Optogenetic manipulation of neural circuitry in vivo

Recent advances in optogenetics have permitted investigations of specific cell types in the nervous system with unprecedented precision and control. This review will discuss the use of optogenetic techniques in the study of mammalian neural circuitry in vivo, as well as practical and theoretical considerations in their application.     

In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker

Macroautophagy mediates the bulk degradation of cytoplasmic components. It accounts for the degradation of most long-lived proteins: cytoplasmic constituents, including organelles, are sequestered into autophagosomes, which subsequently fuse with lysosomes, where degradation occurs. Although the possible involvement of autophagy in homeostasis, development, cell death, and pathogenesis has been repeatedly pointed out, systematic in vivo analysis has not been performed in mammals, mainly because of a limitation of monitoring methods. To understand where and when autophagy occurs in vivo, we have generated transgenic mice systemically expressing GFP fused to LC3, which is a mammalian homologue of yeast Atg8 (Aut7/Apg8) and serves as a marker protein for autophagosomes. Fluorescence microscopic analyses revealed that autophagy is differently induced by nutrient starvation in most tissues. In some tissues, autophagy even occurs actively without starvation treatments. Our results suggest that the regulation of autophagy is organ dependent and the role of autophagy is not restricted to the starvation response. This transgenic mouse model is a useful tool to study mammalian autophagy.     

In vivo metabolic imaging of cardiac bioenergetics in transgenic mice

Recent advances in transgenic technology have made the mouse a particularly interesting small animal in cardiovascular research. Increasingly sophisticated experimental methods and tools are needed for detailed characterization of cardiovascular physiology and biochemistry in the mice. The objective of this study was to develop a method for noninvasive evaluation of cardiac energy metabolism in the mouse. Cardiac gated (31)P magnetic resonance spectroscopy using Image Selected in Vivo Spectroscopy (ISIS) method was applied in old mice overexpressing bovine growth hormone (bGH) (n = 5) and control mice (n = 5). The localized volumes of interest were 128 and 112 microL, respectively. Phosphocreatine-to-ATP ratio was 1.5 +/- 0.13 in the bGH mice and 2.1 +/- 0.04 in the control group (P < 0.01). The study demonstrates the feasibility of application of volume-selective (31)P MRS for evaluation of cardiac energy metabolism in the mouse under maintained physiological conditions.     

Control of neural synchrony using channelrhodopsin-2: a computational study

In this paper, we present an optical stimulation based approach to induce 1:1 in-phase synchrony in a network of coupled interneurons wherein each interneuron expresses the light sensitive protein channelrhodopsin-2 (ChR2). We begin with a transition rate model for the channel kinetics of ChR2 in response to light stimulation. We then define "functional optical time response curve (fOTRC)" as a measure of the response of a periodically firing interneuron (transfected with ChR2 ion channel) to a periodic light pulse stimulation. We specifically consider the case of unidirectionally coupled (UCI) network and propose an open loop control architecture that uses light as an actuation signal to induce 1:1 in-phase synchrony in the UCI network. Using general properties of the spike time response curves (STRCs) for Type-1 neuron model (Ermentrout, Neural Comput 8:979-1001, 1996) and fOTRC, we estimate the (open loop) optimal actuation signal parameters required to induce 1:1 in-phase synchrony. We then propose a closed loop controller architecture and a controller algorithm to robustly sustain stable 1:1 in-phase synchrony in the presence of unknown deviations in the network parameters. Finally, we test the performance of this closed-loop controller in a network of mutually coupled (MCI) interneurons.     

Ca(2+) dysregulation in neurons from transgenic mice expressing mutant presenilin 2

Mutations in amyloid precursor protein (APP), and presenilin‐1 and presenilin‐2 (PS1 and PS2) have causally been implicated in Familial Alzheimer’s Disease (FAD), but the mechanistic link between the mutations and the early onset of neurodegeneration is still debated. Although no consensus has yet been reached, most data suggest that both FAD‐linked PS mutants and endogenous PSs are involved in cellular Ca²⁺ homeostasis. We here investigated subcellular Ca²⁺ handling in primary neuronal cultures and acute brain slices from wild type and transgenic mice carrying the FAD‐linked PS2‐N141I mutation, either alone or in the presence of the APP Swedish mutation. Compared with wild type, both types of transgenic neurons show a similar reduction in endoplasmic reticulum (ER) Ca²⁺ content and decreased response to metabotropic agonists, albeit increased Ca²⁺ release induced by caffeine. In both transgenic neurons, we also observed a higher ER–mitochondria juxtaposition that favors increased mitochondrial Ca²⁺ uptake upon ER Ca²⁺ release. A model is described that integrates into a unifying hypothesis the contradictory effects on Ca²⁺ homeostasis of different PS mutations and points to the relevance of these findings in neurodegeneration and aging.     

