Efferent projections of the infralimbic (area 25) region of the medial prefrontal cortex in the rat: an anterograde tracer PHA-L study

Brain Research, 566 (1991) 26-39 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-89931911503.50

26 o.~LVS17199

Efferent projections of the infralimbic (area 25) region of the medial prefrontal cortex in the rat: an anterograde tracer PHA-L study Masamitsu

Takagishi and Tanemichi

Chiba

The Third Department of Anatomy, Chiba University School of Medicine, Chiba (Japan)

(Accepted 16 July 1991) Key words: Autonomic nervous system; Efferent projection; Infralimbic cortex; Limbic system; Medial prefrontal cortex; Phaseolus vulgaris leukoagglutinin; Rat; Visceral cortex

The efferent projections of the infralimbic region (IL) of the medial prefrontal cortex of the rat were examined by using the anterograde transport of Phaseolus vuigaris leucoagglutinin (PHA-L). Major targets of the IL were found to include the agranular insular cortex, olfactory tubercle, perirhinal cortex, the whole amygdaloid complex, caudate putamen, accumbens nucleus, bed nucleus of the stria terminalis, midline thamalic nuclei, the lateral preoptic nucleus, paraventricular nucleus, supramammillary nucleus, medialmammillary nucleus, dorsal and posterior areas of the hypothalamus, ventral tegmental area, central gray, interpeduncular nucleus, dorsal raphe, lateral parabrachial nucleus and locus coemleus. Previously unreported projections of the IL to the anterior olfactory nucleus, piriform cortex, anterior hypothalamie area and lateroanterior hypothalamic nucleus were observed. The density of labeled terminals was especially high in the agranular insular cortex, olfactory tubercle, medial division of the mediodorsal nucleus of the thalamus, dorsal hypothalamic area and the lateral division of the central amygdaloid nucleus. Several physiological and pharmacological studies have suggested that the IL functions as the 'visceral motor' cortex, involved in autonomic integration with behavioral and emotional events. The present investigation is the first comprehensive study of the IL efferent projections to support this concept.

INTRODUCTION The medial prefrontal cortex (mPFC) in the rat has been the subject of numerous physiological, behavioral and pharmacological studies. Electrical stimulation or lesions of this area have been shown to evoke a wide variety of visceral responses, including changes in blood pressure, heart rate, respiration and gastrointestinal motility6'23'27'~. Lesions in the mPFC have been reported to produce performance deficits in delayed response, delayed alternation and various other behaviors a9'3°'46. In addition, it has been reported that there is a selective activation of the mesocortical dopamine system and increased dopamine release in the mPFC by stress L~. From these results, the mPFC in the rat is thought to be homologous to part of the prefrontal cortex of primates in terms of function 29'3°'46. In anatomical studies, several tract-tracing studies have demonstrated that a certain area in the mPFC and the agranular insular cortex (AI) project directly to the nucleus of the solitary tract (NTS) 3s'¢'49.~'e'~'t~. Together with physiological studies, it has been suggested that this area in the mPFC is a visceral motor region, whereas the A1 is a visceral sensory region regulating

autonomic activities9,3s''tg'62,63,~s, This area in the mPFC is divided cytoarchitectonically into two regions: the prelimbi¢ (area 32) and infralimbic (area 25) regions 3t'~2. The efferent connections of the mPFC including the prelimbic region (PL) have previously been examined by a variety of anterograde tracing techniques, including fiber degeneration methods, wheat germ agglutinin conjugated horseradish peroxidase (WOA-HRP) tracing, autoradiography and most recently Pha~eolus vulgaris leucoagglutinin (PHA-L) s'ta'14' 34,43,s7,7o In these studies the infralimbic region (IL) has mostly been ignored, The reason is as follows. The prefrontal cortex has been defined on the basis of its afferent connection with the mediodorsal nucleus of the thalamus (MD) 42'4s, and by most authors, the IL is not thought to lie within the MD-projection field s'tg'3t. The PHA-L method appears to be the most sensitive and accurate among the various anterograde tracing techniques available now t~'61. Yet the efferent connections of the PL elucidated recently by the PHA-L method did not reveal several prominent autonomic centers sT. Therefore, we assumed that the IL was more important than the PL in terms of visceral motor function, and we examined its efferent connections by the PHA-L method.

