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In this study, artificial neural network (ANN) was used to predict forced convection heat transfer enhancement from microscale pin fin heat sinks under various operating conditions and at different geometries. Experimental data acquired from the literature were employed to train the ANN model. Different numbers of training cycles, layers, and neurons in the network were investigated to design an effective neural network. The performance of the trained network was tested using the mean squared error and the most accurate one was selected to predict the average Nusselt number. Mean absolute percentage errors between the ANN output and the experimental datapoints were calculated and compared with the existing correlations. This machine learning method offers significant improvement for thermal performance prediction and could be a reliable tool for determining convective heat transfer in microscale pin fin heat sinks.

According to the 6th IPCC report, the concentration of greenhouse gases has increased about 20% since pre-industrial revolution, and 17% of them have increased over the last 10 years. The Korea Metoeorological Administration has analyzed satellite-based greenhouse gases to monitor climate change and support government’s achievement of net zero. The KMA has validated satellite-based greenhouse gases using CO2 observed in situ and retrieved TCCON in Anmyeon, the South Korea which is a GAW site. Both ground- and satellite-based CO2 showed a good agreement in their increasing trends with seasonal variations. However, satellite-based CO2 observed total column appear smaller than in situ observations affected by local sources due to observe near the surface, but agree well with TCCON observed the total column. The RMSD of GOSAT, and OCO2 with in situ and TCCON is estimated about 14.85, 16.93 and 2.81, 2.01 ppmv for a 1.0 degree × 1.0 degree spatial resolution on a daily time scale from January 2014 to December 2018. The results show that satellite-based products could be used greenhouse gases monitoring, but it needs to be verified more validation data. We will present the detailed methods and results in the conference.This work was funded by the Korea Meteorological Administration’s Research and Development Program “Technical Development on Weather Forecast Support and Convergence Service using Meteorological Satellites” under Grant (KMA2020-00120).

A comprehensive understanding of functions and performances enables a selection of appropriate alternatives to the existing design and can prevent defective design. A performance-based design quality management can ensure successful project completion. This study proposes a new model for design quality management in order to prevent defective design and to minimize design change. The new quality management model defines the requirement about function and performance based on technical characteristic, and assesses suitability for design alternatives. This study attempts to propose a quality matrix assessment method that can compare the alternative design and requirements defined with the new quality management model. This method can judge conformity and suitability of design quality in accordance with the requirements configured.

Experimental considerations have been performed to obtain the clear optical microscopic images of graphene oxide which are useful to probe its quality and morphological information such as a shape, a size, and a thickness. In this study, we investigated the contrast enhancement of the optical images of graphene oxide after hydrazine vapor reduction on a Si substrate coated with a 300 nm-thick SiO2 dielectric layer. Also, a green-filtered light source gave higher contrast images comparing to optical images under standard white light. Furthermore, it was found that a image channel separation technique can be an alternative to simply identify the morphological information of graphene oxide, where red, green, and blue color values are separated at each pixels of the optical image. The approaches performed in this study can be helpful to set up a simple and easy protocol for the morphological identification of graphene oxide using a conventional optical microscope instead of a scanning electron microscopy or an atomic force microscopy. 초 록: 광학 현미경 관찰을 통해 산화 그래핀의 형상, 크기 및 두께를 쉽게 파악할 수 있는 광학 관찰을 위한 최 적 조건을 확보하고자 하였다. 본 연구에서는 SiO2 절연막이 300 nm 두께로 도포된 실리콘 기판 위의 산화 그래 핀을 하이드라진 증기 환원을 통하여 본래의 모폴로지를 유지한 채 환원된 산화 그래핀의 이미지의 선명도를 증 가시켰고, 녹색 필터를 사용한 관찰을 통해 이미지의 대비값을 보다 증대시켰다. 추가적으로 얻어진 광학 이미지 를 RGB 채널별로 분리하는 방법을 제안하고 이를 통해 이미지를 분석하였다. 그 결과 하이드라진 증기 환원 처 리 및 녹색 파장에서의 광원 하에서 고대비의 이미지 확보가 가능하였으며, 더불어 광학 이미지의 RGB 채널 분 리만으로도 선명한 그래핀 이미지를 얻을 수 있음을 알아내었다.

