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The distribution of calcineurin in rat brain by light and electron microscopic immunohistochemistry and enzyme-immunoassay
Brain Research, 397 (1986) 161-172 Elsevier
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The Distribution of Calcineurin in Rat Brain by Light and Electron Microscopic Immunohistochemistry and Enzyme-Immunoassay SATOSHI GOTO 1, YASUHIKO MATSUKADO l, YOSUKE MIHARA l, NOBUHIRO INOUE 1and EISHICHI MIYAMOTO 2 Departments of INeurosurgery and 2Pharmacology, Kurnamoto University Medical School, Kumamoto 860 (Japan) (Accepted 15 April, 1986) Key words: Calcineurin - - Immunohistochemistry - - Enzyme-immunoassay - - Cerebral hemitransection - Striatonigrai pathway - - Pallidal pathway
Calcineurin is the calcium (divalent cations)-dependent calmodulin-stimulated phosphoprotein phosphatase which is capable of dephosphorylating various substrate proteins. The subcellular and regional distribution of calcineurin in the rat brain has been studied by light and electron microscopic immunohistochemistry using antiserum against calcineurin. Immunoreactivity was observed in many neurons but was not detected in glial cells, such as astrocytes, oligodendrocytes and ependymal cells by the PAP method. Light microscopy demonstrates strong immunoreactivity in neuronal somata and neurites. By electron microscopy, calcineurin immunoreactivity was found to be present in dendrites including postsynaptic densities, somata, spines, axons and terminals. Calcineurin immunoreactivity was present in neurons throughout the brain, but a marked regional variation in strength of the immunoreactivity was observed. The caudatoputamen, hippocampal formation, and substantia nigra were strongly stained. Cerebral and cerebellar neocortex showed moderate immunoreactivity. In substantia nigra and globus paUidus, only neurites were stained, but neuronal somata not. The staining of the substantia nigra was thought to be due to that of the nerve terminals originating from the caudatoputamen, in view of the findings by cerebral hemitransection and electron microscopic immunohistochemistry. We developed an enzyme-immunoassay (EIA) for calcineurin. The sensitivity of the EIA was 1 ng (13 fmol) of calcineurin. We determined the level of calcineurin in various regions of the rat brain. The caudate nucleus, putamen and hippocampal formation showed a high concentration of calcineurin. The results are consistent with those obtained by immunohistochemistry.
INTRODUCTION Calcineurin was first discovered as one of the calmodulin binding proteins in the brain and, therefore, as an inhibitor of the calmodulin-sensitive cyclic nucleotide p h o s p h o d i e s t e r a s e 11'12'25'3°. It has been recently known that calcineurin has an activity of Ca 2+ (divalent c a t i o n s ) - d e p e n d e n t and calmodulin-stimulated p h o s p h o p r o t e i n p h o s p h a t a s e 13'21"23'29'31. It is comprised of a catalytic subunit A with molecular weight of 61,000 D a and regulatory subunit B with 15,000 Da. It is a m a j o r calmodulin binding protein and highly c o n c e n t r a t e d in brain. Until now, several substrates for calcineurin, such as myosin light chain, phosphorylase kinase, inhibitor 1 (ref. 22), type II regulatory subunit of cyclic A M P - d e p e n d e n t protein kinase 13, microtubule proteins 6, p - n i t r o p h e n y l phos-
phate and free phosphotyrosine 19, and p h o s p h o t y r o syl-protein p h o s p h o r y l a t e d by the e p i d e r m a l growth factor receptor/kinase 4, have been r e p o r t e d . W e proposed the possibility that calcineurin could be a new neuronal m a r k e r of h u m a n brain tumors 5. Klee et al.13 suggested that calcineurin plays a pivotal role in turning off the cyclic A M P signal, since several substrates for the cyclic A M P - d e p e n d e n t kinase, such as the type II regulatory subunit of cyclic A M P - d e p e n d e n t kinase, phosphorylase kinase and inhibitor I, are d e p h o s p h o r y l a t e d by calcineurin. W o o d et al. 3° and Wallace et al. 26 r e p o r t e d that CaM-BP80 ( p r o b a b l y identical with calcineurin) was present at a high level particularly in the neostriatum of the mouse brain, d e t e r m i n e d by immunohistochemicai m e t h o d and radioimmunoassay. F r o m these resuits, they suggested that the physiological function
Correspondence: S. Goto, Department of Neurosurgery, Kumamoto University Medical School, Kumamoto 860, Japan. 0006-8993/86/$03.50 (~) 1986 Elsevier Science Publishers B.V. (Biomedical Division)
162 of calcineurin is related to the extrapyramidal system with respect to the generation and regulation of motor command. It was recently reported that calcineurin could dephosphorylate DARPP-32, a dopamine- and adenosine 3',5'-monophosphate-regulated phosphoprotein enriched in dopamine-innervated brain regions 9'17'27. However, the role of calcineurin in the brain is not clear. In this communication, we describe the subcellular and regional distribution of calcineurin throughout the central nervous system and specially focus on the striatonigral pathway of the rat brain, performed by light and electron microscopic immunohistochemistry and enzyme-immunoassay.
MATERIALS AND METHODS
Materials DEAE-cellulose (DE-52) was purchased from Whatman; Sephadex G-150, Sephacryl S-300, Sepharose 4B and CNBr-activated Sepharose 4B from Pharmacia Fine Chemicals; phosphocellulose (Cellex-P) and Affigel blue from Bio-Rad Laboratories; 3,3'-diaminobenzidine and horseradish peroxidase from Sigma; Vectastain biotin-avidin-peroxidase kit from Vector Laboratories; anti-neuron specific enolase serum and D A K O Universal PAP Kit from D A K O ; N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl)-maleimide from Zieben chemicals. Other chemicals were of analytical grade.
Procedures for light microscopic immunohistochemistry We prepared the antisera against calcineurin according to the method described previously 6. Rabbit antisera were further purified by affinity column packed with CNBr-activated Sepharose 4B coupled with purified caicineurin from rat brain, as described by March et al.15. The purified antisera were stored a t - 7 0 °C with 10 mg/ml of bovine serum albumin until use. In the following experiments, we only used these purified antisera. Adult Wistar/albino rats were sacrificed by decapitation, and their brains were quickly removed. The brains were sliced less than 5 mm wide with a razor blade and fixed with 10% formalin overnight, fol-
lowed by paraffin-embedded. Coronal slices (4/~m) of the fixed tissues were prepared from cerebrum, cerebellum and spinal cord. The immunostaining methods were essentially identical with the peroxidase-antiperoxidase technique described by Sternberger et al. 2°, using D A K O Universal PAP Kit. Staining specificity was assessed by the careful examination of tissue sections containing elements other than neuron, or by replacing the primary antiserum with non-immune rabbit serum.
