Proc. Nati. Acad. Sci. USAVol. 88, pp. 2093-2097, March 1991Neurobiology

Dendritic location of neural BC1 RNA(polarity of neurons/RNA polymerase III transcript/in situ hybridization)

HENRI TIEDGE*, ROBERT T. FREMEAU, JR.*t, PETER H. WEINSTOCK*, OTTAVIO ARANCIO*§,AND JURGEN BROSIUS**Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029; and tDepartment ofPhysiology and Cellular Biophysics, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032

Communicated by Richard L. Sidman, December 19, 1990

ABSTRACT In nerve cells, a specialized protein syntheticmachinery is thought to operate in local compartments ofdendrites, in particular beneath synaptic junctions, andthereby to facilitate swift adjustments of the postsynapticprotein repertoire in situ. This notion has been supported bythe identification of polyribosomes and selected mRNAs inthose compartments. In this study, we report the discovery ofa specific RNA polymerase III transcript in dendrites. ThisRNA, a noncoding, 152-nucleotide-long, single-gene transcriptknown as BC1 RNA, is expressed almost exclusively in thenervous system. In adult rats as well as in immature rats in latedevelopmental stages, BC1 RNA has been located in thedendrites and somata of a subset of neurons in the central andperipheral nervous system. The colocalization of BC1 RNAwith dendritic mRNAs and polyribosomes may indicate arole-possibly within the functional unit of a high molecularmass ribonucleoprotein particle-in specific pre- or posttrans-lational processes in postsynaptic compartments of neurons.

The presence of RNA in dendrites constitutes an importantelement of molecular polarity in nerve cells (1-11). RNA isactively transported into dendrites but not into axons ofcultured hippocampal neurons (1). In the dentate gyrus,polyribosomes have been detected in postsynaptic dendriticcompartments, frequently in close association with dendriticspines (2-5). Postsynaptic accumulation of polyribosomeshas been shown to be most dramatic during periods ofdevelopmental synaptogenesis (4), and it has been proposedthat proteins synthesized on these ribosomes may be in-volved in the formation, differentiation, and modification ofsynapses (2-4).Although most neuronal mRNAs seem to be confined to

the perikarya of nerve cells, a small number of mRNAs, allof them coding for dendritic proteins, has recently beendetected in the somatodendritic compartments of neurons inthe rat nervous system. One ofthem is the mRNA for the highmolecular mass form of microtubule-associated protein 2(MAP2, refs. 6-8), a tubulin-binding protein associated withthe dendritic cytoskeleton (see ref. 12 for a review). Anotherdendritic mRNA encodes the a-subunit of Ca2+/calmodulin-dependent protein kinase type II (CaM-KII) (9). CaM-KII ishighly concentrated in postsynaptic densities and has beenimplicated in signal transduction mechanisms and in theinduction of long-term potentiation (see ref. 13 for a review).In addition, the selective targeting of a subset of voltage-dependent calcium channels (VDCCs) to dendrites of hip-pocampal neurons raises the possibility that dendritic VD-CCs might arise from local translation ofVDCC mRNA (14).Based on these observations, it has been proposed (1-10)

that a specialized protein synthetic machinery may operate inpostsynaptic compartments of dendrites. Selected dendritic

proteins would thus be synthesized locally, close to therespective postsynaptic sites where they are required. Thismodel is interesting because it would allow a decentralizedand more flexible regulation of postsynaptic protein pools,facilitating, for example, long-term responses to modulationsof local synaptic activity. Such a mechanism would require aselective sorting and targeting of individual mRNAs to dif-ferent postsynaptic loci. Ribonucleoprotein particles (RNPs)such as the signal recognition particle (15) have been impli-cated in the sorting and targeting of protein-ribosome-mRNA complexes. However, a candidate RNP has not yetbeen identified that would qualify for a role in sorting,targeting, or processing of dendritic mRNAs.

