Joo, F.

Krug, M.

Kuhnt, U.

Matthies, H.

Mihaly, A.

Olah, Z.

Rapp, U.

English

MPG.PuRe

Neuroscience L tters, 116 (1990) 45-50 45 Elsevier Scientific Publishers Ireland Ltd. NSL 07064 Transient increase of rafprotein kinase-like immunoreactivity in the rat dentate gyrus during long-term potentiation Andrf.s Mihfily 1, Zoltfin O1/lh 2, Manfred Krug 3, Ulrich Kuhnt I , Hansj/irgen Matthies 4, Ul f  R. Rapp 5 and Ferenc Jo6 6 ~ Department ofNeurobiology, Max Planck Institute for Biophysical Chemistry, G6ttingen ( F.R.G.), :Laboratory of Cellular Ontology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (U.S.A.), 3Institute of Pharmacology, Medical Academy and 4Institute of Neurobiology and Brain Research, Academy of Sciences of GD R, Magdeburg (G.D.R.), 5Laboratory of Viral Carcinogenesis, NCI-Frederick Cancer Research Facility, Frederick, MD 21701 (U.S,A.) and 6Laboratory of Molecular Neurobiology, Institute of Biophysics, Biological Research Center, Szeged (Hungary) (Received 23 February 1990; Revised version received 18 March 1990; Accepted 23 April 1990) Key words." Immunohistochemistry; Protooncogene; Protein kinase; Long-term potentiation; Dentate fascia Altered levels of cellular afproteins (products of the rafprotooncogenes) have been shown in the neu- rons of the dentate fascia of rats in response to high-frequency stimulation, with light microscopic immu- nohistochemistry b  using polyclonal antibodies. No raf-l-like staining was seen in unstimulated tissue, while the pan-rafantibodies r vealed immunoreactivity n the cytoplasm of neurons in the Ammon's horn and dentate fascia of rats and guinea pigs. The induction of long-term potentiation i the dentate fascia of freely-moving rats triggered the appearance of raf- l-like staining and increased the number of granule cells with pan-raf-like immunoreactivity. Since these proteins are serine/threonine-specific protein kinases, their appearance in long-term potentiation may indicate the activation of important cell membrane - -nucleus transduction pathways. Hippocampal long-term potentiation (LTP) is characterized by a long-lasting in- crease in the efficiency of impulse transmission, following a short train of high-fre- quency stimuli to any one of the monosynaptic excitatory pathways (for review, see ref. 2). The maintenance ofLTP is subserved by several biochemical processes among which the activation [12] and translocation [I] of the CaZ+/phospholipid-dependent protein kinase (PKC) seemed to play a substantial role. Recently, an additional ser- ine/threonine protein kinase, encoded by rafprotooncogenes [11] has been proposed to regulate the downstream transfer of information from the plasma membrane to the nucleus in mitotically activated cell cultures [8, 11]. Extending this proposal to Correspondence." A  Mih~ly, Department of Neurobiology, MPI for Biophysical Chemistry, P.O. Box 2841, D-3400 G6ttingen, F.R.G. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. 46 47 highly differentiated nervous tissue, as a first step, the localization of the products of the raf protooncogenes was investigated in the hippocampus of adult rats and guinea pigs. For this purpose polyclonal antibodies against raf proteins were used [6, 13]. As a second step, we induced LTP in the dentate gyrus of freely moving rats and investigated the light microscopic immunohistochemical localization of raf pro- teins (the products of the rafprotooncogenes) in the dentate gyrus. Two male Wistar rats (130 g) and 2 male guinea pigs (650 g) were anesthetized with pentobarbital (40 mg/kg) and perfused transcardially with 4% paraformaldehyde, 0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). Forty-/~m-thick oronal sec- tions were cut with Vibratome, washed in phosphate-buffered saline (PBS, pH 7.4) and treated with 0.5% sodium borohydride in PBS [16]. Consecutive sections were incubated with polyclonal raf-1 [13] and pan-rafantibodies [6]. The raf-1 antibodies were raised against he synthetic SP 63 peptide [13] which is the C-terminal sequence (12 amino acids) of the raf-1 protein [3]. The pan-rafantibodies were raised against a larger (approx. 30 kDa) C-terminal protein expressed in E. coli, by inserting the v-raf oncogene into the bacterial DNA [6]. The antibodies were diluted to 1:500, !: 1000 and 1:2000, the sections were incubated free-floating for 24 h at 4°C, labelled by means of the avidin-biotin technique [4], and developed with diaminobenzidine- HCI as a substrate. Control sections were incubated without the primary or second- ary antibodies. In order to test the effects of stimulation and LTP, 20 Wistar rats (160-170 g males) were chronically implanted with a pair of stimulating electrodes in the medial ento- rhinal cortex and a recording electrode in the dentate gyrus, as described previously [7]. The animals were conditioned without signs of seizures or afterdischarges until a pronounced LTP could be observed after biphasic stimulation (a train of 300 im- pulses in groups of 15 with 