Effect of long-lasting serotonin depletion on environmental enrichment-induced neurogenesis in adult rat hippocampus and spatial learning

The dentate gyrus of the hippocampal formation produces new neurons throughout adulthood in mammalian species. Several experimental statuses and factors regulating to neurogenesis have been identified in the adult dentate gyrus. For example, exposure to an enriched environment enhances neurogenesis in the dentate gyrus and improves hippocampus-dependent spatial learning. Furthermore, serotonin is known to influence adult neurogenesis, and learning and memory. However, the effects of long-lasting depletion of serotonin over the developing period on neurogenesis have not been investigated. Thus, we examined the influence of long-lasting serotonin depletion on environmental enrichment-induced neurogenesis and spatial memory performance. As reported previously, environmental enrichment significantly increased new neurons in the dentate gyrus. However, there was no improvement of the spatial learning test in adult rats in standard and in environmental enrichment housings. Intracisternal administration of the serotonergic neurotoxin, 5,7-dihydroxytryptamine, on postnatal day 3 apparently reduced serotonin content in the adult hippocampus without regeneration. This experimental depletion of serotonin in the hippocampus of rats housed in an enriched environment had no effect on spatial memory performance, but produced significant decreases in the number of bromodeoxyuridine-labeled new cells in the dentate gyrus. These findings indicate that newly generated cells stimulated by environmental enrichment are not critical for improvements in hippocampus-dependent learning. Furthermore, numbers of bromodeoxyuridine-labeled cells in the dentate gyrus of 5,7-dihydroxytryptamine-injected rats did not differ between 1 day and 4 weeks after bromodeoxyuridine injection. These data suggest that survival of newly generated dentate gyrus cells remains relatively constant under long-lasting serotonin depletion.     

Hedgehog signaling and primary cilia are required for the formation of adult neural stem cells

Neural stem cells that continue to produce neurons are retained in the adult hippocampal dentate gyrus. The mechanisms by which embryonic neural progenitors expand and transform into postnatal neural stem cells, an essential process for the continual production of neurons throughout life, remain unknown. We found that radial astrocytes, the postnatal progenitors in the dentate gyrus, failed to develop after embryonic ablation of ciliary genes or Smoothened (Smo), an essential component for Sonic hedgehog (Shh) signaling. Postnatal dentate neurogenesis failed in these mutant mice, and the dentate gyrus became severely hypotrophic. In contrast, expression of a constitutively active Smo (SmoM2-YFP) resulted in a marked expansion of the dentate gyrus. Double-mutant analyses suggested that both wild-type Smo and SmoM2-YFP function through the primary cilia. We conclude that Shh signaling, acting through the primary cilia, has a critical role in the expansion and establishment of postnatal hippocampal progenitors.     

音猬因子与胰岛素增强子结合蛋白在小鼠胚胎呼吸系统的早期表达

目的 探讨音猬因子信号通路成员与胰岛素增强子结合蛋白在呼吸系统的早期表达与其发育的联系。方法 胚龄9.0～13d小鼠胚胎呼吸系统各年龄段不小于3个,连续石蜡切片,用抗音猬因子(Shh)、抗patched1(Ptcl)、抗smoothened(Smo)、抗胰岛素增强子结合蛋白(ISL1)和抗α-平滑肌肌动蛋白(α-SMA)抗体进行免疫组织化学染色。结果 胚龄10d,Shh表达在前肠内胚层腹侧壁。胚龄11～12d,Ptc1表达在气管、支气管和肺内分支上皮。胚龄13d,Ptc1只表达在肺内分支上皮。胚龄9d,ISL1表达在前肠内胚层腹侧壁。胚龄10～12d,ISL1表达在前肠（气管）腹侧壁和支气管侧壁上皮及邻近的间充质内。胚龄13d,ISL1表达减弱,始终未见在肺内有阳性表达。胎龄12～13d,与气管后壁、支气管内侧壁上皮Ptc1表达减弱处相邻的间充质出现α-SMA阳性平滑肌细胞,其与对侧间充质ISL1阳性细胞的分布呈背-腹侧或内-外侧模式。气管腹侧及肺芽外侧间充质中可见ISL1与Smo共表达细胞。 结论 ISL1在气管及肺芽的发育中可能与Shh信号系统协同发挥作用。     

Loss of vision-guided adaptation of the vestibulo-ocular reflex after depletion of brain serotonin in the rabbit