Correspondence: M. Takagishi,The Third Department of Anatomy, Chiba University School of Medicine, 1-8-I Inohana, Chiba 280, Japan.

27 MATERIALS AND METHODS The experiments were carried out on 31 male Sprague-Dawley rats weighing 200-300 g. The animals were anesthetized with sodium pentobarbital (40 mg/kg i.p.) and placed in a Narishige stereotaxic frame in the 'flat skull' position according to the Paxinos and Watson rat stereotaxic atlas4]. Following surgical exposure of the skull, a small craniotomy was performed over the left medial frontal cortex, and the dura was removed. The position of the injection sites of PHA-L was estimated according to the cytoarchitectonic studies of Krettek and Price 31 and Zillesn, and was cross-checked with the atlas of Paxinos and Watson41, PHA-L was iontophoretically deposited into various sites of the IL through micropipettes with tip diameters of 10-15/~m. A Narishige micromanipulator was used to position the tip of the pipette just lateral to the midline (0.4-0.5 mm), at a depth of 3.5--4.0 mm below the cortical surface, at points 3.5-3.7 mm rostral to the bregma. A 2.5% solution of PHA-L (Vector Labs.) in 0.05 M sodium phosphate buffer (PB, pH 7.4) was injected with 5/aA of pulsed (7 s on, 7 s off) positive current for 20 min. In some cases, PHA-L was pressure-injected. A 1.0-/d Hamilton syringe fitted with a glass pipette (tip diameter 40-50 ,am) was filled with a solution of 2.5% PHA-L in 0.1 M PB. The syringe was positioned i,, the same way as above with a Narishige micromanipulator. Three injections totaling 0.03 #! of PHA-L were given over 20 min. After the i-jections the pipette was left in situ for 10 min to prevent leakage of tracer through the pipette track. Following a 14-day survival time, the animals were reanesthetized and perfused transcardially with 100 ml of 0.9% saline, followed by cold 500 ml of 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M PB. The brains were removed and soaked in 0.1 M PB containing 20% glycerin overnight at 4 °C. The brains were serially cut on a freezing microtome at 50 pm in thickness and the sections were collected into 0.01 M phosphate-buffered saline (PBS, pH 7.4). The brain sections were rinsed in PBS and incubated for 30 min in 1% normal rabbit serum in PBST (PBS containing 0.3% Triton X-100) at 40 °C, The sections were then reacted with the primary antibody (goat anti-PHA-L, Vector Labs.; diluted 1:5000 in PBST) and incubated overnight at room temperature under gentle agitation. The sections were rinsed in PBST and incubated for 3 h with the secondary antibody (biotinylated rabbit anti-goat IgG, Vector Labs.; diluted 1:400 in PBST) at 40 °C, The sections were again rinsed in PBS~ and then incubated for I h in avidin-biotin-peroxidase complex (Vectastain ABC Kit, Vector Labs.; diluted 1:800 in PBST) at room temperature. They were then rinsed in 0.05 M Tris buffer (TB, pH 7.6) and the reaction product was developed in 0,02% 3,Y-diaminobenzidine, 0.3% nickel ammonium sulfate in TB, with 0.015% H 2 0 2. The sections were then rinsed in TB and mounted onto gelatin-coated glass slides, and dried overnight. Finally they were counterstained with Cresyl violet, dehydrated through graded ethanols, and coverslipped with Eukitt.