Procedures for electron microscopic immunohistochemistry Monovalent fragments (Fab') of the antisera against calcineurin were conjugated with horseradish peroxidase (HRP) using N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl)-maleimide32. Wistar/albino rats, weighing about 250 g, were anesthetized with ethyl ether and perfused through the heart for 30 min with an ice-cold fixative containing freshly prepared 4,0% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. After perfusion, the brains were removed and stored for 4 h at 4 °C in the same fixative. After freezing, the sections (10/~m thick) of the fixed tissue were cut in a cryostat. These frozen sections were incubated at 4 °C for 15 h in phosphate-buffered saline (PBS) containing 1% (w/v) BSA and HRP-conjugated Fab'. The sections were fixed in I % glutaraldehyde in PBS after the immunologic reaction and incubated in incomplete Graham-Karnovsky's solution 7 for 30 min, followed by incubation with complete solution for 5 min. After postfixation with 2% osmic acid in PBS for 60 min, the sections were dehydrated by a graded alcohol series and embedded in Epon. The samples of ultrathin sections which were immunostained or non-immunostained were examined by electron microscopy with or without uranyl counterstaining.
Cerebral hemitransection Wistar/albino rats, weighing about 250 g, were anesthetized with diethyl ether and mounted on a stereotaxic frame. After drilling on the left side of the skull 2 mm behind the bregma, the left hemisphere was transected at the level of the globus pallidus with a rectangular knife blade which was lowered to the base of the skull 27. The animals were allowed to survive for 5 days and then decapitated.
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Establishment of enzyme-immunoassay for calcineurin The method rests on the general observation that the majority of the proteins are strongly adsorbed on nitrocellulose s,1°'14'24. Appropriate amounts of purified calcineurin or tissue extracts were adsorbed on nitrocellulose membrane (Schleicher and Schull BA 85) using Micro-Sample Filtration Manifold (Schleicher and Schull). The nitrocellulose membrane was immersed in PBS containing 3% (w/v) bovine serum albumin (BSA) and shaken gently at room temperature for about 1 h to saturate additional protein binding sites. The solution was removed and replaced by PBS containing 0.05% (v/v) Tween 20, 1% (w/v) BSA and diluted (1:500) antiserum (IgG fraction) against calcineurin. After incubation at 4 °C overnight, the membrane was washed for 30 min with several changes of the same buffer without the antiserum, followed by the incubation with PBS containing 3% BSA. The membrane was processed according to the instructions in the Vecstain biotinavidin-peroxidase kit, except for 1 h incubation with biotinyl antibody. After the last wash was performed, the peroxidase reaction was processed for 10-15 min with G r a h a m - K a r n o v s k y ' s solution. When the reaction was complete, the sheet was washed with distilled water and dried. The immunostaining intensity of each spot was determined by dual-wave length TLC scanner (Shimadzu) with a recorder of Shimadzu C-R2A. The area of each spot was integrated by the computer.
Preparation of tissue extracts for enzyme-immunoassay The Wistar/albino rats, weighing about 250 g, were anesthetized with ethyl ether. Brains were immediately removed, frozen in liquid N 2, and sliced. Various regions were punched out with needle on dry-ice. The samples obtained were thawed and homogenized with 10 vols. of 50 mM Tris-HC1 buffer, pH 7.5, containing 1 mM dithiothreitol, 1 mM E G T A and 0.1 mM phenyl-methanesulfonyl fluoride, in a teflonglass homogenizer. The homogenates were centrifuged at 100,000 g for 1 h. The resultant supernatants were dialyzed overnight against PBS containing 0.1 mM PMSF. The samples were stored at -70 °C until use.
Other procedures Calmodulin-Sepharose 4B affinity column was prepared by the method of Klee and Krinks II. Protein was determined by the method of Bradford 1 with bovine serum albumin as standard. RESULTS
Regional distribution Immunoreactivity was observed in many neurons but was not detected in glial cells such as astrocytes, oligodendrocytes, ependymal cells and others, under the experimental conditions with the PAP method. In neurons, calcineurin was detected in somata, dendrites and axons. The caudatoputamen (Fig. 1A), hippocampal formation (Fig. 2A) and substantia nigra (Fig. 2A, B) showed strong immunoreactivity, while the cerebral neocortex (Fig. 1A), cerebellum (Fig. 3A), lateral septum and globus pallidus (Fig. 1E) were moderately stained. In the caudatoputamen (Fig. 1E, F), most neurons showed intense immunostaining in somata and neurites. Some neurons showed immunoreactivity in the nuclei. The dorsolateral part of this region had the strongest immunoreactivity. Bundles of white matter were slightly immunostained and this staining was thought to be due to that of myelinated axons as revealed by electron microscopic immunohistochemical examination (data not shown). The globus pallidus was moderately stained (Fig. 1E). However, in this region, neuronal somata did not show immunoreactivity and only neurites were stained, which were considered to be the axons originating from the caudatoputamen. In the neocortex (Fig. 1B), the most prominently stained neurons were observed in layers III (Fig. 1C), V and VI (Fig. 1D). Both pyramidal and nonpyramidal cells were strongly immunostained. Large cells were stained more strongly than small cells. The olfactory tubercle and bulb were weakly immunoreactive. The rostral hippocampal formation was strongly immunoreactive (Fig. 2C), and especially the stratum pyramidale (Fig. 2E), radiatum and lacunosum moleculare. The dentate gyms showed moderate immunostaining (Fig. 2C). Immunoreactivity was weakly shown in the amygdaloid complex and geniculate body. Most of the thalamic nuclei and hypothal-
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165 neurons did not show any immunoreactivity either. In the cerebellum (Fig. 3A, B), 3 layers i.e. the molecular, Purkinje and granular layers were moderately stained. Each of the granular cells showed immunoreactivity in various degrees. Purkinje cells are divided into two types. One is a strongly stained type and the other is not stained. In general, immunoreacrive Purkinje cells were of a small population. The entire myelencephalon did not show very strong immunoreactivity, and the cerebellar peduncle and pyramidal tract were weakly immunoreactive.
Cellular and electron microscopic distribution
Fig. 1. Light micrographs of coronal sections through a hemisphere of the rat brain at the level of the caudatoputamen. A: strong immunoreactivity is seen in the caudatoputamen, and moderate in the neocortex and lateral septum (x6). B: layers I-VI of the premotor neocortex (x25). Immunoreactivity is relatively strong in layers III, V and VI. Layer III (C) and V (D) are shown (x150). Strong immunoreactivity is present in the pyramidal neurons. The regions of the cytoplasm and neurites (arrows) are the most intensive. Some of the neurons show no immunoreactivity (arrowheads). E: the caudatoputamen (CP)-globus pallidus (GP) (x 40). The globus pallidus is slightly stained, but neuronal bodies are not stained. F: caudate nucleus (x 100). Immunostaining of the cytoplasm of cells is observed. A few neurons show immunoreactivity in the nuclei (curved arrows). In contrast, some of the neurons do not show immunostaining (arrowheads). NC, neocortex; CC, corpus callosum; LS, lateral septum; MS, medial septum; AC, anterior commissure; LOT, lateral olfactory tract.
amus contained neurons which were relatively less immunoreactive than those in other areas. The pars reticulata of the substantia nigra was strongly stained, but neuronal bodies in the substantia nigra as well as the globus pallidus (Fig. 2D) did not show any immunoreactivity. It has been known that this area receives the axons originating from the caudatoputamen. It is considered that the stained structure may be nerve terminals of the axons originating from the caudatoputamen. The pars compacta and pars lateralis of the substantia nigra, containing dopaminergic neurons, did not show any immunoreactivity. The raphe nucleus containing serotoninergic neurons, and locus coeruleus containing adrenergic
The findings by microscopy were as follows. Immunoreactivity was very strong in neuronal somata and dendrites. Axons and nerve terminals also showed strong immunostaining. Weak staining of axons was observed along with fibe.r tracts in the corpus callosum, internal capsule and pyramidal tract of the spinal cord. Some of the neurons appeared to contain immunoreactive products in their nuclei. Under electron microscopy, immunoreactivity was observed in the cytoplasm, on the outer surface of mitochondria, and on microtubules (Fig. 4A, B). The nucleus did not show any immunoreactivity. Dendrites and nerve terminals (Fig. 4 C - F ) were strongly stained. Additionally, postsynaptic densities (PSDs) were strongly stained (Fig. 4C, D).