Altered T cell activation and development in transgenic mice expressing the HIV-1 nef gene.

The nef gene, which encodes related cytoplasmic proteins in both human (HIV) and simian (SIV) immunodeficiency viruses is dispensable for viral replication in vitro. In contrast, in vivo experiments have revealed that SIV nef is required for efficient viral replication and development of AIDS in SIV infected rhesus monkeys, thus indicating that nef plays an essential role in the natural infection. We show that expression of the Nef protein from the HIV-1 NL43 isolate in transgenic mice perturbs development of CD4+ T cells in the thymus and elicits depletion of peripheral CD4+ T cells. Thymic T cells expressing NL43 Nef show altered activation responses. In contrast, Nef protein of the HIV-1 HxB3 isolate does not have an overt effect on T cells when expressed in transgenic animals. The differential effects of the two HIV-1 nef alleles in transgenic mice correlate with down-regulation of CD4 antigen expression on thymic T cells. The differential interactions of the NL43 and HxB3 nef alleles with CD4 were reproduced in a transient assay in human CD4+ CEM T cells. Down-regulation of CD4 by nef in both human and transgenic murine T cells indicates that the relevant interactions are conserved in these two systems and suggests that the consequences of Nef expression on the host cell function can be analyzed in vivo in the murine system. Our observations from transgenic mice suggest that nef-elicited perturbations in T cell signalling play an important role in the viral life cycle in vivo, perhaps resulting in elimination of infected CD4+ T cells.     

In vivo modulation of epidermal growth factor receptor phosphorylation in mice expressing different gangliosides

We studied in this work the in vivo phosphorylation of the epidermal growth factor receptor (EGFr) in skin from knockout mice lacking different ganglioside glycosyltransferases. Results show an enhancement of EGFr phosphorylation, after EGF stimulation, in skin from Sial-T2 knockout and Sial-T2/GalNAc-T double knockout mice as compared with wild-type and Sial-T1 knockout mice. Qualitative analysis of ganglioside composition in mice skin suggest that the increase of EGFr phosphorylation observed in skin from Sial-T2 knockout and Sial-T2/GalNAc-T double knockout mice in response to EGF might not be primary attributed to the expression of GD3 or a-series gangliosides in mice skin. These studies provide, for the first time, an approach for studying the molecular mechanisms involved in the in vivo regulation of EGFr function by gangliosides.     

In vivo transgenic mutation assays

Transgenic rodent gene-mutation models provide relatively quick and statistically reliable assays for gene mutations in the DNA from any tissue. This report summarizes those issues that have been agreed upon at a previous IWGT meeting [Environ. Mol. Mutagen. 35 (2000) 253], and discusses in depth those issues for which no consensus was reached before. It was previously agreed that for regulatory applications, assays should be based upon neutral genes, be generally available in several laboratories, and be readily transferable. For phage-based assays, five to ten animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of approximately 3x10(-5) mutants/locus and 125,000-300,000 plaque or colony forming units (pfu or cfu) per tissue per animal. A full set of data should be generated for a vehicle control and two dose groups. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a blocked design, where samples from negative control, positive control and each treatment group are processed together. The total number of pfus or cfus and the MF for each tissue and animal are reported. Statistical tests should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose-response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is a statistically non-significant change, with all mean MFs within two standard deviations of the control. During the current workshop, a general protocol was agreed in which animals are treated daily for 28 consecutive days and tissues sampled 3 days after the final treatment. This recommendation could be modified by reducing or increasing the number of treatments or the length of the treatment period, when scientifically justified. Normally male animals alone are sufficient and normally at least one rapidly proliferating and one slowly proliferating tissue should be sampled. Although, as agreed previously, sequencing data are not normally required, they might provide useful additional information in specific circumstances, mainly to identify and correct for clonal expansion and in some cases to determine a mechanism associated with a positive response.     