Case R102 Cortical projections. The injection site in case R102 involved labeled cells in layers III-VI of the caudal and central IL, without invading the adjacent PL or tenia tecta (Fig. 1A). In the medial aspect, a dense plexus of labeled axons and terminals was visible throughout the rostrocaudal extent of the IL and PL and in the deep layers of the medial precentral and dorsal peduncular cortices (Fig. 2 A - C ) . Anterograde labeling was also seen in the medial and ventral aspects of the anterior olfactory nucleus (Fig. 2A,B). in midiine cortical structures lying caudal to the injection site, a few labeled fibers and terminals were observed in the dorsal and ventral anterior cingulate cortices and the retrosplenial cortex (Fig. 2E). The IL provided bilateral innervation of cortical structures surroundiag the rhinal sulcus. At rostral lev-

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RESULTS Among 31 experiments, 22 produced satisfactory anterograde labeling and in 13 experiments the injection site involved the IL. Ten of these 13 cases were chosen for description of the efferent projections. The injection sites of these cases are schematically depicted in Fig. 1A, and a photomicrograph demonstrating a representative injection site is shown in Fig. lB. The parcellation of the mPFC used in this paper is based on two cytoarchitectonic studies (refs. 31 and 72).

Fig. 1. A: a schematic drawing of the frontal part of the rat brain indicating the position of PHA-L injection sites reported in the paper. Sagittal section modified from Krettek and Pricesl illustrating the positions of tracer deposits in relation to the cytoarchitectonic divisions of the mPFC. B: low-power photomicrograph of a representative PHA-L injection site in the IL shown in a cross section of the frontal cortex. Note that the contralateral IL is innervated by the commissure fibers, x 10.

28

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els, a dense plexus of labeled axons and terminals was visible in the lateral orbital, ventrolateral orbital and ventral agranular insular cortices, and a few were also visible in the ventral aspect of the dorsal and posterior AI (Figs. 2B-G and 5A,B), No labeling was seen in the granular insular cortex. More caudally, labeled axons appeared in the perirhinal cortex (Figs. 2N and 5E,F). Short, non-varicose fibers predominated in the dorsal endopiriform cortex, and a few were also visible in the caudal aspect of the claustrum and piriform cortex (Figs. 2G-N and 5C,D). All these cortical projections were bilateral, but with ipsilateral predominance. Forebrain projections. Subcortically projecting axons from the IL entered the medial aspect of the caudate putamen and traveled into the internal capsule (Fig. 2DG). Along this course, terminal fibers formed distinct patch-like arrangements in the ventromedial caudate putamen and a dense plexus of labeled axons and terminals was visible in the medial side of the accumbens nucleus and deep layers of the olfactory tubercle (Figs. 2C-E and 6A-D). Labeled fibers in the basal forebrain were seen in the medial aspects of the lateral septum (Fig. 6E), the vertical and horizontal limbs of the diagonal band of Broca, lateral preoptic area, and through-

out the bed nucleus of the stria terminalis (Fig. 6F). Minor inputs to the medial septum, ventral pallidum and substantia innominata were also noted (Fig. 2E-I). These patterns of labeling were bilaterally similar, although of greater density on the ipsilaterai side, In the amygdaloid complex, a dense plexus of anterogradely labeled axons and terminals was visible in the anterior cortical nucleus, basolateral nucleus, basomedial nucleus and the lateral division of the central nucleus (Figs. 2I-L and 7A-C). Lightly labeled fibers and terminals were also seen in the anterior amygdaloid area and medial amygdaloid nucleus (Fig. 7D). These amygdaloid projections were mostly ipsilateral, with the contralaterai projections being very weak. Diencephalic projections. Labeled axons from the IL coursed through the ventrolateral aspect of the medial forebrain bundle to innervate the lateral hypothalamus, particularly the magnt:cellular nucleus (Fig. ?J-M). Anterograde labeling was also seen in the dorsal (Fig. 8A,B) and posterior hypothalamic areas (Fig. 2K-M). A few lightly labeled fibers and terminals were seen in the paraventricular, medial mammillary and supramammillary nuclei (Figs. 2K-N and 8D). Fibers innervating the rostrai thalamus left the inter-

