346
Brain Research, 500 0991) 346-34 t) © 1991 Elsevier Science Publishers B.V. All rights reserved. (~106-8993/91/$03.50 ADONIS (XX~689939124859W
BRES 24859
c-fos protooncogene expression in rat hippocampus and entorhinal
cortex following tetanic stimulation of the perforant path Eugeniusz Nikolaev 1, Wolfgang Tischmeyer 2, Manfred Krug 3, Hansjtirgen Matthies 2,3 and Leszek Kaczmarek I INencki Institute of Experimental Biology, Warsaw (Poland), 21nstituleof Neurobiology and Brain Research, German Academy of Sciences, Magdeburg (ER.G.) and 3Institute of Pharmacology and Toxicology, Medical Academy, Magdeburg (ER.G.) (Accepted 2 July 1991)
Key words: c-Fos; Rat; Perforant path; Hippocampus; Entorhinal cortex
The elevated expression of the c-fos protooncogene has been proposed to be a marker of cell activation leading to a long term cellular response. In this communication we compared the c-fos mRNA accumulation in the hippocampus (i.e. postsynaptic cells) and entorhinal cortex (i.e. presynaptic cells) following high (tetanic) and low frequency electrical stimulation of the pefforant path. Using Northern blot analysis we have found that high frequency stimulation elevates c-los expression in both hippocampus and entorhinal cortex, and the increase of c-fos mRNA levels in the entorhinal cortex is less pronounced, but longer lasting, than in the hippoeampus. Slight increase of c-fos mRNA levels has been also observed in low frequency treated animals in the entorhinal cortex, but not in the hippocampus. These findings raise the question about differences in mechanisms involved in c-los activation in both parts of the brain after stimulation which evokes long term potentiation (LTP) of synaptic efficacy. Long term potentiation (LTP) refers to an increased efficacy of synaptic transmission evoked by high frequency electrical stimulation of afferents in hippocampus 1. This p h e n o m e n o n is believed to serve as a good electro-physiological model of memory formation 2' 25
LTP has been reported to be composed of at least 3 phases of different durability 5"9"17'1s'22. The longest lasting one (more than several hours, we would like to refer to it as L L LTP -- long lasting LTP) has been shown to be dependent on protein biosynthesis and therefore, possibly, on gene expression 9'16'18'21. Several recent reports, in fact, have shown that high frequency electrical stimulation leading to establishment of the LTP provokes elevated expression of some immediate early genes 24 like c-fos, c-jun, jun B and N G F I - A (zif/268, Krox-24, egr-1, TIS-8 a r e synonyms) 4'7's'12-14"26. The same reports, however, were conflicting with regard to the possibility of observing increased c-los protooncogene gene expression after induction of LTP. This apparent discrepancy can be explained by different methods used for LTP induction. In general, the lack of c-los expression was reported when anesthetized rats were used or only relatively weak stimulation was employed to induce L T P 4'7"26. On the other hand, Dragunow et al. 8 and Jeffrey et al. 12 have found consistent c-los activation
in unanesthetized rats. Moreover, the same authors have observed that anesthesia inhibits expression of c-los. Additionally, pentobarbital anesthesia also blocked the formation of L L LTP. Similarly, our experience with LTP suggests that multiple trains of stimulation are needed in order to develop L L LTP consistently. It seemed, therefore, desirable to check whether multiple trains of stimuli induce both L L LTP and c-fos expression. However, the scarcity of available data does not permit to draw any conclusions on a possible functional role of expression of the aforementioned genes in LTP. To extend these data we decided to compare c-fos expression in rat hippocampus and entorhinal cortex following high frequency stimulation of the pefforant path. The procedure for the development of LTP followed the techniques reported previously 16. Briefly, for R N A extraction a total of 90 (9 groups, 10 animals per group) male, 200-250 g Wistar rats were implanted unilaterally with a stimulating electrode into the perforant path of the right hemisphere (AP = 6.9; L = 4.1; H = 4.0). One week following surgery, subjects were either used as controis (0 time), or treated with control low-frequency stimulation (for 45 min, 0.5 Hz), or tetanized (4 trains of 300 impulses at 200 Hz organized in 20 groups of 15 impulses each, separated by 5 s; trains were separated by 15 min intervals). At 45 min, 90 min, 6 h and 24 h
Correspondence: L. Kaczmarek, Nencki Institute of Experimental Biology, 02-093 Warsaw, Pasteura 3, Poland.
