Proc. Nati. Acad. Sci. USA Vol. 83, pp. 8424-8428, November 1986 Neurobiology Localization of sodium channels in hillocks and initial segments of retinal cells (retina//immunocytochemistry/ fiber layer) DEBRA A. WOLLNER AND WILLIAM A. CATTERALL Department of Pharmacology, SJ-30, University of Washington, Seattle, WA 98195 Communicated by Bertil Hille, July 24, 1986

ABSTRACT Affinity-purified antibodies against the sodi- unpublished). However, immunocytochemical methods have um channel from rat brain were employed to localize sodium been successfully used to examine the distribution of sodium channels in the retina by immunocytochemical procedures. In channels in peripheral nerve, skeletal muscle, and eel rat retina, intense staining was observed in the ganglion cell electroplax (8-11). To determine the distribution of sodium axon layer and light staining was detected in fibers of the inner channels in central , we have employed polyclonal plexiform layer. In frog retina, only the ganglion cell axon layer antisera against the purified from rat brain was stained. Examination at higher magnification revealed that (12). These antisera crossreact poorly with sodium channels axon hillocks and initial segments of ganglion cells had a high from peripheral nerve or skeletal muscle, suggesting that they density of immunoreactive sodium channels, whereas the cell are specific for a sodium channel subtype present predomi- bodies were devoid of stain. The sharply defined region of high nantly in the central (13). In this investiga- sodium channel density at the axon hillock is likely to be tion, we use affinity-purified antibodies against this sodium responsible for the low threshold for action potential initiation channel subtype to determine its distribution among the in this region of vertebrate central neurons. different classes of neurons in the retina and its localization in the surface membrane of retinal ganglion cells. A prelim- Voltage-sensitive sodium channels are responsible for the inary report of this work has appeared (14). rising phase of the action potential in electrically excitable cells. Physiological studies indicate that electrical excitability EXPERIMENTAL PROCEDURES is not evenly distributed on the surfaces of central neurons. For example, cat spinal motor neurons and goldfish Materials. Materials were obtained from the following Mauthner cells have a lower threshold for action potential sources: Sprague-Dawley rats, Tyler Laboratories (Belle- generation at their axon initial segments (1-4) and action vue, WA); Rana pipiens, KONS Scientific (Germantown, potentials in cultured and WI); Pansorbin and tetrodotoxin, Calbiochem; goat anti- neurons are initiated preferentially at this site (5). In motor rabbit antiserum, Cappel (Cochranville, PA); rabbit peroxi- neurons, this causes a biphasic action potential as recorded dase-antiperoxidase, Zymed Laboratories (Burlingame, CA); in the cell consisting of an early response in the initial normal goat serum, Vector Laboratories (Burlingame, CA); segment of the axon followed by a larger regenerative spike cresyl violet acetate, Aldrich; diaminobenzidine (Isopac), in the cell soma itself (4). A similar biphasic action potential Sigma; nitrocellulose, Bio-Rad; 125I-labeled protein A (125I- shape has been observed in several different classes of protein A) and [y32P]ATP, New England Nuclear; and the mammalian central neurons, suggesting that a low threshold purified catalytic subunit of the cAMP-dependent protein for action potential initiation at the axon initial segment may kinase prepared by the method of Bechtel et al. (15), Sandra be a widespread characteristic of neurons. This property Rossie of this laboratory. Saxitoxin was tritiated by the allows the initial segment to act as a summing point for graded method ofRitchie et al. (16) and prepared for use as described potentials in the cell body and and plays an (17). important role in information processing at the cellular level. Antibody Purification. Antisera were prepared in female It is therefore of considerable importance in understanding New Zealand White rabbits as described (12) using rat brain the electrical properties and input-output relationships of sodium channels purified to near homogeneity as described neurons to examine directly the distribution of ion channels by Hartshorne and Catterall (18). Antibodies were purified by on the neuronal cell surface and evaluate the role of ion a method similar to that described by Olmsted (19). Approx- channel distribution in determining regional differences in imately 300 pmol of sodium channel from rat brain was electrical excitability. purified through the step of chromatography on wheat germ Previous studies using autoradiographic localization of agglutinin-Sepharose (18) and applied to a 3-10% polyacryl- specifically bound, 125I-labeled scorpion toxin detected a high amide gel under denaturing (20) conditions, but without density of sodium channels in the initial segments of neurites reduction, as a single continuous band over the entire width of cultured spinal cord neurons (6) and a higher density of of the gel. A trace amount of 32P-labeled sodium channels, sodium channels in neurites as compared to cell bodies of prepared as described (21), was added as a marker. After cultured brain neurons (7). These studies suggest that an electrophoresis, sodium channel polypeptides were uneven distribution of sodium channels may be at least electrophoretically transferred to nitrocellulose (22) and the partially responsible for regional differences in electrical disutfide-linked complex of a and 82 subunits was located by excitability of individual neurons. In preliminary experi- autoradiography and excised. The nitrocellulose matrix was ments, scorpion toxin has not proven useful as a label for washed twice with 50 mM Tris HCl/5 mM EDTA/150 mM sodium channels in intact neural tissue or slices (W.A.C., NaCl, pH 7.5, and incubated in 50 mM Tris.HCl/150 mM NaCl, pH 7.4 (TBS) plus 5 mg of bovine serum albumin per The publication costs ofthis article were defrayed in part by page charge ml to block nonspecific protein binding sites. The IgG payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: PAP, peroxidase-antiperoxidase. 8424 Downloaded by guest on September 24, 2021 Neurobiology:Neurobiology:WoRnerWollnerandandCatteraffCatterallProc.Natl. Acad. Sci. USA 83 (1986) 8425 fraction of antiserum 4675 was prepared by precipitation with saturated ammonium sulfate and incubated overnight with the nitrocellulose affinity matrix at 40C. After extensive in the washing TBS, sodium-channel-speciflic antibodies 8080 were eluted in 0.1 M glycine (~pH 2.5) plus 5 mg of bovine .0 serum albumin per ml for is min. The eluate containing pure a- anti-sodium-channel antibodies was collected and the pH was M 60- adjusted to 7.4 by addition of 3 M Tris, pH 8.9. E Immunocytochemistry. Rats and frogs were killed by de- E capitation and the whole eyes were removed. The front ofthe c4o 0 eyes was sliced with a sharp razor and the lenses and vitreous E were removed. The eyecups were immediately immersed in 0 20- ice-cold PLP fixative (23) consisting of 4% paraformalde- 0 hyde, 100 mM lysine, 10 mM NaIO3, and 60 mM sucrose in phosphate-buffered saline (PBS) at pH 8 for 3 hr. The It 0.01 0.1 1 eyecups were sunk successively in 10%, 20%, and 30% Na' channel, pmol solutions of sucrose in PBS with 0.01% NaN3. A few hours prior to cutting, the eyecups were soaked in OCT compound FIG. 1. Immunoreactivity of affinity-purified antibodies. Sodium in the cold. The retinas were then frozen at -200C in a Tissue channels were solubilized and partially purified from rat brain as Tec 1I cryostat, and sections were cut to 10-Atm thickness. described by Hartshorne and Catterall (18) through the step of The sections were collected on room temperature gelatin- chromatography on wheat germ agglutinin-Sepharose. The purified coated slides and allowed to dry. Before staining, the retinas channel was labeled on the a subunit by phosphorylation with were with PBS [y-32PJATP and cAMP-dependent protein kinase (21). Sodium chan- rehydrated containing 0.1% Triton X-100 for nels were partially purified from frog (Ranapipiens) brain as follows. 