In the present study, we used in situ hybridization tech-niques to identify BC1 RNA (16, 17), a short RNA polymer-ase III transcript, in the somata and dendrites of a subset ofneurons in the rat nervous system. Like other RNAs of thistype, BC1 RNA does not code for any protein but appears tobe part of a high molecular mass RNP. The specific locationof BC1 RNA may indicate a functional role related to pre- orposttranslational processes in the somatodendritic compart-ments of neurons.

MATERIALS AND METHODSGeneration of Probes. The 5' part of BC1 RNA shares

sequence homology with the family of repetitive ID elements(16, 17). To prevent hybridization to otherRNAs that containID elements (18), we used probes that correspond to theunique 3' part of BC1 RNA (17). A transcription vector(pMK1) containing a sequence that represents the 60 3'-mostnucleotides of BC1 RNA (17) was constructed by inserting achemically synthesized piece of DNA with the appropriaterestriction site termini between the Kpn I and the Sac I sitesofpBluescript KS (+) (Stratagene). A transcription vector forgenerating probes specific for growth-associated protein 43(GAP-43) mRNA has been described earlier (19). 35S-labeledRNA probes were transcribed from linearized templates,using SP6, T3, or T7 RNA polymerase as recommended bythe manufacturers (Promega and BRL).In Situ Hybridization. Acutely isolated neurons were pre-

pared from spinal cords of8-day-old rats as described (20). Thecells were allowed to settle for 1 hr on poly(D-lysine)-coatedLab-Tek four-chamber slides (Nunc) before fixation. In situhybridization histochemistry was performed as described (21,22). Briefly, formaldehyde-fixed specimens on microscopeslides were illuminated with wide-spectrum UV light (germi-cidal UV lamp, 30 W) for 12 min at a distance of 30 cm to

Abbreviations: RNP, ribonucleoprotein particle; MAP2, microtubule-associated protein 2; CaM-KII, Ca2+/calmodulin-dependent proteinkinase type II; GAP-43, growth-associated protein 43.tPresent address: Department of Neurobiology, Duke UniversityMedical Center, Durham, NC 27710.§Present address: Istituto di Fisiologia, Universita di Verona, 1-37100Verona, Italy.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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postfix the biological material and to stabilize it on gelatin/poly(L-lysine)-coated slides (21). In control experiments, aproteinase K step was included to ascertain accessibility ofthetarget RNA. Prehybridization and hybridization were per-formed at 50'C; the probe concentration was 3 x 106 cpm/ml.After hybridization, dried slides were dipped in NTB2 emul-sion (Kodak) diluted 1:1 in water, air-dried, and exposed at40C. The exposure time for tissue sections hybridized withBC1 probes was 3 days. Tissue sections hybridized withGAP-43 probes were exposed for 18 days to match the labelingintensity of the more abundant BC1 RNA. Slides with acutelyisolated neurons were exposed for 2 weeks (BC1 RNA) or 8weeks (GAP-43 mRNA), respectively.

RESULTSWe used specific single-stranded RNA probes to analyze thedistribution of BC1 RNA in the rat central and peripheralnervous system. The BC1 labeling pattern is nonuniform anddistinct, yet apparently unrelated to specific cell types ortransmitter/receptor systems (Fig. 1). High levels of expres-sion are restricted to gray matter areas including, amongothers, those of the spinal cord and the neocortex. Whitematter areas that contain axonal bundles and glial cells showhybridization signals that are barely above background.Staining patterns obtained with antibodies to glial markerssuch as glial fibrillary acidic protein, myelin basic protein, orproteolipid protein did not correlate with the BC1 RNAhybridization pattern (data not shown).

