Abstract

Neurogranin is a postsynaptic substrate for protein kinase C (PKC). It has been identified in the central nervous system, and the expression has been related to postsynaptic plasticity. Using non-radioactive in situ hybridization histochemistry, we investigated whether mRNA expression of neurogranin varied among the cerebral region and cell types. In most areas of the neocortex excluding area OC (the primary visual area), intense signals were observed in the pyramidal cells in layers III, V and VI. In area OC, intense signals were observed in layers IV as well as layers III and VI. We previously showed that intense signals for GAP-43, a presynaptic PKC substrate, were observed in relay neurons of the lateral geniculate nucleus. From this result and the present result in area OC, we conclude that both pre- and postsynaptic PKC substrates (GAP-43 and neurogranin) are abundant in the geniculocortical synapses. In the hippocampus, intense signals were observed in the pyramidal cells in the subiculum. Taken together with our previous study showing intense signals for GAP-43 in Ammon’s horn, the result indicates that both PKC substrates are abundant in the connections between neurons in Ammon’s horn and in the subiculum.

Introduction

Protein kinase C (PKC) and PKC-associated signal transduction play an important role in synaptic plasticity (for reviews, see Nishizuka, 1986, 1995). Our previous studies investigated the gene expression of GAP-43 and MARCKS (myristoylated alanine-rich C-kinase substrate), which are major PKC-substrates, in the monkey central nervous system (Higo et al., 1998, 1999, 2002d, 2003; Oishi et al., 1998). GAP-43 (also known as B50, F1, neuromodulin, pp46, P-57 and GAP-48; for reviews, see Benowitz and Routtenberg, 1987, 1997) is known to accumulate in presynaptic axon terminals (Nelson and Routtenberg, 1985; Meiri et al., 1986, 1988; Skene et al., 1986). MARCKS has been localized to presynaptic axon terminals and small dendrites (Aderem et al., 1988; Ouimet et al., 1990; Swierczynski and Blackshear, 1995). There are several lines of evidence that relate the expression of both GAP-43 and MARCKS mRNAs with structural synaptic reorganization in the mature nervous system (Van der Zee et al., 1989; Tetzlaff et al., 1991; Linda et al., 1992; Levin and Dunn-Meynell, 1993; Chong et al., 1994; Aigner et al., 1995; Holtmaat et al., 1995; Bendotti et al., 1997; McNamara et al., 2000; McNamara and Lenox, 2000). We previously reported that mRNAs of GAP-43 and MARCKS were highly expressed in specific regions of the adult monkey brain, such as the association areas of the cerebral neocortex and Ammon’s horn of the hippocampus (Higo et al., 1998, 1999, 2002d; Oishi et al., 1998). Furthermore, we previously reported that GAP-43 and MARCKS mRNAs were expressed in the monkey lateral geniculate nucleus (LGN) in an activity-dependent manner (Higo et al., 2000, 2002a). These results have enhanced our understanding of the molecular basis of plasticity in each region of the monkey brain.

In the present study, we focused on the gene expression of neurogranin (also known as RC3, canarigranin, BICKS and p17; for a review, see Gerendasy and Sutcliffe, 1997), another major neuron-specific PKC substrate, which has been localized to postsynaptic dendrites (Represa et al., 1990; Watson et al., 1992). Among the PKC substrates, GAP-43 and neurogranin are the most closely related molecules having identical PKC phosphorylation sites and calmodulin-binding domains (Watson et al., 1990; Baudier et al., 1991; for reviews, see Gerendasy and Sutcliffe, 1997; Chakravarthy et al., 1999). Both GAP-43 and neurogranin bind calmodulin when they are not phosphorylated by PKC and release calmodulin as calcium-calmodulin in response to Ca2+ influx. Thus, GAP-43 and neurogranin are presynaptic and postsynaptic counterparts of calmodulin storage proteins. Like GAP-43, neurogranin and its mRNA are expressed at high levels in the developing rat brain. While the expressions decrease in most regions of the mature rat brain, high levels of expression remain in specific regions such as the hippocampus and cerebral neocortex (Represa et al., 1990; Watson et al., 1990). Although the precise role of neurogranin in the mature nervous system is not yet clear, there are several lines of evidence that relate the expression level with plasticity and memory function in the adult nervous system. Phosphorylation of neurogranin is enhanced during long-term potentiation and depression (Klann et al., 1993; Pasinelli et al., 1995; Ramakers et al., 1995, 2000a,b; Chen et al., 1997) and reduced expression has been observed in Alzheimer’s disease (Chang et al., 1997; Davidsson and Blennow, 1998). Further, neurogranin knockout mice have been shown to have impaired spatial learning (Pak et al., 2000). Thus, the region-specific neurogranin expression in the adult central nervous system might reflect functional specialization which relates to postsynaptic plasticity in each region. In the present study, we performed a non-radioactive in situ hybridization histochemical analysis of the cerebral cortex of the macaque monkey to determine the localization and type of cells expressing neurogranin mRNA. We then quantified the expression levels of neurogranin mRNA in each area of the cerebral neocortex and each subregion of the hippocampal formation. Taken together with our previous studies investigating the expression of GAP-43 mRNA (Higo et al., 1998, 1999; Oishi et al., 1998), we suggest that both pre- and postsynaptic PKC substrates (GAP-43 and neurogranin) are abundant in several specific connections of the monkey brain. The preliminary results of these studies have been reported elsewhere (Higo et al., 2002b,c).

Materials and Methods

Animals and Tissue Preparation

Brain tissue was obtained from 11 macaque monkeys (six Macaca fuscata and five M. mulatta) aged 2 or more years. Monkeys were purchased from a local provider, or were 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. To investigate the effect of visual deprivation on the expression of neurogranin mRNA, five monkeys received tetrodotoxin (TTX, 15 µg in 10 µl of normal saline; Sigma, St Louis, MO), injected intravitreously into the right eye every fifth day for a total of 5 (n = 2), 10 (n = 2), and 30 (n = 1) days before they were killed. The monkeys were anesthetized with ketamine hydrochloride (10 mg/kg, i.m.) and pentobarbital sodium (Nembutal, 20 mg/kg, i.v.), prior to TTX administration. The pupillary light reflex of these monocularly deprived monkeys remained suppressed throughout the deprivation period.

