Neuron,

Vol. 5, W-830,

December,

1990, Copyright

0 1990 by Cell Press

Differential Subcellular Localization of Particular mRNAs in Hippocampal Neurons in Culture Robin Kleiman, Gary Banker, and Oswald Steward Department of Neuroscience University of Virginia Health Sciences Center Charlottesville, Virginia 22908

In situ hybridization was used to assess the subcellular distribution of mRNAs encoding several important neuronal proteins in hippocampal neurons in culture. mRNA encoding GAP-43, a protein that is largely excluded from dendrites, was restricted to nerve cell bodies, as were mRNAs encoding neurofilament-68 and 8-tubulin, which are prominent constituents of dendrites and of axons. In contrast, mRNA encoding MAP-2, a protein that isselectivelydistributed indendritesand cell bodies, was present in both dendrites and cell bodies. These results demonstrate that different mRNAs are differentially distributed within individual hippocampal neurons. Taken together with previous findings from other laboratories, our results suggest that only a limited set of mRNAs are available for local translation within dendrites. Introduction Neurons exhibit a remarkable degree of cytological complexity. For example the postsynaptic surface of a neuron consists of a mosaic of different membrane domains associated with individual synaptic contacts, each with a distinctive and characteristic complement of receptors, ion channels, and associated proteins. The mechanisms that neurons use to establish and maintain this highly differentiated receptive surface are not well understood. Many cells, such as polarized epithelia, possess mechanisms for the selective transport of membrane proteins to different regions of the cell (Rindler et al., 1984), and similar mechanisms are likely to be important in neurons as well (Dotti and Simons, 1990). Another strategy that cells may use to construct domains with different protein compositions is based on the differential distribution of particular mRNAs, so that the synthesisof specific proteins is restricted to particular regions of the cell. A differential distribution of mRNAs has been observed in many cell types, including Xenopus oocytes (Melton, 1987), migrating fibroblasts (Lawrence and Singer, 1986), oligodendroglial cells (Trapp et al., 1987), and muscle fibers (Hall and Ralston, 1989). The selective localization of mRNA can give rise to a differential distribution of the corresponding protein products (Pavlath et al., 1989; Ralston and Hall, 1989). For example, in muscle fibers mRNA for the acetylcholine receptor is selectively localized near the muscle endplate (Merlie and Sanes, 1985; Fontaine et al., 1988), and this localization of mRNA is

believed to contribute to the establishment of receptor-rich domains within the muscle membrane. The possibility that such a mechanism exists in nerve cells was first suggested by observations that polyribosomes are selectively localized beneath postsynaptic membrane specializations (Steward and Levy, 1982; Steward, 1983) and that these synapse-associated polyribosomes are particularly prominent during periods of synaptogenesis (Steward and Falk, 1985). Subsequent studies using in situ hybridization to localize mRNAs in brain sections have provided direct evidence for the differential distribution of specific mRNAs in neurons. Garner et al. (1988) reported that mRNA encoding the dendrite-specific protein MAP2 is localized todendrite-rich qreas, such asthe neuropil layers of the hippocampus and cerebral cortex, whereas mRNAs for (3-tubulin and other microtubule-associated proteins are largely confined to neuronal cell bodies (see alsoTucker et al., 1989; Papandrikopoulou et al., 1989). Burgin et al. (1990) found that mRNA for the a subunit, but not the p subunit, of calcium/ calmodulin-dependent protein kinase is present in the neuropil layers of the hippocampus and in layer 1 of the frontal cortex, suggesting a dendritic localization. Studies of tissue sections can provide only limited information about the distribution of mRNA within individual cells. Hence it is not known how far into the dendritic tree specific mRNAs extend, whether they are uniformly distributed or locally concentrated within dendrites, or what proportion of specific mRNAs are localized in dendrites versus cell bodies. It is particularly difficult to evaluate the possible dendritic distribution of mRNAs that are not neuron specific, because neuropil zones contain glial cells and their processes, as well as dendrites (for example, see Phillips et al., 1987). For these reasons, we have been interested in developing a cell culture approach that permits a more accurate evaluation of the distribution of different mRNAs within individual neurons and that might ultimately be used to examine the cellular mechanisms underlying mRNA sorting. For this purpose we have used low density cultures of hippocampal neurons, since they form well-developed dendrites that can be readily identified based on morphological and immunocytochemical criteria (Bartlett and Banker, 1984; Caceres et al., 1986; Goslin et al., 1988). Cultured hippocampal neurons have also been shown to possess a mechanism for transporting newly synthesized RNA into the dendrites (Davis et al., 1987, 1990), similar to that present in vivo (R. Kleiman, G. Banker, L. Davis, and 0. Steward, 1989, Sot. Neurosci., abstract). In the study presented here, we have examined the subcellular distribution of the mRNAs for four representative neuronal proteins-the high molecular weight form of MAP2, GAP-43, P-tubulin, and neurofil-

