Chronic Renal Failure as a Possible Risk Factor for Allergic Reaction in Therapeutic Plasma Exchange Using Fresh Frozen Plasma.

The incidence of allergic reactions in patients with chronic renal failure during plasma exchange using fresh frozen plasma is not well known. We retrospectively reviewed 62 patients who underwent plasma exchange between January 2013 and May 2018. The most common indication for plasma exchange was desensitization/preconditioning for kidney transplant (61.3%, 38/62). The incidence of allergic reactions was significantly higher in patients with chronic renal failure than patients without (57.1% vs. 25.0%, P = 0.029). Also, the incidence of allergic reactions tended to be higher in peritoneal dialysis patients (75%, 3/4) than in hemodialysis (58.8%, 10/17) and preemptive kidney transplant (58%, 11/19). These results suggested the relationship of chronic renal failure and the incidence of allergic reactions in patients undergoing therapeutic plasma exchange using fresh frozen plasma.

Visualization of Arc promoter-driven neuronal activity by magnetic resonance imaging.

Visualization of direct neuronal activity to understand brain function is one of the most important challenges in neuroscience. We have previously demonstrated that in vivo and in vitro gene expression of the ferritin reporter system could be detected by magnetic resonance imaging (MRI). In addition, increased neuronal activity induces Arc, an immediate early gene, and insertion of a destabilized fluorescent reporter dVenus under Arc promoter control has been used for monitoring neuronal activities in the brain by optical imaging. In this study, to visualize Arc promoter-driven neuronal activities directly, we generated transgenic mice and cell lines that express a destabilized fusion reporter ferritin-mKate2 under Arc promoter control. When transgenic mice and cell lines were treated with pilocarpine, a non-selective muscarinic agonist, an increase in T2-weighted image signal was successfully found in neuronal cells. There was a difference in peak time between MRI and fluorescence imaging, which might result from the binding process of iron with ferritin. Visualization of Arc promoter-driven neuronal activity is essential to understand neural mechanisms underlying cognitive processes and complex behaviors, and could be a useful tool for therapeutic approaches in the brain by MRI.

LC-MS/MS imaging with thermal film-based laser microdissection.

Mass spectrometry (MS) imaging is a useful tool for direct and simultaneous visualization of specific molecules. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to evaluate the abundance of molecules in tissues using sample homogenates. To date, however, LC-MS/MS has not been utilized as an imaging tool because spatial information is lost during sample preparation. Here we report a new approach for LC-MS/MS imaging using a thermal film-based laser microdissection (LMD) technique. To isolate tissue spots, our LMD system uses a 808-nm near infrared laser, the diameter of which can be freely changed from 2.7 to 500 µm; for imaging purposes in this study, the diameter was fixed at 40 µm, allowing acquisition of LC-MS/MS images at a 40-µm resolution. The isolated spots are arranged on a thermal film at 4.5-mm intervals, corresponding to the well spacing on a 384-well plate. Each tissue spot is handled on the film in such a manner as to maintain its spatial information, allowing it to be extracted separately in its individual well. Using analytical LC-MS/MS in combination with the spatial information of each sample, we can reconstruct LC-MS/MS images. With this imaging technique, we successfully obtained the distributions of pilocarpine, glutamate, γ-aminobutyric acid, acetylcholine, and choline in a cross-section of mouse hippocampus. The protocol we established in this study is applicable to revealing the neurochemistry of pilocarpine model of epilepsy. Our system has a wide range of uses in fields such as biology, pharmacology, pathology, and neuroscience. Graphical abstract Schematic Indication of LMD-LC-MS/MS imaging.

Common Hepatic Branch of Vagus Nerve-Dependent Expression of Immediate Early Genes in the Mouse Brain by Intraportal L-Arginine: Comparison with Cholecystokinin-8.

