Involvement of Neuropeptide Galanin Receptors 2 and 3 in Learning, Memory and Anxiety in Aging Mice.

The neuropeptide galanin (GAL), which is expressed in limbic brain structures, has a strong impact on the regulation of mood and behavior. GAL exerts its effects via three G protein-coupled receptors (GAL1-3-R). Little is known about the effects of aging and loss of GAL-Rs on hippocampal-mediated processes connected to neurogenesis, such as learning, memory recall and anxiety, and cell proliferation and survival in the dorsal dentate gyrus (dDG) in mice. Our results demonstrate that loss of GAL3-R, but not GAL2-R, slowed learning and induced anxiety in older (12-14-month-old) mice. Lack of GAL2-R increased cell survival (BrdU incorporation) in the dDG of young mice. However, normal neurogenesis was observed in vitro using neural stem and precursor cells obtained from GAL2-R and GAL3-R knockouts upon GAL treatment. Interestingly, we found sub-strain differences between C57BL/6J and C57BL/6N mice, the latter showing faster learning, less anxiety and lower cell survival in the dDG. We conclude that GAL-R signaling is involved in cognitive functions and can modulate the survival of cells in the neurogenic niche, which might lead to new therapeutic applications. Furthermore, we observed that the mouse sub-strain had a profound impact on the behavioral parameters analyzed and should therefore be carefully considered in future studies.

Enhancing Functional Recovery Through Intralesional Application of Extracellular Vesicles in a Rat Model of Traumatic Spinal Cord Injury.

Local inflammation plays a pivotal role in the process of secondary damage after spinal cord injury. We recently reported that acute intravenous application of extracellular vesicles (EVs) secreted by human umbilical cord mesenchymal stromal cells dampens the induction of inflammatory processes following traumatic spinal cord injury. However, systemic application of EVs is associated with delayed delivery to the site of injury and the necessity for high doses to reach therapeutic levels locally. To resolve these two constraints, we injected EVs directly at the lesion site acutely after spinal cord injury. We report here that intralesional application of EVs resulted in a more robust improvement of motor recovery, assessed with the BBB score and sub-score, as compared to the intravenous delivery. Moreover, the intralesional application was more potent in reducing inflammation and scarring after spinal cord injury than intravenous administration. Hence, the development of EV-based therapy for spinal cord injury should aim at an early application of vesicles close to the lesion.

DCX+ neuronal progenitors contribute to new oligodendrocytes during remyelination in the hippocampus.

A pool of different types of neural progenitor cells resides in the adult hippocampus. Apart from doublecortin-expressing (DCX+) neuronal progenitor cells (NPCs), the hippocampal parenchyma also contains oligodendrocyte precursor cells (OPCs), which can differentiate into myelinating oligodendrocytes. It is not clear yet to what extent the functions of these different progenitor cell types overlap and how plastic these cells are in response to pathological processes. The aim of this study was to investigate whether hippocampal DCX+ NPCs can generate new oligodendrocytes under conditions in which myelin repair is required. For this, the cell fate of DCX-expressing NPCs was analyzed during cuprizone-induced demyelination and subsequent remyelination in two regions of the hippocampal dentate gyrus of DCX-CreERT2/Flox-EGFP transgenic mice. In this DCX reporter model, the number of GFP+ NPCs co-expressing Olig2 and CC1, a combination of markers typically found in mature oligodendrocytes, was significantly increased in the hippocampal DG during remyelination. In contrast, the numbers of GFP+PDGFRα+ cells, as well as their proliferation, were unaffected by de- or remyelination. During remyelination, a higher portion of newly generated BrdU-labeled cells were GFP+ NPCs and there was an increase in new oligodendrocytes derived from these proliferating cells (GFP+Olig2+BrdU+). These results suggest that DCX-expressing NPCs were able to contribute to the generation of mature oligodendrocytes during remyelination in the adult hippocampus.

Functional Integration of Neuronal Precursors in the Adult Murine Piriform Cortex.

