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Home , Amygdala, Nucleus accumbens, Olfactory bulb, Olfactory tract, Olfactory tubercle, Septal area

THE JOURNAL OF COMPARATIVE NEUROLOGY 191~193-212(1980)

An Experimental Study of the Ventral of the Golden Hamster. II. Neuronal Connections of the

RICHARD NEWMAN AND SARAH SCHILLING WINANS Department of , The University of Michigan Medicd School, Ann Arbor, Michigan 48109

ABSTRACT As part of an experimental study of the ventral striatum, the horseradish peroxidase (HRP) method was used to examine the afferent and efferent neuronal connections of the olfactory tubercle. Following iontophoretic applications or hydraulic injections of HRP in the tubercle, neurons labeled by retrograde transport of HRP were observed ipsilaterally in the telencephalon in the main , the medial, lateral, ventral, and posterior divisions of the anterior olfactory , and in the orbital, ventral, and posterior agran- ular insular, primary olfactory, perirhinal, and entorhinal cortices. Labeled cells were also present in the basolateral, basomedial, anterior cortical, and postero- lateral cortical amygdaloid nuclei, and bilaterally in the nucleus of the lateral . In the , ipsilateral HRP-containing neurons were observed in the midline nuclei paraventricularis, parataenialis, and reuniens, and in the parafascicular intralaminar nucleus. Retrograde labeling was present in the ipsilateral in cells of the , , and dorsal raphe. Many of the above projections to the tubercle were found to be topographically organized. Anterograde axonal transport of HRP from the olfactory tubercle labeled terminal fields ipsilaterally in all parts of the anterior olfactory nucleus, in the ventral pallidum, and in the substantia nigra, . Contralaterally, terminal fields were present in the dorsal and lateral divisions of the anterior olfactory nucleus. The projections to the tubercle from the orbital, ventral, and posterior agran- ular insular, and perirhinal neocortices, intralaminar , and - containing areas of the ventral mesencephalon are analogous to the connections of the caudatoputamen, as are the efferents from the tubercle to the ventral and substantia nigra. These connections substantiate the recent suggestion that the olfactory tubercle is a striatal structure, and provide support for the ventral striatal concept. In the present study of the olfactory tubercle, and in the first study in this series on the , the ventral striatum was found to receive projections from a number of structures, including the main olfactory bulb, anterior olfactory nucleus, , , and subicu- lum, and the entorhinal and primary olfactory cortices. These findings suggest that the ventral striatum is concerned with integrating limbic information into the striatal system.

The olfactory tubercle, because it receives structure. Citing embryological, cytological, input from the olfactory bulb, is generally histochemical, and hodological evidence, these considered to be an olfactory area. Recently, investigators have proposed that the olfactory however, Heimer and Wilson ('75) have sug- tubercle, the nucleus accumbens, and the sub- gested that the tubercle is in fact a striatal striatal gray are related striatal structures.

0021-9967/80/1912-0193$03.500 1980 ALAN R. LISS. INC. 194 R. NEWMAN AND S.S. WINANS

Heimer and Wilson refer to these three areas The animals were allowed to survive from and the cell bridges connecting them as the 18-48 hours, then perfused with buffered sa- “ventral striatum,” and have shown that nu- line followed by a fixative solution containing cleus accumbens and the olfactory tubercle glutaraldehyde, and in some cases, parafor- project to a subcommissural rostroventral ex- maldehyde as well. The were removed tension of the globus pallidus they have from the skulls, washed overnight in buffered termed the “ventral pallidurn.” Thus, the ven- sucrose solution, and cut coronally in 40 pm tral striatum and ventral pallidum may to- sections on a freezing microtome. Every fourth gether constitute a ventral striato-pallidal section was reacted for HRP using either dia- system. In contrast to the neocortical input to minobenzidine (DAB), tetramethylbenzidine the caudatoputamen, however, the known te- (TMB), o-dianisidine, or benzidine dihydroch- lencephalic inputs to the ventral striatum are loride (BDHC) as a reagent, then mounted on primarily from limbic structures, including gelatin-coated slides. Sections adjacent to the olfactory cortex, hippocampus, and amyg- those reacted with DAB or BDHC were dala (Heimer and Wilson, ’75). Thus, the ven- mounted and stained with cresyl violet, and tral striatum is one of the few areas in which sections reacted with TMB or dianisidine were the limbic and striatal systems interact di- counter-stained with safranin-o and cresyl vi- rectly. In order to experimentally evaluate the olet, respectively. The reacted sections were ventral striatal hypothesis and to further de- examined microscopically under both bright- fine the anatomy of this area, the afferent and field and darkfield illumination. efferent connections of the olfactory tubercle, which have never been systematically studied RESULTS with modern methods, were examined in the golden hamster using the method of axonal Technical considerations transport of the protein horseradish peroxi- Factors influencing the size and character- dase (HRP). The results of experiments on the istics of an HRP application site, the criteria nucleus accumbens were presented in a pre- by which the limits of an application site were vious paper (Newman and Winans, this issue); defined, and the criteria by which retrograde those pertaining to the tubercle are described neuronal and anterograde terminal labeling here. were identified, have been fully discussed in the first paper in this series (Newman and Winans, this issue) and will only be summa- METHODS rized here. The present study utilized the same group Three regions were observed within the ap- of 50 golden hamsters described in the first plication sites: 1) a dense central area in paper in this series (Newman and Winans, which a large amount of reaction product was this issue). The experimental techniques em- present in neurons and in the extracellular ployed in this study were also fully described space; 2) a middle zone in which there was in that paper. Briefly, in 20 animals HRP only a moderate amount of extracellular ma- (Miles, 0.5 mg/p1 in Tris buffer, pH 8.6) was terial; and 3) a peripheral zone in which a iontophoretically deposited in the olfactory small amount of HRP was present in neuron tubercle or neighboring structures cell bodies only. In this study, it was assumed using a glass micropipette (tip diameter 25-40 that transport of HRP could occur only from pm) and a Grass SD5 or S88 stimulator. In 30 regions (1)and (21, since the concentration of animals, HRP (Sigma I1 or VI, or Miles; HRP in region (3) was very low. Therefore, an 0.1-0.5 mg/p1 in 0.Wi saline) was injected application site was said to be restricted to a into the tubercle or surrounding areas with a given cytoarchitectural area only if the central Hamilton syringe and 26-gauge needle. In and middle regions did not extend beyond the most cases, a direct approach to the tubercle boundaries of that area. was used, the pipette or needle being lowered Neurons were said to be labeled by retro- vertically from the dorsal surface of the . grade transport if they were outside the ap- In some iontophoretic experiments, the micro- plication site and contained discrete granular pipette was directed into the tubercle at an HRF’ reaction product in their cytoplasm. Ter- angle from the contralateral side of the brain minal fields labeled by orthograde transport in order to avoid possible HRP contamination of HRP appeared as clouds of coarsely-granu- of cortical and striatal areas overlying the lar reaction product distributed within the tubercle. of a cytoarchitecturally defined area. CONNECTIONS OF THE OLFACTORY TUBERCLE 195