29 nal capsule and followed a dorsomedial pathway toward midline structures. Labeled fibers and terminals were seen in the rostral and medial aspects of the reticular, anteromedial, anteroventral and ventromedial thalamic nuclei (Fig. 2I,J). Fibers-of-passage were predominant in the caudal aspects of these structures and in the ventrolateral thalamic nucleus. Midline thalamic structures receiving significant terminal projections from the IL included the paratenial, central medial, interanteromedial, intermediodorsal, rhomboid, reuniens, paraventricular nuclei and MD (Figs. 2H-N and 7E,F). Labeling in the MD appeared almost exclusively within the medial division. The projections from the IL to thalamic nuclei were bilateral with ipsilateral predominance. Brainstem projections. Within the ventral tegmental area, a significant number of fibers passing through the medial forebrain bundle were visible. Terminal fibers were also seen throughout this area and in the dorsal and lateral aspects of the interpeduncular nucleus (Fig. 2NP). Labeled terminals and fibers appeared throughout the central gray area, although labeling was most dense in the ventrolateral sector of this region (Figs. 2 0 - Q and 8E).

Within the dorsal pontine tegmentum, labeled terminals and fibers were seen in the laterodorsal tegmental nucleus and the dorsal raphe nucleus (Fig. 2Q,R). Fibers surrounded, but did not enter, the dorsal tegmental nucleus. A small number of terminal-like fibers were also seen in the locus coeruleus (Fig. 8F), Barrington's nucleus, pontine c~entral gray area and medial aspect of the lateral parabrachial nucleus (LPB) (Fig. 2R). The projections caudal to the locus coeruleus were not traced in this case. All these brainstem projections were bilateral with ipsilateral predominance.

Case R107 The ~njection site in case R107 involved layers III-VI of the r¢~stral IL, and the most rostral part of this injection may have included a few cells in the medial orbital cortex (Fig. 1A). The pattern of efferent projections produced by this injection site (Fig. 3) was remarkably similar to that seen in case R102; yet there were some differences as described below. The density and range of terminal fibers increased in the dorsal and ventral anterior cingulate cortices (Fig. 3D-F). The range of labeled fibers in the perirhinal cor-

R107

Fig. 3. A series of line drawings of sections through the brain to illustrate the distribution of terminal labeling seen in case RI07. The small black dots represent the relative density of the labeling.

30 rex increased rostrally (Fig. 3L-N). Terminal labeling was more dense dorsally than ventrally in the caudate putamen (Fig. 3E-H). Within the contralateral amygdala, the density of terminal fibers increased remarkably in the lateral division of the central nucleus (Fig. 3K,L). In the diencephalon, additional labeled terminals and fibers were seen in the medial preoptic area (Fig. 3G,H), rostral anterior hypothalamic area, lab eroanterior hypothalamic nucleus (Fig. 3I and 8C), ventromediai hypothalamic nucleus (Fig. 3J) and the rostral part of the paraventricular thalamic nucleus (Fig. 3I-L). Caudal to the mesencephalon, the density of labeling slightly increased. A few additional labeled terminals were seen in the interstitial nucleus (Fig. 3P), rostral part of the dorsal tegmentai nucleus (Fig. 3Q), caudal part of the laterodorsal tegmental nucleus and subcoeruleus nucleus (Fig. 3R). Anterograde labeling was traced further caudally in this case, and terminal-like fibers were observed in the NTS (not shown in figure). In case R32, the injection was restricted to the IL just caudal to case RI07 (Fig. 1A). The efferent projections from this case were quite similar to case RI07 except

that most labeled terminals shifted caudally. Case R33

The injection site in case R33 involved layers V and VI of the IL at a level slightly dorsal to that of case R102 (Fig. 1A). The most dorsal part of this injection may have included a few cells in the most caudal PL. In general, the pattern of terminal labeling produced by this injection site (Fig. 4) resembled that seen in case RI02, but there were some differences as described below. Anterograde labeling was greatly reduced in the cortical structures surrounding the rhinal sulcus and dorsal endopiriform cortex (Fig. 4C-H). No labeling was seen in the piriform and perirhinal cortices. In the diencephalon, additional labeled terminals and fibers were seen in the medial preoptic area (Fig. 4G,H), dorsomedial hypothalamic nucleus (Fig. 4L), rostral aspect of paraventricular thalamic nucleus (Fig. 4I-L) and zona incerta (Fig. 4J). Caudal to the mesencephalon, no labeling was seen in the interpeduncular nucleus. Other cases

