Riboflavin Homeostasis in the Central Nervous System

Abstract: The mechanisms by which riboflavin, which is not synthesized in mammals, enters and leaves brain, CSF, and choroid plexus were investigated by injecting [¹⁴C]riboflavin intravenously or intraventricularly. Tracer amounts of [¹⁴C]riboflavin with or without FMN were infused intravenously at a constant rate into normal, starved, or probenecid-pretreated rabbits. At 3 h, [¹⁴C]riboflavin readily entered choroid plexus and brain, and, to a much lesser extent, CSF. Over 85% of the [¹⁴C]riboflavin in brain and choroid plexus was present as [¹⁴C]FMN and [¹⁴C]FAD. The addition of 0.2 mmol/kg FMN to the infusate markedly depressed the relative entry of [¹⁴C]riboflavin into brain, choroid plexus, and, less so, CSF, whereas starvation increased the relative entry of [¹⁴C]riboflavin into brain and choroid plexus. After intraventricular injection (2 h), most of the [¹⁴C]riboflavin was extremely rapidly cleared from CSF into blood. Some of the [¹⁴C]riboflavin entered brain, where over 85% of the ¹⁴C was present as [¹⁴C]FMN plus [¹⁴C]FAD. The addition of 1.2³μmol FAD (which was rapidly hydrolyzed to riboflavin) to the injectate decreased the clearance of [¹⁴C]riboflavin from CSF and the phosphorylation of [¹⁴C]riboflavin in brain. Probenecid in the injectate also decreased the clearance of [¹⁴C]riboflavin from CSF. These results show that the control of entry and exit of riboflavin is the mechanism, at least in part, by which total riboflavin levels in brain cells and CSF are regulated. Penetration of riboflavin through the blood-brain barrier, saturable efflux of riboflavin from CSF, and saturable entry of riboflavin into brain cells are three distinct parts of the homeostatic system for total riboflavin in the central nervous system.     

Expression of Neuronal Kinesin Heavy Chain Is Developmentally Regulated in the Central Nervous System of the Rat

Abstract: The kinesin family of motor proteins comprises at least two isoforms of conventional kinesin encoded by different genes: ubiquitous kinesin, expressed in all cells and tissues, and neuronal kinesin, expressed exclusively in neuronal cells. In the present study, we have analyzed the expression of the two kinesin isoforms by immunochemistry at different stages of development of the rat CNS. We have found that the level of expression of neuronal kinesin is five to eight times higher in developing than in adult rat brains, whereas that of ubiquitous kinesin is only ∼2.5 times higher in maturing versus adult brains. Moreover, we have studied the distribution of neuronal kinesin by light microscopic immunocytochemistry in the rat brain at different postnatal ages and have found this protein not only to be more highly expressed in juvenile than in adult rat brains but also to show a different pattern of distribution. In particular, tracts of axonal fibers were clearly stained at early postnatal stages of development but were markedly unlabeled in adult rat brains. Our results indicate that the expression of at least one isoform of conventional neuron-specific kinesin is up-regulated in the developing rat CNS and suggest that this protein might play an important role in microtubule-based transport during the maturation of neuronal cells in vivo.     

Synemin Isoforms in Astroglial and Neuronal Cells from Human Central Nervous System

The intermediate filament (IF) synemin gene encodes three IF proteins (H 180, M 150, L 41 kDa) with overlapping distributions. Synemin M was present early with vimentin and nestin. Synemin H was found later in the nervous system and mesodermic derivatives concomitantly with angiogenesis and the migration of neural crest cells. Synemin L appeared later in neurons. A series of in vitro cell cultures were done to identify the linkage between synemin isoforms and specific cell types of the central nervous system (CNS). The neurons and glia from the brains of humans and rats were cultured and double immunostaining done with antibodies against the H/M or L synemin isoforms and neural cell types (βIII-tubulin or NeuN) or astrocyte intermediate filaments (GFAP or vimentin). In neurons of the CNS, synemin H/M were co-expressed with GFAP, vimentin or nestin in glial cells, whereas synemin L was found in neurons.     

The Role of Microglia in the Homeostasis of the Central Nervous System and Neuroinflammation

Molecular Biology - Recently, much attention has been drawn to unraveling the mechanisms of neurodegenerative and neuroinflammatory disease pathogenesis. A special role in the development of...     

