the distribution of substance p in the cerebral cortex and hippocampal formation: an...

B&n Resew& Liufletin, Vol. 22, pp. 295-303. 0 Pergatnon Press plc, 1989. Printed in the U.S.A. 0~1-923~~ $3.00 -t -00

The Distribution of Substance P in the Cerebral Cortex and Hippocampal Formation: An Immunohistochemical

Study in the Monkey and Rat

S. IRITANI, M. FUJI1 AND K. SATOH

department of Psychiatry, Shiga University of Medical Sciences, Setu Tsukinowa-Gh~, Otsu 520-21, Japan

Received 2 September 1988

IRITANI, S., M. FUJI1 AND K. SATOH. The distribution of substance P in the cerebral cortex and hippocampal formation: An immunohistochemical study in the monkey and rat. BRAIN RES BULL 22(2) 295-303, 1989.-The distribution of substance P-containing fibers in the cerebral cortex and the hippocampal formation of the Japanese monkey (Macaco fuscata fuscata) was studied by immunohistochemistry using a monoclonal antibody raised against substance P. The results were compared with the distribution in homologous regions of the rat brain. Substance P-containing fibers and cell bodies were observed in all regions of the cerebral cortex. In deep layers of the neocortex (IV-VI), substance P-immunoreactive fibers formed arrays that ran perpendicular to the surface. These immunoreactive fibers tended to branch as they approached the cortical surface in layers II and III, at which point they were oriented in many directions. The molecular layer (I) of the monkey nemrtex contained many granular, substance P-immuno~tive structures, resembling terminal boutons. In contrast to the monkey, rat cortical areas contained substantially fewer substance P-containing fibers. The immunoreactive profiles, mostly fine dot-like structures, were seen uuiformly in layers II and IVof the rat neocortex, although in the medial prefrontal cortex many thick, varicose fibers were also observed. Substance P-containing fibers were seen throughout the hippocampai formation of the monkey, including the subiculum and the parahippocampal regions. The regional distribution of immunoreactive fibers was most dense in the molecular layers of dentate gyrus, in the stmtum moleculare of the CA1 region, and in the stratum pyramidalis of the CA2 region. In the rat, the hippocampus and dentate gyrus contained fewer immunoreactive fibers. Moderate densities were observed in the rat subicuhun and entorhinal cortex. These observations indicate that compared to rats substance P-containing neuronal systems in the cerebral cortex and hippocampal formation are highly developed in the monkey, suggesting that the substance P-containing neurons might be more involved in cortical function in primates.

Substance P Distribution Immunocyt~he~st~ Cerebral cortex Hippocampal formation Monkey Species difference

SINCE the discovery of substance P-containing fiber network in the human cerebral cortex (14), the functional role of such peptide-containing system in the cerebral cortex of higher mammalian species has been of considerable interest. The coexistence of substance P with other neurotransmitters in cortical neurons has been shown in rats by recent immunohistochemical studies (20).

Although there have been many ne~~~orni~~ studies on substance Phone neuronal systems in the basal ganglia of some subprimates [for reviews see (13,201 as well as primates (2, 10,ll) including humans (7,9,17), the distribution of substance P-containing fibers in other regions of the telencephalon, particularly the cerebral cortex and hippocampal formation, has not been fully examined in monkeys. Previ- ous studies in the human brain revealed a unique distribution of substance P-containing fibers in the hippocampal formation, which could not be shown in subprimates (7,22). These observations suggest the presence of considerable species dBerences in the organization of substance P neuronal sys-

terns in the telencephalon, and the examination of these differences might provide clues to the role of this peptide in the forebrain. In the present investigation, the distribution and anatomy of substance P-containing fibers and somata in the cerebral cortex and hippocampal formation of the primate and rodent were studied and compared by immunohistochemical techniques using a monoclonal antibody directed against substance P.

METHOD

Five male Japanese monkeys (Macaca fuscafu jiiscaru) and 10 male albino rats were used. The monkeys were deeply anesthetized with sodium pentobarbital (10 mg/kg, IV) and/or with ketamine hydrochloride (25 m&g, IM), and the rats with sodium pentobarbital (20 m&g, IP). The animals were perfused with a Ringer solution (0.9% NaCl) fist, and then with an ice-cold 3% paraformaldehyde, 0.25% glutaral- dehyde, 0.2% picric acid in 0.1 M phosphate bufler (PB; pH

295

296 IRITANI, FUJI1 AND SATOH

FIG. 1. Photomicrographs of substance P-immunoreactive fibers in the medial prefrontal cortex of the Mucacafuscatafuscata. (A) Layer I. (B) Layer II and III. (C) Layer IV. (D and E) Layer VI. Bars: A-C, D, E=50 pm.

