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Home , Basal forebrain, Caudate nucleus, Globus pallidus, Hippocampus, Islands of Calleja, Nucleus accumbens

THE JOURNAL OF COMPARATIVE 191~167-192(1980)

An Experimental Study of the Ventral of the Golden Hamster. 1. Neuronal Connections of the Accumbens

RICHARDNEWMANANDSARAHSCHILLINGWINANS Department of Anatomy, The University of Michigan Medial Schml, 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 . Following iontophoretic applications or hydraulic injections of HRP in nucleus accumbens, cells labeled by retrograde transport of HRP were observed in the ipsilateral telencephalon in the posterior agranular insular, perirhinal, entorhinal, and primary olfactory cortices, in the and hippocampal field CA1, and in the anterior and posterior divisions of the basolateral amygdaloid nucleus. In the , labeled were present ipsilaterally in the central medial, paracentral and parafascicular intralaminar nuclei, and in the midline nuclei parataenialis, paraventricularis, and reuniens. Retrograde labeling was observed in the ipsi- lateral in cells of the and dorsal raphe. Many of these projections to nucleus accumbens were found to be topographically organized. Anterograde transport of HRP from nucleus accumbens demonstrated ipsilat- era1 terminal fields in the ventral pallidum and , . The afferent projections to nucleus accumbens from the posterior insular and perirhinal neocortices, intralaminar , and the -containing ventral tegmental area are analogous to the connections of the caudatoputamen, as are the efferents from nucleus accumbens to the substantia nigra and ventral . These connections substantiate the classification of nucleus accumbens as a striatal structure and provide support for the recently proposed of the ventral striatum. Furthermore, the demonstration that a number of structures, including the , , , and olfactory cortex are important sources of afferents to the nucleus accumbens, suggests that the ventral striatum may serve to integrate limbic information into the striatal system.

It has recently been suggested that the are largely from the , primary nucleus accumbens, , and olfactory cortex, and amygdala, and thus lim- substriatal gray-three apparently unrelated bic in nature, in contrast to the neocortical basal areas -are anatomically and input to the dorsal system. The ventral stria- functionally related striatal structures. Hei- tum, then, is one of the few areas in which mer and Wilson (‘75) have applied the term the limbic and striatal systems interface di- “ventral striatum” to these areas collectively rectly. and have provided evidence that they project The nucleus accumbens is the most promi- to a subcommissural portion of the globus nent component of the ventral striatum. The pallidus, which these investigators have called functional significance of this nucleus has his- the “ventral pallidurn.” Heimer and Wilson torically been a topic of considerable debate; have drawn attention to the fact that while accumbens has been variously included in the the neuronal connections of this ventral stria- olfactory, limbic, and striatal systems because to-pallidal system are in part analogous to of its intermediate location between the cau- those of the classical dorsal system, the known datoputamen and septum, and because of the telencephalic afferents of the ventral striatum multitude of connections ascribed to it by

0021-9967/80/1912-0167$04.400 1980 ALAN R. LISS. INC. 168 R. NEWMAN AND S.S. WINANS early investigators. These reported connec- catalepsy, and Pijenburg et al. ('73) have tions include efferents from nucleus accum- shown that injections of ergometrine cause a bens to the septum and of the substantial increase in motor activity, while (Lauer, '451, the substantia nigra in Smith and Holland ('75) have produced alter- the tamandua and cat (Smith, '30; Rioch, '31), ations in lactation in rats by placing lesions the globus pallidus and the entopeduncular in nucleus accumbens, and Koikegami et al. nucleus of the tamandua (Smith, '30), the rat ('67) have shown electrical stimulation of this and amygdala (Gurdjian, '27, nucleus to induce ovulation in rabbits. '28), and the dorsomedial thalamic nucleus of In summary, nucleus accumbens is appar- the cat (Guillery, '59). Afferents to nucleus ently a striatal structure closely associated accumbens have been described from the cat functionally and anatomically with the limbic hippocampus (Fox, '431, the macaque . The concept of the ventral striatum tubercle (Lauer, '45), the amygdala of the rat may provide a basis for understanding this (Knook, '66), and the parataenial thalamic relationship. In order to test the validity of nucleus in the rabbit and monkey (Cowan and this concept experimentally, and to further Powell, '55; Powell and Cowan, '56). define the anatomy of the ventral striatum, The application of modern experimental the afferent and efferent neuronal connections techniques to this problem has provided infor- of the nucleus accumbens and the olfactory mation suggesting that nucleus accumbens is tubercle, which together compose the majority anatomically related to both the caudatopu- of the ventral striatum, were examined using tamen and the limbic system. Swanson and the method of axonal transport of the protein Cowan ('751, using tritiated thymidine, have horseradish peroxidase (HRP).The results of demonstrated a close ontogenetic relationship HRP experiments in the nucleus accumbens between nucleus accumbens and the caudato- are presented here; those pertaining to the , while Heimer and Wilson ('75) have olfactory tubercle will be described in a second called attention to the histochemical and cy- paper in this series (Newman and Winans, toarchitectural similarities of these two areas. this issue). Furthermore, autoradiographic experiments by a number of researchers have shown the METHODS efferent projections of nucleus accumbens to be largely to the globus pallidus and substan- Fifty male and female golden hamsters tia nigra, and thus similar to the efferents of (Mesocricetus auratus) ranging in weight from the caudatoputamen (Swanson and Cowan, 75 to 130 g received injections or iontophoretic '75; Conrad and Pfaff, '76; Domesick et al., applications of HRP into the nucleus accum- '76). In addition to efferents typical of the bens or neighboring forebrain structures. The striatum, however, projections from nucleus hamsters were anesthetized with sodium pen- tobarbital (75 mg/kg), placed in a Kopf ster- accumbens to limbic areas, including the eotaxic instrument, and subjected to one of and hypothalamus, have been the two procedures described below. proposed (Conrad and Pfaff, '76; Williams et al., '77; Nauta et al., '78). Although the afferents of nucleus accum- Iontophoresis bens have never been systematically studied Horseradish peroxidase (Miles Laboratories, with modern methods, this nucleus, like the Inc., Elkhart, Indiana) was applied iontophor- caudatoputamen, is now known to receive af- etically in 20 hamsters. The HRP was pre- ferent fibers from dopaminergic neurons of the pared at a concentration of 500 pglpl in 0.05 ventral (Ungerstedt, '71; Bjorklund M Tris buffer (pH 8.61 and delivered through and Lindvall, '78; Fallon and Moore, ,781, and a glass micropipette having a tip diameter of projections to the nucleus accumbens have 25-40 pm. Anodal DC pulses of 1.5-3.0 mi- recently been reported from limbic structures croamps (one per second; 500 msec duration) as well, including the subiculum and amyg- were applied with a Grass SD5 or 588 stimu- dala (Swanson and Cowan, '77; Krettek and lator. Current was applied for 10-30 minutes Price, '78a). and the pipette remained in place for 10 min- Pharmacological and electrophysiological utes after the current pulses were stopped studies also suggest that nucleus accumbens before it was withdrawn from the . Dur- is related to both the striatum and the limbic ing this time, a constant cathodal direct cur- system. Dill and Costa ('77) have found that rent of 0.15-0.30 microamps was passed to injections of into accumbens produce prevent diffusion of HRP out of the pipette. CONNECTIONS OF THE NUCLEUS ACCUMBENS 169

