calmodulin-dependent protein kinase phosphatase in the rat central nervous system

Molecular Brain Research 77 (2000) 76–94 www.elsevier.com / locate / bres

Research report

Immunohistochemical study of the distribution of Ca 21 / calmodulindependent protein kinase phosphatase in the rat central nervous system a, b b a a Yasuhisa Nakamura *, Takako Kitani , Sachiko Okuno , Kazuyoshi Otake , Fumi Sato , b Hitoshi Fujisawa a

Section of Neuroanatomy, Graduate School of Medical and Dental Research, Tokyo Medical and Dental University, Tokyo 113 -8519, Japan b Department of Biochemistry, Asahikawa Medical College, Asahikawa 078 -8510, Japan Accepted 7 February 2000

Abstract Distribution of Ca 21 / calmodulin-dependent protein kinase phosphatase (CaM-K Pase) which dephosphorylate multifunctional Ca 21 / calmodulin-dependent protein kinases (CaM-kinases) in the rat brain and spinal cord were examined immunohistochemically by using an antibody against this enzyme. CaM-K Pase was localized only in the cytoplasm as has been investigated in PC 12 cells, and was never observed in the nucleus. Immunostainability varied from cell group to cell group. Mitral cells in the olfactory bulb, pyramidal neurons in the fifth layer of the cerebral cortex, hippocampal and striatal interneurons, dorsal and ventral pallidal, entopeduncular, and the reticular part of the substantia nigra neurons were intensely immunolabeled. Motoneurons in all the cranial nerve nuclei and the anterior horn of the spinal cord also revealed intense immunolabeling. On the contrary, pyramidal neurons in the Ammon’s horn of the hippocampal formation, granule cells in the olfactory bulb, dentate gyrus and cerebellar cortex, Purkinje cells, neurons in the medial habenular nucleus and the inferior olivary nucleus have not shown immunoreactivity. Axons in the white matter or nerve root of the cranial nerve nuclei were immunolabeled. Glial cells in the white matter also showed immunostaining. Because the substrate of CaM-K Pase is multifunctional CaM-kinase II, I and IV, localization of each CaM-kinase was compared with that of CaM-K Pase. The distribution of CaM-K Pase and these CaM-kinases was found to overlap in various regions in the brain and spinal cord. It was concluded, therefore, that CaM-K Pase could regulate the activity of these CaM-kinases by dephosphorylation, when they existed together in neurons.  2000 Elsevier Science B.V. All rights reserved. Themes: Neurotransmitters, modulators, transporters, and receptors Topics: Second messengers and phosphorylation Keywords: CaM-kinase phosphatase; CaM-kinases; Protein phosphatase; Post synaptic density; Striatal cholinergic neuron; Immunohistochemistry

1. Introduction Ca 21 / calmodulin-dependent protein kinases (CaM-kinases) I, II, and IV play important roles as Ca 21 -responsive multifunctional protein kinases, controlling a variety of cellular functions in response to an increase in intracellular Ca 21 . The regulation of their activities, therefore, is quite important in controlling the Ca 21 -signaling system. These protein kinases themselves are regulated by reversible phosphorylation; for example, CaM-kinase II is activated *Corresponding author. Tel.: 181-3-5803-5149; fax: 181-3-58035151. E-mail address: [email protected] (Y. Nakamura)

upon autophosphorylation [9,11,14,17] and CaM-kinase I and IV are activated upon phosphorylation by CaM-kinase kinase [18,21,31,32,47]. On the other hand, CaM-kinases are substrates for protein phosphatases; protein phosphatase 1, 2A and 2C (PP1, PP2A and PP2C) [1,2,5,20,50]. Among them, PP2A which is present in neurons appears to be the major kinase phosphatase that downregulates activated protein kinases [20]. Recently a novel protein phosphatase was found to deactivate CaM-kinases [10,12,16]. These observations suggest that the novel protein phosphatase (CaM-kinase phosphatase; CaM-K Pase) also plays important roles in regulating the activities of the three multifunctional CaM-kinases which are distributed abundantly in the brain and spinal cord. Although

0169-328X / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 00 )00044-9

Y. Nakamura et al. / Molecular Brain Research 77 (2000) 76 – 94

only a few of their relations to brain functions have been reported, CaM-K Pase might be a key enzyme in controlling, at least, part of these brain functions. The distribution of these CaM-kinases has been reported already, and each of them has its characteristic feature [4,7,13,22,30,33,38]; CaM-kinase I and one of the isoforms of CaM-kinase II have been observed to be located only in the cytoplasm, whereas CaM-kinase IV predominantly in the neuronal nucleus; CaM-kinase I was observed almost all areas in the brain, whereas CaM-kinase II and IV were found abundantly in the forebrain. We tried to determine the intraneuronal distribution of CaM-K Pase, and to localize it in the rat central nervous system by means of immunohistochemistry by using newly developed antibody against this enzyme. In addition, we also carried out immunohistochemistry for choline acetyl transferase (ChAT), tyrosine hydroxylase (TH) and glial fibrillary acidic protein (GFAP) on the caudate-putamen, substantia nigra and cerebellar cortex, respectively, where characteristic immunolabeling was observed for CaM-K Pase.

