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Selective regulation of dendritic MAP2 mRNA levels in hippocampal granule cells by nitric oxide
ELSEVIER
Neuroscience Letters 177 (1994) 5 10
NIUROSClENCE LETTERS
Selective regulation of dendritic MAP2 mRNA levels in hippocampal granule cells by nitric oxide H.M. Johnston, B.J. Morris* Department of Pharmacology, University of Glasgow, Glasgow G12 8QQ, UK Received 14 April 1994; Revised version received 5 June 1994; Accepted 7 June 1994
Abstract
Application of NMDA, or agents releasing nitric oxide (NO), onto the dendrites of hippocampal granule cells increased the levels of the mRNA encoding MAP2, a cytoskeletalcomponent induced during periods of neurite outgrowth. Furthermore, local increases in the hybridisation signal in the molecular layer, representing dendritic MAP2 mRNA, occurred independentlyof changes in MAP2 mRNA levels in the cell body layer. The selectivemodulation of MAP2 mRNA in dendrites reveals a mechanismallowinga sustained stimulation of dendritic outgrowth to be confined to those regions of a neuron's dendritic arbour local to glutamate receptor stimulation. Key words: Glutamate; Nitric oxide; Microtubule-associated protein; Dendrite; mRNA
The long-term potentiation "(LTP) of synaptic transmission observed in the hippocampus following glutamate receptor stimulation has become the focus of attention for research into the mechanisms of synaptic plasticity. In the dentate gyrus, N-methyl-D-aspartate (NMDA) receptor stimulation is required for the induction of LTP [4,14,22], and various different biochemical changes are thought to underly its distinct temporal phases [4,28]. The late phase of LTP, which can last for a number of days, is blocked by inhibition of protein synthesis [1,15,32,43]. Since a number of transcription factors are synthesised in the dentate gyrus shortly after LTP induction [10,45], an altered pattern of gene expression in the granule cells, is likely to contribute to the maintenance of LTP. We have demonstrated changes in the levels of prodynorphin and proenkephalin mRNAs in the granule cells after induction of LTP [39], and obtained evidence that release of nitric oxide (NO) may mediate these actions [25,26]. This is consistent with the presence of NO synthase and guanylate cyclase in the dentate gyrus
*Corresponding author. Fax: (44) 41-330-4100. 0304-3940/94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved S S D I 0 3 0 4 - 3 9 4 0 ( 9 4 ) 0 0 4 6 1-I
[6,40], with the evidence that hippocampal NMDA receptor stimulation causes NO release [13,18,31], and with the proposed involvement of NO in dentate gyral LTP [4,5,23,36,41]. We have recently reported that stimulation of NMDA receptors in the molecular layer, or release of NO, leads to a selective increase in the levels of the mRNA encoding the microtubule-associated protein (MAP) MAP2 [27]. A number of other mRNA species encoding components of the cytoskeleton were not affected. This alteration in MAP2 gene expression may underly the prolonged structural changes thought to be associated with LTP [9,11, 19,33], since induction of MAP genes appears, from in vitro evidence, to be both necessary and sufficient for neurite outgrowth [8,12,16]. The mRNA encoding MAP2 is one of a small group ofmRNAs which are found not only in the cell soma, but also distributed along the dendrites [17]. The functional significance of dendritic mRNA is not clear, since other dendritic proteins are synthesised in the cell body and simply transported out into the dendrites. The modula-' tion of somal MAP2 mRNA levels, by NMDA receptor stimulation and by NO release, led us to wonder whether the presence of certain mRNA species in neuronal dendrites might allow a highly localised modulation of the
H.M. Johnston. B.J. Morris/Neuroscience Letters 177 (1994) 5 10
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Fig 1. Percentagechange in hybridisationsignal in hippocampal granule cells followingunilateral injectionof various pharmacologicalagents. The percentagechange is calculatedrelativeto the equivalentregion on the contralateral, uninjectedside, for (a) somal MAP2mRNA; (b) dendriticMAP2 mRNA; or (c) tubulin T26 mRNA. The agents injected were: 1, vehicle:2, NMDA (50/IM); 3, sin-1 (1 mM); 4, sodium nitroprusside (1 mM); 5, 8-bromo-cGMP(500/IM). Valuesare means + S.E.M. *P < 0.05 comparedto group 1, n = 3-7 animals/group,except"n = 2. N.D., not determined. From the volume of tissue affected, it can be assumed that the drugs are effectivewhen the above concentrations are diluted 10- to 100-foldin extracellular fluid.