Information from the peripheral organs is thought to be transmitted to the brain by humoral factors and neurons such as afferent vagal or spinal nerves. The common hepatic branch of the vagus (CHBV) is one of the main vagus nerve branches, and consists of heterogeneous neuronal fibers that innervate multiple peripheral organs such as the bile duct, portal vein, paraganglia, and gastroduodenal tract. Although, previous studies suggested that the CHBV has a pivotal role in transmitting information on the status of the liver to the brain, the details of its central projections remain unknown. The purpose of the present study was to investigate the brain regions activated by the CHBV. For this purpose, we injected L-arginine or anorexia-associated peptide cholecystokinin-8 (CCK), which are known to increase CHBV electrical activity, into the portal vein of transgenic Arc-dVenus mice expressing the fluorescent protein Venus under control of the activity-regulated cytoskeleton-associated protein (Arc) promotor. The brain slices were prepared from these mice and the number of Venus positive cells in the slices was counted. After that, c-Fos expression in these slices was analyzed by immunohistochemistry using the avidin-biotin-peroxidase complex method. Intraportal administration of L-arginine increased the number of Venus positive or c-Fos positive cells in the insular cortex. This action of L-arginine was not observed in CHBV-vagotomized Arc-dVenus mice. In contrast, intraportal administration of CCK did not increase the number of c-Fos positive or Venus positive cells in the insular cortex. Intraportal CCK induced c-Fos expression in the dorsomedial hypothalamus, while intraportal L-arginine did not. This action of CCK was abolished by CHBV vagotomy. Intraportal L-arginine reduced, while intraportal CCK increased, the number of c-Fos positive cells in the nucleus tractus solitarii in a CHBV-dependent manner. The present results suggest that the CHBV can activate different brain regions depending on the nature of the peripheral stimulus.

Original Research: Potential of urinary nephrin as a biomarker reflecting podocyte dysfunction in various kidney disease models.

Urinary nephrin is a potential non-invasive biomarker of disease. To date, however, most studies of urinary nephrin have been conducted in animal models of diabetic nephropathy, and correlations between urinary nephrin-to-creatinine ratio and other parameters have yet to be evaluated in animal models or patients of kidney disease with podocyte dysfunction. We hypothesized that urinary nephrin-to-creatinine ratio can be up-regulated and is negatively correlated with renal nephrin mRNA levels in animal models of kidney disease, and that increased urinary nephrin-to-creatinine ratio levels are attenuated following administration of glucocorticoids. In the present study, renal nephrin mRNA, urinary nephrin-to-creatinine ratio, urinary protein-to-creatinine ratio, and creatinine clearance ratio were measured in animal models of adriamycin nephropathy, puromycin aminonucleoside nephropathy, anti-glomerular basement membrane glomerulonephritis, and 5/6 nephrectomy. The effects of prednisolone on urinary nephrin-to-creatinine ratio and other parameters in puromycin aminonucleoside (single injection) nephropathy rats were also investigated. In all models tested, urinary nephrin-to-creatinine ratio and urinary protein-to-creatinine ratio increased, while renal nephrin mRNA and creatinine clearance ratio decreased. Urinary nephrin-to-creatinine ratio exhibited a significant negative correlation with renal nephrin mRNA in almost all models, as well as a significant positive correlation with urinary protein-to-creatinine ratio and a significant negative correlation with creatinine clearance ratio. Urinary protein-to-creatinine ratio exhibited a significant negative correlation with renal nephrin mRNA. Following the administration of prednisolone to puromycin aminonucleoside (single injection) nephropathy rats, urinary nephrin-to-creatinine ratio was significantly suppressed and exhibited a significant positive correlation with urinary protein-to-creatinine ratio. In addition, the decrease in number of glomerular Wilms tumor antigen-1-positive cells was attenuated, and urinary nephrin-to-creatinine ratio exhibited a significant negative correlation in these cells. In conclusion, these results suggest that urinary nephrin-to-creatinine ratio level is a useful and reliable biomarker for predicting the amelioration of podocyte dysfunction by candidate drugs in various kidney disease models with podocyte dysfunction. This suggestion will also be validated in a clinical setting in future studies.

[Causal relationship between assertiveness and adjustment in children: A short-term longitudinal study].

This study examined the causal relationships between assertiveness and both internal and external adjustment in children. Elementary school children in grades four through six (N = 284) participated in the study, which used a short-term longitudinal design. The children completed questionnaires twice during a 6-months period. They responded to assertiveness questionnaires that included two components: "self-expression" and "consideration of others". They also completed a self-esteem scale as an index of internal adjustment, and the Class Life Satisfaction scale as an index of external adjustment. There was a positive causative relationship between "self-expression" and internal adjustment and between "consideration for others" and external adjustment. In addition, the effects on adjustment varied according to the type of assertiveness. Cluster analysis and MANOVA indicated that the group with high "self-expression" and "consideration for others" had high internal and external adjustment, while the children with poor assertiveness showed the lowest degree of adaptivity.

