In situ hybridization localization of the Na+ channel β1 subunit mRNA in rat CNS neurons

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Neuroscience Letters 176 (1994) 11%122

HHROSClENCE LETTERS

In situ hybridization localization of the N a + channel fll subunit m R N A in rat C N S neurons Youngsuk Oh, Shunsuke Sashihara, Stephen G. Waxman* Department of Neurology, Yale University School of Medicine, New Haven, CT 06510, USA, and Neuroscience Research Center, VA Medical Center, West Haven, CT 06516, USA

Received 25 April 1994; Revised version received 6 June 1994; Accepted 6 June 1994

Abstract

Localization of Na ÷ channel fll subunit (Nafll) mRNA was examined in adult rat hippocampus, cerebellum and spinal cord by in situ hybridization histochemistry. In hippocampus, Nafll mRNA was strongly expressed by CA3 followed by CA1 pyramidal cells and dentate granule cells. In cerebellum, strong Nafll 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 ofNafll mRNA. These results provide the first localization study ofNafll mRNA in the CNS, demonstrating a differential expression in different neurons. Key words: Ion channel; Hippocampus; Cerebellum; Spinal cord

N a ÷ channels play a key role in generating action potentials and the biochemical and molecular characteristics of N a ÷ channels from rat brain are well understood [4,8]. Rat brain N a ÷ channels are composed of three subunits (a, fll and f12) and molecular cloning has demonstrated at least three subtypes of a subunit ( I - I I I ) and one fll subunit [4,8]. R a t brain N a ÷ channel a subunit subtypes are not homogeneously distributed but rather are differentially expressed between different CNS regions and different cell types [4]. F o r example, subtype I m R N A is predominantly expressed in caudal parts of the adult brain and subtype II m R N A in rostral parts [1,6]. Within the hippocampus, subtype II m R N A is the predominantly expressed N a ÷ channel a subunit but is more strongly expressed in pyramidal cells than in granule cells [2]. Although the a subunit alone can encode functional N a ÷ channels in oocytes and m a m m a l i a n cells, the kinetic properties observed in these expression systems dif-

* Corresponding author. Address: Department of Neurology, LCI 707, Yale University School of Medicine, New Haven, CT 06510, USA. Fax: (1) (203) 785-7826. 0304-3940/94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved S S D I 0304-3940(94)00449-K

fer from intact neurons in that N a ÷ currents are inactivated rather slowly and voltage dependence is shifted to more positive potentials [4], suggesting the involvement of other subunits or components in generating normal N a + currents in vivo. The N a + channel fll subunit (Nafll) has been cloned and co-expressed with an ~ subunit in oocytes [3,7] to show that: (1) the expressed N a ÷ currents are very similar to those observed in neurons; and (2) the level of N a + channel expression can be increased. In this study, we localized Nafll m R N A within neurons in adult rat CNS using a non-radioactive in situ hybridization technique. We focused on hippocampus, cerebellum and spinal cord where the distribution of the N a ÷ channel a subunit has been well studied [2,6,15]. Our results indicate that in these tissues most, if not all, neurons which are known to express N a ÷ channel a subunit m R N A s can also express Nafll m R N A but the degree of Nafll m R N A expression is variable between different cell types. Using oligonucleotide primers corresponding to nucleotides 299-316 (numbered according to Genbank) and 1255-1272 of the rat brain Nafll m R N A [7], Nafll m R N A was amplified from rat sciatic nerve total R N A

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via RT-PCR (reverse transcription and polymerase chain reaction) as described previously [9,10]. Amplified PCR products were digested with Alu I and two fragments (nucleotides 299~156 and 457 790), both corresponding to coding regions of Nafll, were cloned into pCR-script SK(+) vector (Stratagene) and confirmed by DNA-sequencing. From these cloned plasmids, digoxigenin-labeled sense or antisense single-strand riboprobes were generated using DIG RNA-labeling (Boehringer Mannheim) and purified using Chroma Spin-100 column (Clontech). These two independent probes gave consistent results with antisense but not sense riboprobes yielding specific staining of neurons (Fig. 1C) but the probe against nucleotides 457 790 yielded better staining, possibly due to probe size and/or secondary structure of the mRNAs. Results using the probe against nucleotides 457 790 are shown in Fig. 1. Adult Sprague-Dawley or Wistar rats (>12 weeks) were used for the experiments and no strain differences were observed. Rats were deeply anesthetized by CO2 narcosis and perfused transcardially with phosphatebuffered saline (PBS; 4°C) followed by a fixative (4% paraformaldehyde in 0.14 M Sorensen's phosphate buffer, pH 7.4, 4°C). Tissues were removed, immersed in a fixative for 2-4 h at 4°C, transferred to 4% paraformaldehyde and 30% sucrose in 0.14 M phosphate buffer and stored overnight at 4°C. 10-/2m cryostat sections were mounted on poly-L-lysine-coated slides, desiccated overnight under vacuum and sequentially incubated in: (1) fixative solution for 5 min; (2) PBS, three changes, 2 min each; (3) proteinase K (10/2g/ml) in 100 mM Tris/ 50 mM EDTA (pH 8.0) for 25 min at 37°C; (4) PBS, three changes, 2 min each; (5) 0.1 M triethanolamine (TEA, pH 8.0) for 2 min; (6) 0.25% acetic anhydride in 0.1 M TEA for 10 rain; (7) 2 × saline-sodium citrate buffer (SSC), three changes, 2 min each; (8) prehybridization solution containing 50% formamide, 5 × SSC, 5 × Denhardt's solution and 100 j2g/ml salmon sperm DNA (Sigma) for 2 h at 58°C; and (9) hybridization solution containing 50% formamide, 10% dextran sulfate, 5 x SSC, 1 x Denhardt's, 100/,tg/ml salmon sperm and 0.5 ng//21 probe overnight at 58°C. The cRNA probes were incubated for 10 min at 65°C before adding into hybridization solution. On each section, 30/21 of freshly prepared hybridization solution was applied, covered with Parafilm and incubated in a humidified chamber. After hybridization, slides were sequentially incubated in: (1) 4 x SSC for 5 min; (2) 2 x SSC, two changes, 10

