A genetically encoded indicator for monitoring intracellular chloride

Inhibitory drives in the nervous system are provided by synapses operating mostly through Cl⁻ ion channels. We describe a novel CFP-YFP-based fluorescence Cl⁻ indicator, Cl-sensor, characterized by a high sensitivity, which can be successfully used as a tool for monitoring intracellular Cl⁻ in various biological preparations. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 380–381, July–October, 2007.     

A genetically encoded, fluorescent indicator for cyclic AMP in living cells

Cyclic AMP controls several signalling cascades within cells, and changes in the amounts of this second messenger have an essential role in many cellular events. Here we describe a new methodology for monitoring the fluctuations of cAMP in living cells. By tagging the cAMP effector protein kinase A with two suitable green fluorescent protein mutants, we have generated a probe in which the fluorescence resonance energy transfer between the two fluorescent moieties is dependent on the levels of cAMP. This new methodology opens the way to the elucidation of the biochemistry of cAMP in vivo.     

Long-term potentiation in cultured hippocampal neurons

Studies performed on low-density primary neuronal cultures have enabled dissection of molecular and cellular changes during N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP). Various electrophysiological and chemical induction protocols were developed for the persistent enhancement of excitatory synaptic transmission in hippocampal neuronal cultures. The characterisation of these plasticity models confirmed that they share many key properties with the LTP of CA1 neurons, extensively studied in hippocampal slices using electrophysiological techniques. For example, LTP in dissociated hippocampal neuronal cultures is also dependent on Ca(2+) influx through post-synaptic NMDA receptors, subsequent activation and autophosphorylation of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and an increase in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor insertion at the post-synaptic membrane. The availability of models of LTP in cultured hippocampal neurons significantly facilitated the monitoring of changes in endogenous postsynaptic receptor proteins and the investigation of the associated signalling mechanisms that underlie LTP. A central feature of LTP of excitatory synapses is the recruitment of AMPA receptors at the postsynaptic site. Results from the use of cell culture-based models started to establish the mechanism by which synaptic input controls a neuron's ability to modify its synapses in LTP. This review focuses on key features of various LTP induction protocols in dissociated hippocampal neuronal cultures and the applications of these plasticity models for the investigation of activity-induced changes in native AMPA receptors.     

The influence of membrane potential on chloride channels activated by GABA in rat cultured hippocampal neurons

Chloride currents were activated by a low concentration of GABA (0.5 m) in neonatal rat hippocampal neurons cultured for up to 14 days. Currents elicited by 0.5 m GABA in neurons, voltage-clamped using the whole-cell technique with pipettes containing 149 mm Cl^–, reversed close to 0 mV whether pipettes contained 144 mm Na⁺ or 140 mm Cs⁺, and were blocked by 100 m bicuculline. Current-voltage curves showed outward rectification. Single channel currents appeared in cell-attached patches when the pipette tip was perfused with pipette solution containing 0.5 m GABA and disappeared when a solution containing 100 m bicuculline plus 0.5 m GABA was injected into the pipette tip. The channels showed outward rectification and, in some patches, had a much lower probability of opening at hyperpolarized potentials. The average chord conductance in 10 patches hyperpolarized by 80 mV was 7.8±1.6 pS (sem) compared with a chord conductance of 34.1±3.5 pS (sem) in the same patches depolarized by 80 mV. Similar single channel currents were also activated in cell-free, inside-out patches in symmetrical chloride solutions when 0.5 m GABA was injected into the pipette tip. The channels showed outward rectification similar to that seen in cell-attached patches, and some channels had a lower probability of opening at hyperpolarized potentials. The average chord conductance in 13 patches hyperpolarized by 80 mV was 11.8±2.3 pS (sem) compared with 42.1±3.1 pS (sem) in the same patches depolarized by 80 mV.We are grateful to B. McLachlan and M. Robertson for their general assistance, to C. McCulloch and M. Smith for writing computer programs and to W. O'Hare for making the pipette injection device.     