30 min. They were then incubated in normal goat serum for The brain was removed and immediately homogenized at 00C with a 30 mmn in PBS and 0.1% Triton X-100. The purified antibody glass homogenizer in 130mM choline chloride/5.4 mM KCl/0.8 mM was diluted inS5 mg of bovine serum albumin per ml and 0.1% MgSO4/50 mM Hepes/Tris.HCl, pH 7.4. The protease inhibitors Triton X-100 in PBS and allowed to incubate overnight at phenylmethylsulfonyl fluoride (1 mM), pepstatin A (0.5 mM), and room temperature or 370C. Samples were allowed to wash for iodoacetamide (1 mM) were added, and the samples were sedimented 15 min at room temperature. The sections with bound for 15 min at 8000 x g in a Beckman Microfuge. Pellets were primary antibody were then stained by the peroxidase- suspended in 75 mM NaCl/2.5 mM EDTA/25 m.M Tris.HCI/50 mM antiperoxidase with a 1:200 Na.H2P04/20 mM KF, pH 7.4 (NET buffer) containing 5% (wt/vol) (PAP) technique (24), incubating Triton X-100 and protease inhibitors, incubated with slow shaking at dilution ofgoat anti-rabbit and rabbit PAP antibodies sequen- 00C for 30 min, and resedimented for 10 min. Solubilized sodium tially at 370C for 30 mmn. The peroxidase reaction was carried channels in the supernatant were used in radioimmunoassay exper- out with 0.4% diaminobenzidine/0.003% H202 in 50 mM iments. For competitive radioimmunoassay experiments, the total Tris-HCI (pH 7.4). Sections were mounted with permount and IgG fraction or affinity-purified antibodies equivalent to 1 1d of examined under oil immersion using a Leitz Dialux micro- antiserum, 4675 were incubated with the indicated concentration of scope with 40x and b00x lenses. Some sections were then sodium channels overnight at 40C in NET buffer containing 1% soaked in xylene to remove the coverslips, rehydrated, and Triton X-100inafinal volume of100 W.. Ten femntomoles of32P-labeled stained with cresyl violet to visualize the cell bodies. sodium channel was added and incubated for 4 hr at 00C. Twenty mnicroliters of 10%6 (wt/vol) fixed Staphylococcus aureus cells (Pansorbin, Calbiochem) was added and, after 30 min, duplicate RESULTS samples were sedimented for 2 min in a Beckman Microfuge and washed twice with NET buffer containing 1% Triton X-100. 32P in the Puriflication and Characterization of Antibodies. The sodi- pellets was determined by liquid scintillation spectroscopy. Percent um channel purified from rat brain is a heterotrimeric of maximum 32P precipitation was plotted vs. sodium channel complex consisting of a (260 kDa), p1 (36 kDa), and 82 (33 concentration as assessed by specific binding of [3H]saxitoxin at 15 kDa) subunits (25). The a and 82 subunits are linked by aM. o, Rat brain with total IgG; o, rat brain with affinlity-purified disuilfide bonds. The simple method for antibody purification antibodies; c, frog brain with total IgG. described above was developed to prepare highly specific antibodies recognizing the a subunit of the brain-type, sistent with the known properties of the a subunit of the rat voltage-sensitive sodium channel. Reactivity of the purified brain sodium channel. There was no appearance of a band of antibodies was determined by radioimmunoassay. The puri- immunoreactivity corresponding to the f32 subunit that was fied antibodies reacted with sodium channels from rat brain released upon reduction, indicating that a majority of anti- in a manner identical to the unpurified IgG fraction (Fig. 1, 0 bodies was directed against the a subunit alone. Fig. 2, lanes and *). The binding of 32P-labeled sodium channels by 3 and 4, illustrate electrophoretic transfer blots ofrat brain P3 purified antibodies and the total IgG fraction was blocked by membranes, prepared as described (18) and analyzed under the same concentrations of unlabeled sodium channels. This reducing and nonreducing conditions. Again, the only visible indicates that the antibody purification procedure provides band of immunoreactivity under nonreducing conditions efficient recovery of active antibodies that react with brain- corresponded to a132 (lane 4) and under reducing conditions type, voltage-sensitive sodium channels. corresponded to a (lane 3). In P3 membranes, sodium Specificity of the purified antibodies was determined by channels comprise <0.1% of the total protein. Thus, affinity electrophoretic transfer blotting. The sodium channel is the purification results in a highly specific probe for voltage- major protein in preparations purified through the step of sensitive sodium channels. Electrophoretic transfer blots chromatography on wheat germ agglutinin-Sepharose as made with whole antiserum showed several additional in- described by Hartshorne and Catterall (18). In electropho- munoreactive bands (not shown), indicating that the purifi- retic transfer blots of this preparation, only one protein band