The animals were pretreated with i.m. injections of ketamine hydrochloride (10 mg/kg) and deeply anesthetized by intravenous 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 U) 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 blocks were postfixed in 4% PFA and 5% sucrose and immersed in 30% sucrose for cryoprotection. 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 –80°C until dissection.

In Situ Hybridization

Brain segments from seven areas of the neocortex and the hippocampal formation were sectioned coronally to a thickness of 16 µm on a cryostat. We identified individual neocortical areas, including the prefrontal area (FD), the temporal association area (TE), the parietal association area (PG), the primary motor area (FA), the primary auditory area (TC), the primay somatosensory area (PB), and the primary visual area (OC), using the cytoarchitectonic criteria of von Bonin and Bailey (1947).

The procedure for in situ hybridization was performed as described in our previous report (Higo et al., 2003). Briefly, the sections on the slides were fixed in 4% PFA, treated with 30 µg/ml proteinase K (Roche Diagnostics, Germany) and dehydrated through a graded series of ethanols. Sections were prehybridized in 50% formamide, 600 mM NaCl, 1× Denhardt’s solution, 0.25% SDS, 10 mM Tris–HCl (pH 7.6), 1 mM EDTA and 200 µg/ml tRNA for 3 h at 50°C. Following prehybridization, sections were transferred to hybridization buffer containing an additional 10% dextran sulfate and 1 µg/ml digoxigenin-labeled RNA probe, which was made from human RC3/neurogranin cDNA (pHRC3, 1.3 kb; a gift from Dr J.B. Watson, University of Calfornia, Los Angeles, CA). Hybridization was performed for at least 16 h at 50°C. The hybridized sections were washed with SSC and then treated with RNase A (30 µg/ml; Roche Diagnostics). Sections were then incubated for 1 h in diluted (1:500) anti-digoxigenin-alkaline phosphatase conjugate (DIG Nucleic Acid Detection Kit; Roche Diagnostics). Finally, the sections were incubated in color-developing buffer containing NBT and BCIP for 5 h.

The specificity of the probes was confirmed by Northern blot analysis, in which a specific 1.3 kb RNA transcript was observed (Fig. 1A). Intense signals for neurogranin mRNA were observed in all neocortical areas examined and weak signals were seen in the hippocampus. No signal was detected in the cerebellum. The lack of expression in the cerebellum is consistent with our previous report using a histochemical technique (Higo et al., 2003). In addition, control sections were hybridized by the method described above, using the sense probe for each mRNA. These control sections showed no specific signals (Fig. 1C).

Quantification and Image Analysis

We quantified the expression patterns in the neocortex and the hippocampus using the same method that we used for the quantification of GAP-43 and MARCKS mRNAs (Higo et al., 1999, 2002d). Briefly, optical density (OD) was measured in each layer of a 300-µm-wide column that sampled all layers of the neocortex or each hippocampal subregion. The OD of the background staining in each section was measured in the subjacent white matter. Six columns from each cortical area and each hippocampal subregion (three columns from each of two sections) were measured for each monkey. We calculated the normalized OD of each layer or each subregion from in situ hybridized sections (in situ ODsignalin situ ODbackground). We also calculated the normalized OD from Nissl-stained sections (Nissl ODsignal – Nissl ODbackground), which represent the cell density in each region. To compensate for differences of cell density among layers or subregions, we determined the ratio as follows (Figs 4, 5E and 8):

graphic

We also represented the relative expression levels of mRNAs among subregions of the hippocampal formation by dividing the OD in each pixel of the digitized images of in situ hybridized sections by that of the adjacent Nissl-stained sections (Fig. 7), as described previously (Higo et al., 1998, 2002a). The OD was measured in each pixel of the digitized in situ hybridized (in situ pODsignal) and Nissl-stained sections (Nissl pODsignal). The ODbackground was estimated as the average of the ODs from five randomly sampled local regions of the white matter.

The OD of each pixel in the in situ hybridized sections was normalized by the following formula:

graphic

Results

Neocortex

The laminar expression patterns of neurogranin mRNA were similar among areas FD, TE, PG, FA, TC and PB (Fig. 2B,D,F,H,J,L). Prominent hybridization signals were observed in the cell body and proximal dendrites of pyramidal cells in layers III, V, and VI (Figs 2B,D,F,H,J,L and 3A). We also observed prominent signals in the horizontal cells (Fig. 3B) and fusiform cells in these layers (Fig. 3C). Cells with the most intense signals were frequently observed in layer III (Fig. 2B,D,F,H,J,L). While the neurons in layers V and VI contained weaker hybridization signals than those in layers III, the proportion of positive neurons in layers V and VI tended to be larger than that in layer III. Signals were weak in the small round cells in layers II and IV. The large pyramidal cells in layer V of area FA also contained the hybridization signals (arrows in Figs 2H and 3D), but the signals were weaker than in neighboring smaller pyramidal cells in layer V and pyramidal cells in layers III and VI. No signal was observed in layer I (Fig. 2B,D,F,H,J,L). Quantitative analysis indicated that the hybridization levels (the neurogranin OD/Nissl OD ratios) in these areas were higher in layers III, V and VI than in the remaining layers (Fig. 4AF). Although detailed quantitative analysis has not been performed, most of the other neocortical areas excluding area OC showed similar laminar expression patterns to those in areas FD, TE, PG, FA, TC and PB.

In area OC, the laminar expression pattern was different from those in the remaining areas. In contrast to the other neocortical areas, the intense hybridization signals were observed in the small round cells in layer IV as well as in the pyramidal cells in layers III and VI (Figs 3E and 5B). Signals were weak in layers II and V. Figure 5C,D shows the results in the border region between area OC and area OB (the secondary visual area). Intense signals were observed in layer IV of area OC, but not in layer IV of area OB. Quantitative analysis indicated that the hybridization level (the neurogranin OD/Nissl OD ratio) was higher in layers IV and VI than in the remaining layers (Fig. 5E). In layer IV, the ratio was higher in layers IVB and IVCα, which receive inputs from magnocellular neurons in the LGN (Lund et al., 1994), than in layers IVA and IVCβ, which receive inputs from parvocellular neurons. As in the other cortical areas, no signal was observed in layer I of area OC (Fig. 5B).