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min each; (3) RNase A (50 pg/ml) in 10 mM Tris/500 mM NaC1 (pH 7.5) for 1 h at 37°C: (4) 2 x SSC, two changes, 10 min each: (5) 0.2 x SSC, three changes, 20 min each at 58°C: (6) buffer 1 (100 mM Tris/150 mM NaCI, pH 7.5) for 2 min; (7) blocking solution containing 2% sheep serum and 1% bovine serum albumin in buffer ! for 30 min; (8) anti-digoxigenin antibody conjugated to alkaline phosphatase (1:50~1000 in blocking solution: Boehringer Mannheim) for 2 h; (9) buffer 1, three changes, 5 min each; (10) buffer 2 (100 mM Tris/150 mM NaC1/50 mM MgCI> pH 9.5), three changes, 5 min each: and (11) color substrate solution containing 420 pg/ml nitro blue tetrazolium (NBT) and 188 pg/ml 5-bromo-4chloro-3-indotyl-phosphate (BCIP) in buffer 2 overnight. The color reaction was stopped by several changes of 10 mM Tris/1 mM EDTA, pH 8.0. Sections were dehydrated and cleared in: (1) 70% ethanol for 1 rain; (2) 95% ethanol, two changes, 1 min each; (3) 100% ethanol, two changes, 1 min each; and (4) xylene, two changes, 2 min each, and were coverslipped with Permount. Control experiments established the specificity of the probes and in situ hybridization protocol: (1) hybridization of sections without addition of a probe; (2) pretreatment of sections with RNase A: and (3) use of adult rat liver, which does not express Na + channels, as a control [7,9]. These control experiments did not reveal any specific labeling. On Northern blot analysis, the riboprobe used in the in situ hybridization was hybridized to an 1.5-kb mRNA in rat brain but not in liver, consistent with a previous report [7] (data not shown). Using this hybridization protocol, Nafll mRNA can be visualized within neurons but not in glial cells of the rat CNS. In the hippocampus, pyramidal layer and dentate gyrus displayed distinct in situ hybridization signals with different intensity (Fig. I A,B); stronger signal was found in the pyramidal layer than in dentate gyrus. The CA3 pyramidal layer was stained more strongly than CA1. Scattered cells in the hilus of the dentate gyrus as well as in stratum oriens and stratum radiatum of the pyramidal layer were also moderately labeled. The pattern of Nafll mRNA distribution in hippocampus is similar to that of Na + channel ~ subunit mRNA, especially subtype II [2,6], which is the most predominantly expressed Na ÷ channel 0~subunit in adult rat brain [4]. Interestingly, similar expression pattern of a K ÷ channel (Kvl.2) mRNA in the hippocampus was reported [11]. Kvl.2 produces delayed rectifier-type K + currents and may be involved in repolarization of action potentials [11]. Thus, CA3 neurons appear to have a high

Fig. 1. Bright-field photomicrographs showing distribution of voltage-sensitive Na + channel fll subumt (Nafil I mRNAs in hippocampus (A,B); cerebellum (D,E): spinal cord (F,G). Areas in circles in A, D and F are shown at higher magnification m B, E and G, respectively. C: hippocampus when hybridized with sense riboprobes. DG, dentate gyrus; m, molecular layer: g, granular layer: w, white matter: df, dorsal funiculus: vf, ventral funiculus. Note labeling of proximal processes in CA3 pyramidal neurons (B: arrow) and spinal cord large motoneurons (G: arrow). Essentially, all Purkinje cells (E: arrow head) were labeled but apparently with a gradient from strong to moderate expression. Bars, 30 (B,E,G) or 210/2m (A,C,D,F).

