THE JOURNAL OF COMPARATIW3 NEUROLOGY 298:215-223 (1990)

Distribution of Neurotein-Containing Fibem in the Frontal Cortex of the

Macaque Monkey

KEIJI SATOH AND HIROKO MATSUMURA Department of Psychiatry, Shiga University of Medical Science, Seta Tsukinowacho,

Otsu 520-21, Japan

ABSTRACT The distribution of neurotensin-containing fibers was examined in the frontal cortex of the

monkey Macaca fuscata using the immunoperoxidase histochemical technique. An extremely dense network of neurotensin-containing fibers was observed in the medial prefrontal regions. The majority of cortical neurotensin fibers was observed in the anterior cingulate cortex (Walker's area 24) and adjacent medial prefrontal regions (areas 6 and 32). In area 24, the fiber density was similar to that in the nucleus accumbens. Immunoreactive fibers were particularly dense in two pyramidal layers (111, V). The medial prefrontal regions, areas 6 and 32, contained a moderate density of immunoreactive fibers. This regional distribution of neurotensin- containing fibers was not observed in other cortical fiber systems that contained substance P, somatostatin, or tyrosine hydroxylase. No neurotensin-containing cell bodies were observed in the frontal cortex.

The present study demonstrates that the laminar and regional distributions of neurotensin- containing fibers are unique when compared to those of substance P- or somatostatin- containing fibers, and also distinct from that of catecholaminergic fibers. The distribution of telencephalic neurotensin fibers points to a relationship with limbic structures.

Key words: cerebral cortex, immunohistochemistry, primate

Neurotensin is one of many neuropeptides that may act as neurotransmitters or neuromodulators (Uhl and Snyder, '77) . This tridecapeptide has been demonstrated in fore- brain limbic structures of many mammalian species, includ- ingprimates (for review see Fuster, '89; Nieuwenhuys, '85). Although biochemical studies have demonstrated only low concentrations of neurotensin in the primate cerebral cortex (Emson et al., '85; Kataoka et al., '79; Manberg et al., '821, receptor binding analysis has revealed a substantial number of neurotensin binding sites in the cingulate cortex as well as in the hippocampus, amygdala, and nucleus accumbens of rats and monkeys (Quirion et al., '82, '87; Young and Kuhar, '81).

Neurotensin interacts with dopamine-containing neu- rons (Nemeroff, '86, '87) and has an effect on dopamine turnover (Cador et al., '89). Further, neurotensin coexists in some mesolimbic dopamine neurons (Hokfelt et al., '84; Kalivas and Miller, '84). Recent pharmacological studies have shown that in rats chronic administration of either haloperidol or clozapine decreases the concentration of neurotensin in both the medial prefrontal and cingulate cortex (Kilts et al., '88), whereas the same treatments increase neurotensin levels in the nucleus accumbens (Gov- oni et al., '80; Kilts et al., '88; Radke et al., '89). Subnormal

concentrations of neurotensin in cerebrospinal fluid (CSF) have been reported for a subgroup of schizophrenic patients and this was reversed by neuroleptic treatment (Widerlow et al., '82). In addition, an increased amount of neurotensin has been observed in one area of the prefrontal cortex (Brodmann area 32) of postmortem schizophrenic brains (Nemeroff et al., '83). These observations suggest that a neurotensin-containing neuronal system might be involved in the pathogenesis of schizophrenia (Nemeroff, '86).

Many neurotensin-containing neuronal systems have recently been studied in subcortical regions of the rat forebrain (Kiyama et al., '86; Roberts et al., '81; Sakamoto et al., '86). Little is known, however, about the anatomical organization of neurotensin-containing fiber networks in the cerebral cortex. In the present study, the distribution of the neurotensin-containing fiber system has been examined in the frontal cortex, including the cingulate cortex of the macaque monkey, by use of an immunohistochemical tech- nique. Further, these observations were compared with the

Accepted May 3,1990. Address reprint requests to Dr. Keiji Satoh, Department of Psychiatry,

Shiga University of Medical Science, Seta Tsukinowacho, Otsu 520-21, Japan.

o 1990 WILEY-LISS, INC.

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distribution of catecholaminergic, substance P-, and soma- tostatin-containing fibers at several levels of the monkey frontal cortex.

K. SATOH AND H. MATSUMURA

MA'IEFUALSAN'DMETHODS Four male Japanese monkeys (Macaca fuscata), weighing

4-6 kg. were used for this experiment. The animals were housed and fed for several months in the university animal care facility under standard conditions prior to the immuno- histochemical procedures.