Eighteen to 48 hours after surgery, the sisted of 250 cc of 0.1 N cacodylate-sucrose animals were reanesthetized and perfused solution (pH 7.2 % sucrose), followed by 250 through the heart with 250 ml of cacodylate- cc of fixative containing 5% glutaraldehyde in buffered saline, followed by 250 ml of a fixa- 0.1 N cacodylate buffer. The were re- tive containing 3% glutaraldehyde, or 1%par- moved from the skulls following perfusion, aformaldehyde and 1.5% glutaraldehyde. The postfixed for four hours in cold (4°C)25% brains were removed from the skulls and sucrose solution prepared in the perfusion washed overnight in cold (4°C)25% sucrose fixative, then washed for 10-15 hours in cold prepared in the cacodylate-buffered saline so- 25% sucrose prepared in 0.1 N cacodylate lution. The pia was then removed with a buffer. Coronal sections were cut on a freezing rubber eraser, the brains cut coronally in 40 microtome and stored in buffered sucroqe for Fm sections on a freezing microtome, and the up to 24 hours before every fifth section was sections stored in a cold solution of 25% su- reacted for HRP. crose in ethylene glycol for up to 24 hours Three different histochemical procedures before every fifth section was reacted for HRP. were used in these experiments. In three cases, Perfusion, pia removal, and of the the diaminobenzidine (DAB) method of Gra- sections followed the procedure described by ham and Karnovsky (‘66) was followed, with Hardy and Heimer (‘77), except that the brains were placed in sucrose solution after removal rather than in fixative, the cut sections were Abbreviations collected in sucrose-ethylene glycol rather AC anterior commiasure than in sodium sulphate, and in a number of ACC nucleus accumbena cases, the fixative was modified by eliminating Bla hasolateral amygdaloid nucleus, anterior division BlP basolateral amygdaloid nucleus, posterior division the paraformaldehyde, as described above. c1 anterior cortical amygdaloid nucleus Sections of these brains were reacted for c2 posterolateral cortical amygdaloid nucleus HRP using the benzidine dihydrochloride c3 posteromedial cortical amygdaloid nucleus (’771, CA1 hippocampal field CA1 (BDHC) method of de Olmos or the Ce central amygdaloid nucleus tetramethylbenzidine (TMB) procedure of CM central medial thalamic nucleus Hardy and Heimer (‘77). Reacted sections were CP caudatoputamen mounted, air dried, and the TMB reacted sec- d anterior olfactory nucleus, pars dorsalis tions were counterstained with 0.1% safranin- DR dorsal raphe FR fasiculus retroflexus 0. Sections adjacent to those reacted for HRP GP glohus pallidus using BDHC were mounted and stained with H2 Forel’s field H2 cresyl violet. All sections were quickly dehy- IPN interpeduncular nucleus 1 anterior olfactory nucleus, pars lateralis drated and coverslipped. The slides were stud- L lateral amygdaloid nucleus ied microscopically, using both brightfield and LOT lateral darkfield illumination. m anterior olfactory nucleus, pars medialis M medial amygdaloid nucleus Pressure injections NLOT nucleus of the lateral olfactory tract On olfactory tubercle Hydraulic injections were made in 30 ham- P anterior olfactory nucleus, pars posterior sters with a Hamilton syringe and 26-gauge PC paracentral thalamic nucleus needle using HRP (Sigma I1 or VI, or Miles) PF parafaaicular thalamic nucleus PIC posterior agranular insular cortex which had been prepared in 0.9% saline at POC primary olfactory cortex concentrations of 100-500 pglpl. The HRP PR (0.025-0.5 pl) was delivered over a period of PT parataenial thalamic nucleus ten minutes with the aid of a microinjector PV paraventricular thalamic nucleus RE thalamic unit attached to the stereotaxic apparatus. Rs rhinal sulcm The needle was left in the brain for ten min- S subiculum utes following the injection, then removed sc suprachiasmatic nucleus slowly. SPt septum so supraoptic nucleus The hamsters were allowed to survive TR entorhinal cortical area TR 18-48 hours after surgery. They were then tt taenia tecta reanesthetized and perfused. In 14 animals, V anterior olfactory nucleus, pars ventralis perfusion and subsequent processing to the VMH ventromedial hypothalamic nucleus VP ventral pallidurn point of reacting for HRP followed the proce- VTA ventral tegmental area dure described above for the iontophoresis 28L entorhinal cortical area 28L experiments. In 16 cases, the perfusion con- 28~‘ entorhinal cortical area 28L’ 170 R. NEWMAN AND S.S. WINANS