ament-68 (NF-68). MAP2 is a neuron-specific microtubule-associated protein that is present in cell bodies and dendrites, but is excluded from axons. As described above, its mRNA has been shown to extend into the dendrites of hippocampal neurons in situ. GAP-43 (also known as Fl or B50) is a neuron-specific phosphoprotein that is concentrated in the axons of hippocampal neurons, especially in the growth cones of developing axons, but is absent from dendrites (Goslin et al., 1988, 1990). NF-68, the low molecular weight neurofilament subunit, isalsoa neuron-specific protein, but it is found in both axons and dendrites (Shaw et al., 1985). B-Tubulin also is present throughout nerve cells and in glial cells. All these proteins are believed to be translated on free polyribosomes, although GAP-43 becomes associated with membranes following posttranslational fatty acylation (Skene and Virag, 1989). We report that the mRNAs for B-tubulin, NF-68, and GAP-43 are all largely restricted to the cell body, occasionally extending for a short distance into the proximal dendrites. In contrast, MAP2 mRNA extends far into the dendritic tree, although its distribution is not uniform Some dendrites are unlabeled, and within labeled dendrites the distribution of MAP2 mRNA often appears patchy. Results mRNA Distribution in the Hippocampus To confirm that the cRNA probes used in the present study produced patterns of labeling comparable to those previously described and to allow comparison of the subcellular distribution of labeling in cultured cells with that observed in situ, we examined the distribution of labeling following hybridization of each probe to rat brain sections. In general, we observed two distinct patterns of mRNA distribution in the hippocampus, one in- which labeling was largely restricted to cell bodies and another in which neuropil layers were also heavily labeled (Figure 1). Hybridization of tissue sections with the probe to 8-tubulin produced heavy labeling over neuronal cell bodylaminae in the hippocampus (Figure IA). Only very light labeling was observed over neuropil layers, which are composed of glial cells, the dendrites of neurons whose cell bodies lie in the pyramidal and granule cell layers, and incoming axons, which contain little or no RNA. Hybridization with the probe for NF-68 produced a pattern of labeling similar to that observed with the B-tubulin probe (Figure IB). A comparable pattern was also seen with the probe for GAP-43 mRNA (Figure ID), except that dentate granule cells exhibited little labeling. Rosenthal et al. (1987) have previously noted that granule cells contain little GAP43 mRNA. In contrast, with the MAP2 probe, labeling over neuronal cell body layers was comparatively light, whereas labeling within neuropil zones was quite prominent (Figure IC). These differences are consistent with those previously described by Garner