Background. The regulation of the epithelial sodium channel (ENaC) is a key determinant of sodium homeostasis. The effect of cyclic AMP (cAMP) on ENaC activity is tissue-specific, and is controversial when ENaC is ex-pressed in Xenopus oocytes. Methods. The modulation of ENaC by cAMP in oocytes expressing human or rat ENaC was performed with two-electrode voltage clamping. Results. 250 μM 8-(4-chlorophenylthio)-adenosine 3′, 5′-cyclic monophosphate (8-CPT-cAMP) added to the bath significantly increased normalized amiloride-sensitive currents within 60 s in oocytes expressing human α, β, and γ subunits (5 ng cRNA each). The cAMP effect was dose-dependent and was partially inhibited by 200 μM Rp-CPT-cAMP, a competitive cAMP antagonist. A transient effect of 8-CPT-cAMP on rat ENaC activity was also observed. Oocytes expressing rat α subunits with γ subunits (which have a putative protein kinase A phosphorylation site) showed similar increases in amiloride-sensitive current with 250 μM 8-CPT-cAMP, while oocytes expressing rat α subunits with β subunits were not activated by 8-CPT-cAMP. Further, rat ENaC (but not human ENaC)-expressing oocytes were not activated by cAMP when oocytes were continuously superfused during electrophysiological recordings, suggesting that rat ENaC activation by cAMP is dependent upon the condition of oocytes during cAMP stimulation. Conclusion. The present results suggest that ENaC expressed in Xenopus oocytes can be activated by cAMP, and that the γ subunit confers sensitivity to cAMP modulation of rat ENaC activity.

In this study, we have identified novel splice variants of the Na+ channel alpha subunit mRNA from cultured rat astrocytes and neuroblastoma cells. These splice variants are characterized by premature truncation or deletion of a segment in the third domain of the Na+ channel alpha subunit. The expression of three splice variants was upregulated by exposure to 1 mM dibutyryl cAMP in spinal cord astrocytes but not in cerebral astrocytes and in B50 and B104 neuroblastoma cells. The calcium ionophore 1 microM A23187, did not influence the expression of splice variants in either astrocytes or neuroblastoma cells. These findings suggest that spinal cord astrocytes may maintain a unique regulatory pathway that participates in the control of Na+ channel mRNA expression.

The alpha-subunit of the amiloride-sensitive epithelial Na+ channel (alpha ENaC) is critical in forming an ion conductive pore in the membrane. We have identified the wild-type and three splice variants of the human alpha ENaC (h alpha ENaC) from the human lung cell line H441, using RT-PCR. These splice variants contain various structures in the extracellular domain, resulting in premature truncation (h alpha ENaCx), 19-amino acid deletion (h alpha ENaC-19), and 22-amino acid insertion (h alpha ENaC + 22). Wild-type h alpha ENaC and splice variants were functionally characterized in Xenopus oocytes by coexpression with hENaC beta- and gamma-subunits. Unlike wild-type h alpha ENaC, undetectable or substantially reduced amiloride-sensitive currents were observed in oocytes expressing these splice variants. Wild-type h alpha ENaC was the most abundantly expressed h alpha ENaC mRNA species in all tissues in which its expression was detected. These findings indicate that the extracellular domain is important to generate structural and functional diversity of h alpha ENaC and that alternative splicing may play a role in regulating hENaC activity.

Using quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR), we found an increased level of Na+ channel beta 1 (Na beta 1) mRNAs in spinal cord astrocytes and in the B50 neuroblastoma cell line after exposure to 1 mM dibutyryl cAMP. In contrast, the calcium ionophore (1 microM A23187) did not affect Na beta 1 mRNA levels in these cells. Further, we amplified full length coding region of Na+ channel beta 2 (Na beta 2) mRNA from rat optic nerve and cultured astrocytes using RT-PCR. It appeared that the Na beta 2 mRNA level was increased by dibutyryl cAMP, but not by A23187, in spinal cord astrocytes. These findings suggest that the Na beta 1 and Na beta 2 mRNA levels in spinal cord astrocytes are influenced by increased cAMP but not by calcium.