The Polarity Protein Scribble Regulates Myelination and Remyelination in the Central Nervous System

The development and regeneration of myelin by oligodendrocytes, the myelin-forming cells of the central nervous system (CNS), requires profound changes in cell shape that lead to myelin sheath initiation and formation. Here, we demonstrate a requirement for the basal polarity complex protein Scribble in CNS myelination and remyelination. Scribble is expressed throughout oligodendroglial development and is up-regulated in mature oligodendrocytes where it is localised to both developing and mature CNS myelin sheaths. Knockdown of Scribble expression in cultured oligodendroglia results in disrupted morphology and myelination initiation. When Scribble expression is conditionally eliminated in the myelinating glia of transgenic mice, myelin initiation in CNS is disrupted, both during development and following focal demyelination, and longitudinal extension of the myelin sheath is disrupted. At later stages of myelination, Scribble acts to negatively regulate myelin thickness whilst suppressing the extracellular signal-related kinase (ERK)/mitogen-activated protein kinase (MAP) kinase pathway, and localises to non-compact myelin flanking the node of Ranvier where it is required for paranodal axo-glial adhesion. These findings demonstrate an essential role for the evolutionarily-conserved regulators of intracellular polarity in myelination and remyelination.     

Expression of Sulfated Gangliosides in the Central Nervous System

Abstract: Several sulfated lipids were detected in the ganglioside fraction isolated from a cell line of oligodendrocyte progenitors that had been metabolically labeled with [³⁵S]sulfate. Separation of the ganglioside fraction by two-dimensional TLC showed that, except for galactosylceramide-sulfate, none of the sulfate-labeled lipids comigrated with those glycosphingolipids visualized by orcinol staining, indicating that these sulfolipids were quantitatively minor components. At least eight sulfate-labeled lipid bands were susceptible to desialylation by Arthrobacter ureafaciens neuraminidase, which resulted in the formation of three new bands that retained the labeled sulfate. Six of the sulfate-labeled lipid bands containing sialic acid were also susceptible to Vibrio cholerae neuraminidase, which generated two labeled bands that appeared identical to the two major products formed after treatment with A. ureafaciens neuraminidase. In vivo labeling of lipids from 14-day-old rat brain with [³⁵S]sulfate demonstrated that the synthesis of sulfated lipids containing sialic acid also occurred in intact brain tissue. These results show that sulfated gangliosides are synthesized in the CNS and that oligodendrocytes are one cell type that contributes to this synthesis.     

Relationship Between Birth Weight and Body Composition,Energy Metabolism,and Sympathetic Nervous System Activity Later in Life

Objective: Epidemiological studies suggest that high birth weight might be associated with an increased risk of obesity later in life. Programming of metabolic, endocrine, and/or autonomic pathways during intrauterine development has been proposed to explain this association. Research Methods and Procedures: To determine the relationship between birth weight and body composition and energy metabolism later in life, we measured fat mass and fat‐free mass (hydrodensitometry or double‐energy X‐ray absorptiometry), 24‐hour energy expenditure, sleeping metabolic rate, and 24‐hour respiratory quotient (respiratory chamber) in 272 adult nondiabetic Pima Indians (161 males/111 females, age 25 ± 5 years, mean ± SD). In these subjects, birth weight varied over a wide range (2000 to 5000 g). Individuals known to be offspring of diabetic pregnancies were excluded. In 44 of the 272 subjects, muscle sympathetic nerve activity was assessed by microneurography. Results: Birth weight was positively correlated with adult height (r = 0.20, p < 0.001) and fat‐free mass (r = 0.21, p < 0.001), but not with fat mass (r = 0.01, not significant). Sleeping metabolic rate, adjusted for age, sex, fat‐free mass, and fat mass, was negatively related to birth weight (r = ?0.13, p < 0.05), whereas adjusted 24‐hour energy expenditure (r = 0.07, not significant) and 24‐hour respiratory quotient (r = ?0.09, not significant) were not. There was no relationship between birth weight and muscle sympathetic nerve activity (r = 0.12, not significant, n = 44). Discussion: In Pima Indians who are not offspring of diabetic pregnancies, high birth weight is associated with increased height and lean body mass, but not with increased adiposity later in life. Although high birth weight may be associated with relatively low resting energy expenditure, it is not associated with major abnormalities in 24‐hour energy metabolism or with low muscle sympathetic nerve activity later in life.     

