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Kindling induced changes in calmodulin kinase II immunoreactivity
Brain Research, 524 (1990) 49-53 Elsevier
49
BRES 15728
Kindling induced changes in calmodulin kinase II immunoreactivity Jeff M. Bronstein 1, Debora B. Farber 2, Paul E. Micevych 3, Robert Lasher 4 and Claude G. Wasterlain 5 Departments of tNeuroscience, 2Ophthalmology, 3Anatomy and 4Neurology, UCLA School of Medicine, Los Angeles, CA 90024 (U.S.A.) and 5Department of Cellular and Structural Biology, University of Colorado School of Medicine, Denver, CO (U.S.A.) (Accepted 6 February 1990) Key words: Kindling; Calmodulin kinase; Immunocytochemistry; Protein phosphorylation; Synaptic plasticity
The distribution of type II calmodulin kinase (CaM kinase) immunoreactivity was studied in control and septally kindled rat brains. CaM kinase was concentrated in limbic structures, such as the hippocampus, lateral septum and amygdala. Within the hippocampus, the molecular layer of the endal limb of the dentate gyrus, the stratum radiatum, and lacunosum moleculare of CA1 were the most heavily stained regions. The cerebellum was stained only in the molecular and Purkinje cell layers, and very low amounts of immunoreactive protein were present in the brainstem and white matter. Kindling resulted in a significant decrease in CaM kinase immunoreactivity in CA3 and in the dentate of the ventral hippocampus but not in the lateral septum. These data suggest that kindling decreases the number of CaM kinase molecules or alters its antigenic distribution, and provides further evidence that alterations of this enzyme may be important in the kindling phenomenon.
INTRODUCTION
32,35. Of particular interest is the report of lasting changes
Kindling is a model of epilepsy and synaptic plasticity in which repeated stimulation of some brain regions by small amounts of electrical current 14 or agonists of excitatory neurotransmitters 34 results in a progressive buildup of seizure activity and culminates in full fledged epileptic seizures. Once established, kindled brains retain the altered state of excitability to stimulation for long periods of time and possibly for the life of the subject. Kindling can be most easily obtained from limbic regions but cannot be elicited from some areas such as cerebelum. Unit discharge patterns and application of some muscarinic agonists which produce kindling indicate that kindling occurs transsynaptically 27"34. This is an attractive model for the study of epilepsy and synaptic plasticity because of the absence of cell necrosis, drugs or poisons, and for its permanence and selectivity for some brain regions. Attempts at identifying molecular processes underlying epileptogenesis and other states of altered excitability have resulted in minimal success. Many modifications in synaptic biochemistry have been reported in kindled animals but most alterations are short-lived and cannot account for persistent changes in behavior4"8'1°'11"21"33. Recent studies suggest that changes in calcium-mediated events may be involved in the kindling phenomenon 22'
in calcium- and calmodulin-dependent protein phosphorylation of synaptic plasma membranes of kindled rats 35. This alteration was most prominent in regions where kindling can be easily elicited (e.g. hippocampus) and absent in areas where kindling cannot be induced (e,g. cerebellum) 28. Furthermore, pretreatment with diazepam, an inhibitor of Ca2÷/calmodulin-dependent phosphorylation, blocks induction of kindling and changes in synaptic membrane phosphorylation (unpublished data). Recently, calmodulin kinase type II (CaM kinase) has been identified as being altered in kindling and is responsible for reported changes in phosphorylation 15. CaM kinase is a Ca2÷/calmodulin-dependent protein kinase which is highly enriched in brain constituting 0.4-2.0% of total brain protein depending on the region 9. It is located both pre- and postsynaptically 25 and appears to modulate neurotransmitter biosynthesis 39 and release 6"19, and may regulate cytoskeletal proteins 38'4°. CaM kinase is identical to the 'major postsynaptic density protein' and therefore probably has important postsynaptic functions as well 17. Forebrain CaM kinase is composed of 50 and 60 kDa subunits which autophosphorylate 1"~6. Self phosphorylation results in dramatic changes in the enzyme's responsiveness to further alterations in calcium 2'18'23'24. Unphosphorylated kinase
Correspondence: C.G. Wasterlain, Department of Neurology, UCLA School of Medicine, Los Angeles, CA 90024, U.S.A.