Experimental results (Fig. 2H). Horseradish peroxidase-containing A number of the connections of the olfactory neurons were observed in parafascicularis tubercle display topographical organization. only in the rostral portion of the nucleus, In order that this may be demonstrated, two medial to the fasciculus retroflexus (Fig. W). experiments in which HRP applications were In the ipsilateral brainstem, many large, made in different parts of the rostral olfactory labeled multipolar were observed in the ven- tubercle will be presented in detail, and the tral tegmental area (Fig. 2M,N). These neu- results of several additional experiments rons were rostrally continuous with similar which help illustrate topographical relation- labeled cells in the area medial to Forel's field ships will be summarized. H2. Horseradish peroxidase was also present in neurons in the caudal mesencephalic por- Rostromedial olfactory tubercle tion of the dorsal raphe (Fig. 2-0). In experiment 791, an iontophoretic appli- Terminal fields were observed in a number cation of HRP was made in the medial third of areas in this experiment. A dense terminal of the rostral olfactory tubercle (Figs. 1A; field was observed in laminae 1A and 1B in 2C,D). all parts (dorsal, ventral, medial, lateral, and Retrograde transport of HRP produced la- beled neurons in a number of ipsilateral telen- A bbreuiations cephalic areas in this brain. The main olfac- AC tory bulb (MOB) was very lightly labeled, as Acc nucleus accumbens were the lateral, medial, and ventral subdi- AIV ventral agranular visions of the anterior olfactory nucleus (AON) Bla basolateral amygdaloid nucleus, (Fig. 2A,B). anterior division BlP basolateral amygdaloid nucleus, In the , =-containing py- posterior division ramidal cells were present along the rhinal c1 anterior cortical amygdaloid in the ventrolateral orbital cortex (ter- nucleus c2 posterolateral cortical amygdaloid nucleus minology of Krettek and Price, '77a) (Fig. 2A), c3 posteromedial cortical amygdaloid nucleus the ventral and posterior agranular insular Ce central amygdaloid nucleus cortices (of Krettek and Price, '77a; Figs. CP caudatoputamen 2B-K; 41, and (Fig. 2GO). d anterior olfactory nucleus, pars dorsalis Pyramidal cells containing reaction product DR dorsal raphe were also observed in the primary olfactory FR fasciculus retroflexus cortex throughout its rostral-caudal extent, GP globus pallidus though these cells were most numerous caudal H2 Forel's field H2 IPN to the level of the nucleus of the lateral olfac- 1 anterior olfactory nucleus, pars tory tract (NLOT) (Figs. 2H-K; 4). Rostra1 to lateralis this level, labeling was mostly confined to L lateral amygdaloid nucleus cells in lamina I1 of the cortex, while caudally, LOT lateral olfactory tract m anterior olfactory nucleus, pars labeled neurons were found in lamina I11 as medialis well. The superficial and deep laminae of en- M medial amygdaloid nucleus torhinal areas 28L, 28L', and TR (of Haug, NLOT nucleus of the lateral olfactory '76) also contained many labeled neurons tract Olh olfactory tubercle (Figs. 2L-0; 6). PF parafascicular thalamic nucleus Retrogradely transported HRP appeared in PIC posterior agranular insular cortex a number of nuclei of the amygdaloid complex. POC Many cells in both the anterior and posterior PR perirhinal cortex PT parataenial thalamic nucleus divisions of the basolateral nucleus (of Krettek PV paraventricular thalamic nucleus and Price, '78b) were filled with granular RE thalamic nucleus reuniens reaction product (Figs. 21-L; 51, as were those RS of the anterior and posterolateral cortical nu- Sept septum SN substantia nigra clei (Fig. 21-K). Smaller numbers of HRP- TR entorhinal cortical area TR containing cells were present in the basome- tt taenia tecta dial nucleus, and bilaterally in the NLOT. V anterior olfactory nucleus, pars Ipsilaterally-labeled thalamic nuclei includ- ventralis VP ventral pallidum ed parataenialis (Fig, 2H-J) and parafascicu- VTA ventral tegmental area laris, which were heavily labeled, as well as 28L entorhinal cortical area 28L paraventricularis (Fig. 2G-I) and reuniens 28L' entorhinal cortical area 28L'

