Impaired sleep and enhanced stress hormone secretion are the hallmarks of stress-related disorders, including major depression. The central neuropeptide, corticotropin-releasing hormone (CRH), is a ...key hormone that regulates humoral and behavioral adaptation to stress. Its prolonged hypersecretion is believed to play a key role in the development and course of depressive symptoms, and is associated with sleep impairment. To investigate the specific effects of central CRH overexpression on sleep, we used conditional mouse mutants that overexpress CRH in the entire central nervous system (CRH-COE-Nes) or only in the forebrain, including limbic structures (CRH-COE-Cam). Compared with wild-type or control mice during baseline, both homozygous CRH-COE-Nes and -Cam mice showed constantly increased rapid eye movement (REM) sleep, whereas slightly suppressed non-REM sleep was detected only in CRH-COE-Nes mice during the light period. In response to 6-h sleep deprivation, elevated levels of REM sleep also became evident in heterozygous CRH-COE-Nes and -Cam mice during recovery, which was reversed by treatment with a CRH receptor type 1 (CRHR1) antagonist in heterozygous and homozygous CRH-COE-Nes mice. The peripheral stress hormone levels were not elevated at baseline, and even after sleep deprivation they were indistinguishable across genotypes. As the stress axis was not altered, sleep changes, in particular enhanced REM sleep, occurring in these models are most likely induced by the forebrain CRH through the activation of CRHR1. CRH hypersecretion in the forebrain seems to drive REM sleep, supporting the notion that enhanced REM sleep may serve as biomarker for clinical conditions associated with enhanced CRH secretion.
Neurons synthesizing the neurotransmitter dopamine exert crucial functions in the mammalian brain. The biggest and most important population of dopamine-synthesizing neurons is located in the ...mammalian ventral midbrain (VM), and controls and modulates the execution of motor, cognitive, affective, motivational, and rewarding behaviours. Degeneration of these neurons leads to motor deficits that are characteristic of Parkinson's disease, while their dysfunction is involved in the pathogenesis of psychiatric disorders including schizophrenia and addiction. Because the aetiology and therapeutic prospects for these diseases include neurodevelopmental aspects, substantial scientific interest has been focused on deciphering the mechanistic pathways that control the generation and survival of these neurons during embryonic development. Researches during the last decade revealed the pivotal role of the secreted Wnt1 ligand and its signalling cascade in the generation of the dopamine-synthesizing neurons in the mammalian VM. Here, we summarize the initial and more recent findings that have unravelled several Wnt1-controlled genetic networks required for the proliferation and commitment of VM progenitors to the dopaminergic cell fate during midgestational embryonic stages, and for the correct differentiation of these progenitors into postmitotic dopamine-synthesizing neurons at late midgestational embryonic and foetal stages.
Abstract The oncogene DJ -1 has been originally identified as a suppressor of PTEN. Further on, loss-of-function mutations have been described as a causative factor in Parkinson's disease (PD). DJ-1 ...has an important function in cellular antioxidant responses, but its role in central metabolism of neurons is still elusive. We applied stable isotope assisted metabolic profiling to investigate the effect of a functional loss of DJ-1 and show that DJ-1 deficient neuronal cells exhibit decreased glutamine influx and reduced serine biosynthesis. By providing precursors for GSH synthesis, these two metabolic pathways are important contributors to cellular antioxidant response. Down-regulation of these pathways, as a result of loss of DJ-1 leads to an impaired antioxidant response. Furthermore, DJ-1 deficient mouse microglia showed a weak but constitutive pro-inflammatory activation. The combined effects of altered central metabolism and constitutive activation of glia cells raise the susceptibility of dopaminergic neurons towards degeneration in patients harboring mutated DJ-1 . Our work reveals metabolic alterations leading to increased cellular instability and identifies potential new intervention points that can further be studied in the light of novel translational medicine approaches.