2. Materials and methods Molecular cloning and sequencing of CaM-K Pase was made [16]. Thereafter, rabbit antibody against CaM-K Pase was prepared by immunization of a peptide corresponding to the carboxyl-terminal 19 amino acids of CaM-K Pase and a cysteinyl residue added to amino terminus for coupling to a carrier protein, keyhole limpet hemocyanin [16]. Western blot analysis was carried out as described elsewhere [16]; in brief, approximately 0.2 mg protein of the crude extract of E. coli cells transformed with pETCaM-K Pase, 25 mg protein of the crude extracts of rat cerebral cortex, brainstem and cerebellum were subjected with approximately 1.7 mg / ml antibody against CaM-K Pase. Six rats (male, Wistar, Nippon Bio-Supp. Center, Tokyo, Japan, 7 weeks) were used in the immunohistochemical study. The rats, under deep pentobarbital anesthesia (50 mg / kg, i.p.) were perfused through the ascending aorta with phosphate buffered 3.8% paraformaldehyde solution with or without 0.1% glutaraldehyde. After removing the brains, they were cut serially at 40 mm into frontal or sagittal sections on a freezing microtome for light microscopy after soaking in 30% phosphate buffered sucrose for 2 days. For electron microscopy, brains were cut at 100– 150 mm with a microslicer immediately after perfusion. Procedures for immunohistochemistry for light and electron microscopy were as reported previously [22,24]. Briefly, free floating sections in 0.1 M phosphate buffer containing 0.1% Triton X-100 were incubated overnight in a refrigerator with purified antibody (1:1000). Then the sections were incubated with biotinylated goat anti-rabbit immunoglobulin G (IgG) (Vector Lab.) and then reacted with Vectastain ABC kit (Elite, Vector Lab.). To detect

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peroxidase, 0.005% 3,39-diaminobenzidine (DAB) was used with 0.003% H 2 O 2 , 0.005% nickel chloride and cobalt acetate in the phosphate buffer. For control, some sections were incubated without antibody against CaM-K Pase overnight, and then they were reacted together with the rest of sections. Brain structures were identified according to the rat stereotaxic atlas by Paxinos and Watson [36]. For light microscopy, the sections were mounted on gelatinized glass slides; some of them were counterstained with 0.5% Cresyl violet. For electron microscopy, target nuclei for the observation were trimmed from the immunostained sections, and embedded in Epon after osmication, dehydration, and block-stain with 2% uranyl acetate in absolute ethanol. Ultrathin sections were examined under a Hitachi H-7100 electron microscope with or without uranium-lead staining. In addition to single immunohistochemical staining for CaM-K Pase, we performed double immunohistochemical staining for CaM-K Pase and ChAT, TH and GFAP. At first, frozen sections selected from the forebrain area were incubated with a mixture of antibodies against CaM-K Pase (1:250) and ChAT (anti-mouse; Chemicon International, INC; 1:200) overnight, then after washing, they were incubated with biotinylated goat anti-mouse IgG (Vector Lab.) for 2 h. After washing, these sections were incubated with a mixture of avidin-D conjugated to TRITC (Vector Lab.) and AMCA conjugated to goat anti-rabbit IgG (Vector Lab.) for 2 h. For double immunohistochemical staining for CaM-K Pase and TH, frozen sections selected from the midbrain region were incubated with a mixture of antibodies against CaM-K Pase (1:500) and TH (anti-mouse; Boehringer Mannheim; 1:5000) overnight, then after washing, they were incubated with biotinylated goat anti-rabbit IgG (Vector Lab.) for 2 h. After washing, these sections were incubated with a mixture of avidin-D conjugated to TRITC (Vector Lab.) and FITC conjugated to donkey anti-mouse IgG (Jackson) for 2 h. The procedure for double immunohistochemical staining for CaM-K Pase and GFAP was the same as that for CaM-K Pase and ChAT, except that antibody against ChAT was replaced with that against GFAP (anti-mouse; Innogenetics; 1:50). These sections were observed under a fluorescence microscope.

3. Results The tissue distribution of CaM-K Pase was first examined by Western blot analysis (Fig. 1). Significant immunoreactivity was detected at the position corresponding to CaM-K Pase in all the examined brain regions, the cerebrum, brainstem and cerebellum. In the next, we examined the immunohistochemical reaction pattern on sections of the rat brain and spinal cord with an antibody against CaM-K Pase. A paramedian sagittal section showed that immunolabeling appears throughout the brain

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3.1. Telencephalon

Fig. 1. Tissue distribution of CaM-K Pase on Western blot analysis. Crude extract of E. coli cells transformed with pETCaM-K Pase (lane 1), crude extracts of cerebral cortex (lane 2), brainstem (lane 3) and cerebellum (lane 4) were subjected.

(Fig. 2a). Control sections which were reacted without primary antibody, and reacted with primary antibody adsorbed by excess amount of a synthetic peptide corresponding to the carboxyl terminal 19 amino acids of CaM-K Pase did not show immunostaining in the central nervous system (Fig. 2b). In this study, immunolabeling was observed only in the cytoplasm, and neuronal nuclei were never stained. The intensity of staining, however, were not uniform and differed from cell group to cell group. The relative intensity of immunohistochemical staining for the antibody against CaM-K Pase in the central nervous system is summarized in Table 1. In the results section, only the degree of immunolabeling of the cell body was described, unless dendrites or neuropile were labeled.

In the main olfactory bulb the cytoplasm of most mitral cells was stained densely, and immunopositive fine strands were observed in the external plexiform layer, which might represent the mitral cell dendrites (Fig. 3). In the granular layer, although granule cells were not immunolabeled, a few neurons were stained weakly in a rather light background. In the glomerular layer, fine fibers surrounding the glomeruli were well stained (Fig. 3). Mitral cells and granule cells in the accessory olfactory bulb showed the same appearance as those in the main olfactory bulb. In the anterior olfactory nucleus, most neurons were labeled weakly, some of them, however,were stained denser than the others not only in the perikarya but also in the primary dendrites. In the cerebral cortex, almost all neurons from the layer I to layer VI in the neocortex were immunolabeled. Among them, layer III and layer V were prominently observed by their dense stainability (Fig. 4). Most of densely stained neurons were pyramidal in shape, but other types such as stellate or fusiform cells were also observed. These large pyramidal neurons showed cytoplasmic staining in their perikarya and apical dendrites which were seen as radial parallel arrays (Fig. 4). In the hippocampal formation, although pyramidal neurons in the subiculum and Ammon’s horn, and granule cells in the dentate gyrus were not immunoreacted, some neurons in these areas showed moderate staining in their perikarya and dendrites; their shape was not pyramidal but variable; round, fusiform or oval (Fig. 5). In the subcortical telencephalic nuclei, such as the caudate-putamen, amygdaloid body and septal nuclei, immunoreactivity did not appear ubiquitously. In the caudate-putamen, only a small number of neurons were moderately stained (Fig. 6). They were distributed throughout the caudate-putamen. The shape of neurons were slender, fusiform, ovoid or triangular, and a few proximal or secondary dendrites of non-spinous type were also stained (Fig. 6b). The longer diameter of these somata

