Major role for tonic GABAA conductances in anesthetic suppression of intrinsic neuronal excitability

Anesthetics appear to produce neurodepression by altering synaptic transmission and/or intrinsic neuronal excitability. Propofol, a widely used anesthetic, has proposed effects on many targets, ranging from sodium channels to GABA(A) inhibition. We examined effects of propofol on the intrinsic excitability of hippocampal CA1 neurons (primarily interneurons) recorded from adult rat brain slices. Propofol strongly depressed action potential production induced by DC injection, synaptic stimulation, or high-potassium solutions. Propofol-induced depression of intrinsic excitability was completely reversed by bicuculline and picrotoxin but was strychnine-insensitive, implicating GABA(A) but not glycine receptors. Propofol strongly enhanced inhibitory postsynaptic currents (IPSCs) and induced a tonic GABA(A)-mediated current. We pharmacologically differentiated tonic and phasic (synaptic) GABA(A)-mediated inhibition using the GABA(A) receptor antagonist SR95531 (gabazine). Gabazine (20 microM) completely blocked both evoked and spontaneous IPSCs but failed to block the propofol-induced depression of intrinsic excitability, implicating tonic, but not phasic, GABA(A) inhibition. Glutamatergic synaptic responses were not altered by propofol (< or =30 microM). Similar results were found in both interneurons and pyramidal cells and with the chemically unrelated anesthetic thiopental. These results suggest that suppression of CA1 neuron intrinsic excitability, by these anesthetics, is largely due to activation of tonic GABA(A) conductances; although other sites of action may play important roles in affecting synaptic transmission, which also can produce strong neurodepression. We propose that for some anesthetics, suppression of intrinsic excitability, mediated by tonic GABA(A) conductances, operates in conjunction with effects on synaptic transmission, mediated by other mechanisms, to depress hippocampal function during anesthesia.     

Cholecystokinin facilitates neuronal excitability in the entorhinal cortex via activation of TRPC-like channels

Cholecystokinin (CCK) is one of the most abundant neuropeptides in the brain, where it interacts with two G protein-coupled receptors (CCK-1 and CCK-2). Activation of both CCK receptors increases the activity of PLC, resulting in increases in intracellular calcium ion (Ca(2+)) release and activation of PKC. Whereas high density of CCK receptors has been detected in the superficial layers of the entorhinal cortex (EC), the functions of CCK in this brain region have not been determined. Here, we studied the effects of CCK on neuronal excitability of layer III pyramidal neurons in the EC. Our results showed that CCK remarkably increased the firing frequency of action potentials (APs). The effects of CCK on neuronal excitability were mediated via activation of CCK-2 receptors and required the functions of G proteins and PLC. However, CCK-mediated facilitation of neuronal excitability was independent of inositol trisphosphate receptors and PKC. CCK facilitated neuronal excitability by activating a cationic channel to generate membrane depolarization. The effects of CCK were suppressed by the generic, nonselective cationic channel blockers, 2-aminoethyldiphenyl borate and flufenamic acid, but potentiated by gadolinium ion and lanthanum ion at 100 μM. Depletion of extracellular Ca(2+) also counteracted CCK-induced increases in AC firing frequency. Moreover, CCK-induced enhancement of neuronal excitability was inhibited significantly by intracellular application of the antibody to transient receptor potential channel 5 (TRPC5), suggesting the involvement of TRPC5 channels. Our results provide a cellular and molecular mechanism to help explain the functions of CCK in vivo.     