Some injections were centered primarily in the medial

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Fig. 4, A series of line drawingsof sections through the brain to illustrate the distribution of terminal labeling seen in case R33. The small black dots represent the relative den6ty of the labeling,

31 orbital cortex (Fig. 1A, cases R219 and R226). In these cases, terminal labeling in the forebrain and diencephalon was seen more laterally in position compared to that with rostral !L injections. In addition, the central division of the MD received more dense terminal fibers

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than seen following injections into the IL, with the density of the labeled terminals concomitantly decreasing in the paratenial thalamic nucleus, anterior hypothalamic area and lateroanterior hypothalamic nucleus. Brainstem projections from the medial orbital cortex were similar

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Fig. S. Photomicrographs of PHA-L-labeled fibers and terminals in the agranular insular, piriform and perirhinal cortex, A: dense labeling

of terminals in the AI. B'. higher magnification of the field shown in A. Note thick distorted axons with beaded varicosities. C: terminals in the piriform cortex. D: higher magnification of the field shown in C. Note the loose network of thin fibers, which rarely branch and are characterized by regularly spaced round varicosities. E: moderate number of fibers and terminals in the perirhinai cortex. F: l-Jgher magnification of the field shown in E. Compared to that seen in D, the density of the network is higher and the varicosities are larger, more deeply stained and more closely spaced. The upper right-hand corner corresponds to the dorsomedial direction of the cross-sectional plane in each photomicrograph. Case R102. A, x25; B, xl00, C, x2S; D, xl00; E, x25; F. xl00.

32 R l l l ) , the pattern of efferent projections closely resembled that seen with injections of the adjacent dorsal IL like in case R33, with the following exceptions. The density of labeled terminals increased in the central division

in pattern, but were reduced in density compared to those observed with IL injection sites• In cases where significant numbers of l~beled cells appeared in the ventral PL (Fig. 1A, cases Rll0 and

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Fig. 6. Photomicrographs demonstrating P H A - L labeled fibers and terminals in the striatum and limbic cortex. A : terminals and fibers in the ventromedial aspect of the caudate putamen• B: a parallel array o f axons with irregularly spaced and variably sized varicosities in the medial aspect of the accumbens nucleus. C: a dense network of axons in the deep layers of the olfactory tubercle. D: higher magnification of the field showi~ in C. Thin tortuous axons with bead-like varicosities form a dense network. E" fibers and terminal labeling in the medial aspect of the lateral septum. Thin varicose axons appear to bifurcate intermittently• Some varicosities have short stalks. F: fibers and terminal labeling in the dorsomediai aspect of the bed nucleus of the stria terminalis. Note the tortuous axons studded with irregularly spaced varicosities of variable size. The upper right-hand corner corresponds to the dorsomedial direction o f the cross.sectional plane in each photomicrograph. Case RI02, A , x25, B, x 100, C, x25; D, x 100; E, x lO0; F, x l00,

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Fig. 7. Photomicrographs of PHA-L-labeled terminals and fibers in the amygdala and thalamus, A: low-power photomicrograph demonstrating the relative distribution of the terminal labeling among subnuclei in the amygdala, B: higher magnification of the terminal labeling in the lateral division of the central nucleus. Note the thin tortuous axons with closely spaced bead-like varicosities, C: higher magnificatio, uf the terminal labeling in the anterior part of the basomedial nucleus. Thin winding axons with widely spaced elongated varicosities form a loose network. D: higher magnification of the terminal labeling in the medial nucleus, The axons morphologically resemble those in the central nucleus but the density of the network is lower, E: a dense network of labeled terminals in the MD. The terminals are localized in the medial division. Sparse labeled terminals are also seen in the contralateral MD, F: higher magnification of the field shown in E, Note that the tortuous axons frequently branch off small grape-like arborizations, which bear closely spaced large and deeply stained varicosities, The upper right-hand corner corresponds to the dorsomedial direction of the cross.sectional plane in each photomicrograph, Case R 102, A, x 10', B, xl~, C, x1(W);D, x100; E, xlO; F, xl~.