Fig. 2. Parasagittal sections of the rat brain show immunohistochemical reaction with an antibody against CaM-K Pase (a) and with the antibody after adsorbed by a synthetic peptide (b). Counterstaining was not done on immunoreacted histological sections of any of the light micrographs. (CblCx, cerebellar cortex; CbrCx, cerebral cortex; CPu, caudate-putamen; dTh, dorsal thalamus; Hip, hippocampal formation; Hyp, hypothalamus; IC, inferior colliculus; OB, olfactory bulb; Pn, pontine nuclei) Scale bar50.5 cm.

Y. Nakamura et al. / Molecular Brain Research 77 (2000) 76 – 94 Table 1. Continued

Table 1 Regions of the CNS examined Olfactory system Main olfactory bulb Mitral cells Granule cells Glomerular layer Accessory olfactory bulb Mitral cells Granule cells Anterior olfactory nucleus Olfactory tubercle Pyramidal cells Islands of Calleja

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Relative intensity of reactivity

111 2 11 111 2 1 2 6

Neocortex Frontal cortex Parietal cortex Temporal cortex Occipital cortex

III, V, III, V, III, V, III, V,

Limbic cortex Piriform cortex Cingulate cortex Retrosplenial cortex Prelimbic cortex Subicular cortex Dentate gyrus Ammons horn

6 V, 11; II, III, IV, VI, 1|11 V, 111; II, III, IV, VI, 1|11 1 111, 6|1 a 6|1 111, 6|1 a

Septum Lateral septal nucleus Medial septal nucleus Nuclei of diagonal band of Broca Bed nucleus of the stria terminalis

111, 6|1 a 111 1111 111

111; 111; 111; 111;

Preoptic area Medial preoptic nucleus Lateral preoptic nucleus

111 111

Amygdala Anterior amygdaloid nucleus Cortical amygdaloid nucleus Medial amygdaloid nucleus Lateral amygdaloid nucleus Basolateral amygdaloid nucleus Basomedial amygdaloid nucleus Central amygdaloid nucleus

111 6 11 11 11 11 11

Claustrum

1

Basal ganglia Caudate-putamen Nucleus accumbens Globus pallidus Ventral pallidum Entopeduncular nucleus Subthalamic nucleus Substantia nigra pars reticulata pars compacta Ventral tegmental area

11, 2 b 11, 2 b 1111 1111 1111 111 1111 6 11

Thalamic reticular nucleus

111

Zona incerta

11

II, II, II, II,

IV, VI, IV, VI, IV, VI, IV, VI,

1 1 1 1

Hypothalamus Anterior hypothalamic area Lateral hypothalamic area Suprachiasmatic nucleus Supraoptic nucleus Paraventricular nucleus Arcuate nucleus Ventromedial nucleus Dorsomedial nucleus Premammillary nucleus Medial mammilary nucleus Lateral mammillary nucleus Posterior hypothalamic area

11 111 11 111 111 11 11 11 11 11 11 11

Habenula Medial habenular nucleus Lateral habenular nucleus

2 11

Dorsal thalamus Anterodorsal nucleus Anteroventral nucleus Anteromedial nucleus Mediodorsal nucleus Ventral anterior and Ventral lateral complex Ventroposteiror nuclei Ventral medial nucleus Laterodorsal nucleus Midline nuclei Intralaminar nuclei Parafascicular nucleus Posterior thalamic nuclei Lateral geniculate nucleus Medial geniculate nucleus Lower brainstem Red nucleus Interstitial nucleus of Cajal Darkschewitsch nucleus Superior colliculus Superficial gray layer Deep gray layers Inferior colliculus External nucleus Central nucleus Pedunculopontine tegmental nucleus Medial parabrachial nucleus Lateral parabrachial nucleus Interpeduncular nucleus Locus coeruleus Trigeminal nuclei Mesencephalic nucleus Motor nucleus Principal sensory nucleus Spinal nucleus Superior olivary complex Trapezoid nucleus Pontine reticular formation Pontine tegmental reticular nucleus Pontine nuclei Central gray Raphe nuclei Medullary reticular formation Lateral reticular nucleus Vestibular nuclei

11 11 1 11 11 11 11 11 11 11 11 11 11 11

111 11 111 1|11 1, 111 c 11 111 11 11 11 1 111 11 111 11 11 111 111 1 1 111 11 11 11 11 11

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Table 1. Continued Cochlear nuclei Dorsal vagal nucleus Inferior olivary complex Prepositus hypoglossi nucleus External cuneate nucleus Dorsal column nuclei Nucleus of the solitary tract Motor nuclei of the cranial nerves

11 11 2 111 11 11 1 111

Cerebellum Molecular layer Purkinje cells Granular layer Granule cell Golgi cell Cerebellar nuclei

2 111 111

Spinal cord Layer I of the posterior horn

111

Other layers of the posterior horn Reticular nucleus Intermediate zone Anterior horn

1 2

11 11 11 111

a

Some neurons show 111 and most neurons show 6 or 1. Some neurons show 11 and medium spiny neurons show 2. c Some neurons show 111 and others show 1. b

Fig. 4. Neurons in the frontal cortex show dense immunoreaction in the layer V where apical dendrites of pyramidal neurons are clearly seen. Some neurons in the layers III and VI are also densely immunolabeled. Scale bar5100 mm.