rate of protein synthesis. This might be a means of spatially restricting the later phases of synaptic plasticity to the region of the original synaptic stimulation. In this study, we have therefore investigated whether stimulation of N M D A receptors, or release of NO,can alter the levels of MAP2 m R N A in the granule cell dendrites as well as in the cell bodies, and if so, whether the changes in the two cellular compartments are likely to be related. Male Wistar rats (180-200 g) were anaesthetised with Equithesin (4 ml/kg) and given a unilateral injection into the molecular layer of the dentate gyrus using standard stereotaxic techniques. Between 3 and 7 animals were used per treatment group. All drugs were delivered in a total volume of 75 nl, dissolved in physiological saline (vehicle), given over a three minute period. The drugs tested were N M D A , sodium nitroprusside (NP), sin-I molsidomine (sin-l, which, like N E spontaneously releases NO) and 8-bromo-cGMP. Bupivocaine was infiltrated as a postoperative analgesic. Twenty-four hours postinjection, 12 ¢tm cryostat sections were cut, and in situ hybridisation techniques carried out at high stringency as previously described [37], using [35S]dATP-labelled 45 mer oligonucleotide probes. The MAP2 oligonucleotide used was complementary to the region coding for the N-terminal 15 amino acids common to all MAP2 isoforms [29], while the T26 oligo was complementary to part of the 3' non-coding region [20] G G G A A A C A G C A T A G A A G C A T C G A T G C CTGCAG C T A G T G C T G G A G C . After high stringency washing, sections were exposed to X-ray film before being dipped in Ilford K5 photographic emulsion and exposed for 7 days. Sense probes, labelled to identical specific
activities, Northern blots, and displacement studies with a 25-fold excess of unlabelled probe, were used as controls for non-specific hybridisation. Under the conditions used the hybridisation signal is totally specific [37]. For quantitation of the hybridisation signal, a 2 x 2 cm grid was laid over photographs, taken at x 100 magnification in the region under the injection site, either over the molecular layer or over the cell body layer. The silver grains in 20 randomly selected squares were counted and compared with those from an equivalent area on the contralateral side. Results were analysed using ANOVA with post-hoc one-sided Dunnett's test for multiple comparisons with the control mean. For immuno-histochemistry, either 2 or 72 h after microinjection of sin-1 or vehicle, following intracardiac perfusion with 4% paraformaldehyde, brains were infiltrated with 30% sucrose, snap-frozen in liquid nitrogen, and sectioned at 50/~m. Sections were then processed for immunohistochemistry according to standard procedures, using a mouse monoclonal anti-MAP2 antibody (clone HM-2, Sigma) and the avidin-biotin-peroxidase visualisation system (Vector). No evidence for any granule cell necrosis was observed, and the absence of any changes in the majority of m R N A species monitored (tubulin isoforms T26 and Tal, the MAP isoforms tau-1 and tau-2, the neurotrophic factors nerve growth factor, neurotrophin-3 and epidermal growth factor, GAP-43, and protease nexin I) following injection of any of the drugs used, argues strongly against any toxic or traumatic events being responsible for the changes observed. As previously reported [27], we observed that NMDA, NP, sin-1 and 8-bromo-cGMP increased the MAP2
H.M. Johnston, B.J. Morris/Neuroscience Letters 177 (1994) 5 10
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Fig. 2. Dendritic changes in MAP2 mRNAs. Hippocampal dentate gyrus contralateral (a,c) and ipsilateral (b,d) to the injection of 75 nl 1 m M sodium nitroprusside, following hybridisation with the MAP2 m R N A probe. The same regions shown in bright field in a and b are shown in dark field in c and d, respectively. The position of the darkly counterstained cell bodies can be seen in the lower portion of a and b. Note that the increase in MAP2 m R N A signal was restricted to the dendritic layer (upper portion of a-d). Bar = 20/.tm.
mRNA signal over the affected granule cell bodies F4.17 = 3.88, P < 0.05 (Fig. la). The effects of NMDA were blocked by NMDA receptor antagonists, and the effects of sin-1 were blocked by inhibition of cGMPdependent protein kinase (PKG, not shown) [27]. However, in some animals, it was apparent that injection of a NO-mimetic agent was increasing MAP2 mRNA levels in the molecular (dendritic) layer of the dentate gyrus as well as in the granule cell (perikaryal) layer. Clear increases in MAP2 hybridisation signal were observed over the molecular layer in the region of the injection, some distance from the cell bodies F4,~7 = 3.00, P ~< 0.05 (Fig. lb). In some cases, an increase in dendritic MAP2 hybridisation signal was observed in the absence of any comparable change in the cell soma (Fig. 2a-d). To determine whether the elevation in dendritic mRNA levels was simply a reflection of increased cell body mRNA levels which had overflowed into the dendrites, we looked for a correlation between the somal MAP2 mRNA changes and the dendritic changes in 16 animals injected with agents able to increase MAP2 mRNA levels (NP, sin-1, 8-bromo-cGMP, or NMDA). The ratio of the dendritic change to the cell body change (each expressed as the percentage signal in the injected region relative to the equivalent contralateral area), varied from 2 to 0.7. No significant correlation was observed between the degree of change in the two cellular compartments (r = 0.266), implying that the dendritic change occurs by a mechanism unrelated to the changes in the cell body. This can be seen particularly clearly when the mean ef-
