Abstract

Myristoylated alanine-rich C-kinase substrate (MARCKS) is a major substrate for protein kinase C, and is involved in synaptic plasticity. Using both Northern blot and in situ hybridization techniques, we investigated whether MARCKS expression varied according to the cerebral region, including the hippocampal formation, or according to the type of neuron. Northern blot analysis showed that the MARCKS mRNA level was higher in the association areas than in the primary sensory and motor areas of the cerebral neocortex. MARCKS mRNA levels in the hippocampus and the amygdala were as high as those in the association areas. The in situ hybridization experiments confirmed the Northern blot results and showed the distribution and characteristics of MARCKS mRNA-positive neurons. In the association areas of the neocortex, prominent signals were observed in neurons in layers II–VI. In the primary areas, prominent signals were restricted to neurons in layers IV-VI. In the hippocampus, the most intense hybridization signals were observed in neurons in the granule cell layer of the dentate gyrus. The observed region-specific expression might reflect functional specialization for plasticity in individual regions of the monkey cerebral cortex.

Introduction

Protein kinase C (PKC)-associated signal transduction plays an important role in many cellular responses, including the plastic change of synapses [reviewed in (Nishizuka, 1986, 1995; Kaczmarek, 1987; Tanaka and Nishizuka, 1994)]. Many studies have shown that several PKC subtypes are highly expressed in specific regions of the monkey brain (Tominaga et al., 1993; Fukuda et al., 1994; Saito et al., 1994). Furthermore, we previously reported that the mRNA of GAP-43, a major PKC substrate, was also highly expressed in specific regions of the monkey brain, such as the association areas of the cerebral neocortex and CA3 subfield of the hippocampus (Higo et al., 1998, 1999a; Oishi et al., 1998). These results have enhanced our understanding of synaptic plasticity in each region of the monkey brain.

Myristoylated alanine-rich C-kinase substrate (MARCKS) is another major substrate for PKC. Dephosphorylated MARCKS associates with the membranes of axon terminals and small dendrites (Aderem et al., 1988; Ouimet et al., 1990; Swierczynski and Blackshear, 1995). In the dephosphorylated form, MARCKS cross-links filamentous actin (Hartwig et al., 1992). When phosphorylated by PKC or bound to calcium-calmodulin, MARCKS is translocated from the plasma membrane to the cytoplasm (Wang et al., 1989; Thelen et al., 1991; Arbuzova et al., 1997, 1998) and loses the capacity to cross-link filamentous actin (Hartwig et al., 1992). Thus, MARCKS has a role in PKC- and calcium-calmodulin-mediated structural alterations of neurons by regulating the interaction between actin filaments and the plasma membrane [reviewed in (Aderem, 1992; Chakravarthy et al., 1999)]. High-level MARCKS expression has been observed in the developing rat brain (Patal and Klingman, 1987; McNamara and Lenox, 1998). Although MARCKS protein and mRNA expression levels decrease in most regions of the mature rat brain (Patal and Klingman, 1987; McNamara and Lenox, 1998), high levels of expression remain in specific regions, such as the hippocampus, amygdala and olfactory cortex, which may be associated with a high degree of plasticity (Ouimet et al., 1990; McNamara and Lenox, 1997, 1998). A recent study in transgenic mice has shown that MARCKS expression in the adult hippocampus is related to spatial learning (McNamara et al., 1998). Previous studies of MARCKS expression were performed primarily in the rodent brain (Ouimet et al., 1990; McNamara and Lenox, 1998), and there is little data on MARCKS expression in the monkey brain.

In the present study, we used Northern blot analysis to measure the MARCKS mRNA levels in various regions of the monkey brain, including several areas of the cerebral neocortex, hippocampus, amygdala, striatum and thalamus. We also performed non-radioactive in situ hybridization histochemistry to determine the localization and type of cells expressing MARCKS mRNA in the cerebral neocortex and hippocampus. The preliminary results of these studies have been reported elsewhere (Higo et al., 1999b).

Materials and Methods

Northern Blot Analysis

Animals and Tissue Preparation

For the Northern blot analysis, brain tissue was obtained from three Japanese monkeys (Macaca fuscata), weighing 2.6, 4.4 and 6.3 kg, and two rhesus monkeys (Macaca mulatta), weighing 4.1 and 4.5 kg. The monkeys were either purchased from a local provider or bred in the Primate Research Institute, Kyoto University. All animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals established by the Institute of Laboratory Animal Resources, and the Guide for the Care and Use of Laboratory Primates established by the Primate Research Institute, Kyoto University. The animals were pretreated with i.m. injections of ketamine hydrochloride (10 mg/kg) and deeply anesthetized by i.v. administrations of pentobarbital sodium (35 mg/kg Nembutal; Abbott, Chicago, IL). Animals were then perfused with ice-cold saline through the ascending aorta. The neocortical areas (determined from sulcal patterns) were quickly dissected on crushed ice using the classification of von Bonin and Bailey (von Bonin and Bailey, 1947) (Fig. 1). The hippocampus, amygdala, caudate nucleus, putamen and thalamus were also dissected. The dissected tissues were frozen immediately in dry ice and stored at –80°C until use.

Probe Synthesis

Human MARCKS cDNA encompassing the whole protein-coding sequence (in the 1.9 kb plasmid pBSH80K1.9B; a gift from Dr D. Stumpo, National Institutes of Environmental Health Sciences) and the 1.1 kb human glyceraldehyde-3′-phosphate dehydrogenase (G3PDH) cDNA (Clontech, Palo Alto, CA) were labeled with digoxigenin, using a random priming method according to the manufacturer's instructions (DIG-DNA Labeling Kit, Roche Diagnostics, Germany). Before use, the labeled probe was precipitated with ethanol and washed to remove unincorporated digoxigenin-labeled nucleotides.

RNA Extraction, Hybridization and Detection

Total RNA was isolated from 150to 300-mg portions of dissected tissues using the method of Chomczynski and Sacchi (Chomczynski and Sacchi, 1987). RNA quantity and purity were assessed by spectroscopic measurements at 230, 260 and 280 nm. Extracted RNA was divided into 15 μg aliquots and denatured by incubating at 60°C for 15 min in the presence of 50% formamide, 2.2 M formaldehyde and 0.5× MOPS buffer (pH 7.0), and was then stored at –30°C until use. The samples were electrophoresed on a 0.9% agarose gel containing 2.2 M formaldehyde and transferred to a nylon membrane (Hybond-N, Amersham, UK) by capillary blotting in 20× SSC (standard saline citrate, a mixture of 3 M sodium chloride and 0.3 M sodium citrate).