Breast cancer incidence and the number of breast cancer survivors have been rapidly increasing among Chinese and Korean women in the United States. However, few data are available regarding quality of life in Asian American breast cancer survivors. This qualitative study aims to describe Asian American women’s perceptions of quality of life and their breast cancer experiences. In-depth interviews with four Chinese and five Korean American breast cancer survivors and three oncologists were conducted in Chinese, Korean, or English. Interviews were recorded and transcripts were translated into English. Qualitative analyses were performed by two independent coders and then discussed and agreed upon by the research team. The respondents reported that the breast cancer experience had affected various domains of quality of life, but women reported having limited resources with which to cope effectively. Depression, anxiety, and stress were commonly reported, but women rarely discussed these issues with family and friends or sought professional help. As immigrants, women’s loneliness and a lack of social support and culturally relevant resources seemed to be major barriers to maintaining good quality of life. Women also expressed interest in learning more about alternative therapies and relaxation skills. These findings can be used to help inform the development of a culturally appropriate intervention for Asian American breast cancer survivors. Future programs may provide information in women’s native languages to teach skills to cope with stress and anxiety, increase women’s self-efficacy within the context of their cultural background, and enhance social support among women from the same ethnic group.

Activity-dependent mechanisms have been implicated in olfactory system development but, although such activity requires ion channels, few reports have described their expression in the olfactory system. We investigated the developmental and denervation-induced regulation of the Na(+)-channel beta 1 subunit (Na beta 1) in rat olfactory bulb (OB) and piriform cortex (PC). In situ hybridization shows that Na beta 1 mRNA expression is upregulated developmentally, but with different time courses in mitral, tufted, and pyramidal cells. In mitral cells, label was detected at postnatal day 4 (P4) and gradually increased to P14. Tufted cells were devoid of Na beta 1 mRNA before P14, when most cells expressed adult levels. In pyramidal cells of PC, Na beta 1 expression was not detectable clearly until P14, with maximal expression at P28. To examine the regulation of Na beta 1 mRNA, we surgically deafferented the OB at P30 and compared the effects on Na beta 1 with those for Na(+)-channel alpha-subunit (Na alpha) mRNAs. Within 5 d of surgery, the Na beta 1 and Na alpha II signals within tufted cells disappeared almost completely. Na beta 1 and Na alpha II expression was decreased in mitral cells to low-to-moderate levels. In pyramidal cells, Na beta 1 mRNA expression was decreased moderately without significant changes in Na alpha II mRNA. Deafferentation had no detectable effects on Na alpha I or III mRNAs in either OB or PC. These data indicate that Na beta 1 mRNA is expressed differentially in subpopulations of cells in the olfactory system during development and after deafferentation and suggest that the expression of Na beta 1 is regulated independently of Na alpha mRNAs via cell-specific and pathway-specific mechanisms.

We have cloned cDNA encoding a mouse nuclear receptor mROR alpha which is a homolog of human retinoic acid receptor-related orphan receptor (hROR alpha). Cotransfection experiments revealed that mROR alpha activates transcription through a retinoic acid responsive element of the laminin B1 gene (lamRARE), but not through a RARE of RAR beta gene (beta RARE) or a synthetic palindromic thyroid hormone responsive element (TREpal). The most distal AGGTCA half-site among the three half-sites of lamRARE was sufficient for binding of mROR alpha and consequently for activation of transcription. Transactivation by mROR alpha was dependent on serum in culture medium after transfection, suggesting the presence of a possible ligand. Northern hybridization and in situ hybridization analyses revealed that mROR alpha is expressed in specific areas of the brain including thalamus and olfactory bulb as well as cerebellum where it is present at highest levels in Purkinje cells. In addition to regionally heterogeneous expression in brain, its expression was temporally regulated during differentiation of P19 cells into neural cells, but not into muscle cells. These observations suggest that mROR alpha plays important roles as a transcription factor not only in differentiation of neural cell lineages but also in the mature brain.

The brain Na+ channel beta 1 subunit (Na beta 1) mRNA has recently been localized within rat central nervous system where it is expressed at differing levels in different types of neurons. In the present study, we have studied the expression pattern of Na beta 1 mRNA in rat dorsal root ganglion (DRG) neurons using non-radioactive in situ hybridization histochemistry. Na beta 1 mRNA is differentially expressed in adult DRG, with higher levels in intermediate-to-large (approximately 25 microns in diameter) DRG neurons than in small (25 microns) DRG neurons. This cell body size-related Na beta 1 mRNA expression is consistently observed beginning at postnatal day 4 and continues throughout development to adulthood. The present results indicate that (i) Na beta 1 mRNA is expressed in neurons in the peripheral nervous system and (ii) Na beta 1 gene expression is differentially regulated in DRG neurons in relation to their cell body sizes.

