Transgenic mice expressing a fluorescent in vivo label in a distinct subpopulation of neocortical layer 5 pyramidal cells

The neuronal components of cortical circuits have been characterized on the basis of their morphological and functional properties, and further refined by correlation of marker proteins with particular cell types. This latter approach has been very fruitful for GABA-containing neurons, but comparable diagnostic markers for subpopulations of excitatory pyramidal cells have been more elusive. An emerging new approach consists of transgenic mice that express fluorescent proteins under the control of promoters that are active in specific cell types. Here, we analyzed a line of transgenic mice that carries a transgene consisting of regulatory sequences of the potassium channel Kv3.1 and enhanced yellow fluorescent protein (EYFP). In these mice, a set of neurons in neocortical layer 5 expresses high levels of the transgenic marker protein. EYFP-expressing, and nonexpressing layer 5 cells were easily identified in living tissue under conditions suitable for patch-clamp electrophysiology. By using immunolabeling, retrograde Fast Blue labeling and electrophysiological recordings with biocytin injections, we identified the fluorescent neurons as a population of pyramidal cells with distinct morphological and electrophysiological properties when compared with nonfluorescent neighboring layer 5 pyramidal cells. The most prominent morphological difference between these two populations was a much smaller number of apical oblique dendrites in EYFP-positive as compared with the EYFP-negative cells. The most prominent electrophysiological feature was a steady spike frequency adaptation in EYFP-positive cells, whereas EYFP-negative cells responded to a depolarizing current injection with a closely spaced spike doublet followed by constant frequency firing. The in vivo labeled transgenic mice provide an experimental tool for further functional differentiation of these populations of layer 5 pyramidal cells.     

Transgenic mice expressing the betaAPP695SWE mutation: effects on exploratory activity,anxiety, and motor coordination

The functional consequences of the betaAPP transgene with the Swedish mutation in mice were assessed in tests of exploratory activity and motor coordination. The betaAPP(695)SWE (Tg2576) transgenic mice are characterized by Abeta plaque formation in the neocortex and hippocampus. By comparison to non-transgenic mice controlled for age and gender, 17-month-old betaAPP(695)SWE transgenic mice displayed impaired spontaneous alternation, increased activity levels in the peripheral part of the open-field, and reduced anxiety in the elevated plus-maze. These results are similar to the loss of inhibitory control observed in some patients with Alzheimer's disease. These measures may be added to cognitive dysfunctions as testing ground for Abeta vaccination and other attempts at experimental therapies.     

Transgenic mice expressing the PS1-A246E mutation: effects on spatial learning,exploration, anxiety,and motor coordination

The functional consequence of the PS1-A246E mutation was assessed in transgenic mice on a background lacking the endogenous PS1 gene. These mice have elevated concentrations of A-beta protein (Abeta(42)) in the absence of plaque formation. By comparison to a mixed background strain (50% B6, 25% SJl, 25% 129Sv) controlled for age and gender, PS1-A246E transgenic mice displayed disinhibitory tendencies, as indicated by increased entries and duration in the open arms of the elevated plus-maze. Despite normal spontaneous alternation rates in a T-maze, latencies before responding were higher in PS1-A246E transgenic mice than controls. Moreover, the PS1-A246E transgenic mice fell more often from two stationary beams, but not from the coat-hanger and the rotorod. By contrast, ambulation in an automated photocell chamber and in an open-field was not affected. Nor was acquisition of place learning in the Morris water maze task. These results indicate that elevated Abeta(42) levels were insufficient for causing spatial defects but caused disinhibition, psychomotor slowing, and loss of motor skills in this model of familial Alzheimer's disease.     