and colleagues (Garner et al., 1988; Tucker et al., 1989). Tracts of axons, which contain little or no mRNA, were labeled at background levels with all probes, except for the labeling of occasional cell bodies (presumably glial) with the 8-tubulin probe. Hybridization with sense probes produced a uniform pattern of labeling over the entire tissue section, with no evidence of selective labeling of cell bodies or neuropil zones (data not shown). Because we were concerned about possible crosshybridization of the probe for MAP2 mRNA with ribosomal RNAs (see Experimental Procedures), we examined the pattern of labeling obtained using a probe for rRNA (see Figures IE and IF). In contrast to the pattern of labeling produced by the MAP2 probe, hybridization with the cDNA probe to rRNA gave rise to labeling that was heaviest in cell body layers and light in neuropil zones. In fact, the distribution of labeling with the rRNA probe might give the false impression that rRNA was not present in neuropil layers. This is not true, however, since ribosomes are present in dendrites and in glial cells and their processes. The heavy labeling of cell bodies, compared with dendritic laminae, presumably reflects the extremely dense packing of cells in the pyramidal and granule cell layers. Thus the pattern of labeling produced by probes for 8-tubulin, GAP-43, and NF-68 mRNAs in sections of the hippocampus, which resembles that seen with the rRNA probe, does not necessarily imply that these mRNAs are excluded from dendrites. Pattern of Hybridization to Neurons in Culture The pattern of labeling of hippocampal neurons in culture following hybridization with each of the probes is illustrated in Figure 2. In general, we observed two patterns of labeling, which were comparable to the two patterns seen in sections of the hippocampus. Autoradiographs of cells that were hybridized with the cRNA probes for 8-tubulin, NF-68, and GAP-43 exhibited a tight localization of silver grains over the cell body (Figures 2A-2F). In contrast, both cell bodiesand processeswere labeled following hybridization with the probe to MAP2 (Figures 2G and 2H). As judged by their number, length, and orientation, the labeled processes corresponded to dendrites. The MAP2 probe produced approximately equivalent labeling of cell body and dendrites across a broad range of exposure times. The great majority of cells that showed cell body labeling also showed dendritic labeling. Even when cell bodies were only lightly labeled, labeling of dendrites was still evident. Hybridization with sense probes was used to assess the specificity of labeling. As shown in Figure 3F, hybridization with a sense probe from the MAP2 clone produced no labeling of neurons or glial cells. The pattern of cell types labeled with each probe also served as a measure of cross-hybridization and nonspecific binding. The expression of MAP2, NF-68, and GAP-43 is generally restricted to neurons, although glial cells have been reported to express MAP2 and

Subcellular 823

Figure

Localization

of mRNA

1. In Situ Hybridization

in Cultured

to Localize

Neurons

mRNAs

in the

Rat Hippocampus

(A-D) Dark-field photomicrographs illustrating sections through the hippocampus that were hybridized with probes to detect mRNAs encoding the following proteins: (A) P-tubulin; (B) NF-68; (C) MAPZ; (D) CAP-43. (E and F) Higher magnification views of the CA1 field comparing the distribution of MAP2 mRNA (E) and rRNA (F). (A)-(E) are horizontal sections; (F) is a coronal section. Abbreviations: pcl, pyramidal cell layer; hf, hippocampal fissure; ml, molecular layer; gel, granule cell layer; DC, dentate gyrus. Bar, 330 pm (A-D); 130 urn (E); 100 urn (F).

Subcellular

Localization

of mRNA

in Cultured

Neurons

a25

Figure

3. Specificity

of In Situ Hybridization

(A-D) Dark-field images of nonneuronal cells mRNA, 22 day exposure; (B) NF-68 mRNA, 23 Arrows denote the location of nonneuronal probes derived from MAP2 cDNA. Exposure

Figure

2. In Situ Hybridization

Cultured exposure; hybridized resultant

to Localize

neurons were hybridized (E and F) CAP-43, 19 day with 35S-labeled probes autoradiographs. Bar, 25

present in hippocampal cultures following hybridization with cRNA probes for (A) B-tubulin day exposure; (C) CAP-43 mRNA, 19 day exposure; and (D) MAP2 mRNA, 29 day exposure. cells. (E and F) Dark-field images of neurons hybridized with (E) antisense and (F) sense times were 29 and 28 days, respectively. Bar, 25 urn.

mRNAs

in Hippocampal

Cell Cultures

with probes for the mRNAs indicated: (A and B) B-tubulin, 18 day exposure; (C and D) NF-68,22 day exposure; (G and H) MAP2, 17 day exposure. Cells were fixed after about 2 weeks in culture and as described in Experimental Procedures. Shown are dark-field and phase-contrast images of the urn.

Figure

4. The Distribution

of MAP2

mRNA

within

Dendrites

of Cultured

Hippocampal

Neurons

The distribution of grains over labeled dendrites is patchy (arrowheads), and some dendrites are unlabeled (arrows). The Image-l Analysis System (Universal Imaging) was used to estimate the extent of labeling. In (A), the white line superimposed over the dendrite shows the distance measured, 140 urn in this case. Note that the glial cells in thus field (g) are unlabeled. (A) Dark-field; (B) phasecontrast.