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fects of sin-1 and sodium nitroprusside on somal and dendritic MAP2 mRNA levels are compared (Fig. 1). No significant changes in either somal or dendritic MAP2 mRNA signal were seen in a group of vehicle-injected animals. In order to determine whether the highly localised increases in MAP2 mRNA levels we have detected are responsible for a sustained alteration in cytoskeletal organisation, we studied the levels of immunoreactive MAP2 protein following injection of vehicle or sin-1. No alteration in MAP2 immunostaining was observed in the dentate gyrus molecular layer 2 h, or 72 h (Fig. 3a) after the injection of vehicle, or 2 h after the injection of sin-1. However, 72 h after injection of sin-l, a pronounced increase in immunoreactive MAP2 was noted in the outer molecular layer, restricted to the vicinity of the injection site (Fig. 3b). This halo of increased immunostaining around the needle tip following sin-1 injection was noted in each of three animals at this time point. While vehicle injection produced no changes in mRNA content, the injection of NMDA, or NO-releasing agents, caused an increase in the levels of MAP2 mRNA in both the cell body layer and the molecular layer of the dentate gyrus. There was no correlation between the degree of change in the two cellular compartments, and in fact, in some cases increases were observed in one layer in the absence of any change in the other layer. This is difficult to reconcile with the hypothesis that the dendritic increases are merely an overflow of increased mRNA levels in the cell body, particularly when an increase can be observed in the molecular layer with no change in the cell body layer. Rather, it would appear that dendritic MAP2 mRNA levels can be varied independently of somal MAP2 mRNA content. It is possible that the dendritic increase is effected transcriptionally, and that the extra mRNA produced is then somehow channelled selectively out to the dendrites in the vicinity of the stimulation. However, this is not supported by our preliminary studies on the time course of these effects, where the changes in somal and dendritic mRNA levels appear simultaneously (Johnston and Morris, in preparation). It seems more likely that the dendritic increase is mediated by a post-transcriptional action on the population of MAP2 mRNA molecules already present in the dendrite. This would imply that NO can increase MAP2 mRNA stability. The regulation of mRNA stability is a well-characterised phenomenon for other mRNA species [38]. A number of potential mechanisms can be envisaged, including a local inhibition of RNAse activity by PKG. Alternatively, an autoregulatory effect has been observed for fl-tubulin, another microtubule component, where unpolymerised fl-tubulin decreases the stability offl-tubulin mRNA [46]. It is conceivable that a similar autoregulatory mechanism could operate for MAP2, allowing phosphorylated, depolymerised MAP2 molecules to decrease MAP2
8
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Fig 3. a,b: bright-lield photomicrographs of sections processed for MAP2 immunohistochemistry, showing the injection site 3 days after unilateral injection of 75 nl (a) vehicle, or (b) sin-I, st, stratmn radiatum: HiF, hippocampal fissure: mol, dentate gyrus molecular layer: gr, granule cell layer. Note the increase in MAP2 immunoreactivity in the immediate vicinity of the needle tip in b), that is not seen in a). c,d: profiles of optical densit~ measurements taken through the molecular layer of the dentate gyrus, from the dorsal edge of the granule cell layer to the ventral edge of the hippocampal fissure. Profile c corresponds to the mid-portion of the section shown in at, while profile d corresponds to the mid-portion of the section shown in b. Note the increase in staining intensity on the dorsal side of the molecular layer fin the vicinity of the needle tip) detected in d but not in c. Equivalent results were obtained in two further animals from each group. Bar = 10 ,urn.
m R N A stability. There is evidence that glutamate receptor stimulation induces MAP2 dephosphorylation [21] and polymerisation [2,3], arguably removing this inhibitiom and increasing local MAP2 m R N A levels. The Ca-'+-activated protease calpain I has been suggested as a possible intracellular mediator of post-synaptic plasticity [28]. Calpain can hydrolyse MAP2 in vitro [24,44], and presumably only the synthesis of new protein can reverse the effects of this proteolysis. The local induction of MAP2 gene expression could, therefore, be a mechanism to restore locally depleted MAP2 protein levels. However, no alteration in MAP2 immunostaining was observed in the dentate gyrus 2 h after the injection of sin-l. This would imply that the subsequent mRNA increase is not a response to restore locally depleted MAP2 protein. In contrast, 72 h after injection of sin-l, an increase in immunoreactive MAP2 was noted in the outer molecular layer, restricted to the vicinity of the injection site. Changes in MAP2 immunostaining have been observed in cultured neurones following glutamate receptor stimu-
lation [2,3,34,35], and in the hippocampus following ischaemic damage [30]. These changes may reflect rapid alterations in the phosphorylation state of MAP2, which are likely to affect the properties of the dendritic cytoskeleton. Our results show that NO may be involved in these actions, and that the effects on MAP2 expression can be extended over a long period of time following the original stimulation. The highly localised increases in immunostaining three days after stimulation strongly suggest that the increases in dendritic MAP2 m R N A have caused a long-term alteration in dendritic structure that is limited to the region of NO release. Furthermore, since there is a strong correlation, both in vitro and in vivo, between induction of MAP2 gene expression at the mRNA level, and neuritic outgrowth [7,8,12,42], the data reported here reveal a mechanism allowing synaptic activity to change dendritic morphology for a sustained period. Overall, our results illustrate a further level of complexity in the plasticity of hippocampal neurotransmission induced by glutamate receptor stimulation. The
H. M. Johnston, B.J. Morris I Neuroscience Letters 177 (1994) 5-10