50 is a l m o s t c o m p l e t e l y calcium- a n d c a l m o d u l i n - d e p e n d e n t while p h o s p h o r y l a t e d kinase is calcium- a n d c a l m o d u l i n independent. A l t e r e d C a M k i n a s e activity associated with k i n d l i n g m a y result from changes in e n z y m e activity or in the n u m b e r of m o l e c u l e s present. T h e r e f o r e , we studied the d i s t r i b u t i o n of C a M kinase in septally k i n d l e d rats a n d f o u n d a decrease in C a M k i n a s e i m m u n o r e a c t i v i t y in the ventral hippocampus. MATERIALS AND METHODS Male Holtzman rats were stereotaxically implanted into the medial septal area with bipolar twisted stainless steel electrodes. Some animals received both septal and dorsal hippocampal electrodes and were used for electrographic recording only. Septal electrodes were located close to midline so that both hippocampi showed after discharges. After 2 weeks of handling, experimental animals received 3 stimulations (400 pA for 1 s at 60 Hz) 3 h apart, 5 days per week through the septai electrode. Full kindling was defined as 12 consecutive stage 5 seizures and there was a rest period of at least 2 weeks after the last stimulation before sacrifice. Previous experiments have shown that rest periods up to 2 months do not reduce either the kindled seizure or the change in protein phosphorylation associated with it35. Controls were paired littermates. Kindled rats were anesthetized with phenobarbital by i.p. injection and rapidly perfused under pressure through the aorta for 25 min with modified Zamboni's fixative (4% picric acid, 4% paraformaldehyde in phosphate buffer). The brains were removed and stored at 4 °C overnight in 20% sucrose, 4% paraformaldehyde, in Sorensen's phosphate buffer (pH 7.6). Brains were mounted, frozen and sectioned (40/~M) on a sliding microtome. Sections were extensively washed with phosphate-buffered saline (PBS, pH 7.6) by gentle agitation and incubated in 2% normal horse serum (NHS) in PBS for 30 min at room temperature and 48 h at 4 °C in C37 monoctonal antibody diluted 1:2000 in 1% NHS. This antibody is specific for the 50 kDa subunit of CaM kinase3. After washing with PBS for 30 min with 3 changes, sections were processed according to instructions provided with Vectastain biotin-avidin-peroxidase kit (Vector Laboratory, Burlingame, CA). Peroxidase was developed 4-6 rain with 1% diaminobenzidine and 0.03% H20 2. The sections were counterstained with Toluidine blue and coverslipped for viewing. For control sections, primary antibody was replaced with 1% NHS. Staining was performed simultaneously on kindled brain sections and their paired controls. In some experiments, adjacent sections were thionin stained29 in order to identify anatomical regions. Stained sections were viewed with a Zeiss model microscope equipped with a blue filter. Images were digitalized and stored using an IBAS imaging system and optical densities were measured in a blind manner in specified regions. Statistical comparisons were made using the Student's t-test for paired data. RESULTS
Fig. 1. Thionin (A) and immunocytochemical (B and C) stained sections of the dorsal hippocampus. In control sections (B), immunoreactivity was concentrated in the endal limb of the dentate and in the stratum radiatum and lacunosum moleculare of CA1. Kindling (C) resulted in a dramatic decrease in immunoreactivity almost in all areas of the hippocampus. A tuft of immunoreactivity was spared from these decreases in the CA2 region of the hippocampus in 3 pairs (designated by the arrow), s, subiculum; r, stratum radiatum; lm, lacunosum moleculare; p, pyramidal cell layer; g, granule cell layer of dentate; m, molecular layer of dentate; h, hilus of dentate.
Regional distribution of CaM kinase in control brains T h e d i s t r i b u t i o n of C a M k i n a s e o b s e r v e d using C37 a n t i b o d i e s was similar to that previously r e p o r t e d 9'25. D r a m a t i c differences in staining intensity were o b s e r v e d a m o n g b r a i n regions with the strongest staining in the n e o c o r t e x a n d limbic system, a n d the w e a k e s t in midbrain and brainstem.
T h e h i p p o c a m p u s was highly i m m u n o r e a c t i v e . T h e e n d a l limb of the d e n t a t e gyrus was heavily s t a i n e d as was C A 1 (Fig. 1). T h e m o l e c u l a r layer of the d e n t a t e c o n t a i n e d m o s t of the i m m u n o r e a c t i v e p r o t e i n although g r a n u l e cell somas were also stained. R e g i o n s c o n t a i n i n g h i p p o c a m p a l p y r a m i d a l cell p r o j e c t i o n s , such as the
51 TABLE I
Comparison of CaM kinase immunoreactivity in kindled and control rats Optical densities were measured as described in Materials and Methods and values are expressed as mean differences in optical density units.
OD difference
S.E.M.