Retrograde amnesia and brain seizure activity in mice: Strain differences

The relationship between retrograde amnesia and brain seizures was examined in two inbred strains of mice, $\text{DBA}\text{2J}$ and $\text{C57BL}\text{6J}$, and their F₁ hybrids. Mice were treated with transcorneal electroshock stimulation 45 sec after training on an inhibitory avoidance task. The mice were either anesthetized with diethyl ether or unanesthetized prior to electroshock administration. In unetherized mice of all strains, the electroshock intensities required to produce retrograde amnesia were comparable to those necessary to elicit electrographic seizures. A similar relationship was seen in F₁ hybrids etherized prior to electroshock, in spite of the fact that both brain seizure and retrograde amnesia thresholds were elevated by diethyl ether. In etherized mice of the $\text{DBA}\text{2J}$ and $\text{C57BL}\text{6J}$ strains, the thresholds for retrograde amnesia were much higher than the brainseizure thresholds. These data indicate that brain seizures are not always a sufficient condition for producing retrograde amnesia.     

Transgenic mice expressing mutant A53T human alpha-synuclein show neuronal dysfunction in the absence of aggregate formation

Alpha-synuclein was implicated in Parkinson's disease when missense mutations in the alpha-synuclein gene were found in autosomal dominant Parkinson's disease and alpha-synuclein was shown to be a major constituent of protein aggregates in sporadic Parkinson's disease and other synucleinopathies. We have generated transgenic mice expressing A53T mutant and wild-type human alpha-synuclein. The mutant transgenic protein was distributed abnormally to the axons, perikarya, and dendrites of neurons in many brain areas. In electron microscopic immunogold studies, no aggregation of alpha-synuclein was found in these mice. However, behavior analysis showed a progressive reduction of spontaneous vertical motor activity in both mutant lines correlating with the dosage of overexpression. In addition, deficits of grip strength, rotarod performance, and gait were observed in homozygous PrPmtB mice. Transgenic animals expressing mutant alpha-synuclein may be a valuable model to assess specific aspects of the pathogenesis of synucleinopathies.     

Transgenic mice expressing a pH and Cl- sensing yellow-fluorescent protein under the control of a potassium channel promoter

During the last few years a variety of genetically encodable optical probes that monitor physiological parameters such as local pH, Ca2+, Cl-, or transmembrane voltage have been developed. These sensors are based on variants of green-fluorescent protein (GFP) and can be synthesized by mammalian cells after transfection with cDNA. To use these sensor proteins in intact brain tissue, specific promoters are needed that drive protein expression at a sufficiently high expression level in distinct neuronal subpopulations. Here we investigated whether the promoter sequence of a particular potassium channel may be useful for this purpose. We produced transgenic mouse lines carrying the gene for enhanced yellow-fluorescent protein (EYFP), a yellow-green pH- and Cl- sensitive variant of GFP, under control of the Kv3.1 K+ channel promoter (pKv3.1). Transgenic mouse lines displayed high levels of EYFP expression, identified by confocal microscopy, in adult cerebellar granule cells, interneurons of the cerebral cortex, and in neurons of hippocampus and thalamus. Furthermore, using living cerebellar slices we demonstrate that expression levels of EYFP are sufficient to report intracellular pH and Cl- concentration using imaging techniques and conditions analogous to those used with conventional ion-sensitive dyes. We conclude that transgenic mice expressing GFP-derived sensors under the control of cell-type specific promoters, provide a unique opportunity for functional characterization of defined subsets of neurons.     

Transgenic mice expressing HIV-1 envelope protein gp120 in the brain as an animal model in neuroAIDS research

HIV-1 infection causes injury to the central nervous system (CNS) and is often associated with neurocognitive disorders. One model for brain damage seen in AIDS patients is the transgenic (tg) mouse expressing a soluble envelope protein gp120 of HIV-1 LAV in the brain in astrocytes under the control of the promoter of glial fibrillary acidic protein. These GFAP-gp120tg mice manifest several key neuropathological features observed in AIDS brains, such as decreased synaptic and dendritic density, increased numbers of activated microglia, and pronounced astrocytosis. Several recent studies show that brains of GFAP-gp120tg mice and neurocognitively impaired HIV patients share also a significant number of differentially regulated genes, activation of innate immunity and other cellular signaling pathways, disturbed neurogenesis, and learning deficits. These findings support the continued relevance of the GFAP-gp120tg mouse as a useful model to investigate neurodegenerative mechanisms and develop therapeutic strategies to mitigate the consequences associated with HIV infection of the CNS, neuroAIDS, and HAND.     