selective induction of dendritic MAP2 gene expression additionally demonstrates, for the first time, a mechanism whereby NO release can cause a protracted alteration in dendritic structure that is limited to those parts of the neurone in the vicinity of the stimulation [19]. This research was supported by the Medical Research Council (UK) and the Wellcome Trust. We would additionally like to thank Drs. S.P. Hunt and W. Wisden for helpful discussions, and Prof. J. Garthwaite for the gift of sin-1 molsidomine. [1] Bailey, C.H., Montarolo, E, Chen, M., Kandel, E.R. and Schacher, S., Inhibitors of protein and RNA synthesis block structural changes that accompany long-term heterosynaptic plasticity in Aplysia, Neuron 9, (1992) 749-758. [2] Bigot, D. and Hunt, S.P., Effect of excitatory amino-acids on microtubule-associated proteins in cultured cortical and spinal neurones, Neurosci. Lett., 111 (1990)275-280. [3] Bigot, D. and Hunt, S.P., Reorganisation of the cytoskeleton in rat neurons following stimulation with excitatory amino acids in vitro, Eur. J. Neurosci., 3 (1992) 551-558. [4] Bliss, T.V.P. and Collingridge, G.L., A synaptic model of memory: long-term potentiation in the hippocampus, Nature, 361 (1993) 31-38. [5] B6hme, G.A., Bon, C., stutzmann, J.-M., Doble, A. and Blanchard, J.-C., Possible involvement of nitric oxide in long-term potentiation, Eur. J. Pharmacol., 199 (1991) 37%381. [6] Bredt, D.S., Hwang, P.M. and Snyder, S.H. Localisation of nitric oxide synthase indicating a neural role for nitric oxide, Nature, 347 (1990) 768 771. [7] Caceres, A., Busciglio, J., Ferreira, A. and Steward, O., An immunocytochemical and biochemical study of MAP2 during postlesion dendritic remodelling in the central nervous system, Mol. Brain Res., 3 (1988) 233-246. [8] Caceres, A., Mautino, J. and Kosik, K.S., Suppression of MAP2 in cultured cerebellar macroneurons inhibits minor neurite formation, Neuron, 9 (1992) 607-618. [9] Chang, F.-L. and Greenough, W.T., Transient and enduring morphological correlates of synaptic activity and efficacy change in the rat hippocampal slice, Brain Res., 309 (1984) 35~,6. [10] Cole, A.J., Saffen, D.W., Baraban, J.M. and Worley, P.F., Rapid increase of an immediate-early gene mRNA in hippocampal neurons by synaptic NMDA receptor activation, Nature, 340 (1989) 474-476. [11] Desmond and Levy, Changes in the numerical density of synaptic contacts with long-term potentiation in the hippocampal dentate gyrus, J. Comp. Neurol., 253 (1986) 476~82. [12] Dinsmore, J.H. and Solomon, F., Inhibition of MAP2 expression affects both morphological and cell division phenotypes of neuronal differentiation, Cell, 64 (1991) 817-826. [13] East, S.J. and Garthwaite, J., NMDA receptor activation in rat hippocampus induces cyclicGMP formation through the L-arginine-nitric oxide pathway, Neurosci. Lett., 123 (1991) 17-19. [14] Errington, M.L., Lynch, M.A. and Bliss, T.V.P., Long-term potentiation in the dentate gyrus: induction and glutamate release are blocked by APV, Neuroscience, 23 (1987) 279 284. [15] Fazeli, M.S., Corbet, J., Dunn, M.J., Dolphin, A.C. and Bliss, T.V.P., Changes in protein synthesis accompanying long-term potentiation in the dentate gyrus in vivo, J. Neurosci., 13 (1993) 1346-1353. [16] Ferreira, A., Busciglio, J., Landa, C. and Caceres, A., Gangliosideenhanced neurite outgrowth: evidence for a selective induction of high molecular weight MAP2, J. Neurosci., 10 (1990) 293-302.
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[17] Garner, C.C., Tucker, R.P. and Matus, A., Selective localisation of messenger RNA for cytoskeletal protein MAP2 in dendrites, Nature, 336 (1988) 674-677. [18] Garthwaite, J., Charles, S.L. and Chess-Williams, R., Endothelium-derived relaxing factor release on activation of NMDA receptors suggests a role as intercellular messenger in the brain, Nature, 336 (1988) 385-388. [19] Geinisman, Y., de Toledo-Morrell, L. and Morrell, E, Induction of long-term potentiation is associated with an increase in the number of axospinous synapses with segmented postsynaptic densities, Brain Res., 566 (1991) 77-88. [20] Ginzburg, J.,Behar, L., Givol, D. and Littauer, U.Z., The nucleotide sequence of rat ~-tubulin, Nucl. Acid. Res., 9 (1986) 26912697. [21] Halpain, S. and Greengard, E, Activation of NMDA receptors induces rapid dephosphorylation of the cytoskeletal protein MAP2, Neuron, 5 (1990) 237-246. [22] Harris, E.W., Ganong, A.H. and Cotman, C.W., Long-term potentiation in the hippocampus involves activation of NMDA receptors, Brain Res., 323 (1984) 132-137. [23] Iga, Y., Yoshioka, M., Togashi, H. and Saito, H., Inhibitory action of N-nitro-L-arginine methyl ester on in vivo long-term potentiation in the rat dentate gyrus, Eur. J. Pharmacol., 238 (1993) 395-398. [24] Johnson, G.V.W., Litersky, J.M. and Jope, R.S., Degradation of microtubule-associated protein 2 and brain spectrin by calpain, J. Neurochem., 56 (1991) 1630-1638. [25] Johnston, H.M. and Morris, B.J. Increased c-fos expression does not cause the elevated proenkephalin mRNA levels in the dentate gyrus following NMDA stimulation, Mol. Brain Res., in press. [26] Johnston, H.M. and Morris, B.J., Nitric oxide alters proenkephalin and prodynorphin gene expression in hippocampal granule cells, Neuroscience, in press. [27] Johnston, H.M. and Morris, B.J. NMDA and nitric oxide increase MAP2 gene expression in hippocampal granule cells, J. Neurochem., in press. [28] Kennedy, M.B., Regulation of synaptic transmission in the central nervous system: long-term potentiation, Cell, 59 (1989) 777-787. [29] Kindler, S., Schwanke, B., Schulz, B. and Garner, C.C., Complete cDNA sequence encoding rat high and low molecular weight MAP2, Nucl. Acid. Res., 18 (1990) 222-228. [30] Kitagawa, K., Matsumoto, M., Niinobe, M., Mikoshiba, K., Hata, R., Ueda, H., Handa, N., Fukunaga, R., Isaka, Y., Kimura, K. and Kamada, T., MAP2 as a sensitive marker for cerebral ischaemic damage, Neuroscience, 31 (1989) 401~,11. [31] Knowles.R.G., Palacios, M., Palmer, R.M.J. and Moncada, S., Formation of nitric oxide from L-arginine in the central nervous system, Proc. Natl. Acad. Sci. USA, 89 (1989) 5159-5162. [32] Krug, M., Lossner, 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~,2. [33] Lisman, J.E. and Harris, K.M., Quantal analysis and synaptic anatomy - - integrating two views of hippocampal plasticity, TINS, 16 (1993) 141-145. [34] Mattson, M.E, Duo, E and Kater, S.B., Outgrowth-regulating actions of glutamate in isolated hippocampal pyramidal neurones, J. Neurosci., 8 (1988) 2087-2100. [35] Mattson, M.P., Guthrie, EB. and Kater, S.B., Intracellular messengers in the generation and degeneration of hippocampal neuroarchitecture, J. Neurosci. Res., 20 (1988) 447464. [36] Mizutani, A., Saito, H. and Abe, K., Involvement of nitric oxide in long-term potentiation in the dentate gyrus, Brain Res., 605 (1993) 309-311. [37] Morris, B.J., Neuronal localisation of neuropeptide Y gene expression in rat brain, J. Comp. Neurol., 290 (1989) 358-368. [38] Morris, B.J., Control of receptor sensitivity at the mRNA level, Mol. Neurobiol., 7 (1993) 1-17.