SUBSTANCE P IN THE MONKEY TELENCEPHALON

FIG. 2. Series of reconstructions of substance P-immunoreactive fibers in the cerebral cortex of the Japanese monkey. (A) Parahip- pocampat gyrus. (B) Insular cortex. (C) Medial prefrontal cortex. These illustrations were drawn by tracing the immunoreactive profile in the original photomicrographs. Note the high density of substance P-containing fibers in the superficial layers (1-3) of the prefrontal and insular cortices. Bars: 200 pm.

7.4) (28). The monkeys were perfused through the ascending aorta (anterograde infusion) and also through the descending aorta (retrograde infusion) using two elastic cannulae con- nected to a rate constant pump. The descending aorta was ligated below the level of cannulation. Rats were perfused through the left ventricle. The brains were immediately re- moved, cut into 5-g mm thick slices and postfixed overnight (4°C) in another picric-acid/paraformaldehyde containing fixative without ~ut~~dehyde. Then the brain slices were rinsed in 15% sucrose-PB solution for 2-3 days 4°C with at least 2 changes.

Thirty micron-thick sections were made from the fixed brain slices using a freezing microtome or a cryostat. Sec- tions were collected, rinsed and stored in phosphate- bufferred saline (PBS), 0.9% NaCl in 0.1 M PB (pH 7.4), for at least 3 days and for up to two weeks prior to the immuno~stochemic~ staining procedures.

Immunohistochemical Procedures

Brain sections were rinsed in 0.1 M Tris-Cl buffered

297

saline (TBS; pH 7.4, O.% NaCI), containing 0.3% Triton X-100 (TX) and 2% normal goat serum (NGS), for 30-60 min, at room temperature. The sections were incubated 1) in a commercially available monoclonal antisubstance P antibody (Sera Lab.; dilutions of l/800-1/3200 in 2% NGS-O.3% TX-O. 1 M TBS solution) (5), overnight (4*C), 2) in a medium containing biotinylated anti-rat IgG (Vector: diluted l/100 in NGS-TX-TBS) at room temperature (45 min), and then 3) in a medium that contained avidin-biotin peroxidase complex [ABC method (LS)] for 45 min. After each incubation step, the sections were rinsed in NGS-TX-TBS solution. Finally, the sections were rinsed in TBS twice for 10 min and reacted with 0.05%, 3,3’ diamino~nzidine-HCl in 0.05 M Tris-Cl bufler (pH 7.6) containing 0.02% hydrogen peroxide at room temperature for 10 min. Following the reaction, the sections were rinsed in 0.05 M Tris-Cl buffer (pH 7.6) two or three times and mounted onto chrome-hum-gelatin coated slide glasses. After drying, the sections were dehydrated and coverslipped with Permount. Some of the sections were soaked in 0.1% osmium tetroxide-PB solution for 1 min at room temperature to intensify the immunoreaction end- product. For anatomical verifications of the structures, some irnmunoreacted sections were counterstained with cresylechtviolet. Some adjacent sections were also stained either with cresylechtviolet or by Kluver-Ban-era method (16a).

Control studies were conducted in both species exactly under the same immunostaining procedure as above except that prior to immunocytochemical labeling the diluted antisubstance P antibody was preadsorbed with 10 pglml or 50 &ml of substance P (Sigma).

RESULTS

monkey Cerebral Cortex

Many substance P-containing cell bodies and fibers were observed throughout the cerebral cortex in the frontal, parietal, temporal, and occipital lobes. Substance P-containing cell bodies were observed in layers II-VI, and the majority of cells were bipolar or multipolar in shape. Addi- tional immunoreactive structures included small caliber fibers with varicosities along the axons (Fig. IC, D), and fine granular puncta (Fig. 1A). The laminar distribution pattern of substance P-immunoreactive fibers was relatively uniform between cortical regions, with only some minor variations (Fig. 2).