Observations at the cellular level in various nervous tissuesindicate that BC1 RNA is expressed by many, but not all,types of neurons. In dorsal root ganglia, the density of silvergrains is highest over the somata of large sensory neurons(Fig. 2 A and B). Adjacent cells show much lower expressionlevels, and the lowest grain density is seen over fiber tractareas that contain axons of the sensory neurons and glialsupport cells. The subcellular distribution of BC1 RNA wasanalyzed in stratified neural tissues. In the retina (Fig. 2 C andD), a high concentration of silver grains was seen over theinner plexiform layer, whereas a weaker hybridization signalwas evident in the ganglion cell layer and the innermost partof the inner nuclear layer. Little or no specific labeling wasobserved over the outer nuclear layer and the outer plexiformlayer. The high levels of expression in the inner plexiformlayer indicate an extrasomatic location of BC1 RNA. As theinner plexiform layer contains a dense synaptic network (25),with interactions among processes arising from bipolar cells,amacrine cells, and ganglion cells, the observed labelingpattern suggests that BC1 RNA may be located in at leastsome of these processes.Analogous expression patterns were observed in laminated

brain areas including the neocortex, the olfactory bulb, andthe hippocampal formation. In the CA3 field of the hippo-campus (Fig. 3A), BC1 labeling was most intense in thestratum pyramidale, the layer that contains the somata andthe proximal dendrites of pyramidal cells. Additionally, ahigh grain density was evident over the stratum oriens and thestratum radiatum. These layers contain the basal and thedistal apical dendrites, respectively, of the CA3 pyramidalcells (26) and have previously been shown to contain thedendritic mRNAs for high molecular mass MAP2 (6) and forthe a-subunit ofCaM-KII (9). In contrast to these layers, thestratum lucidum, a layer that contains the proximal segmentsof the apical CA3 pyramidal dendrites and axons of thedentate granule cells (26), shows a much lower BC1 hybrid-ization signal. This indicates a higher concentration of BC1RNA in the distal relative to the proximal parts of the apicaldendrites of CA3 pyramidal cells. Although the significanceof this parcellation of BC1 RNA remains to be established, itshould be noted that it parallels an interesting physiological

A

OB

HipCx SC

CPu Cb

AA

B

C

FIG. 1. Distribution of BC1 RNA in the adult rat brain and spinalcord, revealed by in situ hybridization with a single-stranded 35S-labeled cRNA probe. (A and B) A complementary ("antisensestrand") RNA probe was used to locate BC1 RNA in a parasagittalbrain section (A) and in three transverse sections through the spinalcord (B). (C) A "sense strand" RNA probe was used in a controlexperiment with a parasagittal brain section. The parasagittal sectionscorrespond to plates 81a/82 in the atlas of Paxinos and Watson (23).Labeling intensities are indicated by the darkness of the autoradio-graphic signal. Strongly labeled brain regions include the neocortex,several thalamic and hypothalamic nuclei, the amygdaloid complex,the superior colliculus, and several brainstem areas. In the spinal cord,only the central gray matter is labeled. White matter areas in brain andspinal cord show little or no specific labeling. (Bar = 0.5 cm.) OB,olfactory bulb; Cx, neocortex; Hip, hippocampal formation; CPu,caudate putamen; AA, anterior amygdaloid area; SC, superior colli-culus; Cb, cerebellum.

difference between the proximal and distal segments of thesedendrites: the proximal parts of apical CA3 pyramidal den-drites, innervated by the mossy fibers in the stratum lucidum,seem to be sparse in N-methyl-D-aspartate receptors, com-pared with hippocampal regions (e.g., the stratum radiatumand stratum oriens) that are innervated by other excitatorypathways (27, 28).The mRNA for GAP-43 was chosen as a representative of

the large number of neuronal mRNAs that are predominantlylocated in the somata of nerve cells. GAP-43, a neuronalprotein that is associated with outgrowth, maintenance, andmodification of axonal nerve terminals (29-31), is located inthe axons of a subset of neurons in the adult rat (32, 33).Interestingly, the in situ hybridization labeling patterns indi-cate that hippocampal neurons that express BC1 RNA alsocontain GAP-43 mRNA; in contrast to BC1 RNA, however,GAP-43 mRNA labeling is largely confined to areas thatcontain neuronal somata. In the CA3 field, GAP-43 mRNA ispresent in the stratum pyramidale (Fig. 3B); however, thereis little or no specific labeling in the stratum oriens and thestratum radiatum, dendritic layers in which BC1 labeling issignificant (Fig. 3A). In conclusion, our results show thatalthough BC1 RNA and GAP-43 mRNA are expressed by thesame type of neuron in the CA3 field, GAP-43 mRNA is

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located almost exclusively in the layer of the cell bodies (seeref. 19 for similar data from the human brain), whereas BC1RNA is present throughout the dendritic fields. Similarobservations were made in other brain areas, including theneocortex and the olfactory bulb.