cation provides a significant improvement in the specificity was labeled by the affinity-purified antibody preparation and utility of the antibodies. (Fig. 2, lanes 1 and 2). The band in lane 1 was 300 kDa and Immunocytochemical Lclization of Sodium Channels in corresponded to the a/32 complex observed under nonreduc- the Retina. Photoreceptors, located in the outer nuclear layer ing conditions. In lane 2, the band of immunoreactivity of the retina, and bipolar and horizontal cells, located in the shifted to 260 kDa upon reduction of disulfide bonds, con- inner nuclear layer, generate only graded potentials in ver- Downloaded by guest on September 24, 2021 8426 Neurobiology: Wollner and Catterall Proc. Nad. Acad. Sci. USA 83 (1986)

1 2 3 4 tials that propagate down the optic nerve. Thus, electrophys- iological experiments detect transient sodium currents typi- aU02- -a-do cal of voltage-sensitive sodium channels primarily in the amacrine cells and the ganglion cells of vertebrate retina. Fig. 3 shows 10-Lm sections of rat retina incubated with affminty-purified antibody and stained using the PAP tech- nique. The immunoreactivity was localized to the ganglion cell axon region (Fig. 3A, label a) with some diffuse stain in the inner plexiform layer (Fig. 3A, label ip). No labeling ofthe inner or outer nuclear layers or the outer plexiform layer was observed. This pattern of immunoreactivity is specific, since it was completely blocked (Fig. 3B) by preincubating the antibodies with sodium channels purified to near homogene- FIG. 2. Electrophoretic transfer blot analysis of antibody spec- ificity. A crude synaptosomal fraction (P3, ref. 18) and sodium ity as described (18). Omission ofthe primary antiserum also channels purified through the step ofchromatography on wheat germ prevented specific labeling of the inner plexiform and gan- agglutinin-Sepharose (18) were prepared as described. Their poly- glion cell axon layers (Fig. 3C). These results indicate that the peptide components were separated by NaDodSO4/polyacrylamide staining observed in Fig. 3A corresponded to sodium chan- gel electrophoresis in 7-20% gradient gels using gel system 2 as nels. A higher magnification of the ganglion cell described (25) without (lanes 1 and 4) or with (lanes 2 and 3) reduction revealed that the stain was concentrated on the axons by boiling with 5 mM 2-mercaptoethanol and alkylation by reaction themselves and was not found on the ganglion cell bodies with 15 mM iodoacetamide at pH 10 for 1 hr at room temperature. whose positions are indicated by open arrows (Fig. 3D). The gel was electrophoretically transferred to nitrocellulose paper, Thus, the major area of sodium channel immunoreactivity in and the nitrocellulose was blocked in TBS containing 5% bovine the rat retina is in the ganglion cell axon layer, but not serum albumin for 30 min. The nitrocellulose was incubated over- night at 40C in the antibodies, diluted 1:10 in the blocking solution, ganglion cell bodies. There is also some immunoreactivity in and washed twice in TBS, twice in TBS with 0.05% Triton X-100, and the inner plexiform layer. Immunoreactivity in the axons of then twice in TBS again. This was followed by a 30-min incubation the ganglion cells is expected since these axons form the optic in 125I-protein A in the blocking solution and washing as before. nerve, a central tract whose sodium channels have been Autoradiography was performed to visualize immunoreactive shown to react with our antiserum with high avidity (13). proteins. The retina of the frog (Rana pipiens) is a good choice for subcellular localization of sodium channels, since ganglion tebrate retina (26). The first cells in the pathway of visual cells and their axons are present in large numbers to mediate signal transduction that generate action potentials are the the extensive visual processing that occurs in the frog eye. amacrine cells (27), located at the boundary of the inner The antiserum IgG fraction generated against rat brain nuclear and inner plexiform layers, which generate single, spikes and regulate the activity ofthe only pe on in ip ga sodium-dependent A output neurons of the retina, the ganglion cells. Ganglion k, Iel A m - cells generate rapidly rising, sodium-dependent action poten- t49 1 1 A B C B | ros I axon ip g layer on E

in 3_

a CF *)a _

'p Di- _ 1

axon layer D I.