After UV irradiation, the membranes were prehybridized overnight at 42°C in 250 μg/ml sheared salmon sperm DNA, 50% formamide, 5× SSC, 50 mM phosphate buffer (pH 6.5) and 1× Denhardt's solution. Hybridization was performed overnight at 42°C in 2 ng/ml of digoxigenin-labeled MARCKS and G3PDH probes, 250 μg/ml sheared salmon sperm DNA, 50% formamide, 5× SSC, 50 mM phosphate buffer (pH 6.5) and 1× Denhardt's solution. The membranes were subsequently rinsed several times at room temperature with 2× SSC and 0.2% SDS, then twice at 55°C with 0.1× SSC and 0.2% SDS.

The buffer was exchanged for a solution of 0.1 M maleic acid, 0.15 M NaCl and 0.2% Tween 20 (pH 7.5) for 10 min at room temperature. Membranes were then incubated in 2% blocking reagent (DIG Nucleic Acid Detection Kit, Roche Diagnostics), 0.1 M maleic acid and 0.15 M NaCl (pH 7.5) for 1 h at room temperature, and further incubated for 3 h at room temperature in diluted (1:2000) anti-digoxigenin Fab-fragments, conjugated with alkaline phosphatase (DIG Nucleic Acid Detection Kit, Roche Diagnostics), 2% blocking reagent, 0.1 M maleic acid and 0.15 M NaCl (pH 7.5). Unbound antibody conjugate was removed by washing three times for 10 min each with 0.1 M maleic acid, 0.15 M NaCl and 0.2% Tween 20 (pH 7.5). Finally, the membranes were pre-incubated for 5 min in 0.1 M Tris–HCl buffer (pH 9.5) containing 0.1 M NaCl and 0.05 M mgCl2, and then incubated for 5 h in the dark in the same buffer containing the substrates nitroblue tetrazolium (NBT, 340 μg/ml; Roche Diagnostics) and 5-bromo-4-chloro-3 indolyl phosphate (BCIP, 170 μg/ml; Roche Diagnostics).

Quantification

Hybridized membrane images were acquired with a 3CCD color video camera (DXC-950; Sony, Tokyo, Japan) and digitized using an image analysis system (MCID; Imaging Research Inc., St Catharines, Canada). While the MARCKS probe hybridized to a specific 2.6 kb RNA transcript in all regions of the monkey brain, the signal intensity was different for each region observed (Fig. 2A). In some cases, the probe also hybridized to the unspliced 4.7 kb form of the RNA transcript (data not shown), as previously observed by others (Harlan et al., 1991). We quantified each 2.6 kb RNA transcript by measuring its optical density. To calibrate the amount of applied total RNA, we employed a two-step standardization method, as described previously (Oishi et al., 1998). In the first step, in order to compensate for differences in optical density caused by different reactivities in the color development of each membrane, we electrophoresed total RNA standards (30, 15, 7.5 and 3.75 μg), extracted from a large amount of cerebral cortex, on the same plate as that for the experimental samples. The amount of MARCKS mRNA in each sample was represented as a multiple of the amount of standard total RNA. In the second step, to compensate for the error in the amount of total RNA applied, we used G3PDH mRNA as an internal control. In the present study, we divided the value obtained for MARCKS mRNA by the value for G3PDH mRNA. Thus, the normalized amount of MARCKS mRNA is indicated as a multiple of the ratio of MARCKS mRNA to G3PDH mRNA in the standard total RNA from brain homogenate. Measurements were performed three times for each RNA sample.

In Situ Hybridization Histochemistry

Animals and Tissue Preparation

For in situ hybridization histochemistry, brain tissues were obtained from five Japanese monkeys (Macaca fuscata) weighing between 5 and 13 kg. The monkeys were purchased from a local provider, and all animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals established by the Institute of Laboratory Animal Resources. The animals were pretreated with i.m. injections of ketamine hydrochloride (10 mg/kg) and deeply anesthetized by i.v. administrations of pentobarbital sodium (Nembutal; 35 mg/kg). The animals were then perfused through the ascending aorta with 0.5 l of ice-cold saline containing 2 ml (2000 units) of heparin sodium, followed by 2–5 l of ice-cold fixative containing 2% paraformaldehyde (PFA) and 0.5% glutaraldehyde in 0.15 M phosphate buffer (pH 7.4), or 2% PFA, 0.5% glutaraldehyde and 0.2% picric acid in phosphate buffer. During perfusion, the heads were chilled on crushed ice.

After perfusion, the brains were immediately removed and blocked in the coronal plane (5 mm thick). The sections were immersed in a post-fixative solution containing 4% PFA and 5% sucrose in phosphate buffer for several hours, followed by successive immersions in phosphate buffer solutions containing 10, 20 and 30% sucrose. The brain blocks were mounted in O.C.T. compound (Miles Inc., Elkhart, IN), rapidly frozen in a dry ice–acetone bath and stored at –80ºC until dissection.

Synthesis of a Probe

The 484 bp fragment of the human MARCKS cDNA that encompasses 308 bp of the 5′-untranslated region and 176 bp of the protein-encoding region was labeled with digoxigenin using a random priming method according to the manufacturer's instructions (DIG-DNA Labeling Kit, Roche Diagnostics). Before use, the labeled probe was precipitated with ethanol and washed to remove unincorporated digoxigenin-labeled nucleotides.

Pretreatment of Sections

Brain segments, including the hippocampal formation and representative association areas (FD, TE and PG), the primary sensory and motor areas (PB, OC and FA), and neighboring areas (LA, PC, PEm, PF and PE, and areas 35, 36 and 51 of Brodmann) of the cortex (Fig. 3A,B) were sectioned coronally to a thickness of 10 μm on a cryostat (CRYOCUT 3000, Leica, Nussloch, Germany). The sections were mounted on slides coated with Vectabond Reagent (VECTOR Laboratories, Burlingame, CA), dried and pretreated for in situ hybridization by successive incubations in 4% PFA in 0.15 M phosphate buffer for 15 min, in 30 μg/ml Proteinase K (Roche Diagnostics; pH 8.0) for 30 min at 37°C and in 4% PFA in 0.15 M phosphate buffer for 10 min. After washing in 0.15 M phosphate buffer, the sections were dehydrated through a series of ethanol treatments (70, 80, 90 and 100% ethanol for 1 min each) and then dried.

