Silke Keiner

Age-related effects on hippocampal precursor cell subpopulations and neurogenesis.

Hippocampal neurogenesis continuously declines in the aging brain but only little is known about age-related alterations in the subgranular zone (SGZ) of the dentate gyrus which accommodates different subpopulations of precursor cells. Here, we examined the age-related effects on total number and proliferation rate of distinct precursor cell populations in the dentate gyrus of 3 and 16 months old transgenic pNestin-GFP mice. Following a single injection of bromodeoxyuridine (BrdU) we observed a significant reduction of all proliferating precursor subtypes in aged mice compared to young controls. Stereological analysis further revealed that this decreased proliferation was not only caused by a general reduction in total number of precursor subtypes but also by a subtype-specific alteration of the proliferation rate. Whereas radial glia-like and early neuronal precursor cells demonstrate decreased proliferation rates, no difference was found for doublecortin-positive precursors. Additional long-term experiments further revealed that these age-related alterations in the proliferative zone were accompanied by a strongly decreased neurogenesis while hippocampal function was not impaired.

Effects of Skilled Forelimb Training on Hippocampal Neurogenesis and Spatial Learning After Focal Cortical Infarcts in the Adult Rat Brain

Background and Purpose— Environmental stimulation consistently increases dentate neurogenesis in the adult brain and improves spatial learning. We tested the hypothesis whether specific rehabilitative training of an impaired forelimb influences these processes after focal cortical infarcts. Methods— Focal cortical infarcts were induced in the forelimb sensorimotor cortex using the photothrombosis model. One group of infarcted animals and sham-operated controls housed in standard cages received one daily session of skilled reaching training of the impaired or dominant forelimb, respectively. A second group was transferred to an enriched environment, whereas a third group remained in the standard cages without further treatment. Bromodeoxyuridine was administered from day 2 until day 6 postinfarct. Proliferation and differentiation of newborn cells was analyzed at day 10 and 42 using immunocytochemistry with neuronal and glial markers and confocal laser scanning microscopy. Spatial learning was tested in the Morris water maze between days 35 and 41. Results— After cortical infarcts in the forelimb sensorimotor cortex, environmental enrichment as well as daily reaching training of the impaired paw both increase dentate neurogenesis and improve functional performance in the Morris water maze. Nevertheless, the reaching training-induced neurogenic response was significantly greater in nonlesioned controls associated with the best spatial learning performance in the water maze. Conclusions— Skilled forelimb training effectively stimulates dentate neurogenesis and spatial learning in the infarcted and healthy brain. However, this reaching training-induced increase in neurogenesis was reduced after cortical infarcts.

Aberrant Neurogenesis After Stroke A Retroviral Cell Labeling Study

Background and Purpose— Adult neurogenesis in the dentate gyrus is a unique form of brain plasticity that is strongly stimulated after stroke. We investigate the morphological properties of new granule cells, which are born and develop after the ischemic insult, and query whether these adult-born neurons properly integrate into the pre-existing hippocampal circuitries. Methods— Two well-established models were used to induce either small cortical infarcts (photothrombosis model) or large territorial infarcts (transient middle cerebral artery occlusion model). New granule cells were labeled 4 days after the initial insult by intrahippocampal injection of a retroviral vector encoding green fluorescent protein and newborn neurons were morphologically analyzed using a semiautomatic Neurolucida system and confocal laser scanning microscopy at 6 weeks. Results— Approximately 5% to 10% of newborn granule cells displayed significant morphological abnormalities comprising additional basal dendrites and, after middle cerebral artery occlusion, also ectopic cell position. The extent of morphological abnormalities was higher after large territorial infarcts and seems to depend on the severity of ischemic damage. An increased portion of mushroom spines in aberrant neurons suggests stable synaptic integration. However, poststroke generated granule cells with regular appearance also demonstrate alterations in dendritic complexity and spine morphology. Conclusions— The remarkable stimulation of dentate neurogenesis after stroke coincides with an increased rate of aberrantly integrated neurons, which may contribute to functional impairments and, hypothetically, favor pathogenesis of adjustment disorders, cognitive deficits, or epilepsy often seen in stroke patients.

Effects of rehabilitative training and anti-inflammatory treatment on functional recovery and cellular reorganization following stroke.

