Efferent projections of the basolateral amygdala in the opossum, Didelphis virginiana

Brain Research Bulletin,

Vol. 17, pp. 335-350.

1986. 0 Ankho Intcmatio~A

Inc. Printed in the U.S.A.

0361-923w86 $3.00 + .oa

Efferent Projections of the Basolateral Amygdala in the Opossum, Didelphis virginiana ALEXANDER Department

of Anatomy,

J. MCDONALD’

University of South Carolina, School of Medicine, Columbia, SC 29208 AND JAMES L. CULBERSON

Department of Anatomy, West Virginia University School of Medicine West Virginia University Medical Center, Morgantown, WV 26505 Received

19 March 1986

MCDONALD, A. J. AND J. L. CULBERSON. Efferent projections of the basolateral amygdala in the opossum, Didelphis virginiana. BRAIN RBS BULL 17(3) 335-350, 1986.-The autoradiogmphic anterogmde- axonal transport technique was used to study efferent projections of the opossum basolateral amygdala. All nuclei of the basoktend amygdala send topolpaphically organixed fibers to the bed nucleus of the stria terminalis (BST) via the s&k terminalis (ST). Injections into rostrolateral portions of the basal nuclei label fibers that surround the commissural bundle of the ST, cross the midline by passing along the outer aspect of the anterior commissure, and terminate primarily in the contrakteral BST, anterior subdivision of the basokteral nucleus (BLa), ventral putamen, and olfactory cortex. Bach of the basal nucki project ipsilaterally to the anterior amygdaloid area, substantia innominata and topographically to the ventral part of the striatum and adjacent olfactory tubercle. The posterior subdivision of the basolateral nuckus (BLp), but not the basomedial nucleus (BM), projects to the ventromedial hypothalamic nucleus. BLa and BLp have projections to the nuckus of the lateral olfactory tract and also send fibers to the central nucleus, as does the lateral nuckus (L). The lateral nucleus also has a strong projection to BM and both nuclei project to the amygdalo-hippocampal area. BLa and BLp send axons to the ventral subiculum and ventral lateral entorhmal area whereas L projects only to the latter area. The kteral nuckus and BLp project to the perirhinal cortex and the posterior agranular insular area. The BLa sends efferents to the anterior agranukr insular area. Rostmlly this projection is continuous with a projection to the entire frontal cortex located rostral and medial to the orbital sulcus. All ofthe nuclei of the basolateral amygdala project to areas on the medial wall ofthe frontal lobe that appear to correspond to the prelimbic and infralimbic areas of other mammals. Despite the great phylogenetic distance separating the opossum from placental mammals, the projections of the opossum basolateral amygdala are very similar to those seen in other mammals. The unique frontal projections of the opossum BLa to the dorsolateral prefrontal cortex appear to be related to the distinctive organization of the mediodorsal thalamic nucleus and prefrontal cortex in this species. Amyedala

Opossum

Efferents

Stria terminalis

THE amygdala is a nuclear complex located in the forebrain of mammals and lower vertebrates that plays an important role in the generation of appropriate behavioral responses to both internal and external stimuli [20,52]. Although the amygdala was examined by several pioneering neuroanatomists including Meynert, Ganser, Kolliker and Cajal[31], the Grst detailed investigations of this region were by Volsch [83,84] and Johnston [25]. Johnston’s seminal descriptive and theoretical analysis of the amygdala focused primarily on the opossum. Johnston first recognized that the amygdaloid nuclei of the opossum, and other animals, could be divided into two groups: a primitive corticomedial group, which is well developed in lower vertebrates, and a

Prefrontal cortex

phylogenetically newer basolateral group, which is best developed in mammals, particularly in primates. He also provided the first detailed description of the two main pathways associated with the amygdala: a dorsal pathway, the stria terminalis, and a ventral pathway, now called the ventral amygdalofugal pathway, associated with the longitudinal association bundle. In his classic account of the robust stria terminalis of the opossum Johnston stated that this species was “a form which is favorable for study and at the same time presents in fairly complete form the elements typical of the stria in mammals.*’ In addition to the stria terminalis, other portions of the olfactory system and limbic system (“paleomammalian

‘Requests for reprints should be addressed to Alexander J. McDonald, Department of Anatomy, University of South Carolina School of Medicine, VA, Bldg. 1, Rm B-30, Columbia, SC 29208.

335

MCDONALD

33h

AN 1) C’ULBERSON

ABBREVIATIONS

AA AC AI AH AHA BLa BLP BM BST Ce Ce, Ce, C C C!Z

Cl Coa Cop DMF DS En GP IC IL

Anterior amygdaloid area Anterior commissure Agranular insular cortex Anterior hypothalamic nucleus Amygdalo-hippocampal area Basolateral amygdaloid nucleus, anterior subdivision Basolateral amygdaloid nucleus, posterior subdivision Basomedial amygdaloid nucleus Bed nucleus of the stria terminalis Central amygdaloid nucleus Central amygdaloid nucleus, lateral subdivision Central amygdaloid nucleus, medial subdivision Caudate nucleus Commissural bundle of the stria terminalis Cingulate cortex Claus&urn Cortical amygdaloid nucleus, anterior subdivision Cortical amygdaloid nucleus, posterior subdivision Dorsomedial frontal cortex Dorsal subiculum Endopiriform nucleus Globus pallidus Internal capsule Infralimbic cortex

in brain,” [43]), including the amygdala, are well developed the opossum whereas more recent acquisitions of the vertebrate forebrain, such as the neocortex (“neomammalian [43]), are poorly represented. The cortex of the brain,” opossum exhibits several characteristics which suggest that

it is relatively undifferentiated [37,64]. These and other “primitive” features of the opossum brain reflect the phylogenetic position of this species [30], for Didclphis is a generalized marsupial that closely resembles primitive mammals of the Cretaceous period [12]. Traditionally, it has been the connections of the amygdala with the hypothalamus and other subcortical areas that have received the most attention. Recently, however, considerable interest has focused on amygdalocortical connections. Studies in the rat, cat, and monkey have demonstrated that animals with better developed cortex have more extensive and better differentiated connections between the basolatera1 amygdala and areas in the temporal, insular and frontal cortex [56]. Because the structure of the opossum forebrain, with its well developed limbic system and poorly developed neocortex, suggests that it may represent a more primitive stage of forebrain evolution than that of the rat, it is of interest to examine the projections of the basolateral amygdala in Didclphis. METHOD

of experiments in 24 adult (male and female) North American opossums (Didelphis virginiuna) that received injections of tritiated leucine into the basolateral amygdala or surrounding regions. Eighteen animals received unilateral injections and six animals received bilateral injections. Opossums were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mgikg) and positioned in a stereotaxic apparatus (David Kopf) that was slightly modified for the opossum [14]. Stereotaxic coordinates were obtained from an unpublished This study