3 5 CONNECTIONS OF THE NUCLEUS ACCUMBENS 171 172 R. NJZWMAN AND S.S. wI"s N 174 R. NEWMAN AND S.S. WWANS

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a m CONNECTIONS OF THE NUCLEUS ACCUMBENS 175

ca t! LI 176 R. NEWMAN AND S.S. WINANS the exception that 0.1 M sodium cacodylate of that area. In a number of cases all three (pH 7.2) was substituted for Tris buffer. Sec- regions were within the target area. tions of 13 other brains were processed with the DAB procedure described by Colman et al. Identification of labeled cell bodies and ('76), and an additional 14 brains were reacted terminal fields for HRF' using Colman and co-workers' o-dian- Neurons were said to be labeled by retro- isidine technique ('76). Reacted sections and grade transport of HRP if they were outside sections adjacent to those treated with DAB the application site and contained discrete were mounted on gelatin-coated slides and air granular HRP reaction product in the perin- dried. Both the unreacted sections and those uclear cytoplasm. developed with o-dianisidine were stained Terminal fields labeled by orthograde trans- with cresyl violet. All sections were then rap- port of HRP appeared as clouds of coarsely idly dehydrated and coverslipped prior to granular reaction product distributed through darkfield and brightfield microscopical exam- the in a cytoarchitecturally defined ination. area. They were seen in the same locations regardless of survival time in animals having RESULTS HRP applications in the same areas of the Technical considerations brain, and at considerable distances from the application site, indicating that HRF' reached Factors influencing the size and these areas by axonal transport and not by characteristics of an HRP application site; diffusion within damaged . identification of the application sites The size of an HRP application site depend- Experimental results ed on the amount and concentration of the Analysis of the experiments performed in HRP introduced and on the method of appli- the present study demonstrates that many of cation. Very small application sites restricted the connections of nucleus accumbens are top- to nucleus accumbens were more reliably pro- ographically organized. In order to deinon- duced when HRP was applied iontophoretical- strate these topographical relationships, four ly than when injected under pressure. In ad- HRP applications varying in location within dition, much less HRP was deposited along nucleus accumbens will be described. The ap- the pipette tract than the needle tract. The plication sites are shown in detail in Figure 1. apparent size of an application site also de- pended on the histochemical technique used Rostra1 nucleus accumbens to visualize the HRP, as reported by de Olmos Brain 7714 (Fig. 2) is an example of a large ('77) and Hardy and Heimer ('77). iontophoretic application of HRP involving At the end of the 18 to 48 hour survival the rostral pole of nucleus accumbens as well periods, three regions were observed within as the caudal-most portion of the anterior the HRP application sites (Fig. 10): 1) a cen- olfactory nucleus, pars posterior. The center tral area in which the cells were densely filled of the iontophoresis site was medial to the with granules of HRP reaction product, and anterior limb of the anterior commissure, at in which a large amount of dense, finely gran- a level rostral to the genu of the corpus cal- ular reaction product was present in the ex- losum (Figs. 1B; 2B,C). The survival time in tracellular space; 2) a middle region surround- this experiment was 36 hours. ing the central zone in which cellular labeling In the ipsilateral telencephalon, a sparse was similar to that described above, but in band of labeled neurons was present along the which only a moderate amount of reaction caudal three-fourths of the rhinal sulcus. Per- product was present in the extracellular space; oxidase-containing pyramidal cells were ob- and 3) a peripheral area in which small served in the posterior agranular insular cor- amounts of granular reaction product were tex (terminology of Krettek and Price, '77a) present in cells only. from the level of the application site to the In this study, it was assumed that transport level of the entorhinal cortex (Fig. 2C, H-J), of HRP could occur only from regions (1) and and in the perirhinal cortex (Fig. 2K-M), (2), since the HRP concentration in region (3) caudal to the posterior insular cortex. Lamina was very low. Therefore, an application site III of the primary olfactory cortex (Fig. 2F,H) was described as restricted to a given cytoar- and the deep layers of entorhinal cortical area chitectonic area only if the central and middle 28L (of Haug, '76) also contained occasional regions did not extend beyond the boundaries labeled pyramidal cells (Fig. 2M). CONNECTIONS OF THE NUCLEUS MCUMBENS 177