FIG. 3. Localization of sodium channel immunoreactivity in the rat retina. (A) Ten-micrometer sections of rat retina were stained with the purified antiserum. (B) Similar section ofretina stained with FIG. 4. Localization of immunoreactivity in frog retina. (A) antibodies that had previously been blocked with highly purified Ten-micrometer section of frog retina stained with affinity-purified sodium channels. (C) Section of retina stained with no antibody antibodies and counterstained with cresyl violet. (B) Section stained present during the first antibody incubation. (D) Detail ofA at higher with affmnity-purified antibodies. (C) Section stained with affinity- magnification showing that stain is found in only the axons of the purified antibodies blocked by highly purified sodium channels. (D) ganglion cells. Cell bodies (indicated by open arrows) are not stained Section stained with no antibody in the first incubation. (Bar = 40 above background. The layers of the retina are labeled as follows: a, Aum.) (E) Detail ofB at higher magnification showing that the stain in layer containing axons of the retinal ganglion cells; g, ganglion cell the layer ofthe ganglion cell axons is localized to the individual fibers body layer; ip, inner plexiform layer; in, inner nuclear layer; on, in this layer. Cell bodies (indicated by open arrows) are not stained. outer nuclear layer; and ros, rod outer segment. (Bar: A-C, 40 ,um; (Bar = 10 Am.) Retinal layers are labeled as in Fig. 3; pe, pigment D, 10 pm.) epithelium. Downloaded by guest on September 24, 2021 Neurobiology: WoUner and CatteraU Proc. NatL. Acad. Sci. USA 83 (1986) 8427 sodium channels crossreacts with frog brain sodium channels cells were cut so that the point oforigin ofthe axon on the cell with a decrease in immunoreactivity by a factor of =5 (Fig. body could be visualized. Of these, four rat (Fig. 5 A-G) and 1, o). This is sufficient immunoreactivity to obtain specific three frog (Fig. 5 H-L) cells are shown in Fig. 5. The cell labeling in the present immunocytochemical studies. bodies showed no detectable immunoreactivity on any por- Fig. 4 A and B show the localization of immunoreactivity tion except the periaxonal region. The boundaries of some of in 10-,um sections of the frog retina. As for rat retina, there the cell bodies in the plane of focus presented are illustrated was intense immunoreactivity in the ganglion cell axon layer. in Fig. 5 C, F, I, and L. The axon hillock region of the cells No detectable stain was observed in any other area ofthe frog (large arrow), where the axon originates from the cell body, retina, including the inner plexiform layer. The immunoreac- was intensely immunoreactive. This circular area ofimmuno- tivity in the ganglion cell axon layer can be completely reactivity ranged from about 0.5 to 1.5 ,am in diameter, and blocked by preincubating the antibodies with highly purified the axon appeared to leave the cell from the middle of it, in sodium channels from rat brain (Fig. 4C) or carrying out the the direction opposite to the bold arrow. The initial segment labeling reactions without a primary antibody (Fig. 4D). More of the axon as it leaves this region was also intensely detailed analysis at higher magnification (Fig. 4E) revealed immunoreactive. In optimal sections, as many as 10-15 that the individual fibers in the axon layer were intensely ganglion cells with this pattern of stain could be observed. stained, although the ganglion cell bodies were not. The Adjacent sections stained with blocked antibodies showed no positions of three ganglion cell bodies are indicated by open immunoreactivity. The cells shown in Fig. 5 were selected to arrows. The stain was confined to the axonal layer, indicating illustrate this pattern without interference from the axon that the brain-type sodium channel recognized by our anti- layer or from surrounding cells. These results indicate that bodies was concentrated only in the axons of the ganglion the high-intensity immunoreactivity observed on axon hill- cells. ocks and initial segments ofganglion cells represents specific Specific Localization of Sodium Channel Immunoreactivity staining of sodium channels in those cellular regions. The on Axon Hillocks and Initial Segments of Retinal Ganglion sodium channel subtype recognized by these antibodies is Cells. In these sections ofrat and frog retinas, some ganglion discretely localized to the axon hillock and axon, and this A B C

axon hillock cellA body axon

D E F

axon hillock / ^ BAKE A___ __ axon, cell body A;.. _.