Cerebral hemitransection Fig. 5 shows calcineurin staining of the transverse section at the midbrain level in the left-side-transected rat brain. It is clear that the staining of the pars reticulata in the substantia nigra on the ipsilateral side almost completely disappeared, compared with the contralateral side. NSE staining of the same section, as one of the control studies, revealed no difference between the left and right sides (data not shown). In the same hemitransected brain, intensity and distribution of calcineurin staining in the caudatoputamen on the ipsilateral side were not remarkably changed (data not shown).
N- methyl- 4-phenyl- 1,2, 3, 6 - tetrahydropyridine (NMPTP) treatment The brains of NMPTP-treated rats were examined by immunohistochemistry. The intensity and distribution of calcineurin staining in the substantia nigra
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Fig. 3. Light micrograph of a sagittal section of the rat cerebellar hemisphere (A, ×40; B, × 160). The molecular layer (ML) and granular layer (GL) are moderately stained. Some of the Purkinje cells are strongly stained (black arrows) and others show no immunoreactivity (white arrows). The granular cells are also stained.
were the same as those of n o n - t r e a t e d rats. The caud a t o p u t a m e n also showed similar immunoreactivity to that of n o n - t r e a t e d rat brain ( d a t a not shown).
Measurement of calcineurin in various rat brain regions Fig. 6 shows a s t a n d a r d curve for the enzyme-immunoassay ( E I A ) for calcineurin. The sensitivity of the E I A is 1 ng (13 fmol) of calcineurin, and is comparable to that of the r a d i o i m m u n o a s s a y ( R I A ) rep o r t e d previously 26. Using the E I A , the level of calcineurin in a variety of rat brain regions was m e a s u r e d (Table I). Calci-
neurin was d e t e c t e d in all regions of the brain examined. The level of calcineurin varied from region to region. The caudate nucleus, p u t a m e n and hippocampus showed the high level, while the globus pallidus, cerebral gray m a t t e r , substantia nigra and cerebellum were relatively lower. The results were generally consistent with the findings o b t a i n e d by immunohistochemistry described above. DISCUSSION Calcineurin immunoreactivity was present only in neurons throughout the brain, but m a r k e d regional
Fig. 2. Light micrographs of coronal sections through a hemisphere of the rat brain at the level of the substantia nigra (A) and raphe nucleus (B) (x 6). In A and B, the hippocampus, substantia nigra and neocortex show strong immunoreactivity, whilst the dentate gyrus and geniculate body show weak immunostaining. The raphe nucleus shows no immunoreactivity. CLF, central longitudinal fissure; NC, neocortex; SCC, superior collicular commissure; FLD, fasciculus longitudinalis dorsalis; FLM, fasciculus longitudinalis medialis; CA, cerebral aqueduct; GD, geniculate body; DG, dentate gyrus; HI, hippocampus; RN, red nucleus; RS, rhinal sulcus; EC, entorhinal cortex; ML, medial lemniscus; SN, substantia nigra; CC, crus cerebri; R, raphe. C: the rostral hippocampal formation (x 25). Immunoreactivity is strong in the stratum oriens (so), stratum pyramidale (sp), stratum radiatum (sr) and stratum lacunosum moleculare (slm). ec, ectal limb of dentate gyrus; en, endal limb of dentate gyrus. D: pars reticulata of the substantia nigra (x200). Neuronal bodies are not stained (white arrows). E: stratum pyramidale of the hippocampal formation shows immunoreactivity in the cytoplasm ( × 140).
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Fig. 5. Light micrograph of coronal section through the cerebral hemisphere of the hemitransected rat brain at the level of the substantia nigra. Immunostaining of the substantia nigra on the ipsilateral side (left side) is not observed in comparison to that on the contralateral side. Rt, right side; Lt, left side; SN, substantia nigra (x 10).
variation in the intensity of immunoreactivity was observed. The c a u d a t o p u t a m e n and h i p p o c a m p a l formation were m a r k e d l y stained. The results were supp o r t e d by those of E I A . The substantia nigra was also strongly stained. It was considered to be staining of the nerve terminals originating from the caudatoputamen. The reasons are as follows. (1) Only neurites were stained, but n e u r o n a l s o m a t a in the substantia nigra were not (Fig. 2D). (2) The immunoreactivity of the neurites d i s a p p e a r e d when the striatonigral
pathway had previously been cut off by cerebral hemitransection (Fig. 5). (3) In this region, immunoreactivity was observed in axons u n d e r the electron microscopic immunohistochemistry (data not shown). If our assumption is right, the immunostaining in the globus pallidus m a y also be due to the staining of the nerve terminals of the striatopallidal pathway. In view of the regional distribution, calcineurin may play an i m p o r t a n t role in the functions of extra-
Fig. 4. Electron micrographs of ultrathin sections through the caudate nucleus without uranyl counterstaining. A: immunoreactivity in the cytoplasm (Cy) of a neuron (x6500). PSD showed immunoreactivity (between arrowheads). The nucleus (Nu) showed faint immunoreactivity. B: an immunoreactive dendrite (D) and PSD (arrow) are shown (x 13,000). The curved arrow points to lightly labeled PSD. Immunoreactive microtubules are also shown (long arrows). C: immunoreactive dendrites (D) and PSDs (arrows) (x7800). D: immunoreactive PSDs (between arrowheads) are shown (x32,500). The curved arrow points to lightly labeled PSD in comparison with the labeled PSD as shown in B. E: immunoreactive nerve terminals are shown (x26,000). F: an axodendritic synapse is shown. Strong immunoreactivity is observed on the synaptic vesicles and PSDs (x 26,000).