Y. Oh et al./Neuroscience Letters 176 (1994) 119-122

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level of mRNA expression for the key proteins involved in producing action potentials, i.e., voltage-sensitive Na ÷ and K + channels. These findings suggest that different excitability of CA3 and CA1 neurons [12] may be due to, at least in part, different levels of Na ÷ and K ÷ channel expression. In the cerebellum, Nafll mRNA was expressed in essentially all Purkinje cells with a gradient from strong to moderate expression (Fig. 1D,E). Most granule cells and scattered cells in the molecular layer were moderately stained. This distribution is similar to that of Na + channel a subunits, subtype II [2]. Very weak or no Nafll mRNA-labeling was observed in cerebellar white matter. In the spinal cord, large motoneurons were stained strongly and scattered neurons throughout the gray matter exhibited moderate to strong expression of Nafll mRNA (Fig. IF, G). No specific labeling was observed in the white matter. The similar distribution of Na ÷ channel a (subtype II) and fll subunit mRNAs in neurons in spinal cord, hippocampus and cerebellum [2] suggest that expression of these subunits is regulated in a coordinated manner. Although the non-radioactive in situ hybridization method provides good cellular resolution, it has limitations of low abundance mRNA detection [5]. Astrocytes have been shown to express rat brain Na ÷ channels both in vivo and in vitro [9,14]. We did not detect any distinct labeling of Nafll mRNA in white matter of cerebellum and spinal cord in this study, suggesting that Na ÷ channel expression in glial cells in vivo is very low compared with neurons; however, in situ hybridization studies on cultured astrocytes have demonstrated Nafll mRNA [10], consistent with modulation of Na ÷ channel expression by neuronal factors [13]. Taken together, these resuits indicate that Nafll mRNA is differentially expressed throughout the nervous system. We thank J.A. Black for helpful discussions. This work was supported in part by the Medical Research Service, VA, and by a grant from the National Multiple Sclerosis Society. Y. Oh was supported by a Multiple Sclerosis Fellowship and S. Sashihara was supported by a Spinal Cord Research Fellowship from the Eastern Paralyzed Veterans Association.

[1] Beckh, S., Noda, M., Lubbert, H. and Numa, S., Differential regulation of three sodium channel messenger RNAs in the rat central nervous system during development, EMBO J., 8 (1989) 3611-3616. [2] Black, J.A., Yokoyama, S., Higashida, H., Ransom, B.R. and Waxman, S.G., Sodium channel mRNAs I, II and III in the CNS: cell-specific expression, Mol. Brain Res., 22 (1994) 275-289. [3] Cannon, S.C., McClatchey, A.I. and Gusella, J.F., Modification of the Na ÷ current conducted by the rat skeletal muscle a subunit by coexpression with ~ human brain fl subunit, Pfluegers Arch., 423 (1993) 155-157. [4] Catterall, W.A., Cellular and molecular biology of voltage-gated sodium channels, Physiol. Rev., 72 (1992) S15-$48. [5] Emson, P.C., In-situ hybridization as a methodological tool for the neuroscientist, Trends Neurosci., 16 (1993) 9-16. [6] Furuyama, T., Morita, Y., Inagaki, S. and Takagi, H., Distribution of I, II and III subtypes of voltage-sensitive Na + channel mRNA in the rat brain, Mol. Brain Res., 17 (1993) 169-173. [7] Isom, L.L., De Jongh, K.S., Patton, D.E., Reber, B.F.X., Offord, J., Charbonneau, H., Walsh, K., Goldin, A.L. and Catterall, W.A., Primary structure and functional expression of the fl~ subunit of the rat brain sodium channel, Science, 256 (1992) 839-842. [8] Mandel, G., Tissue-specific expression of the voltage-sensitive sodium channel, J. Memb. Biol., 125 (1992) 193-205. [9] Oh, Y., Black, J.A. and Waxman, S.G., The expression of rat brain voltage-sensitive Na ÷ channel mRNAs in astrocytes, Mol. Brain Res., 23 (1994) 57 65. [10] Oh, Y. and Waxman, S.G., Thefll subunit mRNA of the rat brain Na ÷ channel is expressed in glial cells, Proc. Natl. Acad. Sci., in press. [11] Sheng, M., Tsaur, M.-L., Jan, Y.N. and Jan, L.Y., Contrasting subcellular localization of the Kv1.2 K ÷ channel subunit in different neurons of rat brain, J. Neurosci., 14 (1994) 2408-2417. [12] Steinhauser, C., Tennigkeit, M., Matthies, H. and Gundel, J., Properties of the fast sodium channels in pyramidal neurons isolated from the CA~ and CA3 areas of the hippocampus of postnatal rats, Pfluegers Arch., 415 (1990) 756-761. [13] Thio, C.L., Waxman, S.G. and Sontheimer, H., Ion channels in spinal cord astrocytes in vitro. III. Modulation of channel expression by coculture with neurons and neuron-conditioned medium, J. Neurophysiol., 69 (1993) 819-831. [14] Waxman, S.G., Sontheimer, H., Black, J.A., Minturn, J.E. and Ransom, B.R., Dynamic aspects of sodium channel expression in astrocytes. In Advances in Neurology, New York, NY, 1993, pp. 135-155. [15] Westenbroek, R.E., Merrick, D.K. and Catterall, W.A., Differential subcellular localization of the R~ and Rn Na + channel subtypes in central neurons, Neuron, 3 (1989) 695-704.