To investigate the effect of visual input on the expression of neurogranin mRNA in area OC, we performed in situ hybridization histochemistry in monkeys that had been deprived of monocular visual input via intraocular injections of TTX. After monocular deprivation for 5, 10, or 30 days, we observed a periodic pattern of staining for cytochrome oxidase in layer IV of area OC (Fig. 6B), indicating reduced neuronal activity of the ocular dominance columns that received visual input from the TTX-injected eye. However, we did not detect such a periodic ocular dominance pattern of staining for neurogranin mRNA after monocular deprivation for each period (Fig. 6C). Unequal distribution of neurogranin mRNA in layer IV correlated with the cell density revealed by Nissl-stained section (Fig. 6A). The hybridization pattern for neurogranin mRNA was identical to those for normal monkeys. These results indicate that the expression of neurogranin mRNA in area OC is not affected after monocular deprivation for these periods.

Hippocampal Formation and Surrounding Regions

In the entorhinal cortex and perirhinal cortex (areas 35 and 36), the hybridization signals were weaker than in the neighboring neocortical areas (areas TE and TA; Fig. 7B,C). In the amygdala, intense hybridization signals for neurogranin mRNA were observed in specific nuclei, such as the basal and lateral nuclei (arrowheads and double arrowheads in Fig. 7B,C). Intense signals were also observed in the claustrum (Fig. 7B,C).

In the hippocampal formation, the most intense hybridization signals were observed in the subiculum (Fig. 7E,F; see Fig. 3F). In the parahippocampal cortex, the signals were weaker than in the neighboring neocortical area (TEO) and the prominent signals were restricted to the deeper layers. Intense signals were observed in the striatum such as the caudate nucleus, but not in the thalamus such as the LGN (Fig. 7E,F). Quantitative analysis in the principal layers of the hippocampus indicated that the neurogranin OD/Nissl OD ratio was highest in the subiculum, higher in the CA3 and CA1 subfields of Ammon’s horn and weak in the dentate gyrus and CA4 subfield of Ammon’s horn (Fig. 8).

Discussion

Comparison with other Animals

At least two previous studies reported the expression of neurogranin and its mRNA in the mature rat brain (Represa et al., 1990; Watson et al., 1990). In general, our results in the monkey brain are consistent with the results in the rat brain in that the prominent mRNA expression for neurogranin was observed in the cerebral neocortex, hippocampus and striatum, but not in the thalamus and the cerebellum (Represa et al., 1990; Watson et al., 1990).

In the rat neocortex, the most intense hybridization signals for neurogranin mRNA were observed in large neurons in layers III, V and VI (Watson et al., 1990). The previous results in the rat neocortex are similar to the present result in most cortical areas of the monkey brain (areas FD, TE, PG, FA, TC and PB). However, the expression of neurogranin mRNA in layer IV of the visual area (area OC), which was observed in the monkey neocortex, has not been reported in the rat neocortex (Watson et al., 1990). The specific laminar distribution of neurogranin mRNA in the visual area may be a characteristic of animals that have highly developed vision, such as primates. In the human, the distribution of neurons expressing neurogranin mRNA has been studied in only a few neocortical areas (temporal and frontal cortex; Chang et al., 1997). The previous results in these areas of the human neocortex showing prominent hybridization signals in the pyramidal cells throughout layers II–VI are similar to our result in the monkey neocortex. A further experiment in the visual cortex is necessary to determine whether the specific laminar distribution of neurogranin mRNA is also present in the human primary visual area (area OC).

In the rat hippocampal formation, prominent signals were observed in the pyramidal cell layer of all regions. Among the hippocampal subregions, the CA3 subfield of Ammon’s horn has shown the most intense hybridization signals (Watson et al., 1990). Previous findings in the rat hippocampus differ from those of the present study in the monkey hippocampus, which showed the highest expression in the subiculum. Moreover, Northern and Western blot analyses in the rat have shown that the expression levels of both neurogranin mRNA and neurogranin protein were almost similar between the cerebral neocortex and hippocampus (Represa et al., 1990; Watson et al., 1990). The results in the rat are inconsistent with the present result from both the Northern blot analysis and in situ hybridization histochemistry in the monkey brain, which showed more intense hybridization signals in the cerebral neocortex than in the hippocampus. We previously reported that the expression patterns of GAP-43 and MARCKS, which are other PKC substrates, in the monkey hippocampus (Higo et al., 1998, 2002d) were also different from those in the rat hippocampus (Kruger et al., 1992; Yao et al., 1993; McNamara and Lenox, 1997). The phylogenetically different expression of these PKC-substrates may be related to the differential hippocampal function between these species.

Comparison with PKC and Type II Calcium/Calmodulin-dependent Protein Kinase

PKC consists of at least 11 isoforms. Previous histochemical studies of the monkey cerebral neocortex reported the region-specific distributions of several isoforms of PKC (Tominaga et al., 1993; Fukuda et al., 1994). Among them, the expression of PKCγ is similar to the expression of neurogranin. PKCγ immunoreactivity is observed in the dendrites of pyramidal cells in layers II, III and VI of the primary motor area (area FA; Tominaga et al., 1993). In the primary visual area (area OC), PKCγ immunoreactivity is observed in layers II, IV and VI (Fukuda et al., 1994). A previous knockout mouse study showed that stimulation of PKC with a phorbol ester increased neurogranin phosphorylation in wild type mice but failed to affect the phosphorylation in mice lacking PKCγ (Ramakers et al., 1999). Thus, phosphorylation of neurogranin in cortical neurons may be accomplished by PKCγ.

Neurogranin is involved in the activity regulation of type II calcium/calmodulin-dependent protein kinase (CAMKII; Pak et al., 2000). CAMKII consists of four isoforms (α, β, γ, δ) encoded by separate genes. Among them, the expression of CAMKIIα in the monkey cerebral neocortex is similar to the expression of neurogranin mRNA. CAMKIIα immunoreactivity is observed in the dendrites of pyramidal cells in layers II–VI of the monkey sensory-motor cortex (areas FA and PB; Jones et al., 1994). In the primary visual area (area OC), CAMKIIα immunoreactivity is observed in layer IV nonpyramidal cells as well as in pyramidal cells (Tighilet et al., 1998). Thus, neurogranin may regulate CAMKIIα activation in monkey cortical neurons. When monocular visual input is deprived for several (7–16) days by TTX injection, CAMKIIα mRNA expression is increased in deprived ocular dominance columns (Tighilet et al., 1998). This previous result is in contrast to our present result showing no deprivation effect on the expression of neurogranin mRNA. The expression level of CAMKIIα mRNA and neurogranin mRNA may be regulated by different mechanisms.

