ARTICLE |
Correspondence to: Karoly Gulya, Dept. of Zoology and Cell Biology, University of Szeged, 2 Egyetem St., POB 659, Szeged H-6722, Hungary.
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Summary |
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To investigate the pattern of expression of the three calmodulin (CaM) genes by in situ hybridization, gene-specific [35S]-cRNA probes complementary to the multiple CaM mRNAs were hybridized in rat brain sections and subsequently detected by quantitative film or high-resolution nuclear emulsion autoradiography. A widespread and differential area-specific distribution of the CaM mRNAs was detected. The expression patterns corresponding to the three CaM genes differed most considerably in the olfactory bulb, the cerebral and cerebellar cortices, the diagonal band, the suprachiasmatic and medial habenular nuclei, and the hippocampus. Moreover, the significantly higher CaM I and CaM III mRNA copy numbers than that of CaM II in the molecular layers of certain brain areas revealed a differential dendritic targeting of these mRNAs. The results indicate a differential pattern of distribution of the multiple CaM mRNAs at two levels of cellular organization in the brain: (a) region-specific expression and (b) specific intracellular targeting. A precise and gene-specific regulation of synthesis and distribution of CaM mRNAs therefore exists under physiological conditions in the rat brain. (J Histochem Cytochem 47:583600, 1999)
Key Words: calmodulin mRNAs, in situ hybridization, quantitative autoradiography, dendritic targeting, mRNA localization, gene expression, image analysis, rat brain
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Introduction |
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The multifunctional intracellular calcium ion (Ca2+) receptor protein calmodulin (CaM) is involved in the regulation of multiple processes (
At least three distinct bona fide CaM genes (CaM I, CaM II, and CaM III) and four pseudogenes have been described in the rat (
The CaM genes exhibit tissue-specific expression with differential regulation during development in the rat (
Because several lines of evidence point to the differential neuronal expression of the three CaM genes, a thorough quantitative comparison of the mRNA levels corresponding to the three CaM genes in the brain under physiological conditions would clarify our understanding of the detailed regional distribution and the real proportions of these transcripts. The complex role of CaM in cytoplasmic and synaptic functions suggests a precise intracellular transport, possibly involving the targeting of CaM mRNAs to the relevant subcellular compartments such as dendritic and/or axonal processes. In fact, the dendritic localization of CaM I mRNA during early postnatal development in the rat brain (
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Materials and Methods |
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Experimental Animals and Tissue Preparation
All animal experiments were carried out in strict compliance with the European Communities Council Directive (86/609/EEC) regarding the care and use of laboratory animals for experimental procedures. Male SpragueDawley rats (200250 g) maintained under standard housing conditions were decapitated and the brains were quickly removed, embedded in Cryomatrix embedding resin (Shandon Scientific; Pittsburgh, PA), and frozen immediately at -70C. Serial coronal cryostat sections (15 µm) from selected brain areas were cut and thaw-mounted onto Cr-Al-gelatin-coated glass slides. Sections were air-dried and stored at -70C until further processing.
cRNA Probes
[35S]-cRNA probes were prepared as previously described (S (1100 µCi/nmol; Isotope Institute, Budapest, Hungary) was incorporated. Labeled probes were purified by size-exclusion chromatography on a ProbeQuant G-50 Sephadex microcolumn (Pharmacia Biotech; Uppsala, Sweden) and the probe-specific activity was determined to be 3.86.1 x 108 cpm/µg.