P<
Dorsal hippocampus CA1 CA2 CA3 CA4 Dentate (endal limb)
-0.019 -0.006 -0.012 -0.024 -0.022
0.008 0.008 0.005 0.008 0.006
0.025 n.s. 0.05 0.01 0.005
Ventral hippocampus CA1 CA2 CA3 Dentate (endal limb) Lateral septum
-0.066 -0.066 -0.097 -0.072 +0.014
0.019 0.016 0.018 0.022 0.016
0.005 0.005 0.005 0.01 n.s.
stratum radiatum and lacunosum moleculare, were heavily labeled, particularly in CA1. CaM kinase immunoreactivity in kindled brain CaM kinase immunoreactivity in 5 kindled animals was compared to that in paired controls. Staining was decreased the most in ventral hippocampus, although reductions in dorsal regions were also observed (Fig. 1, Table I). Within the hippocampus, CA3 and the endal limb of the dentate gyrus contained pronounced differences in staining. Smaller alterations were present in CA1 and CA4, while immunoreactivity in CA2 of the dorsal hippocampus was preserved. This preferential sparing of CA2 appeared as a tuft of CaM kinase immunoreactivity and was very apparent in 3 of the 5 pairs tested (Fig. 1). In contrast to the hippocampus, the lateral septal immunoreactivity was increased compared to controls, although differences did not reach statistical significance (Table I). Measurements in this region were made away from the implanted electrodes, which did not appear to alter staining. DISCUSSION
Septal kindling is associated with long-lasting decreases in CaM kinase activity in hippocampal synaptic plasma membranes 15'35. In this report, we demonstrated a decrease in CaM kinase immunoreactivity in most hippocampal regions. This suggests that the lowered activity reflects a decrease in the number of CaM kinase molecules, implying that the kindled trace is associated with acquired changes in gene expression or in post-
transcriptional processing of CaM kinase. Despite failure of early attempts at demonstrating morphological alterations in kindled brains 27, a selective loss of nonperforated synapses 12 and a change in spine morphology26 have been demonstrated in the hippocampus of kindled rats 12. Because of CaM kinase's possible structural role 31, decreased CaM kinase immunoreactivity may be a cause or an effect of the altered synaptic morphology. Further studies using electron microscopy and immunocytochemistry of CaM kinase in the hippocampus and other regions could help clarify this association. Although the biochemical basis of kindling is still unclear, CaM kinase is ideally designed both functionally and anatomically to alter synaptic excitability. CaM kinase appears to modulate transmitter biosynthesis and r e l e a s e 6'19'39, cytoskeletal proteins 38'4°, and probably has an important postsynaptic role given its extremely high concentration in the postsynaptic density17. Although increases in CaM kinase-mediated phosphorylation of synapsin I enhances transmitter release, so that one would predict an increase in CaM kinase to be associated with hyperexcitable states such as kindling, this is likely to be oversimplistic. The distribution of CaM kinase in excitatory and inhibitory, perforated and non-perforated synapses is still unclear as well as the complex circuitry of kindling. It can easily be imagined that a relative decrease in inhibitory synapses (as suggested by Geinisman et al) 2) could result in an overall increase in excitability. The regional distribution of CaM kinase in the brain and the changes induced by kindling are quite striking. The areas with the highest concentration of enzyme, such as the limbic system, are also the areas in which kindling can most easily be induced 28. In the cerebellum, which is totally resistant to kindling, a separate isoenzyme of CaM kinase is found23. Furthermore, changes in CaM kinase activity and immunoreactivity showed regional specificity. Decreases in hippocampal immunoreactivity induced by kindling were most pronounced in the ventral hippocampus. It is interesting to note that kindling proceeds at a faster rate following ventral than dorsal stimulation29. Anatomical29, neurochemical 3°, and electrophysiologiCal 7'29 investigations have provided strong evidence for functional dissociation between dorsal and ventral aspects of the rat hippocampus. The ventral hippocampus shows a greater tendency to generate epileptiform burst responses 13. The reduction of hippocampal immunoreactivity is absent in CA2 of the dorsal region. The sparing of CA2 was particularly striking in 3 of the 5 pairs tested. It is unknown why this region was different, or why some pairs showed a greater effect than others, but it is interesting to note that CA2 appears to be an excellent region to initiate burst activity in hippocampal slices37
