Anxiolytic effects of muscarinic acetylcholine receptors agonist oxotremorine in chronically stressed rats and related changes in BDNF and FGF2 levels in the hippocampus and prefrontal cortex.

RATIONALE: In depressive disorders, one of the mechanisms proposed for antidepressant drugs is the enhancement of synaptic plasticity in the hippocampus and cerebral cortex. Previously, we showed that the muscarinic acetylcholine receptor (mAChR) agonist oxotremorine (Oxo) increases neuronal plasticity in hippocampal neurons via FGFR1 transactivation. OBJECTIVES: Here, we aimed to explore (a) whether Oxo exerts anxiolytic effect in the rat model of anxiety-depression-like behavior induced by chronic restraint stress (CRS), and (b) if the anxiolytic effect of Oxo is associated with the modulation of neurotrophic factors, brain-derived neurotrophic factor (BDNF) and fibroblast growth factor-2 (FGF2), and phosphorylated Erk1/2 (p-Erk1/2) levels in the dorsal or ventral hippocampus and in the medial prefrontal cortex. METHODS: The rats were randomly divided into four groups: control unstressed, CRS group, CRS group treated with 0.2 mg/kg Oxo, and unstressed group treated with Oxo. After 21 days of CRS, the groups were treated for 10 days with Oxo or saline. The anxiolytic role of Oxo was tested by using the following: forced swimming test, novelty suppressed feeding test, elevated plus maze test, and light/dark box test. The hippocampi and prefrontal cortex were used to evaluate BDNF and FGF2 protein levels and p-Erk1/2 levels. RESULTS: Oxo treatment significantly attenuated anxiety induced by CRS. Moreover, Oxo treatment counteracted the CRS-induced reduction of BDNF and FGF2 levels in the ventral hippocampus and medial prefrontal cerebral cortex CONCLUSIONS: The present study showed that Oxo treatment ameliorates the stress-induced anxiety-like behavior and rescues FGF2 and BDNF levels in two brain regions involved in CRS-induced anxiety, ventral hippocampal formation, and medial prefrontal cortex.

Sonic hedgehog released from scratch-injured astrocytes is a key signal necessary but not sufficient for the astrocyte de-differentiation.

Recent studies demonstrated that mature atrocytes have the capacity for de-differentiating into neural stem/progenitor cells (NSPCs) in vitro and in vivo. However, it is still unknown what signals endow astroglial cells with a de-differentiation potential. Furthermore, the signaling molecules and underlying mechanism that confer astrocytes with the competence of NSPC phenotypes have not been completely elucidated. Here, we found that sonic hedgehog (Shh) production in astrocytes following mechanical injury was significantly elevated, and that incubation of astrocyes with the injured astrocyte conditioned medium (ACM) causes astrocytes to gradually lose their immunophenotypical profiles, and acquire NSPC characteristics, as demonstrated by down-regulation of typical astrocytic markers (GFAP and S100) and up-regulation of markers that are generally expressed in NSCs, (nestin, Sox2, and CD133). ACM treated astrocytes exhibit self-renewal capacity and multipotency similar to NSPCs. Concomitantly, in addition to Ptc, there was a significant up-regulation of the Shh downstream signal components Gli2 and Cyclin D1 which are involved in cell proliferation, dramatic changes in cell morphology, and the disruption of cell-cycle G1 arrest. Conversely, the depletion of Shh by administration of its neutralizing antibody (Shh n-Ab) effectively inhibited the de-differentiation process. Strikingly, Shh alone had little effect on astrocyte de-differentiation to NSPCs. These data above suggest that Shh is a key instructive molecule while other molecules secreted from insulted astrocytes may synergistically promote the de-differentiation event.

In vitro beneficial activation of microglial cells by mechanically-injured astrocytes enhances the synthesis and secretion of BDNF through p38MAPK.