Synaptic plasticity associated with a memory engram in the basolateral amygdala.

Synaptic plasticity is a cellular mechanism putatively underlying learning and memory. However, it is unclear whether learning induces synaptic modification globally or only in a subset of neurons in associated brain regions. In this study, we genetically identified neurons activated during contextual fear learning and separately recorded synaptic efficacy from recruited and nonrecruited neurons in the mouse basolateral amygdala (BLA). We found that the fear learning induces presynaptic potentiation, which was reflected by an increase in the miniature EPSC frequency and by a decrease in the paired-pulse ratio. Changes occurred only in the cortical synapses targeting the BLA neurons that were recruited into the fear memory trace. Furthermore, we found that fear learning reorganizes the neuronal ensemble responsive to the conditioning context in conjunction with the synaptic plasticity. In particular, the neuronal activity during learning was associated with the neuronal recruitment into the context-responsive ensemble. These findings suggest that synaptic plasticity in a subset of BLA neurons contributes to fear memory expression through ensemble reorganization.

Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.

Systems-level identification and analysis of cellular circuits in the brain will require the development of whole-brain imaging with single-cell resolution. To this end, we performed comprehensive chemical screening to develop a whole-brain clearing and imaging method, termed CUBIC (clear, unobstructed brain imaging cocktails and computational analysis). CUBIC is a simple and efficient method involving the immersion of brain samples in chemical mixtures containing aminoalcohols, which enables rapid whole-brain imaging with single-photon excitation microscopy. CUBIC is applicable to multicolor imaging of fluorescent proteins or immunostained samples in adult brains and is scalable from a primate brain to subcellular structures. We also developed a whole-brain cell-nuclear counterstaining protocol and a computational image analysis pipeline that, together with CUBIC reagents, enable the visualization and quantification of neural activities induced by environmental stimulation. CUBIC enables time-course expression profiling of whole adult brains with single-cell resolution.

Globular adiponectin induces LKB1/AMPK-dependent glucose uptake via actin cytoskeleton remodeling.

Previous studies have shown that many metabolic actions of adiponectin are mediated via the activation of AMP kinase and that adiponectin stimulates GLUT4 translocation and glucose uptake in the muscle. In this study, we demonstrate that adiponectin stimulates actin cytoskeleton remodeling, with increased phosphorylation of cofilin, and that blocking of cytoskeletal remodeling with cytochalasin D prevents adiponectin-stimulated AMPK phosphorylation in L6 myoblasts. LKB1 is an upstream kinase of AMPK, and we observed the colocalization of LKB1 with filamentous actin in response to adiponectin. Adiponectin-stimulated translocation of LKB1 from a nuclear to a cytoplasmic location to activate AMPK was also dependent on actin cytoskeleton remodeling. Cytoskeletal remodeling visualized by rhodamine-phalloidin immunofluorescence indicated that adiponectin-stimulated reorganization resulted in the formation membrane ruffles, which were also clearly visible by scanning electron microscopy in L6-GLUT4(myc) myoblasts. The stimulation of glucose uptake, but not of GLUT4-myc translocation to the cell surface, by adiponectin was also dependent on actin cytoskeleton remodeling. These results suggest that actin remodeling induced by adiponectin is essential for mediating LKB1/AMPK signaling and glucose uptake in skeletal muscle cells.

Diabetes influences cardiac extracellular matrix remodelling after myocardial infarction and subsequent development of cardiac dysfunction.

This study was conducted to examine the influence of acute streptozotocin-induced diabetes on cardiac remodelling and function in mice subjected to myocardial infarction (MI) by coronary artery ligation. Echocardiography analysis indicated that diabetes induced deleterious cardiac functional changes as demonstrated by the negative differences of ejection fraction, fractional shortening, stroke volume, cardiac output and left ventricular volume 24 hrs after MI. Temporal analysis for up to 2 weeks after MI showed higher mortality in diabetic animals because of cardiac wall rupture. To examine extracellular matrix remodelling, we used fluorescent molecular tomography to conduct temporal studies and observed that total matrix metalloproteinase (MMP) activity in hearts was higher in diabetic animals at 7 and 14 days after MI, which correlated well with the degree of collagen deposition in the infarct area visualized by scanning electron microscopy. Gene arrays indicated temporal changes in expression of distinct MMP isoforms after 1 or 2 weeks after MI, particularly in diabetic mice. Temporal changes in cardiac performance were observed, with a trend of exaggerated dysfunction in diabetic mice up to 14 days after MI. Decreased radial and longitudinal systolic and diastolic strain rates were observed over 14 days after MI, and there was a trend towards altered strain rates in diabetic mouse hearts with dyssynchronous wall motion clearly evident. This correlated with increased collagen deposition in remote areas of these infarcted hearts indicated by Masson's trichrome staining. In summary, temporal changes in extracellular matrix remodelling correlated with exaggerated cardiac dysfunction in diabetic mice after MI.

