Localization of gene expression of calbindin in the brain of adult rats

Neuroscience Letters, 138 (1992) 211-215 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

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NSL 08564

Localization of gene expression of calbindin in the brain of adult rats Hiroshi A b e a, M a s a h i k o W a t a n a b e a, Tohru Yamakuni b, R y o z o K u w a n o b, Yasuo Takahashi b and Hisatake K o n d o a aDepartment of Anatorny, Tohoku University School of Medicine, Sendai, (Japan) and bDepartment of Neuropharmacology, Brain Research Institute, Niigata University, Niigata (Japan) (Received 20 December 1991; Revised version received 31 January 1992; Accepted 2 February 1992)

Key words: Calbindin; mRNA; Brain; Adult rat; In situ hybridization Localization of gene expression of calbindin, a cytosolic calcium-binding protein, was examined throughout the adult rat brain by in situ hybridization with cDNA probes. The gene was expressed most intensely in the Purkinje cells in the cerebellum, intensely in the granule cells of the dentate gyrus, and moderately in the inferior olivary nucleus, in the nuclei of the trapezoid body, in the medial part of the lateral habenular nuclei, entorhinal cortex and in the mammillary nuclei. In addition, weak expression of the gene was widespread in the forebrain and brainstem gray matter, and also in small cells in the spinal posterior horn as well as the ependymal cells. The widespread and heterogeneous expression of the gene in the brain suggests that calbindin is differentially involved in calcium-regulated phenomena in different neurons.

The resting free calcium concentration in neurons is maintained around 10 7 M by several mechanisms such as buffering in the cytosol, sequestering into and release from internal organellar compartments, and extrusion and influx across the plasma membrane [3]. Calbindin, which was originally found in the chick intestine as vitamin D-dependent calcium-binding protein and later identified in mammalian brain as vitamin D-independent [6, 17, 18], is a good candidate for the cytosolic calcium buffer [2]. The detailed localization of given substances is a necessary step to understand their functional significance and there have been a number of immunohistochemical studies on the localization of calbindin in the brain [2, 4, 5, 9]. However, the immunohistochemical information is not always sufficient to identify neurons synthesizing a given protein in the brain mainly because of the specificity of antibodies used, the existence of axoplasmic transport and the posttranslational modification of the protein. Therefore it is important to localize neurons expressing mRNAs for the protein. Concerning the localization of mRNA for calbindin, the expression of the mRNA in the cerebellar Purkinje cells and the dentate granule cells has been briefly described in previous studies [10, 14, 16]. Thus, the present in situ

Correspondence: H. Abe, Department of Anatomy, Tohoku University School of Medicine, Seiryo-machi 2-1, Sendai 980, Japan. Fax: (81) 22-272-7273.

hybridization analysis addressed this point and clarified the detailed localization of mRNA for calbindin in the entire brain of adult rats. The cDNA for rat cerebellar calbindin, termed spot 35-calbindin, was isolated from a cDNA library prepared from rat cerebella as previously described [1, 19]. A BgIII-PvuII fragment, about 400 bp in length, of the Y-non-coding region of calbindin-cDNA was selected as a cDNA probe (probe A) specific to calbindin. In addition, another BgllI-PvuII fragment, about 700 bp in length, was cut into two fragments by RsaI and a second cDNA probe (probe B), also about 400 bp in length, was obtained. A mixture of these probes A and B was used for the analysis of calbindin mRNA expression. The probe was labeled with [35S]dCTP or [32p]dATP by random-primed DNA labeling kit (Boehringer Mannheim, FRG). Northern blot analysis was performed to determine the size of the mRNA detected. Poly(A) RNA of the brain was isolated from adult rats by homogenization in 5 M guanidinium thiocyanate followed by direct precipitation of RNA from the guanidinium solution with 4 M lithium chloride and oligo(dT) cellulose chromatography. The poly(A) RNA (2/2g) was electrophoresed on a horizontal 1.5% agarose/2.2 M formamide gel and transferred onto a nitrocellulose filter membrane. Blots were prehybridized at 37°C for 2 h in buffer containing 50% formamide, 5 x SSC (0.15 M NaC1 and 0.015 M sodium citrate), 1 x Denhardt's solution, 50 mM sodium phos-

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Fig. 1 12. (For

phate buffer (pH 6.5) and 250 pg/ml of heat denatured salmon sperm DNA. After hybridization in the same buffer containing [32p]dATP-labeled probe, the filter was washed three times in 2 x SSC-0.1% SDS (sodium dodecyl sulfate) at room temperature, and then in 0.1 x

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SSC 0.1% SDS at 42°C for 30 min, and autoradiographed for two days. The probe for calbindin was hybridized to the band of 1.8 kb as reported in the previous study [19] (Fig. 1). For in situ hybridization histochemistry male albino