198 R. NEWMAN AND S.S. WINANS CONNECTIONS OF THE OLFACTORY TUBERCLE 199 200 R. NEWMAN AND S.S. WINANS posterior) of the ipsilateral AON (Fig. 2A), produced by the rostromedial application in and lighter fields were present in the contra- brain 791, while that in the NLOT was similar lateral pars dorsalis and pars lateralis. in these two experiments. A terminal field was also present in the In the thalamus of this brain, HRP was ispsilateral ventral pallidum. Labeled fibers found within cells in nucleus parataenialis were observed to course caudally in the medial (Fig. 3G), nucleus reuniens (Fig. 3H), and forebrain bundle from the application site to nucleus parafascicularis (Fig. 31). As in the the level of the body of the anterior commis- cortex and amygdala, fewer neurons contain- sure, where many of the fibers broke into a ing HRP were present in these areas than in dense terminal field in the medial ventral brain 791. pallidum (Fig. 2E,F). The remainder of the The pattern of labeling in the brainstem in fibers entered the medial portion of the cere- this experiment was similar to that observed bral peduncle, in which they could be traced after the rostromedial HRP application in ex- caudally as far as the level of the mammillary periment 791, except that labeling was lighter bodies. These fibers apparently contributed to in the ventral tegmental area, and a few an ipsilateral terminal field in the medial neurons in the medial substantia nigra, pars substantia nigra, pars reticulata (Fig. 2M). compacta (Fig. 3K,L), contained reaction prod- This field was considerably less dense than uct. those observed in the ventral pallidum or Terminal fields were observed in this exper- anterior olfactory nucleus. iment in the AON (Fig. 3A) as described for brain 791, and in the lateral ventral pallidum Rostrolateral olfactory tubercle (Fig. 3E,F). A terminal field was not present Experiment 795 is an example of an HRP in the substantia nigra of this brain. application in the lateral third of the olfactory tubercle, at a rostrocaudal level similar to Intermediate rostral olfactory tubercle that of brain 791 (Figs. 1B; 3C,D). As in brain 791 in which HRP was deposited Comparison of Figures 2 and 3 shows that in the rostromedial tubercle (Fig. lA), the efferents to the rostral olfactory tubercle from AON and MOB were only sparsely labeled. In the cortex, amygdala, thalamus, and ventral this case, HRP-containing cells in the AON tegmental area primarily terminate medially were confined to the pars lateralis. within the rostral tubercle, while projections Cortical labeling in this brain differed from from the AON, NLOT, and dorsal raphe (and that observed in experiment 791 in that no the MOB) terminate throughout the rostral labeling was observed in the orbital cortex, tubercle. This suggests that there is a medial- perirhinal cortex, or entorhinal area TR. Also, lateral gradient in the density of these projec- fewer HRP-filled neurons were present in the tions to the rostral tubercle. Evidence that ventral agranular insular (Fig. 3B,C), and this is indeed the case is provided by experi- posterior agranular insular cortices (Fig. ment 794. In this experiment, HRP was de- 3E-H), and in entorhinal areas 28L and 28L' posited in the portion of the rostral tubercle (Fig. 3K-N). Only in the primary olfactory (Fig. lB), lying between the areas of the me- cortex (Fig. 3B-J) was the amount of labeling dial application in brain 791 (Fig. 1A) and comparable to that resulting from retrograde lateral application in brain 795 (Fig. 1C). transport from the medial rostral tubercle The locations of labeled neurons in the (brain 791). However, in this brain, the ma- MOB, AON, NLOT, and dorsal raphe, and in jority of HRP-filled neurons in the olfactory all cortical areas but the orbital cortex, which cortex were found rostral to the level of the was not labeled, were similar to those ob- NLOT, rather than caudal. As in brain 791, served after an HRP application in the medial labeling in the rostral olfactory cortex was rostral olfactory tubercle (brain 791). How- largely restricted to lamina 11, while caudally, ever, with the exception of the olfactory cor- significant labeling was also present in lamina tex, the number of labeled neurons in the 111. cortical areas projecting to the olfactory tu- In the amygdaloid complex, HRP-filled neu- bercle was substantially less in this brain. On rons were present in only the anterior division the other hand, labeling in those cortical areas of the basolateral nucleus, the posterolateral demonstrated in experiment 795 (Fig. 3) to cortical nucleus, and bilaterally in the NLOT also project to the lateral tubercle, was greater (Fig. 3G-I). Labeling in the basolateral and in this experiment than in 795, in which HRP cortical nuclei was much lighter than that was deposited in the lateral tubercle. Only the CONNECTIONS OF THE OLFACTORY TUBERCLE 20 I