Hybridization and Detection

The sections were pre-hybridized in 45% formamide, 4× SSC, 1× Denhardt's solution, 0.25% SDS, 10 mM Tris–HCl (pH 7.6) and 500 μg/ml denatured salmon sperm DNA for 3 h at 45°C. Following prehybridization, the sections were transferred to fresh hybridization buffer containing an additional 10% dextran sulfate and 200 ng/ml digoxigeninlabeled DNA probe, placed under cover slips, heated on a dry block at 95°C for 10 min and chilled on ice for 10 min. Hybridization was performed for at least 16 h at 45°C.

The hybridized sections were incubated in 2× SSC and 0.2% SDS to remove the cover slips, rinsed four times at room temperature with 2× SSC and 0.2% SDS to remove the hybridization mixture, and then washed for four periods of 15 min in preheated 0.5× SSC and 0.2% SDS at 55°C.

The buffer was changed to 0.1 M maleic acid, 0.15 M NaCl and 0.2% Tween 20 (pH 7.5) for 10 min at room temperature. Sections were incubated in 2% blocking reagent (DIG Nucleic Acid Detection Kit, Roche Diagnostics), 0.1 M maleic acid and 0.15 M NaCl (pH 7.5) for 1 h at room temperature, and then further incubated for 3 h at room temperature in diluted (1:500) anti-digoxigenin Fab-fragments, conjugated with alkaline phosphatase (DIG Nucleic Acid Detection Kit, Roche Diagnostics), 2% blocking reagent, 0.1 M maleic acid and 0.15 M NaCl (pH 7.5). Unbound antibody conjugate was removed by washing three times for 10 min each with 0.1 M maleic acid, 0.15 M NaCl and 0.2% Tween 20 (pH 7.5).

Finally, the sections were pre-incubated for 5 min in 0.1 M Tris–HCl buffer (pH 9.5) containing 0.1 M NaCl and 0.05 M mgCl2, then incubated for 20 h in the dark in the same buffer containing NBT and BCIP. Color development was stopped by incubation for 10 min in 0.1 M Tris–HCl buffer (pH 7.5) containing 0.01 M EDTA. The sections were incubated in 4% PFA in 0.15 M phosphate buffer for 10 min at room temperature to prevent fading. The sections were then dehydrated through a series of ethanol treatments (70, 80, 90 and 100% ethanol for 1 min each), dipped in xylene three times for 5 min each time, then placed under a coverslip with Permount histological mounting medium (Fisher Scientific, Fair Lawn, NJ).

Normalization for Cell Density Measurement

Due to the extreme variations in cell density, it is difficult to compare the relative mRNA expression levels among the different areas and layers of the monkey cortex. We evaluated the relative expression levels of mRNAs among subregions by dividing the optical density (OD) in each pixel of the digitized images of in situ hybridized sections by that of the adjacent Nissl (cresyl violet)-stained sections, as described previously in the in situ hybridization study of GAP-43 (Higo et al., 1998). Although this normalization is premised on the assumption that the OD of Nissl-stained sections represents the cell density, this method is valid for measuring relative mRNA expression levels, since it takes into account both the proportion of positive cells and the signal intensity within each cell.

Images were acquired using an Olympus BX60 (Olympus, Tokyo, Japan) microscope with a 3CCD color video camera (DXC-950; Sony, Tokyo, Japan), and were digitized using an image analysis system (MCID; Imaging Research Inc.). The OD was measured in each pixel of the digitized in situ hybridized (in situ OD) and Nissl-stained sections (Nissl OD). The measured OD was the sum of the OD originating from the hybridization signal (ODsignal) and that of the background staining (ODbackground). ODbackground was estimated as the average of the ODs from five randomly sampled local regions of the white matter.

The original image resolution was so high (1.03 × 102 mm2/4096 × 4096 pixels) that the contour of each cell could be observed. For this image analysis, the resolution was reduced to 1.64 × 105 mm2/4096 × 4096 pixels in order to eliminate fine differences in the distribution of cell clusters between in situ hybridized and Nissl-stained sections. The OD of each pixel in the in situ hybridized sections was normalized by the formula: 

\[OD_{normalized}{=}\ (\mathit{in\ situ}\ OD_{signal}\ {\mbox{--}}\ \mathit{in\ situ}\ OD_{background})/(Nissl\ OD_{signal}\ {\mbox{--}}\ Nissl\ OD_{background})\]

When the Nissl ODsignal value was very close to the value for Nissl ODbackground (i.e. the difference was <0.01), we substituted 0.01 for the denominator to avoid generating extreme values in cell-free regions such as white matter. This calculation was carried out using MATLAB version 5.0.0 (The Math Works Inc., Natick, MA). The ratio of ODnormalized to the highest ODnormalized in the section (i.e. the relative expression level) was superimposed in pseudocolor on the digitized Nissl-stained section (Fig. 7).

To clarify the difference in the laminar expression patterns between association and primary areas, we compared the expression levels of the outer pyramidal layer (layer III) and the inner pyramidal layer (layer V) of the representative association area (FD) and primary area (PB). OD was measured in a 300-μm-wide column that sampled all layers of the cortex (Fig. 9A,B). Three columns from each cortical area were measured for each monkey (four monkeys for FD, and five monkeys for PB). We determined the ratio: 

\[III_{OD}/V_{OD}\ {=}\ OD_{normalized}\ in\ layer\ III/OD_{normalized}\ in\ layer\ V\]

When the expression of MARCKS mRNA is higher in layer V than in layer III, this ratio is <1. The ratio confirms a difference in the laminar expression patterns between association and primary areas (Fig. 9C).