GAP-43 under some conditions (Geisert et al., 1990; daCunha and Vitkovic, 1990). We observed little or no glial labeling with the probes for NF-68 or GAP-43 at exposure intervals that produced heavy labeling of neurons (Figures 3B and 3C). Glial labeling following hybridization with MAP2 probes varied somewhatsome cells were lightly labeled and others were unlabeled (Figure 3D; Figure4). However, the level of labeling over glia was always much lower than that over neurons. In contrast, hybridization with the probe for B-tubulin mRNA, which is found in all cell types, labeled glial cells to about the same extent as neurons (Figure 3A). These results indicate that the labeling observed following hybridization was specific. Because the entire dendritic tree of an individual cell is visible in culture, considerably more detail concerning the dendritic distribution of mRNA could be

Figure

5. The

Distribution

Neurons were hybridized range of labeling patterns

of GAP-43 with the observed.

mRNA

in Cultured

probe to GAP43 Bar, 25 urn.

Hippocampal

and exposed

discerned compared with that evident in sections. Following hybridization with the MAP2 probe, the pattern of labeling generally reflected the morphology of the dendrites, but it was notably inhomogeneous. Labeling was generally heavier over the thicker, proximal portions of the dendrites and often could be followed intodendritic branches(see Figure4), but rarely extended into theveryfine, distal dendritic processes. Moreover, the pattern of labeling in dendrites was often patchy, with clusters of silver grains separated by regions without grains (Figures 2G and 2H; Figure 3E; Figure 4). Even in heavily labeled cells, 1 or 2 dendrites were frequently unlabeled. In some neurons hybridized with the GAP-43 or B-tubulin probes and exposed for periods sufficient to produce heavy labeling of cell somata, labeling extended over proximal dendrites (Figure 5). The limited dendritic labeling

Neurons for 19 days.

Two

cells,

both

from

the same

coverslip,

illustrate

the

Subcellular 027

Localization

of mRNA

in Cultured

Neurons

with GAP-43 and 8-tubulin was distinctly different from that observed for MAP2 mRNA in that labeling extended for only a short distance along dendrites and often only 1 or 2 dendrites were labeled. To estimate how far into the dendrites MAP2 mRNA extended, we measured the distance of continuous labeling over 28 labeled dendrites from 10 cells. A dendrite was considered to be continuously labeled if there were no unlabeled segments greater than 20 vrn in length. On average, labeling with the MAP2 probe extended for 113 f 51 urn (mean f SD). This is a conservative estimate of the distance of labeling, because we did not include more distal segments if the labeling was discontinuous. In hippocampal cultures at this age, the cells’ longest dendrites are about 160 urn long (Davis et al., IVVO), suggesting that MAP2 mRNA is present along most of the length of labeled dendrites. In contrast, in that minority of cells that exhibited dendritic labeling following hybridization with the probe for GAP-43 mRNA, labeling extended for only 32 & 20 Pm (25 dendrites from 12 cells). To provide an estimate of the relative abundance of MAP2 message in the dendrites as compared with the cell body, we selected 12 neurons and counted the number of grains over cell bodies and dendrites. On average, 69% f 7% of the total silver grains associated

following in situ hybridization derives from incomplete fixation of mRNAs, or some other technical artifact. If not, it suggests that specific mRNAs may be differentially distributed within the dendrites. Polyribosomes are selectively localized within dendrites at sites near synapses (Steward and Levy, 1982; Steward, 1983), and it has been hypothesized that the selective localization of mRNAs may contribute to the development of the mosaic pattern of postsynaptic receptors (Steward et al., 1988). The demonstration that neuronal mRNAs are differentially distributed immediately raises questions concerning the cellular mechanisms responsible for their selective segregation. Because only a limited number of mRNAs appear to be present in dendrites, it is tempting to suppose that in the default case mRNA is simply nottransported outof the cell body.According to this hypothesis, the presence of a specific signal sequence, either within the mRNA itself or within the encoded protein, would be required to target the mes-

Discussion

sage for transport into the dendrites. Restriction of mRNAs to specific sites within the dendrites would require some additional mechanism, such as interaction with the dendritic cytoskeleton. In Xenopus oocytes, translocation of a specific message, Vgl, toward the vegetal pole requires microtubules, and actin filaments are responsible for maintaining it in that location (Yisraeli et al., 1990). Alternatively, it is possible that signal sequences cause some mRNAs to be restricted to the cell body, perhaps by blocking their interaction with the dendritic transport machinery. The availability of cell culture systems suitable for studying the transport and localization of RNA should be useful for resolving such issues.