Gadolinium-staining reveals amyloid plaques in the brain of Alzheimer's transgenic mice

Detection of amyloid plaques in the brain by in vivo neuroimaging is a very promising biomarker approach for early diagnosis of Alzheimer's disease (AD) and evaluation of therapeutic efficacy. Here we describe a new method to detect amyloid plaques by in vivo magnetic resonance imaging (MRI) based on the intracerebroventricular injection of a nontargeted gadolinium (Gd)-based contrast agent, which rapidly diffuses throughout the brain and increases the signal and contrast of magnetic resonance (MR) images by shortening the T1 relaxation time. This gain in image sensitivity after in vitro and in vivo Gd staining significantly improves the detection and resolution of individual amyloid plaques in the cortex and hippocampus of AD transgenic mice. The improved image resolution is sensitive enough to demonstrate an age-dependent increase of amyloid plaque load and a good correlation between the amyloid load measured by μMRI and histology. These results provide the first demonstration that nontargeted Gd staining can enhance the detection of amyloid plaques to follow the progression of AD and to evaluate the activity of amyloid-lowering therapeutic strategies in longitudinal studies.     

Altered intrinsic neuronal excitability and reduced Na+ currents in a mouse model of Alzheimer's disease

Transgenic mice that overproduce beta-amyloid (Aβ) peptides can exhibit central nervous system network hyperactivity. Patch clamp measurements from CA1 pyramidal cells of PSAPP and wild type mice were employed to investigate if altered intrinsic excitability could contribute to such network hyperfunction. At approximately 10 months, when PSAPP mice have a substantial central nervous system Aβ load, resting potential and input resistance were genotype-independent. However, PSAPP mice exhibited a substantially more prominent action potential (AP) burst close to the onset of weak depolarizing current stimuli. The spike afterdepolarization (ADP) was also larger in PSAPP mice. The rate of rise, width and height of APs were reduced in PSAPP animals; AP threshold was unaltered. Voltage-clamp recordings from nucleated macropatches revealed that somatic Na⁺ current density was depressed by approximately 50% in PSAPP mice. K⁺ current density was unaltered. All genotype-related differences were absent in PSAPP mice aged 5-7 weeks which lack a substantial Aβ load. We conclude that intrinsic neuronal hyperexcitability and changes to AP waveforms may contribute to neurophysiological deficits that arise as a consequence of Aβ accumulation.     

Ontogeny of hyperactivity and circling behavior in a transgenic insertional mutant mouse.

In a previous study (Ratty et al., 1990), the discovery of a transgenic mutant mouse that displayed abnormal circling behavior was reported. Mice homozygous for the transgene display this phenotype, whereas heterozygotes are phenotypically normal. In this study, circling mutants displayed excessive lateralized circling behavior and locomotor hyperactivity by Postnatal Days (PND) 14 and 16, respectively. These abnormalities persisted unattenuated through adolescence and adulthood. Disturbances in rearing and grooming were also observed in circling mutants. Surface-righting ability in the mutants was normal, and the age of eye opening was only marginally delayed. However, body weights of the mutants were reduced compared with normal mice from PND 15 to adulthood. Possible relationships between the behaviors that are exhibited by circling mutants and previous neurochemical findings are discussed.     

Age-related functional changes of high-voltage-activated calcium channels in different neuronal subtypes of mouse striatum

By means of whole-cell patch-clamp recordings, we characterized the developmental profile of high-voltage-activated (HVA) calcium (Ca(2+)) channel subtypes in distinct neuronal populations of mouse striatum. Acutely dissociated medium spiny neurons (MSNs) and cholinergic interneurons (ChIs) were recorded from mice at five developmental stages: postnatal-days (PD) 14, 23, 40, 150 and 270. During ageing, total HVA Ca(2+) current recorded from both MSNs and ChIs was unchanged. However, the pharmacological analysis of the differential contribution of HVA Ca(2+) channel subtypes showed a significant rearrangement of each component. In both neuronal subtypes, a large fraction of the total HVA current recorded from PD14 mice was inhibited by the L-type HVA channel blocker nifedipine. This dihydropyridine-sensitive component accounted for nearly 50%, in MSNs, and 35%, in ChIs, of total current at PD14, but its contribution was down-regulated up to 20-25% at 9 months. Likewise, the N-type, omega-conotoxin GVIA-sensitive component decreased from 35% to 40% to about 25% in MSNs and 15% in ChIs. The P-type, omega-agatoxin-sensitive fraction did not show significant changes in both neuronal subtypes, whereas the Q-type, omega-conotoxin MVIIC-sensitive channels did show a significant up-regulation at 9 months. As compared with striatal neurons, we recorded pyramidal neurons dissociated from cortical layers IV-V and found no significant developmental change in the different components of HVA Ca(2+) currents. In conclusion, our data demonstrate a functional reconfiguration of HVA Ca(2+) channels in striatal but not cortical pyramidal neurons during mouse development. Such changes might have profound implications for physiological and pathophysiological processes of the striatum.     