Fig. 3. The main olfactory bulb shows dense immunoreaction in the olfactory glomerulus (Gl), external plexiform layer (EPl) and the mitral cell layer (Mi). In the granular layer, weak immunoreactivity is shown in some neurons (arrows). (GrO, olfactory granular layer) Scale bar5100 mm.

was often between 20 and 30 mm, which was somewhat larger than the so-called medium spiny neurons (Fig. 6b). They resembled neurons stained with an antibody against ChAT in size and shape. A double immunolabeling study showed that most CaM-K Pase positive neurons were labeled with the ChAT antibody, and most ChAT positive neurons were immunostained with CaM-K Pase antibody (Fig. 6c,d). In the nucleus accumbens, the appearance of stainability was the same as in the caudate-putamen. In the ventral pallidum, which extended from the rostral part of the olfactory tubercle level to the optic chiasm level, numerous neurons of pyramidal, fusiform or triangular shape were also stained densely in the somata and dendrites (Figs. 6a and 7). Neurons in the globus pallidus (homologous to the primate external segment of the globus pallidus) and entopeduncular nucleus (the internal segment of the primate globus pallidus) were very densely immunoreacted both in the somata and dendrites some of which were traced more than 100 mm (Figs. 6a and 8). In addition, small round particles like axon terminals were also stained among the labeled somata and dendrites. Electron microscopic observations confirmed that these densely labeled particles were cut ends of small caliber dendrites (Fig. 9). In the lateral septal nuclei, most neurons were stained moderately, not only somata but also proxim-

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Fig. 5. A frontal section of the dorsal hippocampal formation. Granule cells in the dentate gyrus (GrD) and pyramidal cells in the Ammon’s horn (PyH) are not immunolabeled. Labeled neurons in these layers might be interneurons. (CA1 and CA3, CA1 and CA3 sectors of the hippocampal formation; DG, dentate gyrus) Scale bar5500 mm.

al dendrites, whereas in the medial septal nuclei immunoreactivity was much denser. In the amygdaloid body, neurons in the anterior nucleus were most densely stained, and those in the central nucleus were moderately or densely stained, and those in the basolateral and basomedial nuclei were weakly or moderately stained. Those in the cortical nucleus were very weakly stained. In the claustrum a few neurons were weakly labeled.

3.2. Diencephalon In the dorsal thalamus, including the anterior, medial, ventral, lateral, midline, intralaminar, lateral geniculate and medial geniculate nuclei and posterior group, labeling was, on the whole, observed moderately only in the cytoplasm of neuronal somata (Fig. 10). In the hypothalamus, neurons of the suprachiasmatic, paraventricular and mammillary nuclei were somewhat densely labeled in the neuronal somata. In the lateral hypothalamic area, although the neuronal labeling was moderate, primary dendrites were well observed extending more than 50 mm. In the habenular nuclei, most neurons in the medial nuclei were not immunostained, but neurons in the lateral nucleus were moderately labeled (Fig. 10). Neurons in the thalamic reticular nucleus were moderately to densely labeled in both neuronal somata and the initial part of dendrites (Figs. 8 and 10). In the subthalamic nucleus, neuronal somata was stained more densely than in the thalamic reticular nucleus.

3.3. Mesencephalon Neurons in the superior colliculus showed weak to moderate labeling in the superficial and deep layers, and many neurons in the lateral half of the intermediate gray layer were labeled more densely in their somata and primary dendrites (Fig. 11). In the inferior colliculus, although variety of labeling was observed in the central nucleus, larger neurons, in general, appeared denser (Fig. 12). In the external nucleus, neurons were weakly stained. Most neurons in the central gray matter were moderately labeled (Figs. 11 and 12). In the Darkschewitsch nucleus neurons were densely stained in the somata and proximal dendrites. In the tegmentum, magnocellular red nucleus neurons were moderately to densely labeled in their somata and dendrites, some of which could be traced more than 50 mm (Fig. 13). Most of the substantia nigra pars reticulata neurons were densely stained (Fig. 14). Dendrites seemed to form a network, and sometimes well stained dendrites could be traced about 100 mm. On the other hand, most substantia nigra pars compacta neurons were not stained. Double immunolabeling for CaM-K Pase and TH showed that a few neurons were doubly labeled for both antibodies. Neurons in the ventral tegmental area (VTA) were moderately stained only in their somata. The TH-immunopositive neurons in the VTA were not doubly labeled by the antibody against CaM-K Pase. At the pontomesencephalic junction, the pedunculopontine tegmental nucleus neurons were moderately to densely stained. Observations about motoneurons will be described later.

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Fig. 6. (a) Immunolabeled cells (see b) are dispersed in the rostral part of the caudate-putamen (CPu). In the globus pallidus (GP) and the ventral pallidum (VP), neurons are densely labeled. On the right side of the figure, the lateral ventricular wall is densely stained. (b) An enlarged photomicrograph shows labeled neurons in the caudate-putamen, some of which, indicated with arrows, have non-spinous slender processes. (c, d) Epifluorescence photomicrographs showing CAM-K Pase (c) and ChAT (d) immunoreactive cells observed in the same field in the caudate-putamen. Dually labeled cells are indicated with arrowheads. (ac, anterior commissure) Scale bars in a and b5100 mm, c and d520 mm.