The Expression of PKC Substrates in the Specific Connection of the Monkey Brain

In previous studies, we investigated the gene expression of GAP-43, another PKC substrate that accumulates only in presynaptic axon terminals (Nelson and Routtenberg, 1985; Meiri et al., 1986, 1988; Skene et al., 1986). From the subcellular distribution of neurogranin and GAP-43 proteins and our findings summarized in Table 1, we suggest that both postsynaptic PKC substrate (neurogranin) and presynaptic PKC substrate (GAP-43) are abundant in some specific connections (Fig. 9). (i) Intense hybridization signals for GAP-43 mRNA were observed in pyramidal cells in superficial layers as well as deep layers of the association areas (Higo et al., 1999). The present result showed high levels of expression of neurogranin mRNA in layers III, V and VI of the neocortical areas. Thus, GAP-43 may be abundant in the presynaptic membrane and neurogranin may be abundant in the postsynaptic membrane of the corticocortical connections that originate from the association areas of the neocortex and terminate outside layer IV, which are the presumed feedback connections (Felleman and Van Essen, 1991). (ii) We previously showed that intense hybridization signals for GAP-43 were observed in excitatory relay neurons in the LGN (Higo et al., 2000), indicating that GAP-43 protein is localized in the geniculocortical axon terminals. Taken together with the present result in area OC showing intense hybridization signals for neurogranin mRNA in thalamic recipient neurons in layer IV, we concluded that both pre- and postsynaptic PKC substrates are abundant in the geniculocortical connections. (iii) In the hippocampus, intense signals for GAP-43 mRNA were observed in the pyramidal cell layer of Ammon’s horn (Higo et al., 1998). In the present study, we observed intense hybridization signals for neurogranin mRNA in the subicular pyramidal cells. These results indicate that both PKC substrates are abundant in the connections between Ammon’s horn and the subiculum.

There is accumulating evidence indicating that the formation of new synapses is the structural basis of long-term potentiation and memory function (Chang and Greenough, 1984; Buchs and Muller, 1996; Kleim et al., 1996; Toni et al., 1999). GAP-43 and neurogranin are thought to be involved in structural changes accompanying the induction of long-term potentiation and depression (Ramakers et al., 1995, 2000a,b). Although further understanding of the intracellular roles of neurogranin and GAP-43 is a prerequisite for an appropriate interpretation, we suggest that connections containing both presynaptic GAP-43 and postsynaptic neurogranin may be capable of forming new synapses in the mature cerebral cortex, because new synapse formation should involve both pre- and postsynaptic morphological changes.

We are grateful to Drs S. Yamane and K. Kawano for valuable discussion and continuous encouragement during this study and Mr T. Takasu, Ms A. Kameyama and Ms A. Muramatsu for excellent technical assistance. We thank Dr J.B. Watson for providing the neurogranin/RC3 cDNA clone. This work was supported by the National Institute of Advanced Industrial Science and Technology of METI, the Japan Society for the Promotion of Science, a Grant-in-Aid for the 21st Century COE Research (A2) and the Cooperation Research Program of the Primate Research Institute of Kyoto University.

Figure 1. (A) Northern hybridization study to confirm probe specificity. The probe was hybridized to specific RNA transcripts (1.3 kb). Intense signals were observed in all neocortical areas examined (areas PB, TE and OC) and weak signals were observed in the hippocampus. No signal was detected in the cerebellum. (B, C) Control experiments to confirm the specificity of the signals for neurogranin mRNA. The adjacent coronal sections including cortical area TE are shown. Normal reaction for neurogranin mRNA (B) produced positive hybridization signals. No signal was observed in the section hybridized with a sense probe (C). Scale bar = 500 µm.

Figure 1. (A) Northern hybridization study to confirm probe specificity. The probe was hybridized to specific RNA transcripts (1.3 kb). Intense signals were observed in all neocortical areas examined (areas PB, TE and OC) and weak signals were observed in the hippocampus. No signal was detected in the cerebellum. (B, C) Control experiments to confirm the specificity of the signals for neurogranin mRNA. The adjacent coronal sections including cortical area TE are shown. Normal reaction for neurogranin mRNA (B) produced positive hybridization signals. No signal was observed in the section hybridized with a sense probe (C). Scale bar = 500 µm.

Figure 2. Areas FD (A, B), TE (C, D), PG (E, F), FA (G, H), TC (I, J) and PB (K, L) of the cortex. (A, C, E, G, I, K) Nissl-stained sections. (B, D, F, H, J, L) Localization of neurogranin mRNA. In areas FD, TE, PG, FA, TC and PB, intense hybridization signals for neurogranin mRNA were observed in layers III, V and VI. No signal was observed in layer I. Arrows in H indicate the large pyramidal cells (Betz cells) in layer V of area FA, in which the signals were weaker than in neighboring smaller pyramidal cells in layer V and in pyramidal cells in layers III and VI. Scale bar = 200 µm.

Figure 2. Areas FD (A, B), TE (C, D), PG (E, F), FA (G, H), TC (I, J) and PB (K, L) of the cortex. (A, C, E, G, I, K) Nissl-stained sections. (B, D, F, H, J, L) Localization of neurogranin mRNA. In areas FD, TE, PG, FA, TC and PB, intense hybridization signals for neurogranin mRNA were observed in layers III, V and VI. No signal was observed in layer I. Arrows in H indicate the large pyramidal cells (Betz cells) in layer V of area FA, in which the signals were weaker than in neighboring smaller pyramidal cells in layer V and in pyramidal cells in layers III and VI. Scale bar = 200 µm.