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[39] Morris, B.J., Feasey, K.J., ten Bruggencate, G., Herz, A. and H611t. V., Electrical stimulation in vivo increases the expression of proenkephalin mRNA and decreases the expression of prodynorphin mRNA in rat hippocampal granule cells, Proc. Natl. Acad. Sci. USA, 85 (1988) 3226 3230. [40] Nakane, M., lchikawa, M. and Deguchi, T., Light and electron microscopic demonstration of guanylate cyclase in rat brain, Brain Res., 273 (1983) 9 15. [41] O'Dell, T.J., Hawkins, R.D., Kandel, E.R. and Arancio, O., Tests of the roles of two diffusible substances in long-term potentiation. Proc. Natl. Acad. Sci. USA, 88 (1991) 11285 11289. [42] Sano, M., Katoh-Semba, R., Kitajima, S. and Sato, C., Changes in levels of MAPs in relation to the outgrowth of neurites l¥om PCI2 cells, Brain Res.. 510 (1990) 269 276.
[43] Sargent-Jones, L., Grooms, S.Y., Lapadula, D.M. and Lewis, D.V.. Protein synthesis inhibition blocks maintenance but not induction of epileptogenesis in hippocampal slice, Brain Res., 599 (t992) 338 344. [44] Siman, R. and Noszek, J.C., Excitatm'y amino-acids activate calpain 1 and induce structural protein breakdown in vivo, Neuron. I (1988)279 287. [45] Wisden, W., Errington. M.L., Williams, S., Dunnett, S.B., Waters. C., Hitchcock, D., Evan, G., Bliss, T.V.R and Hunt, S.R, Differential expression of immediate-early genes in the hippocampus and spinal cord, Neuron, 4 {1990) 603 6t4. [46] Yen, T.J., Machlin, P.S. and Cleveland, D.W., Autoregulated mstability offl-tubulin mRNAs by recognition of the nascent amino terminus offl-tubulin, Nature, 334 11988) 580 585.
Neuroscience Letters 177 (1994) 5 10
NIUROSClENCE LETTERS
Selective regulation of dendritic MAP2 mRNA levels in hippocampal granule cells by nitric oxide H.M. Johnston, B.J. Morris* Department of Pharmacology, University of Glasgow, Glasgow G12 8QQ, UK Received 14 April 1994; Revised version received 5 June 1994; Accepted 7 June 1994
Abstract
Application of NMDA, or agents releasing nitric oxide (NO), onto the dendrites of hippocampal granule cells increased the levels of the mRNA encoding MAP2, a cytoskeletalcomponent induced during periods of neurite outgrowth. Furthermore, local increases in the hybridisation signal in the molecular layer, representing dendritic MAP2 mRNA, occurred independentlyof changes in MAP2 mRNA levels in the cell body layer. The selectivemodulation of MAP2 mRNA in dendrites reveals a mechanismallowinga sustained stimulation of dendritic outgrowth to be confined to those regions of a neuron's dendritic arbour local to glutamate receptor stimulation. Key words: Glutamate; Nitric oxide; Microtubule-associated protein; Dendrite; mRNA
The long-term potentiation "(LTP) of synaptic transmission observed in the hippocampus following glutamate receptor stimulation has become the focus of attention for research into the mechanisms of synaptic plasticity. In the dentate gyrus, N-methyl-D-aspartate (NMDA) receptor stimulation is required for the induction of LTP [4,14,22], and various different biochemical changes are thought to underly its distinct temporal phases [4,28]. The late phase of LTP, which can last for a number of days, is blocked by inhibition of protein synthesis [1,15,32,43]. Since a number of transcription factors are synthesised in the dentate gyrus shortly after LTP induction [10,45], an altered pattern of gene expression in the granule cells, is likely to contribute to the maintenance of LTP. We have demonstrated changes in the levels of prodynorphin and proenkephalin mRNAs in the granule cells after induction of LTP [39], and obtained evidence that release of nitric oxide (NO) may mediate these actions [25,26]. This is consistent with the presence of NO synthase and guanylate cyclase in the dentate gyrus
*Corresponding author. Fax: (44) 41-330-4100. 0304-3940/94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved S S D I 0 3 0 4 - 3 9 4 0 ( 9 4 ) 0 0 4 6 1-I
[6,40], with the evidence that hippocampal NMDA receptor stimulation causes NO release [13,18,31], and with the proposed involvement of NO in dentate gyral LTP [4,5,23,36,41]. We have recently reported that stimulation of NMDA receptors in the molecular layer, or release of NO, leads to a selective increase in the levels of the mRNA encoding the microtubule-associated protein (MAP) MAP2 [27]. A number of other mRNA species encoding components of the cytoskeleton were not affected. This alteration in MAP2 gene expression may underly the prolonged structural changes thought to be associated with LTP [9,11, 19,33], since induction of MAP genes appears, from in vitro evidence, to be both necessary and sufficient for neurite outgrowth [8,12,16]. The mRNA encoding MAP2 is one of a small group ofmRNAs which are found not only in the cell soma, but also distributed along the dendrites [17]. The functional significance of dendritic mRNA is not clear, since other dendritic proteins are synthesised in the cell body and simply transported out into the dendrites. The modula-' tion of somal MAP2 mRNA levels, by NMDA receptor stimulation and by NO release, led us to wonder whether the presence of certain mRNA species in neuronal dendrites might allow a highly localised modulation of the