Doxorubicin Influences the Expression of Glucosylceramide Synthase in Invasive Ductal Breast Cancer

Introduction

Glucosylceramide synthase (GCS) is one enzyme that provides a major route for ceramide clearance. Recent evidence has indicated an important role for GCS in multidrug resistance (MDR) tumors. Doxorubicin (DOX)can modulate the expression of GCS in leukemia and ovary cell lines. However, few studies have investigated their relationship in breast cancer;

Methods

We collected 84 excision biopsies from patients with invasive ductal breast cancer of whom 33 patients had undergone preoperative chemotherapy. Immunohistochemistry was used to analyze the expression of GCS protein and significantly showed that the expression of GCS was higher in the samples from patients treated with preoperative chemotherapy(p = 0.018). In order to investigate the underlying mechanism, breast cancer cell lines were cultured with different concentrations of DOX, and mRNA and protein levels of GCS were then detected;

Results

DOX significantly upregulated the expression of GCS at both the mRNA and protein level in ERα-positive MCF-7 cells.We then block down the Sp1 site of GCS promoter, which inhibited the DOX-mediated increase in GCS expression; and after Erα was inhibited in MCF-7 cells, the up-regulation of GCS by DOX also been inhibited.

Conclusions

In conclusion, our data demonstrated the novel finding that DOX could modulate the expression of GCS through the Sp1 site of GCS promoter in ERα-positive breast cancer cells.     

The Influence of Fluorine on the Disturbances of Homeostasis in the Central Nervous System

Fluorides occur naturally in the environment, the daily exposure of human organism to fluorine mainly depends on the intake of this element with drinking water and it is connected with the geographical region. In some countries, we can observe the endemic fluorosis—the damage of hard and soft tissues caused by the excessive intake of fluorine. Recent studies showed that fluorine is toxic to the central nervous system (CNS). There are several known mechanisms which lead to structural brain damage caused by the excessive intake of fluorine. This element is able to cross the blood-brain barrier, and it accumulates in neurons affecting cytological changes, cell activity and ion transport (e.g. chlorine transport). Additionally, fluorine changes the concentration of non-enzymatic advanced glycation end products (AGEs), the metabolism of neurotransmitters (influencing mainly glutamatergic neurotransmission) and the energy metabolism of neurons by the impaired glucose transporter—GLUT1. It can also change activity and lead to dysfunction of important proteins which are part of the respiratory chain. Fluorine also affects oxidative stress, glial activation and inflammation in the CNS which leads to neurodegeneration. All of those changes lead to abnormal cell differentiation and the activation of apoptosis through the changes in the expression of neural cell adhesion molecules (NCAM), glial fibrillary acidic protein (GFAP), brain-derived neurotrophic factor (BDNF) and MAP kinases. Excessive exposure to this element can cause harmful effects such as permanent damage of all brain structures, impaired learning ability, memory dysfunction and behavioural problems. This paper provides an overview of the fluoride neurotoxicity in juveniles and adults.     

Neuronal Chemokines: Versatile Messengers In Central Nervous System Cell Interaction

Whereas chemokines are well known for their ability to induce cell migration, only recently it became evident that chemokines also control a variety of other cell functions and are versatile messengers in the interaction between a diversity of cell types. In the central nervous system (CNS), chemokines are generally found under both physiological and pathological conditions. Whereas many reports describe chemokine expression in astrocytes and microglia and their role in the migration of leukocytes into the CNS, only few studies describe chemokine expression in neurons. Nevertheless, the expression of neuronal chemokines and the corresponding chemokine receptors in CNS cells under physiological and pathological conditions indicates that neuronal chemokines contribute to CNS cell interaction. In this study, we review recent studies describing neuronal chemokine expression and discuss potential roles of neuronal chemokines in neuron–astrocyte, neuron–microglia, and neuron–neuron interaction.     

Cytokine Gene Expression Within the Central Nervous System

1. The identification of cytokine genes expressed in the central nervous system is critical to understanding the immune network in various diseases of brain, such as infection, degeneration, and malignancy.2. Expression of cytokine genes in human astrocytoma cell lines and in fresh brain specimens was studied by the reverse-transcribed/polymerase chain reaction method.3. The correlation between clinical malignancy and cytokine gene expression within malignant glioma was examined, especially regarding the relevancy of inhibitory cytokines, such as transforming growth factor- and interleukin-10.     