Conditional recombination reveals distinct subsets of epithelial cells in trachea, bronchi, and alveoli

To identify relationships amongst tracheal and alveolar epithelial cells during lung development, we used conditional systems controlled by the rat CCSP and human SFTPC gene promoters to express Cre-recombinase in the developing mouse lung, thereby permanently labeling cells by expression of alkaline phosphatase or green fluorescent protein. When controlled by the rat CCSP promoter, continuous exposure of the fetus to doxycycline caused widespread recombination in conducting airway epithelial cells, including cells of the trachea, bronchi, and bronchioles before birth, and in both conducting and peripheral airways after birth. Neuroepithelial cells, identified by CGRP staining, were never labeled. Recombination and permanent labeling were observed in both ciliated and nonciliated respiratory epithelial cells, demonstrating their derivation from common progenitor cells during lung morphogenesis. Remarkable dorsal-ventral and cephalo-caudal labeling patterns, established before birth, were identified by recombination controlled by the rat CCSP gene promoter. In the trachea, subsets of epithelial cells labeled by the CCSP promoter were organized horizontally along the dorsal-ventral axis of the trachea, where selective labeling of cells juxtaposed to tracheal and bronchial cartilage was observed. In sharp contrast, recombination controlled by the human SFTPC gene promoter identified related cells that were organized in linear patterns along the cephalo-caudal axis of the conducting airways. Conditional expression of Cre-recombinase in the respiratory epithelium provides a useful model for the study of gene expression and function in the mouse respiratory tract and in the lung.     

Quantitative histological analysis of amyloid deposition in Alzheimer's double transgenic mouse brain

The development of transgenic mice has created new opportunities for the generation of animal models of human neurodegenerative diseases where previously there was no animal counterpart. The first successful transgenic mouse model of Alzheimer's disease expressed increased levels of mutant human amyloid precursor protein, exhibiting neuritic-type amyloid deposits and behavioral deficits at six to nine months of age. More recently, it was shown that transgenic mice expressing both mutant human amyloid precursor protein and presenilin 1 exhibit neuritic-type amyloid deposits and behavioral deficits in as little as 12 weeks. This accelerated Alzheimer phenotype greatly reduces the time necessary to conduct preclinical drug trials, as well as animal housing costs. The purpose of this study was to quantify the deposition of amyloid in five regions of the cortex and two regions of the hippocampus of transgenic mice expressing amyloid precursor protein (K670N, M671L) and presenilin 1 (M146L) mutations at various ages, using quantitative methods of confocal laser scanning microscopy and image analysis. Amyloid burden, expressed as the percentage area occupied by thioflavin S-positive amyloid deposits, increased an average of 179-fold from 12 to 54 weeks of age (0.02+/-0.01% to 3.57+/-0.29%, mean+/-S.E.M., respectively) in five regions of the cortex and two of the hippocampus. This was a function of increases in both deposit number and size.This transgenic mouse provides an ideal animal model for evaluating the efficacy of potential therapeutic agents aimed at reducing amyloid deposition, such as inhibitors of amyloid fibril formation or secretase inhibitors.     

Identification and characterization of inebriated, a gene affecting neuronal excitability in Drosophila.