Brain serotonin in pigmented rabbits was depleted by intraventricular injection of 5,7-dihydroxytryptamine (5,7-DHT). Before depletion, an adaptive increase in the gain of the horizontal vestibulo-ocular reflex (HVOR) could be induced regularly by continuously rotating the animals in combination with optokinetic stimulus. After the depletion, such an increase in HVOR gain did not occur, even though dynamic characteristics of the HVOR and of the optokinetic eye movement were not altered. 5,7-DHT treatment also reduced brain norepinephrine, but depletion of norepinephrine to a similar extent by intraventricular injection of 6-hydroxydopamine in other rabbits did not affect the HVOR adaptation. These results suggest that brain serotonin plays an important role in maintaining adaptive modifiability of the HVOR.     

Shh maintains dermal papilla identity and hair morphogenesis via a Noggin-Shh regulatory loop

During hair follicle morphogenesis, dermal papillae (DPs) function as mesenchymal signaling centers that cross-talk with overlying epithelium to regulate morphogenesis. While the DP regulates hair follicle formation, relatively little is known about the molecular basis of DP formation. The morphogen Sonic hedgehog (Shh) is known for regulating hair follicle epithelial growth, with excessive signaling resulting in basal cell carcinomas. Here, we investigate how dermal-specific Shh signaling contributes to DP formation and hair growth. Using a Cre-lox genetic model and RNAi in hair follicle reconstitution assays, we demonstrate that dermal Smoothened (Smo) loss of function results in the loss of the DP precursor, the dermal condensate, and a stage 2 hair follicle arrest phenotype reminiscent of Shh(-/-) skin. Surprisingly, dermal Smo does not regulate cell survival or epithelial proliferation. Rather, molecular screening and immunostaining studies reveal that dermal Shh signaling controls the expression of a subset of DP-specific signature genes. Using a hairpin/cDNA lentiviral system, we show that overexpression of the Shh-dependent gene Noggin, but not Sox2 or Sox18, can partially rescue the dermal Smo knockdown hair follicle phenotype by increasing the expression of epithelial Shh. Our findings suggest that dermal Shh signaling regulates specific DP signatures to maintain DP maturation while maintaining a reciprocal Shh-Noggin signaling loop to drive hair follicle morphogenesis.     

Molecular and cellular analysis of embryonic avian tongue development.

Signalling cascades first described in Drosophila have been found to regulate patterning and outgrowth in a number of structures in higher vertebrates. We sought to determine whether the evolutionarily conserved genes were important during the development of the tongue. In situ hybridisation was used to determine the temporo-spatial expression of a panel of conserved genes. Histological examination and incorporation of BrdU were used to determine the mechanism by which the tongue develops. We show that evolutionarily conserved genes were expressed in distinct dynamic patterns during tongue development. Sonic Hedgehog (Shh) and Patched (Ptc) were found only in the dorsal tongue epithelium. Shh expression was only observed in the suprabasal layers, whereas Ptc was observed in both basal and suprabasal layers. Cell division in the epithelium was concentrated in regions devoid of Shh. Expression of bone morphogenetic protein-7 (BMP) was identical to that of Shh. Shh and Ptc expression were never detected in the mesenchyme. Ectopic expression of Noggin (a potent antagonist of the BMPs) caused severe abnormalities in tongue morphology, including swelling of the mesenchymal component and a thickening of the epithelial layer. Data from this study suggests that the epithelium and mesenchyme express quite different genes during development. However BMP activity acts to inhibit growth in both tissues.     