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Fig. 6. Standard curve for the assay of calcineurin by enzymeimmunoassay. Various amounts of purified calcineurin indicated were adsorbed on the nitrocellulose membrane, followed by immunostaining with antiserum against calcineurin as a primary antibody. Immunostaining intensity of each spot was analyzed by the TLC scanner with a computer. pyramidal system, as Wallace et ai. previously suggested 26, and hippocampal formation. Calcineurin has been reported to dephosphorylate D A R P P - 3 2 (a dopamine-regu|ated neuronal phosphoprotein) as substrate 9. Calcineurin may be related to the function of the dopaminoceptive neurons. Calcineurin was present in the axons of the striatonigral pathway. This pathway contains the G A B A e r TABLE I Level of calcineurin in various rat brain regions as determined by enzyme-immunoassay
An appropriate volume (50-200 ~1) of 100,000 g supernatant fluid from each brain region was assayed for calcineurin. The data represent the mean _+S.D. on regions from two rats. Each determination was duplicated. Regions
Cerebrum Neocortical gray matter White matter Caudate nucleus Putamen Globus pallidus Hippocampus Thalamus Hypothalamus Substantia nigra Olfactory bulb Cerebellum Pons Medulla oblongata Spinal cord
Calcineurin (l~g/mg of protein)
0.54 _+0.05 0.23 + 0.03 1.73 _+0.42 1.65 _+0.50 0.71 ___0.05 1.45 _+0.39 0.26 _+0.04 0.14 _+0.04 0.50 + 0.11 0.19 _+0.05 0.42 + 0.11 0.25 _+0.04 0.24 + 0.05 0.19 + 0.02
gic and substance P-ergic neurons 2. It is under investigation that calcineurin exists in G A B A - and/or substance P-ergic neurons. These finding may be of interest, since the relationships between the striatum and substantia nigra, and the striatum and globus pallidus are elucidated. Calcineurin may not be present in the adrenergic and serotonergic neurons, since immunostaining was not observed in the neurons of the locus coeruleus and raphe nucleus. W o o d et al. reported that CaM-BPs0 (probably identical with calcineurin) was associated only with neuronal elements, such as PSDs and dendritic microtubules, primarily at postsynaptic sites within neuronal somata and dendrites 3°. We used HRP-conjugated Fab' of antiserum to calcineurin in the investigation by electron microscopic immunohistochemistry. This direct method has proved to be the most sensitive method at present ~6. The findings by the method indicated that calcineurin immunoreactivity was present in dendrites including PSDs, somata, spines, axons and nerve terminals (synaptic vesicles). Calcineurin may be involved in a variety of Ca 2+ and calmodulin-dependent physiological processes via dephosphorylation of various substrate proteins. We have reported that calcineurin can dephosphorylate the microtubule proteins phosphorylated by Ca 2+ and calmodulin-dependent protein kinase with a high molecular weight 6. Immunostaining of calcineurin is observed on the microtubules (the present study) and Ca 2+- and calmodulin-dependent protein kinase is associated with the microtubule fraction 3, indicating that the microtubules are regulated by both enzymes through phosphorylation and dephosphorylation. On the other hand, both enzymes can be activated by calmodulin. It is a very interesting problem which regulation system is more activated in which either phosphorylation or dephosphorylation may occur. The immunohistochemical findings of calcineurin (the present study) and calcium/caimodulin-dependent protein kinase II TM showed that there was no prominent difference in the regional distribution of both enzymes in the brain. We developed an enzyme-immunoassay for calcineurin. It was a useful method for measurement of calcineurin content as compared to radioimmunoassay, in view of its sensitivity, safety, simplicity and rapidity. The results were generally consistent with the findings obtained by immunohistochemistry de-
171 scribed above. Exceptionally, the substantia nigra and globus pallidus had lower levels of calcineurin in comparison to the intensities of the immunostaining. The difference m a y be due to that of the localization of calcineurin in subcellular fractions, since the E I A was p e r f o r m e d for the 100,000 g supernatant. Calcineurin m a y occur in the particulate fractions m o r e than in the s u p e r n a t a n t in these regions. The findings by electron microscopic immunohistochemistry also revealed that calcineurin is present in the particulate fractions such as the nerve terminals and PSDs (Fig. 4). In addition, using E I A , we m e a s u r e d the levels of calcineurin content in o t h e r various rat tissues, such as the pituitary gland, pancreas, skeletal muscle, spleen, thyroid gland, testis, lung, heart and liver. H o w e v e r , these tissues showed significantly low values of calcineurin content (data not shown), as consistent with a previous r e p o r t using r a d i o i m m u noassay 26. Recently, Stewart et al. r e p o r t e d that a Ca 2+ and c a l m o d u l i n - d e p e n d e n t protein phospha-
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tase n a m e d protein p h o s p h a t a s e 2B was purified from rabbit skeletal muscle. The estimated concentration of the enzyme in the skeletal muscle was 12.5-25 mg/kg, which was c o m p a r a b l e to the concentration of calcineurin in the brain 22. H o w e v e r , the concentration of calcineurin in the skeletal muscle was extremely lower than that of brain by E I A . W e incubated sections of the skeletal muscle with the antiserum against calcineurin, but could not detect immunoreactivity (data not shown). These results indicate that calcineurin and p r o t e i n p h o s p h a t a s e 2B antigenically differ from each other. ACKNOWLEDGEMENTS This work was s u p p o r t e d by a G r a n t - i n - A i d for Scientific Research from the Ministry of Education, Science, and Culture of Japan. W e are grateful to Y. Sonoda for technical service and to H. G o t o for secretarial service.
tase-1, Nature (London), 310 (1984) 503-505. 10 Huet, J., Sentenac, A. and Fromageot, P., Spot-immunodetection of conserved determinants in eukaryotic RNA polymerases. Study with antibodies to yeast RNA polymerases subunits, J. Biol. Chem., 257 (1982) 2613-2618. 11 Klee, C.B. and Krinks, M.H., Purification of cyclic 3',5'nucleotide phosphodiesterase inhibitory protein by affinity chromatography on activator protein coupled to Sepharose, Biochemistry, 17 (1978) 120-126. 12 Klee, C.B., Crough, T.H. and Krinks, M.H., Calcineurin: a calcium- and calmodulin-binding proteins of the nervous system, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 6270-6273. 13 Klee, C.B., Krinks, M.H., Manalan, A.S., Cohen, P. and Stewart, A . A . , Isolation and characterization of bovine brain calcineurin: A calmodulin-stimulated protein phosphatase, Meth. Enzymol., 102 (1983) 227-244. 14 Kuno, H. and Kihara, H., Simple microassay of protein with membrane filter, Nature (London), 215 (1967) 974-975. 15 March, S.C., Parikh, I. and Cuatrecasas, P., A simplified method of cyanogen bromide activation of agarose for affinity chromatography, Anal. Biochem., 60 (1974) 149-152. 16 Mitani, F., Ishimura, Y., Izumi, S. and Watanabe, K., Immunohistochemical localization of adrenodoxin and adrenodoxin reductase in bovine adrenal cortex, Acta Endocrinol., 90 (1979) 317-327. 17 Ouiment, C.C., Miller, P.E., Hemmings, H.C., Jr., Walaas, S.I. and Greengard, P., DARPP-32, a dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein enriched in dopamine-innervated brain regions. III. Immunocytochemical localization, J. Neurosci., 4 (1984) 111-124. 18 Ouimet, C.C., McGuinness, T.L. and Greengard, P., Im-