Results

Northern Blot Analysis

Using Northern blot analysis, we measured the amount of MARCKS mRNA in various monkey brain regions, including several areas of the cerebral neocortex (Fig. 1), hippocampus, amygdala, striatum, and thalamus. The results showed that MARCKS mRNA was expressed in all regions of the monkey brain, and that the expression levels varied with the regions studied (Fig. 2A,B). In the neocortex, the amount of MARCKS mRNA was higher in the association areas (0.798 ± 0.108 in FD, 0.776 ± 0.073 in TE and 0.696 ± 0.162 in PG; Fig. 2B) than in the primary motor and sensory areas (0.447 ± 0.083 in PB, 0.436 ± 0.088 in OC and 0.414 ± 0.151 in FA). The Mann–Whitney U-test revealed that the amount of MARCKS mRNA in FD and TE was significantly higher than that in each primary area (Fig. 2C). The levels of MARCKS mRNA in the premotor (0.560 ± 0.108 in FB) and secondary visual (0.485 ± 0.287 in OB) areas were intermediate in value to those in the association and primary areas.

The MARCKS mRNA levels in the hippocampus (0.875 ± 0.083) and amygdala (0.727 ± 0.426) were as high as those in the association areas of the neocortex. Large individual variation was observed in OB of the neocortex, amygdala and caudate nucleus. As for the amygdala, the individual variation may have been due to differences in sampled regions within the amygdala, since high levels of MARCKS mRNA expression were restricted to specific nuclei, such as the lateral nucleus, as shown in the in situ hybridization results (Figs 5 and 7).

In Situ Hybridization Histochemistry

Control Experiments

We used a 484 bp fragment of the human MARCKS cDNA as a probe for in situ hybridization histochemistry. The probe specificity was confirmed by Northern blot analysis, which showed a specific 2.6 kb band for MARCKS mRNA (Fig. 4A). In certain cases the probe also hybridized to the unspliced 4.7 kb form of the RNA transcript (Fig. 4A), as observed previously (Harlan et al., 1991). In addition, two control experiments were carried out as in our previous in situ hybridization studies (Higo et al., 1998, 1999a). A series of adjacent sections that included the hippocampus were treated with ribonuclease A (20 μg/ml) for 30 min at 37°C before in situ hybridization (Fig. 4C). Another series of adjacent sections was used for a competition control (Fig. 4D), in which a >250-fold excess of non-labeled probe was added to the hybridization buffer together with the digoxigenin-labeled probe (200 ng/ml). Only background signals were observed in the sections pretreated with ribonuclease A (Fig. 4C), and the signals were dramatically reduced in the competition-control sections (Fig. 4D). The positive hybridization signals were mainly located in the neuronal cytoplasm in sections that were treated in the normal manner with the digoxigenin-labeled probe (Fig. 4B). These results confirmed that the latter signals were specific for MARCKS mRNA. Though we used two different fixative solutions to perfuse the monkeys, the results of in situ hybridization histochemistry were consistent among them.

Neocortex

In the neocortex, we identified individual cortical areas, including both the association areas (FD, TE and PG) and the primary sensory and motor areas (PB, OC and FA), using the cytoarchitectonic criteria of von Bonin and Bailey (von Bonin and Bailey, 1947). The results of the in situ hybridization histo-chemistry indicated characteristic expression of MARCKS mRNA in each cortical area. For example, both the expression level and pattern of MARCKS mRNA in FD were different from those in the neighboring area LA (Figs 5B and 7A).

The results of the in situ hybridization experiment were consistent with those of Northern blot analysis; that is, the hybridization signals were more abundant in the association areas (FD, TE and PG) than in the primary sensory and motor areas (PB, OC and FA). In the association areas (FD in Figs 5B and 7A; TE in Figs 5D and 7B; PG in Figs 6D and 7E) of the neocortex, prominent hybridization signals for MARCKS mRNA were observed in all layers except layer I. Figure 8B shows the laminar distribution pattern of MARCKS mRNA-positive cells in the representative association area (FD), wherein cells with intense hybridization signals were scattered almost equally in layers II–VI. In the primary sensory and motor areas (PB and FA in Figs 5F and 7C; OC in Figs 6F and 7F), the hybridization signals for MARCKS mRNA were generally weak, and the prominent signals were restricted to deeper layers. Figure 8D shows the laminar distribution pattern of MARCKS mRNA-positive cells in the representative primary area (PB); cells with intense hybridization signals were concentrated in layers IV–VI. To clarify the difference in the laminar expression patterns between association and primary areas, we compared expression levels of the outer pyramidal layer (layer III) and the inner pyramidal layer (layer V) of FD and PB. Though neuronal density was higher in layer III than in layer V of PB (the OD of the Nissl-stained section in Fig. 9B), the OD of in situ hybridized sections was lower in layer III than in layer V. The IIIOD/VOD ratio was <1 in PB of all monkeys examined (n = 5). The Mann– Whitney U-test revealed that the IIIOD/VOD ratio was significantly lower in PB than in FD (P < 0.01; Fig. 9C). Moderate to weak hybridization signals were sometimes observed in the small neurons in layer I (Fig. 11A) and the white matter neurons of all areas.

Intermediate expression patterns were observed in PE, PF (Figs 5F, 6D and 7C,E) and OB (Figs 6F and 7F), i.e. the prominent signals were observed in layers II–VI, and more intense signals were found in layers IV–VI than in layers II and III. The in situ hybridization result for OB was consistent with that from Northern blot analysis, showing values for OB that were intermediate between those of the association areas and the primary areas. In LA (Figs 5B and 7A), TH and TF (Figs 6B and 7D), the signals were weaker than in neighboring cortical areas, and the prominent signals were restricted to layers IV–VI.

In the cerebral neocortex, we observed prominent MARCKS mRNA signals mainly in the pyramidal cells (5–40 μm in diameter; Fig. 11B). We confirmed that certain distinctive types of pyramidal cells, such as Betz cells in FA (Fig. 11C) and Meynert cells in OC (Fig. 11D), contained intense signals. We did not detect any glial cells containing positive signals.

Hippocampal Formation

The hippocampal formation consists of the dentate gyrus and Ammon's horn of the hippocampus, along with the subicular and entorhinal cortices. The most intense hybridization signals were observed in the granule cells of the dentate gyrus (Figs 6B, 7D and 10B). Signals tended to be more intense in the granule cell layer near the end of the hippocampal fissure (Figs 6B and 7D). Many small cells (5–15 μm in diameter) with moderateintensity signals were observed in the molecular layer of the dentate gyrus (Fig. 10B). There were also many positive cells with moderate-intensity signals in the polymorphic layer (Fig. 10B). These had a variety of cell body shapes and sizes (20–50 μm in length), as described in previous Golgi studies (Lorente de No, 1934; Amaral, 1978).