The Role of Taurine in the Central Nervous System and the Modulation of Intracellular Calcium Homeostasis

The effects of taurine in the mammalian nervous system are numerous and varied. There has been great difficulty in determining the specific targets of taurine action. The authors present a review of accepted taurine action and highlight recent discoveries regarding taurine and calcium homeostasis in neurons. In general there is a consensus that taurine is a powerful agent in regulating and reducing the intracellular calcium levels in neurons. After prolonged L-glutamate stimulation, neurons lose the ability to effectively regulate intracellular calcium. This condition can lead to acute swelling and lysis of the cell, or culminate in apoptosis. Under these conditions, significant amounts of taurine (mM range) are released from the excited neuron. This extracellular taurine acts to slow the influx of calcium into the cytosol through both transmembrane ion transporters and intracellular storage pools. Two specific targets of taurine action are discussed: Na⁺-Ca²⁺ exchangers, and metabotropic receptors mediating phospholipase-C.     

中枢神经系统靶向性CuZn—SOD的构建和表达

SOD对中风等由氧自由基毒性引起的神经性紊乱有保护作用,但因血脑屏障使血液中的SOD不能进入中枢神经系统。靶向性SOD可能是进入该系统的途径之一。将人CuZn-SODcDNA与破伤风毒素C部分基因融合,分别整合进pET－22b（＋）及pFastBacHTb载体中,并分别在E.coli及粉纹夜蛾Tn-5B1-4细胞中表达。表达产物分子量为68kD,与理论计算值。蛋白质印迹实验证实,其表达产物能与人CuZn－SOD多克隆抗人本及抗破伤毒素全毒互抗体有免疫反应。在Tn细胞中高效表达,表达产物占可溶性总蛋白质的20％,表达产物有SOD活性,且具有逆行轴突运输的能力。这为靶向性SOD的进一步应用创造了条件。     

Gamma Interferon Signaling in Macrophage Lineage Cells Regulates Central Nervous System Inflammation and Chemokine Production