H.M. Johnston. B.J. Morris/Neuroscience Letters 177 (1994) 5 10
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TUBULIN T 2 6 m R N A
Fig 1. Percentagechange in hybridisationsignal in hippocampal granule cells followingunilateral injectionof various pharmacologicalagents. The percentagechange is calculatedrelativeto the equivalentregion on the contralateral, uninjectedside, for (a) somal MAP2mRNA; (b) dendriticMAP2 mRNA; or (c) tubulin T26 mRNA. The agents injected were: 1, vehicle:2, NMDA (50/IM); 3, sin-1 (1 mM); 4, sodium nitroprusside (1 mM); 5, 8-bromo-cGMP(500/IM). Valuesare means + S.E.M. *P < 0.05 comparedto group 1, n = 3-7 animals/group,except"n = 2. N.D., not determined. From the volume of tissue affected, it can be assumed that the drugs are effectivewhen the above concentrations are diluted 10- to 100-foldin extracellular fluid.
rate of protein synthesis. This might be a means of spatially restricting the later phases of synaptic plasticity to the region of the original synaptic stimulation. In this study, we have therefore investigated whether stimulation of N M D A receptors, or release of NO,can alter the levels of MAP2 m R N A in the granule cell dendrites as well as in the cell bodies, and if so, whether the changes in the two cellular compartments are likely to be related. Male Wistar rats (180-200 g) were anaesthetised with Equithesin (4 ml/kg) and given a unilateral injection into the molecular layer of the dentate gyrus using standard stereotaxic techniques. Between 3 and 7 animals were used per treatment group. All drugs were delivered in a total volume of 75 nl, dissolved in physiological saline (vehicle), given over a three minute period. The drugs tested were N M D A , sodium nitroprusside (NP), sin-I molsidomine (sin-l, which, like N E spontaneously releases NO) and 8-bromo-cGMP. Bupivocaine was infiltrated as a postoperative analgesic. Twenty-four hours postinjection, 12 ¢tm cryostat sections were cut, and in situ hybridisation techniques carried out at high stringency as previously described [37], using [35S]dATP-labelled 45 mer oligonucleotide probes. The MAP2 oligonucleotide used was complementary to the region coding for the N-terminal 15 amino acids common to all MAP2 isoforms [29], while the T26 oligo was complementary to part of the 3' non-coding region [20] G G G A A A C A G C A T A G A A G C A T C G A T G C CTGCAG C T A G T G C T G G A G C . After high stringency washing, sections were exposed to X-ray film before being dipped in Ilford K5 photographic emulsion and exposed for 7 days. Sense probes, labelled to identical specific
activities, Northern blots, and displacement studies with a 25-fold excess of unlabelled probe, were used as controls for non-specific hybridisation. Under the conditions used the hybridisation signal is totally specific [37]. For quantitation of the hybridisation signal, a 2 x 2 cm grid was laid over photographs, taken at x 100 magnification in the region under the injection site, either over the molecular layer or over the cell body layer. The silver grains in 20 randomly selected squares were counted and compared with those from an equivalent area on the contralateral side. Results were analysed using ANOVA with post-hoc one-sided Dunnett's test for multiple comparisons with the control mean. For immuno-histochemistry, either 2 or 72 h after microinjection of sin-1 or vehicle, following intracardiac perfusion with 4% paraformaldehyde, brains were infiltrated with 30% sucrose, snap-frozen in liquid nitrogen, and sectioned at 50/~m. Sections were then processed for immunohistochemistry according to standard procedures, using a mouse monoclonal anti-MAP2 antibody (clone HM-2, Sigma) and the avidin-biotin-peroxidase visualisation system (Vector). No evidence for any granule cell necrosis was observed, and the absence of any changes in the majority of m R N A species monitored (tubulin isoforms T26 and Tal, the MAP isoforms tau-1 and tau-2, the neurotrophic factors nerve growth factor, neurotrophin-3 and epidermal growth factor, GAP-43, and protease nexin I) following injection of any of the drugs used, argues strongly against any toxic or traumatic events being responsible for the changes observed. As previously reported [27], we observed that NMDA, NP, sin-1 and 8-bromo-cGMP increased the MAP2
H.M. Johnston, B.J. Morris/Neuroscience Letters 177 (1994) 5 10
"4
•
Fig. 2. Dendritic changes in MAP2 mRNAs. Hippocampal dentate gyrus contralateral (a,c) and ipsilateral (b,d) to the injection of 75 nl 1 m M sodium nitroprusside, following hybridisation with the MAP2 m R N A probe. The same regions shown in bright field in a and b are shown in dark field in c and d, respectively. The position of the darkly counterstained cell bodies can be seen in the lower portion of a and b. Note that the increase in MAP2 m R N A signal was restricted to the dendritic layer (upper portion of a-d). Bar = 20/.tm.