5 s intervals and 200 Hz). LTP was induced in 10 animals. The controls (I0 animals) were stimulated with constant current square wave pulses (0.1 ms, 100-400/~A, 0.2 Hz). Altogether, 16 animals in pentobarbital nesthesia (40 mg/kg) were transcardially perfused with 4% paraformaldehyde and 0.1% glutaralde- hyde in 0.1 M phosphate buffer (pH 7.4) at different imes (5, 30, 120 and 240 min) following the control and the high-frequency stimulations (2-2 animals at each time). Coronal plane serial frozen sections (40/Lm) were cut from the whole hippocampus between the electrodes, washed in PBS for 12-16 h and incubated free-floating with the two antisera - -  both diluted to 1:3000 - -  for 60 h at 4°C. The other steps of the reaction were identical with that described above. On average 30 sections per brain . I  Fig. 1. Immunohistochemical detection ofRPI with pan-raf(a,b) and raf-I (c,d) antibodies in the rat hip- pocampus (M, molecular layer; asterisk, hilus of the dentate fascia), a: CA3 pyramidal cells containing RPI. Arrow point to interneuron i  stratum oriens (O). The neurons at the arrowhead are not immuno- reactive, b: granule cells of the dentate fascia containing RPI (arrows). Arrowheads point to unstained neurons. Bar for a and b = 100 pm. c: composit photograph ofthe dentate fascia 120 min after the induc- tion of LTP. Strong immunostaining can be observed in the granular layer (arrows). Arrowheads point to red blood cells, d: no immunoreactivity canbe seen on the contralateral side of the same section (arrows: granular layer; arrowheads: red blood cells). Bar for c and d = 0.5 cm. 48 were investigated. The immunostained cell bodies in the granular layer of the dentate gyrus were plotted at a 100 × magnification with the help of an xy plotter, then a 1 mm 2 rectangle with one side parallel to the interhemispheric f ssure was layed over the dentate fascia and the immunostained cells of the granular layer were counted. Since the electrodes were implanted stereotaxically, the serial frozen sections between the electrode tracks represented comparable series. The counting has been done on parallel sections, i.e. on sections bearing the same serial number. From each animal 5 sections were used for counting. In the case of raf-l-like immunoreactivity, no counting was performed. The raf protein-like immunoreactivity (RPI) could be detected in neuronal cell bodies and dendrites in every subfield of the unstimulated hippocampal formation both in guinea pigs and rats, with the help of the pan-rafantibodies (Fig. 1). In the dentate gyrus the staining was mainly confined to neuronal cell bodies and sometimes neuronal cell nuclei of the granular layer (Fig. 1). Although most of the immunoreac- rive neurons were located in the pyramidal and granular layers, not all neurons of these layers contained RPI (Fig. 1). No attempts were made to estimate the ratio of stained:unstained neurons. Increasing dilutions of the antiserum resulted in a de- crease of the staining intensity, indicating the specificity of the method [10]. The con- trol sections incubated without the primary or secondary antibodies did not stain (except for some red blood cells). No RPI could be detected in the normal, unstimu- lated hippocampuses with the help of the raf-I antibodies. The overall pattern of RPI did not change after the different stimulation para- digms. No increase of staining was observed in the molecular layer of the dentate gyrus containing the nerve terminals of the perforant path [9]. However, stronger RPI was seen in the granular layer 120 min after the induction of LTP. The increase of staining was due to the increased number of granule cells containing RPI (Fig. 2). Interestingly enough, the number of immunostained cell bodies decreased there- after, suggesting that the increase of RPI was transient (Fig. 2). No significant changes in the number of immunoreactive granule cells were seen in the contralateral, non-stimulated hippocampuses and following control stimulation (Fig. 2). No RPI could be detected in the hippocampuses stimulated with control stimuli with the help of the raf- 1 antibodies. However, strong immunostaining appeared 120 rain after the induction of LTP, on the side of stimulation (Fig. 1). The staining out- lined the granular layer in the otherwise unstained hippocampal formation (Fig. 1) and was due to a population of granule cells. The staining also disappeared at 240 rain and was not seen on the contralateral side (Fig. 1). Our immunohistochemical experiments for the first time localized raf proteins in the highly differentiated, adult nervous tissue. The immunoreactivity was mainly con- fined to the cytoplasm of the neurons which finding is in agreement with recent bio- chemical data [8, 14], and immunohistochemical localization studies performed on NIH 3T3 fibroblast cultures [11]. Our experiments with tetanized animals suggested the elevation of neuronal rafprotein levels during the maintenance of LTP. The tran- sient elevation was very obvious in the case of pan-raf antibodies, but not so with raf-l-like staining, since the latter could not be detected in non-stimulated neurons. 