In vivo transgenic mutation assays

Transgenic rodent gene-mutation models provide relatively quick and statistically reliable assays for gene mutations in the DNA from any tissue. This report summarizes those issues that have been agreed upon at a previous IWGT meeting [Environ. Mol. Mutagen. 35 (2000) 253], and discusses in depth those issues for which no consensus was reached before. It was previously agreed that for regulatory applications, assays should be based upon neutral genes, be generally available in several laboratories, and be readily transferable. For phage-based assays, five to ten animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of 3×10⁻⁵ mutants/locus and 125,000–300,000 plaque or colony forming units (pfu or cfu) per tissue per animal. A full set of data should be generated for a vehicle control and two dose groups. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a blocked design, where samples from negative control, positive control and each treatment group are processed together. The total number of pfus or cfus and the MF for each tissue and animal are reported. Statistical tests should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose–response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is a statistically non-significant change, with all mean MFs within two standard deviations of the control. During the current workshop, a general protocol was agreed in which animals are treated daily for 28 consecutive days and tissues sampled 3 days after the final treatment. This recommendation could be modified by reducing or increasing the number of treatments or the length of the treatment period, when scientifically justified. Normally male animals alone are sufficient and normally at least one rapidly proliferating and one slowly proliferating tissue should be sampled. Although, as agreed previously, sequencing data are not normally required, they might provide useful additional information in specific circumstances, mainly to identify and correct for clonal expansion and in some cases to determine a mechanism associated with a positive response.     

Light-evoked somatosensory perception of transgenic rats that express channelrhodopsin-2 in dorsal root ganglion cells

In vertebrate somatosensory systems, each mode of touch-pressure, temperature or pain is sensed by sensory endings of different dorsal root ganglion (DRG) neurons, which conducted to the specific cortical loci as nerve impulses. Therefore, direct electrical stimulation of the peripheral nerve endings causes an erroneous sensation to be conducted by the nerve. We have recently generated several transgenic lines of rat in which channelrhodopsin-2 (ChR2) transgene is driven by the Thy-1.2 promoter. In one of them, W-TChR2V4, some neurons were endowed with photosensitivity by the introduction of the ChR2 gene, coding an algal photoreceptor molecule. The DRG neurons expressing ChR2 were immunohistochemically identified using specific antibodies to the markers of mechanoreceptive or nociceptive neurons. Their peripheral nerve endings in the plantar skin as well as the central endings in the spinal cord were also examined. We identified that ChR2 is expressed in a certain population of large neurons in the DRG of W-TChR2V4. On the basis of their morphology and molecular markers, these neurons were classified as mechanoreceptive but not nociceptive. ChR2 was also distributed in their peripheral sensory nerve endings, some of which were closely associated with CK20-positive cells to form Merkel cell-neurite complexes or with S-100-positive cells to form structures like Meissner's corpuscles. These nerve endings are thus suggested to be involved in the sensing of touch. Each W-TChR2V4 rat showed a sensory-evoked behavior in response to blue LED flashes on the plantar skin. It is thus suggested that each rat acquired an unusual sensory modality of sensing blue light through the skin as touch-pressure. This light-evoked somatosensory perception should facilitate study of how the complex tactile sense emerges in the brain.     

Enhanced shutoff of phototransduction in transgenic mice expressing palmitoylation-deficient rhodopsin

Palmitoylation is a reversible, post-translational modification observed in a number of G-protein-coupled receptors. To gain a better understanding of its role in visual transduction, we produced transgenic knock-in mice that expressed a palmitoylation-deficient rhodopsin (Palm(-/-)). The mutant rhodopsin was expressed at wild-type levels and showed normal cellular localization to rod outer segments, indicating that neither rhodopsin stability nor its intracellular trafficking were compromised. But Palm(-/-) rods had briefer flash responses and reduced sensitivity to flashes and to steps of light. Upon exposure to light, rhodopsin became phosphorylated at a faster rate in mutant than in wild-type retinas. Since quench of rhodopsin begins with its phosphorylation, these results suggest that palmitoylation may modulate rod photoreceptor sensitivity by permitting rhodopsin to remain active for a longer period.     

Aberrant phenotypes of transgenic mice expressing dimeric human erythropoietin

Background  

Dimeric human erythropoietin (dHuEPO) peptides are reported to exhibit significantly higher biological activity than the monomeric form of recombinant EPO. The objective of this study was to produce transgenic (tg) mice expressing dHuEPO and to investigate the characteristics of these mice.     