In the last years, RNA interference (RNAi)-mediated gene knockdown has developed into a routine method to assess gene function in cultured mammalian cells in a fast and easy manner. For the use of ...this technique in developing or adult mice, short hairpin (sh)RNA vectors expressed stably from the genome are a faster alternative to conventional knockout approaches. Here we describe an advanced strategy for conditional gene knockdown in mice, where we used the Cre/loxP system to activate RNAi in a time and tissue dependent manner in the adult mouse brain. By placing conditional RNAi constructs into the defined genomic Rosa26 locus and by using recombinase mediated cassette exchange (RMCE) instead of laborious homologous recombination, we developed a fast, easy and reproducible approach to assess gene function in adult mice. We applied this technique to three genes of the MAPK signaling pathway--Braf, Mek1 and Mek2--and demonstrate here the potential of this new tool in mouse mutagenesis.
Hypersecretion of central corticotropin-releasing hormone (CRH) has been implicated in the pathophysiology of affective disorders. Both, basic and clinical studies suggested that disrupting CRH ...signaling through CRH type 1 receptors (CRH-R1) can ameliorate stress-related clinical conditions. To study the effects of CRH-R1 blockade upon CRH-elicited behavioral and neurochemical changes we created different mouse lines overexpressing CRH in distinct spatially restricted patterns. CRH overexpression in the entire central nervous system, but not when overexpressed in specific forebrain regions, resulted in stress-induced hypersecretion of stress hormones and increased active stress-coping behavior reflected by reduced immobility in the forced swim test and tail suspension test. These changes were related to acute effects of overexpressed CRH as they were normalized by CRH-R1 antagonist treatment and recapitulated the effect of stress-induced activation of the endogenous CRH system. Moreover, we identified enhanced noradrenergic activity as potential molecular mechanism underlying increased active stress-coping behavior observed in these animals. Thus, these transgenic mouse lines may serve as animal models for stress-elicited pathologies and treatments that target the central CRH system.
Recent evidence showed that the endocannabinoid system plays an important role in the behavioral adaptation of stress and fear responses. In this study, we chose a behavioral paradigm that includes ...criteria of both fear and stress responses to assess whether the involvement of endocannabinoids in these two processes rely on common mechanisms. To this end, we delivered a footshock and measured the fear response to a subsequently presented novel tone stimulus. First, we exposed different groups of cannabinoid receptor type 1 (CB1)‐deficient mice (CB1−/−) and their wild‐type littermates (CB1+/+) to footshocks of different intensities. Only application of an intense footshock resulted in a sustained fear response to the tone in CB1−/−. Using the intense protocol, we next investigated whether endocannabinoids mediate their effects via an interplay with corticotropin‐releasing hormone (CRH) signaling. Pharmacological blockade of CB1 receptors by rimonabant in mice deficient for the CRH receptor type 1 (CRHR1−/−) or type 2 (CRHR2−/−), and in respective wild‐type littermates, resulted in a sustained fear response in all genotypes. This suggests that CRH is not involved in the fear‐alleviating effects of CB1. As CRHR1−/− are known to be severely impaired in stress‐induced corticosterone secretion, our observation also implicates that corticosterone is dispensable for CB1‐mediated acute fear adaptation. Instead, conditional mutants with a specific deletion of CB1 in principal neurons of the forebrain (CaMK‐CB1−/−), or in cortical glutamatergic neurons (Glu‐CB1−/−), showed a similar phenotype as CB1−/−, thus indicating that endocannabinoid‐controlled glutamatergic transmission plays an essential role in acute fear adaptation.