Two neuronal cell lines expressing the myelin basic protein gene display differences in their in vitro survival and in their response to glia

We have generated two conditionally immortalized neuronal cell lines from primary cultures of embryonic day 13 (E13) and postmitotic (postnatal day 0; P0) cortical neurons transformed with the temperature-sensitive SV-40 large-T antigen. Two clonal cell lines (CN1.4 from E13 cultures and SJ3.6 from P0 cultures) were isolated and stable maintained in vitro. Both cell lines expressed a number of neuronal markers such as the neurofilaments, glutamic acid decarboxylase 67, neuron-specific enolase, and the BG21 isoform of the myelin basic protein gene. At 34°C, the CN1.4 cell line had elaborated short processes, whereas the SJ3.6 cell line produced long processes that formed a delicate network. When these cell lines were cultured at 39°C, some of the cellular processes grew longer, adopting a more mature neuronal morphology. Interestingly, at 39°C, the in vitro survival of these cell lines differed significantly. Whereas the survival of CN1.4 cell line was greatly unaffected, SJ3.6 cells died soon after they were cultured at 39°C. The cell death of SJ3.6 cells was accompanied by fragmentation and condensation of DNA in their nuclei, indicative of an apoptotic event. Under these conditions, SJ3.6 showed an upregulation of the p75 receptor. When this cell line was cocultured with oligodendrocytes, astrocytes, or glial conditioned media (GCM), there was a marked increase in survival. In contrast, little effect of glial cells or GCM was observed on the CN1.4 cell line. These lines appear to be useful models to study neuronal–glial interactions in addition to neuronal cell death and the effects of glial factors that promote the survival of neurons. J. Neurosci. Res. 54:309–319, 1998. © 1998 Wiley-Liss, Inc.     

Nerve growth factor promoter activity revealed in mice expressing enhanced green fluorescent protein

Nerve growth factor (NGF) and its precursor proNGF are perhaps the best described growth factors of the mammalian nervous system. There remains, however, a paucity of information regarding the precise cellular sites of proNGF/NGF synthesis. Here we report the generation of transgenic mice in which the NGF promoter controls the ectopic synthesis of enhanced green fluorescent protein (EGFP). These transgenic mice provide an unprecedented resolution of both neural cells (e.g., neocortical and hippocampal neurons) and non-neural cells (e.g., renal interstitial cells and thymic reticular cells) that display NGF promoter activity from postnatal development to adulthood. Moreover, the transgene is inducible by injury. At 2 days after sciatic nerve ligation, a robust population of EGFP-positive cells is seen in the proximal nerve stump. These transgenic mice offer novel insights into the cellular sites of NGF promoter activity and can be used as models for investigating the regulation of proNGF/NGF expression after injury.     

Stress-induced opioid analgesia and activity in deer mice: sex and population differences

We compared restraint stress-induced opioid, analgesic and locomotory responses of 4 different populations of male and female deer mice, Peromyscus maniculatus artemisiae and P. m. nebrascensis from mainlands, and P. m. angustus and P. m. triangularis from small islands. All of the deer mice displayed immobilization-induced analgesia which was blocked by the prototypical mu-opiate antagonist, naloxone (1.0 mg/kg). In all of the populations males displayed significantly greater levels of analgesia than females. In addition, the levels of opioid-induced analgesia were significantly greater in the insular than in the mainland male and female deer mice. Restraint also induced significant increases in the locomotor activity of the mainland deer mice, while significantly decreasing the activity of the insular animals. Males displayed significantly greater stress-induced changes in locomotor activity than did females. The stress-induced increases in activity were blocked by the delta-opiate antagonist, ICI 154, 129 (10 mg/kg), while the decreases in activity were inhibited by naloxone. These results demonstrate that there are marked sex and population differences in the stress-induced, opioid-mediated responses of deer mice. These 'pharmaco-ecological' findings also suggest that the island-mainland population differences in behavioral responses and ecological characteristics may, in part, be related to differences in the activity of mu-, delta- and possibly other opioid systems.     