213 Fig. 1. Northern blot analysis of expression of calbindin mRNAs in the brain. Poly (A) RNA (2,ug) from rat brain is subjected to RNA blot analysis. Upper and lower arrowheads indicate the positions of 28S and 18S ribosomal RNAs, respectively. Figs. 2 and 3. A coronal section of the olfactory bulb in dark field (Fig. 2) and bright field (Fig. 3). Weak expression of calbindin mRNA is seen in the periglomerular cells (PG). Arrows indicate mitral cells. IG, the internal granule cells. Bar = 0.1 ram. Fig. 4. A coronal section through the frontal lobe and anterior olfactory nucleus (AO). Calbindin mRNA is expressed weakly in the neocortex layer II and III, in the anterior olfactory nucleus (AO), and in the piriform cortex (Pi). Bar = 1 mm. Fig. 5. A coronal section through the caudate-putamen. Calbindin mRNA is expressed weakly in the caudate-putamen (CP) and in the ependyma of the lateral ventricle (arrows). No expression signals of the gene were detected in the anterior commissure (arrowheads). Pi, piriform cortex. Bar - 1 mm.

Fig. 6. A coronal section through the thalamus. Calbindin mRNA is expressed intensely in the dentate gyrus (D), whereas CAI-3 regions of the hippocampus express calbindin mRNA faintly (arrows). Medial part of the lateral babenular nucleus (h) expresses the gene moderately. No significant levels of expression signals were detected in the corpus callosum (*) and the internal capsule (ic). LD, laterodorsal thalamic nucleus. MD, mediodorsal thalamic nucleus; R, reuniens thalamic nucleus; V, ventromedial hypothalamic nucleus. Bar - 1 mm. Fig. 7. A coronal section through the mammillary nucleus. Calbindin mRNA is expressed moderately in the mammillary nucleus (M) and weakly in the superior colliculus (SC), medial geniculate nucleus (MG), and in the amygdaloid nucleus (A). D, the dentate gyrus of the hippocampus. Bar = 1 mm.

Fig. 8. A coronal section through the pons. The entorhinal cortex (E) expresses calbindin mRNA moderately, but no expression of the gene was discerned in the pontine nuclei (Po). Aq, aqueductus cerebri; 3, oculomotor nucleus; PB, pineal body. Bar 1 mm. Fig. 9. A coronal section of cerebellum and medulla oblonga'ta. Purkinje cells (Pu) expresses calbindin mRNA very intensely. Moderate expression of the gene is detected in the inferior olive (IO), while no expression signals were seen in the pyramidal tract (arrows). Cu, cuneate nucleus; So, nucleus of the solitary tract; Sp5, spinal trigeminal nucleus. Bar = 1 mm. Figs. 10 and 11. A coronal section through the locus coeruleus (*) in dark field (Fig. 10) and bright field (Fig. 11). The locus coeruleus and the mesencephalic trigeminal nucleus (arrows) express calbindin mRNA weakly. Pu, cerebellar Purkinje cells. Bar = 0.5 mm. Fig. 12. A cross-section of the cervical spinal cord. Calbindin mRNA is expressed weakly in neurons of the dorsal horn (arrows). No expression signals of the gene were detected in the white matter. CC, central canal. Bar - 0.5 mm.

adult rats (Wistar) were sacrificed by decapitation under ether anesthesia. Brains were removed, frozen on dry ice and stored at - 8 0 ° C until use. F r o z e n sections, 20-30 p m thick, were m a d e on a cryostat and m o u n t e d on gelatin-coated slide glasses. The slides were dipped into 4% p a r a f o r m a l d e h y d e in 0.1 M sodium phosphate buffer (pH 7.2) for 20 min and 2 mg/ml o f glycine in phosphatebuffered saline for 20 min, and subsequently acetylated in 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0). These slides were subject to incubation for 2 h at r o o m temperature in prehybridization mixture containing 4 x SSC, 50% deionized formamide, 1 x D e n h a r d t ' s solution, 2% Sarcosyl, and 250 p g / m l o f heat-denatured salmon sperm D N A in 0.1 M sodium phosphate buffer (pH 7.2). The prehybridization mixture on each slide was replaced with 50 pl hybridization mixture, consisting o f fresh prehybridization mixture with the addition o f 10% dextran sulfate, 100 m M dithiothreitol and 5 x 105 cpm per slide o f each c D N A p r o b e labeled with [35S]dATE After incubation over night at 42°C, the slides were washed 3 times in 0.1 x SSC-0.1% Sarcosyl at 42°C for 40 min and then a u t o r a d i o g r a p h e d using N T B 2 nuclear track emulsion ( K o d a k ) for 8 weeks. In the olfactory bulb, calbindin m R N A was detected weakly only in the periglomerular cells (Figs. 2 and 3). The gene was expressed weakly to moderately in the an-