mRNA signal over the affected granule cell bodies F4.17 = 3.88, P < 0.05 (Fig. la). The effects of NMDA were blocked by NMDA receptor antagonists, and the effects of sin-1 were blocked by inhibition of cGMPdependent protein kinase (PKG, not shown) [27]. However, in some animals, it was apparent that injection of a NO-mimetic agent was increasing MAP2 mRNA levels in the molecular (dendritic) layer of the dentate gyrus as well as in the granule cell (perikaryal) layer. Clear increases in MAP2 hybridisation signal were observed over the molecular layer in the region of the injection, some distance from the cell bodies F4,~7 = 3.00, P ~< 0.05 (Fig. lb). In some cases, an increase in dendritic MAP2 hybridisation signal was observed in the absence of any comparable change in the cell soma (Fig. 2a-d). To determine whether the elevation in dendritic mRNA levels was simply a reflection of increased cell body mRNA levels which had overflowed into the dendrites, we looked for a correlation between the somal MAP2 mRNA changes and the dendritic changes in 16 animals injected with agents able to increase MAP2 mRNA levels (NP, sin-1, 8-bromo-cGMP, or NMDA). The ratio of the dendritic change to the cell body change (each expressed as the percentage signal in the injected region relative to the equivalent contralateral area), varied from 2 to 0.7. No significant correlation was observed between the degree of change in the two cellular compartments (r = 0.266), implying that the dendritic change occurs by a mechanism unrelated to the changes in the cell body. This can be seen particularly clearly when the mean ef-
7
fects of sin-1 and sodium nitroprusside on somal and dendritic MAP2 mRNA levels are compared (Fig. 1). No significant changes in either somal or dendritic MAP2 mRNA signal were seen in a group of vehicle-injected animals. In order to determine whether the highly localised increases in MAP2 mRNA levels we have detected are responsible for a sustained alteration in cytoskeletal organisation, we studied the levels of immunoreactive MAP2 protein following injection of vehicle or sin-1. No alteration in MAP2 immunostaining was observed in the dentate gyrus molecular layer 2 h, or 72 h (Fig. 3a) after the injection of vehicle, or 2 h after the injection of sin-1. However, 72 h after injection of sin-l, a pronounced increase in immunoreactive MAP2 was noted in the outer molecular layer, restricted to the vicinity of the injection site (Fig. 3b). This halo of increased immunostaining around the needle tip following sin-1 injection was noted in each of three animals at this time point. While vehicle injection produced no changes in mRNA content, the injection of NMDA, or NO-releasing agents, caused an increase in the levels of MAP2 mRNA in both the cell body layer and the molecular layer of the dentate gyrus. There was no correlation between the degree of change in the two cellular compartments, and in fact, in some cases increases were observed in one layer in the absence of any change in the other layer. This is difficult to reconcile with the hypothesis that the dendritic increases are merely an overflow of increased mRNA levels in the cell body, particularly when an increase can be observed in the molecular layer with no change in the cell body layer. Rather, it would appear that dendritic MAP2 mRNA levels can be varied independently of somal MAP2 mRNA content. It is possible that the dendritic increase is effected transcriptionally, and that the extra mRNA produced is then somehow channelled selectively out to the dendrites in the vicinity of the stimulation. However, this is not supported by our preliminary studies on the time course of these effects, where the changes in somal and dendritic mRNA levels appear simultaneously (Johnston and Morris, in preparation). It seems more likely that the dendritic increase is mediated by a post-transcriptional action on the population of MAP2 mRNA molecules already present in the dendrite. This would imply that NO can increase MAP2 mRNA stability. The regulation of mRNA stability is a well-characterised phenomenon for other mRNA species [38]. A number of potential mechanisms can be envisaged, including a local inhibition of RNAse activity by PKG. Alternatively, an autoregulatory effect has been observed for fl-tubulin, another microtubule component, where unpolymerised fl-tubulin decreases the stability offl-tubulin mRNA [46]. It is conceivable that a similar autoregulatory mechanism could operate for MAP2, allowing phosphorylated, depolymerised MAP2 molecules to decrease MAP2
8
H.M. Johnston, B.J MorrLs / Neurosciencc Le/ter,~ 177 , 1994) 5 10
' '--'LJ '--' rJ Ln
l'~
1421 gr
,n ,U-,h
• h,b~ , nl~ h rn/L,
I
~h.~ Hi F
142t gr
A HI F
Fig 3. a,b: bright-lield photomicrographs of sections processed for MAP2 immunohistochemistry, showing the injection site 3 days after unilateral injection of 75 nl (a) vehicle, or (b) sin-I, st, stratmn radiatum: HiF, hippocampal fissure: mol, dentate gyrus molecular layer: gr, granule cell layer. Note the increase in MAP2 immunoreactivity in the immediate vicinity of the needle tip in b), that is not seen in a). c,d: profiles of optical densit~ measurements taken through the molecular layer of the dentate gyrus, from the dorsal edge of the granule cell layer to the ventral edge of the hippocampal fissure. Profile c corresponds to the mid-portion of the section shown in at, while profile d corresponds to the mid-portion of the section shown in b. Note the increase in staining intensity on the dorsal side of the molecular layer fin the vicinity of the needle tip) detected in d but not in c. Equivalent results were obtained in two further animals from each group. Bar = 10 ,urn.