is based

on the results

[PC L LAB LEA M NA NOT OS OT P PAC Ped PC PR PrO RS S SI ST TO TT VMN VP X

Interstitial nucleus of the posterior limb of thr anterior commissure Lateral amygdaloid nucleus Longitudinal association bundle Lateral entorhinal area Medial amygdaloid nucleus Nucleus accumbens Nucleus of the lateral olfactory tract Orbital sulcus Olfactory tubercle Putamen Periamygdaloid cortex Cerebral peduncle Piriform cortex Perirhinal cortex Preorbital cortex Rhinal sulcus Subiculum Substantia innominata Stria terminalis Olfactory tubercle Tenia tecta Ventromedial hypothalamic nucleus Ventral putamen Area X

atlas of the opossum brain 1141. Tritiated leucine (L-14,5“H(N)] leucine, sp. act. 30-50 Wmmole; New England Nuclear) was desiccated by freeze-drying or evaporation and reconstituted in 0.9% saline to a final concentration of 15-50 &i//ll. Small amounts of labeled amino acid (0.02-0.5 ~1) were injected stereotaxically over a 20-30 minute period using a 5 ~1 Hamilton syringe attached to an electrode carrier. The needle was left in place for 30 minutes to prevent spread of isotope along the needle track. After a 24 hour survival period animals were anesthetized with sodium pentobarbital, injected with 0.1 cc of heparin through the left ventricle of the heart, and perfused through the ascending aorta with 0.M saline followed by 10% unbuffered fonnalin. Brains were blocked in the stereotaxic plane. removed, and embedded in paraffin. Serial sections were cut at 10-15 pm in the coronal plane and two adjacent sections at 200 pm intervals were mounted on acid-cleaned slides. Slides were then processed for autoradiography [20] and following an exposure period of 3-6 weeks at 4°C were developed in Kodak D-19, stained with cresylecht violet, and examined under brightfield and darkfield illumination. Injection sites and labeled efferent projections were plotted on tracings of brain sections drawn using a Bausch and Lomb microprojector. Under brightfield illumination two fairly distinct areas were recognized at injection sites. A central region surrounding the end of the needle tract exhibited dense accumulations of silver grains over neuronal cell bodies and less label over the neuropil. Surrounding the central region was a peripheral zone where silver grains were equally distributed over cell bodies and neuropil. The central region, which can probably be assumed to be the origin of most labeled efferent projections [19,70], is indicated as a black area in drawings of efferent pathways. The peripheral Lone of diffusion is indicated in drawings by a gray shaded area of uniform stipple surrounding the central region. Although light diffuse label was seen along needle tracts in the striatum dorsal to the amygdala in some animals. neither

PROJE~IONS

OF THE OPOSSUM AMYGDALA

337 at 15 pm in coronal and sag&al planes. In addition, one coronally-sectioned Weigert stained brain fromthematerial used by Johnston 1251(made available by Dr. Alvin B&z, University of Mirmesota) was used to study amygdaloid fiber tracts. The atlas by Oswaldo-Crux and Rocha-Miranda [53] and papers by Gray [21] and Loo [39] were used to identify regions of the opossum brain. RESULTS

Cytoarc~itect~re

of the ~~0s~~~ Basolaterai A~~g~la

Examination of Nissl-stained axons corroborated the Sndings of previous investigations 125, 31, 821 that the nuclear organir&on of the opossum amygdala conforms to the basic mammalian pattern. On the basis of similarities in cytoarchitecture and position the nuclei of the basolateral amygdala in the rat and cat descriid by Rrettek and Price [36] (lateral, anterior basolateral, posterior basolateral and basomedial nuclei) were also recognized in the opossum (Fig. 1). Although these homologies must be regarded as tentative until further information concerning the anatomy, connections and chemistry of these cell groups in the opossum is obtained, the terminology of Rrettek and Price has been used in the present study to denote the nuclei of the opossum basolateral amygdala. Projections of the Anterior Division of the Basolaterul Nucleus (BLa)

FIG. 1. Drawings of two series of coronal sections through the opossum amygdala arrangedfrom rostral (A and F) to caudal (E and I) that iflustrrtteinjection sites cited in the present study. Note that most inbctions extend through more than one level. Sections were drawn at 600 pm intervals. Only the cent4 portion of the injection site is represented (see the Method section). Scale= 1 mm.

striatal cell bodies nor the classic projections of the striatum were labeled in these cases. Labeled fibers and terminal fields were recognized using established criteria [ 19,701.Patterns of silver grains that clearly represent labeled fibers are indicated by large dots in drawings of autoradiographic experiments. Terminal field label, and accumulations of silver grains that could not be unequivocally identified as labeled fibers, are indicated in drawings by small dots. Since the amyg~ consists of a tightly packed nuclear cluster, few injections were confined to individual nuclei. Determination of the projections of a particular nucleus usually required comparison of several cases that received injections in and around the nucleus in question. Because animals with unilateral injections revealed that contralateral amygdaloid e&rents arc relatively light and terminate primarily in the same areas that receive ipsilateral efferents some animals with bilateral amygdaloid injections were used in this study. The nuclear configuration of the opossum amygdala was studied in several cresylecht violet stained brains sectioned

Five animals received injections that involved si8nificant portions of BLa. The injection in O-18 was confined to BLa but was a small injection that was restricted to the rostml pole of the nucleus (Fig. 1B). Although the projections seen in this animal almost certainly arise from BLa, it seems likely that many projections of the nucleus may not be labeled. Animal O-36L received an injection that filled ail parts of BLa and did not impinge upon other nuclei of the basolateral amygdala (Fiis. lB-D, 2G, 3C). This injection also included signet portions of the central nucleus. However, an injection that involved principally the central nucleus (O-4, not illustrated) revealed that the projections of the latter nucleus, primarily to the bra&tern, were strikingly different from BLa. Thus, the projections of BLa can be determined by examining animal O-18 and by comparing cases O-4 and 0-36L. Animal O-36 also received a contralateral injection (case 0-36R) that was identical to 0-36L. Analysis of other animals with unilateral injections that involved BLa reveale_d that this nucleus may have light contralateral projections to the same areas that receive stronger ipsilateral BLa projections (e.g., see case G-6 in Fii. 7). Possible contralateral projections in O-36, therefore, do not complicate the analysis of ipsilateral efferents. Case O-36L will be described in detail. From the injection site in O-36L (Fig. 2G) diffuse silver grains could be followed caudalward to the posterior division of the basolateral nucleus (BLp) where label was uniformly distributed within the confines of the nucleus. This pattern of silver grains indicates that labeled axons probably terminate in BLp. Rows of silver grains, representing labeled fibers, were observed running between BLp and the lateral nucleus (L) to reach the external capsule. These fibers passed caudally and projected to the ventrolateral portion of the entorhinal area (LEA, Fig. 2H). Silver grains were found over layers Ib (deep part of layer I), IV-VI, and to a lesser