347 following the beginning of stimulation the animals were sacrificed, and their right dorsal hippocampus (approx. one third of the hippocampus) as well as their fight entorhinal cortex were collected. No recording electrode was placed in the hippocampus and therefore no recording was done on the animals sacrificed for R N A extraction in order not to interfere with integrity of tissue used for molecular analysis. We have previously reported that implantation of a cannula into the hippocampus activates c-los expression 15. / On the other hand, additional animals were tested electrophysiologically for the duration of LTP evoked in the same experimental conditions 13'16 and they proved that the stimulation paradigm used produces LTP lasting up to at least 3 days. In our hands, LTP is obtained in 9 out of 10 animals of the same age and weight implanted stereotactically as described above. The large number of animals needed for Northern analysis precluded collection of material separately from each individual and therefore precluded also statistical analysis. However, at the same time the pooling of tissue from 10 animals per group provided an averaging of the results. Moreover, the experiment was repeated on an additional group of 50 rats with essentially the same results. For molecular biology analysis the tissue samples collected from all animals of each group were pooled together and used for R N A extraction using the procedure of Chirgwin et al. 3 and processed for Northern analysis as described previously '5. For relative comparison of c-los m R N A abundance after electrical stimulation, the Northern blot and corresponding ethidium bromide picture of 28S rRNA were scanned densitometrically with aid of an LKB Ultrascan laser densitometer and the c-fos m R N A levels were divided per corresponding 28S rRNA level and those values were expressed relatively to the 0 time value taken as 1.0. The results of gene expression analysis following development of LTP are presented in Fig. 1. The top panel of the figure shows a dramatic increase of c-los m R N A levels in the hippocampal samples of tetanized subjects at 45 min after the onset of stimulation (lane 2), followed by a return to the prestimulation levels 24 h later (lane 5). Conversely, there was no corresponding increase in c-los m R N A levels found in the low frequency stimulated groups (lanes 6-9), when compared to the control animals. There was also a clear increase of c-fos m R N A levels in the entorhinal cortex of the animals treated with high frequency stimulation. Interestingly, this m R N A accumulation was longer lasting, but less pronounced than in the hippocampus. Low c-fos m R N A accumulation was
1234
567891011
12131415161718 ii~i!
-2.2
Fig. l. Increase of c-los mRNA levels following LTP induction, Lane 1: RNA extracted from control, non-stimulated hippocampi; lanes 2-5: tetanized animals, 2-45 rain following initiation of tetanization; 3-90 min; 4-6 h; 5-24 h; lanes 6-9: low frequency treated animals, 6-45 min followingthe beginning of low frequency stimulation; 7-90 min; 8-6 h; 9-24 h. Lanes 10-18 contain RNA extracted from entorhinal cortex. 10: control; 11-14: after tetanization (45 min, 90 rain, 6 h, 24 h, resp.; 15-18: low frequency treated animals (45 min, 90 min, 6 h, 24 h, resp.). Top panel: c-los mRNA levels, the number to the right indicates the size of the depicted message in kilobases. Bottom panel: ethidium bromide picture of RNAs after blotting and before hybridization to c-los probe.
also observed in the entorhinal cortex of the low frequency stimulated rats; this was clearly visible after an overexposure of the blots. The bottom panel of Fig. 1 presents the R N A staining of the gel used for the Northern experiment described above, demonstrating that the observed phenomena were not due to different amounts of RNA. Fig. 2
Hippocampus
Ent. Cortex
Relative c - l o s m R N A levels 14 12 10
a!
4 2 I1
o
o.75
1.5
Time
I
a
after
Comx..ol I
~4 onset
~
o
o.75
of stimulation
~queaey
~
t.5
e
~
(hours)
Lowfr'equ,m~
Fig. 2. Relative c-.fos mRNA levels following induction of LTP.