Orchestrated experience-driven Arc responses are disrupted in a mouse model of Alzheimer's disease.

Experience-induced expression of immediate-early gene Arc (also known as Arg3.1) is known to be important for consolidation of memory. Using in vivo longitudinal multiphoton imaging, we found orchestrated activity-dependent expression of Arc in the mouse extrastriate visual cortex in response to a structured visual stimulation. In wild-type mice, the amplitude of the Arc response in individual neurons strongly predicted the probability of reactivation by a subsequent presentation of the same stimulus. In a mouse model of Alzheimer's disease, this association was markedly disrupted in the cortex, specifically near senile plaques. Neurons in the vicinity of plaques were less likely to respond, but, paradoxically, there were stronger responses in those few neurons around plaques that did respond. To the extent that the orchestrated pattern of Arc expression reflects nervous system responses to and physiological consolidation of behavioral experience, the disruption in Arc patterns reveals plaque-associated interference with neural network integration.

[Relationship between assertiveness including consideration for others and adjustment in children].

The relationship between assertiveness and internal and external adjustment was investigated. Elementary school children in grades four to six (n=207) and their classroom teachers (n=8) participated in the study. Internal and external adjustments were measured by using self-ratings, and self- and other- ratings respectively. The children responded to a questionnaires inquiring about assertiveness that included two components of assessment: "self expression" and "consideration for others". Then, the children were divided into 4 groups according to their scores on these two components of assertiveness. The results indicated that children scoring high on both components of assertiveness had higher self-rating scores than those scoring low on both components. Moreover, children that scored high on "consideration for others" tended to have high external adjustment. Also, boys that scored low on "self expression" had lower external adjustment as indicated by the negative ratings of teachers. Furthermore, girls that scored high on "consideration for others" had high external adjustment as indicated by positive ratings of teachers and same-sexed classmates.

Ischemia-reperfusion injury leads to distinct temporal cardiac remodeling in normal versus diabetic mice.

Diabetes is associated with higher incidence of myocardial infarction (MI) and increased propensity for subsequent events post-MI. Here we conducted a temporal analysis of the influence of diabetes on cardiac dysfunction and remodeling after ischemia reperfusion (IR) injury in mice. Diabetes was induced using streptozotocin and IR performed by ligating the left anterior descending coronary artery for 30 min followed by reperfusion for up to 42 days. We first evaluated changes in cardiac function using echocardiography after 24 hours reperfusion and observed IR injury significantly decreased the systolic function, such as ejection fraction, fractional shortening and end systolic left ventricular volume (LVESV) in both control and diabetic mice. The longitudinal systolic and diastolic strain rate were altered after IR, but there were no significant differences between diabetic mice and controls. However, a reduced ability to metabolize glucose was observed in the diabetic animals as determined by PET-CT scanning using 2-deoxy-2-((18)F)fluoro-D-glucose. Interestingly, after 24 hours reperfusion diabetic mice showed a reduced infarct size and less apoptosis indicated by TUNEL analysis in heart sections. This may be explained by increased levels of autophagy detected in diabetic mice hearts. Similar increases in IR-induced macrophage infiltration detected by CD68 staining indicated no change in inflammation between control and diabetic mice. Over time, control mice subjected to IR developed mild left ventricular dilation whereas diabetic mice exhibited a decrease in both end diastolic left ventricular volume and LVESV with a decreased intraventricular space and thicker left ventricular wall, indicating concentric hypertrophy. This was associated with marked increases in fibrosis, indicted by Masson trichrome staining, of heart sections in diabetic IR group. In summary, we demonstrate that diabetes principally influences distinct IR-induced chronic changes in cardiac function and remodeling, while a smaller infarct size and elevated levels of autophagy with similar cardiac function are observed in acute phase.

