Alterations in BDNF Protein Concentrations in the Hippocampus do not Explain the Pro-Neurogenic Effect of Citalopram on Adult Neurogenesis.

INTRODUCTION: Brain-derived neurotrophic factor (BDNF) has been implicated in the pro-neurogenic effect of selective serotonin reuptake inhibitors. In this study, we used Tph2 -/- mice lacking brain serotonin to dissect the interplay between BDNF and the serotonin system in mediating the effects of antidepressant pharmacotherapy on adult neurogenesis in the hippocampus. METHODS: Besides citalopram (CIT), we tested tianeptine (TIA), an antidepressant whose mechanism of action is not well understood. Specifically, we examined cell survival and endogenous concentrations of BDNF following daily injection of the drugs. RESULTS: Twenty-one days of CIT, but not of TIA, led to a significant increase in the survival of newly generated cells in the dentate gyrus of wild-type mice, without a significant effect on BDNF protein levels by either treatment. In Tph2 -/- mice, adult neurogenesis was consistently increased. Furthermore, Tph2 -/- mice showed increased BDNF protein levels, which were not affected by TIA but were significantly reduced by CIT. DISCUSSION: We conclude that the effects of CIT on adult neurogenesis are not explained by changes in BDNF protein concentrations in the hippocampus.

Depletion of angiotensin-converting enzyme 2 reduces brain serotonin and impairs the running-induced neurogenic response.

Physical exercise induces cell proliferation in the adult hippocampus in rodents. Serotonin (5-HT) and angiotensin (Ang) II are important mediators of the pro-mitotic effect of physical activity. Here, we examine precursor cells in the adult brain of mice lacking angiotensin-converting enzyme (ACE) 2, and explore the effect of an acute running stimulus on neurogenesis. ACE2 metabolizes Ang II to Ang-(1-7) and is essential for the intestinal uptake of tryptophan (Trp), the 5-HT precursor. In ACE2-deficient mice, we observed a decrease in brain 5-HT levels and no increase in the number of BrdU-positive cells following exercise. Targeting the Ang II/AT1 axis by blocking the receptor, or experimentally increasing Trp/5-HT levels in the brain of ACE2-deficient mice, did not rescue the running-induced effect. Furthermore, mice lacking the Ang-(1-7) receptor, Mas, presented a normal neurogenic response to exercise. Our results identify ACE2 as a novel factor required for exercise-dependent modulation of adult neurogenesis and essential for 5-HT metabolism.

Distinguishing features of microglia- and monocyte-derived macrophages after stroke.

After stroke, macrophages in the ischemic brain may be derived from either resident microglia or infiltrating monocytes. Using bone marrow (BM)-chimerism and dual-reporter transgenic fate mapping, we here set out to delimit the responses of either cell type to mild brain ischemia in a mouse model of 30 min transient middle cerebral artery occlusion (MCAo). A discriminatory analysis of gene expression at 7 days post-event yielded 472 transcripts predominantly or exclusively expressed in blood-derived macrophages as well as 970 transcripts for microglia. The differentially regulated genes were further collated with oligodendrocyte, astrocyte, and neuron transcriptomes, resulting in a dataset of microglia- and monocyte-specific genes in the ischemic brain. Functional categories significantly enriched in monocytes included migration, proliferation, and calcium signaling, indicative of strong activation. Whole-cell patch-clamp analysis further confirmed this highly activated state by demonstrating delayed outward K+ currents selectively in invading cells. Although both cell types displayed a mixture of known phenotypes pointing to the significance of 'intermediate states' in vivo, blood-derived macrophages were generally more skewed toward an M2 neuroprotective phenotype. Finally, we found that decreased engraftment of blood-borne cells in the ischemic brain of chimeras reconstituted with BM from Selplg-/- mice resulted in increased lesions at 7 days and worse post-stroke sensorimotor performance. In aggregate, our study establishes crucial differences in activation state between resident microglia and invading macrophages after stroke and identifies unique genomic signatures for either cell type.