348 presents a densitometry-based quantification of the Northern blot data. It is widely assumed that the induction of the LTP is acting on the H e b b i a n principle, i.e. involving both presynaptic and postsynaptic elements. It is not, however, clear whether both sides of the synapse contribute, and if so, to which extent, to the maintenance of the LTP. One of our laboratories has previously r e p o r t e d that separating the postsynaptic cell bodies from dendrites leads to the LTP lasting only up to a few hours, i.e. the same time as LTP induced in the presence of inhibitors of protein biosynthesis 1°. M o r e o v e r , Otani and A b r a h a m 21 have r e p o r t e d that injection of a protein biosynthesis inhibitor into the dentate gyrus blocked late phase of LTP. These findings, together with the a f o r e m e n t i o n e d elevated expression of regulatory genes would favor nuclei of the postsynaptic neurons to be the locus of formation of L L LTP. In this communication, however, we present an evidence that there is also c-fos m R N A accumulation on a presynaptic site, namely in the entorhinal cortex of high frequency stimulated animals. We have to stress that the interpretation of this finding requires caution. It is important to note that mechanisms of elevated c-fos expression in entorhinal cortex differ significantly from the ones inducing the c-los ex-
1 Bliss, T.V.P. and Lomo, T.J., Long lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path, J. Physiol., 232 (1973) 331-356. 2 Brown, T.H., Chapman, P.E, Kairiss, EW. and Keenan, C.L., Long-term synaptic potentiation, Science, 242 (1988) 724-728. 3 Chirgwin, J., Przybyla, A., MacDonald, R. and Rutter, W., Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease, Biochemistry, 18 (1979) 5294-5299. 4 Cole, A.J., Saffen, D.W., Baraban, J.M. and Worley, P.E, Rapid increase of an immediate early gene messenger RNA in hippocampal neurons by synaptic NMDA receptor activation, Nature, 340 (1989) 474-476. 5 Colley, P.A., Sheu, E-S. and Routenberg, A., Inhibition of protein kinase C blocks two components of LTP persistence, leaving initial potentiation intact, J. Neurosci., 10 (1990) 33533360. 6 Cui-Wei, X., Clifford, L.M. and Jau-Shyong, H., Perforant path stimulation differentially alters prodynorphin mRNA and proenkephalin mRNA levels in the entorhinal cortex-hippocampal region, Mol. Brain Res., 7 (1990) 199-205. 7 Douglas, R.M., Dragunow, M., and Robertson, H.A., High frequency discharge of dentate granule cells, but not long term potentiation, induces c-los protein, Mol. Brain Res., 4 (1988) 259-262. 8 Dragunow, M., Abraham, W.C., Goulding, M., Mason, S.E., Robertson, H.A. and Faull, R.L., Long-term potentiation and the induction of c-los mRNA and proteins in the dentate gyrus of unanesthetized rats, Neurosci. Lea., 101 (1989) 274-280. 9 Frey, U., Krug, M., Reymann, K.G. and Matthies, H., Anismomycin, an inhibitor of protein synthesis, blocks late phases of LTP phenomena in the hippocampal CAl-region in vitro, Brain Research, 452 (1988) 57--65. 10 Frey, U., Krug, M., Brodeman, R., Reymann, K. and Matthies, H., Long term potentiation induced in dendrites sepa-
pression in the hippocampus. While the last ones arc d e p e n d e n t on neurotransmitter release from the perforant path fibers, the entorhinal cortex p h e n o m e n o n is driven p r o b a b l y by extensive depolarization induced by tetanic stimulation acting antidromically. The role of depolarization in the induction of c-los expression has been shown for the PC 12 cells in culture 11'19. The depolarization effect may explain the slight increase of c-fos m R N A levels observed in entorhinal cortex of low frequency treated rats. Additionally, it is worth mentioning studies showing that high frequency electrical stimulation leads to m R N A accumulation for c-fos and p r o e n k e p h a l i n 6'2°'23. Similarly, increased c-fos m R N A accumulation in the hippocampus was r e p o r t e d by Cole et al. 4 and Wisen et al. 26 after commissural stimulation which p r e v e n t e d LTP formation. Again, a functional relationship of this p h e n o m e n o n to the long term changes in the h i p p o c a m p a l activity is not clear. In conclusion, our results suggest a necessity for further studies on c-fos expression during LTP formation as well as they raise a possibility of different mechanisms and different roles played by c-fos m R N A accumulation in presynaptic vs postsynaptic elements following high frequency electrical stimulation of neurons.