Finally, we used acutely isolated spinal cord neurons for adirect analysis ofthe subcellular location ofBC1 RNA. In situhybridization to such neurons shows that although GAP-43mRNA is restricted to the cell bodies, BC1 RNA is locatedin somata and in neurites (Fig. 4). Almost all of the neuronalprocesses are labeled by the BC1 probe, leaving at most oneneurite per cell unlabeled. Most of the labeled neurites showmorphological characteristics typical of dendrites; they aretapered, relatively broad at the base, and branch occasion-

AIN-X, : FIG. 2. Expression patterns of BC1 RNA inAd = 84>i dorsal root ganglia (A and B) and in the retina (C

and D). A 35S-labeled complementary ("an-tisense strand") RNA probe was used in A and

shy idJ< C; a control ("sense strand") RNA probe was; used in B and D. (A) The large somata of spinal^4W"E§; ganglion cells (two arrows) show an intense BC1

0 s hybridization signal. [Two equivalent neuronsG il are marked by arrows in the control tissue

section (B).] The density of silver grains is verylow over fiber tract areas that contain axons andvarious types of glial cells (asterisks). (C) In theretina, remarkably high amounts of BC1 RNAare found in the inner plexiform layer, which isthe territory of ganglion cell dendrites, of axonsof bipolar cells, and of dendritic processes ofamacrine cells. ONL, outer nuclear layer; OPL,outer plexiform layer; INL, inner nuclear layer;IPL, inner plexiform layer; GCL, ganglion celllayer. In both tissues, expression patterns of

Kvr k BC1 RNA and MAP2 (24) are strikingly similar.Sections were counterstained with hematoxy-lin/eosin. Epiluminescence micrographs weretaken with a Leitz Orthoplan microscope.(x250.)

ally. Although we cannot rule out the possibility that BC1RNA may also be located in the axons of these or other cells,the low signal seen in nerve fiber tracts in tissue sectionsseems to indicate that if BC1 RNA is present in axons insignificant amounts, it is restricted to axonal terminals and/orto the vicinities of somata.

DISCUSSIONThe results of this study demonstrate that BC1 RNA islocated in the dendritic and somatic fields of a subset ofneurons in the nervous system ofthe adult rat. We have madeanalogous observations in immature animals where, duringthe first postnatal week, substantial amounts of BC1 RNA

FIG. 3. Distribution of BC1 RNA (A) and ofGAP-43 mRNA (B) in serial sections through theCA3 field of the hippocampal formation. (C) Acontrol RNA probe ("BC1 sense strand") wasused to determine nonspecific background la-beling. Coronal sections correspond to platenumber 29 in the atlas of Paxinos and Watson(23). Sections were counterstained with cresylviolet. Dark-field micrographs were taken witha Zeiss Axiophot microscope. (x50.) (D) Dia-gramatic representation of the CA3 field. Hip-

N pocampal pyramidal neurons are represented bytriangles. Dendritic arrays of a single pyramidal

11mf cell are indicated. Axons of pyramidal cells areA X 7~ £\ ( not shown. 0, stratum oriens; P, stratum pyra-

midale; L, stratum lucidum; R, stratum radia-tum; mf, mossy fibers (containing bundles ofdentate granule cell axons).