olfactory cortex was much more heavily la- HRP filled neurons, but the pars ventralis and beled by retrograde transport from the inter- lateralis did not. Similar numbers of cells mediate tubercle than from either the medial were labeled in the NLOT, thalamus, and or lateral tubercle. dorsal raphe by the caudal intermediate ap- A similar pattern was observed in the plication in this experiment, and by the rostral amygdala, thalamus, and ventral tegmental intermediate application in experiment 794. area; labeling in these areas was lighter in The cerebral cortex, amygdala, and ventral this experiment than when HRP was applied tegmental area were less heavily labeled in in the medial olfactory tubercle (brain 791), this brain. Terminal fields in the AON and but greater than that produced by an appli- ventral pallidum were similar to those de- cation in the lateral tubercle (brain 795). scribed for brain 794. Terminal fields like those described earlier These results demonstrate that the cortical, were present in the AON in this brain. A amygdaloid, and ventral tegmental area affer- terminal field was also observed in the ventral ents of the olfactory tubercle terminate more pallidum, located lateral to that observed after heavily in the rostral that the caudal tubercle. the medial application in brain 791, but me- Furthermore, comparison of the pattern of dial to that labeled by the lateral application labeling observed in the two brains in which in brain 795. HRP was deposited in the caudal olfactory tubercle (brains 792 and 77161, suggests that Caudal olfactory tubercle the medial-lateral topography noted in the A medial-lateral gradient was observed in cortical, amygdaloid, thalamic, and ventral the cortical, amygdaloid, thalamic, and ven- tegmental area efferents to the rostral tuber- tral tegmental projections to the caudal olfac- cle, also exists in these projections to the tory tubercle, as well as to the rostral tubercle. caudal tubercle. There is also a rostral-caudal component to the topography of certain of these projections. Contralateral labeling 'Ityo experiments illustrating these points will In addition to the ipsilateral labeling de- be briefly described. scribed above, small numbers of contralateral In experiment 792, an iontophoretic appli- HRP-containing cells were observed in various cation of HRP was placed in the medial caudal brains in one or more of the ipsilaterally tubercle (Fig. 1D). The distribution of labeling labeled cortical areas, and in the basolateral observed in this experiment was the same as amygdaloid nucleus. that seen after HRP was deposited in the medial rostral tubercle (brain 791), with the Labeling in other areas exception of the AON. As in brain 791, the Areas in which occasional labeled neurons pars medialis of the AON contained HRP- were observed include the posteromedial cor- filled cells, but the pars lateralis and pars tical nucleus of the amygdala, the magnocel- ventralis did not. In addition, however, HRP lular , the diagonal band nuclei, was present in cells of the AON pars posterior the medial and lateral preoptic areas, and in this brain. "he number of labeled cells in thalamic nuclei rhomboidalis, anteromedialis, the thalamus, NLOT, and dorsal raphe in this and dorsomedialis. The results of the control experiment was similar to that resulting from experiments described below suggest that la- the rostromedial application in brain 791. beling in these thalamic nuclei may have been However, fewer HRP-containing neurons were due to cortical contamination. However, present in the amygdala, cerebral cortex, and sparse projections to the olfactory tubercle ventral tegmental area. Terminal fields in this from these nuclei and from the other areas brain were similar to those described for ex- mentioned above cannot be ruled out. periment 791. A few labeled cells were also observed in a Experiment 7716 is an example of an HRP number of brains adjacent to the needle or application in the intermediate portion of the pipette tract in the anterior cingulate or in- caudal olfactory tubercle (Fig. 1E). With the fralimbic cortices. These neurons were most exception of the AON, areas labeled in brain likely labeled by direct uptake of HRP drawn 794, the rostral counterpart of this experi- into the cortex along the needle or pipette. ment, were labeled here as well. The AON Finally, numerous labeled cells were found labeling was similar to that observed after a among the fibers of the terminal field in the caudal medial application (brain 792); the ventral pallidum in all the brains described AON pars medialis and posterior contained above. However, these cells were so densely 202