of the M D and concomitantly decreased in the paratenial thalamic nucleus and medial division of the MD. Terminal labeling was greatly reduced throughout the

amygdaloid complex except in the basolateral amygdaloid nucleus, and almost no labeling was seen in the AI and LPB.

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Fig. 8. Photomicrographs of PHA-L-labeled terminals and fibers. A: a dense network of labeled terminals in the dorsal hypothalamic area. B : higher magnification of the labeling in the dorsal hypothalamic area. The axons are characterized by numerous short branches furnished with varicosities. C: terminals in the anterior hypothalamic area. Thin tortuous axons with a few irregularly spaced varicosities form a loose network. D: labeled terminals and fibers in the medial mammillary nucleus. Note the thin tortuous axons with regularly spaced bead-like varicosities. E: moderate number of fibers and terminals in the central gray of the mesencephalon. F: several scattered terminals in the locus coeruleus. The upper right-hand corner corresponds to the dorsomedial direction of the cross-sectional plane in each photomicrograph. Case RI02 except for C from case RI07. A, ×25; B, ×I00; C, ×100; D, ×I00: E, x30; F, ×$0,

Some injections were centered in the dorsal tenia tecta and included varying small amounts of the ventral IL (Fig. IA, cases R206 and R218), The pattern of efferent projections of these cases was similar to that with injections of the IL but the density of anterograde labeling was significantly lower in all projection areas,

DISCUSSION

Comparison with previous results Whether the IL is part of the mPFC has been under dispute. The prefrontal cortex in several species has been defined on the basis of its connection with the

35

(,./" BSTf ',':f

•.

, Thalamus PTMDPV vucuns

LPB

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"""

Hypothalamus LPO DA MPA DMHPHSuM J PVXL,,, / y"

'~

\

~ _

Fig. 9. Summary diagram of the efferent projections from the IL.

MD 42'45. And generally, the IL is not thought to lie

PL

IL • "Ai

.........

~

"*" P l r

:

Amygdala Ce

"-

...........

: .~

BLA~BM 9

. . . . . . . . .

•~

Me

-

-"

Thalamus

-

I

PT PV M D CM :

Re

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Hypothalamus

MPA LPO PVH

_-

OMH OA LH

CG

-"

I

SuM

MM PH

~

L D T "-

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-

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-

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Fig. 10. Schematic diagram summarizing the major differences of

projection patterns between the IL and the PL. Solid and broken lines indicate brain areas receiving heavy and sparse innervation, respectively.

within the MD-projection field 5'x9'31, although a few retrograde tracing studies have suggested that it receives some MD input 13'52'53. Since this definition has been widely accepted, very little attention has been paid to the IL in various studies of the mPFC in the rat Accordingly, in a variety of anterograde tracing studies of the efferent connections of mPFC, those of the IL have been noted briefly only when the injection site accidentally included marginal areas of the IL. As far as we know, the investigations where an injection site was actually centered and limited to the IL were very few69'7° and even in those studies the efferent projections were only briefly described. The present study is therefore the first comprehensive investigation of the projections of the II. by the use of PHA-L as an anterograde tracer. The prese,tt fi,dings generally resembled the results of previous anterograde tracing studies of mPFC efferent projections s'n'14'34'43's~' To. Furthermore, our observations are consistent with the descriptions of mPFC efferent projections to a' number of individual cortical 4'2t'49'54, forebrain 7'8'16'2°'4°'st, diencephalic3'tt'25,26'2s's6'Ss'e9 and brainstem structures 2J°'22' 33,36.38,50,55,62,63,66. The following discussion will, therefore, focus on the discrepancies between the present findings and previous reports. Cortical projections. Projections from the IL to the anterior olfactory nucleus and piriform cortex were seen in the present study but have not been reported previously. Projection to AI was seen in accordance with the previous results 21'49, but the difference in density of the terminals in 3 (dorsal, ventral and posterior) areas of this cortex have not been reported. This projection was not seen in the study performed by Wyss et al. 7°, and the higher sensitivity by the PHA-L method appears to account for this discrepancy. Forebrain projections. We observed dense terminal la-