Other ipsilaterally labeled telencephalic 7714 (Fig. 2) and was completely restricted to areas included the caudal portion of the an- this nucleus (Figs. lA, 3B,C). The survival terior division of the basolateral amygdaloid time in this case was 18 hours. nucleus (terminology of Krettek and Price, With certain exceptions, the distribution of '18b) (Fig. 211 and the rostral portion of the labeled cells in this experiment was similar to poterior division of this nucleus (Fig. 25). that observed after the HRP application in the Peroxidase-containing pyramidal cells were rostral pole of nucleus accumbens in brain also present in the stratum pyramidale of field 7714. Differences included the absence of la- CA1 of the intermediate hippocampus (Figs. beling in this brain in the primary olfactory 2K,L; 9) and in the dorsal and intermediate cortex, entorhinal cortex, paracentral tha- subiculum (Figs. 2J-M 9), from the level of lamic nucleus, hippocampal field CA1, and the posterior commissure caudally. medial raphe. Also, fewer cells were labeled In the diencephalon, labeled cells were lo- in the ventral tegmental area (Fig. 3L,M). In cated ipsilaterally in the central medial, par- contrast to experiment 7714 in which the acentral, and parafascicular intralaminar nu- insular area and amygdala were only lightly clei (Figs. 2G,I,4 7). Labeled neurons within labeled, the posterior insular cortex was heav- nucleus parafascicularis were medial and ven- ily labeled in this brain (Fig. 3E-J), and the tral to the fasciculus retroflexus, and located basolateral amygdaloid nucleus contained nu- in the most rostral portion of the nucleus only. merous labeled cells, especially the posterior Labeled midline nuclei included parataenialis division, which was labeled throughout its (Figs. 2F-H, 7), which contained many heav- rostrocaudal extent (Fig. 31-K). ily labeled cells throughout its rostrocaudal As in experiment 7714, a prominent termi- extent, paraventricularis (Figs. 2G,H; 71, nal field was present in the ventral pallidum which was also well labeled, and reuniens, in (Figs. 3E; 11).A terminal field was not present which a number of large, densely labeled mul- in the substantia nigra, however. tipolar cells were observed (Fig. 2G,H). The areas mentioned above as being labeled Labeling was observed in the ipsilateral in brain 7714, but not in this brain (77111, brainstem in the medial raphe, in the caudal may have been labeled only in brain 7714 mesencephalic portion of the dorsal raphe because of the more rostral application site in (Fig. 2M,N), and in the ventral tegmental that experiment. Alternatively, these areas area, which contained many large, heavily may have been labeled by transport of HRP labeled neurons spreading medially over the from the anterior olfactory nucleus, the cau- interpeduncular nucleus to the midline (Figs. dal-most part of which was involved in the 2L; 8). Cells similar in appearance to those in application site in experiment 7714. The re- the ventral tegmental area, and continuous sults of control experiments in which HRP with them, extended rostrally into the sub- was deposited in the anterior olfactory nucle- thalamus in the region medial to Forel's field us, and of additional experiments in which H2 (Fig. 2K). applications of HRP were made in nucleus A dense terminal field was labeled in the accumbens (two of these are described below) ipsilateral ventral pallidum of this brain. demonstrate that the paracentral nucleus, pri- Small bundles of HRP-containing fibers with- mary olfactory cortex, hippocampal field CA1, in the dorsal portion of the medial forebrain and entorhinal cortex are indeed efferent to bundle passed caudally from the iontophoresis accumbens, and that nucleus accumbens does site to the level of the body of the anterior project to the substantia nigra. The medial commissure where many of them terminated raphe was labeled in brain 7714 by transport within the ventral pallidum (Fig. 2E,F). The of HRP from the anterior olfactory nucleus. remainder of these fibers continued past the ventral pallidum, but the number of labeled Caudolateral nucleus accumbens axons was much reduced caudal to this ter- The application site in experiment 7715 minal field. A faint terminal field was also (Fig. 4) was centered in the lateral half of present ipsilaterally in the medial substantia nucleus accumbens (Figs. 1C; 4B,C) at a level nigra, pars reticulata (Fig. 2L). caudal to that of either of the two experiments described above (brains 7711 and 7714). The Intermediate nucleus accumbens survival time in this experiment was 36 hours. The iontophoretic application of HRP in With the exception of hippocampal field brain 7711 (Fig. 3) was centered more caudally CA1, areas labeled by retrograde transport of in nucleus accumbens than that in experiment HRP from more rostral areas of nucleus ac- 178 R. NEWMAN AND S.S. WINANS