The extent of functional maturation and integration of nonproliferative neuronal precursors, becoming neurons in the adult murine piriform cortex, is largely unexplored. We thus questioned whether precursors eventually become equivalent to neighboring principal neurons or whether they represent a novel functional network element. Adult brain neuronal precursors and immature neurons (complex cells) were labeled in transgenic mice (DCX-DsRed and DCX-CreERT2 /flox-EGFP), and their cell fate was characterized with patch clamp experiments and morphometric analysis of axon initial segments. Young (DCX+) complex cells in the piriform cortex of 2- to 4-month-old mice received sparse synaptic input and fired action potentials at low maximal frequency, resembling neonatal principal neurons. Following maturation, the synaptic input detected on older (DCX-) complex cells was larger, but predominantly GABAergic, despite evidence of glutamatergic synaptic contacts. Furthermore, the rheobase current of old complex cells was larger and the maximal firing frequency was lower than those measured in neighboring age-matched principal neurons. The striking differences between principal neurons and complex cells suggest that the latter are a novel type of neuron and new coding element in the adult brain rather than simple addition or replacement for preexisting network components.

Extracellular Vesicles Can Deliver Anti-inflammatory and Anti-scarring Activities of Mesenchymal Stromal Cells After Spinal Cord Injury.

Spinal cord injury is characterized by initial neural tissue disruption that triggers secondary damage and extensive non-resolving inflammation, which aggravates loss of function and hinders recovery. The early onset of inflammation following traumatic spinal cord injury underscores the importance of acute intervention after the initial trauma. Injections of mesenchymal stromal cells (MSCs) can reduce inflammation following spinal cord injury. We asked if extracellular vesicles (EVs) can substitute the anti-inflammatory and anti-scarring activities of their parental MSCs in a rat model of contusion spinal cord injury. We report that MSC-EVs were as potent as the parental intact cells in reducing the level of neuroinflammation for up to 2 weeks post-injury. Acute application of EVs after spinal cord injury was shown to robustly decrease the expression of pro-inflammatory cytokines in the spinal cord parenchyma in the very early phase of secondary damage. Moreover, the anti-scarring impact of MSC-EVs was even more efficient than the parental cells. We therefore conclude that anti-inflammatory and anti-scarring activities of MSC application can be mediated by their secreted EVs. In light of their substantial safety and druggability advantages, EVs may have a high potential in early therapeutic treatment following traumatic spinal cord injury.

Retinal Pericytes: Characterization of Vascular Development-Dependent Induction Time Points in an Inducible NG2 Reporter Mouse Model.

Purpose/aim of the study: In the retina, defects in pericytes (PCs) function/loss are associated with various complications; however, the exact pathological mechanisms are still not fully elucidated. Following the behavior of retina-resident PCs during health and disease will reveal new insights for both the understanding of pathological mechanisms and the development of new regenerative therapies for the treatment of retinopathies. The main goal of this study is to determine whether the NG2-reporter mouse (NG2CreERTM-eGFP) is a suitable model to study the fate of retina-resident PCs. MATERIAL AND METHODS: Vascular development-dependent reporter induction in retinal PCs was evaluated at different time points [(a) > P21, (b) < P21, and (c) P1 to > P21)] and additionally four different modes of application were tested. Reporter expression was evaluated by enhanced green fluorescent protein (eGFP) immunofluorescence by confocal microscopy and induction efficiency was calculated by analyzing NG2-expressing PCs in comparison to eGFP-labeled PCs in the three capillary layers. RESULTS: eGFP-positive PCs were detected in the three retinal capillary layers at all time points and administration routes tested. Multiple tamoxifen (TAM) applications in adult (> P21) NG2CreERTM-eGFP mice resulted in 3.59% eGFP-positive PCs. 2.37% eGFP-labeled PCs were detected after single intraperitoneal TAM injections at early postnatal days (P2/P5); however, just 1.61% PCs revealed reporter expression upon activation via the lactating mother (P4-P7). The highest number of eGFP-labeled PCs (7.09%) was detected following triple TAM administrations (P10-P12). The number of reporter-positive PCs doubled using homozygous animals. CONCLUSION: Despite low recombination efficiency in the used PC-specific fate mapping mouse model, changes in NG2 promoter activity of PCs during vascular development are indicated by single and multiple TAM inductions at different developmental time points. Nevertheless, these findings need further confirmation in up-coming studies by using homozygous NG2CreERTM-eGFP mice and additionally by mating the NG2CreERTM with a different reporter mouse to increase the low recombination efficiency.

Cellular Plasticity in the Adult Murine Piriform Cortex: Continuous Maturation of Dormant Precursors Into Excitatory Neurons.