3.4. Pons and cerebellum In the pons, neurons in the pontine nuclei were stained densely (Fig. 15). Pontine tegmental reticular nucleus neurons were moderately stained. Although neurons in the medial and lateral parabrachial nuclei were stained moderately to densely, background labeling in this region was denser than neuronal labeling (Fig. 16). In the locus coeruleus, most neurons were densely stained only in their somata (Fig. 16). As to the trigeminal nuclei, large neurons

of the mesencephalic trigeminal nucleus showed heavy staining in the cytoplasm (Fig. 16). In the main sensory nucleus, neurons were moderately to densely labeled. Most neurons in the superior olivary nuclear complex and the nucleus of trapezoid body were labeled moderately to densely both in the somata and proximal dendrites. Immunostaining of the cerebellar cortex appeared rather dense (Fig. 2a). In the molecular layer, immunolabeled compact parallel arrays of processes from the Purkinje cell layer to the pial surface were observed in the sagittal or

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Fig. 7. Neurons in the ventral pallidum (VP) show dense labeling of both somata and dendrites. At the upper right part of the figure, the third ventricular wall is densely stained. (Tu, olfactory tubercle) Scale bar5100 mm.

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oblique sections. Among these strands weakly labeled cell bodies were scattered, which are known as stellate cells. Moderate staining was observed in the Purkinje cell layer as if it surrounded Purkinje cells (Fig. 17). The diameter of this labeled contour was from 20 to 30 mm. It seemed that these labeled processes continued to labeled strands in the molecular layer. Electron microscopy proved that the labeling was not in the Purkinje cell bodies but out side of them. In these labeled processes, no glial filaments were observed, and around these labeled processes no accurate synaptic sites were found either (Fig. 18). Double immunolabeling for CaM-K Pase and GFAP in the cerebellar cortex showed that GFAP positive filaments were clearly extended from the Purkinje cell layer to the pial surface, and none of them were overlapped with CaM-K Pase positive figures which were not stained filamentous but diffuse in the molecular layer. In the granular layer, moderate to dense labeling of non-granule cells was seen (Fig. 17). Judging from their diameters ranging between 10 and 15 mm, and their ubiquitous distribution in this layer, they might be Golgi neurons. Electron microscopic observations revealed the different features of nuclear profiles and larger cytoplasmic areas of these labeled Golgi neurons from those of granule cells. Many labeled nerve fibers were seen in the cortical white matter. In the cerebellar medial, interpositus and lateral nuclei, neurons were labeled densely not only in the cytoplasm but also in primary and sometimes secondary dendrites (Fig. 19). Abundant labeled fibers were crossing in these nuclei like a network.

3.5. Medulla oblongata and spinal cord

Fig. 8. Neurons of the entopeduncular nucleus (EP), globus pallidus (GP) and thalamic reticular nucleus (Rt) show dense immunostaining. (ic, internal capsule; ot, optic tract; st, stria terminalis) Scale bar5500 mm.

The inferior olivary nuclear complex revealed almost no immunoreactivity (Fig. 20). In the vestibular nuclei, neurons in the superior nucleus showed dense staining in the somata and dendrites; neurons in the medial nucleus showed moderate to dense staining; in the lateral nucleus neurons were labeled moderately to densely in the somata and primary dendrites (Fig. 19), whereas neurons in the inferior nucleus were only weakly stained. In the dorsal and ventral cochlear nuclei, neurons were densely stained. Neurons in the dorsal vagal nucleus showed dense labeling. Immunoreactivity of the nucleus of the solitary tract was moderate. In the spinal trigeminal nucleus, neurons were labeled moderately to densely in the somata and primary dendrites (Fig. 21). In addition, axons in the spinal trigeminal tract were also well stained. Dorsal column nuclei neurons were labeled moderately to densely both in the somata and dendrites. In the medullary reticular formation, neurons were stained moderately to densely; some dendrites could be traced to secondary ones. In the upper cervical spinal cord, most neurons in the posterior and anterior horns and the intermediate region were immunolabeled moderately to densely.

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Fig. 9. Electron micrograph of neuropile of the globus pallidus shows many immunolabeled dendrites of small diameter, some of which are indicated with asterisks. Microtubules in these dendrites are labeled with reaction product. Arrow heads point to synaptic sites of these dendrites with axon terminals. Block-stain with uranyl acetate only. Scale bar51 mm.

3.6. Motoneurons in the cranial nerve nuclei and the spinal cord Motoneurons in the oculomotor, trochlear (Fig. 22a), motor trigeminal (Fig. 22b), abducens, facial (Fig. 22c), ambigual, hypoglossal nuclei and the anterior horn of the upper cervical cord (Fig. 22d) showed clear immunoreactivity in the cytoplasm of their somata and primary dendrites.

3.7. Other labeling Abundant axons in many regions of the central nervous system such as the corpus callosum, pyramidal tract or nerve roots of motor cranial nerve nuclei (Fig. 13) were also immunolabeled. In addition to neurons, probable glial cells were observed in the white matter, namely corpus callosum, anterior commissure, corticospinal or spinocerebellar tracts. Some processes of these cells reached blood vessels, which suggests that these glial cells were astrocytes (Fig. 23). The ependymal layer of the ventricular systems was moderately to densely stained (Figs. 6a, 7, 10, 16, 19).

3.8. Electron microscopic observations Electron microscopy was carried out on the cerebral

cortex, CA 3 sector of the hippocampal formation, caudate-putamen, ventral pallidum, substantia nigra pars reticulata and cerebellar cortex. The immunopositive neuronal cell body revealed ribosomal heavy labeling (data not shown) and no reaction product was found in the membrane system of cell organelles and cellular nucleus. In the labeled dendrites, microtubules were selectively immunostained (Figs. 9 and 18). Axon terminals showed no immunolabeling in the synaptic vesicles or presynaptic membrane specialization (Fig. 9), although postsynaptic density (PSD) was occasionally manifest in all above observed regions (Figs. 9 and 24). Postsynaptic membrane specialization and dense bodies were conspicuous among other membrane systems after block-stain with uranyl acetate alone. It is not known at present, however, what kind of cell types these PSDs belong to.