11 12
13 14 15 16 17 18 19 20
21
rated from their CA1 somata does not establish a late phase, Neurosci. Lett., 97 (1989) 135-139. Greenberg, M.E., Ziff, E.B. and Greene, L.A., Stimulation of neuronal acetylcholine receptors induces rapid gene transcription, Science, 234 (1986) 80-83. Jeffrey, K.J., Abraham, W.C., Dragunow, M. and Mason, S.E., Induction of los-like immunoreactivity and the maintenance of long term potentiation in the dentate gyrus of unanesthetized rats, Mol. Brain Res., 8 (1990) 267-274. Kaczmarek, L., Nikolaev, E., c-Fos protooncogene expression and neuronal plasticity, Acta Neurobiol. Exp., 50 (1990) 173179. Kaczmarek, L., Nikolaev, E., Tiehmeyer, W., Krug, M. and Matthies, H., Expression of genes encoding transcription factors in long term potentiation, submitted. Kaczmarek, L., Siedlecki, J.A. and Danysz, W., Proto-oncogene c-los induction in rat hippocampus, Mol. Brain Res., 3 (1988) 118-186. Krug, M., Loessner, B. and Ott, T., Anisomycin blocks the late phase of long-term potentiation in the dentate gyrus of freely moving rats, Brain Res. Bull., 13 (1984) 39-43. Malinow, R., Schulman, H. and Tsien, R.W., Inhibition of postsynaptic PKC or CaMK II blocks induction but not expression of LTP, Science, 245 (1989) 862-866. Matthies, H., Neurobiological aspects of learning and memory, Annu. Rev. Psychol., 40 (1989) 381-404. Morgan, J.I. and Curran, T., Role of ion fluxes in the control of c-los expression, Nature, 322 (1986) 552-555. Morris, B.J., Feasey, K.J., Bruggencate, G., Herz, A. and Holt, V., Electrical stimulation in vivo increases the expression of proenkephalin mRNA and decreases the expression of prodynorphin mRNA in hippocampal granule cells, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 3226-3230. Otani, S. and Abraham, W.C., Inhibition of protein synthesis in the dentate gyrus, but not the entorhinal cortex, blocks main-
349 tenance of long-term potentiation in rats, Neurosci. Lett., 106 (1989) 175-180. 22 Reymann, K.G., Frey, U., Jork, R. and Matthies, H., Polymyxin B., an inhibitor of protein kinase C, prevents the maintenance of synaptic long-term potentiation in hippocampal CA1 neurons, Brain Research, 440 (1988) 305-314. 23 Sagar, S.M., Sharp, ER. and Curran, T., Expression of c-los protein in brain. Metabolic mapping at the cellular level, Science, 240 (1988) 1328-1331.
24 Sheng, M. and Greenberg, M.E., The regulation of function of c-los and other immediately early genes in the nervous system, Neuron, 4 (1990) 477--485. 25 Teyler, T.J. and DiScenna, P., Long-term potentiation as a candidate mnemonic device, Brain Res. Rev., 7 (1984) 15-28. 26 Wisden, W., Errington, M.L., Williams, S., Dunnett, S.B., Waters, C., Hitchcock, D., Evan, G., Bliss, T.V.P. and Hunt, S.P., Differential expression of immediate early genes in the hippocampus and spinal cord, Neuron, 4 (1990) 603-614.