Brain serotonin critically contributes to the biological effects of electroconvulsive seizures.

Compounds targeting serotonin (5-HT) are widely used as antidepressants. However, the role of 5-HT in mediating the effects of electroconvulsive seizure (ECS) therapy remains undefined. Using Tph2-/- mice depleted of brain 5-HT, we studied the effects of ECS on behavior and neurobiology. ECS significantly prolonged the start latency in the elevated O-Maze test, an effect that was abolished in Tph2-/- mice. Furthermore, in the absence of 5-HT, the ECS-induced increase in adult neurogenesis and in brain-derived neurotrophic factor signaling in the hippocampus were significantly reduced. Our results indicate that brain 5-HT critically contributes to the neurobiological responses to ECS.

Increased brain-derived neurotrophic factor (BDNF) protein concentrations in mice lacking brain serotonin.

The interplay between BDNF signaling and the serotonergic system remains incompletely understood. Using a highly sensitive enzyme-linked immunosorbent assay, we studied BDNF concentrations in hippocampus and cortex of two mouse models of altered serotonin signaling: tryptophan hydroxylase (Tph)2-deficient (Tph2 (-/-)) mice lacking brain serotonin and serotonin transporter (SERT)-deficient (SERT(-/-)) mice lacking serotonin re-uptake. Surprisingly, hippocampal BDNF was significantly elevated in Tph2 (-/-) mice, whereas no significant changes were observed in SERT(-/-) mice. Furthermore, BDNF levels were increased in the prefrontal cortex of Tph2 (-/-) but not of SERT(-/-) mice. Our results emphasize the interaction between serotonin signaling and BDNF. Complete lack of brain serotonin induces BDNF expression.

Discovery and characterization of alamandine: a novel component of the renin-angiotensin system.

RATIONALE: The renin-angiotensin system (RAS) is a key regulator of the cardiovascular system, electrolyte, and water balance. Here, we report identification and characterization of alamandine, a new heptapeptide generated by catalytic action of angiotensin-converting enzyme-2 angiotensin A or directly from angiotensin-(1-7). OBJECTIVE: To characterize a novel component of the RAS, alamandine. METHODS AND RESULTS: Using mass spectrometry we observed that alamandine circulates in human blood and can be formed from angiotensin-(1-7) in the heart. Alamandine produces several physiological actions that resemble those produced by angiotensin-(1-7), including vasodilation, antifibrosis, antihypertensive, and central effects. Interestingly, our data reveal that its actions are independent of the known vasodilator receptors of the RAS, Mas, and angiotensin II type 2 receptor. Rather, we demonstrate that alamandine acts through the Mas-related G-protein-coupled receptor, member D. Binding of alamandine to Mas-related G-protein-coupled receptor, member D is blocked by D-Pro(7)-angiotensin-(1-7), the Mas-related G-protein-coupled receptor, member D ligand ß-alanine and PD123319, but not by the Mas antagonist A-779. In addition, oral administration of an inclusion compound of alamandine/ß-hydroxypropyl cyclodextrin produced a long-term antihypertensive effect in spontaneously hypertensive rats and antifibrotic effects in isoproterenol-treated rats. Alamandine had no noticeable proliferative or antiproliferative effect in human tumoral cell lines. CONCLUSIONS: The identification of these 2 novel components of the RAS, alamandine and its receptor, provides new insights for the understanding of the physiological and pathophysiological role of the RAS and may help to develop new therapeutic strategies for treating human cardiovascular diseases and other related disorders.

Serotonin is required for exercise-induced adult hippocampal neurogenesis.