MCDONALD AND (‘ULBERSON

338

O-36L

05

FIG. 2. Tracings of coronal sections arranged from rostra1 (A) to caudal (H) that illustrate the results of case O-36L

layer III. Light label was also observed over the middle third of the plexiform layer of the ventral subiculum at these levels (Fig. 2H). Labeled fibers from the injection site also ran dorsally to enter the stria terminalis (ST). Some fibers covered the dorsal, lateral and medial aspects of the commissural bundle whereas other fibers occupied the lateral half of the ST (Fig. 2F). The fibers encapsulating the commissural bundle, which may be termed “pericommissural bundle” fibers, ran rostralward and followed the commissural bundle to the anterior commissure (AC) (Fig. 2D,E). Rows of silver grains mixed with diffuse label were found bilaterally along the caudal aspect of the AC just outside of the stria terminalis component of the AC. At the midline this label was continuous with transported label from the contralateral injection site. However, in O-18, in which BLa was injected unilaterally, label was also observed bilaterally along the caudal aspect of the AC. No label was seen in other contralateral areas in O-18 but the small size of the injection in this animal may have precluded visualization of these projections. In 0-36L, and extent,

also in O-18. labeled fibers in the lateral part of the ST projected to the lateral portion of the bed nucleus of the stria terminalis (BST) (Fig. 2E). Along the entire course of the ST there was diffuse label located just lateral to this fiber bundle. adjacent to the medial aspect of the caudate nucleus (Figs. 2E.F). These silver grains appear to represent a projection to a portion of the lateral BST that extends from the region of the AC back to the central amygdaloid nucleus [S3]. Cells in this portion of the bed nucleus, which may be termed the “parastrial” portion of the BST. are similar lo, but slightly smaller than. neurons of the caudate nucleus and the border between these two nuclei is not clearly defined. The parastrial BST also appears to extend dorsal and rostra1 to the AC. where it is located along the lateral wall of the lateral ventricle. and can be followed as far anteriorly as the nucleus accumbens. Diffuse label was also seen in this rostra1 part of the parastrial BST in 0-361~ (Fig. 2B-D). Numerous labeled fibers were observed exiting the injection site and coursing dorsolaterally toward the external cap-

PROJECTIONS

OF THE OPOSSUM

AMYGDALA

339

along the timdus and dorsal bank of the rhinal sulcus (Fig. 2A-F). This cortex, most of which is located superficial to the claustrum, includes the ventral portion of the insular cortex and the adjacent perirhinal cortex of Gray [21]. In accordance with recent studies in rodents and cat [32,33,58] these areas will be termed agranular insular cortex (AI). In case 0-36L, and in other animals in which BLa was involved in the injection site, terminal label was observed in layers Ib (deep portion of layer I), V and VI of the ventral (perirhinal) and dorsal (ventral insular) portions of AI (Fig. 2C-E). The density of the layer I projection attenuated from dorsal to ventral and from rostral to caudal within AI. The caudal portion of the AI projection was confined to the perirhinal cortex in the fundus of the rhinal sulcus (Fig. 2E,F). Diffuse label was also observed over a deep arm of the endopirifonu nucleus located deep to AI and the claustrum, but not over the claustrum itself (Fig. 2B,C). This label may represent, in part, fibers projecting to the preorbital cortex (Pro; Fig. 2A,B), which is contiguous with the rostral part of AI. Diffuse silver grains in the deep arm of the endopiriform nucleus and deep layers of AI were continuous with label in layers V and VI of PrO whereas the projection to layer Ib of AI was continuous with the projection to layer Ib of PrO (Fig. 2B,C). In the rostral three fourths of PrO and in frontal polar cortex silver grains were observed over layer VI but not over more superlkial cortical layers (Fii. 2A). This deep label was continuous ventrally with light label in the deep portion of the cortex located along the far rostral part of the rhinal sulcus (Fig. 2A). The projection to PrO also extended onto the caudal and medial bank and lip of the orbital sulcus (Fig. 2B,C). Near the rostral pole of the orbital sulcus this projection shifted mediiy to include an area on the dorsomedial shoulder of the frontal lobe that merges with the rostral cingulate cortex on the medial wall of the hemisphere (Fig. 2A). Silver grains were observed primarily over layers I, V, and VI of this dorsomedial frontal cortex (DMF). This projection to DMF could be followed rostrally to the frontal pole. There was also a light projection in 0-36L to layers I, V and VI of the anterior cingulate cortex (Cg) of Gray [21] and to au area of indistinct lamination ventral to the cingulate area and dorsal to the tenia tecta (Fig. 2A). The area of indistinct lamination will be termed infralimbic cortex (IL) since it closely resembles the infmlimbic cortex of the rat and cat [32,33] in both position and cytoarchitecture. The anterior cingulate area and IL occupy the ventral half of the medial wall of the frontal lobe as far rostrally as the frontal pole. Caudally the infralimbic area appears to blend with the rostral Dart of the dorsal subiculum and liaht label in this

1 FIG. 3. BrightfieId (A) and darkfiekl (B) photomicrographs of the rostrolatcrai part of the olfactory tubercie in case 0-36L. Silver grains are found over layers III, II, and the deepest part of layer I. Background grain densities arc seen over the islands of CaIIeja (arrows in A), pnrvocekiar parts of layer II, and most of layer I. ScaIe=u)o pm. (C) Photomicrograph of the injection site in case 0-36L. Compare with Figs. 1C and 2G. Scale=500 pm.

received projections from BLa. Labeled fibers exited the rostral pole of the injection site in 0-36L and coursed diffusely through the anterior amygdaloid area (AA, Fig. 2F). Diffuse label also filled the substantia innominata (SI) and extended medially to become continuous with label in the . .._- ,.-.. a-. _. . . . .. . . .-_ . the boundaries

represent

of these areas, these silver grains probably

terminal label as well as labeled fibers. In the nu-

cleus of the lateral olfactory tract label was seen over layer Ib and the underlying cellular layer (Fig. 2E). Layer Ib of the adjacent pirifotm cortex also received a projection (Fig. 2EF). Labeled fibers were also observed coursing dorsalward to

MCDONALD AND CU I .BERSOh

340

FIG. 4. Tracings of coronal sections arranged from rostra1 (A) to caudal (F) that illustrate the results of case G-3.