Figure 3. High-magnification photomicrographs of cortical neurons that have hybridization signals for neurogranin mRNA. (A) The pyramidal cell in layer III of area TE. (B) The horizontal cell in layer VI of area TE. (C) The fusiform cell in layer VI of area TE. (D) The large pyramidal cell in layer V of area FA. (E) The small round cells in layer IV of area OC. (F) The pyramidal cell in the subiculum. Scale bar = 20 µm.

Figure 3. High-magnification photomicrographs of cortical neurons that have hybridization signals for neurogranin mRNA. (A) The pyramidal cell in layer III of area TE. (B) The horizontal cell in layer VI of area TE. (C) The fusiform cell in layer VI of area TE. (D) The large pyramidal cell in layer V of area FA. (E) The small round cells in layer IV of area OC. (F) The pyramidal cell in the subiculum. Scale bar = 20 µm.

Figure 4. To quantify the hybridization signal, the neurogranin OD/Nissl OD ratios in each of six cortical areas were calculated (see Materials and Methods for details). The ratios were higher in layers III and VI than in the remaining layers. Asterisks indicate significant difference from the lowest value in each area (*P < 0.02; **P < 0.01, one sample t-test, n = 6).

Figure 4. To quantify the hybridization signal, the neurogranin OD/Nissl OD ratios in each of six cortical areas were calculated (see Materials and Methods for details). The ratios were higher in layers III and VI than in the remaining layers. Asterisks indicate significant difference from the lowest value in each area (*P < 0.02; **P < 0.01, one sample t-test, n = 6).

Figure 5. (A, B) Nissl-stained (A) and in situ hybridized sections (B) of area OC. In the primary visual area, intense signals for neurogranin mRNA were observed in the small round cells in layer IV as well as in the pyramidal cells in layers III and VI. No signal was observed in layer I. Scale bar = 200 µm. (C, D) Nissl-stained (C) and in situ hybridized sections (D) of the border region between area OC and area OB (the secondary visual area). Arrowheads indicate the border. Intense signals were observed in layer IV of area OC, but not in layer IV of area OB. Scale bar = 500 µm. (E) The neurogranin OD/Nissl OD ratio in area OC. The ratio was higher in layers IV and VI than in the remaining layers. *Significantly different from the lowest value (layer II; P < 0.02, one sample t-test, n = 6).

Figure 5. (A, B) Nissl-stained (A) and in situ hybridized sections (B) of area OC. In the primary visual area, intense signals for neurogranin mRNA were observed in the small round cells in layer IV as well as in the pyramidal cells in layers III and VI. No signal was observed in layer I. Scale bar = 200 µm. (C, D) Nissl-stained (C) and in situ hybridized sections (D) of the border region between area OC and area OB (the secondary visual area). Arrowheads indicate the border. Intense signals were observed in layer IV of area OC, but not in layer IV of area OB. Scale bar = 500 µm. (E) The neurogranin OD/Nissl OD ratio in area OC. The ratio was higher in layers IV and VI than in the remaining layers. *Significantly different from the lowest value (layer II; P < 0.02, one sample t-test, n = 6).

Figure 6. Surface parallel sections through area OC of a monkey that had been monocularly deprived for 30 days. (A) Nissl-stained section. (B) Cytochrome oxidase-stained section. (C) Localization of neurogranin mRNA. No deprivation effect was observed for neurogranin mRNA. Scale bar = 1 mm.

Figure 6. Surface parallel sections through area OC of a monkey that had been monocularly deprived for 30 days. (A) Nissl-stained section. (B) Cytochrome oxidase-stained section. (C) Localization of neurogranin mRNA. No deprivation effect was observed for neurogranin mRNA. Scale bar = 1 mm.

Figure 7.  Photographs of Nissl-stained (A, D) and in situ hybridized (B, E) sections for the detection of neurogranin mRNA. The relative expression levels of neurogranin mRNA in each section of the cerebral cortex are also shown (C, F). The relative expression levels (0.1–1) of neurogranin mRNA were superimposed in pseudocolor on the digitized images of Nissl-stained sections (see Materials and Methods for details). (AC) Sections including the amygdala, the entorhinal cortex, areas 35 and 36 (the perirhinal cortex), TE, TA and claustrum. The arrowheads and double arrowheads in B and C indicate hybridization signals in the basal and lateral nuclei of the amygdala, respectively. (DF) Sections including the dentate gyrus, Ammon’s horn, subiculum, presubiculum, TF and TH (the parahippocampal cortex), TEO, TA, the caudate nucleus and the lateral geniculate nucleus. Am, amygdala; Cd, caudate nucleus; Cl, claustrum; DG, dentate gyrus; Ent, entorhinal cortex; LGN, lateral geniculate nucleus; PreS, presubiculum; Sub subiculum; amts, anterior middle temporal sulcus; ots, occipitotemporal sulcus; rs, rhinal sulcus; ts, temporal sulcus. Scale bar = 2 mm.

Figure 7.  Photographs of Nissl-stained (A, D) and in situ hybridized (B, E) sections for the detection of neurogranin mRNA. The relative expression levels of neurogranin mRNA in each section of the cerebral cortex are also shown (C, F). The relative expression levels (0.1–1) of neurogranin mRNA were superimposed in pseudocolor on the digitized images of Nissl-stained sections (see Materials and Methods for details). (AC) Sections including the amygdala, the entorhinal cortex, areas 35 and 36 (the perirhinal cortex), TE, TA and claustrum. The arrowheads and double arrowheads in B and C indicate hybridization signals in the basal and lateral nuclei of the amygdala, respectively. (DF) Sections including the dentate gyrus, Ammon’s horn, subiculum, presubiculum, TF and TH (the parahippocampal cortex), TEO, TA, the caudate nucleus and the lateral geniculate nucleus. Am, amygdala; Cd, caudate nucleus; Cl, claustrum; DG, dentate gyrus; Ent, entorhinal cortex; LGN, lateral geniculate nucleus; PreS, presubiculum; Sub subiculum; amts, anterior middle temporal sulcus; ots, occipitotemporal sulcus; rs, rhinal sulcus; ts, temporal sulcus. Scale bar = 2 mm.

Figure 8. The neurogranin OD/Nissl OD ratio in the hippocampal principal layers. Asterisks indicate significant difference from the lowest value (DG; *P < 0.02; **P < 0.01, one sample t-test, n = 6). Abbreviations as in Figure 7.