Voluntary wheel running has long been known to induce precursor cell proliferation in adult hippocampal neurogenesis in rodents. However, mechanisms that couple activity with the promitotic effect are not yet fully understood. Using tryptophan hydroxylase (TPH) 2 deficient (Tph2-deficient) mice that lack brain serotonin, we explored the relationship between serotonin signaling and exercise-induced neurogenesis. Surprisingly, Tph2-deficient mice exhibit normal baseline hippocampal neurogenesis but impaired activity-induced proliferation. Our data demonstrate that the proproliferative effect of running requires the release of central serotonin in young-adult and aged mice. Lack of brain serotonin further results in alterations at the stage of Sox2-positive precursor cells, suggesting physiological adaptations to changes in serotonin supply to maintain homeostasis in the neurogenic niche. We conclude that serotonin plays a direct and acute regulatory role in activity-dependent hippocampal neurogenesis. The understanding of exercise-induced neurogenesis might offer preventive but also therapeutic opportunities in depression and age-related cognitive decline.

The α crystallin domain of small heat shock protein b8 (Hspb8) acts as survival and differentiation factor in adult hippocampal neurogenesis.

Adult hippocampal neurogenesis is to a large degree controlled at the level of cell survival, and a number of potential mediators of this effect have been postulated. Here, we investigated the small heat shock protein Hspb8, which, because of its pleiotropic prosurvival effects in other systems, was considered a particularly promising candidate factor. Hspb8 is, for example, found in plaques of Alzheimer disease but exerts neuroprotective effects. We found that expression of Hspb8 increased during differentiation in vitro and was particularly associated with later stages (48-96 h) of differentiation. Gain-of-function and loss-of-function experiments supported the hypothesis that Hspb8 regulates cell survival of new neurons in vitro. In the dentate gyrus of adult mice in vivo, lentiviral overexpression of Hspb8 doubled the surviving cells and concomitantly promoted differentiation and net neurogenesis without affecting precursor cell proliferation. We also discovered that the truncated form of the crystallin domain of Hspb8 was sufficient to affect cell survival and neuronal differentiation in vitro and in vivo. Precursor cell experiments in vitro revealed that Hspb8 increases the phosphorylation of Akt and suggested that the prosurvival effect can be produced by a cell-autonomous mechanism. Analysis of hippocampal Hspb8 expression in mice of 69 strains of the recombinant inbred set BXD revealed that Hspb8 is a cis-acting gene whose expression was associated with clusters of transcript enriched in genes linked to growth factor signaling and apoptosis. Our results strongly suggest that Hspb8 and its α-crystallin domain might act as pleiotropic prosurvival factor in the adult hippocampus.

Modification of pax6 and olig2 expression in adult hippocampal neurogenesis selectively induces stem cell fate and alters both neuronal and glial populations.

The generation of new neurons in the mammalian hippocampus continues throughout life, and lineage progression is regulated by transcription factors, local cues, and environmental influences. The ability to direct stem/progenitor cell fate in situ may have therapeutic potential. Using an in vivo retroviral delivery and lineage tracing approach, we compare the lineage-instruction factors, Pax6 and Olig2, and demonstrate that both participate in regulation of adult hippocampal neurogenesis in adult rats. We show that overexpression of the proneuronal factor Pax6 pushes neuronal precursor cells to early maturation and increases the frequency of neuronal phenotypes. However, Pax6 overexpression results in no net increase in neurogenesis at 3 weeks. Blocking of Olig2 function reduces and slows neuronal commitment and differentiation and decreases net neurogenesis. Altering expression of both factors also changes gliogenesis. Our results establish that Pax6 decreases the number of Neuron-Glia 2 progenitor cells and prevents oligodendrocytic lineage commitment, while repression of Olig2 results in an expanded astrocytic lineage. We conclude that selectively modifying transcriptional cues within hippocampal progenitor cells is sufficient to induce a cell fate switch, thus altering the neurogenesis-gliogenesis ratio. In addition, our data show the competence of multiple progenitor lineages to respond divergently to the same signal. Therefore, directing instructive cues to select phenotype and developmental stage could be critical to achieve precise outcomes in cell genesis. Further understanding the regulation of lineage progression in all progenitor populations within the target region will be important for developing therapeutic strategies to direct cell fate for brain repair.

Enriched environment and physical activity reduce microglia and influence the fate of NG2 cells in the amygdala of adult mice.