In vivo hyperoxic preconditioning prevents myocardial infarction by expressing bcl-2

Preconditioning with oxidative stress has been demonstrated in vitro to stimulate the cellular adaptation to subsequent severe oxidative stress. However, it is uncertain whether this preconditioning works in vivo. In the present study, we examined in vivo the beneficial effect of oxidative preconditioning. After rats were pretreated with whole-body hyperoxygenation (100% O(2) at 3 atmosphere for 20 mins, four cycles with 20-min intermission), isolated hearts were subjected to 45-min ischemia followed by 90-min reperfusion. This hyperoxic preconditioning significantly reduced infarct size, cytochrome-c release, DNA fragmentation, and terminal deoxynucleotidyl transferase-mediated dUTD nick-end labeling-positive cell frequency in the left ventricle, biphasically with an early (30-min) and a delayed (48-hr) effect after the hyperoxygenation. Mechanistically, the NF-kappaB activity and Bcl-2 expression were enhanced in the hearts, and a NF-kappaB inhibitor, pyrrolidine dithiocarbamate, abolished the Bcl-2 induction as well as the infarct-limiting effect. An antioxidant, N-acetylcysteine, and protein kinase C (PKC) inhibitors chelerythrine and G? 6983 also blocked the preconditioning effects. These results indicate that hyperoxia induces myocardial tolerance against ischemia-reperfusion injury in association with Bcl-2 induction by NF-kappaB activation through reactive oxygen species and PKC-dependent signaling pathway.     

Experimental Helicobacter felis infection in transgenic mice expressing human group IIA phospholipase A2

BACKGROUND: Both various virulence factors of Helicobacter pylori and host factors influence the clinical outcome of H. pylori infection. In animal experiments with Helicobacter felis, large variations in the severity of disease have been observed between different mouse strains infected with a single isolate of H. felis. C57BL/6 J mouse strain that lacks the expression of group IIA phospholipase A2 has been shown to develop more severe gastric inflammation than other mouse strains. Thus, group IIA phospholipase A2 has been suggested to play a role in regulating inflammation in gastric mucosa. The aim of this study was to examine the possible role of group IIA phospholipase A2 in experimental Helicobacter infection. MATERIALS AND METHODS: Transgenic mice expressing human group IIA phospholipase A2 and their group IIA phospholipase A2 deficient nontransgenic C57BL/6 J littermates were infected with H. felis. The mice were killed 3, 8, and 19 weeks after inoculation of bacteria to determine the histopathological changes in gastric mucosa. RESULTS: The infected mice developed chronic inflammation in gastric mucosa. We found no differences in the colonization of bacteria between transgenic and nontransgenic mice. At 3 and 8 weeks, no difference was found in the severity of inflammation between the two groups. Nineteen weeks after the administration of bacteria the inflammation was more marked in nontransgenic than transgenic mice. Group IIA phospholipase A2 was expressed by in situ hybridization in the neck cells of the glandular stomach in transgenic mice. CONCLUSIONS: The results of the present study suggest that the endogenous expression of group IIA phospholipase A2 diminishes chronic inflammation in gastric mucosa in experimental H. felis infection in mice.     

绿色荧光蛋白转基因小鼠模型的建立及胚胎冷冻保种

目的建立绿色荧光蛋白转基因小鼠模型,并采取胚胎冷冻的方法进行保种。方法通过原核显微注射法,把线性化、纯化后的外源基因pEGFP注射入BDF1小鼠受精卵中,胚胎移植给同期发情的假孕受体母鼠,获得子代小鼠。经鉴定对有表达的转基因鼠进行胚胎冷冻保种。结果移植注射胚胎385枚给30只假孕小鼠共出生了306只后代鼠,经PCR和southern blot检测得到5只阳性小鼠。F2代转基因鼠胚胎冷冻240枚胚胎。结论通过显微注射法使外源基因pEGFP在小鼠基因组中得到整合,建立了转pEGFP的转基因小鼠模型。     

In vivo selection of neutralization-resistant virus variants but no evidence of B cell tolerance in lymphocytic choriomeningitis virus carrier mice expressing a transgenic virus-neutralizing antibody