4. Discussion CaM-K IV [31,32,47] and CaM-K I [18,21] are activated by CaM-K kinases. On the other hand, CaM-K II is activated by itself by means of autophosphorylation [9,11,14,17]; CaM-K II does not need CaM-kinases for its activation. Conversely CaM-K Pase deactivates CaM-K II, I and IV by dephosphorylation. CaM-K Pase, therefore, might be involved in regulation of the activity of multi-

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Fig. 10. A photomicrograph shows immunolabeling of the rostral part of the dorsal thalamus. (AD, anterodorsal nucleus; AV, anteroventral nucleus; CM, central medial nucleus; ic, internal capsule; LHb, lateral habenular nucleus; MD, thalamic medial dorsal nucleus; MHb, medial habenular nucleus; Rh, rhomboid nucleus; Rt, thalamic reticular nucleus; VA, ventral anterior nucleus) Scale bar5500 mm.

Fig. 11. Immunolabeled neurons in the superior colliculus and the periaqueductal gray (PAG). A curved arrow points to gathering of moderately labeled neurons of the intermediate gray layer (IGL). (SGL, superficial gray layer) Scale bar5500 mm.

Fig. 12. Densely labeled large neurons (arrows) are dispersed throughout the central nucleus of the inferior colliculus. (PAG, periaqueductal gray) Scale bar5500 mm.

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Fig. 13. Magnocellular red nucleus neurons are densely labeled. Labeled fiber bundles of the oculomotor nerve (arrow heads) run through the red nucleus. Scale bar5100 mm.

functional CaM-kinases. Although CaM-K Pase distributes ubiquitously including the adrenal, brain, lung, pancreas, thymus, spleen, uterus and so forth [16], in this section we discuss the distribution of CaM-K Pase in the central nervous system, because substrates for CaM-K Pase, CaMK II, I, IV, are abundantly present in the brain and spinal

Fig. 15. Neurons in the pontine nuclei (Pn) are labeled densely. Scale bar5100 mm.

cord [10,12,16]. At first, the relationship between the distribution of CaM-K Pase and CaM-kinases will be discussed.

4.1. Subcellular distribution of CaM-K Pase

Fig. 14. Most labeled neurons in the substantia nigra are restricted in the pars reticulata (SNr). Some dendrites are traced more than 100 mm. (cp, cerebral peduncle; SNc, substantia nigra pars compacta) Scale bar5100 mm.

CaM-K Pase in the brain and spinal cord was observed exclusively in the cytoplasm and was never observed in the nucleus in the present study as revealed previously in the PC 12 cells [16]. Among these substrates, CaM-K I [38] and CaM-K II b [4,7,33] have been shown to distribute in the cytoplasm but not in the nucleus. Distribution of CaM-K II a, however, appears both in the cytoplasm and nucleus [26]. On the other hand, CaM-K IV is present predominantly in the neuronal nucleus [13,22,30]. As shown in the present study, CaM-K Pase located only in the cytoplasm, and CaM-K I has been known to locate also in the cytoplasm of neurons in almost all areas in the brain except in the globus pallidus and substantia nigra pars reticulata [38]. These facts mean that CaM-K Pase can dephosphorylate CaM-K I in the brain when CaM-K I should be deactivated, except in the globus pallidus and substantia nigra pars reticulata. Subcellular distribution of CaM-K II has revealed that subunit a located both in the cytoplasm and cellular nucleus, and subunit b only in the cytoplasm [26]. Concerning these data, CaM-K II is present in the cytoplasm of most neurons in the brain and spinal cord. CaM-K Pase, therefore, can

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Fig. 16. A photomicrograph shows the dorsal part of the pontine area. Labeled neurons are shown in the motor trigeminal nucleus (mo5), locus coeruleus (LC), mesencephalic trigeminal nucleus (me5), and medial and lateral parabrachial nuclei (MPB and LPB). At the upper right part of the section, forth ventricular well is densely stained. (scp, superior cerebellar peduncle) Scale bar5100 mm.

deactivate CaM-K II in most neurons too. That means that CaM-K Pase may be involved in the regulation of the activity of CaM-K II in most parts of the central nervous system where CaM-K Pase exists. CaM-K II has been reported to be a major component in the postsynaptic densities (PSDs) which is translocated from CaM-K II in the cytosol by phosphorylation-dependent manner [43,51]. Other kinds of protein phosphatases, PP1 and PP2A, have been suggested to exist at synaptic sites of the central nervous system neurons [1,2,42,44], and PP1 was proved to dephosphorylate CaM-K II and released it from the PSD, which might regulate the long term potentiation [51]. On the other hand, in the present study we found that CaM-K Pase was located in the PSDs in various regions of the brain. Because CaM-K Pase has been observed to dephosphorylate CaM-K II a [10,12,16], this enzyme might also dephosphorylate CaM K II at synaptic sites. Difference of functional role of CaM-K Pase and PP1 on the synaptic site should be solved by future studies. On the other hand, although CaM K IV is located mainly in the cellular nucleus [13,30], the cytoplasm of cell bodies of many neurons such as in the locus coeruleus, red nucleus, pontine, cochlear, vestibular or cerebellar nuclei showed weak immunolabeling [22]. In addition, electron microscopic observation revealed the immuno-

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Fig. 17. An enlarged view of the cerebellar cortex shows immunolabeled Golgi cells (arrows in the granular layer, GrC), radial processes in the molecular layer (Mol) and round contour in the Purkinje cell layer (P). Scale bar5100 mm.

staining of small caliber dendritic processes of cerebral cortical neurons or cerebellar granule cells [22]. Accordingly, CaM-K Pase in the neuronal cytoplasm can also deactivate CaM-K IV in many neuron groups. CaM-K IV is also dephosphorylated by PP2C [5] and PP2A [34,50]. Although it has not been known yet whether CaM-K Pase and PP2A or PP2C cooperate to dephosphorylate in neurons, PP2A and PP2C are able to react with CaM-K IV at least in some brain regions where CaM-K IV does not coexist with CaM-K Pase.