Many positively stained pyramidal cells (10–20 μm in diameter) were observed throughout Ammon's horn (Figs 6B and 7D). The normalized expression levels confirmed that the hybridization signals in the CA3 pyramidal cell layer were more intense than in the CA4, CA2 and CA1 subfields of Ammon's horn (Fig. 7D). In addition, intense hybridization signals were observed in stratum oriens cells that had various shapes and sizes (10–50 μm in diameter; Fig. 10D). Cells that resembled the fusiform-shaped neurons described in the Golgi study (Rosene and Van Hoesen, 1987) often had intense hybridization signals (Figs 10D and 11E). There were many round positive cells with moderate-intensity hybridization signals throughout the stratum radiatum and the stratum lacunosum moleculare (5–20 μm in diameter; Figs 10D and 11F).

Higher intensity signals were observed in the subicular complex than in either the adjacent CA1 subfield of Ammon's horn (Figs 6B and 7D) or the adjacent parahippocampal cortex (TF and TH; Figs 6B and 7D). These signals were observed in both pyramidal and non-pyramidal cells in every layer. The listed features for the dentate gyrus, Ammon's horn and subicular complex were consistent throughout the rostral to caudal levels of the hippocampal formation (data not shown).

We identified each region of the entorhinal cortex and the adjacent perirhinal (areas 35 and 36 of Brodmann) and parahippocampal cortex (TF and TH) using the cytoarchitectonic criteria of Amaral et al. (Amaral et al., 1987). In the entorhinal cortex, prominent signals were observed in layers II, V and VI (Figs 5D and 7B). The signals in the perirhinal cortex were as intense as those in the entorhinal cortex (Figs 5D and 7B). The expression patterns were consistent through the rostral to caudal fields of the entorhinal cortex (data not shown). In the amygdala, intense signals were localized to a specific nucleus, such as the lateral (Figs 5D and 7B), accessory basal and medial nuclei (data not shown). As in the cerebral cortex, we did not detect MARCKS mRNA-positive glial cells in the hippocampal formation.

Discussion

The Northern blot analysis showed that MARCKS mRNA was highly expressed in certain regions of the macaque monkey brain, such as the association areas of the neocortex (FD, TE and PG), the hippocampus and the amygdala. The results from in situ hybridization histochemistry were consistent with those from Northern blot analysis, and allowed us to determine the distribution and characteristics of the MARCKS mRNA-positive cells.

Comparison with GAP-43

In previous studies, we investigated the gene expression of GAP-43, another PKC substrate, in the monkey brain (Oishi et al., 1994, 1995, 1998; Higo et al., 1998, 1999a). The Northern blot analysis confirmed that the amount of GAP-43 mRNA as well as MARCKS mRNA was higher in the association areas (FD, TE and PG) than in the lower sensory areas (PC, OB and OC) (Oishi et al., 1994, 1995, 1998). In situ hybridization histochemistry revealed prominent hybridization signals for GAP-43 mRNA in layers II–VI of the association areas (Higo et al., 1999a). In the lower sensory areas, the GAP-43 mRNA signals were weak in layers I–III, and cells with more intense signals were observed in layers IV–VI (Higo et al., 1999a). These findings combined with the present results confirmed that mRNAs of both the major PKC-substrates, MARCKS and GAP-43, were abundant in the association areas (FD, TE and PG) of the monkey cortex. Both mRNAs were abundant in the supragranular layers as well as in the infragranular layers of the association areas (Table 1).

Recent in situ hybridization studies have shown that GAP-43 mRNA hybridization signals were weaker in area 24 of Brodmann [the cingulate area, corresponding to the LA region of von Bonin and Bailey (McFarland et al., 2000)] and the parahippocampal cortex [corresponding to the TF and TH regions of von Bonin and Bailey (Eastwood and Harrison, 1998)] than in neighboring cortical areas, and intense signals were restricted to the deep layers (layers IV–VI). These results are also similar to our result for MARCKS mRNA (Figs 5B, 6B and 7A,D). Thus, the expression pattern of MARCKS mRNA in the monkey cerebral neocortex is almost identical to that of GAP-43 mRNA. Since transgenic mouse studies have shown that the expression levels of both MARCKS and GAP-43 in the adult brain are related to learning ability (McNamara et al., 1998; Routtenberg et al., 2000), neurons expressing these molecules may have a role in memory function.

In the hippocampus, the MARCKS mRNA expression pattern was different from that of GAP-43 mRNA. The hybridization signals for GAP-43 mRNA were more intense in the CA4 and CA3 subfields of Ammon's horn than in the granule cell layer of the dentate gyrus (Higo et al., 1998). In contrast, the signals for MARCKS mRNA were more intense in the granule cell layer of the dentate gyrus than in the CA4 and CA3 subfields of Ammon's horn (Figs 6B, 7D and Table 1). In the subicular complex, the hybridization signals for GAP-43 mRNA were weak (Higo et al., 1998), whereas the signals for MARCKS mRNA were intense (Figs 6B, 7D and Table 1). The differential gene expression of MARCKS and GAP-43 has also been reported in rat (McNamara and Lenox, 1997) and chicken (Meberg et al., 1996) brains. This differential gene expression may be related to the different cellular distributions of MARCKS and GAP-43 proteins. In contrast to GAP-43 protein, which accumulates only in presynaptic axonal terminals (Nelson and Routtenberg, 1985; Meiri et al., 1986, 1988; Skene et al., 1986), MARCKS protein is localized to postsynaptic dendritic spines as well as presynaptic axon terminals (Ouimet et al., 1990; Lu et al., 1998). One possibility is that MARCKS protein exists in the dendritic spines of neurons in the granule cell layer of the dentate gyrus and the subicular complex of the monkey. Immunohistochemical studies to localize MARCKS protein would be required to test this possibility.