干扰维生素D3上调蛋白1通过调控Shh影响高糖诱导的肾小管上皮细胞凋亡

目的:探讨维生素D3上调蛋白1(VDUP-1)对高糖诱导的肾小管上皮细胞凋亡的影响及机制。方法:人近端肾小管上皮HK-2细胞用高糖处理后,real-time PCR和Western blot检测细胞中VDUP-1的水平。HK-2细胞转染VDUP-1小干扰RNA,real-time PCR和Western blot检测抑制效果。高糖条件培养VDUP-1表达下调的HK-2细胞,流式细胞术检测细胞凋亡,试剂盒检测细胞中caspase-3和caspase-9的活性,ELISA法测定培养上清液中肿瘤坏死因子α(TNF-α)含量,Western blot检测细胞中音猬因子(Shh)信号通路关键蛋白Patched 1 (Ptch1)、Smoothened (Smo)、锌指蛋白Gli2和Shh的水平。用外源性Shh处理HK-2细胞,Western blot检测Ptch1、Smo和Gli2的水平。用外源性Shh处理VDUP-1表达下调的HK-2细胞,高糖处理后,流式细胞术检测细胞凋亡,试剂盒检测细胞中caspase-3和caspase-9的活性,ELISA法测定培养液上清中TNF-α含量。结果:高糖处理后,HK-2细胞中VDUP-1的mRNA和蛋白水平升高(P 0. 05)。转染VDUP-1小干扰RNA后,HK-2细胞中VDUP-1的mRNA和蛋白水平下降(P 0. 05)。与正常培养的细胞相比,高糖处理后HK-2细胞凋亡率显著升高,细胞中caspase-3和caspase-9活性明显升高,TNF-α含量亦明显升高(P 0. 05);下调VDUP-1表达后的HK-2细胞经高糖处理后细胞凋亡率显著降低,细胞中caspase-3和caspase-9活性也明显降低(P 0. 05)。高糖培养后细胞中Ptch1、Smo、Gli2和Shh的蛋白水平下降,而下调VDUP-1表达部分拮抗高糖对HK-2细胞中Ptch1、Smo、Gli2和Shh表达的影响。外源性Shh可以促进细胞中Ptch1、Smo和Gli2的表达,抑制高糖诱导的HK-2细胞凋亡和分泌TNF-α,与下调VDUP-1共同抑制高糖诱导的肾小管上皮细胞凋亡。结论:干扰VDUP-1表达可能通过激活Shh信号通路抑制高糖诱导的肾小管上皮细胞凋亡和分泌TNF-α。     

Sonic Hedgehog及其受体Patched在小鼠视交叉发育过程中的表达

在小鼠胚胎发育过程中,胚胎第13d(E13)至15d(E15)是视交叉发育的主要阶段。在本研究中,我们观察了在E13～E15,Sonic Hedgehog(Shh)及其受体Patched(Ptc)在视觉传导通路的表达。结果发现:在视交叉和视束中,Shh在视神经纤维接近中线时表达上调,越过中线后表达下调,并且主要表达在较深的区域。Ptc在E13～E14的视网膜和E14～E15的视束中有表达,但在视交叉中无表达。Ptc,而不是Shh,表达在体外培养的生长锥中。Shh和Ptc在视觉传导通路发育中的表达提示Shh可能在引导视神经生长方面发挥一定作用。     

Depletion in serotonin decreases neurogenesis in the dentate gyrus and the subventricular zone of adult rats

During adulthood, neuronal precursor cells persist in two discrete regions, the subventricular zone[19]and the hippocampal subgranular zone,[11]as recently demonstrated in primates.[10]To date, a few factors such as adrenal steroids[9]and trophic factors[13]are known to regulate adult neurogenesis. Since neuronal activity may also influence cellular development and plasticity in brain, we investigated the effects of serotonin depletion on cell proliferation occurring in these regions. Indeed, in addition to its role as a neurotransmitter, 5-hydroxytryptamine (serotonin) is considered as a developmental regulatory signal.14, 22Prenatal depletion in 5-hydroxytryptamine delays the onset of neurogenesis in 5-hydroxytryptamine target regions[14]and 5-hydroxytryptamine promotes the differentiation of cortical and hippocampal neurons.15, 23Although in the adult brain, a few studies have suggested that 5-hydroxytryptamine may play a role in neuronal plasticity by maintaining the synaptic connections in the cortex and hippocampus,3, 6, 16no information is actually available concerning the influence of 5-hydroxytryptamine on adult neurogenesis. If further work confirms that new neurons can be produced in the adult human brain as is the case for a variety of species, it is particularly relevant to determine the influence of 5-hydroxytryptamine on neurogenesis in the hippocampal formation, a part of the brain largely implicated in learning and memory processes. Indeed, lack of 5-hydroxytryptamine in the hippocampus has been associated with cognitive disorders, such as depression,[1]schizophrenia and Alzheimer's disease.[7]In the present study, we demonstrated that both inhibition of 5-hydroxytryptamine synthesis and selective lesions of 5-hydroxytryptamine neurons are associated with decreases in the number of newly generated cells in the dentate gyrus, as well as in the subventricular zone.     