Post-ischemic inflammation plays a critical role in cellular reorganization and functional recovery after stroke. We therefore address the hypothesis whether anti-inflammatory treatment with either indometacin or minocycline combined with rehabilitative training improve functional recovery and influence perilesional cellular response following focal cortical infarcts. Using the photothrombosis model in adult rats, focal cortical infarcts were induced in the fore- and hindlimb sensorimotor cortex. Inflammatory processes were blocked by intraperitoneal application of indometacin or minocycline twice daily during the first 2 weeks of the experiment. Immediately after the infarct, the animals received a daily session of skilled reaching training of the impaired forelimb. In addition, Bromodeoxyuridine (BrdU) was administrated for 5 sequential days post infarct. Proliferation and differentiation of astrocytes, microglia, immature and mature neurons in the perilesional zone were immunocytochemically quantified at days 14 and 42. Functional recovery was assessed in a sensorimotor walking task preoperatively and 4, 14 and 28 days post surgery. Combined rehabilitative training and indometacin or minocycline strongly improved sensorimotor performance and significantly reduced the number of proliferating microglia compared to reaching training alone. Furthermore, the combination increased the survival of proliferating astrocytes and, moreover, minocycline increased the doublecortin-positive cells in the perilesional zone. Anti-inflammatory drug application combined with rehabilitative training demonstrates improved functional recovery and significantly modifies proliferation and survival of distinct glial and neuronal subpopulations in the direct vicinity of cortical infarcts compared to reaching training alone.

Immunocytochemical Detection of Newly Generated Neurons in the Perilesional Area of Cortical Infarcts After Intraventricular Application of Brain-Derived Neurotrophic Factor

The adult brain responds to focal infarction with proliferation of glial subpopulations. In addition, cells that express the immature neuronal marker doublecortin have been found consistently in the perileisonal zone. We investigated whether application of brain-derived neurotrophic factor (BDNF) would influence this perilesional proliferative response. Photothrombotic infarcts were induced in the sensorimotor forelimb and hindlimb cortex of adult rats. Brain-derived neurotrophic factor or vehicle was continuously infused intraventricularly for 2 weeks after the infarct using osmotic minipumps. Proliferating cells were labeled by daily intraperitoneal injections of bromodeoxyuridine during the first 2 weeks and were quantified at days 14 and 42 using semiautomatic stereology. Triple immunofluorescence with antibodies against immature and mature neuronal and glial markers was used to identify the proliferating cell populations. On day 14 after intraventricular BNDF application, the numbers of doublecortin-positive cells were doubled in the perilesional zone. On day 42, BDNF-treated animals had a small number of mature neurons in these areas, whereas vehicle-treated controls did not. Behavioral analysis with a battery of sensorimotor tests revealed, however, that the alterations in the perilesional cellular response were not associated with an improved functional outcome.

CXCR4 prevents dispersion of granule neuron precursors in the adult dentate gyrus.

Neurogenesis in the adult dentate gyrus (DG) generates new granule neurons that differentiate in the inner one‐third of the granule cell layer (GCL). The migrating precursors of these neurons arise from neural stem cells (NSCs) in the subgranular zone (SGZ). Although it is established that pathological conditions, including epilepsy and stroke, cause dispersion of granule neuron precursors, little is known about the factors that regulate their normal placement. Based on the high expression of the chemokine CXCL12 in the adult GCL and its role in guiding neuronal migration in development, we addressed the function of the CXCL12 receptor CXCR4 in adult neurogenesis. Using transgenic reporter mice, we detected Cxcr4‐GFP expression in NSCs, neuronal‐committed progenitors, and immature neurons of adult and aged mice. Analyses of hippocampal NSC cultures and hippocampal tissue by immunoblot and immunohistochemistry provided evidence for CXCL12‐promoted phosphorylation/activation of CXCR4 receptors in NSCs in vivo and in vitro. Cxcr4 deletion in NSCs of the postnatal or mature DG using Cre technology reduced neurogenesis. Fifty days after Cxcr4 ablation in the mature DG, the SGZ showed a severe reduction of Sox2‐positive neural stem/early progenitor cells, NeuroD‐positive neuronal‐committed progenitors, and DCX‐positive immature neurons. Many immature neurons were ectopically placed in the hilus and inner molecular layer, and some developed an aberrant dendritic morphology. Only few misplaced cells survived permanently as ectopic neurons. Thus, CXCR4 signaling maintains the NSC pool in the DG and specifies the inner one‐third of the GCL as differentiation area for immature granule neurons.

Functional recruitment of newborn hippocampal neurons after experimental stroke.