Fast inactivating potassium current in cultured hippocampal neurons

Using a voltage-clamp whole-cell technique, we studied transmembrane currents in hippocampal neurons after their long-lasting cultivation. Based on the activational characteristics and data on pharmacological sensitivity, we isolated and described an A-type voltage-activated fast inactivating potassium current (FIPC). This transient FIPC was activated at −50… −40 mV and was rather sensitive to 4-aminopyridine (4-AP). Extracellular application of 5 mM 4-AP decreased the FIPC amplitude by 75%, while application of 10 mM tetraethylammonium evoked no significant changes in it. Kinetics of FIPC activation could be described by one exponent in the fourth degree. With variations of the membrane potential from −40 to 30 mV, the activation time constant varied from 2.8 to 1.5 msec. Inactivation kinetics was described by one exponent with the time constant varying from 37 msec at −45 mV to 18 msec at 40 mV. Stationary activation and inactivation curves could be described by Boltzmann's equation; a half value of the level of stationary inactivation was reached at −80 mV, while stationary activation was attained at −25 mV. Kinetics of deinactivation (from stationary inactivation) was monoexponential with the time constant of 41 msec. It is supposed that FIPC through the membrane of hippocampal neurons is provided by the type Kv4.2 potassium channels.     

Presynaptic CaMKII is necessary for synaptic plasticity in cultured hippocampal neurons

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional enzyme that is very critical for synaptic plasticity and memory formation. Although significant progress has been made in understanding the role of postsynaptic CaMKII in synaptic plasticity, very little is known about its presynaptic function during plasticity changes. Here we report that KN-93, a membrane-permeable CaMKII inhibitor, blocked glutamate-induced increases in the frequency of miniature excitatory postsynaptic currents (mEPSCs) and the number of presynaptic functional boutons in cultured hippocampal pyramidal neurons. In addition, presynaptic injection of the membrane-impermeable CaMKII inhibitor peptide 281-309 blocked synaptic plasticity induced by tetanus, glutamate, or NO/cGMP pathway activation as expressed by long-lasting increases in EPSC amplitude and functional presynaptic boutons. Presynaptic injection of CaMKII itself coupled with weak tetanus produced an immediate and long-lasting enhancement of EPSC amplitude. Thus, the present results conclusively prove that presynaptic CaMKII is essential for synaptic plasticity in cultured hippocampal neurons.     

肾上腺髓质素降低培养海马神经元胞内游离钙离子浓度

经荧光探针Fluo 3-AM标记细胞内游离钙后,用激光共聚焦显微镜检测肾上腺髓质素(adrenomedullin,ADM)对原代培养大鼠海马神经元内游离钙浓度([Ca^2 ]1)的影响。实验结果如下：(1)ADM(0．01-1．0μmol／L)浓度依赖性地降低细胞内钙浓度。(2)降钙素基因相关肽受体阻断剂(calcitonin gene-related peptide,CGRP8-37)预处理可部分抑制ADM的效应。(3)ADM可显著抑制高钾引起的[Ca^2 ]1增加。(4)ADM可显著抑制三磷酸肌醇(inositol 1,4,5-trisphosphate,IP3)引起的内钙释放,而对兰尼定(ryanodine)引起的内钙释放无显著影响。以上结果提示,ADM降低培养海马神经元内游离钙浓度,此作用与其抑制IP,引起的内钙释放有关,ADM对静息状态下的Ca^2 内流无影响,但可显著抑制高钾引起的Ca^2 内流,CGRP受体介导了ADM的上述效应。     