In the deeper layers of the cortex, particularly layers V and VI, many arrays of substance P-containing fibers were observed. These fibers were oriented perpendicular to the surface, and appeared to be fibers of passage with few varicosities (Fig. lD, E). In layers III and IV, the number of immunoreactive fibers increased and exhibited considerable branching, although fibers running toward the pial surface were still prominent features in these regions (Fig. 1C). From the upper zone of layer III dorsally, numerous granular immunoreactive puncta appeared among the fibres. These were oriented in various directions and were the dominant feature of layers II and III (Fig. 1s). Many substance P-containing boutons were uniformly distributed in the molecular layer (Fig. 1 A), and occasionally a few parallel-running fibers were seen among them, running parallel to the surface. Single immunoreactive fibers were observed in the subcortical white matter.

Although the pattern of distribution of substance P-containing fibers in the neocortex was similar among dif-


FIG. 3. Reconstruction of substance P-immunoreactive fibers in the hippocampal formation of the macaque monkey. High densities of substance P-containing fibers are observed in the stratum pyramidalis (SP) of CA2 region, the molecular layer (ML) of fascia dentata and the subiculum (SUB). Photomicrographs of bracketed areas (a,b) are shown on Fig. 4. Abbreviations; H: hilus of the fascia dentata; SLM, SO and SR: stratum lacunosum-moleculare, stratum oriens and stratum radiatum of the h~p~campus. Bar: 1 mm.

FACING PAGE

FIG. 4. Photomicro~aphs of substance P-immunoreactive fibers in the hippocampal formation of the Japanese monkey. The photomicrographs of two bracketed areas in Fig. 3, a and b, are shown here in A and B, respectively. (A) Granular layer (GL) and molecular layer (ML) of the dentate gyrus, and stratum lacunosum-moleculare (SLM) of CA1 region. (B) Stratum radiatum (SR) and stratum pyramidalis (SP) of CA2 region of the hippocampus. (C) Adjacent section to B stained by Khiver Barrera method. A zone of dense substance P-containing terminals lies at the boundary between stratum pyramidalis and stratum radiatum, as indicated by open stars. (D) Subiculum. A, B, C, I)=50 pm.

SUBSTANCE P IN THE MONKEY TELENCEPHALON 299

300

A- l

.,_ ‘. . -, ,\ - $?( I 9, _ q

.-_ :’ y_-.: . . -

:,* _ -.I Q

5-6

I ;:\ _.’ c WM

. 5-6

FIG. 5. Series of reconstruction of substance P-immunoreactive Ii- bers in the temporal cortex (A), dorsal prefrontal cortex (B) and medial mefrontal cortex (C) of the rat. Note the difference of distribution of substance P-containing fibers between the medial prefrontal cortex (C) and other cortical regions (A, B). Numerals 1-6 repre- sent cortical-layer numbers. Abbreviations; WM: white matter. Bars: 200 pm.

ferent lobes of the cerebral cortex, the highest densities of immunoreactive fibers were seen in the prefrontal cortex, particularly the ‘medial prefrontal cortex’ that appears to correspond to area 24 of Walker (27) (Fig. 2C), and the temporal lobe. Other regions of the cerebral cortex, e.g., cingu- late, insular (Fig. 2B) and parahippocampal (Fig. 2A) cortices showed a similar but less dense distribution of substance P-containing fibers.

Monkey Hippocampal Formation

Substance P-immunoreactive fibers were observed throughout the hippocampal formation (Fig. 3). The highest concentration of immunoreactive fibers was seen in the molecular layer of the area dentata (Figs. 3, 4A), in the stratum lacunosum-moleculare of CA1 (Fig. 4A), in the stratum pyramidalis of CA2 (Fig. 4B), and in the subiculum (Figs. 3, 4D). The immunoreactive profiles in the outer third of the molecular layer of facia dentata consisted of fine puncta (Fig. 4A). In the pyramidal layer of the hippocampus, granular immunoreactive puncta were observed very close to the compactly arranged-pyramidal cells (Fig. 4B, C). Such characteristic distribution of substance P-containing structure was observed in CA2 region but not in CA1 or CA3 area (Fig. 3).

Rat Telencephalon

Many fine, granular immunoreactive structures were observed in layers II and IV of the cerebral cortex as two bands (Figs. SA, B, 6A, B). This bilaminar pattern was consistently observed in all parts of the rat neocortex, except for the

IRITANI, FUJI1 AND SATOH

‘medial prefrontal cortex’ (Figs. 5C, 6C, D). In the latter region, which appears to correspond roughly to the prelimbic area (Krettek and Price), many coarse fibers were observed in layers II-IV in addition to uniform granular immunoreactive puncta.