In Situ Hybridization
The protocol for hybridization with [35S]-cRNA probes was carried out according to
RNA Isolation and Northern Blot Analysis
Total RNA was prepared by the method of
Membrane Standard Scale
A scale consisting of 12 halving dilutions of radiolabeled and size-exclusion chromatography-purified riboprobe was prepared (final activity range 12000 cpm/mm2), as described by
Autoradiography
Tissue sections and membrane standards or Northern blots were apposed to Hyperfilm-ßmax (Amersham; Arlington Heights, IL) autoradiographic film for 36 hr at 4C, as described by
Densitometry
Quantification was performed exclusively on film autoradiographic images, as described previously (
For standardization, co-exposed nylon membrane standard scales were used. Radioactivity/pixel ± SD was plotted vs ROD ± SD corresponding to the data points of the standard scales, and nonlinear regression analysis using an exponential model was applied:
where p1m and p2m are parameters corresponding to the membrane and x is the radioactivity. The membrane standards were calibrated to tissue-equivalent radioactivity by a mathematical transformation of their curves ( (corresponding to brain paste standards) as:
Film background-corrected RODs (the nonspecific hybridization was indistinguishable from the background) of anatomically defined brain areas were determined in duplicate measurements in five animals. Radioactivity/pixel values corresponding to ROD ± SD measurements of the brain areas were calculated by interpolating with the exponential equations of the co-exposed calibrated membrane standard scales. The mRNA contents of different brain areas were estimated via the formula:
where the Avogadro no. = 6.0225 x 1023 and f = 37792.9 is a correction factor to scale up a pixel volume (42 µ x 42 µ x 15 µ) to 1 mm3. Final results were expressed in mRNA copy number ± SD.
Data Processing and Statistics
Nonlinear regression analysis was performed with the computer program Statgraphics 6.0 (Manugistics; Rockville, MD). Other data reduction was accomplished with Microsoft Excel 5.0a (Microsoft; Redmond, WA). Analysis of significance was carried out with the two-tailed Student's t-test. The differences between mRNA copy numbers of the same brain area were considered significant when p was less than 0.01.
Microscopy
Developed emulsion-coated tissue sections were air-dried and coverslipped with Entellan (Merck; Darmstadt, Germany). Some of the sections were counterstained with toluidine blue. Specimens were examined in a Leica DM LB microscope (Leica Mikroskopie und Systeme GmbH; Wetzlar, Germany) equipped for brightfield and darkfield microscopy. Microscopic images (1600 x 1200 pixel, 8-bit gray scale) were captured with a Polaroid DMC 1 digital microscope camera (Polaroid; Cambridge, MA) connected to a Power Macintosh 8100/80 AV. Defined brain areas in the autoradiographs (X-ray films or emulsion-coated sections) were identified according to toluidine blue-counterstained sections (
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Results |
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Control Experiments
High specificity of the three probes for CaM I, II, and III mRNAs was expected, because no significant homology among the three probes could be determined by sequence alignment. Northern blot analysis of rat brain total RNA revealed two transcripts for CaM I (approximately 4.0 KB and 1.7 KB), a single transcript for CaM II (approximately 1.4 KB), and three transcripts for CaM III (approximately 2.3 KB, 1.9 KB, and 0.9 KB; Figure 1). No labeling was observed when the sense probes were hybridized (not shown). In situ hybridization of antisense probes to tissue sections established a specific and unique distribution, whereas hybridization with sense probes resulted in a very low labeling with nonspecific distribution (Figure 10E). Pretreatment of sections with RNase A resulted in a complete loss of the measurable signals (not shown).
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Calmodulin mRNA Distribution in the Rat Brain
The three distinct CaM genes were widely expressed throughout the rat brain (Figure 2 Figure 3 Figure 4; Table 1). An overall similar pattern and quantity of CaM gene expression was observed to be localized mainly in gray matter areas enriched in neuronal cell bodies, in contrast with the low hybridization signals of white matter structures. In spite of the similar general outline, significant differences in the distribution of the multiple CaM mRNA species were found in certain brain areas. A detailed, quantitative description and comparison of the differential distribution of the three bona fide CaM gene transcripts in the rat brain is given below.
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Olfactory Bulb
The CaM genes exhibited a differential expression pattern and a relatively high mRNA occurrence in this region (Figure 2A2C; Table 1). The CaM mRNAs were generally relatively abundant in the granular and mitral cell layers. The CaM I and CaM II mRNA contents of these areas were similar, but the CaM III mRNA levels were higher in the mitral cell layer and lower in the granular layer (Figure 5A5C). The glomerular layer had lower hybridization levels; similar mRNA copy numbers were detected for CaM I and II but a significantly higher copy number for CaM III. The lowest mRNA levels in the bulb were measured in the external plexiform layer, where the majority of the transcripts were derived from the CaM I and III genes. Diffuse labeling, and also patchy labeling clearly corresponding to cell bodies, was evident for all CaM mRNAs.
