21
Neuroscience Letters, 131 (1991) 21-26 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 0304394091005454 NSL 08051
The effects of quisqualate and nocodazole on the organization of MAP2 and neurofilaments in spinal cord neurons in vitro Dominique Bigot and Stephen P. H u n t MRC Neurobiology Unit, MRC Centre, Cambridge ( U.K.) (Received 7 April 1991; Revised version received 13 June 1991; Accepted 18 June 199 I)
Key words: Excitatory amino acid; Nocodazole; MAP2, Neurofilament The relationship between microtubules, neurofilaments and microtubule-associated protein (MAP)2 was investigated in spinal cord neurons grown for up to 14 days in vitro. Neurons were labelled using antibodies against MAP2, neurofilaments and tubulin, and immunofluorescence analyzed by confocal microscopy. A well-structured network of neurofilaments and microtubules was observed in unstimulated cultures. MAP2 staining was poorly structured but became more filamentous following depolymerization of microtubules with nocodazole. Double-staining experiments suggested that MAP2 was now closely associated with neurofilaments in cell bodies and dendrites. Stimulation of cultures with excitatory amino acids increased the resistance of the microtubular cytoskeleton to depolymerization by nocodazole. Again double-labelling experiments demonstrated an increased association between neurofilaments and MAP2 immunofluorescence. Previous results suggested that the stability of the neuronal cytoskeleton could be modulated by glutamate receptors acting through an increased binding of MAP2 to microtubules. From the results presented here, we further suggest that cross-linking of neurofilaments to microtubules may also play a role in this process.
The neuronal cytoskeleton is composed of 3 major components, microfilaments made up of G-actin, neurofilaments composed of a triplet of high, medium and heavy molecular weight proteins and microtubules composed of polymerized tubulin. The stability of microtubules is thought to be under the control of microtubuleassociated proteins (MAPs) such as MAP2, which is restricted to dendrites [3, 18], and tau, found predominantly in axons. We have previously demonstrated [4,4a] that excitatory amino acid (EAA) stimulation of spinal cord and cortical neurons in culture induces stabilization of the microtubular network by increasing MAP2 binding directly or indirectly to microtubules. We now present evidence that MAP2 also binds to neurofilaments. This suggests that MAP2 is also involved in cross-linking microtubules and neurofilaments during the process of stabilization induced by EAA stimulation. Spinal cord neurons were prepared from rat embryos at El4 [4a]. Tissues chopped into small pieces were disaggregated with trypsin and further dissociated with Pasteur pipettes. Cells, resuspended in DMEM (Gibco) + 10% foetal calf serum (FCS, Sera Lab.), were plated Correspondence: D. Bigot, MRC Neurobiology Unit, MRC Centre, Hills Road, Cambridge CB2 2QH, U.K.