6 m CONNECTIONS OF THE NUCLEUS ACCUMBENS 179 180 R. NEWMAN AND S.S. WINANS

6 m CONNECTIONS OF THE NUCLEUS ACCUMBENS 181 182 R. NEWMAN AND S.S. WI"S cumbens in experiments 7711 or 7714 con- eral or rostral parts of nucleus accumbens tained labeled cells in this experiment as well. with the following exceptions: Within the Certain quantitative differences, however, thalamus, the paraventricular and central were striking. The primary olfactory, poste- medial nuclei were not labeled; the basolateral rior insular, and perirhinal cortices and en- nucleus of the amydala contained labeled neu- torhinal area 28L all contained many more rons throughout its posterior division, but only labeled neurons in this brain. Also, fewer caudally in its anterior division; the ventral labeled cells were present in the subiculum, tegmental area was poorly labeled no labeling and the majority of these were in the inter- was found in the dorsal raphe. A terminal mediate subiculum (Fig. 4K), in contrast to field was present in this brain only in the brains 7711 and 7714 in which both the dorsal ventral pallidum (Fig. 5D,E). and intermediate subiculum were heavily la- beled by HRF' applications in the rostral half Contralateral labeling of nucleus accumbens. In the amygdala, label- In each of the brains described above, a ing was observed throughout the anterior di- number of labeled neurons were observed in vision of the basolateral nucleus (Fig. 4F,G), one or more contralateral cortical areas. These but only in the rostral portion of the posterior regions included the posterior insular, perir- division (Fig. 4H). Finally, the ventral teg- hinal, and entorhinal cortices. In experiments mental area was poorly labeled, especially the in which HRP was deposited in rostral, inter- caudal half (Fig. 4K,L). mediate, and caudomedial nucleus accumbens Terminal fields were observed in this brain (brains 7711, 7714, and 7814), this labeling in the ventral pallidum (Figs. 4D; 11) and in was rather sparse, but in brain 7715, in which the medial pars reticulata of the substantia the HRP application was centered in caudo- nigra (Fig. 4K). lateral nucleus accumbens and in which the ipsilateral cortical labeling was most exten- Caudomedial nucleus accumbens sive, these contralateral cortical areas con- The application site in experiment 7814 tained a more substantial number of labeled (Fig. 5) was only slightly caudal to that of cells. Occasional contralateral HRP-contain- experiment 7715 (Fig. 4)) but was centered ing neurons were also present in several medial to the anterior limb of the anterior brains in the basolateral nucleus of the amyg- commissure, rather than lateral to it (Figs. dala. 1D; 5B,C). The survival time in this case was 36 hours. Labeling in other areas The pattern of labeling in the cortical areas In addition to the labeled areas discussed of this brain differed remarkably from that above, a few HRP-labeled cells were found in observed in the three experiments described various brains in the dorsomedial, dorsal an- above. No labeled neurons were observed in terior, anteromedial, and rhomboid thalamic the primary olfactory, posterior insular, or nuclei. Other lightly labeled areas included perirhinal cortices in this brain. Within the the magnocellular preoptic area and the per- entorhinal cortex, labeling was present in area iventricular central gray at the level of the 28L' (Fig. 5L-N), but not in the more lateral diencephalic-mesencephalic junction. The re- area 28L, in contrast to brains having HRP sults of control experiments described below applications in rostral, intermediate, or cau- suggest that cortical contamination probably dolateral nucleus accumbens (brains 7714, produced the labeling observed in the tha- 7711, and 77151, in which labeling was almost lamic nuclei. However, limited projections to entirely confined to area 28L. Entorhinal area nucleus accumbens from these nuclei and from TR (of Haug, ,761, medial to area 28L', also the preoptic area and central gray could nei- contained numerous well-labeled pyramidal ther be proven nor ruled out. cells in this brain (Fig. 5L). The intermediate Occasional labeled cells were also observed subiculum contained many more labeled cells in many brains in the anterior cingulate and than were observed in brains having more infralimbic cortices, adjacent to the needle or lateral or rostral application sites, and the pipette tract. It seems likely that these neu- ventral subiculum was labeled as well (Fig. rons were labeled by direct uptake of HRP 5L-N). No labeling was present in the dorsal drawn into the cortex along the needle or subiculum or hippocampus. pipette tract. Labeling in subcortical areas was similar to Finally, in each brain described above, that observed after HRP applications in lat- large, densely labeled neurons were present CONNECTIONS OF THE NLTCLEUS ACCUMBENS 183 in the ventral pallidum, scattered within the HRP has failed to demonstrate connections terminal field there. Because these cells were which have been shown to exist by other so heavily packed with reaction product, it techniques (Nauta et al., '74). Therefore, it is was felt that their axons had been damaged possible that afferents to nucleus accumbens by the micropipette, allowing HRP to enter might exist in addition to those demonstrated the cells by diffusion. While it is possible that here. The use of orthograde transport of HRP these cells are efferent to nucleus accumbens, to demonstrate efferent connections must be it seems more likely that they project through presumed to have the same possible