Comparison with Other Animals

At least one previous study has attempted to determine the MARCKS mRNA distribution in the rat brain (McNamara and Lenox, 1997). In rat cerebral neocortex, MARCKS mRNA expression is enriched in the piriform, temporal and parietal areas, and is scarce in the cingulate area. In the temporal and parietal areas, moderate to high intensity hybridization signals were observed in all layers, except for layer I. These results in the rat cortex are similar to our present results in the monkey cortex, in which we observed prominent MARCKS mRNA expression in layers II–VI of the temporal (TE; Figs 5D and 7B) and parietal association areas (PG; Figs 6D and 7E) and weak expression in the cingulate area (LA; Figs 5B and 7A). However, the finding that MARCKS mRNA was present in astrocytes in the most superficial aspect of layer I of the rat cerebral cortex (McNamara and Lenox, 1997) is not consistent with the lack of MARCKS mRNA-positive glial cells in the monkey cortex observed in the present study.

That MARCKS mRNA is highly expressed in the rat amygdala and hippocampus (McNamara and Lenox, 1997) is consistent with our results for the monkey brain (Fig. 2A,B). The expression pattern within the hippocampus was, however, slightly different in the rat compared with the monkey. In the rat hippocampus, the MARCKS mRNA hybridization signals were more intense in the CA1 than in the CA3 subfield of Ammon's horn (McNamara and Lenox, 1997). We have shown that the hybridization signals for MARCKS mRNA were more intense in the CA3 than in the CA1 subfield of monkey hippocampus (Figs 5B, 6D, 10 and Table 1). The GAP-43 mRNA expression pattern in the hippocampus was also different in the monkey compared with the rat (Kruger et al., 1992; Yao et al., 1993; Higo et al., 1998). The phylogenetically different distribution of these molecules may be related to differences in hippocampal function between these species.

In humans, MARCKS mRNA was expressed in all layers of the dorsolateral prefrontal cortex (McNamara et al., 1999). This result is similar to our results concerning the prefrontal area of the monkey cortex (Figs 5B and 7A). Further studies of the human cerebral neocortex are needed to determine whether the amount of MARCKS mRNA is also higher in the association areas than in the primary areas.

The mRNA Signal Intensity among Different Areas and Cell Types

In the present study, we determined that the amount of MARCKS mRNA was higher in the association areas (FD, TE and PG) than in the primary sensory and motor areas (PB, OC and FA) of the monkey cortex (Fig. 2B,C). The amounts of MARCKS mRNA in the premotor (Fig. 2B) and secondary visual (Fig. 2B) areas were intermediate between those in the association areas and the primary motor and sensory areas. A previous study reported a gradient of phosphorylation for an 81 kDa (pI 4.0) protein, probably MARCKS, in the occipitotemporal visual processing pathway of macaque monkeys. This phosphorylation was higher in the secondary visual area (OB) than in the primary visual area (OC), and was highest in the temporal association area (TE) (Nelson and Routtenberg, 1985). Our result suggests that this gradient of phosphorylation is regulated, at least in part, by the level of mRNA.

We confirmed that both of the major PKC-substrates, MARCKS and GAP-43, were abundant in the supragranular layers, as well as in the infragranular layers of the association areas (Table 1). Since MARCKS mRNA, as well as GAP-43 mRNA, was localized in most neurons in the supragranular layers of the association areas (Higo et al., 1999a) (Fig. 8), some of the neurons in these layers should contain both MARCKS and GAP-43 mRNA. The abundance of these PKC-substrates suggests that PKC-associated signal transduction plays an important role in the plastic change of the neurons in the supragranular layers of the association areas, which supply both cortico-cortical connections (Barbas and Mesulam, 1981; Schwartz and Goldman, 1984; Friedman et al., 1986; Shiwa, 1987; Vogt and Pandya, 1987; Barbas, 1988; Johnson et al., 1989; Seltzer and Pandya, 1989; Andersen et al., 1990) and intrinsic connections (Amir et al., 1993; Levitt et al., 1993; Kritzer and Goldman-Rakic, 1995; Fujita and Fujita, 1996).

Signals were observed mainly in pyramidal (Fig. 11B,C,D) cells of the cerebral neocortex. Among pyramidal cells, we confirmed that certain types of cells contained the most prominent signals. Betz cells in layer V of FA contained MARCKS mRNA (Fig. 11C), suggesting that MARCKS was expressed in the long-projecting neurons. In OC, MARCKS mRNA was highly expressed in the Meynert cells in layers V or VI (Fig. 11D). Previous studies showed that some of these Meynert cells project to area MT of the cortex or the superior colliculus (Lund and Boothe, 1975; Tigges et al., 1981; Fries and Distel, 1983; Sipp and Zeki, 1989; Peters, 1994). Our findings might reflect a functional specialization regarding the plasticity of these projections.

In the hippocampus, we observed several distinctive subtypes of hippocampal non-principal neurons that expressed MARCKS mRNA, such as the fusiform-shaped neurons in the stratum oriens (Fig. 11E) and small neurons in the stratum radiatum of Ammonic subfields (Fig. 11F). This suggests that these hippocampal non-principal neurons, as well as the principal neurons, bear PKC-mediated plasticity.

Notes

This work was supported by grants from the Agency of Industrial Science and Technology, Ministry of Economy, Trade and Industry, Japan. We are grateful to Drs T. Arikuni and K. Kawano for valuable discussion and continuous encouragement during this study. We thank Dr D. Stumpo for providing the MARCKS cDNA clone.

Table 1

Summary of MARCKS and GAP-43 mRNA expression in the monkey cerebral neocortex and hippocampus

 MARCKS GAP-43 
The relative levels of MARCKS and GAP-43 mRNA expression were classified as follows: ++++, very intense; +++, intense; ++, moderate; +, weak. The relative expression of GAP-43 mRNA was taken from our previous reports (Oishi et al., 1994, 1995, 1998; Higo et al., 1998, 1999a). 
Cerebral neocortex 
    Association areas (FD, TE and PG) 
        Layer I 
        Layers II–III +++ +++ 
        Layers IV–VI +++ +++ 
    Primary areas (PB, OC and FA) 
        Layer I 
        Layers II–III 
        Layers IV–VI +++ +++ 
Hippocampus 
    Dentate gyrus ++++ ++ 
    CA4 ++ +++ 
    CA3 +++ ++++ 
    CA2 ++ 
    CA1 ++ ++ 
    Subiculum +++ 
    Presubiculum +++ 
 MARCKS GAP-43 
The relative levels of MARCKS and GAP-43 mRNA expression were classified as follows: ++++, very intense; +++, intense; ++, moderate; +, weak. The relative expression of GAP-43 mRNA was taken from our previous reports (Oishi et al., 1994, 1995, 1998; Higo et al., 1998, 1999a). 
Cerebral neocortex 
    Association areas (FD, TE and PG) 
        Layer I 
        Layers II–III +++ +++ 
        Layers IV–VI +++ +++ 
    Primary areas (PB, OC and FA) 
        Layer I 
        Layers II–III 
        Layers IV–VI +++ +++ 
Hippocampus 
    Dentate gyrus ++++ ++ 
    CA4 ++ +++ 
    CA3 +++ ++++ 
    CA2 ++ 
    CA1 ++ ++ 
    Subiculum +++ 
    Presubiculum +++ 
Figure 1.