m R N A stability. There is evidence that glutamate receptor stimulation induces MAP2 dephosphorylation [21] and polymerisation [2,3], arguably removing this inhibitiom and increasing local MAP2 m R N A levels. The Ca-'+-activated protease calpain I has been suggested as a possible intracellular mediator of post-synaptic plasticity [28]. Calpain can hydrolyse MAP2 in vitro [24,44], and presumably only the synthesis of new protein can reverse the effects of this proteolysis. The local induction of MAP2 gene expression could, therefore, be a mechanism to restore locally depleted MAP2 protein levels. However, no alteration in MAP2 immunostaining was observed in the dentate gyrus 2 h after the injection of sin-l. This would imply that the subsequent mRNA increase is not a response to restore locally depleted MAP2 protein. In contrast, 72 h after injection of sin-l, an increase in immunoreactive MAP2 was noted in the outer molecular layer, restricted to the vicinity of the injection site. Changes in MAP2 immunostaining have been observed in cultured neurones following glutamate receptor stimu-
lation [2,3,34,35], and in the hippocampus following ischaemic damage [30]. These changes may reflect rapid alterations in the phosphorylation state of MAP2, which are likely to affect the properties of the dendritic cytoskeleton. Our results show that NO may be involved in these actions, and that the effects on MAP2 expression can be extended over a long period of time following the original stimulation. The highly localised increases in immunostaining three days after stimulation strongly suggest that the increases in dendritic MAP2 m R N A have caused a long-term alteration in dendritic structure that is limited to the region of NO release. Furthermore, since there is a strong correlation, both in vitro and in vivo, between induction of MAP2 gene expression at the mRNA level, and neuritic outgrowth [7,8,12,42], the data reported here reveal a mechanism allowing synaptic activity to change dendritic morphology for a sustained period. Overall, our results illustrate a further level of complexity in the plasticity of hippocampal neurotransmission induced by glutamate receptor stimulation. The
H. M. Johnston, B.J. Morris I Neuroscience Letters 177 (1994) 5-10
selective induction of dendritic MAP2 gene expression additionally demonstrates, for the first time, a mechanism whereby NO release can cause a protracted alteration in dendritic structure that is limited to those parts of the neurone in the vicinity of the stimulation [19]. This research was supported by the Medical Research Council (UK) and the Wellcome Trust. We would additionally like to thank Drs. S.P. Hunt and W. Wisden for helpful discussions, and Prof. J. Garthwaite for the gift of sin-1 molsidomine. [1] Bailey, C.H., Montarolo, E, Chen, M., Kandel, E.R. and Schacher, S., Inhibitors of protein and RNA synthesis block structural changes that accompany long-term heterosynaptic plasticity in Aplysia, Neuron 9, (1992) 749-758. [2] Bigot, D. and Hunt, S.P., Effect of excitatory amino-acids on microtubule-associated proteins in cultured cortical and spinal neurones, Neurosci. Lett., 111 (1990)275-280. [3] Bigot, D. and Hunt, S.P., Reorganisation of the cytoskeleton in rat neurons following stimulation with excitatory amino acids in vitro, Eur. J. Neurosci., 3 (1992) 551-558. [4] Bliss, T.V.P. and Collingridge, G.L., A synaptic model of memory: long-term potentiation in the hippocampus, Nature, 361 (1993) 31-38. [5] B6hme, G.A., Bon, C., stutzmann, J.-M., Doble, A. and Blanchard, J.-C., Possible involvement of nitric oxide in long-term potentiation, Eur. J. Pharmacol., 199 (1991) 37%381. [6] Bredt, D.S., Hwang, P.M. and Snyder, S.H. Localisation of nitric oxide synthase indicating a neural role for nitric oxide, Nature, 347 (1990) 768 771. [7] Caceres, A., Busciglio, J., Ferreira, A. and Steward, O., An immunocytochemical and biochemical study of MAP2 during postlesion dendritic remodelling in the central nervous system, Mol. Brain Res., 3 (1988) 233-246. [8] Caceres, A., Mautino, J. and Kosik, K.S., Suppression of MAP2 in cultured cerebellar macroneurons inhibits minor neurite formation, Neuron, 9 (1992) 607-618. [9] Chang, F.-L. and Greenough, W.T., Transient and enduring morphological correlates of synaptic activity and efficacy change in the rat hippocampal slice, Brain Res., 309 (1984) 35~,6. [10] Cole, A.J., Saffen, D.W., Baraban, J.M. and Worley, P.F., Rapid increase of an immediate-early gene mRNA in hippocampal neurons by synaptic NMDA receptor activation, Nature, 340 (1989) 474-476. [11] Desmond and Levy, Changes in the numerical density of synaptic contacts with long-term potentiation in the hippocampal dentate gyrus, J. Comp. Neurol., 253 (1986) 476~82. [12] Dinsmore, J.H. and Solomon, F., Inhibition of MAP2 expression affects both morphological and cell division phenotypes of neuronal differentiation, Cell, 64 (1991) 817-826. [13] East, S.J. and Garthwaite, J., NMDA receptor activation in rat hippocampus induces cyclicGMP formation through the L-arginine-nitric oxide pathway, Neurosci. Lett., 123 (1991) 17-19. [14] Errington, M.L., Lynch, M.A. and Bliss, T.V.P., Long-term potentiation in the dentate gyrus: induction and glutamate release are blocked by APV, Neuroscience, 23 (1987) 279 284. [15] Fazeli, M.S., Corbet, J., Dunn, M.J., Dolphin, A.C. and Bliss, T.V.P., Changes in protein synthesis accompanying long-term potentiation in the dentate gyrus in vivo, J. Neurosci., 13 (1993) 1346-1353. [16] Ferreira, A., Busciglio, J., Landa, C. and Caceres, A., Gangliosideenhanced neurite outgrowth: evidence for a selective induction of high molecular weight MAP2, J. Neurosci., 10 (1990) 293-302.
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