36 beling in the lateral division of the central nucleus of the amygdala in agreement with the previous study4°, but the difference in terminal density between the medial and the lateral divisions in the nucleus was not seen in this study. This projection was not seen in some previous studies?'57. The difficulty in restricting retrograde tracer injections to individual nuclei, lower sensitivity and difference of injection sites may account for these discrepancies. Diencephalic projections. Projections from the IL to the anterior hypothalamic area and lateroanterior hypothalamic nucleus were seen in the present study. These projections have not been reported previously. Brainstem projections. Projections from the IL to the brainstem structures were more extensive than previously reported, particularly within the dorsal raphe nucleus and the locus coeruleus2't°. We observed the IL projections to the parabrachial nucleus (PB) to be consistent with some previous findings, but the distribution and density of the terminals were different from those seen in those studies 36's°'63'66. In the previous studies using horseradish peroxidase, labeled terminals were seen diffusely throughout the PB 5°, diffusely within the LPB 63, or in the most medial portion of the medial PB ss. A recent PH.A-L study showed that this projection terminates diffusely within the PB, with little organization 36, We did, however, observe that the terminal labeling is mostly restricted to the medial aspect of the LPB. We did not pay particular attention to the NTS in the present study, as extensive studies have already been performed in this nucleus 3s'62'63'~', and we simply confirmed those results. In addition~ the other striking feature of the projection pattern is that the most intensive projections were seen to the AI, olfactory tubercle, MD and dorsal hypothalamic area. A summary diagram of the efferent projections from the IL is presented in Fig. 9, and a diagram comparing the projection patterns of the IL and PL is shown in Fig. 10.

Morphology of PHA-L-containing axons A recently recognized morphological feature of the PHA-L labeled projections is the variety in the arrangement of the fibers and varicosities in each nucleus, In our study, 3 types of terminal nerve fibers presented such a morphological feature according to their light microscopic appearance, The network of thin fibers, which rarely branched and were characterized by regularly spaced bead-like varicosities, were seen in the piriform cortex (Fig. 5D). On the other hand, a parallel array of axons with irregularly spaced and variable sized varicositics were observed in the medial aspect of the accumbens nucleus (Fig. 6B), In the medial division of the

MD, tortuous axons frequently branched off small grapelike arborizations, which bore closely spaced large and deeply stained varicosities (Fig. 7E). In most other projection areas, variably modified types of these axons were observed. The variety of axon morphology appears to reflect the different types of synaptic contact to the target neurons. The ultrastructural study of the synaptic input to these projection areas is in progress.