Neurons Exhibit at least Two Distinct Patterns of mRNA Distribution Our observations of hippocampal neurons confirm and extend previous studies demonstrating a differential subcellular distribution of mRNAs within neurons. We found that mRNAs for 8-tubulin, GAP-43, and NF-68 are localized almost exclusively to the cell body, whereas mRNA for MAP2 extends far into the dendritic tree. In fact, our results suggest that the majority of the MAP2 mRNA present in a neuron is localized within the dendritic compartment. Similar findings have recently been reported for sympathetic neurons in culture (Bruckenstein et al., 1990). Additional studies will be required to determine whether all mRNAs are distributed in one of these two distinct patterns, or whether there are other patterns of message distribution as well. Our results also suggest that MAP2 message is not uniformlydistributedwithin thedendritictree. Labeling often has a patchy distribution, and some dendrites are not labeled at all. The dendritic labeling observed following incorporation and transport of [3H]uridine also often has a patchy character, although all dendrites appear to be labeled (Davis et al., 1990). It is possible that the uneven appearance of labeling

The Significance of Dendritic Protein Synthesis No clear pattern has yet emerged concerning the types of proteins that are synthesized within dendrites. What seems most surprising is the limited number of mRNAs that have been identified in dendrites so far. Certainly many proteins present in dendrites are not synthesized there. For example, we found that mRNAs encoding 8-tubulin and NF-68, two prominent constituents of the dendritic cytoskeleton, appear to be localized exclusively in the cell body. This suggests that dendritic protein synthesis may play some special role in neuronal function. One obvious possibility is that the dendritic localization of specific mRNAs is related to protein compartmentalization. For example, the synthesis of MAP2 in dendrites and its rapid association with dendritic microtubules may prevent its entry into the axon (Garner et al., 1988). It is difficult, however, to extend such arguments to calciumlcalmodulin-dependent protein kinase, an enzyme presumably present throughout thecell.ThemRNAsencodingthetwosubunitsofthis enzyme are differentially distributed, one extending into the dendrites and the other apparently restricted to the cell body (Burgin et al., 1990). This would suggest that the selective targeting of mRNAs in neurons

with these cells were localized over dendrites. Because the cell body is thicker than the dendrites, limiting the efficiency of grain production, this value probably underestimates the amount of somatic mRNA. Nonetheless it is apparent that dendrites contain a significant proportion of the cell’s MAP2 message.

Neuron 828

may have functions other than simply restricting the distribution of the proteins they encode. For example, the dendritic localization of a limited subset of mRNAs may permit the local regulation of their translation, perhaps including regulation by local synaptic activity. The significance of dendritic protein synthesis should become clearer as a more complete catalog of the mRNAs present in dendrites emerges. Experimental

Procedures

Cell Culture System Hippocampal cultures were prepared as previously described (Banker and Cowan, 1977; Coslin and Banker, 1991). Briefly, hippocampi from El&E19 rat fetuses were collected and incubated in 0.25% trypsin for 15 min at 37OC. Neurons were dispersed by trituration and plated onto polylysine-treated glass coverslips at a density of 50,000 cells per 60 mm dish. After the neurons had attached, the coverslips were transferred to dishes containing monolayer cultures of astroglia and maintained in serum-free medium. Some cultures were treated with cytosine arabinoside (5 x 1O-b M) on day 4 or 5 to reduce glial proliferation. Preparation of Cultured Neurons for Hybridization The procedure for fixation and storage followed that recommended for cultured cells by Lawrence and Singer (1985). Coverslips that had been maintained for 14-15 days in vitro were fixed for IO;15 min in 4% paraformaldehyde, 4% sucrose in PBS (warmed to 37OC). Coverslips were then rinsed in PBS, transferred to 70% ethanol, and stored at 4’C until prehybridization. Preparation of Tissue Sections for Hybridization Adult male rats were perfused with 0.4% or 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). Their brains were removed, placed overnight in 30% sucrose, 4% paraformaldehyde, and sectioned at 20 lrn on a cryostat. Sections were thawmounted onto polylysine-coated microscope slides and stored at -8OOC. Preparation of cRNA Probes The cDNA for the MAP2 probe was provided by A. Matus and has been characterized previously(clonel9a; Garner et al., 1988). The cDNA was a 1.3 kb fragment complementary to the 3’ end of the coding region of the 9.2 kb mRNA for MAPL. It hybridizes selectively with the mRNA encoding the high molecular weight form, but not the low molecular weight form, of MAP2 (Garner et al., 1988). The cDNA was cloned into the EcoRl site of a pBluescript I I KS vector and was linearized with the restriction enzyme Pstl, which also cuts the insert. Transcription from the T3 promoter produced 600 baseantisense probes, containing 510 bases of insert. Linearization with Sal1 and transcription from the SP6 promoter produced 1315 base sense probes. The chicken g-tubulin cDNA was a gift from M. Kirschner (Cleveland et al., 1980). The 1.25 kb Pstl-Pstl fragment is complementary to part of the coding region of the 1.85 kb B-tubulin mRNA. It was cloned into the Pstl site of a pGEM-2 plasmid and supplied to us by D. Chikaraishi. This plasmid was linearized with Hindlll, and 1305 base antisense cRNA probes were transcribed from the SP6 promoter. The 1.25 kb cDNA for mouse NF-68, a gift from N. Cowan, is complementary to a portion of the coding region (Lewis and Cowan, 1985). It was cloned into the EcoRl site of a Bluscript Ml3 plasmid and linearized with Xmalll, which also cuts the insert. Transcription from the T3 promoter produced a 653 base antisense probe. The plasmid containing the GAP-43 cDNA was a gift from A. Routtenberg~Rosenthaletal.,1987).ThiscDNA,a1.3kbfragment complementary to the coding region of the 1.5 kb mRNA, was cloned into the EcoRl site of a pCEM-3 vector. The plasmid was linearized with PVAII, which also cuts the insert, yielding a cRNA of 743 bases, containing 695 bases of insert when transcribed using the T7 promoter.