On the basis of behavioral interactions with mutations in a potassium channel gene of Drosophila--Shaker (Sh)--we have isolated mutations in a new gene called inebriated (ine). In a wildtype background, ine mutants display no observable behavioral defects. However, in a Sh mutant background, ine mutations cause downturned wings and an indented thorax. This distinctive phenotype is also exhibited by flies of other genotypes that cause extreme neuronal hyperexcitability. We utilized the potassium channel blocking drugs quinidine and dideoxy forskolin (DDF) to test the effects of ine on synaptic transmission. DDF and ine mutations each potentiated the effects of quinidine on synaptic transmission, but neither had any observable effects in the absence of quinidine. Application of DDF to ine mutants had no effects either in the presence or absence of quinidine. We conclude that ine mutations increase neuronal membrane excitability and perhaps block a DDF-sensitive potassium channel.     

Treadmill exercise represses neuronal cell death in an aged transgenic mouse model of Alzheimer's disease

The present study was undertaken to further investigate the protective effect of treadmill exercise on the hippocampal proteins associated with neuronal cell death in an aged transgenic (Tg) mice with Alzheimer's disease (AD). To address this, Tg mouse model of AD, Tg-NSE/PS2m, which expresses human mutant PS2 in the brain, was chosen. Animals were subjected to treadmill exercise for 12 weeks from 24 months of age. The exercised mice were treadmill run at speed of 12 m/min, 60 min/day, 5 days/week on a 0% gradient for 3 months. Treadmill exercised mice improved cognitive function in water maze test. Treadmill exercised mice significantly reduced the expression of Aβ-42, Cox-2, and caspase-3 in the hippocampus. In parallel, treadmill exercised Tg mice decreased the phosphorylation levels of JNK, p38MAPK and tau (Ser404, Ser202, Thr231), and increased the phosphorylation levels of ERK, PI3K, Akt and GSK-3α/β. In addition, treadmill exercised Tg mice up-regulated the expressions of NGF, BDNF and phospho-CREB, and the expressions of SOD-1, SOD-2 and HSP-70. Treadmill exercised Tg mice up-regulated the expression of Bcl-2, and down-regulated the expressions of cytochrome c and Bax in the hippocampus. The number of TUNEL-positive cells in the hippocampus in mice was significantly decreased after treadmill exercise. Finally, serum TC, insulin, glucose, and corticosterone levels were significantly decreased in the Tg mice after treadmill exercise. As a consequence of such change, Aβ-dependent neuronal cell death in the hippocampus of Tg mice was markedly suppressed following treadmill exercise. These results strongly suggest that treadmill exercise provides a therapeutic potential to inhibit both Aβ-42 and neuronal death pathways. Therefore, treadmill exercise may be beneficial in prevention or treatment of AD.     

A systematic RNAi screen reveals involvement of endocytic pathway in neuronal dysfunction in alpha-synuclein transgenic C. elegans

Mutations or multiplications in alpha-synuclein gene cause familial forms of Parkinson disease or dementia with Lewy bodies (LB), and the deposition of wild-type alpha-synuclein as LB occurs as a hallmark lesion of these disorders, collectively referred to as synucleinopathies, implicating alpha-synuclein in the pathogenesis of synucleinopathy. To identify modifier genes of alpha-synuclein-induced neurotoxicity, we conducted an RNAi screen in transgenic C. elegans (Tg worms) that overexpress human alpha-synuclein in a pan-neuronal manner. To enhance the RNAi effect in neurons, we crossed alpha-synuclein Tg worms with an RNAi-enhanced mutant eri-1 strain. We tested RNAi of 1673 genes related to nervous system or synaptic functions, and identified 10 genes that, upon knockdown, caused severe growth/motor abnormalities selectively in alpha-synuclein Tg worms. Among these were four genes (i.e. apa-2, aps-2, eps-8 and rab-7) related to the endocytic pathway, including two subunits of AP-2 complex. Consistent with the results by RNAi, crossing alpha-synuclein Tg worms with an aps-2 mutant resulted in severe growth arrest and motor dysfunction. alpha-Synuclein Tg worms displayed a decreased touch sensitivity upon RNAi of genes involved in synaptic vesicle endocytosis, and they also showed impaired neuromuscular transmission, suggesting that overexpression of alpha-synuclein caused a failure in uptake or recycling of synaptic vesicles. Furthermore, knockdown of apa-2, an AP-2 subunit, caused an accumulation of phosphorylated alpha-synuclein in neuronal cell bodies, mimicking synucleinopathy. Collectively, these findings raise a novel pathogenic link between endocytic pathway and alpha-synuclein-induced neurotoxicity in synucleinopathy.     