172 munocytochemical localization of calcium/calmodulin-dependent protein kinase II in rat brain, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 5604-5608. 19 PaUen, C.J. and Wang, H., Calmodulin-stimulated dephosphorylation of p-nitrophenyl phosphate and free phosphotyrosine by calcineurin, J. Biol. Chem., 258 (1983) 8550-8553. 20 Sternberger, L.A., Hardy, P.H., Cuculis, J.J. and Meter, H.G., The unlabelled antibody enzyme method of immunohistochemistry - - preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes,J. Histochem. Cytochem., 18 (1970)314-333. 21 Stewart, A . A . , Ingebritsen, T.S., Manalan, A., Klee, C.B. and Cohen, P., Discovery of a Ca 2÷- and calmodulin-dependent protein phosphatase. Probable identity with calcineurin (CaM-BP80), FEBS Lett., 137 (1982) 80-84. 22 Stewart, A.A., Ingebritsen, T.S. and Cohen, P., The protein phosphatase involved in cellular regulation. 5. Purification and properties of a Ca2+/calmodulin-dependent protein phosphatase (2B), Eur. J. Biochem., 132 (1983) 289-295. 23 Tonks, N.K. and Cohen, P., Calcineurin is a calcium ion dependent, calmodulin-stimulated protein phosphatase, Biochim. Biophys. Acta, 747 (1983) 191-193. 24 Towbin, H., Staehelin, T. and Gordon, J., Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 4350-4354. 25 Wallace, R.W., Lynch, T.J. and Tallant, E.A., Purification and characterization of an inhibitory protein of brain ade-
26
27
28
29
30
31
32
nylate cyclic nucleotide phosphodiesterase, J. Biol. Chem., 254 (1979) 377-382. Wallace, R.W., Tallant, E.A. and Cheung, W.Y., High levels of a heat-labile calmodulin-binding protein (CAMBPs0) in bovine neostriatum, Biochemistry, 19 (1980) 1831-1837. Wallaas, S.I. and Greengard, P., DARPP-32, a dopamineand adenosine 3':5'-monophosphate-regulated phosphoprotein enriched in dopamine-innervated brain regions. I. Regional and cellular distribution in the rat brain, J. Neurosci., 4 (1984) 84-98. Wang, J.H. and Desai, R., Modulator binding protein: bovine brain protein exhibiting the Ca 2+ dependent association with the protein modulator of cyclic nucleotide phosphodiesterase, J. Biol. Chem., 252 (1977) 4175-4184. Winkler, M.A., Merat, D.L., Tallant, E.L., Hawkins, S. and Cheung, W.Y., Catalytic site of calmodulin-dependent protein phosphatase from bovine brain resides in subunit A, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 3054-3058. Wood, J.G., Wallace, R.W., Whitaker, J.N. and Cheung, W.Y., Immunohistochemical localizaton of calmodulin and a heat-labile calmodulin-binding protein (CaM-BPs~I) in basal ganglia of mouse brain, J. Cell Biol., 84 (1980) 66-76. Yang, S.D., Tallant, E.A. and Cheung, W.Y., Calcineurin is a calmodulin-dependent protein phosphatase, Biochim. Biophys. Res. Commun., 106(1982) 1419-1425. Yoshitake, S., Yamada, Y., Ishikawa, E. and Masseyeff, R., Conjugation of glucose oxidase from aspergillus niger and rabbit antibodies using N-Hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl)-maleimide, Eur. J. Biochem., 101 (1979) 395-399.
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BRE 12149
The Distribution of Calcineurin in Rat Brain by Light and Electron Microscopic Immunohistochemistry and Enzyme-Immunoassay SATOSHI GOTO 1, YASUHIKO MATSUKADO l, YOSUKE MIHARA l, NOBUHIRO INOUE 1and EISHICHI MIYAMOTO 2 Departments of INeurosurgery and 2Pharmacology, Kurnamoto University Medical School, Kumamoto 860 (Japan) (Accepted 15 April, 1986) Key words: Calcineurin - - Immunohistochemistry - - Enzyme-immunoassay - - Cerebral hemitransection - Striatonigrai pathway - - Pallidal pathway
Calcineurin is the calcium (divalent cations)-dependent calmodulin-stimulated phosphoprotein phosphatase which is capable of dephosphorylating various substrate proteins. The subcellular and regional distribution of calcineurin in the rat brain has been studied by light and electron microscopic immunohistochemistry using antiserum against calcineurin. Immunoreactivity was observed in many neurons but was not detected in glial cells, such as astrocytes, oligodendrocytes and ependymal cells by the PAP method. Light microscopy demonstrates strong immunoreactivity in neuronal somata and neurites. By electron microscopy, calcineurin immunoreactivity was found to be present in dendrites including postsynaptic densities, somata, spines, axons and terminals. Calcineurin immunoreactivity was present in neurons throughout the brain, but a marked regional variation in strength of the immunoreactivity was observed. The caudatoputamen, hippocampal formation, and substantia nigra were strongly stained. Cerebral and cerebellar neocortex showed moderate immunoreactivity. In substantia nigra and globus paUidus, only neurites were stained, but neuronal somata not. The staining of the substantia nigra was thought to be due to that of the nerve terminals originating from the caudatoputamen, in view of the findings by cerebral hemitransection and electron microscopic immunohistochemistry. We developed an enzyme-immunoassay (EIA) for calcineurin. The sensitivity of the EIA was 1 ng (13 fmol) of calcineurin. We determined the level of calcineurin in various regions of the rat brain. The caudate nucleus, putamen and hippocampal formation showed a high concentration of calcineurin. The results are consistent with those obtained by immunohistochemistry.
INTRODUCTION Calcineurin was first discovered as one of the calmodulin binding proteins in the brain and, therefore, as an inhibitor of the calmodulin-sensitive cyclic nucleotide p h o s p h o d i e s t e r a s e 11'12'25'3°. It has been recently known that calcineurin has an activity of Ca 2+ (divalent c a t i o n s ) - d e p e n d e n t and calmodulin-stimulated p h o s p h o p r o t e i n p h o s p h a t a s e 13'21"23'29'31. It is comprised of a catalytic subunit A with molecular weight of 61,000 D a and regulatory subunit B with 15,000 Da. It is a m a j o r calmodulin binding protein and highly c o n c e n t r a t e d in brain. Until now, several substrates for calcineurin, such as myosin light chain, phosphorylase kinase, inhibitor 1 (ref. 22), type II regulatory subunit of cyclic A M P - d e p e n d e n t protein kinase 13, microtubule proteins 6, p - n i t r o p h e n y l phos-
phate and free phosphotyrosine 19, and p h o s p h o t y r o syl-protein p h o s p h o r y l a t e d by the e p i d e r m a l growth factor receptor/kinase 4, have been r e p o r t e d . W e proposed the possibility that calcineurin could be a new neuronal m a r k e r of h u m a n brain tumors 5. Klee et al.13 suggested that calcineurin plays a pivotal role in turning off the cyclic A M P signal, since several substrates for the cyclic A M P - d e p e n d e n t kinase, such as the type II regulatory subunit of cyclic A M P - d e p e n d e n t kinase, phosphorylase kinase and inhibitor I, are d e p h o s p h o r y l a t e d by calcineurin. W o o d et al. 3° and Wallace et al. 26 r e p o r t e d that CaM-BP80 ( p r o b a b l y identical with calcineurin) was present at a high level particularly in the neostriatum of the mouse brain, d e t e r m i n e d by immunohistochemicai m e t h o d and radioimmunoassay. F r o m these resuits, they suggested that the physiological function
Correspondence: S. Goto, Department of Neurosurgery, Kumamoto University Medical School, Kumamoto 860, Japan. 0006-8993/86/$03.50 (~) 1986 Elsevier Science Publishers B.V. (Biomedical Division)
162 of calcineurin is related to the extrapyramidal system with respect to the generation and regulation of motor command. It was recently reported that calcineurin could dephosphorylate DARPP-32, a dopamine- and adenosine 3',5'-monophosphate-regulated phosphoprotein enriched in dopamine-innervated brain regions 9'17'27. However, the role of calcineurin in the brain is not clear. In this communication, we describe the subcellular and regional distribution of calcineurin throughout the central nervous system and specially focus on the striatonigral pathway of the rat brain, performed by light and electron microscopic immunohistochemistry and enzyme-immunoassay.