terminate in the ventral portion of the putamen (Fig. 2E,F). This projection field extended rostralward and included the portion of the ventral putamen located deep to the olfactory tubercle as well as layer III of adjacent portions of the olfactory tubercle (Fig. 2A-D). In a continuous zone along the lateral and rostra1 edges of the olfactory tubercle silver grains were also seen over layers Lb and II (Figs. 2A-D, 3A,B). There was no projection to the islands of Calleja of the olfactory tubercle (Fig. 3A,B), the nucleus accumbens, or the medial portion of the olfactory tubercle. Since the central nucleus was involved in the injection site in case 0-36L, it was necessary to determine the extent that it contributed to the projections observed in this animal. In case O-4, which received an injection that was fairly well

confined to the central nucleus, and in other animals in which this nucleus was injected, projections were seen to the substantia innominata (SD, the lateral part of BST. and numerous brainstem areas. Since projections to SI and the lateral part of BST were also observed in O-18, where the injection site was confined to BLa. it appears that both BLa and the central amygdaloid nucleus contributed to these projections in case 0-36L. Because the projection in animal 18 to the BST was sparse compared to the AC projection, it appears that the central nucleus is largely responsible for the BST projection seen in 0-36L. Brainstem projections, which were lightly labeled in 0-36L (Fig. 2G). were only observed with injection sites that involved the central nucleus. In 0- 18, in which the injection site was confined to BLa and did

PROJECTIONS OF THE OPOSSUM AMYGDALA

FIG. 5. Tracings of coronal sections arrangedfrom rostral (A) to caudal (E) that illustrate the results of case O-15.

not involve the central nucleus, diffuse silver grains were distributed over both lateral and medial subdivisions of the central nucleus. Projections of the Posterior Division of the Basolateral Nucleus (BLp) Six animals received injections that involved significant portions of BLp. The injection in case O-3 included most of the rostrocaudal extent of the nucleus and only impinged slightly on the dorsally adjacent lateral nucleus (Figs. 1, 4&E). This case will be described in detail. From the caudal edge of the injection site label extended caudalward and was difisely distributed over the caudal pole of BLp (Fig. 4F) and the amygdalo-hippocampal area. Since silver grain density dropped to background levels near the boundaries of these nuclei the label in these areas probably represents terminal field label rather than diffusion from the injection site. IX&se silver grains were also observed over the plexiform and cellular layer of the rostral portion of the ventral subiculum (Fig. 4F). Labeled fibers ran caudolaterally to project to layer IV-VI and, to a lesser extent, layer III and Ib of the lateral entorhinal area (Fig. 4F). There was also a projection to the dorsally adjacent perirhinal cortex (PR). This portion of the perirhinal cortex, located between the entorhinal area and the temporal area of Gray 1211,is very thin and compressed, and does not exhibit distinct laminae. It probably corresponds to the perirhinal cortex described by Krettek and Price [33] in the rat and cat. Silver grains were distributed lightly over layer Ib and the deep part of this cortex. This cortical projection was continuous rostraIly with efferents to the portion of perirhinal cortex located at amygdaloid levels.

At caudal amygdaloid levels the perirhinal cortex is thicker than at levels through the entorhinal area. At rostral amygdalar levels distinct laminar bands become evident as this cortex becomes continuous with the agranular instdar cortex that receives a projection from BLa (see above). The BLa projection cortex may correspond to the anterior agranular insular cortex seen in the rat and cat (dorsal and ventral agranular insular areas of [32,33]) whereas the perirhmal cortex found at amygdaloid levels would appear to correspond roughly to the posterior agranuhu insular area of the rat and cat [33]. For ease of description, therefore, the cortex in the fundus of the rhinal sulcus at amygdaloid levels will be referred to as posterior agranular insular cortex whereas more rostral portions of AI will be termed antetior agranular insular cortex. In animal O-3 silver grains were observed over layer Ib and the deep part of the posterior AI (Fig. 4B-E). Silver grains were also seen over the deep arm of the endopirifotm nucleus located deep to AI. The BLp also has projections that course through the ST. In case O-3 labeled fibers ran dorsomedially from the injection site, coursed aloug the medial aspect of the central nucleus, projected through the lateral portion of the ST, and terminated primarily in the lateral part of BST (Fig. 4B-D). IX&se label was also observed over the parastrial portion of BST. Further rostrally the projection to the lateral BST was continuous with label in the ventromedhd portion of the nucleus accumbens (Fig. 4A). There was aIso light label over layer III of the adjacent portion of the olfactory tubercle. Extending dorsalward from the nucleus accumbens along the medial wall of the lateral ventricle (Fig. 4A) were sparse silver grains that probably represent labeled fibers. This pro-

MCDONALD

AND (‘ULBERSON

FIG. 6. Tracings of coronal sections arranged from rostra1 (A) to caudal (G) that illustrate the results of case O-5.

jection terminated in layer I of IL and the adjacent cingulate area (Fig. 4A).

anterior

Diffusely arranged fibers, perhaps of ST origin, ran through the anterior hypothalamus and terminated in the ventromedial nucleus of the hypothalamus (Fig. 4D,E). This projection was seen in all animals in which BLp was included in the injection site but was never observed with injections of the basomedial amygdaloid nucleus. Additional efferents observed in animal O-3 included projections to the central nucleus, BLa, anterior amygdaloid area, substantia innominata, the nucleus of the lateral olfactory tract and the laterally adjacent piriform cortex (layer IB) (Fig. 4B-E). Projecticms of the Basomedial

Nucleus (BM)

Five animals received injections which included significant portions of BM. No single animal received an injection that involved all portions of BM but animals O-15 and O-5 had injection sites that occupied, respectively, medial and lateral portions of BM with little involvement of adjacent nuclei (Fig. 1). These cases will be discussed in detail and compared to animals that received injections of adjacent nuclei. The injection site of O-15 involved the medial portion of BM throughout its rostrocaudal extent but did not impinge upon other nuclei (Figs. 1, 5D). Caudal to the injection site there were silver grains in the amygdalo-hippocampal area and posterior cortical nucleus. but it appeared that most of this label was due to diffusion from the injection site (Fig.