Distribution of single-channel conductances in cultured rat hippocampal neurons

1. The nonhomogeneous spatial distribution of ionic channels in neurons has been implied from intracellular recordings at somatic and dendritic locations. These reports indicate that Na- and Ca-dependent regenerative currents are distributed differently throughout the neuron. Although a variety of K conductances and a noninactivating Na conductance have been described in intracellular studies, little is known about the spatial distribution of inward and outward currents throughout different regions of the neuron. 2. We recorded from cell-attached patches from cultured hippocampal cells from 1-day-old rats. The cells were cultured for 3-21 days. The spatial distribution of a variety of ionic channels was determined by comparing the conductances from somatic and dendritic membranes. Single-channel currents obtained from cell-attached patches were identified by the time course of ensemble (averaged) responses, voltage dependence, and the effect of channel blocking agents. 3. We consistently observed that only the rapidly inactivating inward current was localized to the soma. The other channel types that we studied, including an inward noninactivating, delayed rectifier and transient A-type currents, were observed in both the somatic and dendritic regions. 4. We suggest that the distribution of ionic conductances that we have observed may be functional in limiting excitability during development of neurons.     

An improved genetically encoded red fluorescent Ca2+ indicator for detecting optically evoked action potentials

Genetically encoded Ca(2+) indicators (GECIs) are powerful tools to image activities of defined cell populations. Here, we developed an improved red fluorescent GECI, termed R-CaMP1.07, by mutagenizing R-GECO1. In HeLa cell assays, R-CaMP1.07 exhibited a 1.5-2-fold greater fluorescence response compared to R-GECO1. In hippocampal pyramidal neurons, R-CaMP1.07 detected Ca(2+) transients triggered by single action potentials (APs) with a probability of 95% and a signal-to-noise ratio >7 at a frame rate of 50 Hz. The amplitudes of Ca(2+) transients linearly correlated with the number of APs. The expression of R-CaMP1.07 did not significantly alter the electrophysiological properties or synaptic activity patterns. The co-expression of R-CaMP1.07 and channelrhodpsin-2 (ChR2), a photosensitive cation channel, in pyramidal neurons demonstrated that R-CaMP1.07 was applicable for the monitoring of Ca(2+) transients in response to optically evoked APs, because the excitation light for R-CaMP1.07 hardly activated ChR2. These technical advancements provide a novel strategy for monitoring and manipulating neuronal activity with single cell resolution.     

Real-time monitoring of cyclic nucleotide signaling in neurons using genetically encoded FRET probes

Signaling cascades involving cyclic nucleotides play key roles in signal transduction in virtually all cell types. Elucidation of the spatiotemporal regulation of cyclic nucleotide signaling requires methods for tracking the dynamics of cyclic nucleotides and the activities of their regulators and effectors in the native biological context. Here we review a series of genetically encoded FRET-based probes for real-time monitoring of cyclic nucleotide signaling with a particular focus on their implementation in neurons. Current data indicate that neurons have a very active metabolism in cyclic nucleotide signaling, which is tightly regulated through a variety of homeostatic regulations.     

Transcytosis of the polymeric immunoglobulin receptor in cultured hippocampal neurons

BACKGROUND: A wide variety of proteins are transported across epithelial cells by vesicular carriers. This process, transcytosis, is used to generate cell surface polarity and to transport macromolecules between the luminal and serosal sides of the epithelial layer. The polymeric immunoglobulin receptor is a well-characterized transcytotic molecule in epithelia. It binds to its ligand, polymeric immunoglobulin, at the basolateral surface, and the receptor-ligand complex is transcytosed to the apical surface, where the ligand is released. Our previous studies have shown that hippocampal neurons may employ mechanisms similar to those of epithelial cells to sort proteins to two plasma membrane domains. The machinery used for axonal delivery recognizes proteins that are targeted apically in epithelia, whereas basolaterally destined proteins are delivered to the dendrites. It has not been clear, however, whether transcytosis occurs in neurons. RESULTS: We report expression of the polymeric immunoglobulin receptor in cultured hippocampal neurons, using a Semliki Forest Virus expression system, and show by immunofluorescence microscopy that the newly synthesized receptor is targeted from the Golgi complex predominantly to the dendrites - only about 20% of the infected neurons display axonal immunofluorescence. Addition of ligand leads to significant redistribution of the receptor to the axons, shown by an approximately three-fold increase in axonal immunoreactivity with the anti-receptor antibodies. CONCLUSIONS: Our results suggest that a transcytotic route, analogous to that in epithelia, exists in neurons, where it transports proteins from the somatodendritic to the axonal domain. Cultured neurons expressing the polymeric immunoglobulin receptor offer an experimental system that should be useful for further characterization of this novel neuronal pathway at the molecular and functional level.     