Neurogenesis in the healthy adult murine brain is based on proliferation and integration of stem/progenitor cells and is thought to be restricted to 2 neurogenic niches: the subventricular zone and the dentate gyrus. Intriguingly, cells expressing the immature neuronal marker doublecortin (DCX) and the polysialylated-neural cell adhesion molecule reside in layer II of the piriform cortex. Apparently, these cells progressively disappear along the course of ageing, while their fate and function remain unclear. Using DCX-CreERT2/Flox-EGFP transgenic mice, we demonstrate that these immature neurons located in the murine piriform cortex do not vanish in the course of aging, but progressively resume their maturation into glutamatergic (TBR1+, CaMKII+) neurons. We provide evidence for a putative functional integration of these newly differentiated neurons as indicated by the increase in perisomatic puncta expressing synaptic markers, the development of complex apical dendrites decorated with numerous spines and the appearance of an axonal initial segment. Since immature neurons found in layer II of the piriform cortex are generated prenatally and devoid of proliferative capacity in the postnatal cortex, the gradual maturation and integration of these cells outside of the canonical neurogenic niches implies that they represent a valuable, but nonrenewable reservoir for cortical plasticity.

Motor deficits following dorsal corticospinal tract transection in rats: voluntary versus skilled locomotion readouts.

Following spinal cord injury, severe deficits result from damages to ascending and descending tracts, such as the corticospinal tract (CST) which is highly relevant for the motor execution in humans. Unfortunately, no curative treatment is available and intensive efforts are deployed in animal models, such as the CST transection model, to identify interventions providing functional regeneration after spinal cord injury. The CatWalk XT is a system for multi-parameter gait analysis of voluntary locomotion. In this study, the performance of the CatWalk XT for monitoring of functional deficits associated with dorsal CST lesion in rats was compared to skilled locomotion tests. Motor deficits associated with dorsal CST transection could be reliably monitored over seven weeks based on skilled locomotion testing, i.e. Horizontal Ladder Walk and Grid Walk. The collateral lesion to the overlaying gracile and cuneate funiculi occurring during dorsal CST transection resulted in slight hyposensitivity and proprioceptive deficit, which likely contributed to the lowered performance in skilled locomotion. In contrast, parameters of voluntary locomotion were not significantly affected by dorsal CST transection. Finally, an abnormal adduction reflex was detected immediately after lesion of the CST and could be conveniently used to confirm successful CST lesion in rats of experimental groups. The functional relevance of the dorsal CST in locomotion of rats is not as prominent as compared to in humans and thus challenging the motor execution is mandatory to reliably investigate CST function. A detailed analysis of voluntary walking using the CatWalk XT is not adequate to detect deficits following dorsal CST lesion in rats.

Pericytes Stimulate Oligodendrocyte Progenitor Cell Differentiation during CNS Remyelination.

The role of the neurovascular niche in CNS myelin regeneration is incompletely understood. Here, we show that, upon demyelination, CNS-resident pericytes (PCs) proliferate, and parenchymal non-vessel-associated PC-like cells (PLCs) rapidly develop. During remyelination, mature oligodendrocytes were found in close proximity to PCs. In Pdgfbret/ret mice, which have reduced PC numbers, oligodendrocyte progenitor cell (OPC) differentiation was delayed, although remyelination proceeded to completion. PC-conditioned medium accelerated and enhanced OPC differentiation in vitro and increased the rate of remyelination in an ex vivo cerebellar slice model of demyelination. We identified Lama2 as a PC-derived factor that promotes OPC differentiation. Thus, the functional role of PCs is not restricted to vascular homeostasis but includes the modulation of adult CNS progenitor cells involved in regeneration.

Tamoxifen Activation of Cre-Recombinase Has No Persisting Effects on Adult Neurogenesis or Learning and Anxiety.