a m V n CONNECTIONS OF THE OLFACMRY TUBERCLE 203

c

H

c 204 CONNECTIONS OF THE OLFACTORY TCTBERCLE 205

Fig. 6. Darkfield photomicrograph of a coronal section of brain 791, showing labeled neurons in the anterior (A) and posterior (P)divisions of the basolateral nucleus. Photograph corresponds to the area enclosed by the rectangle in Figure W. filled with reaction product that it was felt to the cortical areas traversed by the micro- that their had been damaged by the pipette (Jones and Leavitt, '74; Beckstead, '76; pipette, allowing HRP to enter them by dif- Krettek and Price, '77b), in one experiment fusion. While cells in this area may indeed be (7716, Fig. lF), the micropipette was intro- efferent to the olfactory tubercle, it seems duced into the tubercle at an angle from the more likely that these neurons project past opposite side of the brain to avoid contami- the tubercle to more rostra1 regions such as nating the cortex dorsal to the tubercle, and the AON (Haberly and Price, '78a), MOB (de in several brains small applications of HRP Olmos, et al., '781, or frontal cortex (Heimer, were made in the cortex overlying the tuber- '78). cle. The amount of HRP deposited in the cortex of the latter brains was much greater Control experiments than that present in the cortex of the experi- In the experiments presented in this paper, mental brains. only small amounts of HRP were drawn from Areas containing labeled cells in the exper- the application site into the cortex dorsal to imental brains and in the control brains in the olfactory tubercle as the micropipette was which HRP was deposited in the frontal cortex withdrawn from the brain. However, since included the paraventricular thalamic nucleus several of the areas described above, in which and the dorsal raphe. Labeling in these two labeled cells were found, are known to project areas after injection of HRP directly into the frontal cortex was quantitatively similar to taken into account, these findings are in that produced by HRP applications in the agreement with the results of Haberly and tubercle, in which only minimal amounts of Price ('78) for the caudal olfactory tubercle of HRP were deposited in the cortex. Therefore, rat, except that in the hamster, no HRP-con- these nuclei must project to the olfactory tu- taining neurons were observed in the area of bercle as well as to the frontal cortex. the "dorsal peduncular cortex" of Haberly and Price. DISCUSSION In agreement with the results of this study, the olfactory cortex (Heimer, '72; de Olmos, Technical considerations '72; Price, '73; Haberly and Price, '78a) and The HRP technique has several qualifica- (Haberly and Price, '78a) tions which must be considered when inter- have been reported to project to the olfactory preting data obtained by the use of this meth- tubercle in the rat. In the hamster, the projec- od. These include the possibility that not all tions to the whole of the tubercle from the neurons may be capable of transporting HRP, anterior part of the olfactory cortex were transport by fibers-of-passage, and the diffi- found to be primarily from pyramidal cells in culty in precisely defining the effective size of cortical lamina 11, while efferents from more an HRP application site. These matters and posterior regions of the olfactory cortex arise others relating to the use of the HRP tech- in lamina I11 as well, as described by Haberly nique have been discussed in the first paper and Price ('78a) for the rat caudal tubercle. in this series (Newman and Winans, this issue. Brainstem nuclei known to project to the olfactory tubercle of the rat include the sub- Afferent connections of the olfactory tubercle stantia nigra and ventral tegmental area (Un- Previously unreported projections to the ol- gerstedt, '71; Bjorklund and Lindvall, '78; Fal- factory tubercle identified in the present study lon and Moore, '78), and the dorsal and medial include those from the parataenial thalamic (Bobillier et al., '76). In the ham- nucleus, the posterior agranular insular cor- ster, ventral mesencephalic efferents to the tex, and perirhinal cortex. In addition, contra- tubercle are largely from the ventral tegmen- lateral labeling was observed in various tal area; only a few labeled neurons were ever brains in these cortical areas, as well as in the observed in the substantia nigra. In partial entorhinal and primary olfactory cortices, and agreement with the findings of Bobillier et al. the basolateral amygdaloid nucleus. These ('76) in the , the dorsal raphe, but not the contralateral projections to the olfactory tu- medial raphe, was found to project to the bercle are consistent with the findings of van olfactory tubercle of the hamster. Species var- Alphen ('69) and de Olmos and Ingram ('721, iation may account for this discrepancy. and are similar to the results observed in our Other areas previously reported to project study of the afferents of nucleus accumbens to the olfactory tubercle, which were also (Newman and Winans, this issue). observed to do so here, include the rostra1 Although the afferent connections of the sulcal cortex (Leonard, '69; Beckstead, '791, olfactory tubercle and their topography have the anterior and posterolateral cortical, baso- never been systematically examined prior to lateral, and basomedial amygdaloid nuclei this study in the hamster, many of the con- (Krettek and Price, '78a; Haberly and Price, nections described here have been reported in '78a), the NLOT (Haberly and Price, '78a), anterograde tracing experiments in various and the paraventricular, parafascicular, and species. The olfactory tubercle has been shown reuniens thalamic nuclei (Haberly and Price, to receive fibers from both the MOB and AON '78a; Herkenham, '78). (Price, '73; Scalia and Winans, '75; Broadwell, Although