terior olfactory nucleus, a m y g d a l o i d nucleus, and the piriform and entorhinal cortices (Figs. 4, 5, 7 and 8). Calbindin m R N A was expressed in the h i p p o c a m p a l formation, but their intensity was variable in different regions. It was expressed intensely in the granule cells o f the dentate gyrus, faintly in the C A 1 - 2 regions and actually nothing in the CA3 region (Figs. 6 and 7). In the cerebral neocortex, calbindin m R N A was weakly expressed in the layers II and III (Figs. 4-8). In the c a u d a t e - p u t a m e n , accumbens nucleus, septum, thalamic nuclei, and medial part o f the lateral habenular nuclei, calbindin m R N A was expressed weakly to moderately (Figs. 5 and 6). Weak expression o f the m R N A was discerned in the supraoptic and paraventricular nuclei o f the h y p o t h a l a m u s . The mammillary nucleus expressed calbindin m R N A moderately (Fig. 7). The pineal b o d y expressed the gene weakly (Fig. 8). In the midbrain, pons and medulla oblongata, weak expression o f the gene was detected in several discrete nuclei and zones such as the locus coeruleus (Figs. 10 and 11), the oculom o t o r nucleus (Fig. 8), the mesencephalic and spinal trigeminal nucleus (Figs. 9-11), the substantia nigra, the prepositus hypoglossal nucleus, the superficial layer o f the superior colliculus (Fig. 7), the central gray (Fig. 8), the solitary nucleus, the gracilis and cuneate nuclei (Fig. 9), and parabrachial nuclei, medial geniculate nuclei (Fig. 7), nuclei o f the lateral lemniscus. The inferior olive

214 (Fig. 9) and nuclei of the trapezoid body expressed calbindin m R N A moderately, while no expression of the gene was observed in the pontine nuclei (Fig. 8), the raphe nuclei or the inferior colliculi. In the cerebellum, calbindin m R N A was expressed most intensely in all Purkinje cells (Fig. 9). N o distinct expression of the m R N A was detected in the granule cell layer or the deep cerebellar nuclei. In the spinal cord weak expression of calbindin m R N A was detected in cells of the layers I and II of the posterior horn (Fig. 12). The choroid plexuses of all the ventriculi did not express calbindin mRNA, whereas calbindin m R N A was expressed weakly in portions of the ependyma (Fig. 5). No significant levels of expression signals of the m R N A were detected in glial cells of the white matter of the entire brain such as corpus callosum, the internal capsule (Fig. 6), the anterior commissure (Fig. 5), the pyramidal tract (Fig. 9), and spinal cord white matter (Fig. 12). The present study revealed for the first time that the gene for calbindin is expressed not only in the cerebellar Purkinje cells and dentate granule cells but also widely and differentially throughout the entire brain of adult rats. There have been a number of studies describing the widespread and heterogeneous localization of calbindinlike immunoreactivity in the brain [4, 5, 9]. However, recent studies have suggested a considerable cross-reactivity of some calbindin antisera with such a closely related but distinct calcium binding protein as calretinin [12, 13]. Thus the final identification of true nature of proteins attributed to calbindin-like immunoreactivity has remained to be elucidated by in situ hybridization analysis. Since the present findings on the localization of calbindin m R N A are largely consistent with that of calbindin-immunoreactivity by the previous studies [2, 4, 5, 9], the synthesis of authentic calbindin in almost all the neurons revealed by immunohistochemistry is confirmed by the present study. Among the immunopositive neurons, the immunoreactivity in the inferior olivary neurons seems not to have attracted much attention, because there has been discrepancy concerning the immunoreactivity in axons and axonal endings of these neurons in the cerebellar cortex [1, 4, 5, 9]. Since the present study revealed clearly for the first time moderate levels of expression of calbindin m R N A s in the olivary neuron somata, it is likely that the posttranslational modification may occur in the axons or such a cytosolic calbindin protein may be transported into their axons by some unknown regulatory, but not simple diffusive, mechanisms, resulting in a content of immunoreactive calbindin in their axons below the detection level by the immunoreaction in the olivary neurons. Some discrepancy concerning the localization of cal-

bindin-immunoreactivity has been noticed in several brain loci from different studies: the absence or presence of the immunoreactive neuron somata have been reported in the locus ceruleus, and the gracilis and cuneate nuclei [4, 5]. Since the expression of calbindin m R N A was clearly shown in these loci by the present study, it is now clear that this discrepancy is simply due to the specificity of antisera used. Several recent studies have suggested the possibility that calbindin may protect neurons against excitotoxicity by its buffering intracellular calcium rise. For example, calbindin protein and its m R N A level are specifically reduced in neuronal populations particularly affected in aging and some neurodegenerative diseases [7, 15]. Corticosterone has been shown to induce calbindin and its m R N A levels specifically in the hippocampus [8]. A reduction in calbindin-immunoreactivity has been reported in the hippocampus, particularly in the dentate gyrus cells and their mossy fiber system of rats which had been kindled, although no reduction in its m R N A has been detected in the same specimens [1 1, 16]. The present findings on the detailed localization of calbindin m R N A in the entire brain offer the important morphological basis for further studies to understand the exact functional significance of this protein more clearly. The authors wish to thank Mr. H. Iwasa for his photographic assistance. This work was supported by Ooi Maternity Clinic Foundation, Shiogama, Miyagi prefecture, Japan.

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