Figure 8. The neurogranin OD/Nissl OD ratio in the hippocampal principal layers. Asterisks indicate significant difference from the lowest value (DG; *P < 0.02; **P < 0.01, one sample t-test, n = 6). Abbreviations as in Figure 7.

Figure 9. Schematic diagram showing the expression of neurogranin and GAP-43 mRNAs in the neurons subserving the specific connections. The expression of GAP-43 mRNA was taken from our previous report (Higo et al., 1998, 1999, 2000). Taken together with the present results about the expression of neurogranin mRNA, we suggest that both postsynaptic PKC substrate (neurogranin) and presynaptic PKC substrate (GAP-43) are abundant in the connections shown above.

Figure 9. Schematic diagram showing the expression of neurogranin and GAP-43 mRNAs in the neurons subserving the specific connections. The expression of GAP-43 mRNA was taken from our previous report (Higo et al., 1998, 1999, 2000). Taken together with the present results about the expression of neurogranin mRNA, we suggest that both postsynaptic PKC substrate (neurogranin) and presynaptic PKC substrate (GAP-43) are abundant in the connections shown above.

Table 1


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

 Neurogranin GAP-43 
Cerebral neocortex   
Areas FD, TE and PG   
 Layer I – 
 Layer II ++ 
 Layer III +++ +++ 
 Layer IV ++ 
 Layer V ++ +++ 
 Layer VI +++ +++ 
Areas FA, TC and PB   
 Layer I – 
 Layer II 
 Layer III +++ 
 Layer IV ++ 
 Layer V ++ +++ 
 Layer VI +++ +++ 
Area OC   
 Layer I – 
 Layer II 
 Layer III ++ 
 Layer IV +++ ++ 
 Layer V +++ 
 Layer VI +++ +++ 
Hippocampus   
 Dentate gyrus ++ 
 CA4 ++ 
 CA3 ++ +++ 
 CA1 ++ ++ 
 Subiculum +++ 
 Presubiculum 
 Neurogranin GAP-43 
Cerebral neocortex   
Areas FD, TE and PG   
 Layer I – 
 Layer II ++ 
 Layer III +++ +++ 
 Layer IV ++ 
 Layer V ++ +++ 
 Layer VI +++ +++ 
Areas FA, TC and PB   
 Layer I – 
 Layer II 
 Layer III +++ 
 Layer IV ++ 
 Layer V ++ +++ 
 Layer VI +++ +++ 
Area OC   
 Layer I – 
 Layer II 
 Layer III ++ 
 Layer IV +++ ++ 
 Layer V +++ 
 Layer VI +++ +++ 
Hippocampus   
 Dentate gyrus ++ 
 CA4 ++ 
 CA3 ++ +++ 
 CA1 ++ ++ 
 Subiculum +++ 
 Presubiculum 

The relative levels of neurogranin and GAP-43 mRNA expression were classified as follows: +++, intense; ++, moderate; +, weak; –, no signal. The expression pattern of neurogranin mRNA is different between area OC and the remaining areas of the cerebral neocortex. The expression pattern of GAP-43 mRNA is different between the association areas (FD, TE and PG) and the primary motor and sensory areas (FA, TC, PB and OC). The relative expression of GAP-43 mRNA was taken from our previous reports (Higo et al., 1998, 1999; Oishi et al., 1998).

References

Aderem AA, Albert KA, Keum MM, Wang JKT, Greengard P, Cohn ZA (
1988
) Stimulus-dependent myristoylation of a major substrate for protein kinase C.
Nature
 
332
:
362
–364.
Aigner L, Arber S, Kapfhammer JP, Laux T, Schneider C, Botteri F, Brenner H-R, Caroni P (
1995
) Overexpression of the neural growth-associated protein GAP-43 induces nerve sprouting in the adult nervous system of transgenic mice.
Cell
 
83
:
269
–278.
Baudier J, Deloulme JC, Van Dorsselaer A, Black D, Matthes HW (
1991
) Purification and characterization of a brain-specific protein kinase C substrate, neurogranin (p17). Identification of a consensus amino acid sequence between neurogranin and neuromodulin (GAP43) that corresponds to the protein kinase C phosphorylation site and the calmodulin-binding domain.
J Biol Chem
 
266
:
229
–237.
Bendotti C, Baldessari S, Pende M, Southgate T, Guglielmetti F, Samanin R (
1997
) Relationship between GAP-43 expression in the dentate gyrus and synaptic reorganization of hippocampal mossy fibres in rats treated with kainic acid.
Eur J Neurosci
 
9
:
93
–101.
Benowitz LI, Routtenberg A (
1987
) A membrane phosphoprotein associated with neural development, axonal regeneration, phospholipid metabolism, and synaptic plasticity.
Trends Neurosci
 
10
:
527
–532.
Benowitz LI, Routtenberg A (
1997
) GAP-43: an intrinsic determinant of neuronal development and plasticity.
Trends Neurosci
 
20
:
84
–91.
Buchs PA, Muller D (
1996
) Induction of long-term potentiation is associated with major ultrastructural changes of activated synapses.
Proc Natl Acad Sci USA
 
93
:
8040
–8045.
Chakravarthy B, Morley P, Whitfield J (
1999
) Ca2+-calmodulin and protein kinase Cs: a hypothetical synthesis of their conflicting convergences on shared substrate domains.
Trends Neurosci
 
22
:
12
–16.
Chang F-L, Greenough WT (
1984
) Transient and enduring morphological correlates of synaptic activity and efficacy change in the rat hippocampal slice.
Brain Res
 
309
:
35
–46.
Chang JW, Schumacher E, Coulter PM II, Vinters HV, Watson JB (
1997
) Dendritic translocation of RC3/neurogranin mRNA in normal aging, Alzheimer disease and fronto-temporal dementia.
J Neuropathol Exp Neurol
 
56
:
1105
–1118.
Chen SJ, Sweatt JD, Klann E (
1997
) Enhanced phosphorylation of the postsynaptic protein kinase C substrate RC3/neurogranin during long-term potentiation.
Brain Res
 
749
:
181
–187.
Chong MS, Reynold ML, Irwin N, Coggeshall RE, Emson PC, Benowitz LI, Woolf CJ (
1994
) GAP-43 expression in primary sensory neurons following central axotomy.
J Neurosci
 