new connections were identified '75a,b; Devor, '76; Rosene and Heimer, '77; for the olfactory tubercle and several previ- Haberly and Price, '78a,b), but in the present ously described connections were confirmed in study, the MOB was only very lightly labeled this study, some previously reported projec- by HRP applications in the olfactory tubercle, tions to the olfactory tubercle were not found despite the tubercle's reputation as an impor- in the hamster. Leonard ('69) and Beckstead tant olfactory center. The AON also contained ('79), using anterograde techniques in the rat, only occasional labeled neurons. Portions of found the medial frontal cortex to be efferent the AON efferent to the tubercle include the to the olfactory tubercle. Although only occa- medial, lateral, ventral, and posterior subdi- sional HRP-containing neurons were observed visions. When differences in nomencature are here in the medial cortex of the hamster, CONNECTIONS OF THE OLFACTORY TUBERCLE 207 projections from these cortical areas cannot be that the question of whether tubercular cells ruled out. The degeneration observed by Leon- project to the bulb, is a problem of definition: ard ('69) was in lamina I11 of the tubercle, but if cells deep to lamina I11 are considered to the HRP applications in the experiments de- belong to the olfactory tubercle, then the tu- scribed here were centered in laminae I and bercle may indeed be efferent to the bulb. This 11. Therefore, the medial cortex may have connection may therefore not have been dem- been only lightly labeled by the relatively low onstrated in the experiments presented here, HRP concentration of lamina 111. since the concentration of HRP deep to lamina Johnson ('65), Raisman ('66), and Siegel and I11 was certainly too low for anterograde Tassoni ('71b), using degeneration methods, transport to occur from this area. have suggested that the septum is efferent to Projections from the olfactory tubercle of the olfactory tubercle, and Siegel and Tassoni the rat have been described to the dorsomedial ('71a), de Olmos and Ingram ('721, and Siegel thalamic nucleus, the nuclei gemini of the et al. ('75) have observed degeneration in the , and the ventral pallidum, olfactory tubercle after placing lesions in the (Scott and Leonard, '71; Heimer et al., '75). hippocampus. In the present study, no labeling Only the projection to the ventral pallidum was present in these areas after HRP appli- was observed in the present study. However, cations in the tubercle. It is probable that the the efferents to the nuclei gemini and dorso- degenerating fibers observed by the above medial nucleus have been shown to originate investigators were entorhinal efferents pass- in cells of lamina I11 of the tubercle (Scott and ing to the tubercle in the fimbria or , Chafin, '75; Heimer et al., '75; Siegel et al., which were interrupted by hippocampal or '77). These connections may not have been septa1 lesions. This interpretation is in agree- demonstrated in the preset study because the ment with the autoradiographic experiments HRP applications usually involved the full of Swanson and Cowan ('77, '79). thickness of laminae I and 11, but only the Fallon et al. ('78), using the HRP method, superficial part of lamina 111. have reported the in the rat Fallon et al. ('78) have reported the olfactory to be reciprocally connected with the septum tubercle to be efferent to the septum, nucleus and the nucleus accumbens. However, their accumbens, and the amygdala. However, their HRP injections were quite large, and it is evidence for these conclusions is not altogeth- probable that the labeling they observed in er convincing, since their HRP applications the tubercle resulted from diffusion of HRP were quite large. Terminal fields were not from the injection sites. Figure 13B in their observed in these areas in the present study. paper strongly suggests that this is true in the case of the septum. Topographical organization of the connections of the olfactory tubercle Efferent connections of the olfactory tubercle Evidence has been obtained in a number of species that the main olfactory bulb projec- In agreement with the results of the present tions to the olfactory tubercle may be bpo- study, Swanson ('76) has reported that the graphically organized (Price, '73; Devor, '76; olfactory tubercle projects to the AON and Rosene and Heimer, '77; Scott and McBride, substantia nigra of the rat. A number of other '78). The results of this study show that tu- efferentswhich have been reported to arise in bercular afferents from a variety of other the olfactory tubercle, however, were not con- areas are also topographically arranged. firmed here, probably because of differences in The pattern of labeling observed in the AON the laminae of the tubercle involved in the in the experiments described above may be various studies. Dennis and Kerr ('76) have summarized as follows: The pars medalis of reported that efferents from the olfactory tu- the AON projects to all but the rostrolateral bercle innervate the main olfactory bulb of portion of the tubercle; the pars posterior was the rat. This connection was not noted in the labeled only in brains in which HRP was present study or by other investigators deposited in the caudal tubercle; the pars (Broadwell and Jacobwitz, '76; Haberly and lateralis projects to all parts of the rostral Price, '78a,b; Davis et al., '78). However, de tubercle, while the pars ventralis was labeled Olmos et al. ('78) have recently shown cells only after HRP applications in the medial and deep to lamina I11 of the tubercle to project to intermediate rostral tubercle; labeling was the rat's olfactory bulb, and have suggested never observed in the pars dorsalis or externa. 208 R. NEWMAN AND S.S. WINANS