B cell tolerance is maintained by active deletion and functional anergy of self-reactive B cells depending on the time, amount, and site of the self-antigen expression. To study B cell tolerance toward a transplacentally transmitted viral Ag, we crossed transgenic mice expressing the mu heavy and the kappa light chain of the lymphocytic choriomeningitis virus (LCMV)-neutralizing mAb KL25 (HL25-transgenic mice) with persistently infected LCMV carrier mice. Although HL25-transgenic LCMV carrier mice exhibited the same high virus titers as nontransgenic LCMV carrier mice, no evidence for B cell tolerance was found. In contrast, enhanced LCMV-neutralizing Ab titers were measured that, however, did not clear the virus. Instead, LCMV isolates from different tissues turned out to be neutralization resistant Ab escape variants expressing different substitutions of amino acid Asn119 of the LCMV-glycoprotein 1 that displays the neutralizing B cell epitope. Virus variants with the same mutations were also selected in vitro in the presence of the transgenic mAb KL25 confirming that substitutions of Asn119 have been selected by LCMV-neutralizing Abs. Thus, despite abundant expression of viral neo-self-antigen in HL25-transgenic LCMV carrier mice, transgenic B cells expressing LCMV-neutralizing Abs were rather stimulated than tolerized and neutralization resistant Ab escape variants were selected in vivo.     

Diabetes and tumor formation in transgenic mice expressing Reg I

To examine the effect of overexpressed regenerating gene (Reg) I on pancreatic beta-cells, we generated transgenic mice expressing Reg I in islets (Reg-Tg mice). Three lines of Reg-Tg mice were established. In line-1 Reg-Tg mice, the expression level of Reg I mRNA in islets was 7 times higher than those in lines 2 and 3 of Reg-Tg mice, and line 1 mice developed diabetes by apoptosis of beta-cells, as well as various malignant tumors. In addition to the decrease in beta-cells, compensatory islet regeneration and proliferation of ductal epithelial cells were observed in line-1 Reg-Tg mice. Because Reg I protein was secreted primarily into pancreatic ducts from acinar cells, it may primarily stimulate the proliferation of ductal epithelial cells, and not beta-cells, and their differentiation into islets. Moreover, the tumor-promoting activity of Reg I protein should be considered for its possible clinical applications.     

Neonatal bleeding in transgenic mice expressing urokinase-type plasminogen activator.

Spontaneous intestinal and intra-abdominal bleeding was observed in a high percentage of newborn transgenic mice carrying the murine urokinase-type plasminogen activator (uPA) gene linked to the albumin enhancer/promoter. These hemorrhagic events were directly related to transgene expression in the liver and the development of high plasma uPA levels. Two lines were established from surviving founder mice that displayed multigenerational transmission of the bleeding phenotype. Fatal hemorrhaging developed between 3 and 84 hr after birth in about half of the transgenic offspring of these lines; transgenic pups that did not bleed nevertheless passed the phenotype to their young. The phenotypic variability could not be explained by differences in transgene expression. All transgenic neonates were severely hypofibrinogenemic and displayed loss of clotting function that extended beyond the risk period for bleeding. These mice provide a means of studying the pathophysiology of plasminogen hyperactivation and evaluating therapeutic protocols designed to prevent bleeding.     

Dose-dependent lung remodeling in transgenic mice expressing transforming growth factor-alpha

Transgenic mice overexpressing human transforming growth factor-alpha (TGF-alpha) develop emphysema and fibrosis during postnatal alveologenesis. To assess dose-related pulmonary alterations, four distinct transgenic lines expressing different amounts of TGF-alpha in the distal lung under control of the surfactant protein C (SP-C) promoter were characterized. Mean lung homogenate TGF-alpha levels ranged from 388 +/- 40 pg/ml in the lowest expressing line to 1,247 +/- 33 pg/ml in the highest expressing line. Histological assessment demonstrated progressive alveolar airspace size changes that were more severe in the higher expressing TGF-alpha lines. Pleural and parenchymal fibrosis were only detected in the highest expressing line (line 28), and increasing terminal airspace area was associated with increasing TGF-alpha expression. Hysteresis on pressure-volume curves was significantly reduced in line 28 mice compared with other lines of mice. There were no differences in bronchoalveolar lavage fluid cell count or differential that would indicate any evidence of lung inflammation among all transgenic lines. Proliferating cells were increased in line 28 without alterations of numbers of type II cells. We conclude that TGF-alpha lung remodeling in transgenic mice is dose dependent and is independent of pulmonary inflammation.