MATERIALS AND METHODS
Materials DEAE-cellulose (DE-52) was purchased from Whatman; Sephadex G-150, Sephacryl S-300, Sepharose 4B and CNBr-activated Sepharose 4B from Pharmacia Fine Chemicals; phosphocellulose (Cellex-P) and Affigel blue from Bio-Rad Laboratories; 3,3'-diaminobenzidine and horseradish peroxidase from Sigma; Vectastain biotin-avidin-peroxidase kit from Vector Laboratories; anti-neuron specific enolase serum and D A K O Universal PAP Kit from D A K O ; N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl)-maleimide from Zieben chemicals. Other chemicals were of analytical grade.
Procedures for light microscopic immunohistochemistry We prepared the antisera against calcineurin according to the method described previously 6. Rabbit antisera were further purified by affinity column packed with CNBr-activated Sepharose 4B coupled with purified caicineurin from rat brain, as described by March et al.15. The purified antisera were stored a t - 7 0 °C with 10 mg/ml of bovine serum albumin until use. In the following experiments, we only used these purified antisera. Adult Wistar/albino rats were sacrificed by decapitation, and their brains were quickly removed. The brains were sliced less than 5 mm wide with a razor blade and fixed with 10% formalin overnight, fol-
lowed by paraffin-embedded. Coronal slices (4/~m) of the fixed tissues were prepared from cerebrum, cerebellum and spinal cord. The immunostaining methods were essentially identical with the peroxidase-antiperoxidase technique described by Sternberger et al. 2°, using D A K O Universal PAP Kit. Staining specificity was assessed by the careful examination of tissue sections containing elements other than neuron, or by replacing the primary antiserum with non-immune rabbit serum.
Procedures for electron microscopic immunohistochemistry Monovalent fragments (Fab') of the antisera against calcineurin were conjugated with horseradish peroxidase (HRP) using N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl)-maleimide32. Wistar/albino rats, weighing about 250 g, were anesthetized with ethyl ether and perfused through the heart for 30 min with an ice-cold fixative containing freshly prepared 4,0% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. After perfusion, the brains were removed and stored for 4 h at 4 °C in the same fixative. After freezing, the sections (10/~m thick) of the fixed tissue were cut in a cryostat. These frozen sections were incubated at 4 °C for 15 h in phosphate-buffered saline (PBS) containing 1% (w/v) BSA and HRP-conjugated Fab'. The sections were fixed in I % glutaraldehyde in PBS after the immunologic reaction and incubated in incomplete Graham-Karnovsky's solution 7 for 30 min, followed by incubation with complete solution for 5 min. After postfixation with 2% osmic acid in PBS for 60 min, the sections were dehydrated by a graded alcohol series and embedded in Epon. The samples of ultrathin sections which were immunostained or non-immunostained were examined by electron microscopy with or without uranyl counterstaining.
Cerebral hemitransection Wistar/albino rats, weighing about 250 g, were anesthetized with diethyl ether and mounted on a stereotaxic frame. After drilling on the left side of the skull 2 mm behind the bregma, the left hemisphere was transected at the level of the globus pallidus with a rectangular knife blade which was lowered to the base of the skull 27. The animals were allowed to survive for 5 days and then decapitated.
163
Establishment of enzyme-immunoassay for calcineurin The method rests on the general observation that the majority of the proteins are strongly adsorbed on nitrocellulose s,1°'14'24. Appropriate amounts of purified calcineurin or tissue extracts were adsorbed on nitrocellulose membrane (Schleicher and Schull BA 85) using Micro-Sample Filtration Manifold (Schleicher and Schull). The nitrocellulose membrane was immersed in PBS containing 3% (w/v) bovine serum albumin (BSA) and shaken gently at room temperature for about 1 h to saturate additional protein binding sites. The solution was removed and replaced by PBS containing 0.05% (v/v) Tween 20, 1% (w/v) BSA and diluted (1:500) antiserum (IgG fraction) against calcineurin. After incubation at 4 °C overnight, the membrane was washed for 30 min with several changes of the same buffer without the antiserum, followed by the incubation with PBS containing 3% BSA. The membrane was processed according to the instructions in the Vecstain biotinavidin-peroxidase kit, except for 1 h incubation with biotinyl antibody. After the last wash was performed, the peroxidase reaction was processed for 10-15 min with G r a h a m - K a r n o v s k y ' s solution. When the reaction was complete, the sheet was washed with distilled water and dried. The immunostaining intensity of each spot was determined by dual-wave length TLC scanner (Shimadzu) with a recorder of Shimadzu C-R2A. The area of each spot was integrated by the computer.
Preparation of tissue extracts for enzyme-immunoassay The Wistar/albino rats, weighing about 250 g, were anesthetized with ethyl ether. Brains were immediately removed, frozen in liquid N 2, and sliced. Various regions were punched out with needle on dry-ice. The samples obtained were thawed and homogenized with 10 vols. of 50 mM Tris-HC1 buffer, pH 7.5, containing 1 mM dithiothreitol, 1 mM E G T A and 0.1 mM phenyl-methanesulfonyl fluoride, in a teflonglass homogenizer. The homogenates were centrifuged at 100,000 g for 1 h. The resultant supernatants were dialyzed overnight against PBS containing 0.1 mM PMSF. The samples were stored at -70 °C until use.