Right-hemispheric dominance of spatial memory in split-brain mice.

Left-right asymmetry of human brain function has been known for a century, although much of molecular and cellular basis of brain laterality remains to be elusive. Recent studies suggest that hippocampal CA3-CA1 excitatory synapses are asymmetrically arranged, however, the functional implication of the asymmetrical circuitry has not been studied at the behavioral level. In order to address the left-right asymmetry of hippocampal function in behaving mice, we analyzed the performance of "split-brain" mice in the Barnes maze. The "split-brain" mice received ventral hippocampal commissure and corpus callosum transection in addition to deprivation of visual input from one eye. In such mice, the hippocampus in the side of visual deprivation receives sensory-driven input. Better spatial task performance was achieved by the mice which were forced to use the right hippocampus than those which were forced to use the left hippocampus. In two-choice spatial maze, forced usage of left hippocampus resulted in a comparable performance to the right counterpart, suggesting that both hippocampal hemispheres are capable of conducting spatial learning. Therefore, the results obtained from the Barnes maze suggest that the usage of the right hippocampus improves the accuracy of spatial memory. Performance of non-spatial yet hippocampus-dependent tasks (e.g. fear conditioning) was not influenced by the laterality of the hippocampus.

Lipocalin-2 induces cardiomyocyte apoptosis by increasing intracellular iron accumulation.

Our objective was to determine whether lipocalin-2 (Lcn2) regulates cardiomyocyte apoptosis, the mechanisms involved, and the functional significance. Emerging evidence suggests that Lcn2 is a proinflammatory adipokine associated with insulin resistance and obesity-related complications, such as heart failure. Here, we used both primary neonatal rat cardiomyocytes and H9c2 cells and demonstrated for the first time that Lcn2 directly induced cardiomyocyte apoptosis, an important component of cardiac remodeling leading to heart failure. This was shown by detection of DNA fragmentation using TUNEL assay, phosphatidylserine exposure using flow cytometry to detect annexin V-positive cells, caspase-3 activity using enzymatic assay and immunofluorescence, and Western blotting for the detection of cleaved caspase-3. We also observed that Lcn2 caused translocation of the proapoptotic protein Bax to mitochondria and disruption of mitochondrial membrane potential. Using transient transfection of GFP-Bax, we confirmed that Lcn2 induced co-localization of Bax with MitoTracker® dye. Importantly, we used the fluorescent probe Phen Green SK to demonstrate an increase in intracellular iron in response to Lcn2, and depleting intracellular iron using an iron chelator prevented Lcn2-induced cardiomyocyte apoptosis. Administration of recombinant Lcn2 to mice for 14 days increased cardiomyocyte apoptosis as well as an acute inflammatory response with compensatory changes in cardiac functional parameters. In conclusion, Lcn2-induced cardiomyocyte apoptosis is of physiological significance and occurs via a mechanism involving elevated intracellular iron levels and Bax translocation.

Equilibrative nucleoside transporter 1 plays an essential role in cardioprotection.

To better understand the role of equilibrative nucleoside transporters (ENT) in purine nucleoside-dependent physiology of the cardiovascular system, we investigated whether the ENT1-null mouse heart was cardioprotected in response to ischemia (coronary occlusion for 30 min followed by reperfusion for 2 h). We observed that ENT1-null mouse hearts showed significantly less myocardial infarction compared with wild-type littermates. We confirmed that isolated wild-type adult mouse cardiomyocytes express predominantly ENT1, which is primarily responsible for purine nucleoside uptake in these cells. However, ENT1-null cardiomyocytes exhibit severely impaired nucleoside transport and lack ENT1 transcript and protein expression. Adenosine receptor expression profiles and expression levels of ENT2, ENT3, and ENT4 were similar in cardiomyocytes isolated from ENT1-null adult mice compared with cardiomyocytes isolated from wild-type littermates. Moreover, small interfering RNA knockdown of ENT1 in the cardiomyocyte cell line, HL-1, mimics findings in ENT1-null cardiomyocytes. Taken together, our data demonstrate that ENT1 plays an essential role in cardioprotection, most likely due to its effects in modulating purine nucleoside-dependent signaling and that the ENT1-null mouse is a powerful model system for the study of the role of ENTs in the physiology of the cardiomyocyte.