Astrocytes have been shown to express voltage-sensitive Na+ channels, but the molecular structure of these channels is not yet known. Recent studies have demonstrated the expression of rat brain voltage-sensitive Na+ channel mRNAs in astrocytes. In this study, we used a combined non-radioactive in situ hybridization/immunocytochemistry method to investigate the expression of voltage-sensitive Na+ channel beta 1 subunit (Na beta 1) mRNA in definitively identified, GFAP-positive astrocytes cultured from two different regions of the rat brain, cerebrum and cerebellum. In general, two morphologically distinct types of GFAP-positive astrocytes were observed in culture: flat, fibroblast-like and stellate, process-bearing. We observed a differential expression of Na beta 1 mRNA in GFAP-positive astrocytes: 1) stellate astrocytes expressed Na beta 1 mRNA, although the level of Na beta 1 mRNA expression was variable, and 2) flat astrocytes generally did not express Na beta 1 mRNA. Moreover, Bergmann-like cells from cerebellum did not express Na beta 1 mRNA, while the granule cells associated with Bergmann-like cell expressed Na beta 1 mRNA. These observations indicate that Na beta 1 mRNA is differentially expressed in rat astrocytes with various morphologies in vitro.

Although the molecular characteristics of glial Na+ channels are not well understood, recent studies have shown the presence of mRNA for rat brain Na+ channel alpha subunits in astrocytes and Schwann cells. In this study, we asked whether the mRNA for the rat brain Na+ channel beta 1 subunit is expressed in glial cells. We performed in situ hybridization using a complementary RNA probe for the coding regions of the rat brain Na+ channel beta 1 subunit mRNA and detected beta 1 subunit mRNA in cultured rat optic nerve astrocytes and sciatic nerve Schwann cells. The beta 1 subunit was amplified by reverse transcription-polymerase chain reaction in rat optic and sciatic nerves, which lack neuronal somata but contain astrocytes and Schwann cells, respectively. Doublet bands of the beta 1 subunit mRNA were amplified from both optic and sciatic nerves. Through the cloning and sequencing of these bands, we confirmed the amplification of a mRNA highly homologous to the previously cloned rat brain Na+ channel beta 1 subunit (beta 1.1) and a novel form of the beta 1 subunit mRNA (beta 1.2), which is closely homologous to beta 1.1 but contains an additional 86-nucleotide insert in 3' noncoding regions. Two beta 1 subunit mRNAs were also amplified from rat brain and skeletal muscle, but not from rat liver or kidney. These results indicate that rat brain Na+ channel beta 1 subunit mRNAs are expressed in glial cells as well as in neurons.

Changes in Na+ transport in rat alveolar type II (ATII) cells during culture were quantified and related to alterations in spatial distribution of proteins antigenically related to amiloride-sensitive Na+ channels. Adult rat ATII cells were cultured for periods ranging from 24 to 96 h. When patch clamped in the whole cell mode, both freshly isolated and cultured ATII cells exhibited outwardly rectified Na+ currents. At 0 and 24 h in culture, these currents were equally inhibited by amiloride, benzamil, and 5-(N-ethyl-N-isopropyl)-2',4'-amiloride (inhibitory constant approximately 1 microM). These conductive pathways were equally permeable to Na+ and K+. Immunocytochemical localization at 0 or 24 h in culture revealed the presence of plasma membrane antigenic sites; after 48 h, the appearance of intracellular antigenic sites increased significantly. A single band of molecular mass 135 kDa in membrane proteins of freshly isolated ATII cells was recognized in Western blots; at 48 h in culture, two lower bands with molecular masses of 75 and 65 kDa were detected in either membrane or cytoplasmic proteins. Photolabeling with 2'-methoxy-5'-nitrobenzamil showed that the 135-, 75-, and 65-kDa bands contained amiloride-binding sites. These results suggest the presence of low amiloride affinity conductive pathways in freshly isolated and cultured ATII cells. Culturing ATII cells resulted in internalization and possible breakdown of these pathways and decreased Na+ transport.