4.2. Distribution of CaM-K Pase in the brain and spinal cord CaM-K Pase was distributed throughout the central nervous system not only in neuronal cell bodies and dendrites but also in axons. As to the corticospinal tract, weakly labeled fibers were observed in the internal capsule, cerebral peduncle, pyramis and posterior column of the spinal cord. CaM-K Pase might be transported by the axonal flow from pyramidal neurons of the cerebral cortex to the spinal cord as like as CaM-K II a which was demonstrated in this tract [45,46]. Although the density of

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Fig. 18. An electron micrograph shows part of a Purkinje cell in the lower field; nucleus (Nu) and cell bodies are free of immunolabeling. Near this neuron, immunolabeled processes (asterisks) are shown. They do not directly contact the Purkinje cell, and have no synaptic contact with other processes. Block-stain with uranyl acetate only. (Ni, Nissl substance) Scale bar51 mm.

Fig. 19. A photomicrograph shows immunolabeled neurons in the cerebellar nuclei and lateral vestibular nucleus (LVe). (Int, cerebellar interpositus nucleus; Lat, cerebellar lateral nucleus; Med, cerebellar medial nucleus) Scale bar5500 mm.

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caudate-putamen and granular layer of the olfactory bulb, dentate gyrus and cerebellar cortex, labeled neurons were not pyramidal, medium spiny and granule cells, respectively, but they seemed to be so-called intrinsic neurons.

Fig. 20. In the inferior olive, neither principal (IOP) nor dorsal and medial accessory nuclei (IOD and IOM) have immunolabeled neurons. Neurons of the medullary reticular formation dorsal to the IOD show moderate immunolabeling. (py, pyramis) Scale bar5100 mm.

immunoreactivity of CaM-K Pase varied from cell group to cell group, neurons in the nuclei of the diagonal band of Broca, dorsal and ventral pallidum, entopeduncular nucleus and substantia nigra pars reticulata showed densest immunostaining. On the contrary, almost no reactivity was observed in the granule cells of the olfactory bulb, the dentate gyrus and the cerebellar cortex, pyramidal cells of the Ammon’s horn, neurons in the medial habenular nucleus and the inferior olivary nucleus, and Purkinje cells. In the Ammon’s horn of the hippocampal formation,

Fig. 21. Neurons in the interpolar spinal trigeminal nucleus show moderate labeling. (spt, spinal trigeminal tract) Scale bar5100 mm.

4.2.1. Distribution of CaM-K Pase in the hippocampal formation and caudate-putamen Hippocampal interneurons have been identified using antibodies against calcium binding proteins, such as parvalbumin, calretinin, or calbindin, or other neuropeptides, such as somatostatin, neuropeptide Y, vasoactive intestinal polypeptide (VIP) or cholecystochinin (CCK) [6,35]. Because what kind of peptides are involved in these CaM-K Pase labeled neurons are unknown at present, it should be solved in the future. Striatal interneurons have also been classified by means of immunocytochemistry by using antibodies against acetylcholine, calcium binding proteins and so forth. Thereafter, four classes of interneurons have been identified: 1) the large cholinergic neurons; 2) GABAergic interneurons that contain parvalbumin; 3) GABAergic interneurons that contain calretinin; and 4) a class of interneurons that contain somatostatin, NADPH-diaphorase and nitric oxide synthase, and that perhaps employ GABA as well [15]. Considering the cell size, shape and distribution of caudate-putamen neurons of the present and previous immunohistochemical studies for ChAT [40,41], the CaM-K Pase labeled caudate-putamen neurons might be cholinergic interneurons. Double immunostaining technique confirmed this hypothesis; most CaM-K Pase positive neurons were also labeled with the antibody against ChAT. Cholinergic interneurons in the caudate-putamen have been paid attention by their involving in the long-term depression and long-term potentiation [3]. Because CaM-K Pase was localized in the cholinergic neurons in the caudate-putamen, there is a possibility that CaM-K Pase regulate the synaptic plasticity in this nucleus at a neuronal level in addition to a synaptic level. 4.2.2. CaM-K Pase in the cerebellar Golgi cells and other neurons As to Golgi cells of the cerebellum, they have been known to express metabotropic glutamate receptor type 2 / 3 (mGluR 2 / 3) in the rat cerebellum [28,29]. The distribution pattern of mGluR2 / 3 in developmental stages has also been demonstrated. It appeared in the Golgi cell and Bergmann glia after postnatal day 3 [19], and although Golgi cell labeling remained in the adult rat, glial labeling lost at postnatal day 21. The population of CaM-K Pase labeled strands in the molecular layer was similar to that of mGluR 2 / 3 labeled Golgi cell processes [19]. Because these strands seemed to continue to the round features surrounding Purkinje cells, and we could not find any glial filaments in strands and processes surrounding Purkinje cells by electron microscopy, these labeled processes might not be of astrocytes but Golgi cell dendrites. Furthermore the function of Golgi cells in relation to

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Fig. 22. Immunolabeled motoneurons in the trochlear (4, a), trigeminal (mo5, b), facial (7, c) and anterior horn of the spinal cord (AH, d) are shown. Labeling of cytoplasm, not nucleus, are clearly shown. (af, anterior funiculus) Scale bars5100 mm.

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granule cells [38]. On the contrary, CaM-K Pase was observed only in Golgi cells in the present study. Dephosphorylation of CaM-kinases in Purkinje cells or granule cells, therefore, might be carried out by other protein phosphatases such as PP1 or PP2.