Neural activity changes underlying the working memory deficit in alpha-CaMKII heterozygous knockout mice.

The alpha-isoform of calcium/calmodulin-dependent protein kinase II (alpha-CaMKII) is expressed abundantly in the forebrain and is considered to have an essential role in synaptic plasticity and cognitive function. Previously, we reported that mice heterozygous for a null mutation of alpha-CaMKII (alpha-CaMKII+/-) have profoundly dysregulated behaviors including a severe working memory deficit, which is an endophenotype of schizophrenia and other psychiatric disorders. In addition, we found that almost all the neurons in the dentate gyrus (DG) of the mutant mice failed to mature at molecular, morphological and electrophysiological levels. In the present study, to identify the brain substrates of the working memory deficit in the mutant mice, we examined the expression of the immediate early genes (IEGs), c-Fos and Arc, in the brain after a working memory version of the eight-arm radial maze test. c-Fos expression was abolished almost completely in the DG and was reduced significantly in neurons in the CA1 and CA3 areas of the hippocampus, central amygdala, and medial prefrontal cortex (mPFC). However, c-Fos expression was intact in the entorhinal and visual cortices. Immunohistochemical studies using arc promoter driven dVenus transgenic mice demonstrated that arc gene activation after the working memory task occurred in mature, but not immature neurons in the DG of wild-type mice. These results suggest crucial insights for the neural circuits underlying spatial mnemonic processing during a working memory task and suggest the involvement of alpha-CaMKII in the proper maturation and integration of DG neurons into these circuits.

In vivo and in vitro visualization of gene expression dynamics over extensive areas of the brain.

In vivo monitoring of gene expression using promoter-destabilized fluorescence protein constructs is a powerful method for examining the expression dynamics of immediate-early genes in the brain. However, weak fluorescence signals derived from such constructs have hampered analyses of gene expression over extensive areas of the brain. We succeeded in producing transgenic mice with brains exhibiting high level expression of the reporter gene driven by the Arc gene promoter, which is activated in association with various brain functions (reporter mRNA abundance was near 100-fold greater than endogenous Arc mRNAs). This high expression of the reporter gene enabled us to monitor Arc gene expression dynamics in vivo, over an area that included the whole of the dorsal cerebral cortex. Moreover, we were able to perform three-dimensional analyses of activated regions using paraformaldehyde-fixed brains. In addition to the visual cortex, we found that the cingulate cortex was strongly activated by light stimuli. These mice are extremely useful for the functional analysis of gene expression over extensive areas of the brains in both wild-type mice and mutants with impaired brain function.

Leptin protects H9c2 rat cardiomyocytes from H2O2-induced apoptosis.

Obesity is a known risk factor for induction of myocardial infarction, but, paradoxically, may also confer a protective effect against subsequent remodeling leading to heart failure. In this study, we investigated the effect of leptin, the product of the obese (ob) gene, on cardiomyocyte apoptosis, a well-characterized component of cardiac remodeling after myocardial infarction. Exposing H9c2 cells to H(2)O(2) decreased cell viability, and this was attenuated by pretreating cells with leptin for 1 h, but not 24 h. Leptin also attenuated the ability of H(2)O(2) to increase phosphatidylserine exposure and annexin V binding. Further investigation of underlying mechanisms of leptin's protective effect demonstrated that the H(2)O(2)-induced decrease in mitochondrial membrane potential (Psi) leading to cytochrome c release was attenuated by leptin pretreatment, and this was associated with reduced translocation of the pro-apoptotic Bax protein to the mitochondrial membrane. Finally, leptin prevented H(2)O(2)-induced increases in caspase-3 cleavage and activity, although again 24 h leptin pretreatment did not confer significant protection. In summary, we have demonstrated that acute leptin pretreatment mediates anti-apoptotic effects in H9c2 rat cardiomyocytes, which may be of significance in clarifying the direct impact of leptin on the heart.

Double transfection into primary dissociated neurons.

Synapses play a fundamental role in the processing of information in a neuronal network by mediating communication between neurons. To monitor presynaptic and postsynaptic structures simultaneously in living neurons, we developed double transfection method on the basis of calcium phosphate coprecipitation and introduced two fluorescent reporters into cultured neurons. The optimized protocol allowed different populations of neurons to be visualized by different color fluorescences, and presynaptic and postsynaptic structures to be observed in the same field of view of a microscope.