Cortical projections to the olfactory tubercle rostral-caudal components. The anterior cor- are topographically arranged with respect to tical, basomedial, and posterior basolateral both the medial-lateral and rostral-caudal nuclei were observed to contain labeled neu- axes of the tubercle. In an experiment in rons only when HRP was deposited in the which HRP was deposited in the rostromedial medial portion of the rostral or caudal tubercle tubercle (brain 791; Fig. 2), the amount of (brains 791 and 792). The anterior basolateral labeling in all labeled cortical areas (orbital, and posterolateral cortical nuclei were labeled ventral agranular insular, posterior agranular both in these medial tubercle experiments and insular, perirhinal, and enthorhinal cortices), in experiments in which HRP applications with the exception of the olfactory cortex, was were made in more lateral areas of the rostral much greater than that produced by an HRP (brains 794 and 795) or caudal (brain 7716) application in the rostrolateral tubercle (case tubercle. However, the amount of labeling in 795; Fig. 3), while labeling after HRP appli- these latter nuclei decreased progressively as cations in the intermediate tubercle (brain the application site became increasingly more 794) was intermediate in density. The olfac- lateral. tory cortex, on the other hand, was more A less-marked rostral-caudal topography is heavily labeled by retrograde transport of present in the amygdaloid efferents, particu- HRP from the intermediate tubercle than from larly those from the basolateral nucleus. Both either medial or lateral tubercular areas. This divisions of the basolateral nucleus were la- medial-lateral pattern is also apparent in beled more heavily after an HRP application brains having HRP applications in the caudal in the rostromedial tubercle (brain 791) than tubercle (brains 792 and 7716). in brain 792, in which HRP was applied in the A rostral-caudal component is also evident caudomedial tubercle. A similar, though less- in the topography of the projections of the marked pattern of labeling was observed in primary olfactory, entorhinal, and posterior the cortical nuclei. The NLOT efferents were insular cortices; these areas contained many distributed evenly throughout the olfactory more labeled cells in the brains having rostral tubercle of the hamster. HRP applications (experiments 791, 794, and The topographical organization of the amyg- 795) than in brains which had caudal appli- daloid afferents described above is in poor cations (experiments 792 and 7716). Th’is ros- agreement with the results of earlier studies. tral-caudal topography is less-marked in the Krettek and Price (‘78a) have reported the projections of the other labeled cortical areas. anterior division of the basolateral nucleus of The primary olfactory cortical efferents dis- the rat and cat to project laterally within the play point-to-point topography, in contrast to tubercle, and the posterior division to project the projections of the other cortical areas, medially. Haberly and Price (‘78a) reported which are topographic only with respect to that the projection from the rat NLOT was their termination within the tubercle. The directed primarily to the lateral tubercle, and olfactory cortex caudal to the NLOT projects did not find that fibers from the anterior and primarily to the rostromedial tubercle (brain posterolateral cortical nuclei terminate topo- 791; Fig. 21, while the cortex rostral to NLOT graphically within the tubercle as reported projects most heavily to the rostrolateral tu- here. Since the topographical characteristics bercle (brain 795, Fig. 3). In brain 794, in observed here in the hamster are quite pro- which the HRP application site was centered nounced, especially the medial-lateral com- in the intermediate part of the rostral tuber- ponent, species variation appears to be a likely cle, an intermediate level of the primary ol- explanation for these discrepancies. factory cortex, overlapping the level of the The tubercular projections from the thala- NLOT, was labeled most densely. Thus, suc- mus are mainly to the medial olfactory tuber- cessively more rostral olfactory cortical areas cle. Labeling in the thalamus decreased suc- project most heavily to correspondingly more cessively in brains in which HRP was lateral tubercular areas. This same pattern deposited in the medial, intermediate, and was seen in experiments in which HRP appli- lateral portions of the rostral tubercle (brains cations were made in the medial (brain 792) 791,794, and 795). This pattern was also seen and intermediate (brain 7716) caudal olfacto- in the caudal olfactory tubercle (experiment ry tubercle. 792 and 7716). A rostral-caudal component to The amygdaloid projections to the olfactory the topography of these efferents was not tubercle, like the cortical projections, display observed. With the exception of that from a topography having both medial-lateral and nucleus parafascicularis, the thalamic projec- CONNECTIONS OF THE OLFACTORY TUBERCLE 209 tions to the tubercle are topographic only in exposure of brain sections to during their termination within the tubercle; they dehydration. Therefore, projections from the appear to arise in all parts of their respective lateral tubercle to the nigra cannot be ruled nuclei. Efferents from parafascicularis, how- out. ever, are largely from cells in the rostral portions of the nucleus only, ventromedial to Evidence supporting the ventral striatal the fasciculus retroflexus. concept The ventral tegmental area (VTA), like the In recent years it has become apparent that cerebral cortex, thalamus, and amygdala, the limbic and striatal systems are not as projects most heavily to the medial olfactory anatomically isolated from each other as was tubercle. Labeling in this area was greater in once believed. Heimer and Wilson (‘75) and brains having medial HRP applications (brains Heimer (‘78) have proposed that the nucleus 791 and 792) than in brains in which HRP accumbens, olfactory tubercle, substriatal was deposited in more lateral parts of the gray (the portion of the caudatoputamen ven- rostral or caudal tubercle (brains 795 and tral to the posterior limb of the anterior com- 7716). There is also a rostral-caudal compo- missure), and the cell bridges connecting these nent to this topography; labeling in the VTA areas, together referred to as the “ventral was heavier in experiments in which HRP striatum,” are functionally related striatal was deposited in the rostral olfactory tubercle structures which collectively provide a means (brains 791, 794, and 795) than in brains through which the allocortical components of having caudal applications (brains 792 and the limbic system may influence the striatum. 7716). Fallon et al. (‘78) have reported that The results of the present study on the con- cells of the VTA of the rat project to the medial nections of the olfactory tubercle and of the tubercle, while the lateral tubercle is inner- first study in this series on the connections of vated by neurons in the lateral VTA and the nucleus accumbens (Newman and Winans, medial substantia nigra. In the hamster, only this’issue) support these ideas. a few labeled neurons were present in the Both the olfactory tubercle and nucleus ac- nigra after HRP applications in the lateral cumbens were found to receive afferents from tubercle; the ventral mesencephalic projection areas which project to the caudatoputamen, to the lateral as well as medial tubercle was including the cerebral cortex, intralaminar primarily from the VTA. Apparently the top- thalamus, and the brainstem ographic organization of the brainstem dopa- nuclei. Examination of the organization of the minergic nuclei differs between these species. cortical projections to the ventral striatum Of the efferent projections of the olfactory reveals a number of parallels in the cortical tubercle, only that to the ventral pallidum connections of the dorsal and ventral striatal was found to display topographical organiza- areas. The basic arrangement of newortical tion in the present study. Medial tubercular afferents to the dorsal striatum is such that areas project to the medial ventral pallidum cortical areas project most strongly to the (brains 791, 7921, lateral areas to the lateral portions of the caudatoputamen lying