Immunohistochemistry. Under deep anaesthesia, each monkey was perfused through the ascending and descendingaorta with 500 ml of saline (0.9% NaCl) followed by 3 liters of a fixative solution that contained 0.25% glutaraldehyde, 3-470 paraformaldehyde, 0.2% picric acid, 0.1 M phosphate buffer (PB), pH 7.4 (Iritani et al., '89). The brain was immediately removed and cut into several small blocks. Tissue blocks were immersed and postfixed in a similar, picric acid-containing fixative without glutaralde- hyde (12-24 hr, PC), followed by a rinse for 1-3 days in 15% sucrose in phosphate buffer (4°C). The blocks were cut using a cryostat or a freezing microtome (20 Fm) in coronal or horizontal planes. These sections were collected and stored in phosphate-buffered saline (0.1 M PR, pH 7.4,0.9% NaC1) (PBS).

The primary antiserum against neurotensin was raised in rabbits following injections of neurotensin-KLM (key hole limpet hemosianin) conjugate. The specificity of the antiserum was characterized by radioimmunoassay and by immunohistochemical testing (Fukami et al., '88). After rinsing in 0.2-0.3% Triton X-100 in PBS for several days, the brain sections were incubated in the primary antibody against neurotensin (dilution of 1:500-1:8,000,48 hr, 4"C), and were then treated with biotinylated antirabbit I&, followed by the avidin-biotin-peroxidase complex (ABC method) and reacted with 3,3'diaminobenzidine-HCl (Hsu et al., '81). Some of the sections were intensified by osmium or were counterstained with cresylechtviolet. The speci- ficity of the immunoreaction was examined by an absorp- tion test. No immunoreactivity was observed when the primary antiserum was preadsorbed with excess neuro- tensin (50 pg/ml). Adjacent sections were processed for tyrosine hydroxylase- or other neuropeptide-immunocyto- chemistry. Tyrosine hydroxylase-immunoreactivity was demonstrated by using a monoclonal antibody directed against this enzyme (Boehringer-Mannheim), and soma- tostatin- or substance P-immunoreactivity by using mono- clonal antibodies to cyclic somatostatin (provided by Dr. J.C. Brown, Dept. of Physiology, University of British Columbia) or substance P (Sera Lab), followed by the stan- dard ABC immunoperoxidase method as described above.

RESULTS Many neurotensin-containing fibers were identified in

the frontal cortex, including the cingulate cortex. These fibers were either short-branching processes or puncta (Fig. 1B-D), and were morphologically similar to those observed in the nucleus accumbens (Fig. IA). They were densely distributed particularly in the medial aspects of the cerebral hemisphere, especially in the anterior cingulate cortex (Fig. 2).

In the anterior cingulate cortex (corresponding to area 24 of Walker; see Insausti et al., '871, neurotensin-containing

fibers were observed in all layers, but with some differences in the density of fibers between layers (Fig. 3). The highest concentration of immunoreactive fibers was seen in two adjoining pyramidal layers, i.e., the deeper zone of layer 111 and layer V (Figs. lC,D, 3). In these areas, the immunoreac- tive fibers were of large caliber and oriented in various directions (Fig. 1C,D). Superficial layers contained a moder- ate number of fibers, with layer I containing a relatively higher density (Figs. IB, 3). This molecular layer was characterized by many granular, neurotensin immunoreac- tive puncta and also by horizontally oriented fibers. Layer VI also contained neurotensin-containing fibers with smaller caliber and their axis oriented perpendicular to the surface in coronal sections (Fig. 3).

A moderate density of fibers was seen in adjacent cortical regions, i.e., medial prefrontal cortex (area 32; see Insausti et al., '87) and mediodorsal prefrontal cortex (areas 6 and 9; see Insausti et al., '87) (Fig. 2). The distribution of neuroten- sin-containing fibers in the mediodorsal prefrontal and medial prefrontal cortices was principally similar to those in the anterior cingulate cortex, except that it was less dense. In other regions of the frontal cortex, such as the lateral prefrontal, dorsolateral prefrontal, or orbitofrontal cortex, there were scant or isolated immunoreactive fibers or puncta (Figs. 2,3).

In the caudal half of the anterior cingulate cortex, a zone of dense deposits of granular, neurotensin-containing puncta was seen in a wedgelike form in coronal sections (Fig. 2). In a horizontal plane, many smooth-surfaced fibers running parasagittal to the midline were observed in the deep layers of the cingulate cortex. During their course, these fibers appeared to give off fine fibers toward the cortical layers. No immunoreactive cell bodies were detected in the frontal cortex or in the underlying white matter.