Functional implications Electrical stimulation or lesions of the mPFC are known to evoke a variety of autonomic responses, including changes in blood pressure, heart rate, respiration and gastrointestinal motility6'23'27'6s. Several recent studies have reported a direct descending projection from the rat mPFC to the NTS 3s'~2'6s'~s. The NTS is a visceroceptive cell group in the dorsal medulla and integrates a number of autonomic reflexes35. The NTS receives primary terminations of the facial, glossopharyngeal and vagal nerves, which carry primarily gustatory and visceral information ~s. The insular cortex is reciprocally connected with the NTS and maintains significant connections with central autonomic nuclei49' 66 Electrical stimulation of this cortex is known to evoke a variety of visceral responses similar to those seen in the case of mPFC 4s'71. Furthermore, several combined electrophysiological-anatomical investigations have suggested that the insular cortex may serve as a visceral sensory cortex ~'7~, Although little is known of the details of prefrontal cortex involvement in autonomic function, the results to date led several researchers to propose that the mPFC (PL, IL) are involved in 'motor' aspects of visceral control, whereas the insular region is involved in sensory aspects o'3s'4o'62'63.ss, The mPFC projection to the NTS originates primarily from the IL, with a minor contribution from the ventral PL 3s'62'63'~, The mPFC projection to the insular cortex, especially to the AI, also originates mainly from the IL 2t'4~. This connection is reciprocaP 9, Thus there is significant difference between the PL and the IL efferent projections. A schematic diagram summarizing the major differences in projection pattern between the IL (our results) and the PL (according to Sesack et al, sT) is shown in Fig, 10, The central amygdaloid nucleus (Ce) maintains significant connections which contain a wide variety of neuropeptides, with central autonomic nuclei and influences on various visceral functions tg. A role in cardiovascular regulation of the Ce, in which the PB and the NTS are also involved4~, is particularly well documented 37. The LPB constitutes a major terminal field for ascending fibers from caudal portions of the NTS, which are concerned with general visceral afferent information 24'39'44,

37 and ~nnervates the autonomic nuclei of the hypothalamus and related portions of the amygdala and the bed nucleus of the stria terminalis is. Comparing the present data with previous findings 36's7, it is clear that the mPFC projections to the Ce and LPB also originate primarily from the IL. Thus, the efferent projections to prominent autonomic centers from the IL are most extensive and dense than those from the PL as shown in Fig. 10. Accordingly, the present results support the concept of ours and Neafsey et al. 27'e~'~ and provide further evidence that the IL is more important than the PL in visceral motor function. In addition, the IL projects to a number of olfactoryassociated structures, including the anterior olfactory nucleus, horizontal limb of the diagonal band of Broca, olfactory tubercles and piriform cortex. A projection from the IL to the olfactory bulb has also been demonstrated 3a. Furthermore, the IL appears to receive olfactory afferent information relayed through the piriform cortex-endopiriform nucleus complex 32. Accordingly, the

IL may be involved in generating autonomic or visceral responses to odors such as urine, feces and food. Another interesting aspect of the role of the mPFC in autonomic function comes from evidence of its role in stress responses. For example, it has been reported that there is a selective activation of the mesocortical dopamine system and increased dopamine release in the mPFC by stress TM. Furthermore, there is a report that states that removal of the mPFC reduces gastri~ lesions produced by stress s9. On the other hand, the IL receives direct projection from the ]imbic system, area CA1 of the hippocampus 6°'67. Therefore, together with these findings, the present data also provide a possible morphological substrata for explaining how emotional stress produces autonomic response.

Acknowledgements.We would like to thank Mrs. F. Saito and Mr. K. Miyama for their technical assistance in these experiments and also Dr. K. Tanaka for his assistance in the preparation of this manuscript.

ABBREVIATIONS AA ac

Acb ACo Aq AI BLA BM BMA BST Ce CeL CO CM CPu DA dAC DB DEn DMH DR IL IMD IP LC LDT LH LPB LPO

anterior amygdaloid area anterior commissure accumbens nucleus anterior cortical amygdaloid nucleus aqueduct agranular insular cortex basolateral amygdaloid nucleus, anterior part basomedial amygdaloid nucleus basomedial amygdaloid nucleus, anterior part bed nucleus of the stria terminalis central amygdaloid nucleus central amygdaloid nucleus, lateral division central gray (of midbrain) central medial thalamic nucleus caudate putamen dorsal hypothalamic area dorsal anterior cingulate cortex diagonal band of Broca dorsal endopiriform nucleus dorsomedial hypothalamic nucleus dorsal raphe nucleus infralimbic cortex (infralimbic region of the mPFC) intennediodorsal thalamic nucleus interpeduncular nucleus locus coeruleus latemdorsal tegmentai nucleus lateral hypothalamic area lateral parabrachial nucleus lateral preoptic area

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