Brain Research, 500 0991) 346-34 t) © 1991 Elsevier Science Publishers B.V. All rights reserved. (~106-8993/91/$03.50 ADONIS (XX~689939124859W
BRES 24859
c-fos protooncogene expression in rat hippocampus and entorhinal
cortex following tetanic stimulation of the perforant path Eugeniusz Nikolaev 1, Wolfgang Tischmeyer 2, Manfred Krug 3, Hansjtirgen Matthies 2,3 and Leszek Kaczmarek I INencki Institute of Experimental Biology, Warsaw (Poland), 21nstituleof Neurobiology and Brain Research, German Academy of Sciences, Magdeburg (ER.G.) and 3Institute of Pharmacology and Toxicology, Medical Academy, Magdeburg (ER.G.) (Accepted 2 July 1991)
Key words: c-Fos; Rat; Perforant path; Hippocampus; Entorhinal cortex
The elevated expression of the c-fos protooncogene has been proposed to be a marker of cell activation leading to a long term cellular response. In this communication we compared the c-fos mRNA accumulation in the hippocampus (i.e. postsynaptic cells) and entorhinal cortex (i.e. presynaptic cells) following high (tetanic) and low frequency electrical stimulation of the pefforant path. Using Northern blot analysis we have found that high frequency stimulation elevates c-los expression in both hippocampus and entorhinal cortex, and the increase of c-fos mRNA levels in the entorhinal cortex is less pronounced, but longer lasting, than in the hippoeampus. Slight increase of c-fos mRNA levels has been also observed in low frequency treated animals in the entorhinal cortex, but not in the hippocampus. These findings raise the question about differences in mechanisms involved in c-los activation in both parts of the brain after stimulation which evokes long term potentiation (LTP) of synaptic efficacy. Long term potentiation (LTP) refers to an increased efficacy of synaptic transmission evoked by high frequency electrical stimulation of afferents in hippocampus 1. This p h e n o m e n o n is believed to serve as a good electro-physiological model of memory formation 2' 25
LTP has been reported to be composed of at least 3 phases of different durability 5"9"17'1s'22. The longest lasting one (more than several hours, we would like to refer to it as L L LTP -- long lasting LTP) has been shown to be dependent on protein biosynthesis and therefore, possibly, on gene expression 9'16'18'21. Several recent reports, in fact, have shown that high frequency electrical stimulation leading to establishment of the LTP provokes elevated expression of some immediate early genes 24 like c-fos, c-jun, jun B and N G F I - A (zif/268, Krox-24, egr-1, TIS-8 a r e synonyms) 4'7's'12-14"26. The same reports, however, were conflicting with regard to the possibility of observing increased c-los protooncogene gene expression after induction of LTP. This apparent discrepancy can be explained by different methods used for LTP induction. In general, the lack of c-los expression was reported when anesthetized rats were used or only relatively weak stimulation was employed to induce L T P 4'7"26. On the other hand, Dragunow et al. 8 and Jeffrey et al. 12 have found consistent c-los activation
in unanesthetized rats. Moreover, the same authors have observed that anesthesia inhibits expression of c-los. Additionally, pentobarbital anesthesia also blocked the formation of L L LTP. Similarly, our experience with LTP suggests that multiple trains of stimulation are needed in order to develop L L LTP consistently. It seemed, therefore, desirable to check whether multiple trains of stimuli induce both L L LTP and c-fos expression. However, the scarcity of available data does not permit to draw any conclusions on a possible functional role of expression of the aforementioned genes in LTP. To extend these data we decided to compare c-fos expression in rat hippocampus and entorhinal cortex following high frequency stimulation of the pefforant path. The procedure for the development of LTP followed the techniques reported previously 16. Briefly, for R N A extraction a total of 90 (9 groups, 10 animals per group) male, 200-250 g Wistar rats were implanted unilaterally with a stimulating electrode into the perforant path of the right hemisphere (AP = 6.9; L = 4.1; H = 4.0). One week following surgery, subjects were either used as controis (0 time), or treated with control low-frequency stimulation (for 45 min, 0.5 Hz), or tetanized (4 trains of 300 impulses at 200 Hz organized in 20 groups of 15 impulses each, separated by 5 s; trains were separated by 15 min intervals). At 45 min, 90 min, 6 h and 24 h
Correspondence: L. Kaczmarek, Nencki Institute of Experimental Biology, 02-093 Warsaw, Pasteura 3, Poland.