It has long been promulgated that microglial cells serve beneficial roles in the central nervous system (CNS). The beneficial role of microglial cells is considered to be linked with microglial activation and consequent up-regulation of various trophic factors. However, what triggers microglial activation and consequent elevated level of trophic factors, especially brain-derived neurotrophic factor (BDNF), following traumatic CNS injury has become a crucial but elusive issue. Furthermore, an effort still remains in understanding of the cellular and molecular mechanisms underlying the endogenous neuroprotection of activated microglial cells. In this study, we demonstrated that mechanically-injured astrocyte conditioned medium (ACM) could provoke beneficial activation of microglial cells and thus promote the transcription, synthesis and release of BDNF in cultured microglial cells. The microglia-derived BDNF can exerted a demonstrable biological role in promoting neurite outgrowth and intimate terminal contacts of dorsal root ganglion (DRG) neurons co-cultured with microglial cells. Moreover, ACM induced remarkable p38MAPK phosphorylation in cultured microglial cells that preceded the burst of BDNF. Activating p38-MAPK by anisomycin resulted in salutary effects similar to those seen with ACM, whereas specific inhibition of the p38MAPK by SB203580 abrogated all the positive effects of ACM, including BDNF promotion and subsequent neurite outgrowth of DRG neurite outgrowth of DRG neurons and their intimate terminal contacts with microglial cells. Together, our results indicated that the neuroprotection of the microglial source is mainly caused by micro-environmental soluble molecules released from injured astrocytes, and ACM-induced BDNF production and release from microglial cells may be mediated through p38-MAPK signaling pathway. Therefore, these findings may lay a foundation to further investigations on the microglial beneficial activation role in the repair of traumatic CNS injury and neurodegenerative diseases.

ErbB2 activation contributes to de-differentiation of astrocytes into radial glial cells following induction of scratch-insulted astrocyte conditioned medium.

Radial glial cells play a significant role in the repair of spinal cord injuries as they exert critical role in the neurogenesis and act as a scaffold for neuronal migration. Our previous study showed that mature astrocytes of spinal cord can undergo a de-differentiation process and further transform into pluripotential neural precursors; the occurrence of these complex events arise directly from the induction of diffusible factors released from scratch-insulted astrocytes. However, it is unclear whether astrocytes can also undergo rejuvenation to revert to a radial glial progenitor phenotype after the induction of scratch-insulted astrocytes conditioned medium (ACM). Furthermore, the mechanism of astrocyte de-differentiation to the progenitor cells is still unclear. Here we demonstrate that upon treating mature astrocytes with ACM for 10 days, the astrocytes exhibit progressive morphological and functional conversion to radial glial cells. These changes include the appearance of radial glial progenitor cells, changes in the immunophenotypical profiles, characterized by the co-expression of nestin, paired homeobox protein (Pax6) and RC2 as well as enhanced capability of multipotential differentiation. Concomitantly, ErbB2 protein level was progressively up-regulated. Thereby these results provide a potential mechanism by which ACM could induce mature astrocytes to regain the profile of radial glial progenitors due to activating the ErbB2 signaling pathways.

Multiple conserved 5' elements are required for high-level pollen expression of the Arabidopsis reproductive actin ACT1.

The Arabidopsis thaliana actin gene family comprises eight genes, which are divided into two ancient classes, vegetative and reproductive. We dissected various 5' elements and the conserved expression pattern of the reproductive actin gene ACT1, which is the most strongly expressed pollen actin gene. A basal construct containing only 310 bp of sequence upstream of the major transcriptional start site showed essentially full promoter activity in pollen and ovules. Further truncations of the 5'-flanking region and two different 10 bp replacements within a 55 bp conserved domain each caused a several-fold reduction in mature pollen expression. Intron L, located in the 5'-untranslated region (5'-UTR) was also required for high-level expression of pollen and organ primordia, and it had the properties of an enhancer. Pollen expression was not preserved when intron L was precisely replaced by intron L2 from the vegetatively expressed actin gene ACT2. ACT1 reporter gene constructs were strongly expressed in both pollen and ovules of tobacco and in the pollen of rice. Promoter-reporter fusions of the most distantly related Arabidopsis reproductive actin gene ACT1 showed strong expression in tobacco pollen and ovules indistinguishable from that directed by ACT1. Thus, multiple conserved cis-sequence elements within the 5'-flanking region and 5'-UTR of ACT1 direct high levels of reproductive tissue-specific expression.