onto astrocyte beds at 200,000 cells/well in 24 well plates. After 1 or 2 days the medium was replaced by serum free medium [6], as previously described [4]. The medium was changed twice a week. After 13-15 days in culture neurons were stimulated for a period of 2 h with 50/~M quisqualate dissolved in Earle's balanced salt solution (EBSS) without magnesium. When necessary 3.3-33/tM nocodazole was applied for 35 min to 2 h after the 2 h stimulation. The cultures were then washed with phosphate buffer (0.1 M pH 7.4), fixed for 10 min with methanol at -20°C and washed carefully again with phosphate buffer (PB). MAP2 proteins were identified by using either a monoclonal antibody which recognizes high and low molecular weight MAP2 [15] or by using a polyclonal antibody raised against porcine MAP2 and microtubules labelled with a rat monoclonal anti-tubulin antibody, a gift from Dr. J. Kilmartin [18]. Initially, different monoclonal antibodies were used to identify neurofilaments: 3 mouse antibodies raised against pig spinal cord neurofilament polypeptides and specific for each phosphorylated subunit (68 kDa, 160 kDa and 200 kDa) [8, 23] (Amersham) and a mouse antibody recognizing the nonphosphorylated 200 kDa subunit [22] obtained by immunization with rat hypothalamus. A 3-step fluorescent immunohistochemical procedure
22 was chosen to amplify the staining. MAP2, neurofilaments and tubulin antibodies were recognized by biotinylated anti-mouse or anti-rat IgG, which were then identified with avidin-FITC complex (Vector). The antibodies were diluted in 0.1 M Tris-HCl pH 7.4 + 0.95% NaC1 + 0.3% Triton X-100. For double staining, polyclonal MAP2 antibody and mouse monoclonal neurofilament antibody were used sequentially. MAP2 antibody was labelled by an anti-rabbit IgG directly linked to fluorescein, while neurofilament antibody was recognized by biotinylated anti-mouse IgG which was then identified with avidin-Texas red complex (all Vector). To check the specificity of the staining, controls lacking the first or the second antibody were used. In all controls no staining was observed. Cultures were examined with a Polyvar fluorescent/light microscope or a Biorad confocal laser microscope [9]. Breakthrough of Texas red fluorescence into the F I T C channel on the confocal
microscope was removed by a subtraction program (Biorad). The pattern of neurofilament staining in mature cultures (14 days in vitro (DIV)) depended on the antibody used. The monoclonal antibodies raised against the phosphorylated 68 kDa or 160 kDa subunits gave the same pattern, staining axons and a small number of cell bodies. Cell bodies which were brightly stained, showed a very well-defined filamentous network. However, the antibody against non-phosphorylated 200 kDa subunit, gave excellent cell body and neurite staining in 35.9_+ 2.05% of all neurons in culture, and was used exclusively in this study. In untreated cultures at 14 DIV, a fine network of neurofilaments and microtubules was visible (Fig. l B,C). MAP2 staining was, however, less structured (Fig. 1A). Depolymerization o f microtubules with nocodazole (33 /~M for 35 min) (Fig. 1B1) resulted in a more fibrous
Fig. 1. A: spinal cord neuron at 14 DIV stained with monoclonal anti-MAP2 antibody. AI: spinal cord neuron at 13 DIV stained with anti-MAP2 antibody after 35 min treatment with 33 gM nocodazole. MAP2 staining has become more fibrous. B: spinal cord neuron at 13 DIV stained with anti-tubulin antibody, showing a well structured cytoplasmicnetwork. B1: tubulin staining of spinal cord neuron at 12 DIV after 35 min exposure to 33 gM nocodazole. Tubulin labelling is now diffuse, suggesting that microtubules are completely depolymerized. Bar=8.3 gm. C, CI: spinal cord neuron double-stained with antibodies against the 200 kDa non-phosphorylatedsubunit of neurofilament (C) and against MAP2 (C1) after 35 min exposure to 33 #M nocodazole. Most of the filaments labelled with anti-neurofilament antibody are also MAP2-positive, as shown by the arrows. Bar = 6.0 gm.
23
MAP2 network (Fig. 1AI). Using double-labelling techniques, neurofilaments were found to be in close association with similar filaments stained with MAP2 (Fig. 1C, C l). In general, all neurofilaments appeared to double label with MAP2. We were previously able to show that treatment of neuronal cultures with EAAs resulted in a reorganization of MAP2 staining and a stabilization of microtubules, shown in part by an increased resistance to depolymerization with nocodazole. Because of the association between neurofilaments and MAP2 described following nocodazole treatment, we asked whether the sti-
mulation of MAP2 seen following EAA treatment also resulted in an increased or closer association between MAP2 and neurofilaments, in parallel to that seen previously between microtubules and MAP2. Neuronal cultures treated with nocodazole (3.3 or 33 /tM for 2 h) with or without prior stimulation with quisqualate (50/zM for 2 h) were stained with MAP2 antibody. Considerably preserved morphology was seen both at low and high nocodazole concentration treatments when cultures had been previously stimulated with quisqualate (Fig. 2). Staining of comparable cultures with tubulin antibodies showed a complete collapse
Fig. 2. Spinal cord cultures at 13 DIV stained with polyclonal anti-MAP2 antibody. Considerably preserved morphology is seen at low and high nocodazole concentration treatments when cultures have been previously stimulated with quisqualate. A: unstimulated culture. B: culture treated for 2 h with 3.3/zM nocodazole. C: culture treated for 2 h with 33/zM nocodazole. D: culture which has been stimulated for 2 h with 50/~M QA. E: stimulated culture which has been exposed for 2 h to 3.3/zM nocodazole. F: stimulated culture which has been exposed for 2 h to 33/~M nocodazole. Bar = 200/an.