Proliferative cells expressing proteoglycan neuron-glia 2 (NG2) are considered to represent parenchymal precursor cells in the adult brain and are thought to differentiate primarily into oligodendrocytes. We have studied cell genesis in the adult amygdala and found that, up to 1 year after the labeling of proliferating cells with bromodeoxyuridine, most proliferating NG2 cells remain NG2 cells, and only a few slowly differentiate into mature oligodendrocytes, as assessed by the expression of 2',3'-cyclic nucleotide 3'-phosphodiesterase. We have detected no signs of neurogenesis but have confirmed the expression of "neuronal" markers such as Doublecortin in NG2 cells. Nestin-expressing NG2 cells in the amygdala show electrophysiological properties known for oligodendrocyte precursor cells in the corpus callosum. Application of the glutamate agonist kainate elicits a "complex" response consisting of a rapid and long-lasting blockade of the resting K(+) conductance, a transient cationic current, and a transient increase of an outwardly directed K(+) conductance, suggesting the responsiveness of NG2 cells to excitation. Proliferation of NG2 cells increases in response to behavioral stimuli of activity, voluntary wheel running, and environmental enrichment. In addition to reducing the number of newborn microglia, behavioral activity results in a decrease in S100ß-expressing newborn NG2 cells in the amygdala. Because S100ß expression in NG2 cells ceases with oligodendrocyte maturation, this finding suggests that NG2 cells in the amygdala undergo activity-dependent functional alterations, without resulting in a measurable increase in new mature oligodendrocytes over the time period covered by the present study. The adult amygdala thus shows signs of mixed activity-dependent plasticity: reduced numbers of microglia and, presumably, an altered fate of NG2 cells.

Microglia Function on Precursor Cells in the Adult Hippocampus and Their Responsiveness to Serotonin Signaling.

Microglia are the resident immune cells of the adult brain that become activated in response to pathogen- or damage-associated stimuli. The acute inflammatory response to injury, stress, or infection comprises the release of cytokines and phagocytosis of damaged cells. Accumulating evidence indicates chronic microglia-mediated inflammation in diseases of the central nervous system, most notably neurodegenerative disorders, that is associated with disease progression. To understand microglia function in pathology, knowledge of microglia communication with their surroundings during normal state and the release of neurotrophins and growth factors in order to maintain homeostasis of neural circuits is of importance. Recent evidence shows that microglia interact with serotonin, the neurotransmitter crucially involved in adult neurogenesis, and known for its role in antidepressant action. In this chapter, we illustrate how microglia contribute to neuroplasticity of the hippocampus and interact with local factors, e.g., BDNF, and external stimuli that promote neurogenesis. We summarize the recent findings on the role of various receptors in microglia-mediated neurotransmission and particularly focus on microglia's response to serotonin signaling. We review microglia function in neuroinflammation and neurodegeneration and discuss their novel role in antidepressant mechanisms. This synopsis sheds light on microglia in healthy brain and pathology that involves serotonin and may be a potential therapeutic model by which microglia play a crucial role in the maintenance of mood.

Enduring Effects of Conditional Brain Serotonin Knockdown, Followed by Recovery, on Adult Rat Neurogenesis and Behavior.

Serotonin (5-hydroxytryptamine, 5-HT) is a crucial signal in the neurogenic niche of the hippocampus, where it is involved in antidepressant action. Here, we utilized a new transgenic rat model (TetO-shTPH2), where brain 5-HT levels can be acutely altered based on doxycycline (Dox)-inducible shRNA-expression. On/off stimulations of 5-HT concentrations might uniquely mirror the clinical course of major depression (e.g., relapse after discontinuation of antidepressants) in humans. Specifically, we measured 5-HT levels, and 5-HT metabolite 5-HIAA, in various brain areas following acute tryptophan hydroxylase 2 (Tph2) knockdown, and replenishment, and examined behavior and proliferation and survival of newly generated cells in the dentate gyrus. We found that decreased 5-HT levels in the prefrontal cortex and raphe nuclei, but not in the hippocampus of TetO-shTPH2 rats, lead to an enduring anxious phenotype. Surprisingly, the reduction in 5-HT synthesis is associated with increased numbers of BrdU-labeled cells in the dentate gyrus. At 3 weeks of Tph2 replenishment, 5-HT levels return to baseline and survival of newly generated cells is unaffected. We speculate that the acutely induced decrease in 5-HT concentrations and increased neurogenesis might represent a compensatory mechanism.