To determine the mechanism by which vasopressin increases apical membrane Na+ entry, we evaluated whether or not this hormone could recruit Na+ channels from a subapical membrane pool using specific polyclonal antibodies raised against high amiloride affinity bovine renal papillary Na+ channels. We also studied the effect of protein kinase A (PKA)-mediated phosphorylation on single-channel activity of highly purified Na+ channels incorporated into planar lipid bilayer membranes. PKA induced a significant increase in open-channel probability (Po) with no change in single-channel conductance. As shown previously and reconfirmed in the present work, PKA catalyzed the phosphorylation of a single subunit of this Na+ channel protein, namely, a 300-kDa polypeptide. On the other hand, protein kinase C, in combination with diacylglycerol, Ca2+, and phosphatidylserine, phosphorylated both the 130- and 55-kDa subunits of this purified Na+ channel, with a concomitant decrease in Po of both untreated and previously PKA-treated channels. We also found, in expression studies conducted in confluent monolayers of amphibian renal A6 cells, that vasopressin did not induce the apical insertion of new channel proteins. These observations support the hypothesis that vasopressin increases the apical Na+ permeability by activating Na+ channels already resident in the apical membrane by a direct phosphorylation mechanism rather than by cytoplasmic recruitment of latent Na+ channels.

An amiloride binding protein in adult rat and rabbit alveolar type II (ATII) cells was characterized using three different antibodies against epithelial Na+ channel proteins. We found that 1) polyclonal antibodies raised against epithelial Na+ channel proteins from bovine kidney cross-react with a 135-kDa protein in ATII membrane vesicles on Western blots; 2) using the photoreactive amiloride analog, 2'-methoxy-5'-nitrobenzamil (NMBA), in combination with anti-amiloride antibodies, we found that NMBA specifically labeled the same M(r) protein; and 3) monoclonal anti-idiotypic antibodies directed against anti-amiloride antibodies also recognized this same M(r) protein on Western blots. We also demonstrated a low benzamil affinity binding site (apparent Kd = 370 nM) in rabbit ATII cell membranes and both high and low benzamil affinity binding sites (apparent Kd = 6 nM and 230 nM) in bovine kidney membranes using [3H]Br-benzamil as a ligand. Pharmacological inhibitory profiles for displacing bound [3H]Br-benzamil were also different between ATII cells and bovine kidneys. These observations indicate that adult ATII pneumocytes express a population of epithelial Na+ channels having a low affinity to benzamil and amiloride and a pharmacological inhibitory profile different from that in bovine kidney.

The purpose of this study was to document the existence, assess the spatial localization, and characterize some of the transport properties of proteins antigenically related to epithelial Na+ channels in freshly isolated rabbit and rat alveolar type II (ATII) cells. ATII cells, isolated by elastase digestion of lung tissue and purified by density-gradient centrifugation, were incubated with polyclonal antibodies raised against Na+ channel protein purified from beef kidney papilla (NaAb), followed by a secondary antibody (goat antirabbit immunoglobulin G conjugated to fluorescein isothiocyanate). Rat ATII cells exhibited specific staining with NaAb at the level of the plasma membrane, which, in most cells, colocalized with that of the lectin Maclura pomiferra agglutinin, an apical surface marker. In Western blots, NaAb specifically recognized a 135 +/- 10-kDa protein in rat ATII membrane vesicles. When patch clamped in the whole cell mode using symmetrical solutions (150 mM Na+ glutamate), ATII cells exhibited outwardly rectified Na+ currents that were diminished by amiloride (10-100 microM) instilled into the bath solution. Ion substitution studies showed that the conductive pathways were three times more permeable to Na+ than K+. Amiloride, benzamil, and 5-(N-ethyl-N-isopropyl)-2',4'-amiloride were equally effective in diminishing 22Na+ flux into rabbit and rat ATII cells (45% inhibition at 100 microM, with IC50 of approximately 1 microM for all inhibitors). Tetraethylammonium chloride (10 mM) or BaCl2 (2 mM), well-known K+ channel blockers, had no effect on 22Na+ uptake. These results indicate that ATII cells express an amiloride-sensitive Na+ conductance, probably a channel, with a lower affinity for amiloride and its structural analogues than the well-established amiloride-sensitive Na+ channels found in bovine renal papila and cultured amphibian A6 kidney cells.

The upregulated expression of tetrodotoxin sensitive (TTXs) Na+ channels is thought to play an important role in the development of ectopic discharges (EDs) in axotomized sensory neurons. The present study examined the levels of mRNAs of three subtypes of TTXs Na(+) channels, Na(v)1.7, Na(v)1.6, and Na(x), in the dorsal root ganglion (DRG) after segmental spinal nerve ligation. Following nerve ligation, the level of mRNAs of Na(v)1.7 and Na(v)1.6 was decreased, while the Nax mRNA level was increased at 5 days, but not at 1 day, postoperatively compared with the normal levels. Thus, if upregulated expression of TTXs Na+ channels contributes to the generation of EDs in axotomized DRG neurons, Na(x) is the most likely contributor among the three tested subtypes.