24 o f m i c r o t u b u l a r c y t o s k e l e t o n at b o t h c o n c e n t r a t i o n s o f n o c o d a z o l e . D o u b l e - s t a i n i n g o f n e u r o n s before a n d after t r e a t m e n t with either n o c o d a z o l e , E A A o r E A A followed by n o c o d a z o l e s h o w e d a close a s s o c i a t i o n between M A P 2 a n d n e u r o f i l a m e n t s staining (Fig. 3). A f t e r stimulation with E A A alone, m o s t n e u r o f i l a m e n t s were closely related to M A P 2 b u t m a n y M A P 2 positive filaments were n o t o b v i o u s l y in register with stained neurofila-
ments, suggesting that as p r e v i o u s l y d e m o n s t r a t e d , M A P 2 is also closely associated with m i c r o t u b u l e s . U s i n g a variety o f t r e a t m e n t s , we have s h o w n t h a t M A P 2 can be closely a s s o c i a t e d with neurofilaments. This c o m p l e m e n t s a previous study [4] s h o w i n g a similar relationship between M A P 2 a n d m i c r o t u b u l e s a n d suggests t h a t d u r i n g the process o f E A A - m e d i a t e d stabilization, m i c r o t u b u l e s a n d n e u r o f i l a m e n t s m a y , in p a r t , be
Fig. 3. Spinal cord neurons at 14 DIV double-stained with polyclonal anti-MAP2 antibody (FITC: A,C,E) and monoclonal antibody raised against the non-phosphorylated 200 kDa subunit of neurofilaments (Texas red: B,D,F). A,B: unstimulated neuron treated for 2 h with 33/aM nocodazole. C,D: neuron which has been stimulated for 2 h with 50/aM QA. MAP2 appears more closely associated with neurofilaments, as indicated by the arrows. E,F: neuron which has been stimulated for 2 h with 50/aM QA and then treated for two more hours with 33 FtM nocodazole. Most of the filaments stained with anti-neurofilament antibody are also MAP2 positive, as shown by the arrows. Bar = 5.6/am.
