basal forebrain function.     

Increase of nestin-immunoreactive neural precursor cells in the dentate gyrus of pediatric patients with early-onset temporal lobe epilepsy.

A considerable potential for neurogenesis has been identified in the epileptic rat hippocampus. Here, we explore this feature in human patients suffering from chronic mesial temporal lobe epilepsy. Immunohistochemical detection of the neurodevelopmental antigen nestin was used to detect neural precursor cells, and cell-type specific markers were employed to study their histogenetic origin and potential for neuronal or glial differentiation. The ontogenetic regulation of nestin-positive precursors was established in human control brains (week 19 of gestation-15 years of age). A striking increase of nestin-immunoreactive cells within the hilus and dentate gyrus could be observed in a group of young patients with temporal lobe epilepsy (TLE) and surgical treatment before age 2 years compared to adult TLE patients and controls. The cellular morphology and regional distribution closely resembled nestin-immunoreactive granule-cell progenitors transiently expressed during prenatal human hippocampus development. An increased Ki-67 proliferation index and clusters of supragranular nestin-immunoreactive cells within the molecular layer of the dentate gyrus were also noted in the group of young TLE patients. Confocal studies revealed colocalization of nestin and the betaIII isoform of tubulin, indicating a neuronal fate for some of these cells. Vimentin was consistently expressed in nestin-immunoreactive cells, whereas cell lineage-specific markers, i.e., glial fibrillary acidic protein, MAP2, neurofilament protein, NeuN, or calbindin D-28k failed to colocalize. These findings provide evidence for increased neurogenesis in pediatric patients with early onset of temporal lobe epilepsy and/or point towards a delay in hippocampal maturation in a subgroup of patients with TLE.     

Regeneration of granule neurons after lesioning of hippocampal dentate gyrus: evaluation using adult mice treated with trimethyltin chloride as a model

The hippocampal dentate gyrus in adult animals is known to contain neural progenitors that proliferate and differentiate into neurons in response to brain injury. Little has been observed, however, on regeneration of the granule cell layer of the dentate gyrus that has been directly injured. Using trimethyltin (TMT)-treated mice as an in vivo model, we evaluated the ability of this layer to regenerate after injury. The administration of TMT induced neuronal death in the dentate gyrus selectively 2 days later, with recovery of granule neurons on day 14 and thereafter. At an early stage (days 2-5) after the damage by TMT treatment, 5-bromo-2'-deoxyuridine (BrdU) incorporation into at least two different types of cells was facilitated in the dentate gyrus: BrdU-positive/neuronal nuclear antigen (NeuN)-negative cells were found predominantly in the subgranular zone and granule cell layer, whereas BrdU-positive/NeuN-positive cells were numerous in the dentate molecular layer and hilus. In addition, expression of proliferating cell nuclear antigen, nestin, NeuroD3, and doublecortin, which are markers for proliferating cells and neural progenitors/neuronal precursors, was extremely enhanced in the dentate gyrus at the early stage after treatment. Double staining revealed that BrdU was colocalized with nestin and doublecortin in the subgranular zone. Behavioral analysis revealed that TMT-induced cognition impairment was ameliorated by day 14 after the treatment. Taken together, our data indicate that the hippocampal dentate gyrus itself is capable of regenerating the neuronal cell layer through rapid enhancement of neurogenesis after injury.     

NPY mediates basal and seizure-induced proliferation in the subcallosal zone

Stem cell niches exist around the lateral ventricle and in the subgranular layer of the dentate gyrus, supporting adult neurogenesis. Recently, a third germinal layer, the subcallosal zone has been identified supporting the generation of oligodendrocytes in the adult brain. We have previously described a proliferative role for neuropeptide Y on precursors in the dentate gyrus, caudal subventricular zone and subcallosal zone under basal conditions and in the dentate gyrus after seizures. Here we sought to determine a role for neuropeptide Y in seizure-induced proliferation in the subcallosal niche. Using the chemoconvulsant kainate and neuropeptide Y(-/-) mice with controls, we show an effect of neuropeptide Y on basal proliferation and demonstrate a significant reduction in seizure-induced proliferation in the subcallosal zone.     

Differential expression of Musashi1 and nestin in the adult rat hippocampus after ischemia

Both nestin and the neural RNA-binding protein Musashi1 (Msi1) are expressed in neural stem cells in the subventricular zone. Neurogenesis in the hippocampus has received much attention, so we evaluated the expression of Msi1 and nestin in the adult rat hippocampus after transient forebrain ischemia. Both Msi1 and nestin were induced in the reactive astrocytes after ischemia, especially in the CA1 region, until 35 days after ischemia. Induction of both molecules suggested that reactive astrocytes might have immature characteristics. In the subgranular zone (SGZ) of the hippocampal dentate gyrus, Msi1-positive cells formed clusters after ischemia. These cells were labeled by bromodeoxyuridine (BrdU) but did not express glial fibrillary acidic protein. In contrast, very few nestin-positive cells were labeled by BrdU. Our results suggest that neuronal progenitor cells in the SGZ expressed Msi1 but not nestin.     

Ionizing radiation-induced apoptosis of proliferating stem cells in the dentate gyrus of the adult rat hippocampus.

The occurrence of radiation-induced apoptosis in normal brain was investigated using an animal model of radiosurgery. Adult male Fischer rats aged 3 to 4 months were subjected to single dose convergent beam irradiation (10 Gy). Apoptotic cell death was determined by in situ labeling of DNA nick ends (TUNEL) and light microscopic evaluation of cell morphology. Five hours after irradiation, a highly significant increase of apoptotic cells in the subgranular zone of the dentate gyrus was paralleled by a corresponding significant decrease of cells immunoreactive for the proliferation marker Ki-67. Morphology, location and distribution of cells affected by radiation-induced apoptosis in the dentate gyrus subgranular zone, together with NeuN-immunohistochemistry, support the contention that these cells belong to the immature progenitor population responsible for neurogenesis in the adult rat hippocampus.     