Transgenic mice ectopically expressing HOXA5 in the dorsal spinal cord show structural defects of the cervical spinal cord along with sensory and motor defects of the forelimb

Mutation of murine Hoxa5 has shown that HOXA5 controls lung, gastrointestinal tract and vertebrae development. Hoxa5 is also expressed in the spinal cord, yet no central nervous system phenotype has been described in Hoxa5 knockouts. To identify the role of Hoxa5 in spinal cord development, we developed transgenic mice that express HOXA5 in the dorsal spinal cord in the brachial region. Using HOXA5-specific antibodies, we show this expression pattern is ectopic as the endogenous protein is expressed only in the ventral spinal cord at this anterio-posterior level. This transgenic line (Hoxa5SV2) also displays forelimb-specific motor and sensory defects. Hoxa5SV2 transgenic mice cannot support their body weight in a forelimb hang, and forelimb strength is decreased. However, Rotarod performance was not impaired in Hoxa5SV2 mice. Hoxa5SV2 mice also show a delayed forelimb response to noxious heat, although hindlimb response time was normal. Administration of an analgesic significantly reduced the hang test defect and decreased the transgene effect on forelimb strength, indicating that pain pathways may be affected. The morphology of transgenic cervical (but not lumbar) spinal cord is highly aberrant. Nissl staining indicates superficial laminae of the dorsal horn are severely disrupted. The distribution of cells and axons immunoreactive for substance P, neurokinin-B, and their primary receptors were aberrant only in transgenic cervical spinal cord. Further, we see increased levels of apoptosis in transgenic spinal cord at embryonic day 13.5. Our evidence suggests apoptosis due to HOXA5 misexpression is a major cause of loss of superficial lamina cells in Hoxa5SV2 mice.     

WAR-5、Fasudil治疗EAE的疗效比较及作用机制研究

目的比较WAR-5与盐酸法舒地尔(Fasudil)对实验性自身免疫性脑脊髓炎(EAE)的治疗效果,并探究其作用机制。方法采用小鼠髓鞘少突胶质细胞糖蛋白35-55多肽(MOG35-55)免疫雌性C57BL/6小鼠建立慢性EAE模型,并随机分为EAE对照组、WAR-5治疗组和Fasudil治疗组。于免疫后第3天开始,分别腹腔注射WAR-5、Fasudil及生理盐水,比较各组小鼠体质量变化和临床评分。于免疫后第28天处死各组动物,制作脊髓冰冻切片进行CD4+T细胞、CD68+巨噬细胞及核转录因子-κB(p-NF-κB/p65)染色,观察其在脊髓中的表达。提取脑组织的蛋白用Western blot法检测Rho相关卷曲螺旋蛋白激酶(ROCK)Ⅱ和p-NF-κB/p65蛋白表达,并采用Griess法检测脾细胞培养上清液的一氧化氮(NO)释放水平。结果与EAE对照组(108.70±20.26、399.00±25.94)比较,WAR-5治疗组(13.67±4.041、52.67±13.43)及Fasudil治疗组(15.33±5.033、31.00±13.45)脊髓内CD4+T细胞和CD68+巨噬细胞数目均减少(均P0.01),WAR-5与Fasudil两治疗组相比,差异无统计学意义。与EAE对照组(1.9500±0.3976、0.5885±0.05982)比较,WAR-5组(0.6137±0.1778、0.0096±0.0015)及Fasudil组(1.1960±0.2225、0.3574±0.1496)脑内ROCKⅡ、p-NF-κB/p65表达均降低,WAR-5治疗组脑内ROCKⅡ、p-NF-κB/p65表达亦均低于Fasudil治疗组(均P0.05)。WAR-5和Fasudil治疗组上清液NO含量分别为(16.40±0.5247)μmol/L、(17.69±0.2833)μmol/L,均低于EAE对照组〔(20.07±0.6116)μmol/L;均P0.001〕,且WAR-5治疗组低于Fasudil治疗组(P0.01)。与Fasudil治疗组比较,800μg及1600μg药物注射后,WAR-5治疗组小鼠的存活状态明显较好;800μg药物注射后,WAR-5治疗组小鼠的脚趾无明显血管扩张反应。结论 WAR-5对EAE的治疗效果与Fasudil相近,且其治疗安全性较好,扩张血管作用较小,其作用机制可能与WAR-5抑制小鼠ROCKⅡ的表达及抑制炎性反应相关。     