25 cross-linked directly or indirectly by M A P 2 . Indeed, there is evidence in the literature indicating the formation o f a three-dimensional n e t w o r k between microtubules and neurofilaments [11-14]. Immuno-electronmicr o s c o p y o f adult m o t o r spinal cord neurons has also d e m o n s t r a t e d that M A P 2 constituted cross-links between microtubules and neurofilaments [12] and M A P 2 has been shown to localize with intermediate filaments in glial cells after prolonged microtubule depolymerisation [5]. F r o m these and previous results we suggest that the E A A - m e d i a t e d stabilization o f the cytoskeleton by increased association o f M A P 2 with microtubules and cross-linking o f M A P 2 with neurofilament m a y play a role in the morphological fine tuning o f neuronal structure that occurs during development and perhaps also in the adult brain. U n p h o s p h o r y l a t e d M A P 2 has a greater ability to polymerize and stabilize microtubules [16] and hence p h o s p h o r y l a t e d M A P 2 allows for a m o r e labile and plastic cytoskeleton. Also several in vitro studies have shown an increase o f viscosity or gelation o f microtubule solution when neurofilaments were added [1, 20]. D e p h o s p h o r y l a t i o n o f M A P 2 occurs following stimulation o f adult rat brain slices with glutamate receptor agonist N-methyl-D-aspartate ( N M D A ) [10] and a switch f r o m highly p h o s p h o r y l a t e d to d e p h o s p h o r y l a t e d f o r m o f M A P 2 during development o f the cat visual cortex is t h o u g h t to herald the end o f the critical period for neuronal plasticity [2]. F u r t h e r m o r e a n u m b e r o f groups have shown that E A A s can influence the differentiation o f neurons in culture. D e v e l o p m e n t o f neurites is facilitated by N-methyl-D-aspartate activation [7, 21], while quisqualate and kainate stimulation decreases the growth o f dendrites o f h i p p o c a m p a l neurons [19]. T a k e n together with these observations, our results emphasize the influence o f E A A activation on the M A P 2 - m e d i a t e d control o f neurite stability and growth b o t h t h r o u g h stabilization o f microtubules and cross-linking with neurofilament. D. Bigot was supported by a grant f r o m R h 6 n e - P o u lenc. We would like to thank M a r g a r e t M o l l o y for typing the manuscript and Drs. A. M a t u s and E. M o n t e j o de Garcini for M A P 2 antisera.
I Aamodt, E.J. and William, R.C., Microtubule-associated proteins connect microtubules and neurofilaments in vitro, Biochemistry, 23 (1984) 6023~503I. 2 Aoki, C. and Siekevitz, P., Ontogenetic changes in the cyclic adenosine 3',5'-monophosphate-stimulatable phosphorylation of cat visual cortex proteins, particularly of microtubule-associated-prorein 2 (MAP2): effects of normal and dark rearing and of the exposure to light, J. Neurosci., 5 (1985) 2465-2483.
3 Bernhardt, R. and Matus, A., Light and electron microscopic studies of the distribution of microtubule-associated protein 2 in rat brain: a difference between dendritic and axonal cytoskeletons, J. Comp. Neurol., 226 (1984) 203-221. 4 Bigot, D., Matus, A. and Hunt, S.P., Reorganisation of the cytoskeleton in rat neurons following stimulation with excitatory amino acids in vitro, Eur. J. Neurosci., in press. 4a Bigot, D. and Hunt, S.P., Effect of excitatory amino acids on microtubule-associated proteins in cultured cortical and spinal neurones, Neurosci. Len., 111 (1990) 275-280. 5 Bloom, G.S. and Vallee, R.B., Association of microtubule-associated protein 2 (MAP2) with microtubules and intermediate filaments in cultured brain cells, J. Cell Biol., 96 (1983) 1523-1531. 6 Bottenstein, J.E. and Sato, G.H., Growth of a rat neuroblastoma cell line in serum-free supplemented medium, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 514-519. 7 Brewer, G.J. and Cotman, C.W., NMDA receptor regulation of neuronal morphology in cultured hippocampal neurons, Neurosci. Lett., 99 (1989) 268-273. 8 Debus, E., Flugge, G., Weber, K. and Osborn, M., A monoclonal antibody specific for the 200K polypeptide of the neurofilament triplet, EMBO J., 1 (1982) 41-45. 9 Fine, A.V., Amos, W.B., Durbin, R.M. and McNaughton, P.A., Confocal microscopy: applications in neurobiology, Trends Neurosci., 11 (1988) 346-351. I0 Foster, G.A., Dahl, D. and Lee. V.M-Y., Temporal and topographic relationships between the phosphorylated and nonphosphorylated epitopes of the 200 KDa neurofilament protein during development in vitro, J. Neurosci., 7 (1987) 2651-2663. 