limita- accumbens to more rostra1 regions. Cells in tion. In addition, it was found here that the this area, for example, are known to project to amount of HRP required in an area for ortho- the frontal cortex (Heimer, '78), anterior ol- grade transport to occur is greater than that factory nucleus (Swanson, '76; Haberly and necessary for retrograde transport. Conse- Price, '78), and (de Olmos et al., quently, it may not always be possible to '78). restrict an HRP application to the area under investigation and at the same time deposit Control experiments enough HRP to insure successful orthograde In the experiments described above, only transport. trace amounts of HRP were deposited in the Fibers-of-passage may also complicate the frontal cortex overlying nucleus accumbens as interpretation of HRP experiments in some the pipette was withdrawn from the brain. cases, since damaged axons may take up HRP Nevertheless, because many of the labeled and transport it in a retrograde direction (Hal- areas in these experiments are known to pro- perin and LaVail, '79, and Hedreen and ject to the frontal cortex (Jones and Leavitt, McGrath ('78) have shown that HRP may '74; Beckstead, '76; Krettek and Price, '77b), enter undamaged axons and label terminals in several brains, applications of HRP were and perikarya by intraaxonal diffusion. How- made in the cortex dorsal to accumbens in ever, this labeling by diffusion is not likely to order to evaluate the possibility that some have been a factor in the experiments de- labeling resulted from cortical contamination. scribed above, since it disappears in most Although these applications were relatively axons within 24 hours. With the exception of small, the amount of HRP deposited in the the retrograde labeling observed in the ven- frontal cortex in these brains was much great- tral pallidum, it is also unlikely that transport er than that resulting from applications of by damaged fibers-of-passagewas a significant HRP in nucleus accumbens. Areas containing problem. Since the needle used for hydraulic a significant number of labeled cells in both injections caused considerably more tissue the experimental brains and the control brains damage than the micropipettes used for ion- included the paraventricular thalamic nucleus tophoresis, the patterns of cell labeling ob- and the dorsal raphe. However, the quantity served with the two techniques should have of labeled neurons in these areas in the control been different if significant retrograde trans- brains was less than in the experimental port was occurring along damaged fibers-of- brains, despite the much greater amounts of passage. However, differences were not noted. HRP deposited in the cortex in the control Perhaps the most important factor to con- experiments. These control experiments, then, sider when using the HRP technique is the demonstrated that the labeling observed in method of defining the region of an application the experimental brains was not due to retro- site from which transport can occur. As men- grade transport from HRP-contaminated cor- tioned above, the size of this region is probably tex. smaller for orthograde than for retrograde Control HRP applications were also made transport, and undoubtedly differs somewhat in the anterior olfactory nucleus in several for each group of afferent and efferent neurons brains, as was mentioned above. of the area under investigation (de Olmos, '77). In addition, the apparent size of an ap- DISCUSSION plication site is influenced by the survival time and by the particular reaction used to Technical considerations visualize the HRP. In the present study, it was The HRP technique has certain qualifica- assumed that transport occurred only from tions which must be considered when inter- regions (1) and (2) of the application sites, preting data derived by this method. In some since the concentration of HRP in region (3) cases, the method of retrograde transport of was very low. This was appropriate in the case 184 R. “MAN AND S.S. N”s of retrograde transport, since the results ob- tral tegmental area, substantia nigra, and cell tained when all three regions of the applica- group A8 of Diihlstrom and Fuxe (‘64) (Un- tion site were within nucleus accumbens were gerstedt, ’71; Fallon and Moore, ’78; Nauta et similar to those obtained when only regions al., ’78). In the present study, only the ventral (1)and (2) were within the nucleus. However, tegmental area was found to project to nucleus this assumption may have overestimated the accumbens. Fallon and Moore (‘78) and Bjork- area from which orthograde transport could lund and Lindvall (‘78) have described the occur, as will be seen below. dopaminergic cells of the ventral midbrain as a single nuclear group whose neurons project topographically upon the forebrain. The ab- Afferent connections of nucleus accumbens sence of projections to nucleus accumbens from Previously unreported sources of afferents the substantia nigra or area A8 in the hamster to nucleus accumbens identified here include is consistent with this concept, since it may the posterior agranular insular, perirhinal, simply reflect differences in the topographic and entorhinal cortices. These areas, as well organization of the dopaminergic “nucleus” in as the , may project to these two species. both the ipsilateral and contralateral nucleus Other areas reported to project to nucleus accumbens. The possibility of contralateral accumbens on the basis of anterograde exper- cortical afferents to nucleus accumbens is con- iments, which were found to be sources of sistent with