Dissected neocortical regions for Northern blot analysis are shown on the left hemisphere of the cerebral cortex. The indicated regions correspond to the FD (prefrontal area), FB (premotor area), FA (primary motor area), PB (primary somatosensory area), TE (temporal association area), PG (parietal association area), OB (secondary visual area) and OC (primary visual area), named according to von Bonin and Bailey (von Bonin and Bailey, 1947).

Figure 1.

Dissected neocortical regions for Northern blot analysis are shown on the left hemisphere of the cerebral cortex. The indicated regions correspond to the FD (prefrontal area), FB (premotor area), FA (primary motor area), PB (primary somatosensory area), TE (temporal association area), PG (parietal association area), OB (secondary visual area) and OC (primary visual area), named according to von Bonin and Bailey (von Bonin and Bailey, 1947).

Figure 2.

(A) MARCKS mRNA was detected in all regions of the monkey brain, but the signal level differed among the regions. The differences in the amount of G3PDH mRNA may be due to an error in the amount of total RNA applied. (B) The normalized amount of MARCKS mRNA is shown as a multiple of the ratio of MARCKS mRNA to G3PDH mRNA in the standard total RNA from brain homogenate (See Materials and Methods for details). The average value of five monkey brains is shown with the standard error. In the cerebral neocortex, the amount of MARCKS mRNA was higher in the association areas (FD, TE and PG) than in the primary areas (PB, OC and FA). The expression levels in the hippocampus and amygdala were as high as those in the association areas of the neocortex. (C) The amount of MARCKS mRNA in each association area was compared with that in each primary area. The amount of MARCKS mRNA in FD and TE was significantly higher than that in each primary area (Mann–Whitney U-test, P < 0.05).

Figure 2.

(A) MARCKS mRNA was detected in all regions of the monkey brain, but the signal level differed among the regions. The differences in the amount of G3PDH mRNA may be due to an error in the amount of total RNA applied. (B) The normalized amount of MARCKS mRNA is shown as a multiple of the ratio of MARCKS mRNA to G3PDH mRNA in the standard total RNA from brain homogenate (See Materials and Methods for details). The average value of five monkey brains is shown with the standard error. In the cerebral neocortex, the amount of MARCKS mRNA was higher in the association areas (FD, TE and PG) than in the primary areas (PB, OC and FA). The expression levels in the hippocampus and amygdala were as high as those in the association areas of the neocortex. (C) The amount of MARCKS mRNA in each association area was compared with that in each primary area. The amount of MARCKS mRNA in FD and TE was significantly higher than that in each primary area (Mann–Whitney U-test, P < 0.05).

Figure 3.

(A) The coronal sectioning levels shown in (B) are indicated by lowercase letters. (B) Line drawings of coronal sections through the anterior to the posterior levels of monkey brain. The regions that included the representative association areas (FD, TE and PG), the representative primary sensory (PB and OC) and motor areas (FA), the hippocampus, and the entorhinal cortex were selected for in situ hybridization histochemistry (boxes), and the results are shown in Figures 5 and 6. amts, anterior middle temporal sulcus; ca, calcarine sulcus; ci, cingulate sulcus; cs, central sulcus; ip, intraparietal sulcus; l, lunate sulcus; la, lateral fissure; oi, inferior occipital sulcus; ots, occipitotemporal sulcus; po, parieto-occipital sulcus; prcs, precentral sulcus; ps, principal sulcus; rs, rhinal sulcus; ts, temporal sulcus.

(A) The coronal sectioning levels shown in (B) are indicated by lowercase letters. (B) Line drawings of coronal sections through the anterior to the posterior levels of monkey brain. The regions that included the representative association areas (FD, TE and PG), the representative primary sensory (PB and OC) and motor areas (FA), the hippocampus, and the entorhinal cortex were selected for in situ hybridization histochemistry (boxes), and the results are shown in Figures 5 and 6. amts, anterior middle temporal sulcus; ca, calcarine sulcus; ci, cingulate sulcus; cs, central sulcus; ip, intraparietal sulcus; l, lunate sulcus; la, lateral fissure; oi, inferior occipital sulcus; ots, occipitotemporal sulcus; po, parieto-occipital sulcus; prcs, precentral sulcus; ps, principal sulcus; rs, rhinal sulcus; ts, temporal sulcus.

Figure 4.

(A) Northern hybridization study to confirm probe specificity. The probe was hybridized to specific (2.6 and 4.7 kb) RNA transcripts. (B–D) Control experiments to confirm the specificity of the signals for MARCKS mRNA. Three adjacent coronal sections including the hippocampus are shown. Normal reactivities for MARCKS mRNA (B) produced positive hybridization signals. Only background signal levels were observed in the sections pretreated with ribonuclease A (C), and signals were dramatically reduced in the competition-control sections (D). Scale bar = 1 mm.

Figure 4.

(A) Northern hybridization study to confirm probe specificity. The probe was hybridized to specific (2.6 and 4.7 kb) RNA transcripts. (B–D) Control experiments to confirm the specificity of the signals for MARCKS mRNA. Three adjacent coronal sections including the hippocampus are shown. Normal reactivities for MARCKS mRNA (B) produced positive hybridization signals. Only background signal levels were observed in the sections pretreated with ribonuclease A (C), and signals were dramatically reduced in the competition-control sections (D). Scale bar = 1 mm.

Figure 5.