G H I FIG. 5. Detail of stain at the axon hillock and axon initial seg- ments of individual retinal gan- glion neurons. (A-) Rat retinal ganglion cells. (A and B) The I' f }/ s same cell in the plane offocus of /S A* :r: : .. ace the axon and the cell body, re- beU. spectively. (H-L) Different frog ganglion cells. The periaxonal region ofeach cell is indicated by a bold arrow. The axons leave the cells in the direction indicat- ed by the arrow and display in- J K L tense immunoreactivity. (C, F, axon I, and L) Diagrammatic repre- hi llockt,.. sentations of the cells shown in v A.* B, E, H, and K, respectively. of The areas of cell bodies in the 'sheaf e cell -axon plane of section are outlined in ) body the diagrams and show only e background immunoreactivity. (Bar = 10 pm; the photograph in J was magnified 1.5 times the others.) Downloaded by guest on September 24, 2021 8428 Neurobiology: Wollner and Catterall Proc. Natl. Acad Sci. USA 83 (1986) subtype of sodium channels is excluded from the surface of segments, this region also has a high density of sodium the cell body at any other region. channels as established by focal patch clamp recording (33, 34). It is of considerable interest to establish the mechanisms DISCUSSION by which such high-density localizations of sodium channels Sodium Channels Are Present in High Density in Retinal at sites of action potential initiation are established and Ganglion Cell Axons. In previous work, Sarthy et al. (28) maintained. found that retinal ganglion cells, labeled by retrograde transport of injected dye and maintained in dissociated cell We thank Mr. William Downey and Mr. Carl Baker for their culture, are specifically labeled by "251-labeled scorpion excellent technical assistance. We are grateful to Dr. P. V. Sarthy for toxin, confirming the presence of sodium channels in this cell helpful discussions, to Dr. J. A. Beavo for valuable advice and use as inferred from record- of his cryostat, and to Ms. Laurie Hoff for typing the manuscript. type previously electrophysiological This work was supported by Grant T32-GM07750 and R01-NS15751 ings. Other dissociated cells were not labeled above back- from the National Institutes of Health. ground. These results suggested that ganglion cells have the highest density of sodium channels of the various classes of 1. Furshpan, E. J. & Furukawa, T. (1962) J. Neurophysiol. 25, retinal neurons that survive dissociation and cell culture. 732-771. Here we show that ganglion cell axons of adult frog and rat 2. Barrett, J. N. & Crill, W. E. (1980) J. Physiol. 304, 231-249. retina in situ have a high density of sodium channels recog- 3. Coombs, J. S., Eccles, J. C. & Fatt, P. (1955) J. Physiol. nized by our antibodies against rat brain sodium channels. In (London) 130, 291-325. frog retina, no other cell types have detectable levels of this 4. Araki, T. & Terzuolo, C. A. (1962) J. Neurophysiol. 25, sodium channel subtype. In rat retina, fibers in the inner 772-789. plexiform layer are stained. Since amacrine cells 5. Smith, T. J., Jr. (1983) Brain Res. 288, 381-383. specifically 6. Catterall, W. A. (1981) J. Neurosci. 1, 777-783. generate transient sodium currents in some vertebrate spe- 7. Boudier, J. A., Berwald-Netter, Y., Dellmann, H. D., cies (29), it is possible that this lower intensity staining Boudier, J. L., Couraud, F., Koulakoff, A. & Cau, P. (1985) represents sodium channels in the fine, highly branched Dev. Brain Res. 20, 137-142. amacrine cell dendrites, which are reported to generate 8. Haimovich, B., Bonnila, E., Casadei, J. & Barchi, R. (1974) J. conducted action potentials (30). In frog and rat retina, no Neurosci. 4, 2259-2268. neuronal cell bodies were stained by antibodies from serum 9. Fritz, L. C. & Brockes, J. P. (1983) J. Neurosci. 3, 2300-2309. 4675, indicating that the sodium channel subtype recognized 10. Ellisman, M. H. & Levinson, S. R. (1982) Proc. Natl. Acad. by these antibodies is primarily restricted to the axonal region Sci. USA 79, 6707-6711. of these cells. our results do not exclude the 11. Meiri, H., Zeitoun, I., Grunhagen, H. H., Lev-Ram, V., However, Eshhar, Z. & Schlessinger, J. (1984) Brain Res. 310, 168-173. possibility that other sodium channel subtypes may be 12. Costa, M. R. C. & Catterall, W. A. (1984) J. Biol. Chem. 259, present in other cells in the retina or in the cell bodies of 8210-8218. ganglion and amacrine cells. 