5E). Numerous labeled fibers passed dorsalward from the injection site to enter the ST (Fig. 5D). Labeled fibers in the ST were observed in the ventrolateral portion of the stria and along the dorsal and medial surfaces of the commissural bundle (Fig. SD). Some fibers of the ST terminated diffusely in the medial portion of the BST that extends ventromedially behind the AC (Fig. 5C). There were also silver grains over the parastrial portion of BST. Fibers of the stria that surround the commissural bundle of the ST follow that bundle to the AC. Silver grains in both diffuse and linear arrangements were seen just outside of the caudal part of the AC (Fig. 5C). This label, which was heavier ipsilaterally, was continuous rostrally with silver grains that covered the dorsal and ventral surface of the AC (Fig. 5B). Further rostralward, label over the BST became continuous with silver grains over the nucleus accumbens. Most fibers terminated in the ventromedial portion of the nucleus accumbens and layer III of the adjacent olfactory tubercle (Fig. 5A). From the rostral edge of the injection site diffusely arranged labeled fibers projected forward into the substantia innominata and anterior amygdaloid area (Fig. 5C). Since diffuse silver grains were found over all portions of these two areas but did not extend beyond their borders, this label must represent, in part, labeled axon terminals. Diffuse label was also found over the anterior division of the cortical nucleus and the medial nucleus but it could not be determined whether this represents diffusion from the injection site or labeled terminals. Projections to regions adjacent to the medial and lateral

PROJECTIONS OF THE OPOSSUM AMYGDALA borders of the frontal lobe cortex were observed in animal O-15. There was a projection to the deep arm of the endopiriform nucleus located beneath the ventral portion of AI (Fig. 5A-C) and to the plexiform layer of the rostral portion of the dorsal subiculum (Fig. 5B). The latter projection was continuous tostmlly with a light projection to layer Ib of the infralimbic cortex. The injection site in animal O-5 involved lateral portions of BM and also impinged upon the rostrolateral portion of the periamygdaloid cortex (PAC) (Figs. 1, 6). Most of the projections observed in O-15 were also seen in O-5. Additional projections observedin O-5 must originate, therefore, from either lateral portions of BM or from PAC. The extent to which the latter area contributed to the efferents seen in O-5 was estimated by comparing this case to animals that had injections centered in PAC (e.g., O-29, Fig. 1; O-23, not illustrated). The injection site in O-29 was centered in PAC but also extended dorsally into the area of BM that was injected in O-15 (Fig. 1). In animal O-5 there were diffuse silver grains over the AHA (Fig. 6G) which appear to represent a projection to that nucleus. At more caudal levels there was some light label in the LEA and in the super&al part of the plexiform layer of the subiculum. These light projections appear to originate in the PAC since they were seen with injections centered in PAC (e.g., O-29) and were much heavier in PAC animals. The ST projections seen in O-5 were similar to those observed in O-15 but projections to more lateral portions of the BST and to the contralateral central nucleus via the contralateral ST were found in O-5 (Fig. 6GE). These projections were not observed with injections involving PAC or medial portions of BM. As in O-15, projections were seen to the AA and SI iu O-5. There was also a dense accumulation of label over the central nucleus in O-5. However, the needle track passed through the lateral part of the central nucleus in this animal and a few labeled cell bodies were observed. These cells might project to other parts of the central nucleus via local axonal collaterals and thus produce label throughout the nucleus. The projection to ventral portions of the striatum (ventral putamen and nucleus accumbens) and olfactory tubercle in O-5 (Fig. 6B) extended further laterally than in O-15, suggesting that the projections of BM to these areas are topographically organized. In addition to a projection to the deep arm of the endopiriform nucleus, as seen in O-15, there was also a projection to the deep part of layer I of the anterior portion of AI in O-5 (Fig. 6B,C). Further rostrally there were projections to the rostral dorsal subiculum (plexiform layer), and to the iufralimbic and anterior cingulate cortex (6A,B). Projections to these cortical areas were not observed with PAC injections. Projections Observed With a Large Injection Involving Basolateral and Basomedial Nuclei

Animal O-6 received a large injection of tritiated leucine that involved the rostral portions of BLa and BLp as well as the rostrolateral portion of BM and caudolateral part of the AA (Figs. IF-H, 7D-E). The injection also included a region that was rostral to BLp and ventral to the rostral portion of the lateral nucleus. This region is termed area X since it was difficult to determine if it was part of one of the basal nuclei, a ventral portion of L, or a separate nucleus of the amygdalo-piriform region (Fig. 7D). At the rostra1 pole of the basolateral amygdala, area X blends with a cluster of cells

343

located deep to the ventromedial portion of the piriform cortex which suggests that area X may correspond to a portion of the ventral endopiriform nucleus of the rat and cat 1361. The ipsilateral projections seen in this case (Fig. 7), e expected, represent the sum of projections descrii for each individual nucleus of the basal nuclear compkx (see above). Since the rostml pole of this brain was not saved the anticipated projections to the rostral frontal lobe were not observed. However the projection of BLa to caudal portions of the preorbital area was seen (Fii. 7A). Smce no new ipsilateral projections were seen in this animal, it would appear that the AA must project ipsilaterally to areas that receive projections from the basal nuclear complex. Alternatively, the involvement of AA in the injection site was not sufficient for its efferents to be adequately labeled. The most striking aspect of the projections seen in O-6 was the extent of the contralateral efferent projection. Numerous fibers coursed dorsomedially from the injection site to form a compact cuff of labekd fibers that completely surrounded the commissural bundle of the ST (Fig. 7D). These pericommissural bundle fibers retained this position as they ran through the ST and followed the commissural bundle to the AC (Fig. 7C-E). They completely encapsulated the AC as they crossed to the contralatetal side of the brain (Fig. 7B). Some fibers terminated in the contralateral BST (Fig. 7A,B) but others passed caudally in the contralateral ST where they capped the dorsolateral aspect of the commissural bundle and terminated in BLa (Fig. 7C-E). Some of the fibers that decussated did not course in the ST but ran ventral to the posterior limb of the anterior commissure (AC) and projected to two small cell clusters (WC; Fig. 7B) that may correspond to the interstitial nucleus of the posterior limb of the AC of the rat [16]. Other fibers ran caudally, streamed past the rostral pole of L(Fig. 7C), and ran caudally to BLa and area X. There were also contralatera1 projections to layer Ib of the ventral portion of the piriform cortex and the anterior AI (Fig. 7A-D). Further rostrally, diffuse silver grains filled the contralateral ventral putamen but did not spread medially to include the nucleus accumbens (Fig. 7A). The precise origins of the contralateral projections are difficult to determine in this case due to the extent of the injection site. BLp appears to have no contralateral projections (see O-3) and BM has very few contralateral efferents (see O-5). Probably most of the contralateral projection originates in AA, BLa or area X. Since the contralateral efferents to AI, BST and the ventral putamen mirror ipsilateral projections of BLa, it seems likely that at least some of the contralateral projections originate in BLa. On the other hand, in case 0-32L, in which the injection site involved area X, the amygdalo-piriform transition area, and a small portion of the adjacent piriform cortex but not BLa (Fig. IF-G), there were numerous fibers that surrounded the commissural bundle of the ST, crossed to the contralateral side by streaming along the outer aspect of the AC, and entered the contralateral amygdala via the ST. The final destination of these fibers could not be ascertained because animal O-32 also received a contralateral injection of the piriform cortex (case 0-32R). As in other animals that received piriform cortex injections, there was an ipsilateral projection to the basolateral nucleus in case O-32R so possible conttalateral projections to the amygdala from injection 0-32L could not be discerned. Likewise, label observed in the ventral striatum and in other areas contralateral to 0-32L may have arisen from injection O-32R.