14
:
4375
–4385.
Davidsson P, Blennow K (
1998
) Neurochemical dissection of synaptic pathology in Alzheimer’s disease.
Int Psychogeriatr
 
10
:
11
–23.
Felleman DJ, Van Essen DC (
1991
) Distributed hierarchical processing in the primate cerebral cortex.
Cereb Cortex
 
1
:
1
–47.
Fukuda K, Saito N, Yamamoto M, Tanaka C (
1994
) Immunocytochemical localization of the alpha-, beta I-, beta II- and gamma-subspecies of protein kinase C in the monkey visual pathway.
Brain Res
 
658
:
155
–162.
Gerendasy DD, Sutcliffe JG (
1997
) RC3/neurogranin, a postsynaptic calpacitin for setting the response threshold to calcium influxes.
Mol Neurobiol
 
15
:
131
–163.
Higo N, Oishi T, Yamashita A, Matsuda K, Hayashi M (
1998
) Gene expression of growth-associated proteins, GAP-43 and SCG10, in the hippocampal formation of the macaque monkey: non-radioactive in situ hybridization study.
Hippocampus
 
8
:
533
–547.
Higo N, Oishi T, Yamashita A, Matsuda K, Hayashi M (
1999
) Quantitative non-radioactive in situ hybridization study of GAP-43 and SCG10 mRNAs in the cerebral cortex of adult and infant macaque monkeys.
Cereb Cortex
 
9
:
317
–331.
Higo N, Oishi T, Yamashita A, Matsuda K, Hayashi M (
2000
) Expression of GAP-43 and SCG10 mRNAs in lateral geniculate nucleus of normal and monocularly deprived macaque monkeys.
J Neurosci
 
20
:
6030
–6038.
Higo N, Oishi T, Yamashita A, Matsuda K, Hayashi M (
2002
) Expression of MARCKS mRNA in lateral geniculate nucleus and visual cortex of normal and monocularly deprived macaque monkeys.
Vis Neurosci
 
19
:
633
–643.
Higo N, Oishi T, Yamashita A, Matsuda K, Hayashi M (
2002
) Expression of neurogranin mRNA in area 17 of infant and adult macaque monkeys.
Soc Neurosci Abstr
 
28
:
530
.12.
Higo N, Oishi T, Yamashita A, Matsuda K, Hayashi M (
2002
) Gene expression of neurogranin in the cerebral cortex of macaque monkey.
Neurosci Res
 
26
(Suppl.):
S21
.
Higo N, Oishi T, Yamashita A, Matsuda K, Hayashi M (
2002
) Northern blot and in situ hybridization analyses of MARCKS mRNA expression in the cerebral cortex of the macaque monkey.
Cereb Cortex
 
12
:
552
–564.
Higo N, Oishi T, Yamashita A, Matsuda K, Hayashi M (
2003
) Cell type- and region-specific expression of protein kinase C-substrate mRNAs in the cerebellum of the macaque monkey.
J Comp Neurol
 
467
:
135
–149.
Holtmaat AJGD, Dijkhuizen PA, Oestreicher AB, Romijn HJ, Van der Lugt NMT, Berns A, Margolis FL, Gispen WH, Verhaagen J (
1995
) Directed expression of the growth-associated protein B-50/GAP-43 to olfactory neurons in transgenic mice results in changes in axon morphology and extraglomerular fiber growth.
J Neurosci
 
15
:
7953
–7965.
Jones EG, Huntley GW, Benson DL (
1994
) Alpha calcium/calmodulin-dependent protein kinase II selectively expressed in a subpopulation of excitatory neurons in monkey sensory-motor cortex: comparison with GAD-67 expression.
J Neurosci
 
14
:
611
–629.
Klann E, Chen SJ, Sweatt JD (
1993
) Mechanism of protein kinase C activation during the induction and maintenance of long-term potentiation probed using a selective peptide substrate.
Proc Natl Acad Sci USA
 
90
:
8337
–8341.
Kleim JA, Lussnig E, Schwarz ER, Comery TA, Greenough WT (
1996
) Synaptogenesis and FOS expression in the motor cortex of the adult rat after motor skill learning.
J Neurosci
 
16
:
4529
–4535.
Kruger L, Bendotti C, Rivolta R, Samanin R (
1992
) GAP-43 mRNA localization in the rat hippocampus CA3 field.
Brain Res Mol Brain Res
 
13
:
267
–272.
Levin BE, Dunn-Meynell A (
1993
) Regulation of growth-associated protein 43 (GAP-43) messenger RNA associated with plastic change in the adult rat barrel receptor complex.
Mol Brain Res
 
18
:
59
–70.
Linda H, Piehl F, Dagerlind A, Verge VMK, Arvidsson U, Chullheim S, Risling M, Ulfhake B, Hokfelt T (
1992
) Expression of GAP-43 mRNA in the adult mammalian spinal cord under normal conditions and after different types of lesions, with special reference to motoneurons.
Exp Brain Res
 
941
:
284
–295.
Lund JS, Yoshioka T, Levitt JB (
1994
) Substrates for Interlaminar connections in area V1 of macaque monkey cerebral cortex. In: Cerebral cortex (Peters A, Rockland KS, eds), pp.
37
–60. New York: Plenum Press.
McNamara RK, Lenox RH (
1997
) Comparative distribution of myristoylated alanine-rich C kinase substrate (MARCKS) and F1/GAP-43 gene expression in the adult rat brain.
J Comp Neurol
 
379
:
48
–71.
McNamara RK, Lenox RH (
2000
) Differential regulation of primary protein kinase C substrate (MARCKS, MLP, GAP-43, RC3) mRNAs in the hippocampus during kainic acid-induced seizures and synaptic reorganization.
J Neurosci Res
 
62
:
416
–426.
McNamara RK, Jiang Y, Streit WJ, Lenox RH (
2000
) Facial motor neuron regeneration induces a unique spatial and temporal pattern of myristoylated alanine-rich C kinase substrate expression.
Neuroscience
 
97
:
581
–589.
Meiri KF, Pfenninger KH, Willard MB (
1986
) Growth-associated protein, GAP-43, a polypeptide that is induced when neurons extend axons, is a component of growth cones and corresponds to pp46, a major polypeptide of a subcellular fraction enriched in growth cones.
Proc Natl Acad Sci USA
 