Spatial memory deficits in aged rats: contributions of the cholinergic system assessed by ChAT

Activity of choline acetylase (ChAT) was measured in basal forebrain cholinergic nuclei and in projection sites of these cells in the hippocampus and cortex of young rats and of aged rats who showed impaired performance on the radial arm maze. Decreased ChAT activity was found in the vertical diagonal band nucleus, the dentate gyrus and striatum of aged rats.     

GFAP-expressing radial glia-like cell bodies are involved in a one-to-one relationship with doublecortin-immunolabeled newborn neurons in the adult dentate gyrus

The present study examined the relationship between radial glial cells and newborn neurons in the adult dentate gyrus using three different methods. Single labeling immunocytochemistry for newly born neurons using doublecortin, as well as double labeling using an additional antibody to glial fibrillary acidic protein (GFAP) to label astrocytes were used at the light microscopic level. Furthermore, doublecortin immunoelectron microscopy was used to examine the ultrastructural relationship between newborn neurons and astrocytes in the adult dentate gyrus. These data showed an intimate one-to-one relationship between GFAP-expressing radial glia-like cell bodies and their non-radial processes that wrap around the basal and lateral sides of newborn neurons to cradle them in the subgranular zone. A similar relationship is observed for the newborn neurons at the base of the granule cell layer, but the cell body of the GFAP-expressing radial glia-like cells is not as intimately associated with the cell body of the newborn neurons at this site. Furthermore, newborn neurons with apical dendritic processes and growth cones in the granule cell layer extend them along radial glial processes. These newborn neurons do not receive axosomatic or axodendritic synapses indicating the absence of basket cell innervation. These data show that GFAP-expressing radial glia-like cells in the dentate gyrus cradle newborn neurons in the subgranular zone and that their radial processes provide a scaffold for neuronal process outgrowth.     

Migration and distribution of two populations of hippocampal granule cell precursors during the perinatal and postnatal periods

Methacrylate-embedded sections and short-survival thymidine radiograms of the hippocampal dentate gyrus were examined in perinatal and postnatal rats in order to trace the site of origin and migration of the precursors of granule cells and study the morphogenesis of the granular layer. The densely packed, spindle-shaped cells of the secondary dentate matrix (a derivative of the primary dentate neuroepithelium) stream in a subpial position towards the granular layer of the internal dentate limb during the perinatal and early postnatal periods. By an accretionary process, the crest of the granular layer forms on day E21 and on the subsequent days the granular layer of the internal dentate limb expands progressively in a lateral direction. Granule cells differentiation, as judged by the transformation of polymorph, darkly staining small cells into rounder, lightly staining larger granule cells, follows the same gradient from the external dentate limb to the internal dentate limb. The secondary dentate matrix is in a process of dissolution by day P5. This matrix is the source of what will later become the outer shell of the granular layer composed of early generated granule cells. The thicker inner shell of the granular layer, formed during the infantile and juvenile periods, derives from an intrinsic, tertiary germinal matrix. On day E22, the dentate migration of the secondary dentate matrix becomes partitioned into two components: a) the subpial component of extradentate origin, referred to in this context as the first dentate migration, and b) the second dentate migration. The latter is distributed in the basal polymorph layer throughout the entire dentate gyrus and is henceforth recognized as the tertiary dentate matrix. The tertiary dentate matrix is prominent between days P3 and P10. It is postulated that the great increase in granule cell population during the infantile period is principally due to cells derived from this intrinsic matrix of the dentate gyrus. Between days P20 and P30 the tertiary dentate matrix disappears in the basal polymorph layer and henceforth proliferative cells become largely confined to the subgranular zone at the base of the granular layer. The subgranular zone is the source of granule cells produced during the juvenile and adult periods.     

Gender-specific 5-HT1A receptor changes in BrdU nuclear labeling patterns in neonatal dentate gyrus

The actions of 5-HT1A receptors on cell proliferation in the rat neonatal dentate gyrus are unknown. We injected a 5-HT1A receptor agonist (ipsapirone) or antagonist (Way 100635) 1 h before injections of BrdU in neonates of both genders between days 2-4, a peak time of dentate gyrus granule cell proliferation. The BrdU immunoreactive (IR) nuclei in the granule cell layer and subgranular zone were examined after 2 weeks. The BrdU-IR nuclear staining patterns were classified as being either diffuse (homogenous dark BrdU-staining throughout the nucleus) or punctate (multiple distinct small stained spots within the nucleus). Most BrdU-labeled nuclei with a diffuse pattern were seen in the subgranular zone while the punctate pattern nuclei were seen within the granular cell layer of the dentate gyrus. 5-HT1A antagonist showed no overall change in absolute number or pattern of labeled nuclei compared to control animals. After a 5-HT1A agonist, there was also no differences in the total number of BrdU-IR nuclei (punctate and diffuse pattern). However, in both genders, the proportion of the BrdU-labeled nuclei showing a punctate compared to diffuse pattern increased: 33% in females and 18% in males. In females, the 5-HT1A receptor agonist increased the number of nuclei showing a punctate pattern by 41%, while in males the 5-HT1A receptor agonist decreased the number of nuclei showing a diffuse pattern by 29%. These results indicate gender-specific 5-HT1A receptor action on the state of nuclear DNA in the cells of the dentate gyrus, without increasing the total number of BrdU-labeled nuclei.