All the probes were labeled by transcription presence of [??]UTP, as described by Maniatis specific acitivty of the antisense probes ranged 5.6 x IO8 cpm per ug of RNA.

reactions in the et al. (1982). The from 3.1 x 108 to

Northern Blot Analysis Northern blot hybridization was used to assess the specificity of the cRNA probes. RNA was isolated from rat cortex by guanidinium isothiocyanate extraction followed by CsCl gradient centrifugation (Chirgwin et al., 1979; Ausubel et al., 1987). PolyfA) RNA was obtained by passing the total RNA over oligofdn columns. Eithertotal RNAor poly(A) RNAwas separated byelectrophoresis on denaturing 1.4% agarose-glyoxal gels. The gels were then equilibrated in 45 mM Tris-acetate, 1 mM EDTA (pH 8.0), and the RNA was transferred to a nylon membrane (Nytran; Schleicher & Schuell, Inc.) and baked at 6S°C (Maniatis et al., 1983). The membranes were hybridized at 65OC with lw cpm of the labeled probes in a buffer consisting of 50% formamide, 5x SSPE (1 x SSPE consists of 0.18 M NaCI, 1 mM EDTA, ?nd 10 mM phosphate buffer [pH 7.71),5x Denhardt’s solution (5x Denhardt’s solution consists of 0.1% BSA, 0.1% polyvinylpyrrolidone, 0.1% Ficoll), 1% or 7% SDS, 100 pglml tRNA, and 50 Pglml salmon sperm DNA. The membranes were washed once in 1 x SSPE containing 1% SDS (15 min at 6YC) and three times in 1 x SSPE. They were then treated with RNAase (IO pglml in 5x SSC [lx SSC consists of 0.15 M NaCl and 0.015 M sodium citrate]) for 30 min at 40°C and washed in 1 x SSPE, 1% SDS (15 min at 20°C) followed by 0.1x SSPE, 1% SDS (30 min at 60°C). The membranes were dried, sprayed with Amplify (Amersham), and exposed to Kodak X-Omat AR film at -70°C for 72 hr. Following hybridization to blotsof brain RNA, the NF-68 probe labeled two bands of 2.5 and 4.0 kb, the GAP-43 probe labeled a single band of 1.5 kb, and the B-tubulin probe labeled a single band of 1.9 kb, as expected from previous results (Lewis and Cowan, 1985; Rosenthal et al., 1987; Cleveland et al., 1980). We were unable to obtain satisfactory blots using the MAP2 probe. When blots of poly(A) or total RNA were hybridized with the MAP2 probe there was no detectable band at 9.2 kb, although there was labeling of 18s and 28s rRNAs on blots of total RNA. The lack of labeling of a 9.2 kb band presumably is due to technical problems associated with transblotting such a large mRNA. As described in Results, hybridization in tissue sections gave a pattern of labeling identical to that reported by Garner et al. (1988) using an equivalent cDNA probe and quite different from that produced with a probe to rRNA (Figures IE and IF).