52 and is selectively preserved in epileptic brains with hippocampal damage. It should be kept in mind that differences in immunoreactivity can be affected by a number of factors including cell density, intracellular distribution, and differential distribution between different cell types. These factors can alter antibody penetration and antigen accessibility. Since CaM kinase is a labile enzyme with a known vulnerability to ischemic conditions 36, a high pressure method of perfusion fixation was used. AI-
REFERENCES 1 Bennett, M.K., Erondu, N.E. and Kennedy, M.B., Purification and characterization of a calmodulin-dependent protein kinase that is highly enriched in brain, J. Biol. Chem., 258 (1983) 12735-12744. 2 Bronstein, J.M., Farber, D.B. and Wasterlain, C.W., A retinal calmodulin-dependent kinase: calcium/calmodulin-stimulated and -inhibited states, J. Neurochem., 50 (1988) 1438-1446. 3 Bronstein, J.M., Wasterlain, C.G., Bok, D., Lasher, R. and Farber, D.B., Localization of retinal calmodulin kinase, Exp. Eye Res,, 47 (1988) 391-402. 4 Byrne, M.C., Gottlieb, R. and McNamara, J.O., Amygdala kindling induces muscarinic cholinergic receptor decline in a highly specific distribution within the limbic system, Exp. Neurol., 69 (1980) 85-98. 5 Darrow, H. and Emmel, V. In Staining Procedures, Williams and Wilkins, Baltimore, MD 1960, pp. 94-95. 6 DeLorenzo, R.J., Freedman, S.D., Yoke, W.B. and Maurer, S.C., Stimulation of Ca2+-dependent neurotransminer release and presynaptic nerve terminal protein phosphorylation by calmodulin and a calmodulin-like protein isolated from synaptic vesicles, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 1838-1842. 7 Elul, R., Regional differences in the hippocampus of the cat. I. Specific discharge patterns of the dorsal and ventral hippocampus and their role in generalized seizures, Electroencephalogr. Clin. Neurophysiol., 16 (1964) 489-502. 8 Engel, J. Jr. and Sharpless, N.S., Long lasting depletion of dopamine in the rat amygdala induced by kindling stimulation, Brain Research, 136 (1977) 381-386. 9 Erondu, N.E. and Kennedy, M.B., Regional distribution of Type II cae÷/calmodulin-dependent protein kinase in rat brain, J. Neurosci., 5 (1985) 3270-3277. 10 Farjo, I.B. and Blackwood, D.H.R., Reduction in tyrosine hydroxylase activity in the rat amygdala induced by kindling stimulation, Brain Research, 153 (1978) 423-426. 11 Gee, K.W., Hollingger, M.H., Bowyet, J.F. and Killam, E.K., Modification of dopaminergic receptor sensitivity in the rat brain after amygdaloid kindling, Exp. Neurol., 66 (1979) 771-777. 12 Geinisman, Y., Morrell, E and DeToledo-Morrell, L., Remodeling of synaptic architecture during hippocampal 'kindling', Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 3260-3264. 13 Gilbert, M., Racine, R.J. and Smith, G.K., Epileptiform burst responses in ventral versus dorsal hippocampal slices, Brain Research, 361 (1985) 389-391. 14 Goddard, G.V., Mclntyre, D.C. and Leech, C.K., A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 243-330. 15 Goldenring, J.R., Wasterlain, C.G., Ostereicher, A.B., DeGraan, P.N.E., Farber, D.B., Glaser, G.H. and DeLorenzo, R.J., Kindling induces a long-lasting change in the activity of a hippocampal membrane calmodulin-dependent protein kinase system, Brain Research, 377 (1986) 47-53. 16 Goldenring, J.R., Gonzalez, B., McGuire, J.S. Jr. and DeLorenzo, R.J., Purification and characterization of a calmodulin-
though it is possible that CaM kinase exists in different states of autophosphorylation in different brain regions which also could affect staining. C37 antibody binding was found to react identically with native and phosphorylated enzyme 3. In spite of these variables, regional staining was in agreement with enzyme activity localization9. Acknowledgements. Supported by the Medical Scientist Training Program, Research Service of the Veterans Administration, and Research Grants Ns13515 and E402651 from NIH.