The adult brain responds to diverse pathologies such as stroke with increased generation of neurons in the dentate gyrus of the hippocampus. However, only little is known regarding the functional integration of newborn neurons into pre-existing neuronal circuits. In this study, we investigated whether newborn neurons generated after experimental stroke are recruited for different behavioral tasks. Adult mice received photochemical cortical infarcts in the sensorimotor cortex and proliferating cells were labeled using the proliferation marker, bromodeoxyuridine. Eight weeks after stroke induction, the animals were trained to perform either a spatiotemporal task or a sensorimotor task. Immediate early gene expression (c-fos, Zif268) in newborn neurons was analyzed directly after the last session. Using this approach, we demonstrate that post-stroke generated neurons are recruited within the hippocampal networks. The sensorimotor task activates significantly more newborn neurons compared to the spatiotemporal task. Further experiments employing the two well-established stimulators of neurogenesis, enriched environment and voluntary wheel running, both significantly increase post-stroke neurogenesis in the dentate gyrus but do not affect the percentage of recruited neurons compared to controls. Significantly, the spatiotemporal task leads to a higher portion of activated newborn neurons in the granule cell layer, suggesting a specific spatial activation pattern of new neurons in the dentate gyrus.

Differential stroke-induced proliferative response of distinct precursor cell subpopulations in the young and aged dentate gyrus

The capability of the adult brain to generate new hippocampal neurons after brain insults like stroke is decreasing during the aging process. Recent evidence further indicates that the proliferative properties of the precursor cells change in the aged brain. We therefore analyzed the early proliferative response of distinct precursor cell populations in the subgranular zone of the dentate gyrus in 3 and 16 months old transgenic nestin-green-fluorescent protein mice 4 days after ischemic cortical infarcts. A detailed immunocytochemical analysis of proliferating precursors revealed a significant infarct-induced activation of the earliest radial glia-like precursor cells (type 1 cells) and the more differentiated precursor cell subtypes (type 2b cells) in young mice. In contrast the proliferation of early neuronal precursor cells (type 2a cells) was stimulated in the aged brain. Additional long-term experiments further demonstrated that this differential proliferative response of distinct precursor cells is associated with an enhanced number of newborn neurons in the young DG after stroke whereas this increase in neurogenesis was absent in the aged brain. However, our study demonstrates that even precursor cells in the aged hippocampus possess the ability to respond to remote cortical infarcts.

Erratum: Role of Mitochondrial Metabolism in the Control of Early Lineage Progression and Aging Phenotypes in Adult Hippocampal Neurogenesis (Neuron (2017) 93(3) (560–573.e6) (S089662731630959X), (10.1016/j.neuron.2016.12.017))

Ruth Beckervordersandforth,* Birgit Ebert, Iris Sch€ affner, Jonathan Moss, Christian Fiebig, Jaehoon Shin, Darcie L. Moore, Laboni Ghosh, Mariela F. Trinchero, Carola Stockburger, Kristina Friedland, Kathrin Steib, Julia von Wittgenstein, Silke Keiner, Christoph Redecker, Sabine M. Hölter, Wei Xiang, Wolfgang Wurst, Ravi Jagasia, Alejandro F. Schinder, Guo-li Ming, Nicolas Toni, Sebastian Jessberger, Hongjun Song, and D. Chichung Lie* *Correspondence: ruth.beckervordersandforth@fau.de (R.B.), chi.lie@fau.de (D.C.L.) http://dx.doi.org/10.1016/j.neuron.2017.03.008

Two distinct populations of doublecortin-positive cells in the perilesional zone of cortical infarcts

BackgroundRecovery following stroke depends on cellular plasticity in the perilesional zone (PZ). Doublecortin (DCX), a protein mainly labeling immature neurons in neurogenic niches is also highly expressed in the vicinity of focal cortical infarcts. Notably, the number of DCX+ cells positively correlates with the recovery of functional deficits after stroke though the nature and origin of these cells remains unclear.ResultsIn the present study, we aimed to characterize the population of DCX+ cells in the vicinity of ischemic infarcts in a mouse model in detail. Employing a photothrombosis model, distinct immunohistochemical techniques, stereology and confocal microscopy, we show that: i) DCX+ cells in the perilesional zone do not constitute a homogenous population and two cell types, stellate and polar cells can be distinguished according to their morphology. ii) Stellate cells are mainly located in the lateral and medial vicinity of the insult and express astrocytic markers. iii) Polar cells are found almost exclusively in the corpus callosum region including in the preserved deep cortical layers close to the subventricular zone (SVZ). Further, they do not show any colocalisation of glial markers. Polar morphology and distribution suggest a migration towards the lesion.ConclusionsIn summary, our findings provide evidence that in mice DCX+ cells in the perilesional zone of cortical infarcts comprise a distinct cell population and the majority of cells are of glial nature.