Amiloride-sensitive sodium channels mediate sodium entry across the apical membrane of epithelial cells in variety of tissues. The rate of Na(+) entry is controlled by the regulation of the epithelial sodium channel (ENaC) complex. Insertion/retrieval of the ENaC complex into the apical membrane as well as direct kinetic effects at the single channel level are recognized mechanisms of regulation. Recent data suggest that the syntaxin family of targeting proteins interact with and functionally regulate a number of ion channels and pumps. To evaluate the role of these proteins in regulating ENaC activity, we co-expressed rat ENaC cRNA (alpha, beta, gamma subunits) with syntaxin 1A or 3 cRNAs in Xenopus oocytes. Basal ENaC currents were inhibited by syntaxin 1A and stimulated by syntaxin 3. Both syntaxin 1A and syntaxin 3 could be co-immunoprecipitated with ENaC subunit proteins, suggesting physical interaction. Interestingly, immunofluorescence data suggest that with either syntaxin isoform the ENaC-associated epifluorescence on the oocyte surface is enhanced. These data indicate that (i) both syntaxin isoforms increase the net externalization of the ENaC channel complex, (ii) that the functional regulation is isoform specific, and (iii) suggest that ENaC may be regulated through mechanisms involving protein-protein interactions.

The sodium channel beta 1 subunit (Na beta 1) is a component of the rat brain voltage-dependent sodium channel. We have used nonradioactive in situ hybridization cytochemical techniques to demonstrate that transcript levels of Na beta 1 are differentially upregulated during postnatal development of several CNS regions, with selective labeling of specific neuronal populations. In the hippocampus, labeling of the pyramidal cell layer (particularly in the CA3 region) and dentate granule cells was initially observed at postnatal day 2 (P2) and P10, respectively, and became progressively more intense with maturation. Labeled cells were first observed in the hilus at P10. In the developing cerebellum, transient labeling was observed in the external granule cell layer beginning at P1 while label increased in the internal granule cell layer up to P21. Purkinje cells showed significant label beginning at P4 and increasing up to P21. Weak signal was seen in neurons of deep nuclei at P1 and increased up to P21. Na beta 1 labeling in the spinal cord was first observed in the ventral horn at P2, and the intensity of labeling in these large motoneurons gradually increased. In addition, there was a ventral-dorsal gradient in this region, with label appearing subsequently in neurons of Rexed laminae IX, VII and VIII, and in the dorsal horn (Rexed laminae I-VI). In these regions, the labeling reached a plateau within the first 2-3 weeks after birth and persisted into the adult rat. The time course and regional heterogeneity of Na beta 1 expression are consistent with the hypothesis that the expression of mature Na+ channels, including Na beta 1, contributes to the development of circuitry that supports complex patterns of electrogenesis.

The central role of Ranvier is a complex structure involves three cellular elements: the axon, the oligodendrocyte, and the perinodal astrocyte. This chapter examines the interaction of these three cell types at central node of Ranvier. It begins by examining the mechanism that regulates the development of the myelinated tracts that have properties that optimize the number of axonal functions. It then considers the molecular structure of the axon membrane, and the glial-axonal interactions that participate in the development of the axon membrane. It looks at the relationship between astrocytes and the central nervous system myelinated fibers.

Although the pattern of expression of various sodium channel alpha- and beta-subunits changes as development proceeds, the mechanisms that control the expression of these subunits are not yet understood. To study the role of nerve growth factor (NGF) in modulating the expression of sodium channel subunits, we used in situ hybridization cytochemistry to examine the distribution of sodium channel alpha- and beta 1-subunit mRNAs in embryonic day 16 (E16) dorsal root ganglia (DRG) neurons cultured in the absence or presence of NGE. At 4 days in vitro in the absence of NGF, sodium channel alpha-subunit II mRNA was expressed at low-to-moderate levels in DRG neurons, but the transcripts for sodium channel alpha-subunits I, III and NaG and beta 1-subunit were not detectable. In the presence of NGF, DRG neurons expressed low-to-moderate levels of sodium channel alpha-I, high levels of alpha-II and low levels of alpha-III; NaG mRNA was not detectable. Sodium channel beta 1 mRNA was up-regulated and was expressed at high levels in DRG neurons in NGF-containing media. These observations demonstrate that the NGF exerts a differential up-regulation of sodium channel alpha- and beta-subunit mRNAs in DRG neurons derived from E16 embryos.