dependent protein kinase from brain cytosol able to phosphorylate tubulin and microtubule associated protein, J. Biol. Chem., 258 (1983) 12632-12640. 17 Kennedy, M.B., Bennett, M.K. and Erondu, N.E., Biochemical and immunocytochemical evidence that the 'major postsynaptic density protein' is a subunit of a calmodulin-dependent protein kinase, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 7357-7361. 18 Lai, Y., Narin, A.C. and Greengard, P., Autophosphorylation reversibly regulated the Ca2+/calmodulin-dependent protein kinase II, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 4253-4257. 19 Llinas, P., McGuinness, T.L., Leonard, C.S., Sugimori, M. and Greengard, P., Intraterminal injection of synapsin I or calcium/ calmodulin-dependent protein kinase II alters neurotransmitter release at the squid giant axon, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 3035-3039. 20 Lou, L.L., Lloyd, S.J. and Schulman, H., Activation of the multifunctional Ca2+/calmodulin-dependent protein kinase by autophosphorylation: ATP modulates production of an autonomous enzyme, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 9497-9501. 21 McNamara, J.O., Selective alterations of regional beta-adrenergic receptor binding in the kindling model of epilepsy, Exp. Neurol., 61 (1978) 582-591. 22 Miller, J.J. and Bainbridge, K.G., Biochemical and immunohistochemical correlates of kindling-induced epilepsy: role of calcium binding protein, Brain Research, 278 (1983) 322-326. 23 Miller, S.G. and Kennedy, M.B., Distinct forebrain and cerebellar isoenzymes of type II Ca2+/calmodulin-dependent kinase associate differently with the postsynaptic density fraction, J. Biol. Chem., 260 (1985) 9039-9046. 24 Miller, S.G. and Kennedy, M.B., Regulation of calcium/ calmodulin-dependent protein kinase by autophosphorylation. A calcium-triggered switch, Cell, 44 (1986) 861-870. 25 Ouimet, C.C., McGuinness, T.L. and Greengard, P., Immunocytochemical localization of calcium/calmodulin-dependent protein kinase II in rat brain, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 5604-5608. 26 Paul, L.A. and Wasterlain, C.G., Golgi studies reveal differences in dendritic spines between septal kindled rats and controls, Soc. Neurosci. Abstr., 12 (1986) 1376. 27 Racine, R., Tuff, L. and Zaide, J., Kindling, unit discharge patterns and neural plasticity, Can. J. Neurol. Sci., 2 (1974) 395-405. 28 Racine, R., Kindling, the first decade, Neurosurgery, 3 (1978) 234-252. 29 Racine, R.J., Rose, P.A. and Burnham, W.M., After discharge thresholds and kindling rates in dorsal and ventral hippocampal and dentate gyrus, Can. J. Neurol. Sci., 4 (1977) 273-278. 30 Storm-Mathisen, .I. In Functions of the Septo-Hippocarnpal System, Ciba Foundation Symposium, 58, 1977, pp. 49-79. 31 Vallano, M.L., Goldenring, J.R., Buckholy, M.T., Larson, R.E. and DeLorenzo, R.J., Separation of endogenous calmodulinand cAMP-dependent kinases from microtubule preparations, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 3202-3206. 32 Wadman, W.J., Heinemann, U., Konnerth, A. and Newhaus,
53
33 34 35
36
S., Hippocampal slices of kindled rats reveal calcium involvement in epileptogenesis, Exp. Brain Res., 57 (1983) 404-407. Wasterlain, C.G., Morin, A.M. and Jonec, F., Interactions between chemical and electrical kindling of the rat amygdala, Brain Research, 247 (1982) 341-346. Wasterlain, C.G. and Jonec, V., Chemical kindling by muscarinic amygdaloid stimulation in the rat, Brain Research, 271 (1983) 311-323. Wasterlain, C.G. and Farber, D.B., Kindling alters the calcium/ calmodulin-dependent phosphorylation of synaptic plasma membrane proteins in rat hippocampus, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 1253-1257. Wasterlain, C.G., Calcium- and calmodulin-dependent phosphorylation in ischemic brain, Soc. Neurosci. Abstr., 10 (1984) 293.17.
37 Wong, R.K.S. and Traud, R.D., Synchronized burst discharge in disinhibited hippocampal slice. 1. Initiation in CA2-Ca3 region, J. Neurophysiol., 49 (1983) 442-458. 38 Yamamoto, H., Fukunaga, K., Tanaka, E. and Miyanoto, E., Calcium and calmodulin-dependent phosphorylation of microtubule associated protein 2 and tau factor and inhibition of microtubule assembly, J. Neurochem., 41 (1983) 119-125. 39 Yamauchi, T. and Fujisawa, H., Purification and characterization of the brain calmodulin-dependent protein kinase (kinase II) which is involved in the activation of tryptophan 5monooxygenase, Eur. J. Biochem., 132 (1983) 15-21. 40 Yamauchi, T. and Fujisawa, H., Phosphorylation of microtubule associated protein 2 by calmodulin-dependent protein kinase (kinase II) which occurs in brain tissue, Biochem. Biophys. Res. Commun., 110 (1983) 287-291.