Intracranial (i.c.) infection of mice with lymphocytic choriomeningitis virus (LCMV) results in anorexic weight loss, mediated by T cells and gamma interferon (IFN-γ). Here, we assessed the role of CD4⁺ T cells and IFN-γ on immune cell recruitment and proinflammatory cytokine/chemokine production in the central nervous system (CNS) after i.c. LCMV infection. We found that T-cell-depleted mice had decreased recruitment of hematopoietic cells to the CNS and diminished levels of IFN-γ, CCL2 (MCP-1), CCL3 (MIP-1α), and CCL5 (RANTES) in the cerebrospinal fluid (CSF). Mice deficient in IFN-γ had decreased CSF levels of CCL3, CCL5, and CXCL10 (IP-10), and decreased activation of both resident CNS and infiltrating antigen-presenting cells (APCs). The effects of IFN-γ signaling on macrophage lineage cells was assessed using transgenic mice, called “macrophages insensitive to interferon gamma” (MIIG) mice, that express a dominant-negative IFN-γ receptor under the control of the CD68 promoter. MIIG mice had decreased levels of CCL2, CCL3, CCL5, and CXCL10 compared to controls despite having normal numbers of LCMV-specific CD4⁺ T cells in the CNS. MIIG mice also had decreased recruitment of infiltrating macrophages and decreased activation of both resident CNS and infiltrating APCs. Finally, MIIG mice were significantly protected from LCMV-induced anorexia and weight loss. Thus, these data suggest that CD4⁺ T-cell production of IFN-γ promotes signaling in macrophage lineage cells, which control (i) the production of proinflammatory cytokines and chemokines, (ii) the recruitment of macrophages to the CNS, (iii) the activation of resident CNS and infiltrating APC populations, and (iv) anorexic weight loss.Immune cell recruitment to and infiltration of the central nervous system (CNS) is central to the pathology of a variety of inflammatory neurological diseases, including infectious meningoencephalitis, multiple sclerosis, and cerebral ischemia (59, 60). Chemokines have been shown to be highly upregulated in both human diseases and animal models of neuroinflammation and are thought to be important mediators of immune cell entry into the CNS (59, 60). For example, during experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS), the chemokines CCL2 (monocyte chemoattractant protein 1 [MCP-1α]), CCL3 (macrophage inflammatory protein 1α [MIP-1α]), CCL5 (regulated upon activation, T-cell expressed and secreted [RANTES]), and CXCL10 (gamma interferon [IFN-γ]-inducible protein 10 [IP-10]) are produced by either resident CNS cells or infiltrating cells (27) and serve to amplify the ongoing inflammatory response (25, 28). However, in some EAE studies, neither CCL3 nor CXCL10 were required for disease (72, 73). During CNS viral infection, CXCL10 and CCL5 are highly produced in several models (2, 41, 48, 82). In addition, mice deficient in CCR5, which binds (among others) CCL3 and CCL5, do not display impaired CNS inflammation after certain viral infections (13). Thus, the role of chemokines in CNS inflammation is likely complex and dissimilar between autoimmune and viral infection models.IFN-γ is present in the CNS during autoimmunity and infection (7, 54, 69). Several studies suggest that IFN-γ can be a potent inducer of CNS chemokine expression. Adenoviral expression of IFN-γ in the CNS strongly induced CCL5 and CXCL10 mRNA and protein, and this induction was dependent on the presence of the IFN-γ receptor (50). In EAE and Toxoplasma infection, mice deficient in IFN-γ or the IFN-γ receptor demonstrated reduced expression of several chemokines, including CCL2, CCL3, CCL5, and CXCL10 (26, 69). However, given the near-ubiquitous expression of the IFN-γ receptor (44), the mechanisms by which IFN-γ regulates CNS chemokine production remain to be elucidated.We studied neuroinflammation and immune-mediated disease using a well-studied mouse model of infection with lymphocytic choriomeningitis virus (LCMV). Intracranial (i.c.) injection of mice with LCMV results in seizures and death 6 to 8 days after inoculation. The onset of symptoms is associated with a massive influx of mononuclear cells into the cerebrospinal fluid (CSF), meninges, choroid plexus, and ependymal membranes (6, 8, 18), as well as the presence of proinflammatory cytokines (7, 38). The immune response is critical for disease, since infection of irradiated or T-cell-depleted mice leads to persistent infection with very high levels of virus in multiple tissues without the development of lethal meningitis (18, 34, 64). i.c. LCMV infection of β₂-microglobulin-deficient mice (β₂m^−/− mice) also results in meningitis and production of proinflammatory cytokines and chemokines; however, meningitis occurs with a later onset and lower severity compared to wild-type mice (17, 24, 53, 57). Interestingly, i.c. LCMV infection of these mice also causes severe anorexia and weight loss (33, 38, 46, 52, 57) that is mediated by major histocompatibility complex (MHC) class II-restricted, CD4⁺ T cells (17, 46, 53, 57). Anorexia and weight loss are also observed in wild-type mice, but they succumb to lethal meningitis shortly thereafter (33), making study of this particular aspect of disease difficult. LCMV-induced weight loss, similar to what we have observed in β₂m^−/− mice also occurs in perforin-deficient mice, which possess CD8⁺ T cells (37). Although some reports have observed weight loss after peripheral LCMV infection (11, 45), we note that these studies used high doses of the clone 13 strain of LCMV, in contrast to our studies which have used the Armstrong strain of LCMV and orders of magnitude less virus (33, 38, 46, 52, 57). Although we cannot exclude a contribution of peripheral cells to weight loss in our i.c. Armstrong infection model, we previously showed that this weight loss does not occur with peripheral infection with LCMV Armstrong (33, 38), indicating that interactions between the CNS and the immune system are contribute substantially to disease.During LCMV infection, there is biphasic production of IFN-γ: a small, early peak of IFN-γ (most likely produced by NK or NKT cells), followed by T-cell-mediated production of IFN-γ (23, 75). Further, both CD4⁺ T cells and CD8⁺ T cells produce large amounts of IFN-γ after LCMV infection and T-cell production of IFN-γ is critical for LCMV-induced weight loss (35). Chemokines, especially CXCL10, CCL5, and CCL2, and their receptors, are upregulated in the brain after i.c. LCMV infection (2, 13). Brain chemokine mRNA expression after i.c. LCMV infection is reduced in IFN-γ-deficient mice and relatively absent in athymic mice (2). However, the mechanism(s) by which T cells and IFN-γ mediate the effects on CNS chemokine expression, cellular infiltration into the CNS, and LCMV-induced anorexic weight loss remain unclear.In the present study, we focused on two major questions. The first question concerned the role of IFN-γ on immune cell recruitment to and chemokine/cytokine production within the CNS? We found that macrophages and myeloid dendritic cells (DCs) require IFN-γ for their accumulation within the CNS. Second, since macrophages and myeloid DCs are the predominant cellular infiltrate, we sought to determine whether IFN-γ signaling on these cells was direct with regard to their recruitment and to chemokine/cytokine production. We found that IFN-γ signaling in macrophage lineage cells contributes significantly to their recruitment, to chemokine production in the CNS, and to anorexic weight loss. Together, these data suggest that much of the proinflammatory effects of IFN-γ in the CNS are mediated by the effects of IFN-γ on CD68-bearing cells.     