the findings of van Alphen (‘69). afferents to accumbens in this study as well, Although the afferent connections of nucle- include the olfactory cortex (de Olmos and us accumbens and their topographical organ- Ingram, ’72 Heimer and Wilson, ,751, hippo- ization have never been systematically stud- campus (Fox, ’43; Raisman et al., ’66; de Olmos ied prior to this investigation in the hamster, and Ingram, ’72; Siegel and Tassoni, ’71a; a number of the projections to nucleus accum- Siegel et al., ’75; Heimer and Wilson, ,751, bens reported here have been noted in anter- thalamic nuclei parataenialis, paraventricu- ograde tracing experiments in other species. laris, and reuniens (Cowan and Powell, ’55; Cowan et al. (‘65) observed degeneration in Powell and Cowan, ’56; Swanson and Cowan, nucleus accumbens after placing lesions in the ’75; Herkenham, ’78), and the dorsal raphe amygdala, and Krettek and Price (‘78a), using (Bobillier et al., ’76). the autoradiographic technique, demonstrated Several previously reported projections to projections to nucleus accumbens of the rat nucleus accumbens were not observed in this and cat from the basolateral and basomedial study. The prelimbic, anterior cingulate, and amygdaloid nuclei, and from the amygdalo- rostral sulcal cortices were reported to be hippocampal area. In the present study, amyg- efferent to the nucleus accumbens of the rat daloid efferents were found to nucleus accum- by Beckstead (‘79). In the present study, no bens from the basolateral nucleus only. The labeling was observed in the rostral sulcal basomedial nucleus of the hamster is very cortex following applications of HRP in ac- poorly developed, and it is likely that the cumbens. Occasional HRP-labeled cells were absence of basomedial afferents reflects this present in the anterior cingulate and infral- lack of development. While no labeling was imbic cortices in most brains, adjacent to the observed in the present study in the amygdalo- needle or pipette tract. While sparse projec- hippocampal area, the adjacent subiculum and tions to nucleus accumbens from these latter entorhinal area TR were shown to be efferent cortical areas in the hamster cannot be ruled to nucleus accumbens. It is possible, therefore, out, it seems likely that this labeling was due that in the experiments of Krettek and Price, to the uptake of HRP drawn into the cortex diffusion of tritiated into these along the needle or pipette tract. areas occurred from injection sites in the Siegel and Tassoni (’71b) have proposed that amygdala. the septum projects to nucleus accumbens, in In the present study in the hamster, the contrast to the findings reported here. It is entire subiculum was shown to project to nu- likely that the degenerating axons observed cleus accumbens. In contrast, Swanson and in nucleus accumbens by these investigators Cowan (’77) found only the ventral portion of were hippocampal, entorhinal, or subicular the subiculum to be efferent to nucleus accum- fibers passing to nucleus accumbens in the bens in the rat. This difference apparently , which were damaged by the lesions represents an interspecific variation. placed in the septum. This interpretation is In the rat. nucleus accumbens is known to supported by the autoradiographic experi- receive dopaminergic afTerents from the ven- ments of Swanson and Cowan (’79). CONNECTIONS OF THE NUCLEUS ACCUMBENS 185 Degeneration in nucleus accumbens follow- rat by Heimer and Wilson ('751, Domesick et ing placement of lesions in the anterior medial al. ('761, and Nauta et al. ('78). thalamic nucleus of the rat was described by Efferents from nucleus accumbens to the Leonard ('69). Such a projection was not con- substantia nigra have also been described by clusively demonstrated in the present study a number of investigators (Swanson and Cow- in the hamster. It is possible that Leonard's an, '75; Conrad and Pfaff, '76 Domesick et al., lesions damaged fibers passing to accumbens '76). In the present study, faint terminal fields from thalamic nuclei adjacent to anteromedi- were present in the pars reticulata of the alis, such as paraventricularis, parataenialis, substantia nigra only in brains 7714 and 7715. or centromedialis. Since these terminal fields were quite faint, it Finally, Fallon et al. ('781, using the HRP is possible that inadvertent slight differences method, have suggested that nucleus accum- in processing prevented similar fields from bens receives afferents from the islands of being demonstrated in brains 7711 and 7814. Calleja. This projection was not observed in Less-rapid dehydration of the brain sections the present study, even though the "MI3 tech- in , for example, could render a faint nique employed here is more sensitive than terminal field undetectable, as causes the DAB technique used by Fallon et al. It fading of the HRP reaction product, especially seems likely that the large injections of HRP product contained in terminals. employed by Fallon et al. allowed HRP to In contrast to the fmdings described here, reach the island cells by diffusion, rather than Nauta et al. ('78), using the autoradiographic by retrograde transport. technique in the rat, reported a projection from the nucleus accumbens to the ventral Efferent connections of nucleus accumbens tegmental area, and Phillipson ('79) observed In the present study, a dense terminal field labeling in nucleus accumbens after HRP ap- was observed in the ventral pallidum in all of plications in the rat ventral tegmental area. the brains, in agreement with findings in the However, ventral tegmental area efferents