Adult neurogenesis is a tightly regulated process continuously taking place in the central nervous system of most mammalian species. In neuroscience research, transgenic animals bearing the tamoxifen-inducible CreERT2-Lox system are widely used. In this study, we made use of a Nestin-CreERT2/R26R-YFP transgenic mouse model in which the CreERT2 activates the expression of YFP in multipotent neural stem cells upon tamoxifen application. Humoral factors, such as the levels of estrogens, have been reported to affect the hippocampal neurogenesis. The application of tamoxifen, a mixed agonist/antagonist of the estrogen receptor that permeates the blood-brain-barrier, could thus influence adult neurogenesis. Although the functions of adult neurogenesis are yet to be fully deciphered, a reciprocal interaction between rates of neurogenesis on the one hand and learning and mood regulation on the other hand, has been suggested. The impact of tamoxifen on neurogenesis and behavior was therefore addressed following five daily applications according to the open field test, the elevated plus maze, and Morris water maze. In addition, the impact of short-term tamoxifen application on progenitor cell proliferation, morphology, and fate in the neurogenic niche of the dentate gyrus were investigated. Finally, the influence of the route of administration (oral vs. intra-peritoneal) and gender-specific response were scrutinized. The sub-acute analysis did neither reveal significant differences in behavior, such as voluntary motor activity, anxiety behavior, and spatial learning, nor in cell proliferation, cell survival, dendritic arborization or maturation rate within the dentate gyrus between saline solution-, corn oil-, and tamoxifen-treated groups. Finally, neither the route of application, nor the gender of treated mice influenced the response to tamoxifen. We conclude that short tamoxifen treatments used to activate the CreERT2 system in transgenic mouse models does not have a measurable impact on adult neurogenesis or the here tested behavior, and is therefore appropriate for most studies in the field.

Expression of Lymphatic Markers in the Adult Rat Spinal Cord.

Under physiological conditions, lymphatic vessels are thought to be absent from the central nervous system (CNS), although they are widely distributed within the rest of the body. Recent work in the eye, i.e., another organ regarded as alymphatic, revealed numerous cells expressing lymphatic markers. As the latter can be involved in the response to pathological conditions, we addressed the presence of cells expressing lymphatic markers within the spinal cord by immunohistochemistry. Spinal cord of young adult Fisher rats was scrutinized for the co-expression of the lymphatic markers PROX1 and LYVE-1 with the cell type markers Iba1, CD68, PGP9.5, OLIG2. Rat skin served as positive control for the lymphatic markers. PROX1-immunoreactivity was detected in many nuclei throughout the spinal cord white and gray matter. These nuclei showed no association with LYVE-1. Expression of LYVE-1 could only be detected in cells at the spinal cord surface and in cells closely associated with blood vessels. These cells were found to co-express Iba1, a macrophage and microglia marker. Further, double labeling experiments using CD68, another marker found in microglia and macrophages, also displayed co-localization in the Iba1+ cells located at the spinal cord surface and those apposed to blood vessels. On the other hand, PROX1-expressing cells found in the parenchyma were lacking Iba1 or PGP9.5, but a significant fraction of those cells showed co-expression of the oligodendrocyte lineage marker OLIG2. Intriguingly, following spinal cord injury, LYVE-1-expressing cells assembled and reorganized into putative pre-vessel structures. As expected, the rat skin used as positive controls revealed classical lymphatic vessels, displaying PROX1+ nuclei surrounded by LYVE-1-immunoreactivity. Classical lymphatics were not detected in adult rat spinal cord. Nevertheless, numerous cells expressing either LYVE-1 or PROX1 were identified. Based on their localization and overlapping expression with Iba1, the LYVE-1+ cell population likely represents a macrophage subpopulation, while a significant fraction of PROX1+ cells belong to the oligodendrocytic lineage based on their distribution and the expression of OLIG2. The response of these LYVE-1+ and PROX1+ cell subpopulations to pathological conditions, especially in spinal cord inflammatory conditions, needs to be further elucidated.

Hippocampal neurogenesis and antidepressive therapy: shocking relations.

Speculations on the involvement of hippocampal neurogenesis, a form of neuronal plasticity, in the aetiology of depression and the mode of action of antidepressive therapies, started to arise more than a decade ago. But still, conclusive evidence that adult neurogenesis contributes to antidepressive effects of pharmacological and physical therapies has not been generated yet. This review revisits recent findings on the close relation between the mode(s) of action of electroconvulsive therapy (ECT), a powerful intervention used as second-line treatment of major depression disorders, and the neurogenic response to ECT. Following application of electroconvulsive shocks, intricate interactions between neurogenesis, angiogenesis, and microglia activation, the hypothalamic-pituitary-adrenal axis and the secretion of neurotrophic factors have been documented. Furthermore, considering the fact that neurogenesis strongly diminishes along aging, we investigated the response to electroconvulsive shocks in young as well as in aged cohorts of mice.