344

MCDONALD

AND CUi..BEKSO!-v

FIG. 7. Tracings of coronal sections arranged from rostra1 (A) to caudal (F) that illustrate the results of case O-6.

Projections

of the Luteral Nuc~1eu.s (L)

Three cases received injections which involved L but only two were centered in this nucleus. Unfortunately, both cases (O-3 1L and O-3 IR) were on opposite sides of the same brain. However, the terminal fields observed in this animal were very similar to those that receive ipsilateral projections from L in other species [33-361. This suggests that the projections seen on either side of brain 3 1 represent terminals of fibers arising primarily from the ipsilateral injection site and that fibers observed crossing in the AC in this brain may augment ipsilateral projections to the same terminal fields. The results of case O-31R will be discussed in detail since the injection involved most of the lateral nucleus, as well as part of the putamen, but did not impinge upon other nuclei of the amygdala (Figs. lB-D, 8F). In this case it is assumed that the projections to the globus pallidus (Fig. 8E) and substantia nigra were efferents of the putamen and that the other projections originated from the lateral nucleus. These striatal projections were not seen in case 0-31L where the injection site did not include the putamen. Caudal to the injection site numerous silver grains were observed over BLp and AHA (Fig. 8G). The AHA was heavily labeled and since silver grains tilled the confines of the nucleus but fell off sharply at its borders, it appears that this label represents a projection to this nucleus. The label in

BLp does not observe nuclear boundaries and may be due to diffusion from the injection site. Silver grains in the subiculum and adjacent comu ammonis (Fig. 8G) also appear to be due to diffision from the injection site since they were diffusely distributed and gradually attenuated at greater distances from the injection site. Although no neuronal cell bodies in these hippocampal areas had a high concentration of label it is possible that some of their connections could have been labeled. The light projection to the dorsal subiculum (Fig. 8) may have originated in part from the hippocampus. At the caudal pole of the amygdala a stream of diffise label and labeled fibers passed dorsolaterally through the deep layers (IV-VI) of LEA and terminated in layer III of LEA and in the rostra1 part of the perirhinal cortex (Fig. 8G,H). The projection to the perirhinal cortex terminated in layer I and the deepest portion of the cortex adjacent to the lateral ventricle. This projection was continuous rostrally with projections to the posterior portion of AI which involved layers I and VI, as well as the deep arm of the endopiriform nucleus (Fig. 8D-F). The anterior portion of AI received a very light projection (Fig. 8B,C). Near the rostra1 pole of the hemisphere there was a strong projection to layer I of the anterior cingulate cortex and lighter label in layer 1of IL (Fig. 8A,9). Numerous labeled fibers passed dorsomedially from the

PROJECTIONS OF THE OPOSSUM AMYGDALA

345

FIG. 8. Tracings of coronal sections arranged from rostral (A) to caudal (H) that illustrate the results of case O-31R.

iqjection site to enter the ST (Fig. SF). Whereas some silver gmins were distributed over the late& ST, a much greater grain density was seen over the parastrial BST (Fig. 8E). The lateral ST projection terminated lightly in the lateral BST (F&g.8D). Dense accumulation of label over the parastrial BST could be followed as far rostral as the nucleus accumbens (Fig. 8C). The label over the parastrial BST gradually attenuated as the nucleus was followed rostralward suggesting that silver grains seen over this area represent diffusely arranged fibers as well as labeled axon terminals. Very sparse label was seen in the nucleus accumbens (Fig. 8C). At the level of the amygdala, L had a strong projection to BM (Fig. 8F). Further rostrally diffuse label was found over AA, Ce and L, and there was also a dense accumulation of label over the lon~tu~~ association bundle (LAB) which runs along the dorsomedial comer of L (Fig. 8E). Labeled fibers in the LAB ran forward, joined the posterior limb of the AC and crossed the midline in the commissure (Fig. 8D). Since these decussating fibers blended with similar fibers from the contmlateral injection site their ultimate destination could not be ascertained.

DISCUSSION Subcortical Projections of the Opossum Basolateral Amy&ala

This investigation confirms the finding of earlier descrip tive studies using the Weigert technique that the projections of the opossum basolateral amygdala reach a number of subcortical areas by way of the stria terminalis and the ventral amygdalofugal pathway [8, 25, 391. The use of the autoradiographic tract tracing technique in the present study, however, has permitted a more accurate and reliable determination of the origin, course and terminations of these tracts. Injections of tritiated leucine into the opossum basolateral amygdala labeled fibers passing through the lateral ST and, in some cases, running along the edges of the commissural bundle of the ST (bundle I of Johnston, {25]). The projections of the opossum basolateral amygdala that course through the lateral ST terminate, in part, in the bed nucleus of the ST (BST). These projections are virtually identical to

McDONALD

FIG. 9. Brighttield (A) and darkfield (B) photomicrographs of the medial frontal cortex in case O-31R at the level of Fig. 8A. Note strong projection to the molecular layer (ML) that avoids its superficial zone. Scale-250 pm.

Ahl)