Comparison with other peptide-containing fibers system. Many substance P- and somatostatin-containing fibers were demonstrated in adjacent sections through the frontal cortex. Anatomical details of these neuropeptide systems are presented elsewhere (Iritani et al., '89; Iritani and Satoh, in preparation). Representative diagrams show- ing the distribution of these neuropeptide-containing fibers are found in Figure 3. Each neuropeptide-containing sys- tem possessed unique features; for example, substance P fibers have a higher density in the superficial layers (I-IIII, whereas somatostatin-containing fibers (Fig. 1E) tend to concentrate in two zones, the superficial layers 11-111 and the deep layers (V). In comparison with neurotensin- containing fibers, substance P- or somatostatin-containing fibers were rather uniformly distributed over the cortical regions of the frontal lobe, although there was a tendency for them to have a higher density in areas 24 and 32 (Fig. 3; compare areas 24 and 46).

Comparison with tyrosine hydroxylase-immunore- active fibers. Comparison of two adjacent sections pro- cessed for tyrosine hydroxylase immunohistochemistry and for neurotensin immunohistochemistry revealed some over- lap in the regions that contained substantial numbers of both fiber networks in the monkey prefrontal cortex. The density of tyrosine hydroxylase-containing fibers was rather high in the anterior cingulate cortex (area 24) and media) prefrontal cortex (area 321, where a high density of neuro- tensin fibers was also observed (Fig. 4). However, there was no correlations between the two neuronal systems in terms of the laminar distributions of immunoreactive fibers in these cortical areas (Fig. 3). In general, the cortical field

NEUROTENSIN IN MONKEY FRONTAL CORTEX

Fig. 1. A-D. Irnmunocytochemistry of neurotensin-containing fibers in the macaque monkey. A. Nucleus accurnbens. B-D. Layer I (B), V (C) and 111 (D) of the anterior cingulate cortex (area 24). An arrow indicates the pial surface. E. Somatostatin-containing fibers in layer VI of the anterior cingulate cortex. Bars = 100 pm.

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B

K. SATOH AND H. MATSUMURA

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Fig. 2. Diagramatic representation of regional distribution of neurotensin-containing fibers in the frontal cortex of the macaque monkey. Numerals indicate the Walker areas, as recently modified by Insausti et al., '87. Levels are indicated by A, B, C, and D, from rostra1 to caudal. (Levels A, B, and C correspond roughly to the diagram for tyrosine hydroxylase-immunoreactive fibers as shown in Fig. 4.) Abbreviations: Ac: nucleus accumbens; Cd: caudate nucleus.

NEUROTENSIN IN MONKEY FRONTAL CORTEX 219

Figure 2 continued

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NEUROTENSIN IN MONKEY FRONTAL CORTEX 22 1

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Fig. 4. Diagramatic representation of the distribution of tyrosine hydroxylase-containing fibers in the frontal cortex of the macaque monkey. Walker's areas (as modified by Insausti) are indicated by numerals. Levels A, B, and C roughly correspond to those in Figure 2. Abbreviation: Ac, nucleus accumbens.

that contained an abundant network of tyrosine hydroxy- lase fibers was larger than that containing neurotensin fibers (compare Figs. 2,4). For example, there was a dense distribution of tyrosine hydroxylase containing fibers in the dorsolateral prefrontal cortex (area 46), where only an iso- lated neurotensin-containing fibers were observed (Fig. 3).

DISCUSSION Previous histochemical and biochemical studies have

shown the existence of neurotensin-containing neuronal systems in the cerebral cortex, including the anterior cingulate cortex (for review, see Nieuwenhuys, '85). The present study has extended these earlier observations to the primate brain by providing a detailed anatomical descrip- tion of the neurotensin immunoreactive fibers in the fron- tal cortex. We have shown that a substantial number of cortical neurotensin-containing fibers was located in Walk-

er's area 24, the anterior cingulate cortex, and the adjacent medial prefrontal cortical regions. This heterogeneous dis- tribution could not be demonstrated for two other peptides, substance P and somatostatin, although areas 24 and 32 (medial prefrontal cortex) contained relatively high concen- trations of two neuropeptide-containing fibers.

The laminar distribution of fibers containing neuroten- sin in area 24 had some similarity but is not identical to that of substance P- or somatostatin-containing fibers. The neurotensin-containing fibers were extremely dense in the two pyramidal layers (111 and V), where relative dense networks of two neuropeptide-containing fibers were dem- onstrated. In the rat, however, it has been shown that neurotensin immunoreactive fibers form a band in the deeper layers of the neocortex, with highest density in layer VI (Jennes et al., '82). Whether this discrepancy between rats and monkeys is due to species difference or not must be determined.