11 Halpain, S. and Greengard, P., Activation of NMDA receptors induces rapid dephosphorylation of the cytoskeletal protein MAP2, Neuron, 5 (1990) 237-246. 12 Heimann, R., Shelanski, M.L. and Liem, R.K.H., Microtubuleassociated-proteins bind specifically to the 70-KDa neurofilament protein, J. Biol. Chem., 260 (1985) 12160-12166. 13 Hirokawa, N., Hisanaga, S.-I. and Shiomura, Y., MAP2 is a component of crossbridges between microtubules and neurofilaments in the neuronal eytoskeleton: quick-freeze, deep-etch immunoelectron microscopy and reconstitution studies, J. Neurosci., 8 (1988) 27692779. 14 Hirokawa, N., Glicksman, M.A. and Willard, M.B., Organization of mammalian neurofitament polypeptides within the neuronal cytoskeleton, J. Cell Biol., 98 (1984) 1523-1536. 15 Hisanaga, S.I. and Hirokawa, N., Structure of the peripheral domains of neurofilaments revealed by low angle rotary shadowing, J. Mol. Biol., 202 (1988) 297-305. 16 Huber, G. and Matus, A., Differences in the cellular distributions of two microtubule-associated proteins, MAPI and MAP2, in rat brain, J. Neurosci., 4 (1984) 151-160. 17 Jameson, L. and Caplow, M., Modification ofmicrotubule steadystate dynamics by phosphorylation of microtubule-associated-proteins, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 3413-3417. 18 Kilmartin, J.V., Wright, B. and Milstein, C., Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line, J. Cell Biol., 93 (1982) 576-582. 19 Matus, A., Bernhardt, R., Bodmer, R. and Alaimo, D., Microtubule-associated-protein 2 and tubulin are differently distributed in the dendrites of developing neurons, Neuroscience, 17 (1986) 371389. 20 Mattson, M.P., Dou, P. and Kater, S.B., Outgrowth-regulating actions of glutamate in isolated hippocampal pyramidal neurons, J. Neurosci., 8 (1988) 2087-2100.
26 21 Minami, Y., Murofushi, H. and Sakai, H., Interaction of tubulin with neurofilaments: formation of networks by neurofilament-dependent tubulin polymerization, J. Biochem., 92 (1982) 889-898. 22 Pearce, I.A., Cambray-Deakin, M.A. and Burgoyne, R.D., Glutamate acting on NMDA receptors stimulates neurite outgrowth from cerebellar granule cells, FEBS Lett., 223 (1987) 143-147. 23 Sternberger, L.A. and Sternberger, N.H., Monoclonal antibodies
distinguish phosphorylated and non-phosphorylated forms of neurofilaments in situ, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 6126-6130. 24 Weber, K., Shaw, G., Osborn, M., Debus, E. and Geisler, N., Neurofilaments, a subclass of intermediate filaments: structure and expression, Cold Spring Harbor, Symp. Quant. Biol, 48 (1983) 71 729.
Neuroscience Letters, 131 (1991) 21-26 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 0304394091005454 NSL 08051
The effects of quisqualate and nocodazole on the organization of MAP2 and neurofilaments in spinal cord neurons in vitro Dominique Bigot and Stephen P. H u n t MRC Neurobiology Unit, MRC Centre, Cambridge ( U.K.) (Received 7 April 1991; Revised version received 13 June 1991; Accepted 18 June 199 I)
Key words: Excitatory amino acid; Nocodazole; MAP2, Neurofilament The relationship between microtubules, neurofilaments and microtubule-associated protein (MAP)2 was investigated in spinal cord neurons grown for up to 14 days in vitro. Neurons were labelled using antibodies against MAP2, neurofilaments and tubulin, and immunofluorescence analyzed by confocal microscopy. A well-structured network of neurofilaments and microtubules was observed in unstimulated cultures. MAP2 staining was poorly structured but became more filamentous following depolymerization of microtubules with nocodazole. Double-staining experiments suggested that MAP2 was now closely associated with neurofilaments in cell bodies and dendrites. Stimulation of cultures with excitatory amino acids increased the resistance of the microtubular cytoskeleton to depolymerization by nocodazole. Again double-labelling experiments demonstrated an increased association between neurofilaments and MAP2 immunofluorescence. Previous results suggested that the stability of the neuronal cytoskeleton could be modulated by glutamate receptors acting through an increased binding of MAP2 to microtubules. From the results presented here, we further suggest that cross-linking of neurofilaments to microtubules may also play a role in this process.