C‘LiIBERSOh

those seen in the rat and cat by Krettek and Price [35]. In all three species the two divisions of the basolateral nucleus project to the lateral portion of BST, the basomedial nucleus projects to both medial and lateral portions of BST, and the lateral nucleus projects to the portion of BST that is located just medial to the head and tail of the caudate nucleus (parastrial BST of the present account). In rhe opossum. however, the basal nuclei also appear to project to the parastrial BST. A strong, topographically organized projection to the BST, therefore, appears to be a characteristic feature of the mammalian basolateral amygdala. This is undoubtedly one of the most important projections of the basolateral nuclear complex since information from the basolateral amygdala can indirectly reach numerous subcortical regions, including almost all portions of the hypothalamus, via the widespread projections of the BST [ 13.711. The labeled “pericommissural bundle” fibers of the present study, which encapsulate the commissural bundle as it runs through the ST, appeared to arise primarily from rostra1 portions of the basal nuclei, and could be followed to the AC where they encapsulated the caudal face, and in some cases also the rostra1 face (case O-6). of the commissure. While some of the label surrounding the AC may represent a projection to the bed nucleus of the commissure. as described by Valverde [78] in the cat, many of the labeled fibersin this location project to contralateral brain areas. Recent axonal transport studies in rat [27, 29, 861 and cat [26,51] have demonstrated that, as in the present study. the basolateral nucleus, ventral endopiriform nucleus (area X of the present study) and, to a lesser extent, the basomedial nucleus have crossed projections that pass through the anterior commissure. As in the opossum many of these decussating fibers are associated with the commissural bundle of the ST. In the opossum the crossed projections of the rostra1 basolateral amygdala terminated in the contralateral BST. striatum, piriform cortex, anterior agranular insular cortex. BLa, and area X. Likewise, using axonal transport techniques in the rat Kelley et cd. [29] have observed a contralatera1 projection to the striatum that originates in BLa and, to a much lesser extent, from BM and the lateral nucleus. Similar projections were detected in the rat and monkey by Russchen and co-workers [62.63]. It seems likely that the contralateral projections of the basolateral amygdala observed in the opossum and other mammals may play a role in interhemispheric transfer of information related to motivation, emotion, and learning [26] as well as provide one route by which epileptiform activity involving the temporal lobe may reach contralateral brain areas [76]. As in other mammals [35,47, 561 the opossum basolateral amygdala projects to several basal forebrain areas including the substantia innominata, rostra1 ventral putamen. nucleus accumbens, and olfactory tubercle. The projections to the rostra1 ventral striatum are topographically organized in the opossum. Portions of the olfactory tubercle overlying the striatal projection fields are also involved in these projections. Similar results have been obtained in rodents, carnivores and primates [35, 49, 50, 62, 631. In addition to these rostra1 ventral striatal projections the opossum basolateral amygdala also projects to more caudal portions of the striatum. Similar projections have recently been described in the rat [29. 63, 801. cat [61] and monkey [62]. In the rat the basolateral amygdala (primarily BLa) sends fibers to the entire striatum at caudal levels and primarily to dorsomedial and ventral striatal regions at rostral levels. In the present study the full extent of the caudal

PROJECTIONS

OF THE OPOSSUM

striatal projection field could not be determined because this region contained diffuse silver grains surrounding the trans-striatal paths of needle tracks. However there is clearly a projection to the ventral part of the caudal striatum in the opossum. The projection to the dorsomedial striatum in the rat appears to correspond to the projection to the parastrial BST seen in the present study. As in the opossum this region in the rat receives projections from the lateral and basomedial nuclei as well as the basolateral nucleus [29]. As pointed out by the latter authors, the striatal projections of the basohtteml amygdala, prefrontal cortex and ventral tegmental area exhibit considerable overlap and can be used to define a “limbic striatum” where emotional and motivational factors can influence somatic motor activity. Since the basolateral amygdala has reciprocal connections with higher order sensory association areas of the cortex and sends efferents to the striatum, it is in a position to translate motivationally significant stimuli into appropriate behavioral responses. The present study demonstrates that BLp projects to the core of the ventromedii hypothalamic nucleus (VMN) in the opossum. A projection to the capsule of VMN in the opossum originates in the amygdalo-hippocampal area (AHA) and/or posterior cortical nucleus (unpublished observations). These results only partially agree with a previous electrophysiological study of opossum amygdaloid projections which found that corticomedial, but not basolateral, amygdaloid stimulation evokes responses in VMN [ 151. In an autoradiographii tract tracing study in the rat and cat Krettek and Price [U] found that the only part of the basolateral amygdaIa that projects to VMN is BM, and this nucleus sends efferents mostly to the core of the nucleus. Likewise, McBride and Sutin [41] found HRP labeled neurons in BM of the cat after injections into the VMN. However in a recent HRP study in the rat labeled neurons were found in all nuclei of the basolateral amygdala after small injections into VMN [40]. Thus it is not clear at the present time the extent to which the varying results described above are related to methodological or species differences. It is clear, however, that VMN plays a very important role in autonomic, endocrine and behavioral activities and that amygdaloid projections to this nucleus may account for many of the responses elicited by amygdaloid stimulation [ 18, 28, 591. The intra-amygdaloid projections of the opossum basolatera1 amygdala are very similar to those of the rat, cat and monkey [I, 16, 36, 54, SS]. However, the topographical organization of basolateral amygdaloid projections to the central nucleus that was observed in the cat [36] was not seen in the opossum. A projection of the lateral nucleus to the amygdalo-hippocampal area (AHA) was observed in the present investigation but not in an autoradiographic study of the cat and rat [36]. In the rat, however, an HRP injection into the posterior cortical nucleus that also may have involved the underlying AHA did label cells in L [54]. It has been suggested that in the rat and cat the lateral nucleus can influence VMN by way of its strong projection to BM [56]. Although BM does not appear to project to VMN in the opossum it is possible that the lateral nucleus may influence VMN by way of its projection to AHA, which projects to the capsule of the opossum VMN. Projections

347

AMYGDALA

to the Entorhinal Cortex and Subiculum

In the opossum the anterior and posterior divisions of the basolateml nucleus project to the ventral subiculum and ven-

tral lateral entorhinal area whereas the lateral nucleus appears to project solely to the latter ama. The origin, course, and laminar terminations of these projections closely resemble those described in the rat and cat [16,341. Similar projections also exist in the monkey [2,56]. The projection of BLa to the subiculum seen in the opossum, however, was not observed by Kmttek and Price [34] in the rat and cat. All nuclei of the opossum basolateral amygdaht appear to have a projection to the rostral portion of the dorsal subiculum that is continuous anteriorly with the infialimbic cortex. In eutherian mammals the corpus callosum is located just caudal to the in6alimbic cortex and there is no structure resembling the rostral dorsal subicular area of the acallosal opossum. The results of the present study, in conjunction with previous studies in other species, demonstrate that basolateral amygdaloid projections to the entorhinal cortex and subiculum are a characterisitc feature of the mammalian limbic system. As pointed out by Price [56], amygdaloid projections to the entorhinal cortex allow the amyg& to modulate inputs to the hippocampal formation whereas projections to the ventral subiculum can influence the output of the hippocampal formation to the hypothalamus. It has been suggested that these amygdaloid projections might provide information regarding the internal state of the organism and the affective significance of stimuli [79]. Projections