49
BRES 15728
Kindling induced changes in calmodulin kinase II immunoreactivity Jeff M. Bronstein 1, Debora B. Farber 2, Paul E. Micevych 3, Robert Lasher 4 and Claude G. Wasterlain 5 Departments of tNeuroscience, 2Ophthalmology, 3Anatomy and 4Neurology, UCLA School of Medicine, Los Angeles, CA 90024 (U.S.A.) and 5Department of Cellular and Structural Biology, University of Colorado School of Medicine, Denver, CO (U.S.A.) (Accepted 6 February 1990) Key words: Kindling; Calmodulin kinase; Immunocytochemistry; Protein phosphorylation; Synaptic plasticity
The distribution of type II calmodulin kinase (CaM kinase) immunoreactivity was studied in control and septally kindled rat brains. CaM kinase was concentrated in limbic structures, such as the hippocampus, lateral septum and amygdala. Within the hippocampus, the molecular layer of the endal limb of the dentate gyrus, the stratum radiatum, and lacunosum moleculare of CA1 were the most heavily stained regions. The cerebellum was stained only in the molecular and Purkinje cell layers, and very low amounts of immunoreactive protein were present in the brainstem and white matter. Kindling resulted in a significant decrease in CaM kinase immunoreactivity in CA3 and in the dentate of the ventral hippocampus but not in the lateral septum. These data suggest that kindling decreases the number of CaM kinase molecules or alters its antigenic distribution, and provides further evidence that alterations of this enzyme may be important in the kindling phenomenon.
INTRODUCTION
32,35. Of particular interest is the report of lasting changes
Kindling is a model of epilepsy and synaptic plasticity in which repeated stimulation of some brain regions by small amounts of electrical current 14 or agonists of excitatory neurotransmitters 34 results in a progressive buildup of seizure activity and culminates in full fledged epileptic seizures. Once established, kindled brains retain the altered state of excitability to stimulation for long periods of time and possibly for the life of the subject. Kindling can be most easily obtained from limbic regions but cannot be elicited from some areas such as cerebelum. Unit discharge patterns and application of some muscarinic agonists which produce kindling indicate that kindling occurs transsynaptically 27"34. This is an attractive model for the study of epilepsy and synaptic plasticity because of the absence of cell necrosis, drugs or poisons, and for its permanence and selectivity for some brain regions. Attempts at identifying molecular processes underlying epileptogenesis and other states of altered excitability have resulted in minimal success. Many modifications in synaptic biochemistry have been reported in kindled animals but most alterations are short-lived and cannot account for persistent changes in behavior4"8'1°'11"21"33. Recent studies suggest that changes in calcium-mediated events may be involved in the kindling phenomenon 22'
in calcium- and calmodulin-dependent protein phosphorylation of synaptic plasma membranes of kindled rats 35. This alteration was most prominent in regions where kindling can be easily elicited (e.g. hippocampus) and absent in areas where kindling cannot be induced (e,g. cerebellum) 28. Furthermore, pretreatment with diazepam, an inhibitor of Ca2÷/calmodulin-dependent phosphorylation, blocks induction of kindling and changes in synaptic membrane phosphorylation (unpublished data). Recently, calmodulin kinase type II (CaM kinase) has been identified as being altered in kindling and is responsible for reported changes in phosphorylation 15. CaM kinase is a Ca2÷/calmodulin-dependent protein kinase which is highly enriched in brain constituting 0.4-2.0% of total brain protein depending on the region 9. It is located both pre- and postsynaptically 25 and appears to modulate neurotransmitter biosynthesis 39 and release 6"19, and may regulate cytoskeletal proteins 38'4°. CaM kinase is identical to the 'major postsynaptic density protein' and therefore probably has important postsynaptic functions as well 17. Forebrain CaM kinase is composed of 50 and 60 kDa subunits which autophosphorylate 1"~6. Self phosphorylation results in dramatic changes in the enzyme's responsiveness to further alterations in calcium 2'18'23'24. Unphosphorylated kinase
Correspondence: C.G. Wasterlain, Department of Neurology, UCLA School of Medicine, Los Angeles, CA 90024, U.S.A.