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FIG. 4. Location of BC1 RNA (A-F) and GAP-43 mRNA (G and H) in acutely isolated spinal cord neurons (20). (B, D-F, and H)Epiluminescence micrographs show the location of autoradiographic silver grains over individual neurons. B, D, F, and H show single neurons;E shows a group of neurons. (A, C, and G) Phase-contrast micrographs, corresponding to epiluminescence micrographs B, D, and H, portray thenerve cells with their processes. Overexposure (exposure times of >8 weeks) of neurons hybridized with the probe complementary to GAP-43mRNA produced little or no specific labeling of neurites although it resulted in heavy labeling of neuronal perikarya and in higher levels ofnonspecific background labeling; furthermore, the respective "sense strand" control probes (BC1 and GAP-43) failed to produce any specificlabeling ofacutely isolated cells (data not shown). Cultured neurons could not be used for this experiment because the specificity of BC1 expressionis lost in tissue culture; primary cultures, even if of nonneuronal origin, will accumulate BC1 RNA a few days after explantation (ref. 34; H.T.and J.B., unpublished observations). Cells were counterstained with cresyl violet and methylene blue. Epiluminescence micrographs were takenwith a Leitz Orthoplan microscope; phase-contrast micrographs were taken with a Nikon Microphot-FX microscope. (x240.)

can be detected in developing dendritic fields in several brainareas (neocortex, cerebellar cortex, olfactory bulb; data notshown). These findings are consistent with a functional roleof BC1 RNA in the somatic and dendritic compartments ofvarious types of developing and mature neurons.A discussion of the possible functional role of BC1 RNA

has to take into account our unpublished results showing thata counterpart with only distant sequence similarity in the 3'domain exists in Homo sapiens. On the other hand, the BC1RNA gene and the regions immediately flanking it are highlyconserved in several rodent species. Furthermore, the BC1expression pattern in mouse closely resembles the one in rat.Though these data may rule out an early phylogenetic originof BC1 RNA, they indicate the presence of strong evolution-ary pressure to maintain BC1 RNA in rodents. As thenervous systems in a number of animal orders have under-gone most drastic developments in the recent phylogenetichistory, a function of BC1 RNA in neurons is conceivabledespite its relative recent origin in evolution.Recent experiments (H.T., J. G. Cheng, K. Eisinger, and

J.B., unpublished) indicate that in vivo BC1 RNA is com-plexed with proteins to form a high molecular mass RNP. The

transport of BC1 RNA into dendrites may therefore dependon specific signal sequences in the RNA and/or the proteinmoiety of the RNP. The presence of a BC1 RNP in dendriteswould also be of considerable interest within the context ofprevious reports (2-5) that polysomes and other cellularelements that may be involved in a local protein biosynthesisare positioned beneath postsynaptic sites in dendrites. Thesecompartments have been suggested as the sites of synthesisand processing of synapse-related proteins such as neuro-transmitter receptors, ion channels, protein kinases, andcytoskeletal proteins (1-10). Just as in the case of generalprotein biosynthesis in the cytoplasm of all eukaryotic cells,pre- or posttranslational processes in special postsynapticcompartments of neurons are likely to require the participa-tion of specific RNPs. Neuronal RNPs may be expected toassist, for example, in the sorting of dendritic mRNAs (or ofthe respective polyribosomes) and in their targeting to thesites of dendritic protein synthesis or in the directionaltransport of dendritic proteins, in statu nascendi, to theirrespective postsynaptic target sites. Although functionalroles such as these are reminiscent of the ubiquitous signalrecognition particle that operates in the cytoplasm of various

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cell types (15), more unconventional neuron-specific fuuic-tions without direct precedence are equally possible.

We thank Dr. R. Neve for the GAP-43 cDNA clone that was usedin this study, R. Woolley for his help in preparing the illustrations,and Drs. I. Black, P. Hof, A. MacDermott, J. Morrison, H. Potter,J. Roberts, and R. Sloviter for comments on earlier drafts of themanuscript. This work was supported by the Deutsche Forschungs-gemeinschaft (H.T.), the National Research Service (R.T.F.), andthe National Institute of Mental Health (J.B.).

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