The Role of Astrocytes in the Neurorepair Process.

Astrocytes are highly specialized glial cells responsible for trophic and metabolic support of neurons. They are associated to ionic homeostasis, the regulation of cerebral blood flow and metabolism, the modulation of synaptic activity by capturing and recycle of neurotransmitters and maintenance of the blood-brain barrier. During injuries and infections, astrocytes act in cerebral defense through heterogeneous and progressive changes in their gene expression, morphology, proliferative capacity, and function, which is known as reactive astrocytes. Thus, reactive astrocytes release several signaling molecules that modulates and contributes to the defense against injuries and infection in the central nervous system. Therefore, deciphering the complex signaling pathways of reactive astrocytes after brain damage can contribute to the neuroinflammation control and reveal new molecular targets to stimulate neurorepair process. In this review, we present the current knowledge about the role of astrocytes in brain damage and repair, highlighting the cellular and molecular bases involved in synaptogenesis and neurogenesis. In addition, we present new approaches to modulate the astrocytic activity and potentiates the neurorepair process after brain damage.

Neurobiology of glycine transporters: From molecules to behavior.

Glycine transporters (GlyTs) are Na+/Cl--dependent neurotransmitter transporters, responsible for l-glycine uptake into the central nervous system. GlyTs are members of the solute carrier family 6 (SLC6) and comprise glycine transporter type 1 (SLC6A9; GlyT1) and glycine transporter type 2 (SLC6A5; Glyt2). GlyT1 and GlyT2 are expressed on both astrocytes and neurons, but their expression pattern in brain tissue is foremost related to neurotransmission. GlyT2 is markedly expressed in brainstem, spinal cord and cerebellum, where it is responsible for glycine uptake into glycinergic and GABAergic terminals. GlyT1 is abundant in neocortex, thalamus and hippocampus, where it is expressed in astrocytes, and involved in glutamatergic neurotransmission. Consequently, inhibition of GlyT1 transporters can modulate glutamatergic neurotransmission through NMDA receptors, suggesting an alternative therapeutic strategy. In this review, we focus on recent progress in the understanding of GlyTs role in brain function and in various diseases, such as epilepsy, hyperekplexia, neuropathic pain, drug addiction, schizophrenia and stroke, as well as in neurodegenerative disorders.

Lack of Brain Serotonin Affects Feeding and Differentiation of Newborn Cells in the Adult Hypothalamus.

Serotonin (5-HT) is a crucial signal in the neurogenic niche microenvironment. Dysregulation of the 5-HT system leads to mood disorders but also to changes in appetite and metabolic rate. Tryptophan hydroxylase 2-deficient (Tph2-/- ) mice depleted of brain 5-HT display alterations in these parameters, e.g., increased food consumption, modest impairment of sleep and respiration accompanied by a less anxious phenotype. The newly discovered neural stem cell niche of the adult hypothalamus has potential implications of mediating stress responses and homeostatic functions. Using Tph2-/- mice, we explore stem cell behavior and cell genesis in the adult hypothalamus. Specifically, we examine precursor cell proliferation and survival in Tph2-/- mice at baseline and following Western-type diet (WD). Our results show a decline in BrdU numbers with aging in the absence of 5-HT. Furthermore, wild type mice under dietary challenge decrease cell proliferation and survival in the hypothalamic niche. In contrast, increased high-calorie food intake by Tph2-/- mice does not come along with alterations in cell numbers. However, lack of brain 5-HT results in a shift of cell phenotypes that was abolished under WD. We conclude that precursor cells in the hypothalamus retain fate plasticity and respond to environmental challenges. A novel link between 5-HT signaling and cell genesis in the hypothalamus could be exploited as therapeutic target in metabolic disease.