83
:
3537
–3541.
Meiri KF, Willard M, Johnson MI (
1988
) Distribution and phosphorylation of the growth-associated protein GAP-43 in regenerating sympathetic neurons in culture.
J Neurosci
 
8
:
2571
–2581.
Nelson RB, Routtenberg A (
1985
) Characterization of protein F1 (47 kDa, 4.5 pI): a kinase C substrate directly related to neural plasticity.
Exp Neurol
 
89
:
213
–224.
Nishizuka Y (
1986
) Studies and perspectives of protein kinase C.
Science
 
233
:
305
–312.
Nishizuka Y (
1995
) Protein kinase C and lipid signaling for sustained cellular responses.
FASEB J
 
9
:
484
–496.
Oishi T, Higo N, Umino Y, Matsuda K, Hayashi M (
1998
) Development of GAP-43 mRNA in the macaque cerebral cortex.
Dev Brain Res
 
109
:
87
–97.
Ouimet CC, Wang JK, Walaas SI, Albert KA, Greengard P (
1990
) Localization of the MARCKS (87 kDa) protein, a major specific substrate for protein kinase C, in rat brain.
J Neurosci
 
10
:
1683
–1698.
Pak JH, Huang FL, Li J, Balschun D, Reymann KG, Chiang C, Westphal H, Huang KP (
2000
) Involvement of neurogranin in the modulation of calcium/calmodulin-dependent protein kinase II, synaptic plasticity, and spatial learning: a study with knockout mice.
Proc Natl Acad Sci USA
 
97
:
11232
–11237.
Pasinelli P, Ramakers GM, Urban IJ, Hens JJ, Oestreicher AB, de Graan PN, Gispen WH (
1995
) Long-term potentiation and synaptic protein phosphorylation.
Behav Brain Res
 
66
:
53
–59.
Ramakers GM, De Graan PN, Urban IJ, Kraay D, Tang T, Pasinelli P, Oestreicher AB, Gispen WH (
1995
) Temporal differences in the phosphorylation state of pre- and postsynaptic protein kinase C substrates B-50/GAP-43 and neurogranin during long-term potentiation.
J Biol Chem
 
270
:
13892
–13898.
Ramakers GMJ, Gerendasy DD, de Graan PNE (
1999
) Substrate phosphorylation in the protein kinase cgamma knockout mouse.
J Biol Chem
 
274
:
1873
–1874.
Ramakers GM, Heinen K, Gispen WH, de Graan PN (
2000
) Long term depression in the CA1 field is associated with a transient decrease in pre- and postsynaptic PKC substrate phosphorylation.
J Biol Chem
 
275
:
28682
–28687.
Ramakers GM, Pasinelli P, van Beest M, van der Slot A, Gispen WH, De Graan PN (
2000
) Activation of pre- and postsynaptic protein kinase C during tetraethylammonium-induced long-term potentiation in the CA1 field of the hippocampus.
Neurosci Lett
 
286
:
53
–56.
Represa A, Deloulme JC, Sensenbrenner M, Ben-Ari Y, Baudier J (
1990
) Neurogranin: immunocytochemical localization of a brain-specific protein kinase C substrate.
J Neurosci
 
10
:
3782
–3792.
Skene JHP, Jacobson RD, Snipes GJ, McGuire CB, Norden JJ, Freeman JA (
1986
) A protein induced during nerve growth (GAP-43) is a major component of growth-cone membranes.
Science
 
233
:
783
–786.
Swierczynski SL, Blackshear PJ (
1995
) Membrane association of the myristoylated alanine-rich C kinase substrate (MARCKS) protein. Mutational analysis provides evidence for complex interactions.
J Biol Chem
 
270
:
13436
–13445.
Tetzlaff W, Alexander SW, Miller FD, Bisby MA (
1991
) Response of facial and rubrospinal neurons to axotomy: changes in mRNA expression for cytoskeletal proteins and GAP-43.
J Neurosci
 
11
:
2528
–2544.
Tighilet B, Hashikawa T, Jones EG (
1998
) Cell- and lamina-specific expression and activity-dependent regulation of type II calcium/calmodulin-dependent protein kinase isoforms in monkey visual cortex.
J Neurosci
 
18
:
2129
–2146.
Tominaga S, Saito N, Tsujino T, Tanaka C (
1993
) Immunocytochemical localization of alpha-, beta I-, beta II- and gamma-subspecies of protein kinase C in the motor and premotor cortices of the rhesus monkey.
Neurosci Res
 
16
:
275
–286.
Toni N, Buchs PA, Nikonenko I, Bron CR, Muller D (
1999
) LTP promotes formation of multiple spine synapses between a single axon terminal and a dendrite.
Nature
 
402
:
421
–425.
Van der Zee CEEM, Nielander HB, Vos JP, Lopes da Silva S, Verhaagen J, Oestreicher AB, Schrama LH, Schotman P, Gispen WH (
1989
) Expression of growth-associated protein B-50 (GAP43) in dorsal root ganglia and sciatic nerve during regenerative sprouting.
J Neurosci
 
9
:
3505
–3512.
von Bonin G, Bailey P (
1947
) The neocortex of Macaca mulatta. In: Illinois monographs in the medical sciences (Allen RB, Kampmeier OF, Schour I, Serles ER, eds), pp.
1
–163. Urbana, IL: University of Illinois Press.
Watson JB, Battenberg EF, Wong KK, Bloom FE, Sutcliffe JG (
1990
) Subtractive cDNA cloning of RC3, a rodent cortex-enriched mRNA encoding a novel 78 residue protein.
J Neurosci Res
 
26
:
397
–408.
Watson JB, Sutcliffe JG, Fisher RS (
1992
) Localization of the protein kinase C phosphorylation/calmodulin-binding substrate RC3 in dendritic spines of neostriatal neurons.
Proc Natl Acad Sci USA
 
89
:
8581
–8585.
Yao GL, Kiyama H, Tohyama M (
1993
) Distribution of GAP-43(B50/F1) mRNA in the adult rat brain by in situ hybridization using an alkaline phosphatase labeled probe.
Mol Brain Res
 
18
:
1
–16.