Divergent Dimethylarginine Dimethylaminohydrolase Isoenzyme Expression in the Central Nervous System

Cellular and Molecular Neurobiology - The endogenous methylated derivative of ʟ-arginine, Nω,Nω′-dimethyl-ʟ-arginine (asymmetric dimethylarginine, ADMA), an independent...     

Polysialic Acid Synthase (ST8Sia II/STX) mRNA Expression in the Developing Mouse Central Nervous System

Abstract: A comparative study was undertaken to correlate the immunohistochemical localization of polysialic acid (PSA) and the in situ localization of ST8Sia II mRNA. In situ hybridization of postnatal day 3 mouse brain showed high levels of ST8Sia II mRNA expression in the cerebral neocortex, striatum, hippocampus, subiculum, medial habenular nucleus, thalamus, pontine nuclei, and inferior colliculus; intermediate-level expression in the olfactory bulb, hypothalamus, superior colliculus, and cerebellum; and low-level expression in other regions. The distribution of ST8Sia II mRNA in the neocortex and cerebellum coincided with the immunohistochemical localization of PSA. During brain development, ST8Sia II mRNA started decreasing and had almost disappeared by postnatal day 14. Comparison between ST8Sia II and IV mRNA expression was also undertaken by northern blot analysis and competitive PCR analysis. During the late embryonic to early postnatal stages of the mouse CNS, the ST8Sia II mRNA showed abundant mRNA expression compared with the ST8Sia IV mRNA. Competitive PCR analysis of the adult mouse CNS showed weak expression of the two genes in the olfactory bulb, thalamus, hippocampus, and eyes. The regional and transient expression of ST8Sia II mRNA coincides with that of PSA, suggesting that ST8Sia II is closely involved in the biosynthesis and expression of PSA in the developing mouse CNS.     

A Neuronal Relay Mediates a Nutrient Responsive Gut/Fat Body Axis Regulating Energy Homeostasis in Adult Drosophila

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Evaluation of Voltage-Sensitive Fluorescence Dyes for Monitoring Neuronal Activity in the Embryonic Central Nervous System

Using an optical imaging technique with voltage-sensitive dyes (VSDs), we investigated the functional organization and architecture of the central nervous system (CNS) during embryogenesis. In the embryonic nervous system, a merocyanine-rhodanine dye, NK2761, has proved to be the most useful absorption dye for detecting neuronal activity because of its high signal-to-noise ratio (S/N), low toxicity and small dye bleaching. In the present study, we evaluated the suitability of fluorescence VSDs for optical recording in the embryonic CNS. We screened eight styryl (hemicyanine) dyes in isolated brainstem–spinal cord preparations from 7-day-old chick embryos. Measurements of voltage-related optical signals were made using a multiple-site optical recording system. The signal size, S/N, photobleaching, effects of perfusion and recovery of neural responses after staining were compared. We also evaluated optical responses with various magnifications. Although the S/N was lower than with the absorption dye, clear optical responses were detected with several fluorescence dyes, including di-2-ANEPEQ, di-4-ANEPPS, di-3-ANEPPDHQ, di-4-AN(F)EPPTEA, di-2-AN(F)EPPTEA and di-2-ANEPPTEA. Di-2-ANEPEQ showed the largest S/N, whereas its photobleaching was faster and the recovery of neural responses after staining was slower. Di-4-ANEPPS and di-3-ANEPPDHQ also exhibited a large S/N but required a relatively long time for recovery of neural activity. Di-4-AN(F)EPPTEA, di-2-AN(F)EPPTEA and di-2-ANEPPTEA showed smaller S/Ns than di-2-ANEPEQ, di-4-ANEPPS and di-3-ANEPPDHQ; but the recovery of neural responses after staining was faster. This study demonstrates the potential utility of these styryl dyes in optical monitoring of voltage changes in the embryonic CNS.