Mutant presenilin 1 alters synaptic transmission in cultured hippocampal neurons

Mutations in presenilins are the major cause of familial Alzheimer disease, but the precise pathogenic mechanism by which presenilin (PS) mutations cause synaptic dysfunction leading to memory loss and neurodegeneration remains unclear. Using autaptic hippocampal cultures from transgenic mice expressing human PS1 with the A246E mutation, we demonstrate that mutant PS1 significantly depressed the amplitude of evoked alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate receptor-mediated synaptic currents. Analysis of the spontaneous miniature synaptic activity revealed a lower frequency of miniature currents but normal miniature amplitude. Both alterations could be rescued by the application of a gamma-secretase blocker. On the other hand, the application of synthetic soluble Abeta42 in wild-type neurons induced the PS1 mutant phenotype on synaptic strength. Together, these findings strongly suggest that the expression of mutant PS1 in cultured neurons depresses synaptic transmission by causing a physical reduction in the number of synapses. This hypothesis is consistent with morphometic and semiquantitative immunohistochemical analysis, revealing a decrease in synaptophysin-positive puncta in PS1 mutant hippocampal neurons.     

Neuroprotective and neurotrophic efficacy of phytoestrogens in cultured hippocampal neurons

Epidemiological data from retrospective and case-control studies have indicated that estrogen replacement therapy (ERT) can decrease the risk of developing Alzheimer's disease. In addition, ERT has been found to promote cellular correlates of memory and to promote neuronal survival both in vivo and in vitro. Phytoestrogens have been proposed as potential alternatives to ERT. To determine whether phytoestrogens exert estrogen agonist effect in neural tissue, investigations of neuroprotective and neurotrophic efficacy of phytoestrogens were conducted. Six phytoestrogens, genistein, genistin, daidzein, daidzin, formononetin, and equol, were tested for their neuroprotective efficacy against two toxic insults, glutamate excitotoxicity and beta-amyloid(25-35). Neuronal membrane damage was quantitatively measured by lactate dehydrogenase (LDH) release, and neuronal mitochondrial viability was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid (MTT) assay. Results of these studies demonstrated that all phytoestrogens induced a modest but significant reduction in LDH release following exposure to glutamate and beta-amyloid(25-35). In contrast, none of phytoestrogens induced a significant increase in reduced MTT levels, which occurred in the presence of a full estrogen agonist, 17beta-estradiol. Analysis of the neurotrophic potential of genistein and daidzein, two phytoestrogens that exerted a significant reduction in LDH release, demonstrated that neither of these molecules promoted hippocampal neuron process outgrowth. Results of these analyses indicate that although phytoestrogens exert a neuroprotective effect at the plasma membrane, they do not sustain neuron mitochondrial viability nor do they induce cellular correlates of memory as neurite outgrowth and synaptogenesis are putative mechanisms of memory. Data derived from these investigations would predict that phytoestrogens could exert some neuroprotective effects analogous to that of antioxidants, but that these molecules are not functional equivalents to endogenously active 17beta-estradiol or to estrogen replacement formulations and, therefore, would raise the concern that they may not reduce the risk of Alzheimer's disease or sustain memory function in postmenopausal women.     