4.2.3. Distribution of CaM-K Pase in small neurons As described above, some neurons such as the hippocampal pyramidal cells, olfactory, dentate and cerebellar granule cells, striatal medium spiny, medial habenular, inferior olivary neurons and Purkinje cells did not show apparent immunolabeling with CaM-K Pase. Except for pyramidal neurons in the Ammon’s horn and Purkinje cells, smaller neurons seemed to lack CaM-K Pase. In these neurons in which CaM-K Pase was not proved immunohistochemically, CaM-K II and CaM-K I were observed in the neuronal cytoplasm, although CaM-K IV was localized only in cellular nuclei [4,13,22,26,30,33,38]. As a consequence multifunctional CaM-kinases in these neurons will not be influenced by CaM-K Pase. However, it is unknown whether CaM-K Pase is actually lacking in these small neurons, or whether the content of this enzyme is too small to be visualized. Because PP1, PP2A, and PP2C can dephosphorylate multifunctional CaM-kinases, there is a possibility that neurons which lack CaM-K Pase might use such protein phosphatases to dephosphorylate these CaM-kinases for deactivation in these neuron groups.

Fig. 23. A blood vessel (BV) in the corpus callosum. Some astrocytes (arrows) extends their processes to the vessel. Scale bar510 mm.

mGluR2 / 3 was examined in transgenic mice, using the immunotoxin-mediated cell targeting technique [49]. In the present study, immunolabeling for CaM-K Pase in the cerebellar cortex resembled that for mGluR2 / 3: labeling of the Golgi cells, and not-labeling of the Purkinje cells and granule cells [19,25,27,28]. The distribution of mGluR2 / 3 has been studied immunohistochemically in the rat [27,37]. In addition to mGluR2 / 3-labeled neurons in the cerebellum, occasional staining of non-medium spiny neurons in the caudate-putamen resembles the staining of CaM-K Pase in the present study. mGluR2 / 3 is a member of metabotropic glutamate receptors which are coupled to G protein and initiate intracellular signaling cascade [39]. CaM-K Pase is an enzyme that dephosphorylates CaMkinases which have a role in intracellular signal transduction. Although there is a possibility that mGluR2 / 3 and CaM-K Pase relate each other in a intracellular signaling cascade, we do not have any evidence at present about this hypothesis. It is interesting, however, to know the similar distribution of these two substances in the cerebellum and the caudate-putamen. In the cerebellum, CaM-K II has been localized in Purkinje cells [7,8,48], CaM-K IV in granule cells [13,22,30] and CaM-K I in Purkinje cells and

4.2.4. Distribution of CaM-K Pase in the basal ganglia other than the caudate–putamen Dense staining of neurons in the brain with CaM-K Pase was observed in the mitral cell layer of the olfactory bulb, nuclei of the diagonal band of Broca, dorsal and ventral pallidum, entopeduncular nucleus and substantia nigra pars reticulata. Among them, both pallidum, entopeduncular nucleus and substantia nigra pars reticulata receive GABAergic inputs from the caudate-putamen or nucleus accumbens. There is, however, no common feature between these basal ganglia structures and olfactory related structures. Some multifunctional CaM-kinases have shown characteristic immunostaining in some of these nuclei. In the olfactory bulb, CaM-K II has reported to be stained densely [33]. In the globus pallidus, entopeduncular nucleus and substantia nigra pars reticulata, CaM-K IV did not show any immunolabeling in either cytoplasm or nucleus [22]. Immunoreactivity for CaM-K I was not shown in the substantia nigra pars reticulata either [38]. The reason why strong immunoreaction for CaM-K Pase was observed in the basal ganglia where CaM-Kinase I and IV do not exist is not known at present. We can not, however, exclude the possibility that CaM-K Pase works mainly for CaM-Kinase II in these nuclei. Neurons in the above basal ganglia and thalamic reticular nucleus and probable Golgi cells in the cerebellum use GABA as the transmitter. We cannot, however, simply relate CaM-K Pase with GABA related enzyme, because striatal projec-

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Fig. 24. Electron micrographs show the conspicuous feature of postsynaptic densities (PSDs, arrows) in the caudate-putamen (a) and CA3 sector of the hippocampal formation (b). (AT, axon terminal). Block-stain with uranyl acetate only. Scale bars50.5 mm.

tion neurons whose transmitter is GABA were not labeled with antibody against CaM-K Pase at all, and many cortical non-pyramidal neurons some of which were also use GABA were only weakly stained.

4.2.5. Concluding remarks Multifunctional CaM-kinases and CaM-K Pase have been thought to play an important role in signal transduction through the cytoplasm and nucleus. Neurons which contain these enzymes are not distributed homogeneously in the brain and spinal cord as has been reported previously [4,7,13,22–24,26,30,33,38]. That is, CaM-kinases and CaM-K Pase might have functional correlation with neuron groups or neuronal circuitries which are composed of CaM-kinases-rich neurons or poor neurons. At present, however, no accurate evidence has been obtained about whether, for example, CaM-kinases and / or CaM-K Pase are related to the basal ganglia circuitry or the function of intrinsic neurons in various neuron groups. The next step of this work will be a study to reveal co-localization of neurotransmitters or neuropeptides with CaM-K Pase in such regions as the neocortex or hippocampal formation, and a gene targeting study to impair the CaM-K Pase containing neurons in some special nuclei to know the

neuronal function of them like an immunotoxin study dealt with cerebellar Golgi cells [49].

Acknowledgements The authors wish to thank Dr. Takeshi Kaneko for his advice about immunohistochemical studies, and Ms. Mie Taguchi for technical assistance. This study was supported by Grants-in-Aid [11680735 for Scientific Research (C) (Y.N.) and [10102002 for Specially Promoted Research (H.F.) from the Ministry of Education, Science, Sports and Culture of Japan.

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