Unilateral lesion of the subthalamic nucleus transiently provokes bilateral subacute glial cell proliferation in the adult rat substantia nigra.

Ablative or functional lesions of the subthalamic nucleus (STN) lead to significant improvements of motor deficits and major levodopa associated motor complications in patients with Parkinson's disease. The biological mechanisms underlying the clinical effectiveness still remain largely unknown. It has been demonstrated previously that the adult substantia nigra (SN) bears the capacity for cellular plasticity throughout adulthood and that this property can be influenced by external stimuli. In the present study we investigated the subacute and chronic effects of unilateral STN-lesion on newly generated neural cells in the adult healthy SN of the rat. With this experimental design we demonstrate a bilateral transient increase in the total numbers of newborn nigral cells following STN-lesion. Additionally, we show a transient bilateral decrease in the number of newborn neuro-glial antigen 2 (NG2)-positive and in the number of new microglia cells. No newborn neurons, however, were detected. Thus, we conclude that unilateral ablative STN lesion transiently changes plasticity of neural cell subpopulations in the healthy adult SN of the rat.

Bradykinin B2 receptor is essential to running-induced cell proliferation in the adult mouse hippocampus.

Physical exercise is a strong external effector that induces precursor cell proliferation in the adult mouse hippocampus. Research into mechanisms has focused on central changes within the hippocampus and we have established that serotonin is the signaling factor that transduces physical activity into adult neurogenesis. Less focus has been given on potential peripheral signals that may cause pro-mitotic running effects. Vasoactive kinin peptides are important for blood pressure regulation and inflammatory processes to maintain cardiovascular homeostasis. Acting via the two receptors termed B1 (B1R) and B2R, the peptides also function in the brain. In particular, studies attribute B2R a role in cell proliferation and differentiation into neurons in vitro. Here, we determined B1R and B2R mRNA expression levels in the adult mouse hippocampus and prefrontal cortex in vivo, and in response to running exercise. Using mice depleted in either or both receptors, B1-knockout (KO), B2KO and B1/2KO we observed changes in running performance overnight and in running distances. However, voluntary exercise led to the known pro-mitotic effect in the dentate gyrus of B1KO mice while it was attenuated in B2KO accompanied by an increase in microglia cells. Our data identify B2R as an important factor in running-induced precursor cell proliferation.

Variability of doublecortin-associated dendrite maturation in adult hippocampal neurogenesis is independent of the regulation of precursor cell proliferation.

BACKGROUND: In the course of adult hippocampal neurogenesis most regulation takes place during the phase of doublecortin (DCX) expression, either as pro-proliferative effect on precursor cells or as survival-promoting effect on postmitotic cells. We here obtained quantitative data about the proliferative population and the dynamics of postmitotic dendrite development during the period of DCX expression. The question was, whether any indication could be obtained that the initiation of dendrite development is timely bound to the exit from the cell cycle. Alternatively, the temporal course of morphological maturation might be subject to additional regulatory events. RESULTS: We found that (1) 20% of the DCX population were precursor cells in cell cycle, whereas more than 70% were postmitotic, (2) the time span until newborn cells had reached the most mature stage associated with DCX expression varied between 3 days and several weeks, (3) positive or negative regulation of precursor cell proliferation did not alter the pattern and dynamics of dendrite development. Dendrite maturation was largely independent of close contacts to astrocytes. CONCLUSION: These data imply that dendrite maturation of immature neurons is initiated at varying times after cell cycle exit, is variable in duration, and is controlled independently of the regulation of precursor cell proliferation. We conclude that in addition to the major regulatory events in cell proliferation and selective survival, additional micro-regulatory events influence the course of adult hippocampal neurogenesis.