线粒体复合物及ATP酶活性在不同月龄帕金森病模型小鼠的差异

目的探讨不同月龄帕金森病(PD)模型小鼠线粒体呼吸功能的差异。方法选用8个月龄、16个月龄雌性C57BL/6小鼠,随机分为8个月龄对照组、8个月龄模型组、16个月龄对照组、16个月龄模型组。模型组小鼠皮下注射MPTP制成PD模型,对照组注射等量生理盐水;进行行为学测试,并检测其黑质多巴胺含量,采用紫外分光吸光度法检测线粒体复合物Ⅰ~Ⅳ的活性,采用荧光素酶发光法检测ATP的合成活力。结果与对照组相比,模型组小鼠行为学成绩降低,黑质多巴胺含量显著下降(P0.05),线粒体复合物Ⅰ的活性明显低于对照组(P0.05),ATP合成活力降低;与8个月龄PD小鼠相比,16个月龄小鼠行为学成绩、多巴胺含量及线粒体复合物Ⅰ的活性及ATP合成活力降低更明显(P0.05)。各组线粒体复合物Ⅱ、Ⅲ、Ⅳ差异无统计学意义。结论小鼠黑质纹状体系统线粒体呼吸功能减弱可能是导致PD发生的重要原因之一,老龄可加速其功能障碍。     

Components of the GABA system in mice selectively bred for differences in open-field activity

Mice selectively bred for differences in open-field activity were utilized to test the hypothesis that differences in open-field behaviour are mediated at least in part by components of the GABA system, including brain glutamic acid decar☐ylase activity (V, V_max, andK_m), as well as glutamate and GABA concentrations. Enzyme velocity was found to be inversely correlated with open-field activity, but it accounted for less than 15% of the variance. Moreover, the rank order of the lines was not as predicted for a genetically correlated character and the magnitude of the differences in enzyme activity among the lines was not large. It was therefore concluded that the GABA system is not an important mediator of differences in open-field behaviour in these lines of mice.     

Spontaneous alternation and exploration in weaver mutant mice

Weaver mutant mice engaged less in motor activity and hole poking. Weaver mice alternated spontaneously in a 4-trial but not a 2-trial test. These results are similar to those of a previous study with staggerer mutants, in that both are less active. However, weaver mutants are less affected in the spontaneous alternation measure.     

Spontaneous alternation and exploration in staggerer mutant mice

Staggerer mutant mice were found to be less active in terms of motor activity in a maze, hole poking and rearing. Staggerer mutant mice also showed a lack of spontaneous alternation in both 2-trial and 4-trial tests. A lack of spontaneous alternation in this mutant may be due to a deficit in response inhibition or in spatial orientation, similar to that of animals with limbic lesions.     