Cerebral Cortex
The expression of the CaM genes was relatively strong in layers 26 of the cerebral cortex (Figure 2D2L, Figure 3A3L, Figure 4A4I, and Figure 6A6C; Table 1), but low in Layer 1. In Layers 26, higher mRNA levels were observed for CaM III in the cingulate, frontal, and parietal cortices (38.244.3 x 106/mm3), but somewhat lower levels in the occipital and temporal cortices (28.830.1 x 106/mm3) compared with the CaM I and II copy numbers (32.536.9 x 106/mm3). The strongest labeling of the pyramidal cells of Layer 5 in the forelimb and hindlimb areas was more evident for CaM II and III mRNAs (Figure 6A6C). The CaM II labeling was mainly associated with cell bodies, whereas the CaM I and III labeling was more diffuse, corresponding not only to perikarya but also to the surrounding neuropil areas (Figure 6D6F). Diffuse labeling was likewise evident in Layer 1 of all cortical regions, where considerable differences were determined (CaM II < CaM III < CaM I; Figure 6D6F). Very strong expression was observed for all CaM genes (CaM I, 82.8 x 106/mm3 CaM II, 81.3 x 106/mm3 < CaM III, 108.4 x 106/mm3) in the piriform cortex (Figure 2D2L and Figure 3A3L).
Basal Ganglia and Associated Areas
Low to moderate CaM mRNA levels were observed in these structures (Table 1). Significantly higher mRNA levels were determined for CaM I in the nucleus accumbens (Figure 2D2F), for CaM II in the caudate putamen (Figure 2G2L), and for CaM III in the globus pallidus (Figure 3A3C). The substantia nigra exhibited lower CaM gene expression (Figure 4A4C), with higher mRNA copy numbers in the pars compacta than in the pars reticulata. Moderate expression for all CaM genes was characteristic in the red nucleus (Figure 4A4C).
Limbic Areas
These areas exhibited moderate to strong CaM gene expression (Table 1). Very strong hybridization signals were detected in the hippocampusdentate gyrus complex (Figure 3D3L and Figure 4A4C), where a differential expression pattern was again evident. Very high CaM mRNA levels for all CaM genes were measured in the pyramidal cell layer in the CA3 subfield (86.387.9 x 106/mm3; Figure 7A7C). Lower mRNA levels were detected in the pyramidal cell layer of the CA1 and CA2 subfields, where the CaM II mRNA copy number (66.569.6 x 106/mm3) was significantly higher than those of CaM I and III (56.661.3 x 106/mm3). Differences in mRNA levels were similarly apparent in the granular cell layer of the dentate gyrus because the CaM I mRNAs were more abundant (90.2 x 106/mm3; a value equal to that for the CA1 pyramidal cell layer), whereas the CaM II (59.3 x 106/mm3) and CaM III (47.6 x 106/mm3) mRNA copy numbers were significantly lower. The molecular layers of the hippocampus displayed relatively low labeling associated with both cell bodies and the neuropil (Figure 7D7F). Corresponding to the variable intensities of diffuse labeling (neuropil) with the three CaM probes, considerably different mRNA contents were determined in this region (CaM II < III < I). The CaM I mRNA copy number was significantly higher than those of CaM II and III in the lateral septal nucleus (Figure 2J2L and Figure 8A8C). The differential expression was obvious in the nuclei of the vertical (Figure 2G2I) and horizontal (Figure 2J2L) diagonal bands, where moderate expression for CaM I, intermediate expression for CaM II and strong expression for CaM III were demonstrated. Considerably higher mRNA contents were detected for CaM III in the basolateral amygdaloid nucleus (Figure 3D3F) and for CaM I in the amygdalohippocampal area (Figure 3J3L). Similar CaM I mRNA copy numbers were found in the medial and lateral habenular nuclei, whereas the CaM II level and especially the CaM III mRNA level were significantly higher in the medial than in the lateral (Figure 3G3L and Figure 8D8F) habenular nuclei. High levels of CaM mRNAs (CaM I II < III) were measured in the tenia tecta (Figure 2D2F) and the nucleus of the lateral olfactory tract (Figure 3A3C). Lower CaM gene expression was observed in the bed nucleus of the stria terminalis (CaM I < II
III; Figure 2J2L). A differential expression pattern was also evident in the subiculum (CaM I < III < II).