Tumor necrosis factor (TNF)-α, a pro-inflammatory cytokine, has been implicated in both neuronal death and survival in Parkinson's disease (PD). The substantia nigra (SN), a CNS region affected in ...PD, is particularly susceptible to inflammatory insults and possesses the highest density of microglial cells, but the effects of inflammation and in particular TNF-α on neuronal survival in this region remains controversial. Using adenoviral vectors, the CRE/loxP system and hypomorphic mice, we achieved chronic expression of two levels of TNF-α in the SN of adult mice. Chronic low expression of TNF-α levels reduced the nigrostriatal neurodegeneration mediated by intrastriatal 6-hydroxydopamine administration. Protective effects of low TNF-α level could be mediated by TNF-R1, GDNF, and IGF-1 in the SN and SOD activity in the striatum (ST). On the contrary, chronic expression of high levels of TNF-α induced progressive neuronal loss (63% at 20
days and 75% at 100
days). This effect was accompanied by gliosis and an inflammatory infiltrate composed almost exclusively by monocytes/macrophages. The finding that chronic high TNF-α had a slow and progressive neurodegenerative effect in the SN provides an animal model of PD mediated by the chronic expression of a single cytokine. In addition, it supports the view that cytokines are not detrimental or beneficial by themselves, i.e., their level and time of expression among other factors can determine its final effect on CNS damage or protection. These data support the view that new anti-parkinsonian treatments based on anti-inflammatory therapies should consider these dual effects of cytokines on their design.
►Chronic up-regulation of TNF-α in the substantia nigra (SN) causes neuronal cell death. ►Low TNF expression protects neurons of the SN and striatal terminals from 6-OHDA. ►Low TNF levels up-regulated GDNF in the SN and SOD activity in the striatum. ►High TNF expression induced monocyte/macrophage but not neutrophil infiltration. ►A system to study the effects of 3 different levels of TNF in the SN was generated.
Urocortin 3 (UCN3) is strongly expressed in specific nuclei of the rodent brain, at sites distinct from those expressing urocortin 1 and urocortin 2, the other endogenous ligands of ...corticotropin-releasing hormone receptor type 2 (CRH-R2). To determine the physiological role of UCN3, we generated UCN3-deficient mice, in which the UCN3 open reading frame was replaced by a tau-lacZ reporter gene. By means of this reporter gene, the nucleus parabrachialis and the premammillary nucleus were identified as previously unknown sites of UCN3 expression. Additionally, the introduced reporter gene enabled the visualization of axonal projections of UCN3-expressing neurons from the superior paraolivary nucleus to the inferior colliculus and from the posterodorsal part of the medial amygdala to the principal nucleus of the bed nucleus of the stria terminalis, respectively. The examination of tau-lacZ reporter gene activity throughout the brain underscored a predominant expression of UCN3 in nuclei functionally connected to the accessory olfactory system. Male and female mice were comprehensively phenotyped but none of the applied tests provided indications for a role of UCN3 in the context of hypothalamic-pituitary-adrenocortical axis regulation, anxiety- or depression-related behavior. However, inspired by the prevalent expression throughout the accessory olfactory system, we identified alterations in social discrimination abilities of male and female UCN3 knock-out mice that were also present in male CRH-R2 knock-out mice. In conclusion, our results suggest a novel role for UCN3 and CRH-R2 related to the processing of social cues and to the establishment of social memories.
Abstract The establishment of the regional subdivisions of the vertebrate CNS is accomplished through the activity of different neuroepithelial organizing centers. The wingless/int (Wnt) family of ...secreted glycoproteins, among other factors, plays a crucial role in signaling from these centers. Wnt1 secreted from the boundary between the mid- and hindbrain, for instance, controls the development of this brain region and of associated neuronal populations. Different Wnts secreted from the caudomedial pallium, the cortical hem, pattern the adjacent hippocampal field. The first step in Wnt signal transduction is binding of the Wnt ligand to its receptors, the seven-pass transmembrane Frizzled proteins. Inactivation of different Frizzled genes in mice have revealed an extensive functional redundancy between these receptors. In order to discriminate between a possible participation of different Frizzled receptors in the transduction of Wnt signals at the mid-/hindbrain boundary and the cortical hem, we have performed a detailed expression study of the 10 known murine Frizzled genes at crucial stages of mouse embryonic development. Our analysis reveals a highly dynamic yet distinct expression pattern of individual Frizzled genes in the anterior neural tube of the developing mouse embryo. The overlapping spatio-temporal expression of at least two and up to six Frizzled genes in any region of the developing mouse brain, however, also suggests a vast functional redundancy of the murine Frizzled receptors. This redundancy has to be taken into consideration for future analyses of Frizzled receptor function at these signaling centers in the mouse.
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