Hybridization of Cultured Neurons Fixed cells were washed for 10 min in PBS containing 5 mM MgCI,, 10 min in 0.2 M Tris with 0.1 M glycine (pH 7.4), and in some cases for an additional 10 min in 2x SSC, 50% deionized formamide. Coverslips were pretreated for 1-3 hr at 42OC in hybridization buffer consisting of 50% formamide, 0.3 M NaCI, 20 mM Tris (pH 8.0), 5 mM EDTA, 1 x Denhardt’s solution, 10% dextransulfate,and10mMdithiothreitol.AlOO~Idropofhybridization buffer was then placed in the center of a 35 mm sterile plastic petri dish. An aliquot of probe containing 10 mglml tRNA was heated to 95OC for 3 min. For each coverslip, 4 x 106 cpm of probe mix was added to the drop of hybridization buffer. Coverslips were placed cell side down on this hybridization mixture. The petri dishes were transferred to a chamber humidified with 4x SSC, 50% formamide and hybridized overnight at 55’C. Each coverslip was transferred to a fresh 35 mm plastic petri dish, cell side up, and rinsed twice in 2 x SSC, 10 mM B-mercaptoethanol, 1 mM EDTA for 10 min. To hydrolyze nonspecifically bound probe, coverslips were treated with RNAase (2 kg/ml in 500 mM NaCI, 10 mM Tris [pH 8.01) for 30 min at room temperature. Following two more 10 min rinses in 2x SSC containing 10 mM B-mercaptoethanol and 1 mM EDTA, coverslips were washed for 2 hr at 55°C in a “stringency” buffer containing 0.1 x SSC, 10 mM B-mercaptoethanol, and 1 mM EDTA. Coverslips were then washed twice in 0.5x SSC, dehydrated through graded alcohols containing 0.3 M ammonium acetate, and allowed to air dry.

Subcellular 829

Localization

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in Cultured

Neurons

Hybridization of Tissue Sections Hybridization was carried out as described by Rosenthal et al. (1987). The mounted sections were postfixed for IO min in 4% paraformaldehyde in phosphate buffer, pretreated with proteinase K for IO-30 min, washed in 0.5x SSC, dried with a Kimwipe, and placed flat on 3 mm thick glass bars in a 150 mm petri dish kept humidified with formamide-containing buffer. The sections were covered with 100 PI of hybridization buffer (without the probe) and incubated for l-3 hr at 42’C. Following heating (as described above), a 20 ~1 aliquot of hybridization buffer containing 1-2 ~1 of probe and 2 kg of tRNA was added to the hybridization buffer. A 22 x 22 mm coverslip was placed over the hybridization mix, and hybridization was carried out overnight at 55OC. The following day, the coverslips were removed, and the slides were treated with RNAase and subjected to a stringency wash as described above. The sections were then dehydrated in graded ethanols containing ammonium acetate and dried. Autoradiography Coverslips with attached cells were mounted cell side up on clean slides with a drop of Cytoseal 280 (Thomas Scientific) and allowed to dry for 24 hr. The slides were coated with Kodak NTB2 emulsion that had been diluted I:1 with water and allowed to dry. They were then placed into slide boxes containing desiccant and stored at 4OC for 2-4 weeks. Autoradiographs were developed with Kodak D-19 for 3 min at 15X, fixed, rinsed in running water, and coverslipped with Kaiser’s glycerol jelly. Similar procedures were used for tissue sections, except that they were stained with cresyl violet after development, dehydrated, and coverslipped with Permount or Histoclad. Acknowledgments We thank Drs. A. Matus, N. Cowan, A. Routtenberg, and M. Kirschner, who provided cDNA clones for MAPZ, NF-68, CAP-43, and J3-tubulin, and to D. Chikaraishi for recloning the neurofilament and tubulin cDNAs into riboprobe vectors. Thanks to Dr. Ted Mifflin and Regina Matulaitis of the Molecular Probe Laboratory of the University of Virginia for producing the labeled cRNA probes used in this study and to Drs. Pat Trimmer and Enrique Torre for their help in developing techniques used in this study. This research was funded by NIH grant NS23094 (to C. B. and 0. 5.); R. K. received support from NIH training grant HD07323. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

August

28, 1990; revised

October

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