347 following the beginning of stimulation the animals were sacrificed, and their right dorsal hippocampus (approx. one third of the hippocampus) as well as their fight entorhinal cortex were collected. No recording electrode was placed in the hippocampus and therefore no recording was done on the animals sacrificed for R N A extraction in order not to interfere with integrity of tissue used for molecular analysis. We have previously reported that implantation of a cannula into the hippocampus activates c-los expression 15. / On the other hand, additional animals were tested electrophysiologically for the duration of LTP evoked in the same experimental conditions 13'16 and they proved that the stimulation paradigm used produces LTP lasting up to at least 3 days. In our hands, LTP is obtained in 9 out of 10 animals of the same age and weight implanted stereotactically as described above. The large number of animals needed for Northern analysis precluded collection of material separately from each individual and therefore precluded also statistical analysis. However, at the same time the pooling of tissue from 10 animals per group provided an averaging of the results. Moreover, the experiment was repeated on an additional group of 50 rats with essentially the same results. For molecular biology analysis the tissue samples collected from all animals of each group were pooled together and used for R N A extraction using the procedure of Chirgwin et al. 3 and processed for Northern analysis as described previously '5. For relative comparison of c-los m R N A abundance after electrical stimulation, the Northern blot and corresponding ethidium bromide picture of 28S rRNA were scanned densitometrically with aid of an LKB Ultrascan laser densitometer and the c-fos m R N A levels were divided per corresponding 28S rRNA level and those values were expressed relatively to the 0 time value taken as 1.0. The results of gene expression analysis following development of LTP are presented in Fig. 1. The top panel of the figure shows a dramatic increase of c-los m R N A levels in the hippocampal samples of tetanized subjects at 45 min after the onset of stimulation (lane 2), followed by a return to the prestimulation levels 24 h later (lane 5). Conversely, there was no corresponding increase in c-los m R N A levels found in the low frequency stimulated groups (lanes 6-9), when compared to the control animals. There was also a clear increase of c-fos m R N A levels in the entorhinal cortex of the animals treated with high frequency stimulation. Interestingly, this m R N A accumulation was longer lasting, but less pronounced than in the hippocampus. Low c-fos m R N A accumulation was
1234
567891011
12131415161718 ii~i!
-2.2
Fig. l. Increase of c-los mRNA levels following LTP induction, Lane 1: RNA extracted from control, non-stimulated hippocampi; lanes 2-5: tetanized animals, 2-45 rain following initiation of tetanization; 3-90 min; 4-6 h; 5-24 h; lanes 6-9: low frequency treated animals, 6-45 min followingthe beginning of low frequency stimulation; 7-90 min; 8-6 h; 9-24 h. Lanes 10-18 contain RNA extracted from entorhinal cortex. 10: control; 11-14: after tetanization (45 min, 90 rain, 6 h, 24 h, resp.; 15-18: low frequency treated animals (45 min, 90 min, 6 h, 24 h, resp.). Top panel: c-los mRNA levels, the number to the right indicates the size of the depicted message in kilobases. Bottom panel: ethidium bromide picture of RNAs after blotting and before hybridization to c-los probe.
also observed in the entorhinal cortex of the low frequency stimulated rats; this was clearly visible after an overexposure of the blots. The bottom panel of Fig. 1 presents the R N A staining of the gel used for the Northern experiment described above, demonstrating that the observed phenomena were not due to different amounts of RNA. Fig. 2
Hippocampus
Ent. Cortex
Relative c - l o s m R N A levels 14 12 10
a!
4 2 I1
o
o.75
1.5
Time
I
a
after
Comx..ol I
~4 onset
~
o
o.75
of stimulation
~queaey
~
t.5
e
~
(hours)
Lowfr'equ,m~
Fig. 2. Relative c-.fos mRNA levels following induction of LTP.
