Optimizing Nervous System-Specific Gene Targeting with Cre Driver Lines: Prevalence of Germline Recombination and Influencing Factors.

The Cre-loxP system is invaluable for spatial and temporal control of gene knockout, knockin, and reporter expression in the mouse nervous system. However, we report varying probabilities of unexpected germline recombination in distinct Cre driver lines designed for nervous system-specific recombination. Selective maternal or paternal germline recombination is showcased with sample Cre lines. Collated data reveal germline recombination in over half of 64 commonly used Cre driver lines, in most cases with a parental sex bias related to Cre expression in sperm or oocytes. Slight differences among Cre driver lines utilizing common transcriptional control elements affect germline recombination rates. Specific target loci demonstrated differential recombination; thus, reporters are not reliable proxies for another locus of interest. Similar principles apply to other recombinase systems and other genetically targeted organisms. We hereby draw attention to the prevalence of germline recombination and provide guidelines to inform future research for the neuroscience and broader molecular genetics communities

Proteomics, ultrastructure, and physiology of hippocampal synapses in a fragile X syndrome mouse model reveal presynaptic phenotype

Fragile X syndrome (FXS), the most common form of hereditary mental retardation, is caused by a loss-of-function mutation of the Fmr1 gene, which encodes fragile X mental retardation protein (FMRP). FMRP affects dendritic protein synthesis, thereby causing synaptic abnormalities. Here, we used a quantitative proteomics approach in an FXS mouse model to reveal changes in levels of hippocampal synapse proteins. Sixteen independent pools of Fmr1 knock-out mice and wild type mice were analyzed using two sets of 8-plex iTRAQ experiments. Of 205 proteins quantified with at least three distinct peptides in both iTRAQ series, the abundance of 23 proteins differed between Fmr1 knock-out and wild type synapses with a false discovery rate (q-value) <5%. Significant differences were confirmed by quantitative immunoblotting. A group of proteins that are known to be involved in cell differentiation and neurite outgrowth was regulated; they included Basp1 and Gap43, known PKC substrates, and Cend1. Basp1 and Gap43 are predominantly expressed in growth cones and presynaptic terminals. In line with this, ultrastructural analysis in developing hippocampal FXS synapses revealed smaller active zones with corresponding postsynaptic densities and smaller pools of clustered vesicles, indicative of immature presynaptic maturation. A second group of proteins involved in synaptic vesicle release was up-regulated in the FXS mouse model. In accordance, paired-pulse and short-term facilitation were significantly affected in these hippocampal synapses. Together, the altered regulation of presynaptically expressed proteins, immature synaptic ultrastructure, and compromised short-term plasticity points to presynaptic changes underlying glutamatergic transmission in FXS at this stage of development. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc

Reduced mGluR5 Activity Modulates Mitochondrial Function

The metabotropic glutamate receptor 5 (mGluR5) is an essential modulator of synaptic plasticity, learning and memory; whereas in pathological conditions, it is an acknowledged therapeutic target that has been implicated in multiple brain disorders. Despite robust pre-clinical data, mGluR5 antagonists failed in several clinical trials, highlighting the need for a better understanding of the mechanisms underlying mGluR5 function. In this study, we dissected the molecular synaptic modulation mediated by mGluR5 using genetic and pharmacological mouse models to chronically and acutely reduce mGluR5 activity. We found that next to dysregulation of synaptic proteins, the major regulation in protein expression in both models concerned specific processes in mitochondria, such as oxidative phosphorylation. Second, we observed morphological alterations in shape and area of specifically postsynaptic mitochondria in mGluR5 KO synapses using electron microscopy. Third, computational and biochemical assays suggested an increase of mitochondrial function in neurons, with increased level of NADP/H and oxidative damage in mGluR5 KO. Altogether, our observations provide diverse lines of evidence of the modulation of synaptic mitochondrial function by mGluR5. This connection suggests a role for mGluR5 as a mediator between synaptic activity and mitochondrial function, a finding which might be relevant for the improvement of the clinical potential of mGluR5

Suprachiasmatic control of melatonin synthesis in rats: inhibitory and stimulatory mechanisms

The suprachiasmatic nucleus (SCN) controls the circadian rhythm of melatonin synthesis in the mammalian pineal gland by a multisynaptic pathway including, successively, preautonomic neurons of the paraventricular nucleus (PVN), sympathetic preganglionic neurons in the spinal cord and noradrenergic neurons of the superior cervical ganglion (SCG). In order to clarify the role of each of these structures in the generation of the melatonin synthesis rhythm, we first investigated the day- and night-time capacity of the rat pineal gland to produce melatonin after bilateral SCN lesions, PVN lesions or SCG removal, by measurements of arylalkylamine N-acetyltransferase (AA-NAT) gene expression and pineal melatonin content. In addition, we followed the endogenous 48 h-pattern of melatonin secretion in SCN-lesioned vs. intact rats, by microdialysis in the pineal gland. Corticosterone content was measured in the same dialysates to assess the SCN lesions effectiveness. All treatments completely eliminated the day/night difference in melatonin synthesis. In PVN-lesioned and ganglionectomised rats, AA-NAT levels and pineal melatonin content were low (i.e. 12% of night-time control levels) for both day- and night-time periods. In SCN-lesioned rats, AA-NAT levels were intermediate (i.e. 30% of night-time control levels) and the 48-h secretion of melatonin presented constant levels not exceeding 20% of night-time control levels. The present results show that ablation of the SCN not only removes an inhibitory input but also a stimulatory input to the melatonin rhythm generating system. Combination of inhibitory and stimulatory SCN outputs could be of a great interest for the mechanism of adaptation to day-length (i.e. adaptation to seasons

Adult mouse eIF2Bϵ Arg191His astrocytes display a normal integrated stress response in vitro

Vanishing white matter (VWM) is a genetic childhood white matter disorder, characterized by chronic as well as episodic, stress provoked, neurological deterioration. Treatment is unavailable and patients often die within a few years after onset. VWM is caused by recessive mutations in the eukaryotic initiation factor 2B (eIF2B). eIF2B regulates protein synthesis rates in every cell of the body. In normal cells, various types of cellular stress inhibit eIF2B activity and induce the integrated stress response (ISR). We have developed a VWM mouse model homozygous for the pathogenic Arg191His mutation in eIF2Bϵ (2b5 ho ), representative of the human disease. Neuropathological examination of VWM patient and mouse brain tissue suggests that astrocytes are primarily affected. We hypothesized that VWM astrocytes are selectively hypersensitive to ISR induction, resulting in a heightened response. We cultured astrocytes from wildtype and VWM mice and investigated the ISR in assays that measure transcriptional induction of stress genes, protein synthesis rates and cell viability. We investigated the effects of short- A nd long-term stress as well as stress recovery. We detected congruent results amongst the various assays and did not detect a hyperactive ISR in VWM mouse astrocytes.</p