49 210- c 190.  .~ 170. -~ 150. 130  =o 110 9o ® + o i i i i # i i I i 5 30  120  240  5 30  120 240  min  A B Fig. 2 Graph showing the average number of cell bodies containing pan-raf-like staining in the granular layer of the dentate fascia. Each point is the average of 10 measurements. A shows the counts in different time intervals after control, low frequency stimulation (11). B contains the counts after tetanization (Q). The counts taken from the contralatera], unstimulated side are also depicted without the standard devia- tions (D). Differences between a and b (t =8.5; P=0.001, and b and c (t =3.2; P=0.05) are significant. The RNA expression patterns of raf-1 in the mouse brain showed the highest level in the cerebellum, and only moderate (just above the detectable) levels in other parts of the brain [15]. This might explain the fact that RPI was not seen in the normal hippocampus. The raf-1 antibodies are very specific, since the SP 63 peptide is not present in other proteins of the raffamily [5, 11]. Our results obtained in tetanized animals suggested the increase of raf-1 protein levels in the dentate gyrus. Due to this increase the RPI became detectable in our experiments. The pan-rafantibodies, on the other hand, reacted not only with the raf-1 protein [14], but also with the B-rafprotein [5] which should be a par excellence brain-related protein as shown by the RNA expression pattern [15]. On the basis of this and the similarities of the amino acid sequences [5], we think that the RPI detected with pan- rafantibodies should mainly be the B-raf-like staining. However, this has to be cor- roborated by reacting the tissue with specific B-raf protein antibodies. This work is in progress in our laboratory. Miss K. Kaiser and Mrs. M. Wagner (Magdeburg) provided excellent echnical assistance. A.M. is a recipient of a fellowship from Alexander von Humboldt Foun- dation. 1 Akers, R.F., Lovinger, D.M., Colley, P.A., Linden, D.J. and Routtenberg, A., Translocation fpro- tein kinase C activity may mediate hippocampal long-term potentiation, Science, 231 (1986) 587 589. 2 Bliss, T.V.P. and Lynch, M.A., Long-term potentiation fsynaptic transmission in the hippocampus: properties and mechanisms. In P.W. Landfield and S.A. Deadwyler (Eds.), Long-Term Potentiation: From Biophysics to Behavior, Alan R. Liss, New York, 1988, pp. 3 72. 50 3 Bonner, T.I., Oppermann, H., Seeburg, P., Kerby, S.B., Gunnell, M.A., Young, A.C. and Rapp, U.R., The complete coding sequence of the human rafoncogene and the corresponding structure of the c-raf- I gene, Nucleic Acids Res., 14 (1986) 100%1015. 4 Hsu, S.-M., Raine, L. and Fanger, H.J., Use of avidin-biotin-peroxidase complex (ABC) in immuno- peroxidase t chniques: a comparison between ABC and unlabelled antibody (PAP) procedures, J. His- tochem. Cytochem., 29 (1981) 577 580. 5 lkawa, S., Fukui, M., Ueyama, Y., Tamaoki, N., Yamamoto, T. and Toyoshima, K., B-raf, a new member of the rqf family, is activated by DNA rearrangement, Mol. Cell. Biol., 8 (1988) 2651 2654. 6 Kolch, W., Schultz, A.M., Oppermann, H. and Rapp, U.R., Preparation ofraf-oncogene-specific anti- serum with rafprotein produced in E. coli, Biochim. Biophys. Acta, 949 (1988) 233 239. 7 Krug, M., Br6demann, R. and Ott, T., Blockade of long-term potentiation i the dentate gyrus of freely moving rats by the glutamic acid antagonist GDEE, Brain Res., 249 (1982) 57~2. 8 Morrison, D.K., Kaplan, D.R., Rapp, U. and Roberts, T.M., Signal transduction from membrane to cytoplasm: growth factors and membrane-bound oncogene products increase raf-1 phosphorylation and associated protein kinase activity, Proc. Natl. Acad. Sci. 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Brain Res., 71 (1988) 227 230. 13 Schultz, A.M., Copeland, T.D., Mark, G.E., Rapp, U.R. and Oroszlan, S., Detection of the myristilated gag-rqf transforming protein with raJ:specific antipeptide s ra, Virology, 146 (1985) 78 89. 14 Schultz, A.M., Copeland, T.D., Oroszlan, S. and Rapp, U.R., Identification and characterization f c-rq[2 phosphoproteins  transformed murine cells, Oncogene, 2 (1988) 187 193. 15 Storm, S.M., Cleveland, J.L. and Rapp, U.R., Expression of raf family proto-oncogenes in normal mouse tissues, Oncogene, 5 (1990) 101 107. 16 Willingham, M.C., An alternative fixation-processing method for preembedding ultrastructural immu- nocytochemistry of cytoplasmic antigens: the GBS (glutaraldehyde-borohydride- saponin) procedure, J. Histochem. Cytochem., 31 (1983) 791- 798. 

Transient increase of raf protein kinase-like immunoreactivity in the rat dentate gyrus during long-term potentiation.

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Transient increase of raf protein kinase-like immunoreactivity in the rat dentate gyrus during long-term potentiation.

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