closest ventral pallidum (brain 7951, and intermedi- to them (Kemp and Powell, ’70). The cortical ate areas (brains 794 and 7716) to interme- efferents to the ventral striatum also follow diate regions of the ventral pallidum. A ros- this pattern; they arise in cortex lying in, and tral-caudal component to the topography of ventral to, the rhinal sulcus, and terminate in this projection was not evident, though this the most ventral part of the striatum-the could not be determined with certainty with- ventral striatum proper. Thus, the whole of out the aid of brains sectioned in the saggital the cortical afferents to the corpus striatum is or horizontal planes. seen to form a continuum. Furthermore, this The substantia nigra was found to contain pattern is observed in the projections of indi- a terminal field only in brains having HRP vidual cortical areas to the ventral striatum. applications in the medial olfactory tubercle For example, dorsal (rostral) subicular areas (experiments 791 and 792). However, this ter- project to rostral nucleus accumbens, while minal labeling was rather faint, and it is ventral (caudal) subicular efferents innervate possible that slight differences in the process- only caudal nucleus accumbens. Similarly, en- ing of brains having lateral HRP applications torhinal area 28L projects only to the lateral might have rendered equally faint nigral portion of caudal accumbens, while fibers from fields undetectable. In particular, terminal la- the more medial areas 28L’ and TR terminate beling seems prone to fading caused by over- in the medial portion of caudal accumbens. 210 R. NEWMAN AND S.S. WINANS The thalamic connections of the ventral to arise from the lateral as well as medial striatum also reflect the continuity of the substantia nigra, but only from the lateral dorsal and ventral striatal areas. Whereas the portion of the ventral tegmental area (Fallon projections from the caudal intralaminar nu- and Moore, '78). This projection, then, like cleus parafascicularis to nucleus accumbens those from the cortex and intralaminar thal- and the olfactory tubercle arise from cells amus, is topographically continuous over the lying ventromedial to the fasciculus retroflex- whole of the striatum. us, Nauta et al. ('74) and Kuypers et al. ('74) The efferents of the ventral striatum, like have presented data from the rat that the the afferent connections discussed above, re- majority of parafascicular neurons projecting flect the continuity of the dorsal and ventral to the caudatoputamen lie in the portion of striatal areas. The projections of the whole of nucleus parafascicularis dorsolateral to the the striatum to the pallidum are topographi- fasciculus retroflexus. A similar example is cally arranged such that the dorsal striatum provided by the finding of this study that the projects to dorsal pallidal areas, while effer- rostral intralaminar thalamic projection to ents from the ventral striatum innervate ven- the ventral striatum is largely from centralis tral regions of the globus pallidus (i.e., the medialis, the most medial of the rostral intra- ventral pallidum). Furthermore, efferents laminar nuclei, together with the observation from the nucleus accumbens, which lies dorsal of Jones and Leavitt ('74) that the medial to the olfactory tubercle, terminate in the rostral intralaminar nuclei project to ventral ventral pallidum dorsal to the efferents from regions of the , while more the tubercle. lateral areas project to dorsal parts of the caudate. These relationships provide support for the belief that the caudatoputamen, nucle- The ventral striatum and the limbic system us accumbens, and the olfactory tubercle are The ventral striatum is one of the few brain related striatal structures, since the intralam- areas in which the limbic and striatal systems inar nuclei are thus seen to project topograph- interact directly. In addition to the cortical, ically to the whole of the corpus striatum. thalamic, and ventral mesencephalic afferents In addition to the intralaminar thalamic it receives in common with the dorsal stria- projections to accumbens and the tubercle, the tum, the ventral striatum receives projections ventral striatum receives input from the mid- from a variety of limbic system structures, line thalamic nuclei parataenialis, paraven- including the primary olfactory cortex, entor- tricularis, and reuniens. Of these, only the hinal cortex, hippocampus, , and connections of reuniens in the rat have been amygdala. Therefore, it seems likely that an thoroughly studied (Herkenham, '78). The ef- important function of the ventral striatum is ferent connections of reuniens are primarily the processing of limbic input to the striatal to those limbic cortical areas which are them- system. More specifically, the fact that a num- selves efferent to the ventral striatum. Per- ber of the limbic areas which project to the haps the midline nuclei function in the ventral ventral striatum receive multimodal sensory striatal system in a manner analogous to the information (Whitlock and Nauta, '56; Kuy- ventral tier nuclei of the dorsal system, pro- pers et al., '65; Jones and Powell, '70; Seltzer viding a means by which ventral striatum can and Pandya, '74; van Hoesen and Pandya, interact reciprocally with cortical areas effer- '75b; Herzog and van Hoesen, '761, suggests ent to it. that the ventral striatum, like the dorsal The ventral striatum, like the caudatopu- striatum, coordinates motor system activity tamen, receives dopaminergic afferents from with somatic sensory input. However, the ven- the ventral , and the topography of tral striatum is also involved in the control of this projection to nucleus accumbens and the visceral function (Koikegami et al., '67; Smith olfactory tubercle is complementary to that to and Holland, '751, and receives direct and the dorsal striatum. In the present study, cells indirect visceral sensory information (Scalia in all parts of the ventral tegmental area were and Winans, '75; van Hoesen and Pandya, found to be efferent to both nucleus accumbens '75a; Norgren '76 Rosene and Heimer, '77). It and the olfactory tubercle, and in addition, is likely, then, that the ventral striatum, rath- the lateral portion of the tubercle was shown er than being exclusively concerned with so- to receive a small projection from the medial matic motor control, is involved with inte- substantia nigra. The dopaminergic efferents grating limbic system information into visceral to the dorsal striatum, in contrast, are known as well as somatic effector systems, including CONNECTIONS OF THE OLFACTORY TUE3ERCLE 211 hormone-secreting cells of the hypothalamus de Olmos, J.S., and W.R. Ingram (1972) The projection and pituitary, and lower motor neurons of the field of the in the rat brain. An experi- brainstem and spinal cord. mental study. J. Comp. Neurol., 146:303-334. Devor, M. (1976) Fiber trajectories of olfactory bulb ef- Further study of the anatomy of the ventral ferents in the hamster. J. Gmp. Neurol., 166:31-48. striatal system will be required to evaluate Fallon, J.H., and R.Y. Moore (1978) in- this hypothesis. Since the ventral pallidum is nervation of the . N.Topography of the dopamine projection to the basal forebrain and neostria- the recipient of the major efferent projection turn. J. Comp. Neurol., 180:545-581). of the ventral striatum, determination of the Fallon, J.H., JN. Riley, J.C. Sip, and RY. Moore (1978) The connections of the ventral pallidum should islands of Calleja: Organization and connections. J. help to clarify the functions of the ventral Comp. Neurol., 181:375-396. Haberly, L.B., and J.L. Price (1978a) hiation and striatal system. systems of the olfactory cortex of the rat. I. Systems originating in the and adjacent areas. J. Comp. Neurol., 178:711-740. ACKNOWLEDGMENTS Haberly, L.B., and J.L. Price (1978b) Association and commissural fiber systems of the olfactory cortex of the We gratefully acknowledge the excellent rat. 11. Systems originating in the olfactory peduncle. J. artistic contributions of William L. Brudon. Comp. Neuml., 181:781-808. This research was supported by grant no. Haug, F.-M.& (1976) Sulphide silver pattern and cy- NS14071 from NINCDS. toarchitectonics of parahippocampal areas in the rat. Adv. Anat. Embryol. 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