50 is a l m o s t c o m p l e t e l y calcium- a n d c a l m o d u l i n - d e p e n d e n t while p h o s p h o r y l a t e d kinase is calcium- a n d c a l m o d u l i n independent. A l t e r e d C a M k i n a s e activity associated with k i n d l i n g m a y result from changes in e n z y m e activity or in the n u m b e r of m o l e c u l e s present. T h e r e f o r e , we studied the d i s t r i b u t i o n of C a M kinase in septally k i n d l e d rats a n d f o u n d a decrease in C a M k i n a s e i m m u n o r e a c t i v i t y in the ventral hippocampus. MATERIALS AND METHODS Male Holtzman rats were stereotaxically implanted into the medial septal area with bipolar twisted stainless steel electrodes. Some animals received both septal and dorsal hippocampal electrodes and were used for electrographic recording only. Septal electrodes were located close to midline so that both hippocampi showed after discharges. After 2 weeks of handling, experimental animals received 3 stimulations (400 pA for 1 s at 60 Hz) 3 h apart, 5 days per week through the septai electrode. Full kindling was defined as 12 consecutive stage 5 seizures and there was a rest period of at least 2 weeks after the last stimulation before sacrifice. Previous experiments have shown that rest periods up to 2 months do not reduce either the kindled seizure or the change in protein phosphorylation associated with it35. Controls were paired littermates. Kindled rats were anesthetized with phenobarbital by i.p. injection and rapidly perfused under pressure through the aorta for 25 min with modified Zamboni's fixative (4% picric acid, 4% paraformaldehyde in phosphate buffer). The brains were removed and stored at 4 °C overnight in 20% sucrose, 4% paraformaldehyde, in Sorensen's phosphate buffer (pH 7.6). Brains were mounted, frozen and sectioned (40/~M) on a sliding microtome. Sections were extensively washed with phosphate-buffered saline (PBS, pH 7.6) by gentle agitation and incubated in 2% normal horse serum (NHS) in PBS for 30 min at room temperature and 48 h at 4 °C in C37 monoctonal antibody diluted 1:2000 in 1% NHS. This antibody is specific for the 50 kDa subunit of CaM kinase3. After washing with PBS for 30 min with 3 changes, sections were processed according to instructions provided with Vectastain biotin-avidin-peroxidase kit (Vector Laboratory, Burlingame, CA). Peroxidase was developed 4-6 rain with 1% diaminobenzidine and 0.03% H20 2. The sections were counterstained with Toluidine blue and coverslipped for viewing. For control sections, primary antibody was replaced with 1% NHS. Staining was performed simultaneously on kindled brain sections and their paired controls. In some experiments, adjacent sections were thionin stained29 in order to identify anatomical regions. Stained sections were viewed with a Zeiss model microscope equipped with a blue filter. Images were digitalized and stored using an IBAS imaging system and optical densities were measured in a blind manner in specified regions. Statistical comparisons were made using the Student's t-test for paired data. RESULTS
Fig. 1. Thionin (A) and immunocytochemical (B and C) stained sections of the dorsal hippocampus. In control sections (B), immunoreactivity was concentrated in the endal limb of the dentate and in the stratum radiatum and lacunosum moleculare of CA1. Kindling (C) resulted in a dramatic decrease in immunoreactivity almost in all areas of the hippocampus. A tuft of immunoreactivity was spared from these decreases in the CA2 region of the hippocampus in 3 pairs (designated by the arrow), s, subiculum; r, stratum radiatum; lm, lacunosum moleculare; p, pyramidal cell layer; g, granule cell layer of dentate; m, molecular layer of dentate; h, hilus of dentate.
Regional distribution of CaM kinase in control brains T h e d i s t r i b u t i o n of C a M k i n a s e o b s e r v e d using C37 a n t i b o d i e s was similar to that previously r e p o r t e d 9'25. D r a m a t i c differences in staining intensity were o b s e r v e d a m o n g b r a i n regions with the strongest staining in the n e o c o r t e x a n d limbic system, a n d the w e a k e s t in midbrain and brainstem.
T h e h i p p o c a m p u s was highly i m m u n o r e a c t i v e . T h e e n d a l limb of the d e n t a t e gyrus was heavily s t a i n e d as was C A 1 (Fig. 1). T h e m o l e c u l a r layer of the d e n t a t e c o n t a i n e d m o s t of the i m m u n o r e a c t i v e p r o t e i n although g r a n u l e cell somas were also stained. R e g i o n s c o n t a i n i n g h i p p o c a m p a l p y r a m i d a l cell p r o j e c t i o n s , such as the
51 TABLE I
Comparison of CaM kinase immunoreactivity in kindled and control rats Optical densities were measured as described in Materials and Methods and values are expressed as mean differences in optical density units.
OD difference
S.E.M.