The neuronal cytoskeleton is composed of 3 major components, microfilaments made up of G-actin, neurofilaments composed of a triplet of high, medium and heavy molecular weight proteins and microtubules composed of polymerized tubulin. The stability of microtubules is thought to be under the control of microtubuleassociated proteins (MAPs) such as MAP2, which is restricted to dendrites [3, 18], and tau, found predominantly in axons. We have previously demonstrated [4,4a] that excitatory amino acid (EAA) stimulation of spinal cord and cortical neurons in culture induces stabilization of the microtubular network by increasing MAP2 binding directly or indirectly to microtubules. We now present evidence that MAP2 also binds to neurofilaments. This suggests that MAP2 is also involved in cross-linking microtubules and neurofilaments during the process of stabilization induced by EAA stimulation. Spinal cord neurons were prepared from rat embryos at El4 [4a]. Tissues chopped into small pieces were disaggregated with trypsin and further dissociated with Pasteur pipettes. Cells, resuspended in DMEM (Gibco) + 10% foetal calf serum (FCS, Sera Lab.), were plated Correspondence: D. Bigot, MRC Neurobiology Unit, MRC Centre, Hills Road, Cambridge CB2 2QH, U.K.
onto astrocyte beds at 200,000 cells/well in 24 well plates. After 1 or 2 days the medium was replaced by serum free medium [6], as previously described [4]. The medium was changed twice a week. After 13-15 days in culture neurons were stimulated for a period of 2 h with 50/~M quisqualate dissolved in Earle's balanced salt solution (EBSS) without magnesium. When necessary 3.3-33/tM nocodazole was applied for 35 min to 2 h after the 2 h stimulation. The cultures were then washed with phosphate buffer (0.1 M pH 7.4), fixed for 10 min with methanol at -20°C and washed carefully again with phosphate buffer (PB). MAP2 proteins were identified by using either a monoclonal antibody which recognizes high and low molecular weight MAP2 [15] or by using a polyclonal antibody raised against porcine MAP2 and microtubules labelled with a rat monoclonal anti-tubulin antibody, a gift from Dr. J. Kilmartin [18]. Initially, different monoclonal antibodies were used to identify neurofilaments: 3 mouse antibodies raised against pig spinal cord neurofilament polypeptides and specific for each phosphorylated subunit (68 kDa, 160 kDa and 200 kDa) [8, 23] (Amersham) and a mouse antibody recognizing the nonphosphorylated 200 kDa subunit [22] obtained by immunization with rat hypothalamus. A 3-step fluorescent immunohistochemical procedure
22 was chosen to amplify the staining. MAP2, neurofilaments and tubulin antibodies were recognized by biotinylated anti-mouse or anti-rat IgG, which were then identified with avidin-FITC complex (Vector). The antibodies were diluted in 0.1 M Tris-HCl pH 7.4 + 0.95% NaC1 + 0.3% Triton X-100. For double staining, polyclonal MAP2 antibody and mouse monoclonal neurofilament antibody were used sequentially. MAP2 antibody was labelled by an anti-rabbit IgG directly linked to fluorescein, while neurofilament antibody was recognized by biotinylated anti-mouse IgG which was then identified with avidin-Texas red complex (all Vector). To check the specificity of the staining, controls lacking the first or the second antibody were used. In all controls no staining was observed. Cultures were examined with a Polyvar fluorescent/light microscope or a Biorad confocal laser microscope [9]. Breakthrough of Texas red fluorescence into the F I T C channel on the confocal