Brain inflammation and oxidative stress in a transgenic mouse model of Alzheimer-like brain amyloidosis

Background  

An increasing body of evidence implicates both brain inflammation and oxidative stress in the pathogenesis of Alzheimer's disease (AD). The relevance of their interaction in vivo, however, is unknown. Previously, we have shown that separate pharmacological targeting of these two components results in amelioration of the amyloidogenic phenotype of a transgenic mouse model of AD-like brain amyloidosis (Tg2576).     

Neonatal neuronal overexpression of glycogen synthase kinase-3 beta reduces brain size in transgenic mice

Glycogen synthase kinase-3beta (GSK-3beta) is important in neurogenesis. Here we demonstrate that the kinase influenced post-natal maturation and differentiation of neurons in vivo in transgenic mice that overexpress a constitutively active GSK-3beta[S9A]. Magnetic resonance imaging revealed a reduced volume of the entire brain, concordant with a nearly 20% reduction in wet brain weight. The reduced volume was most prominent for the cerebral cortex, without however, disturbing the normal cortical layering. The resulting compacted architecture was further demonstrated by an increased neuronal density, by reduced size of neuronal cell bodies and of the somatodendritic compartment of pyramidal neurons in the cortex. No evidence for apoptosis was obtained. The marked overall reduction in the level of the microtubule-associated protein 2 in brain and in spinal cord, did not affect the ultrastructure of the microtubular cytoskeleton in the proximal apical dendrites. The overall reduction in size of the entire CNS induced by constitutive active GSK-3beta caused only very subtle changes in the psychomotoric ability of adult and ageing GSK-3beta transgenic mice.     

Strain and gender differences in the behavior of mouse lines commonly used in transgenic studies

The present study was aimed at establishing behavioral differences between three inbred mouse strains (129S2/SvHsd, C57BL/6JOlaHsd, FVB/NHsd) and two F1 hybrid lines derived from them (129 x C57BL/6 and 129 x FVB). The choice of the given strains was based on the frequent use of these mice in transgenic research. For the behavioral phenotyping, we employed a test battery consisting of the following models: elevated plus-maze (EPM), open field (OF), light-dark exploration, spontaneous locomotor activity, rota-rod (RR), Porsolt's forced-swimming test (FST), and Morris water task. Significant variations between the strains were established in all tests. Anxiety-like behavior was more pronounced in the 129S2/Sv and 129 x C57BL/6 mice, the FVB/N mice were spontaneously hyperactive, the best coordination ability was demonstrated by the C57BL/6 and 129 x C57BL/6 groups. A good performance in the learning test was established in both hybrid lines and the 129S2/Sv mice, whereas the well-known visual impairment of the FVB strain was confirmed by low performance in spatial and non-spatial tasks. Differences related to the gender were revealed occasionally; most importantly, 129 x C57BL/6 males had a higher anxiety level than their female counterparts in the EPM. Several other gender dissociations suggest the strain and task specificity. In conclusion, we would like to highlight the importance of the genetic background and gender of mice for the molecular biological and pharmacological studies and also the need for well-established testing protocols to obtain wide information at the first stage of behavioral screening of genetically modified mice.     