to the Cerebral Cortex

Each of the nuclei of the opossum basolateral amygdala appears to have projections to the cortex, but the exact areas of termination vary. Of particular interest are the widespread projections of BLa to the frontal lobe. These effemnts reach several different frontal areas and the laminar termination patterns vary according to the region. One common characteristic that ties these frontal areas together is that they all appear to receive projections from the mediodorsal nucleus of the thalamus (MD) [5,75] and thus by definition [601 constitute the prefrontal cortex (PFC) of the opossum. The striking overlap in amygdaloid and mediodorsal thalamic projections to the opossum frontal cortex is not altogether surprising since varying degrees of overlap appear to exist in rodents, carnivores, and primates. In primates each of the three subdivisions of MD project to a distinct portion of the PFC. The medial (magnocellular) subdivision of MD, which receives olfactory input [a], projects primarily to the orbitofrontal and medial frontal cortex whereas the lateral (parvocellular) and paralamellar (far lateral) subdivisions project, respectively, to the dorsolateral frontal convexity and frontal eye fields [48, 55,73,74,81]. The basolateral amygdala in primates, in particular the magnocellular and parvocellular divisions of the basal nucleus and accessory basal nucleus, projects primarily to the part of the PFC that has connections with the medial subdivision of MD and to the medial subdivision of MD itself [3,25,5 1,52,60]. Convergence of inputs from the amygdala and MD in the PFC is also exhibited by non-primate placental mammals (e.g., [57]). Autoradiographic tract tracing studies have shown that as in primates, the rat basolateral amygdala (in particular the basolateral nucleus) projects both to the medial part of MD and to cortical areas that receive projections from this portion of MD [32,33]. Cortical areas in the rat that receive converging inputs from both MD and the basolateral amygdaia include the dorsal agranular insular cortex located on the dorsal lip of the rostra1 portion of the rhinal sukus, the

148

prelimbic area located in the medial frontal cortex, and the dorsal frontal polar cortex [32, 33,661. There is evidence that similar connections may exist in the cat [33, 38, 42, 44, 571 but the exact details of these relationships have not been the focus of a single comprehensive investigation. The opossum MD is less differentiated than that of the rat, cat and monkey. It is a large nucleus that contains a homogeneous population of large cells [9]. Unlike other mammals the entire extent of the opossum MD receives olfactory input [23]. It has therefore been suggested that the entire opossum MD may correspond to the magnocellular medial subdivision of primates [7,23]. This dominance of MD by olfactory inputs is undoubtedly a reflection of the importance of this sensory modality in determining the behavior of the opossum. There is reason to believe that the relatively undifferentiated, olfactory-dominated opossum MD may represent a primitive condition. Olfaction appears to be the most important sense for determining learned behaviors in lower mammals and in many non-mammalian vertebrates [20]; in both the opossum and hedgehog [69], conservative metatherian and eutherian mammals, respectively, MD is a homogeneous large-celled nucleus with no lateral parvocellular subdivisions. The results of anterograde and retrograde degeneration studies in the opossum originally suggested that only the preorbital cortex, located on the dorsolateral frontal convexity, represented the prefrontal cortex of the opossum [ 10,751. Recent orthograde and retrograde axonal transport studies, however, reveal that the opossum MD projects not only to the preorbital area but to almost the entire extent of the frontal lobe [5]. The results of the present study indicate that the area1 distribution of BLa and MD projections are nearly identical. Thus, unlike the rat and monkey, the opossum basolateral amygdala appears to project to virtually the entire PFC including its dorsolateral convexity. These findings support the notion that the mammalian amygdala projects most strongly to the areas of the PFC that receive efferents from limbic-related and olfactory-related portions of MD [57], which in the opossum constitute the entire nucleus [7]. There is also evidence that a small number of widelyspaced cells in the opossum basolateral amygdala project to MD [7]. Perhaps because of the diffuse distribution of these neurons, no injection in the present study included enough cells to produce recognizable terminal label in MD. By virtue of similarities in topography and hodology it appears that the anterior agranular insular cortex in the opossum, which received projections from BLa. L and perhaps BM, corresponds to the dorsal and ventral agranular insular cortices (AI) of the rat. In the latter species the dorsal AI receives afferents from the basolateral amygdala whereas the ventral AI receives projections from olfactory cortical areas [33]. It is of interest that in the olfactory dominated brain of the opossum the entire extent of the anterior agranular insular cortex appears to receive direct projections from the main olfactory bulb [45, 68, 721. The opossum basolateral amygdala also has projections to another frontal cortical area that receives projections from the main olfactory bulb. This

McDONAL,D AND C‘Ul,BERSON region, which includes the cortex located along the far I’OStral part of the rhinal sulcus and the inferior surface of the frontal cortex, has been termed the lateral and medial sulcal cortex by Shammah-Lagnado and Negrao [68] and probably corresponds to orbital cortical areas in the rat. In the latter species these areas do not appear to receive amygdaloid projections [33]. The anterior cinpulate and infralimbic cortices on the medial wall of the opossum frontal lobe, which receive projections from all of the nuclei of the basolateral amygdala, appear to correspond to the prelimbic and infralimbic cortices in the rat. Autoradiographic investigations in the rat discerned projections to these medial frontal areas from the basolateral nucleus, but not from the lateral and basomedial nuclei [33]. A recent retrograde axonal transport study. however, demonstrated medial frontal projections arising from all nuclei of the rat basolateral amygdala [67] as in the present study. The present study demonstrates that BLa projects to prefrontal cortex on the frontal pole and preorbital area and to the rostra1 part of the postorbital area located in the caudal limb of the orbital sulcus. The latter area is of particular interest since it is the rostra1 extreme of the opossum motor cortex and may correspond to the frontal eye field of the opossum ] 171. Using the horseradish peroxidase retrograde transport technique Sarter and Markowitsch (661 have recently demonstrated projections of the lateral and basolatera1 nuclei to dorsal frontal polar and rostra1 motor (lateral precentral) cortex in the rat. Basolateral amygdaloid projections to motor and premotor cortex have also been described in the cat [38,42] whereas in the monkey pro.jections were only seen to the latter area [4]. The present study also demonstrated projections from the BLp and L to the posterior agranular insular and caudal perirhinal cortices of the opossum. In the rat and cat BLa. L. and other nuclei of the basolateral amygdala (.personal observation), project to these areas [33]. These cortical areas in the rat (associative insular cortex of 1221) receive sensory information from all the major sensory modalities by both cortico-cortical and thalamo-cortical projections [22. 65. 77). These connections are probably present in the opossum. but in this species they are supplemented by direct projections from the olfactory bulb [68]. The presence of basolateral amygdaloid projections to these sensory association areas in the opossum suggests that these connections are a characteristic feature of the mammalian brain whrch may have existed in primitive mammals. It has been postulated that these amygdalo-cortical projections might allow emotional or motivational states to influence attention IO behaviorallysignificant sensory stimuli [46]. ACKNOWLEDGEMENTS

The authors are grateful for the technical

assistance of Strvcn Prichard, Diane Raymond and Neda Osterman and the secretarial assistance of Judy Lawrence. This work was supported by funds from the University of South Carolina and the West Virginia Medical Corporation.

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OF THE OPOSSUM

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AMYGDALA

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