348 presents a densitometry-based quantification of the Northern blot data. It is widely assumed that the induction of the LTP is acting on the H e b b i a n principle, i.e. involving both presynaptic and postsynaptic elements. It is not, however, clear whether both sides of the synapse contribute, and if so, to which extent, to the maintenance of the LTP. One of our laboratories has previously r e p o r t e d that separating the postsynaptic cell bodies from dendrites leads to the LTP lasting only up to a few hours, i.e. the same time as LTP induced in the presence of inhibitors of protein biosynthesis 1°. M o r e o v e r , Otani and A b r a h a m 21 have r e p o r t e d that injection of a protein biosynthesis inhibitor into the dentate gyrus blocked late phase of LTP. These findings, together with the a f o r e m e n t i o n e d elevated expression of regulatory genes would favor nuclei of the postsynaptic neurons to be the locus of formation of L L LTP. In this communication, however, we present an evidence that there is also c-fos m R N A accumulation on a presynaptic site, namely in the entorhinal cortex of high frequency stimulated animals. We have to stress that the interpretation of this finding requires caution. It is important to note that mechanisms of elevated c-fos expression in entorhinal cortex differ significantly from the ones inducing the c-los ex-
1 Bliss, T.V.P. and Lomo, T.J., Long lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path, J. Physiol., 232 (1973) 331-356. 2 Brown, T.H., Chapman, P.E, Kairiss, EW. and Keenan, C.L., Long-term synaptic potentiation, Science, 242 (1988) 724-728. 3 Chirgwin, J., Przybyla, A., MacDonald, R. and Rutter, W., Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease, Biochemistry, 18 (1979) 5294-5299. 4 Cole, A.J., Saffen, D.W., Baraban, J.M. and Worley, P.E, Rapid increase of an immediate early gene messenger RNA in hippocampal neurons by synaptic NMDA receptor activation, Nature, 340 (1989) 474-476. 5 Colley, P.A., Sheu, E-S. and Routenberg, A., Inhibition of protein kinase C blocks two components of LTP persistence, leaving initial potentiation intact, J. Neurosci., 10 (1990) 33533360. 6 Cui-Wei, X., Clifford, L.M. and Jau-Shyong, H., Perforant path stimulation differentially alters prodynorphin mRNA and proenkephalin mRNA levels in the entorhinal cortex-hippocampal region, Mol. Brain Res., 7 (1990) 199-205. 7 Douglas, R.M., Dragunow, M., and Robertson, H.A., High frequency discharge of dentate granule cells, but not long term potentiation, induces c-los protein, Mol. Brain Res., 4 (1988) 259-262. 8 Dragunow, M., Abraham, W.C., Goulding, M., Mason, S.E., Robertson, H.A. and Faull, R.L., Long-term potentiation and the induction of c-los mRNA and proteins in the dentate gyrus of unanesthetized rats, Neurosci. Lea., 101 (1989) 274-280. 9 Frey, U., Krug, M., Reymann, K.G. and Matthies, H., Anismomycin, an inhibitor of protein synthesis, blocks late phases of LTP phenomena in the hippocampal CAl-region in vitro, Brain Research, 452 (1988) 57--65. 10 Frey, U., Krug, M., Brodeman, R., Reymann, K. and Matthies, H., Long term potentiation induced in dendrites sepa-
pression in the hippocampus. While the last ones arc d e p e n d e n t on neurotransmitter release from the perforant path fibers, the entorhinal cortex p h e n o m e n o n is driven p r o b a b l y by extensive depolarization induced by tetanic stimulation acting antidromically. The role of depolarization in the induction of c-los expression has been shown for the PC 12 cells in culture 11'19. The depolarization effect may explain the slight increase of c-fos m R N A levels observed in entorhinal cortex of low frequency treated rats. Additionally, it is worth mentioning studies showing that high frequency electrical stimulation leads to m R N A accumulation for c-fos and p r o e n k e p h a l i n 6'2°'23. Similarly, increased c-fos m R N A accumulation in the hippocampus was r e p o r t e d by Cole et al. 4 and Wisen et al. 26 after commissural stimulation which p r e v e n t e d LTP formation. Again, a functional relationship of this p h e n o m e n o n to the long term changes in the h i p p o c a m p a l activity is not clear. In conclusion, our results suggest a necessity for further studies on c-fos expression during LTP formation as well as they raise a possibility of different mechanisms and different roles played by c-fos m R N A accumulation in presynaptic vs postsynaptic elements following high frequency electrical stimulation of neurons.
11 12
13 14 15 16 17 18 19 20
21
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