microscope was removed by a subtraction program (Biorad). The pattern of neurofilament staining in mature cultures (14 days in vitro (DIV)) depended on the antibody used. The monoclonal antibodies raised against the phosphorylated 68 kDa or 160 kDa subunits gave the same pattern, staining axons and a small number of cell bodies. Cell bodies which were brightly stained, showed a very well-defined filamentous network. However, the antibody against non-phosphorylated 200 kDa subunit, gave excellent cell body and neurite staining in 35.9_+ 2.05% of all neurons in culture, and was used exclusively in this study. In untreated cultures at 14 DIV, a fine network of neurofilaments and microtubules was visible (Fig. l B,C). MAP2 staining was, however, less structured (Fig. 1A). Depolymerization o f microtubules with nocodazole (33 /~M for 35 min) (Fig. 1B1) resulted in a more fibrous
Fig. 1. A: spinal cord neuron at 14 DIV stained with monoclonal anti-MAP2 antibody. AI: spinal cord neuron at 13 DIV stained with anti-MAP2 antibody after 35 min treatment with 33 gM nocodazole. MAP2 staining has become more fibrous. B: spinal cord neuron at 13 DIV stained with anti-tubulin antibody, showing a well structured cytoplasmicnetwork. B1: tubulin staining of spinal cord neuron at 12 DIV after 35 min exposure to 33 gM nocodazole. Tubulin labelling is now diffuse, suggesting that microtubules are completely depolymerized. Bar=8.3 gm. C, CI: spinal cord neuron double-stained with antibodies against the 200 kDa non-phosphorylatedsubunit of neurofilament (C) and against MAP2 (C1) after 35 min exposure to 33 #M nocodazole. Most of the filaments labelled with anti-neurofilament antibody are also MAP2-positive, as shown by the arrows. Bar = 6.0 gm.
23
MAP2 network (Fig. 1AI). Using double-labelling techniques, neurofilaments were found to be in close association with similar filaments stained with MAP2 (Fig. 1C, C l). In general, all neurofilaments appeared to double label with MAP2. We were previously able to show that treatment of neuronal cultures with EAAs resulted in a reorganization of MAP2 staining and a stabilization of microtubules, shown in part by an increased resistance to depolymerization with nocodazole. Because of the association between neurofilaments and MAP2 described following nocodazole treatment, we asked whether the sti-
mulation of MAP2 seen following EAA treatment also resulted in an increased or closer association between MAP2 and neurofilaments, in parallel to that seen previously between microtubules and MAP2. Neuronal cultures treated with nocodazole (3.3 or 33 /tM for 2 h) with or without prior stimulation with quisqualate (50/zM for 2 h) were stained with MAP2 antibody. Considerably preserved morphology was seen both at low and high nocodazole concentration treatments when cultures had been previously stimulated with quisqualate (Fig. 2). Staining of comparable cultures with tubulin antibodies showed a complete collapse
Fig. 2. Spinal cord cultures at 13 DIV stained with polyclonal anti-MAP2 antibody. Considerably preserved morphology is seen at low and high nocodazole concentration treatments when cultures have been previously stimulated with quisqualate. A: unstimulated culture. B: culture treated for 2 h with 3.3/zM nocodazole. C: culture treated for 2 h with 33/zM nocodazole. D: culture which has been stimulated for 2 h with 50/~M QA. E: stimulated culture which has been exposed for 2 h to 3.3/zM nocodazole. F: stimulated culture which has been exposed for 2 h to 33/~M nocodazole. Bar = 200/an.