TGF-beta1 is increased in a transgenic mouse model of familial Alzheimer's disease and causes neuronal apoptosis

Alzheimer's disease (AD) is a neurodegenerative disorder, due to excess amyloid-beta peptide (Abeta). TGF-beta1 and beta-catenin signaling pathways have been separately implicated in modulating Abeta-neurotoxicity. However, the underlying mechanisms remain unclear. Here, we report that TGF-beta1 and nuclear Smad7 and beta-catenin levels were markedly upregulated in cortical brain regions of the TgCRND8 mice, a mouse model of familial Alzheimer's disease. Coimmunoprecipitation of cortical brain tissue lysates revealed an interaction between Smad7 and beta-catenin. This interaction which was significantly enhanced in the TgCRND8 mice was also associated with an increase in TCF/LEF DNA-shift binding activity. TCF/LEF reporter gene activity was significantly increased in mouse primary cortical neuronal cultures (MCN) from the TgCRND8 mice, compared to controls. Interestingly, exposure of MCN to Abeta(1-42) led to an increase in TGF-beta1 and nuclear levels of both beta-catenin and Smad7. Furthermore, addition of TGF-beta1 to the MCN caused an increase in apoptosis and Smad7 levels. When Smad7 or beta-catenin levels were reduced by siRNA, TGF-beta1-induced apoptosis was suppressed, indicating that both Smad7 and beta-catenin are required for TGF-beta1-induced neurotoxicity. Since Abeta(1-42)-induced TGF-beta1, we suggest that TGF-beta1 may amplify Abeta(1-42)-mediated neurodegeneration in AD via Smad7 and beta-catenin interaction and nuclear localization.     

Impaired neurogenesis, neuronal loss, and brain functional deficits in the APPxPS1-Ki mouse model of Alzheimer's disease

Amyloid-β peptide species accumulating in the brain of patients with Alzheimer's disease are assumed to have a neurotoxic action and hence to be key actors in the physiopathology of this neurodegenerative disease. We have studied a new mouse mutant (APPxPS1-Ki) line developing both early-onset brain amyloid-β deposition and, in contrast to most of transgenic models, subsequent neuronal loss. In 6-month-old mice, we observed cell layer atrophies in the hippocampus, together with a dramatic decrease in neurogenesis and a reduced brain blood perfusion as measured in vivo by magnetic resonance imaging. In these mice, neurological impairments and spatial hippocampal dependant memory deficits were also substantiated and worsened with aging. We described here a phenotype of APPxPS1-Ki mice that summarizes several neuroanatomical alterations and functional deficits evocative of the human pathology. Such a transgenic model that displays strong face validity might be highly beneficial to future research on AD physiopathogeny and therapeutics.     

Genetic analysis of Pten and Tsc2 functional interactions in the mouse reveals asymmetrical haploinsufficiency in tumor suppression

The role of tumor suppressor haploinsufficiency in oncogenesis is still poorly understood. The PTEN and TSC2 tumor suppressors function to antagonize mTOR (mammalian target of rapamycin) activation by Akt; hence, compound heterozygous inactivation of Pten and Tsc2 in the mouse may in principle exacerbate the tumor phenotypes observed in the single mutants in a reciprocal manner. In contrast, we found that while Tsc2 heterozygosity unmasks Pten haploinsufficiency in growth and tumor suppression, tumorigenesis in Tsc2+/- mutants is surprisingly not accelerated by Pten heterozygosity, even though mTOR activation is cooperatively enhanced by compound Pten/Tsc2 heterozygosity. We show that the wild-type alleles of both Pten and Tsc2 are retained in prostate tumors from both Pten+/- and Pten+/-Tsc2+/- mice, whereas TSC-related tumor lesions are invariably associated with Tsc2 loss of heterozygosity (LOH) in both Tsc2+/- and Pten+/-Tsc2+/- mice. These findings demonstrate that inactivation of TSC2 is epistatic to PTEN in the control of tumor initiation and progression and, importantly, that both Pten and Tsc2 are haploinsufficient for suppression of tumorigenesis initiated by Pten heterozygosity, while neither Pten nor Tsc2 is haploinsufficient for repression of carcinogenesis arising from Tsc2 heterozygosity, providing a rationale for the differential cancer susceptibility of the two human conditions associated with PTEN or TSC2 heterozygous mutations.