222 K. SATOH AND H. MATSUMURA

The functional role of the neurotensin fiber system in the prefrontal cortex is still unknown. The present investiga- tion provides evidence that the cortical neurotensin axonal network is concentrated in the associational cortical area, the anterior cingulate cortex, which has connections with many other limbic brain structures. Biological abnormali- ties of this neurotensin system may therefore be associated with mental disorders such as schizophrenia as has been suggested previously (Nemeroff et al., '83; Nemeroff, '87).

The regional distribution pattern of neurotensin-contain- ing axons (as shown in the present study) appears to correspond to that of neurotensin binding sites (Quirion et al., '87) in the monkey cortex; both showing high density in the anterior cingulate cortex. The binding study has shown that the superficial and deep layers are heavily labeled, whereas intermediate layers 111 and IV contained less receptor density. This laminar distribution was somewhat different from that seen in the immunohistochemical mate- rial.

The source of the neurotensin-containing fibers in the prefrontal and cingulate cortex in the monkey brain has not been established. Earlier studies predicted two areas of origin for these peptide-containing fibers. One region was the hippocampal formation (Roberts et al., '81). Others have considered the brainstem parabrachial nucleus as a source (Jennes et al., '82). However, a recent immunohis- tochemical study has revealed that some dopaminergic neurons in the ventral tegmental area (VTA) possesses neurotensin-immunoreactivity in their perikarya (Hokfelt et al., '841, and these dopaminergic neurons project to the rat medial prefrontal cortex (Seroogy et al., '88; Studler et al., '88). Reserpine treatment produces a dose- and time- dependent decrease in both dopamine and neurotensin in the rat prefrontal cortex (Bean et al., '89b). Release of both dopamine and neurotensin are increased in the prefrontal cortex after electrical stimulation of the medial forebrain bundle (Bean et al., '89a). Although the existence of such neurotensin/dopamine-containing pathways to the prefron- tal cortex from the VTA in the primate has yet to be proven, it seems likely that the anterior cingulate cortex and adjoining cortical areas are heavily innervated by mesocor- tical neurotensin neurons that might also contain dopa- mine in their terminal axons.

Although there was some overlap in the distribution of neurotensin-containing fibers and tyrosine hydroxylase- containing fibers, there were many areas where tyrosine hydroxylase-positive axons were found, but few, if any, neurotensin-containing fibers could be observed (see Re- sults, compare Figs. 2,4) . Furthermore, the laminar distri- bution of neurotensin- and tyrosine hydroxylase immuno- reactive fibers differed even at the anterior cingulate cortex (see Fig. 3) . These differences may be due to the fact that some but not all dopaminergic neurons show immunoreac- tivity for neurotensin as seen in the rat (Hokfelt, '84; Seroogy et al., '88), or it may be that some of tyrosine hydroxylase-containing axons in the frontal cortex might be noradrenergic, originating from the locus coeruleus.

Recent immunohistochemical studies have revealed many fiber systems that contain neuropeptides in the primate frontal cortex. These include substance P (Iritani et al., '89), somatostatin (Iritani and Satoh, in preparation), neuropeptide Y, cholecystokinin, and vasoactive intestinal polypeptide (VIP) (for review, see Jones, '86, '88; Nieuwen- huy, '85). Comparison of these peptidergic innervation among mammalian species has shown that axonal network containing substance P is highly developed in the primate prefrontal cortex. The morphology of the neurotensin- containing fiber system differs distinctively from the other peptidergic systems in two ways. First, its source appears to be subcortical, as discussed above, rather than from intrin- sic cortical GABA neurons like those other peptides (Jones, '88). Second, it has a prominent distribution in the medial prefrontal regions, which makes this neurotensin fibers unique in terms of cortical function of the primate frontal lobe.

ACKNOWLEDGMENTS The authors thank Dr. S. Shiosaka (Osaka University

Medical School) for providing the antibody to neurotensin, andDrs. C.R. Gerfen (LCB, NIMH), S.R. Vincent, and H.C. Fibiger (Division of Neurological Sciences, University of British Columbia) for their generosity and comments. The present study was supported by a grant from NCNP of the ministry of Health and Welfare (63-A-7-15), and by a Grant-in-aid for Scientific Research (C-61570523) from the Ministry of Education, Science, and Culture.

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