The expression of rat brain voltage-sensitive Na+ channel mRNAs in Schwann cells was examined using in situ hybridization cytochemistry and RT-PCR. The mRNAs of rat brain Na+ channel subtype II and III, but not subtype I, were detected in cultured Schwann cells from sciatic nerve and in intact sciatic nerve, which contains Schwann cells but not neuronal cell bodies. These results indicate that rat brain Na+ channel mRNAs, which have been considered as mainly neuronal-type messages, are also expressed in glial cells in vitro and in vivo.

Localization of Na+ channel beta 1 subunit (Na beta 1) mRNA was examined in adult rat hippocampus, cerebellum and spinal cord by in situ hybridization histochemistry. In hippocampus, Na beta 1 mRNA was strongly expressed by CA3 followed by CA1 pyramidal cells and dentate granule cells. In cerebellum, strong Na beta 1 mRNA expression was observed in Purkinje cells and moderate expression in granule cells and scattered cells of the molecular layer. In spinal cord, neurons in gray matter exhibited moderate to strong expression of Na beta 1 mRNA. These results provide the first localization study of Na beta 1 mRNA in the CNS, demonstrating a differential expression in different neurons.

The expression of rat brain sodium channel α-subunit mRNAs I, II and III and a putative glial cell-specific sodium channel (NaG) mRNA was examined in cultured astrocytes from P-0 rat spinal cord by RNA blot hybridization and by non-isotope in situ hybridization cytochemistry utilizing two independent sets of isoform-specific RNA probes. Sodium channel mRNA I was not detectable in the cultured astrocytes by RNA blot or in situ hybridization. Sodium channel mRNA II showed negligible-to-low levels of expression in flat, fibroblast-like and ‘pancake’ astrocytes at 4 days in vitro (div), while stellate, process-bearing astrocytes exhibited low-to-moderate levels of mRNA II expression. At 7 div, mRNA II expression ranged from low-to-moderate in flat astrocytes and was moderately high in most process-bearing astrocytes. In RNA blots, a weak band was observed at 9.5 kb. Sodium channel mRNA III expression was negligible in flat astrocytes and was detectable in low-to moderate levels in stellate astrocytes beginning at 4 div; by 7 div, mRNA III was detectable in low levels in flat astrocytes and low-to-moderate levels in stellate astrocytes. RNA blots showed two bands of nearly equal intensity, one at 9.0 kb and one at 7.2 kb. NaG mRNA showed increased expression with time in culture, being detectable in flat and stellate astrocytes at 4 div and becoming very prominent in flat astrocytes at extended times in culture. In RNA blots of cultured astrocytes at 7 div, a strong hybridizing signal with the NaG probe was observed. These observations demonstrate that flat and stellate astrocytes cultured from rat spinal cord express rat brain sodium channel mRNA II and III, and NaG, and suggest that astrocytes in vitro may co-express multiple forms of sodium channel mRNA.

Astrocytes from various regions of CNS have been shown to express voltage-activated Na + currents. To date, three distinct subtypes (I, II and III) of Na + channels have been cloned from rat brain. We have applied a combined technique of reverse transcription and polymerase chain reaction (RT-PCR) to examine the expression of rat brain Na + channels in rat astrocytes in vivo and in vitro. Five PCR primer sets were used to amplify coding or 3′ non-coding regions of subtype I, II, and III Na + channels. We were able to amplify all three of these rat brain Na + channel subtypes from rat optic nerve, which does not have neuronal cell bodies but does contain astrocytes known to express voltage-sensitive Na + channels. In studies on cultured spinal cord astrocytes, we were also able to amplify all three subtypes of rat brain Na + channel mRNAs. In control experiments, RT-PCR was performed on RNAs prepared from several rat tissues, including brain, skeletal muscle, and liver. Rat brain was shown to express the three Na + channel subtypes as expected. In rat skeletal muscle, subtype I and III Na + channel mRNAs, but not subtype II, were amplified. In rat liver, Na + channel messages were not detectable. The present study provides the first direct evidence that astrocytes in vivo and in vitro express rat brain voltage-sensitive Na + channel mRNAs, which have been considered as mainly neuronal-type Na + channel messages.