24 o f m i c r o t u b u l a r c y t o s k e l e t o n at b o t h c o n c e n t r a t i o n s o f n o c o d a z o l e . D o u b l e - s t a i n i n g o f n e u r o n s before a n d after t r e a t m e n t with either n o c o d a z o l e , E A A o r E A A followed by n o c o d a z o l e s h o w e d a close a s s o c i a t i o n between M A P 2 a n d n e u r o f i l a m e n t s staining (Fig. 3). A f t e r stimulation with E A A alone, m o s t n e u r o f i l a m e n t s were closely related to M A P 2 b u t m a n y M A P 2 positive filaments were n o t o b v i o u s l y in register with stained neurofila-
ments, suggesting that as p r e v i o u s l y d e m o n s t r a t e d , M A P 2 is also closely associated with m i c r o t u b u l e s . U s i n g a variety o f t r e a t m e n t s , we have s h o w n t h a t M A P 2 can be closely a s s o c i a t e d with neurofilaments. This c o m p l e m e n t s a previous study [4] s h o w i n g a similar relationship between M A P 2 a n d m i c r o t u b u l e s a n d suggests t h a t d u r i n g the process o f E A A - m e d i a t e d stabilization, m i c r o t u b u l e s a n d n e u r o f i l a m e n t s m a y , in p a r t , be
Fig. 3. Spinal cord neurons at 14 DIV double-stained with polyclonal anti-MAP2 antibody (FITC: A,C,E) and monoclonal antibody raised against the non-phosphorylated 200 kDa subunit of neurofilaments (Texas red: B,D,F). A,B: unstimulated neuron treated for 2 h with 33/aM nocodazole. C,D: neuron which has been stimulated for 2 h with 50/aM QA. MAP2 appears more closely associated with neurofilaments, as indicated by the arrows. E,F: neuron which has been stimulated for 2 h with 50/aM QA and then treated for two more hours with 33 FtM nocodazole. Most of the filaments stained with anti-neurofilament antibody are also MAP2 positive, as shown by the arrows. Bar = 5.6/am.
25 cross-linked directly or indirectly by M A P 2 . Indeed, there is evidence in the literature indicating the formation o f a three-dimensional n e t w o r k between microtubules and neurofilaments [11-14]. Immuno-electronmicr o s c o p y o f adult m o t o r spinal cord neurons has also d e m o n s t r a t e d that M A P 2 constituted cross-links between microtubules and neurofilaments [12] and M A P 2 has been shown to localize with intermediate filaments in glial cells after prolonged microtubule depolymerisation [5]. F r o m these and previous results we suggest that the E A A - m e d i a t e d stabilization o f the cytoskeleton by increased association o f M A P 2 with microtubules and cross-linking o f M A P 2 with neurofilament m a y play a role in the morphological fine tuning o f neuronal structure that occurs during development and perhaps also in the adult brain. U n p h o s p h o r y l a t e d M A P 2 has a greater ability to polymerize and stabilize microtubules [16] and hence p h o s p h o r y l a t e d M A P 2 allows for a m o r e labile and plastic cytoskeleton. Also several in vitro studies have shown an increase o f viscosity or gelation o f microtubule solution when neurofilaments were added [1, 20]. D e p h o s p h o r y l a t i o n o f M A P 2 occurs following stimulation o f adult rat brain slices with glutamate receptor agonist N-methyl-D-aspartate ( N M D A ) [10] and a switch f r o m highly p h o s p h o r y l a t e d to d e p h o s p h o r y l a t e d f o r m o f M A P 2 during development o f the cat visual cortex is t h o u g h t to herald the end o f the critical period for neuronal plasticity [2]. F u r t h e r m o r e a n u m b e r o f groups have shown that E A A s can influence the differentiation o f neurons in culture. D e v e l o p m e n t o f neurites is facilitated by N-methyl-D-aspartate activation [7, 21], while quisqualate and kainate stimulation decreases the growth o f dendrites o f h i p p o c a m p a l neurons [19]. T a k e n together with these observations, our results emphasize the influence o f E A A activation on the M A P 2 - m e d i a t e d control o f neurite stability and growth b o t h t h r o u g h stabilization o f microtubules and cross-linking with neurofilament. D. Bigot was supported by a grant f r o m R h 6 n e - P o u lenc. We would like to thank M a r g a r e t M o l l o y for typing the manuscript and Drs. A. M a t u s and E. M o n t e j o de Garcini for M A P 2 antisera.
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