Thalamus
In general, the thalamic nuclei exhibited moderate CaM mRNA levels (Table 1). Higher and differential expressions were detected in the parafascicular (CaM II < III < I; Figure 3J3L) and the paraventricular (CaM II III < I; Figure 3J3L) thalamic nuclei, while the reticular (CaM II < I
III; Figure 3G3I) and the reuniens (Figure 3G3I) thalamic nuclei exhibited lower mRNA levels.
Hypothalamus
Moderate but differential CaM gene expression was characteristic of the hypothalamic nuclei (Table 1). Slightly lower mRNA levels for CaM I than for CaM II and III were detected in the arcuate, dorsomedial, and ventromedial hypothalamic nuclei (Figure 3G3I). Differences in CaM mRNA copy numbers were characteristic of the medial preoptic area (CaM I < II < III; Figure 2J2L), the suprachiasmatic nucleus (CaM II < III < I; Figure 3A3C and Figure 8G8I), the supraoptic nucleus (CaM II < I III; Figure 3A3F and Figure 8G8I), the retrochiasmatic part of the supraoptic nucleus (CaM II < I < III) and the paraventricular hypothalamic nucleus (CaM I < II < III; Figure 3D3F). A slightly elevated CaM III mRNA content was observed in the tuber cinereum (Figure 3D3F).
MidbrainBrainstem
Moderate CaM mRNA levels, but a differential distribution, were observed in these structures (Table 1). Differential mRNA levels were detected in the superior colliculus (CaM II III < I). The superficial layer exhibited an intermediate gene expression, and the optic nerve layer a stronger one (Figure 4A4F and Figure 8J8L). Moderate differential CaM gene expression was determined in the raphe nuclei (CaM I < II
III; Figure 4D4F), the nucleus trapezoid body (CaM II
III < I; Figure 4G4I), and the parabigeminal nucleus (CaM II < CaM I
III; Figure 4D4F). The CaM mRNA levels were low to moderate, without significant differences, in the central gray (Figure 4D4F), the central nucleus of the inferior colliculus (Figure 4G4I), the dorsal tegmental nucleus (Figure 4G4I), the inferior olive (Figure 4J4L), the interpeduncular nucleus (Figure 4A4C), the medial geniculate nucleus (Figure 4A4C), the pontine nuclei (Figure 4D4F), the pontine reticular nucleus (Figure 4G4I), the prepositus hypoglossal nucleus (Figure 4J4L), and the trochlear nucleus (Figure 4D4F).
Cerebellum
The differential expression pattern of the multiple CaM genes was evident in this area (Table 1). The granular cell layer exhibited strong expression for CaM I (49.4 x 106/mm3) and CaM II (56.1 x 106/mm3), whereas the CaM III mRNA level (23.8 x 106/mm3) was much lower (Figure 4J4L). A low but differential expression (CaM II < CaM III < CaM I) was characteristic in the molecular cell layer (Figure 9D9F). High-resolution autoradiography revealed very strong expression for CaM II but lower mRNA levels for CaM I and III in the Purkinje cells (Figure 9A9F).
White Matter
Although the CaM gene expression dominated in gray matter areas, the autoradiographic signal in white matter areas (e.g. anterior commissure, Figure 2J2L; corpus callosum, Figure 2D2L, Figure 3A3L, and Figure 4A4F; cerebellar white matter, Figure 4J4L) was unambiguous too. Low levels for all CaM mRNAs were determined in these areas (Table 1).
Ventricles
Moderate CaM gene expression was determined in the choroid plexus (CaM I < II < III; Table 1). Microscopic analysis of emulsion-coated sections demonstrated labeling similar to that of the choroid plexus in the ependymal cells lining the ventricles (Figure 10A10C).