Other procedures Calmodulin-Sepharose 4B affinity column was prepared by the method of Klee and Krinks II. Protein was determined by the method of Bradford 1 with bovine serum albumin as standard. RESULTS
Regional distribution Immunoreactivity was observed in many neurons but was not detected in glial cells such as astrocytes, oligodendrocytes, ependymal cells and others, under the experimental conditions with the PAP method. In neurons, calcineurin was detected in somata, dendrites and axons. The caudatoputamen (Fig. 1A), hippocampal formation (Fig. 2A) and substantia nigra (Fig. 2A, B) showed strong immunoreactivity, while the cerebral neocortex (Fig. 1A), cerebellum (Fig. 3A), lateral septum and globus pallidus (Fig. 1E) were moderately stained. In the caudatoputamen (Fig. 1E, F), most neurons showed intense immunostaining in somata and neurites. Some neurons showed immunoreactivity in the nuclei. The dorsolateral part of this region had the strongest immunoreactivity. Bundles of white matter were slightly immunostained and this staining was thought to be due to that of myelinated axons as revealed by electron microscopic immunohistochemical examination (data not shown). The globus pallidus was moderately stained (Fig. 1E). However, in this region, neuronal somata did not show immunoreactivity and only neurites were stained, which were considered to be the axons originating from the caudatoputamen. In the neocortex (Fig. 1B), the most prominently stained neurons were observed in layers III (Fig. 1C), V and VI (Fig. 1D). Both pyramidal and nonpyramidal cells were strongly immunostained. Large cells were stained more strongly than small cells. The olfactory tubercle and bulb were weakly immunoreactive. The rostral hippocampal formation was strongly immunoreactive (Fig. 2C), and especially the stratum pyramidale (Fig. 2E), radiatum and lacunosum moleculare. The dentate gyms showed moderate immunostaining (Fig. 2C). Immunoreactivity was weakly shown in the amygdaloid complex and geniculate body. Most of the thalamic nuclei and hypothal-
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165 neurons did not show any immunoreactivity either. In the cerebellum (Fig. 3A, B), 3 layers i.e. the molecular, Purkinje and granular layers were moderately stained. Each of the granular cells showed immunoreactivity in various degrees. Purkinje cells are divided into two types. One is a strongly stained type and the other is not stained. In general, immunoreacrive Purkinje cells were of a small population. The entire myelencephalon did not show very strong immunoreactivity, and the cerebellar peduncle and pyramidal tract were weakly immunoreactive.
Cellular and electron microscopic distribution
Fig. 1. Light micrographs of coronal sections through a hemisphere of the rat brain at the level of the caudatoputamen. A: strong immunoreactivity is seen in the caudatoputamen, and moderate in the neocortex and lateral septum (x6). B: layers I-VI of the premotor neocortex (x25). Immunoreactivity is relatively strong in layers III, V and VI. Layer III (C) and V (D) are shown (x150). Strong immunoreactivity is present in the pyramidal neurons. The regions of the cytoplasm and neurites (arrows) are the most intensive. Some of the neurons show no immunoreactivity (arrowheads). E: the caudatoputamen (CP)-globus pallidus (GP) (x 40). The globus pallidus is slightly stained, but neuronal bodies are not stained. F: caudate nucleus (x 100). Immunostaining of the cytoplasm of cells is observed. A few neurons show immunoreactivity in the nuclei (curved arrows). In contrast, some of the neurons do not show immunostaining (arrowheads). NC, neocortex; CC, corpus callosum; LS, lateral septum; MS, medial septum; AC, anterior commissure; LOT, lateral olfactory tract.
amus contained neurons which were relatively less immunoreactive than those in other areas. The pars reticulata of the substantia nigra was strongly stained, but neuronal bodies in the substantia nigra as well as the globus pallidus (Fig. 2D) did not show any immunoreactivity. It has been known that this area receives the axons originating from the caudatoputamen. It is considered that the stained structure may be nerve terminals of the axons originating from the caudatoputamen. The pars compacta and pars lateralis of the substantia nigra, containing dopaminergic neurons, did not show any immunoreactivity. The raphe nucleus containing serotoninergic neurons, and locus coeruleus containing adrenergic
The findings by microscopy were as follows. Immunoreactivity was very strong in neuronal somata and dendrites. Axons and nerve terminals also showed strong immunostaining. Weak staining of axons was observed along with fibe.r tracts in the corpus callosum, internal capsule and pyramidal tract of the spinal cord. Some of the neurons appeared to contain immunoreactive products in their nuclei. Under electron microscopy, immunoreactivity was observed in the cytoplasm, on the outer surface of mitochondria, and on microtubules (Fig. 4A, B). The nucleus did not show any immunoreactivity. Dendrites and nerve terminals (Fig. 4 C - F ) were strongly stained. Additionally, postsynaptic densities (PSDs) were strongly stained (Fig. 4C, D).
Cerebral hemitransection Fig. 5 shows calcineurin staining of the transverse section at the midbrain level in the left-side-transected rat brain. It is clear that the staining of the pars reticulata in the substantia nigra on the ipsilateral side almost completely disappeared, compared with the contralateral side. NSE staining of the same section, as one of the control studies, revealed no difference between the left and right sides (data not shown). In the same hemitransected brain, intensity and distribution of calcineurin staining in the caudatoputamen on the ipsilateral side were not remarkably changed (data not shown).
N- methyl- 4-phenyl- 1,2, 3, 6 - tetrahydropyridine (NMPTP) treatment The brains of NMPTP-treated rats were examined by immunohistochemistry. The intensity and distribution of calcineurin staining in the substantia nigra
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Fig. 3. Light micrograph of a sagittal section of the rat cerebellar hemisphere (A, ×40; B, × 160). The molecular layer (ML) and granular layer (GL) are moderately stained. Some of the Purkinje cells are strongly stained (black arrows) and others show no immunoreactivity (white arrows). The granular cells are also stained.
were the same as those of n o n - t r e a t e d rats. The caud a t o p u t a m e n also showed similar immunoreactivity to that of n o n - t r e a t e d rat brain ( d a t a not shown).
Measurement of calcineurin in various rat brain regions Fig. 6 shows a s t a n d a r d curve for the enzyme-immunoassay ( E I A ) for calcineurin. The sensitivity of the E I A is 1 ng (13 fmol) of calcineurin, and is comparable to that of the r a d i o i m m u n o a s s a y ( R I A ) rep o r t e d previously 26. Using the E I A , the level of calcineurin in a variety of rat brain regions was m e a s u r e d (Table I). Calci-
neurin was d e t e c t e d in all regions of the brain examined. The level of calcineurin varied from region to region. The caudate nucleus, p u t a m e n and hippocampus showed the high level, while the globus pallidus, cerebral gray m a t t e r , substantia nigra and cerebellum were relatively lower. The results were generally consistent with the findings o b t a i n e d by immunohistochemistry described above. DISCUSSION Calcineurin immunoreactivity was present only in neurons throughout the brain, but m a r k e d regional
Fig. 2. Light micrographs of coronal sections through a hemisphere of the rat brain at the level of the substantia nigra (A) and raphe nucleus (B) (x 6). In A and B, the hippocampus, substantia nigra and neocortex show strong immunoreactivity, whilst the dentate gyrus and geniculate body show weak immunostaining. The raphe nucleus shows no immunoreactivity. CLF, central longitudinal fissure; NC, neocortex; SCC, superior collicular commissure; FLD, fasciculus longitudinalis dorsalis; FLM, fasciculus longitudinalis medialis; CA, cerebral aqueduct; GD, geniculate body; DG, dentate gyrus; HI, hippocampus; RN, red nucleus; RS, rhinal sulcus; EC, entorhinal cortex; ML, medial lemniscus; SN, substantia nigra; CC, crus cerebri; R, raphe. C: the rostral hippocampal formation (x 25). Immunoreactivity is strong in the stratum oriens (so), stratum pyramidale (sp), stratum radiatum (sr) and stratum lacunosum moleculare (slm). ec, ectal limb of dentate gyrus; en, endal limb of dentate gyrus. D: pars reticulata of the substantia nigra (x200). Neuronal bodies are not stained (white arrows). E: stratum pyramidale of the hippocampal formation shows immunoreactivity in the cytoplasm ( × 140).