Photographs of Nissl-stained (A, C, E) and in situ hybridized sections (B, D, F) for the detection of MARCKS mRNA. (A, B) Sections including FD and LA (the cingulate area). (C, D) Sections including the amygdala, the entorhinal cortex, areas 35 and 36 (the perirhinal cortex), and TE. The arrowhead in (D) indicates hybridization signals in the lateral nucleus of the amygdala. (E, F) Sections including FA, PB, PC, PEm and PF. Abbreviations are the same as those in Figure 3. Scale bar = 1 mm.

Photographs of Nissl-stained (A, C, E) and in situ hybridized sections (B, D, F) for the detection of MARCKS mRNA. (A, B) Sections including FD and LA (the cingulate area). (C, D) Sections including the amygdala, the entorhinal cortex, areas 35 and 36 (the perirhinal cortex), and TE. The arrowhead in (D) indicates hybridization signals in the lateral nucleus of the amygdala. (E, F) Sections including FA, PB, PC, PEm and PF. Abbreviations are the same as those in Figure 3. Scale bar = 1 mm.

Figure 6.

Photographs of Nissl-stained (A, C, E) and in situ hybridized (B, D, F) sections for the detection of MARCKS mRNA. (A, B) Sections including the dentate gyrus, Ammon's horn, subiculum, presubiculum, TF and TH (the parahippocampal cortex), and TEO. (C, D) Sections including PE and PG. (E, F) Sections including OB and OC. Abbreviations are the same as those in Figure 3. Scale bar = 1 mm.

Photographs of Nissl-stained (A, C, E) and in situ hybridized (B, D, F) sections for the detection of MARCKS mRNA. (A, B) Sections including the dentate gyrus, Ammon's horn, subiculum, presubiculum, TF and TH (the parahippocampal cortex), and TEO. (C, D) Sections including PE and PG. (E, F) Sections including OB and OC. Abbreviations are the same as those in Figure 3. Scale bar = 1 mm.

Figure 7.

The relative expression levels of MARCKS mRNA in each coronal section of the cerebral cortex. The relative expression levels (0.1–1) of MARCKS mRNA were superimposed in pseudocolor on the digitized images of Nissl-stained sections (see Materials and Methods for details). (A) A section including FD and LA. (B) A section including the amygdala, entorhinal cortex, areas 35 and 36, and TE. The arrowhead indicates hybridization signals in the lateral nucleus of the amygdala. (C) A section including FA, PB, PC, PEm and PF. (D) A section including the dentate gyrus, Ammon's horn, subiculum, presubiculum, TF and TH, and TEO. (E) A section including PE and PG. (F) A section including both OB and OC. Abbreviations are the same as those in Figure 3. Scale bar = 1 mm.

The relative expression levels of MARCKS mRNA in each coronal section of the cerebral cortex. The relative expression levels (0.1–1) of MARCKS mRNA were superimposed in pseudocolor on the digitized images of Nissl-stained sections (see Materials and Methods for details). (A) A section including FD and LA. (B) A section including the amygdala, entorhinal cortex, areas 35 and 36, and TE. The arrowhead indicates hybridization signals in the lateral nucleus of the amygdala. (C) A section including FA, PB, PC, PEm and PF. (D) A section including the dentate gyrus, Ammon's horn, subiculum, presubiculum, TF and TH, and TEO. (E) A section including PE and PG. (F) A section including both OB and OC. Abbreviations are the same as those in Figure 3. Scale bar = 1 mm.

Figure 8.

Photomicrographs of the association (FD, A, B) and primary (PB, C, D) areas. (A, C) Nissl-stained sections. (B, D) Localization of MARCKS mRNA. In the association area, cells with intense hybridization signals were scattered almost equally in layers II–VI. In the primary area, cells with intense hybridization signals were more abundant in layers IV–VI than in layers I–III. Scale bar = 200 μm.

Figure 8.

Photomicrographs of the association (FD, A, B) and primary (PB, C, D) areas. (A, C) Nissl-stained sections. (B, D) Localization of MARCKS mRNA. In the association area, cells with intense hybridization signals were scattered almost equally in layers II–VI. In the primary area, cells with intense hybridization signals were more abundant in layers IV–VI than in layers I–III. Scale bar = 200 μm.

Figure 10.

Photomicrographs of the dentate gyrus (A, B) and CA1 subfield of Ammon's horn (C, D). (A, C) Nissl-stained sections. (B, D) Localization of MARCKS mRNA. The most intense hybridization signals were observed in the granule cell layer of the dentate gyrus. gc, granule cell layer; lm, stratum lacunosum-moleculare; ml, molecular layer; or, stratum oriens; pm, polymorphic layer; py, pyramidal cell layer; rd, stratum radiatum. Scale bar = 100 μm.

Figure 10.

Photomicrographs of the dentate gyrus (A, B) and CA1 subfield of Ammon's horn (C, D). (A, C) Nissl-stained sections. (B, D) Localization of MARCKS mRNA. The most intense hybridization signals were observed in the granule cell layer of the dentate gyrus. gc, granule cell layer; lm, stratum lacunosum-moleculare; ml, molecular layer; or, stratum oriens; pm, polymorphic layer; py, pyramidal cell layer; rd, stratum radiatum. Scale bar = 100 μm.

Figure 11.

Nomarski-contrast photomicrographs of neurons with hybridization signals for MARCKS mRNA. (A) The small neurons in layer I of TE. (B) Pyramidal cell in layer V of TE. (C) Betz cell in layer V of FA. (D) Meynert cell in layer VI of OC. (E) The fusiform-shaped neuron in the stratum oriens of the CA3 subfield of Ammon's horn. (F) The small neurons in the stratum radiatum of the CA3 subfield of Ammon's horn. Arrows in (B) and (C) show the apical dendrites of the pyramidal cells. Scale bar = 20 μm.

Figure 11.

Nomarski-contrast photomicrographs of neurons with hybridization signals for MARCKS mRNA. (A) The small neurons in layer I of TE. (B) Pyramidal cell in layer V of TE. (C) Betz cell in layer V of FA. (D) Meynert cell in layer VI of OC. (E) The fusiform-shaped neuron in the stratum oriens of the CA3 subfield of Ammon's horn. (F) The small neurons in the stratum radiatum of the CA3 subfield of Ammon's horn. Arrows in (B) and (C) show the apical dendrites of the pyramidal cells. Scale bar = 20 μm.

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