Intracellular Localization
Certain areas in the brain are highly laminated, in which layers containing neuronal perikarya, layers composed of mainly dendrites and their presynaptic afferents, or virtually exclusively axonal tracts, can be distinguished. Layers containing cell bodies, such as cerebral cortical Layers 26 (Figure 6A6F), the cerebellar granular and Purkinje cell layers (Figure 9A9F), the pyramidal cell layer of the hippocampus (Figure 7A7F), and the granular and mitral cell layers of the olfactory bulb (Figure 5A5C), exhibited very strong expression for all three CaM genes (Table 1). Lower but unambiguous CaM mRNA levels were detected in areas containing mainly dendrites and their presynaptic afferents, such as the cerebral (Layer 1; Figure 6D6F), cerebellar (Figure 9D9F) and hippocampal (Figure 7D7F), molecular layers and the external plexiform layer of the olfactory bulb (Figure 5A5C). In these areas, most of the labeling was diffusely distributed, presumably corresponding to structures of the neuropil, although patchy labeling corresponding to cell bodies was also evident (Figure 5A5C, Figure 6D6F, and Figure 7D7F). A dispersed pattern of the silver grains was further evident in areas surrounding the heavily labeled cell bodies in cerebral cortical Layers 26 (Figure 6D6F). The mRNA levels corresponding to the three CaM genes differed significantly in these areas, because relatively high CaM I, intermediate CaM III, and low CaM II mRNA levels were detected (Table 1). White matter structures (Figure 2 Figure 3 Figure 4) containing mainly axonal tracts (and glial cells as well) exhibited very low expression for all CaM genes.
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Discussion |
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Three (CaM I, II, and III) gene-specific cRNA probes complementary to the 3'-nonhomologous (noncoding) regions of their mRNAs were designed. Probe specificity was determined by Northern blot analysis: the lengths of the six detected mRNA species were in good agreement with those previously obtained for the rat brain (
The results of our in situ hybridization study demonstrated that the three CaM genes are widely expressed, with a similar overall occurrence in the rat brain. The detected pattern of mRNA distribution corresponding to each CaM gene generally confirmed those previously described in the rat brain (
By determination of the exact mRNA quantities, a direct and therefore more precise comparison of the expression of the different CaM genes was possible. For each CaM gene, a high regional variance (3540-fold differences) in mRNA copy number was detected. The brain areas most prominently exhibiting a differential distribution of their CaM mRNAs were the external plexiform layer of the olfactory bulb, Layer 1 of the cerebral cortex, the globus pallidus, the diagonal band, the lateral septal nucleus, the suprachiasmatic nucleus, the supraoptic nucleus, the hippocampusdentate gyrus complex, the medial habenular nucleus, the optic nerve layer of the superior colliculus, and the cerebellar cortex. For the first time, evidence has been established here that indicates differential CaM gene expression in several brain areas, most notably the molecular layers in the cerebral and cerebellar cortices and the hippocampus. Strong evidence of non-neuronal CaM gene expression was similarly observed in the ependymal cells and the choroid plexus. CaM II mRNA was detected in the choroid plexus by
Differential labeling of the major structural specializations of the neurons too was also investigated. In agreement with the results of previous studies (
Our results indicate that the CaM mRNAs exhibit a dendritic localization in neurons in the adult rat brain. Moreover, transcripts of the three CaM genes are differentially targeted to the dendritic compartment, because we found mostly a perikaryal localization for CaM II mRNA, whereas the mRNA for CaM III, and especially for CaM I, is under somatodendritic targeting. Although the translocation of neuronal mRNAs to different subcellular domains has been described by several authors (
The results we obtained on CaM mRNA distribution by in situ hybridization exhibit a similar localization to those detected by CaM immunocytochemistry (
Conclusions
In agreement with the pivotal role of CaM in the nervous system, the multiple mRNAs corresponding to the three bona fide CaM genes are widely expressed in the rat brain. Differential expression of the CaM genes was detected at two different cellular organization levels. First, we found a differential area-specific CaM mRNA distribution in several brain areas under physiological conditions in adult animals. Second, differential in vivo intraneuronal CaM mRNA targeting was revealed. Other authors have already reported a differential regulation for the CaM genes under (patho)physiological or experimental conditions in the rodent brain and PC12 cells (
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Acknowledgments |
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Supported by grants from the National Scientific Research Fund, Hungary (OTKA F22658 and T22822 to AP and KG, respectively) and by the National Council on Technical Development (OMFB No. 97-20-MU-0028 to KG).
The skillful technical assistance of Ms Susan Ambrus and Ms Maria Kosztka is highly appreciated.
Received for publication December 30, 1998; accepted January 5, 1999.
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