168
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Fig. 5. Light micrograph of coronal section through the cerebral hemisphere of the hemitransected rat brain at the level of the substantia nigra. Immunostaining of the substantia nigra on the ipsilateral side (left side) is not observed in comparison to that on the contralateral side. Rt, right side; Lt, left side; SN, substantia nigra (x 10).
variation in the intensity of immunoreactivity was observed. The c a u d a t o p u t a m e n and h i p p o c a m p a l formation were m a r k e d l y stained. The results were supp o r t e d by those of E I A . The substantia nigra was also strongly stained. It was considered to be staining of the nerve terminals originating from the caudatoputamen. The reasons are as follows. (1) Only neurites were stained, but n e u r o n a l s o m a t a in the substantia nigra were not (Fig. 2D). (2) The immunoreactivity of the neurites d i s a p p e a r e d when the striatonigral
pathway had previously been cut off by cerebral hemitransection (Fig. 5). (3) In this region, immunoreactivity was observed in axons u n d e r the electron microscopic immunohistochemistry (data not shown). If our assumption is right, the immunostaining in the globus pallidus m a y also be due to the staining of the nerve terminals of the striatopallidal pathway. In view of the regional distribution, calcineurin may play an i m p o r t a n t role in the functions of extra-
Fig. 4. Electron micrographs of ultrathin sections through the caudate nucleus without uranyl counterstaining. A: immunoreactivity in the cytoplasm (Cy) of a neuron (x6500). PSD showed immunoreactivity (between arrowheads). The nucleus (Nu) showed faint immunoreactivity. B: an immunoreactive dendrite (D) and PSD (arrow) are shown (x 13,000). The curved arrow points to lightly labeled PSD. Immunoreactive microtubules are also shown (long arrows). C: immunoreactive dendrites (D) and PSDs (arrows) (x7800). D: immunoreactive PSDs (between arrowheads) are shown (x32,500). The curved arrow points to lightly labeled PSD in comparison with the labeled PSD as shown in B. E: immunoreactive nerve terminals are shown (x26,000). F: an axodendritic synapse is shown. Strong immunoreactivity is observed on the synaptic vesicles and PSDs (x 26,000).
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Fig. 6. Standard curve for the assay of calcineurin by enzymeimmunoassay. Various amounts of purified calcineurin indicated were adsorbed on the nitrocellulose membrane, followed by immunostaining with antiserum against calcineurin as a primary antibody. Immunostaining intensity of each spot was analyzed by the TLC scanner with a computer. pyramidal system, as Wallace et ai. previously suggested 26, and hippocampal formation. Calcineurin has been reported to dephosphorylate D A R P P - 3 2 (a dopamine-regu|ated neuronal phosphoprotein) as substrate 9. Calcineurin may be related to the function of the dopaminoceptive neurons. Calcineurin was present in the axons of the striatonigral pathway. This pathway contains the G A B A e r TABLE I Level of calcineurin in various rat brain regions as determined by enzyme-immunoassay
An appropriate volume (50-200 ~1) of 100,000 g supernatant fluid from each brain region was assayed for calcineurin. The data represent the mean _+S.D. on regions from two rats. Each determination was duplicated. Regions
Cerebrum Neocortical gray matter White matter Caudate nucleus Putamen Globus pallidus Hippocampus Thalamus Hypothalamus Substantia nigra Olfactory bulb Cerebellum Pons Medulla oblongata Spinal cord
Calcineurin (l~g/mg of protein)
0.54 _+0.05 0.23 + 0.03 1.73 _+0.42 1.65 _+0.50 0.71 ___0.05 1.45 _+0.39 0.26 _+0.04 0.14 _+0.04 0.50 + 0.11 0.19 _+0.05 0.42 + 0.11 0.25 _+0.04 0.24 + 0.05 0.19 + 0.02
gic and substance P-ergic neurons 2. It is under investigation that calcineurin exists in G A B A - and/or substance P-ergic neurons. These finding may be of interest, since the relationships between the striatum and substantia nigra, and the striatum and globus pallidus are elucidated. Calcineurin may not be present in the adrenergic and serotonergic neurons, since immunostaining was not observed in the neurons of the locus coeruleus and raphe nucleus. W o o d et al. reported that CaM-BPs0 (probably identical with calcineurin) was associated only with neuronal elements, such as PSDs and dendritic microtubules, primarily at postsynaptic sites within neuronal somata and dendrites 3°. We used HRP-conjugated Fab' of antiserum to calcineurin in the investigation by electron microscopic immunohistochemistry. This direct method has proved to be the most sensitive method at present ~6. The findings by the method indicated that calcineurin immunoreactivity was present in dendrites including PSDs, somata, spines, axons and nerve terminals (synaptic vesicles). Calcineurin may be involved in a variety of Ca 2+ and calmodulin-dependent physiological processes via dephosphorylation of various substrate proteins. We have reported that calcineurin can dephosphorylate the microtubule proteins phosphorylated by Ca 2+ and calmodulin-dependent protein kinase with a high molecular weight 6. Immunostaining of calcineurin is observed on the microtubules (the present study) and Ca 2+- and calmodulin-dependent protein kinase is associated with the microtubule fraction 3, indicating that the microtubules are regulated by both enzymes through phosphorylation and dephosphorylation. On the other hand, both enzymes can be activated by calmodulin. It is a very interesting problem which regulation system is more activated in which either phosphorylation or dephosphorylation may occur. The immunohistochemical findings of calcineurin (the present study) and calcium/caimodulin-dependent protein kinase II TM showed that there was no prominent difference in the regional distribution of both enzymes in the brain. We developed an enzyme-immunoassay for calcineurin. It was a useful method for measurement of calcineurin content as compared to radioimmunoassay, in view of its sensitivity, safety, simplicity and rapidity. The results were generally consistent with the findings obtained by immunohistochemistry de-
171 scribed above. Exceptionally, the substantia nigra and globus pallidus had lower levels of calcineurin in comparison to the intensities of the immunostaining. The difference m a y be due to that of the localization of calcineurin in subcellular fractions, since the E I A was p e r f o r m e d for the 100,000 g supernatant. Calcineurin m a y occur in the particulate fractions m o r e than in the s u p e r n a t a n t in these regions. The findings by electron microscopic immunohistochemistry also revealed that calcineurin is present in the particulate fractions such as the nerve terminals and PSDs (Fig. 4). In addition, using E I A , we m e a s u r e d the levels of calcineurin content in o t h e r various rat tissues, such as the pituitary gland, pancreas, skeletal muscle, spleen, thyroid gland, testis, lung, heart and liver. H o w e v e r , these tissues showed significantly low values of calcineurin content (data not shown), as consistent with a previous r e p o r t using r a d i o i m m u noassay 26. Recently, Stewart et al. r e p o r t e d that a Ca 2+ and c a l m o d u l i n - d e p e n d e n t protein phospha-
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