Altered N-methyl-D-aspartate receptor function in reelin heterozygous mice: male-female differences and comparison with dopaminergic activity

The aim of this study was to investigate the in vivo relationship between reelin and NMDA receptor function in schizophrenia. We assessed the effect of reelin deficiency in behavioral models of aspects of this illness, NMDA receptor subunit levels, and NMDA receptor, dopamine D₂ receptor, and dopamine transporter density. Male, but not female, reelin heterozygous mice showed significantly enhanced MK-801-induced locomotor hyperactivity compared to wildtype controls (7.4-fold vs. 5.2-fold effect of MK-801 over saline, respectively) but there were no genotype differences in the response to amphetamine. Both male and female reelin heterozygous mice showed enhanced effects of MK-801 on startle, but not prepulse inhibition (PPI) of startle. There were no group differences in the effect of apomorphine on startle or PPI. The levels of NMDA receptor subunits were not altered in the striatum. In the frontal cortex, male and female reelin heterozygous mice showed significant up-regulation of NR1 subunits, but down-regulation of NR2C subunits, which was associated with significantly elevated NR1/NR2A and NR1/NR2C ratios. However, there were no differences in [³H]MK-801 binding density in the nucleus accumbens or caudate nucleus, nor in the density of [³H]YM-09151 or [³H]GBR12935 in these brain regions. The enhanced effects of MK-801 in reelin heterozygous mice in this study could be reflective of the role of reelin deficiency in schizophrenia. This genotype effect was male-specific for locomotor hyperactivity, a model of psychosis, but was seen in male and female mice for startle, which could be an indication of changes in anxiety. Changes in NMDA receptor subunit levels and ratios were also seen in both male and female mice. These results suggest that the role of reelin deficiency in schizophrenia may be particularly mediated by altered NMDA receptor responses, with some of these effects being strictly sex-specific.     

Ghrelin receptor‐knockout mice display alterations in circadian rhythms of activity and feeding under constant lighting conditions

Ghrelin is an orexigenic hormone produced by the stomach. Ghrelin, however, may also be a modulator of the circadian system given that ghrelin receptors are expressed in the master clock, the suprachiasmatic nucleus (SCN) and several outputs of this region. To investigate this, we performed analyses of running wheel activity and neuronal activation in wild type (WT) and growth hormone secretagogue receptor‐knockout (GHSR‐KO) mice under various lighting conditions. GHSR‐KO and WT mice were maintained under constant dark (DD) or constant light (LL) with ad libitum access to food before being placed on a schedule of temporally restricted access to food (4 h/day) for 2 weeks. There were no differences between KO and WT mice in free‐running period under DD, but GHSR‐KO mice required more days to develop a high level of food anticipatory activity, and this was lower than that observed in WT mice. Under LL, GHSR‐KO mice showed greater activity overall, lengthening of their circadian period, and more resistance to the disorganisational effects of LL. Furthermore, GHSR‐KO mice showed greater activity overall, and greater activity in anticipation of a scheduled meal under LL. These behavioral effects were not correlated with changes in the circadian expression of the Fos, Per1 or Per2 proteins under any lighting conditions. These results suggest that the ghrelin receptor plays a role in modulating the activity of the circadian system under normal conditions and under restricted feeding schedules, but does so through mechanisms that remain to be determined.     

Effects of subacute administration of methamphetamine and nicotine on locomotor activity in transgenic mice expressing the human tyrosine hydroxylase gene

Summary We produced transgenic (Tg) mice carrying the human tyrosine hydroxylase (TH) gene. To investigate differences in the dopaminergic (DAergic) neuronal activity between the Tg and nTg mice, we examined changes in the locomotor activity induced by methamphetamine (MAP) and nicotine (NIC), which enhances DA release and induces TH enzyme activation, respectively. Surprisingly, however, the intensity of MAP (2.5 mg/kg, once a day for 14 days)-induced hyperlocomotion in the nTg mice was greater than that in the Tg mice, and, furthermore, the Tg mice were less sensitive to subacute administration of NIC (0.5 mg/kg, once a day for 14 days) than the nTg mice. These results suggest that DAergic neuronal function is suppressed in Tg mice to compensate for the overexpression of TH.