Input of orexin/hypocretin neurons revealed by a genetically encoded tracer in mice

The finding of orexin/hypocretin deficiency in narcolepsy patients suggests that this hypothalamic neuropeptide plays a crucial role in regulating sleep/wakefulness states. However, very little is known about the synaptic input of orexin/hypocretin-producing neurons (orexin neurons). We applied a transgenic method to map upstream neuronal populations that have synaptic connections to orexin neurons and revealed that orexin neurons receive input from several brain areas. These include the amygdala, basal forebrain cholinergic neurons, GABAergic neurons in the preoptic area, and serotonergic neurons in the median/paramedian raphe nuclei. Monoamine-containing groups that are innervated by orexin neurons do not receive reciprocal connections, while cholinergic neurons in the basal forebrain have reciprocal connections, which might be important for consolidating wakefulness. Electrophysiological study showed that carbachol excites almost one-third of orexin neurons and inhibits a small population of orexin neurons. These neuroanatomical findings provide important insights into the neural pathways that regulate sleep/wakefulness states.     

Single-channel recordings of chloride currents in primary cultured Drosophila neurons

Single-chloride-channel currents were recorded from primary cultured Drosophila neurons by means of the gigaohm-seal patch-clamp technique. Small inward-going current channels were observed in excised inside-out patches with the external face of the membrane exposed to bathing solutions devoid of K⁺, Na⁺, and Ca²⁺. The inward current was affected by changing the anions but not the cations bathing the cytoplasmic face of the patch. Complete replacement of CI^? by glutamate eliminated the current. The current was maintained with intracellular solutions containing NO₃^? in place of CI^?. The single-channel conductance was estimated to be 7 ps with CI^?, and 11 ps with NO₃^? at 10°C. Possible functions of this anion-selective channel have been discussed.     

Synapse formation proceeds independently of dendritic elongation in cultured hippocampal neurons

In central neurons, dendritic differentiation begins well after axonal elongation and is accompanied by the compartmentation of the microtubule-associated protein 2 (MAP2) in the somatodendritic domain. Whether MAP2 plays a role in the morphological and functional maturation of dendrites remains an open question and is the focus of this study. Cultured hippocampal neurons depleted of MAP2 by means of antisense oligonucleotides failed to elongate their dendrites. On the other hand, MAP2-depleted neurons were capable of receiving synapses within the same time course as their control counterparts. However, both the number of synapses per cell and the synaptic density were markedly reduced in neurons in which dendritic elongation has been impaired. Taken collectively, these results suggest that the expression of MAP2 is required for the morphological differentiation of dendrites. Dendritic elongation, however, is not a prerequisite for synapse formation in cultured hippocampal neurons.     

Suppression of kinesin expression in cultured hippocampal neurons using antisense oligonucleotides

Kinesin, a microtubule-based force-generating molecule, is thought to translocate organelles along microtubules. To examine the function of kinesin in neurons, we sought to suppress kinesin heavy chain (KHC) expression in cultured hippocampal neurons using antisense oligonucleotides and study the phenotype of these KHC "null" cells. Two different antisense oligonucleotides complementary to the KHC sequence reduced the protein levels of the heavy chain by greater than 95% within 24 h after application and produced identical phenotypes. After inhibition of KHC expression for 24 or 48 h, neurons extended an array of neurites often with one neurite longer than the others; however, the length of all these neurites was significantly reduced. Inhibition of KHC expression also altered the distribution of GAP-43 and synapsin I, two proteins thought to be transported in association with membranous organelles. These proteins, which are normally localized at the tips of growing neurites, were confined to the cell body in antisense-treated cells. Treatment of the cells with the corresponding sense oligonucleotides affected neither the distribution of GAP-43 and synapsin I, nor the length of neurites. A full recovery of neurite length occurred after removal of the antisense oligonucleotides from the medium. These data indicate that KHC plays a role in the anterograde translocation of vesicles containing GAP-43 and synapsin I. A deficiency in vesicle delivery may also explain the inhibition of neurite outgrowth. Despite the inhibition of KHC and the failure of GAP-43 and synapsin I to move out of the cell body, hippocampal neurons can extend processes and acquire as asymmetric morphology.