Sonic hedgehog guides commissural axons along the longitudinal axis of the spinal cord

Dorsal commissural axons in the developing spinal cord cross the floor plate, then turn rostrally and grow along the longitudinal axis, close to the floor plate. We used a subtractive hybridization approach to identify guidance cues responsible for the rostral turn in chicken embryos. One of the candidates was the morphogen Sonic hedgehog (Shh). Silencing of the gene SHH (which encodes Shh) by in ovo RNAi during commissural axon navigation demonstrated a repulsive role in post-commissural axon guidance. This effect of Shh was not mediated by Patched (Ptc) and Smoothened (Smo), the receptors that mediate effects of Shh in morphogenesis and commissural axon growth toward the floor plate. Rather, functional in vivo studies showed that the repulsive effect of Shh on postcommissural axons was mediated by Hedgehog interacting protein (Hip).     

Increased expression of preproneuropeptide Y and preprosomatostatin mRNA in striatum after selective serotoninergic lesions in rats

The levels of neuropeptide Y and somatostatin may change when serotoninergic neurotransmission is altered in different brain regions. To assess whether serotonin regulates the synthesis of these peptides, we measured the levels of preproneuropeptide Y (ppNPY) and preprosomatostatin (ppSOM) mRNA in different brain regions after intracerebroventricular injection of 5,7-dihydroxytryptamine (5,7-DHT), a selective serotonin neurotoxin. The mRNA of these peptides significantly increased in the striatum but not in hippocampus and frontal cortex. It thus appears that serotonin has an inhibitory effect on the biosynthesis of neuropeptide Y and somatostatin in striatum whereas it probably acts by stimulating the release of these peptides in hippocampus and frontal cortex.     

Central serotonin depletion modulates the behavioural, endocrine and physiological responses to repeated social stress and subsequent c-fos expression in the brains of male rats.

Intraspecific confrontation has been used to study effect of depleting central serotonin on the adaptation of male rats to repeated social stress (social defeat). Four groups of adult male rats were used (serotonin depletion/sham: stressed; serotonin depletion/sham: non-stressed). Central serotonin was reduced (by 59-97%) by a single infusion of the neurotoxin 5,7-dihydroxtryptamine (150 microg) into the cerebral ventricles; levels of dopamine and noradrenaline were unaltered (rats received appropriate uptake blockers prior to neurotoxic infusions). Sham-operated animals received solute only. Rats were then either exposed daily for 10 days to a second larger aggressive male in the latter's home cage, or simply transferred to an empty cage (control procedure). Rats with reduced serotonin failed to show the increased freezing behaviour during the pre-defeat phase of the social interaction test characteristic of sham animals. There was no change in the residents' behaviour. Core temperature increased during aggressive interaction in sham rats, and this did not adapt with repeated stress. By contrast, stress-induced hyperthermia was accentuated in serotonin-reduced rats as the number of defeat sessions increased. Basal core temperature was unaffected by serotonin depletion. Heart rate increased during social defeat, but this did not adapt with repeated stress; serotonin depletion had no effect on this cardiovascular response. Basal corticosterone was increased in serotonin-depleted rats, but the progressive reduction in stress response over days was not altered. C-fos expression in the brain was not altered in control (non-stressed) rats by serotonin reduction in the areas examined, but there was increased expression after repeated social stress in the medial amygdala of 5-HT depleted rats. These experiments show that reduction of serotonin alters responses to repeated social stress in male rats, and suggests a role for serotonin in the adaptive process.     

Differential recovery of adrenocortical responses to neural stimuli following administration of 5,7-dihydroxytryptamine into the hypothalamus

Summary In view of the role of serotonin in adrenocortical regulation, the effects of depletion of hypothalamic serotonin, using localized injections of the neurotoxin 5,7-dihydroxytryptamine into the hypothalamic paraventricular nucleus, on the rise in plasma corticosterone following afferent neural stimulation, were studied. The neurotoxin caused a significant reduction (p<0.001) in hypothalamic serotonin content of about 50% during the first month and about 30% up to two months later. Basal and ether stress-induced rises in plasma corticosterone levels were unaffected at all times after this treatment, but responses to stimulation of the sciatic nerve were reduced for up to four weeks (p<0.01), recovering at later times. Responses to photic and acoustic stimuli were almost entirely prevented up to four weeks following the treatment (p<0.001) but showed a gradual recovery to full, or almost full, adrenocortical responses at eight weeks, following acoustic and photic stimulation respectively. These results demonstrate a differential recovery of the adrenocortical responses, following the neurotoxin injection and indicate that different neural modalities require different 5-HT concentrations in the PVN for the expression of a full adrenocortical response.