13. Wollner, D. A. & Catterall, W. A. (1985) Brain Res. 331, Specific Localization of Sodium Channels in Axon Hillocks 145-149. and Initial Segments. Because they have few presynaptic 14. Wollner, D. A. & Catterall, W. A. (1986) Fed. Proc. Fed. Am. boutons containing sodium channels that are reactive with Soc. Exp. Biol. 45, 1008 (abstr.). our antibodies, retinal ganglion cells afford an unusual 15. Bechtel, P. J., Beavo, J. A. & Krebs, E. G. (1977) J. Biol. opportunity to examine the distribution ofsodium channels in Chem. 252, 2691-2697. individual neurons in situ. We find that this subtype of 16. Ritchie, J. M., Rogart, R. B. & Strichartz, G. R. (1976) J. sodium channel not Physiol. (London) 261, 477-494. is only present in axons but is also 17. Catterall, W. A., Morrow, C. S. & Hartshorne, R. L. (1979) J. localized in high density in axon hillocks and axon initial Biol. Chem. 254, 11379-11387. segments. The boundary between the high density of sodium 18. Hartshorne, R. P. & Catterall, W. A. (1984) J. Biol. Chem. channels in the axon hillock and the low density in the 259, 1667-1675. surrounding cell body is remarkably sharp, changing from 19. Olmsted, J. B. (1981) J. Biol. Chem. 256, 11955-11957. maximum to minimum density in <0.5 pum. Although the PAP 20. Maizel, J., Jr. (1971) Methods Virol. 51, 179-224. technique used here does not allow quantitative estimates of 21. Costa, M. R. C., Casnellie, J. E. & Catterall, W. A. (1982) J. channel density, the change in density at the boundary seems Biol. Chem. 257, 7918-7921. almost all or none. Such a precise localization of sodium 22. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Nati. channels at this site to Acad. Sci. USA 76, 4350-4354. appears require immobilization of the 23. Fambrough, D. M. & Devreotes, P. N. (1978) J. Cell Biol. 76, channels as observed in skeletal muscle fibers (31). Interac- 237-244. tion with cytoskeleton, extracellular basement membrane, or 24. Sternberger, L. A., Hardy, P. H., Jr., Cuculis, J. J. & Meyer, a similar immobile anchor is likely to be involved. H. G. (1970) J. Histochem. Cytochem. 18, 315-333. Ultrastructural specializations and specific staining patterns 25. Messner, D. J. & Catterall, W. A. (1985) J. Biol. Chem. 260, with carbohydrate reagents have been described in this 10597-10604. region ofthe , suggesting a concentration ofbasement 26. Werblin, F. S. & Dowling, J. E. (1969) J. Neuro. Physiol. 32, membrane and cytoskeletal elements (32). 339-355. Our results add to a growing body of evidence that sodium 27. Werblin, F. S. & Copenhagen, D. R. (1974) J. Gen. Physiol. channels are present in at sites 63, 88-110. high density ofaction potential 28. Sarthy, P. V., Curtis, B. M. & Catterall, W. A. (1983) J. initiation in excitable cells. In many types of central neurons, Neurosci. 3, 2532-2544. the action potential is initiated at the axon hillock (2-4). 29. Barnes, S. & Werblin, F. S. (1986) Proc. Nati. Acad. Sci. USA Previous results (6) showed that the density of sodium 83, 1509-1512. channels in initial segments ofneurites ofcultured spinal cord 30. Miller, R. F. & Dacheux, R. (1976) Brain Res. 104, 157-162. neurons is 7.4-fold greater than the adjacent cell body. The 31. Stuhmer, W. & Almers, W. (1982) Proc. Nati. Acad. Sci. USA sharp localization of sodium channels in this site observed 79, 946-950. here in retinal ganglion neurons in situ further establishes 32. Waxman, S. G. & Ritchie, J. M. (1985) Science 228, of sodium channels in axon hillocks and 1502-1507. clustering initial 33. Beam, K. G., Caldwell, J. H. & Campbell, D. T. (1985) Na- segments as an important determinant of neuronal excitabil- ture (London) 313, 588-590. ity. In skeletal muscle fibers, the action potential is initiated 34. Roberts, W. M. & Almers, W. (1985) Biophys. J. 47, 189a in the peri-endplate region. Like axon hillocks and initial (abstr.). Downloaded by guest on September 24, 2021