The distribution of substance P-containing fibers in the rat hippocampal formation was generally in accord with previous observations made in this species (6) and in the cat (25). In contrast to the monkeys, substance P-immunoreactive structures were less obvious in the hippocampal formation of rats, although immunoreactive fibers could be observed in the pyramidal layer of hippocampus (Fig. 6E) and in the subiculum.

The control study with preadsorbed antibody resulted in no positive staining of structure described in the monkey and rat brain.

DISCUSSION

Substance P-containing cell bodies and axons have been identified in the cerebral cortex of several mammalian species, i.e., rats, cats, monkeys (present study) and humans [for reviews see (16,19)]. However, as demonstrated clearly in the present study, there appears to be a significant species difference in the laminar distribution of substance P-containing axons in the cerebral cortex. In the monkey, the immunoreactive fibers were observed in all cortical layers and significant terminal-like staining could be observed in the molecular layer. In the rat, a bilaminar distribution of substance P-positive fibers was found in most cortical regions; i.e., uniform immunoreactive puncta in layers II and IV, but not in the molecular layer. A similar species difference in the distribution of substance P-immunoreactive fibers was shown in rodent olfactory bulb (1).

There were notable differences in the density of substance P-containing fibers across monkey cortical areas. In the present immunohistochemical study, a relatively high density of substance P-containing fibers was shown in the prefrontal and temporal cortices compared to other cortical areas. The density of fibers was especially high in the superficial layers of the neocortex. In support of this observation, high concentrations of substance P have been found using radioimmunoassay in the prefrontal and temporal cortices of the Japanese monkey (12). The uneven distribution of substance P-containing fibers was also evident in the medial prefrontal cortex of the rat. The reasons for these regional variations is not known. However, in the rats the medial prefrontal cortex has been shown to contain two different substance P-containing neuronal systems, one intrinsic and another extrinsic, by lesion studies [Satoh, Vincent and Fibiger, unpublished observation; see also (24)] (see below for more discussion).

In the monkey neocortex, the laminar distribution of substance P-containing fibers differs markedly from that of tyrosine hydroxylase-containing fibers (4,8), dopamine+- hydroxylase-, serotonin- and choline acetyltransferase-immunoreactive fibers (4,18). However, there are some similarities between the substance P-neuronal system and the

FACING PAGE

FIG. 6. Photomicrographs of substance P-immunoreactive fibers in the rat neocortex and hippocampal formation. (A, B) Parietal cortex. (C, D) Medial prefrontal cortex. The network of varicose fibers is observed in layers II-IV of the medial prefrontal cortex (C, D), whereas such characteristic fiber network is lacking in the par&al cortex (A, B). (E) Dentate gyrus (rat). Abbreviations; GL: granular layer. Bar: A-E=50 pm.

SUBSTANCE P IN THE MONKEY TELENCEPHALON 301


somatostatin-28( 1-12)~immunoreactive fibers in the monkey neocortex (3). This similarity may arise from the fact that both substance P and somatostatin are present within some of the intrinsic GABA neurons in the neocortex (20).

The regional distribution of substance P-containing fibers in the hippocampal formation of the monkey (present study) resembled closely that in the human hippocampal formation (7,22). In our preparations, dense terminal-like labeling was observed in the stratum moleculare of CA2, stratum moleculare of the fascia dentata, as has been demonstrated in the human hippocampal formation (7).

The origin of substance P-containing axons in the monkey cerebral cortex and hippocampal formation is poorly under- stood. One possibility is the intrinsic, cortical substance P neurons, which were observed in the present study as well as in previous studies in other primates (2,23). Another possible source is the laterodorsal tegmental nucleus in the pontine tegmentum. Substance P-immunoreactive neurons in the

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laterodorsal tegmental nucleus has previously been shown to project to the medial prefrontal cortex in the rat (24,26). Whatever the source or sources are, the present observation of dense innervation of monkey telencephalic structures suggests that among all the neurochemically identified fiber systems so far examined the substance P-containing neuronal systems is highly developed in the primate. This peptide might be involved in higher brain functions, such as memory and affect, in the primates.

ACKNOWLEDGEMENTS

We wish to acknowledge the support of Dr. H. C. Fibiger (Uni- versity of British Columbia) during the inital part of this study. Drs. S. R. Vincent and K. Semba (U.B.C.) provided most helpful com- ments on the manuscript. Thanks also to Mr. and Mrs. Hugh Mur- ray, and to Dr. R. Torii, Institute for Experimental Animals (S.U.M.S.). This work was supported by the Ministry of Education (Japan) No. 61570523.

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