P<
Dorsal hippocampus CA1 CA2 CA3 CA4 Dentate (endal limb)
-0.019 -0.006 -0.012 -0.024 -0.022
0.008 0.008 0.005 0.008 0.006
0.025 n.s. 0.05 0.01 0.005
Ventral hippocampus CA1 CA2 CA3 Dentate (endal limb) Lateral septum
-0.066 -0.066 -0.097 -0.072 +0.014
0.019 0.016 0.018 0.022 0.016
0.005 0.005 0.005 0.01 n.s.
stratum radiatum and lacunosum moleculare, were heavily labeled, particularly in CA1. CaM kinase immunoreactivity in kindled brain CaM kinase immunoreactivity in 5 kindled animals was compared to that in paired controls. Staining was decreased the most in ventral hippocampus, although reductions in dorsal regions were also observed (Fig. 1, Table I). Within the hippocampus, CA3 and the endal limb of the dentate gyrus contained pronounced differences in staining. Smaller alterations were present in CA1 and CA4, while immunoreactivity in CA2 of the dorsal hippocampus was preserved. This preferential sparing of CA2 appeared as a tuft of CaM kinase immunoreactivity and was very apparent in 3 of the 5 pairs tested (Fig. 1). In contrast to the hippocampus, the lateral septal immunoreactivity was increased compared to controls, although differences did not reach statistical significance (Table I). Measurements in this region were made away from the implanted electrodes, which did not appear to alter staining. DISCUSSION
Septal kindling is associated with long-lasting decreases in CaM kinase activity in hippocampal synaptic plasma membranes 15'35. In this report, we demonstrated a decrease in CaM kinase immunoreactivity in most hippocampal regions. This suggests that the lowered activity reflects a decrease in the number of CaM kinase molecules, implying that the kindled trace is associated with acquired changes in gene expression or in post-
transcriptional processing of CaM kinase. Despite failure of early attempts at demonstrating morphological alterations in kindled brains 27, a selective loss of nonperforated synapses 12 and a change in spine morphology26 have been demonstrated in the hippocampus of kindled rats 12. Because of CaM kinase's possible structural role 31, decreased CaM kinase immunoreactivity may be a cause or an effect of the altered synaptic morphology. Further studies using electron microscopy and immunocytochemistry of CaM kinase in the hippocampus and other regions could help clarify this association. Although the biochemical basis of kindling is still unclear, CaM kinase is ideally designed both functionally and anatomically to alter synaptic excitability. CaM kinase appears to modulate transmitter biosynthesis and r e l e a s e 6'19'39, cytoskeletal proteins 38'4°, and probably has an important postsynaptic role given its extremely high concentration in the postsynaptic density17. Although increases in CaM kinase-mediated phosphorylation of synapsin I enhances transmitter release, so that one would predict an increase in CaM kinase to be associated with hyperexcitable states such as kindling, this is likely to be oversimplistic. The distribution of CaM kinase in excitatory and inhibitory, perforated and non-perforated synapses is still unclear as well as the complex circuitry of kindling. It can easily be imagined that a relative decrease in inhibitory synapses (as suggested by Geinisman et al) 2) could result in an overall increase in excitability. The regional distribution of CaM kinase in the brain and the changes induced by kindling are quite striking. The areas with the highest concentration of enzyme, such as the limbic system, are also the areas in which kindling can most easily be induced 28. In the cerebellum, which is totally resistant to kindling, a separate isoenzyme of CaM kinase is found23. Furthermore, changes in CaM kinase activity and immunoreactivity showed regional specificity. Decreases in hippocampal immunoreactivity induced by kindling were most pronounced in the ventral hippocampus. It is interesting to note that kindling proceeds at a faster rate following ventral than dorsal stimulation29. Anatomical29, neurochemical 3°, and electrophysiologiCal 7'29 investigations have provided strong evidence for functional dissociation between dorsal and ventral aspects of the rat hippocampus. The ventral hippocampus shows a greater tendency to generate epileptiform burst responses 13. The reduction of hippocampal immunoreactivity is absent in CA2 of the dorsal region. The sparing of CA2 was particularly striking in 3 of the 5 pairs tested. It is unknown why this region was different, or why some pairs showed a greater effect than others, but it is interesting to note that CA2 appears to be an excellent region to initiate burst activity in hippocampal slices37
52 and is selectively preserved in epileptic brains with hippocampal damage. It should be kept in mind that differences in immunoreactivity can be affected by a number of factors including cell density, intracellular distribution, and differential distribution between different cell types. These factors can alter antibody penetration and antigen accessibility. Since CaM kinase is a labile enzyme with a known vulnerability to ischemic conditions 36, a high pressure method of perfusion fixation was used. AI-
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