- caudal SUBIC AS HIPP (CAI) PIC ACC PR - n n

ENT

Fig. 6. A series of schematic diagrams illustrating the topographical organization of the projections to accumbens from (A) the primary olfactory, posterior insular, and perirhinal cortices; (B) the subiculum (SUBIC) and hippocampus (HIPP);(C) the anterior and posterior division of the basolateral amygdaloid nucleus (BL); and (D)entorhinal (END areas 28L, 28L', and TR. Within a given projection system, heavy projections are indicated by wide arrows, lighter projections by thinner arrows. 186 R. NEWMAN AND S.S. wI"S

Fig. 7. Darkfield photomicrograph of a coronal section through the thalamus of brain 7714, corresponding to Figure 4G, showing HRP-labeled neurons in the paraventricular, parataenial, central medial, and paracentral nuclei.

from nucleus accumbens were not noted in niques has clearly yielded no concensus on the autoradiographic studies in the rat by Swan- efferent projections of nucleus accumbens. The son and Cowan ('75) and Conrad and Waf€ studies cited above are in agreement only on ('76). projections to the substantia nigra and palli- Numerous other efferent connections of nu- dum, the two areas also identified here; in cleus accumbens have been described in stud- other respects they often differ markedly. It is ies employing autoradiographic or degenera- likely, therefore, that at least some of the tion techniques, including projections to the projections described in those reports were globus pallidus, septum, hypothalamus, pmptic incorrectly ascribed to accumbens. area, hippocampus, frontal cortex, olfactory tubercle, and thalamus (Swanson and Cowan, Topographical organization of the connections '75; Conrad and Haff, '76; Williams et al., '77; of nucleus accumbens Fallon et al., '78; Herkenham, '78; Nauta et This study demonstrates that a number of al., '78). Some of the limitations of HRP tech- the afferent projections to nucleus accumbens nique discussed earlier may have prevented are topographically organized. As dia- the demonstration of these connections in the grammed in Figure 6A, the primary olfactory, present study. In particular, the method used posterior agranular insular, and perirhinal here for defining the limits of an effective cortical areas project most heavily to caudo- HRP application might have overestimated lateral nucleus accumbens. Labeling in these the size of the area from which orthograde cortices was very light when HRP was depos- transport could occur; perhaps larger HRP ited in the rostra1 pole of the nucleus (brain applications would have labeled additional 7714; Fig. 2), but increased progressively in terminal fields. On the other hand, the use of brains having HRP applications in interme- autoradiographic and degeneration tech- diate (brain 7711; Fig. 3) or caudolateral C0N"IONS OF THE NUCLEUS ACCUMBENS 187

Fig. 8. Darkfield photomicrograph of a coronal section through the midbrain of brain 7714, corresponding to Figure 4L, showing HRP-containing neurons in the ventral tegmental area. 111, oculomotor nerve. Calibration bar, 200 pm for this figure; 500 pm for Figures 7 and 9.

(brain 7715; Fig. 4) nucleus accumbens. No dorsal subiculum and rostral intermediate labeling was observed in these cortical areas subiculum, while caudal areas of the dorsal after an HRP application in caudomedial nu- and intermediate subiculum were labeled by cleus accumbens (brain 7814; Fig. 5). "he HRP deposited in the intermediate portion of projections from these cortices are topographic the nucleus (brain 7711; Fig. 3). After an HRP only with respect to their termination within application in caudolateral nucleus accum- nucleus accumbens. All parts of this nucleus bens (brain 7715; Fig. 41, many HRP-contain- receiving fibers from the insular and perirhin- ing cells were present in the intermediate a1 regions do so from neurons distributed subiculum, but only a few were observed in throughout these areas, and though the olfac- the dorsal subiculum. The ventral subiculum tory cortical projection originates mainly in a and caudal-most intermediate subiculum were restricted region of the olfactory cortex be- labeled only after a caudomedial HRP appli- tween the levels of the rostral and middle cation in nucleus accumbens (brain 7814; Fig. amygdala (Fig. 4F-H), in each brain in which 5K-M). "he entorhinal projection to nucleus the olfactory cortex was labeled, HRP-contain- accumbens originates in lateral areas 28L, ing neurons were present throughout this re- 28L', and TR, and is directed almost exclu- gion. sively at the caudal pole of the nucleus (Fig. In contrast, the efferents to accumbens from 6D). Areas 28L' and TR project only to the the subiculum and entorhinal cortex display caudomedial part of nucleus accumbens (Fig. point-to-point specificity (Fig. 6B,D). An ap- 51-K; brain 7814) while axons from area 28L plication of HRP in rostral nucleus accumbens terminate in the caudolateral portion of the (brain 7714; Fig. 2) labeled cells in the rostral nucleus (Fig. 4F,G, experiment 7715). 188 R. NEWMAN AND S.S. wI"S

Fig. 9. Darkfield photomicrograph of a coronal section of brain 7714,corresponding to Figure 4K,showing HRP-labeled pyramidal cells in the dorsal subiculum (above arrow) and in hippocampal field CA1 (below arrow).

The intermediate portion of hippocampal dal as well as rostral nucleus accumbens. It is field CA1 was found to be efferent to rostral possible that the degeneration they observed nucleus accumbens in the present study (Fig. in the caudal area following ablation of the 6B). Heimer and Wilson ('75, p. 181) have rat hippocampus was due to the destruction of diagrammed a hippocampal projection to cau- subicular or entorhinal fibers-of-passage pro-

Fig. 10. Darkfield photomicrograph of a coronal brain section through the nucleus accumbens, showing a large iontophoretic HRP application centered in accumbens and extending into surrounding regions. 1, 2, and 3, zones of application site, as described in the text. Fig. 11. Darkfield photomicrograph of a coronal section of brain 7711 corresponding to Figure ZE,pointing out a dense terminal field in the ventral pallidum. Calibration bar, 500 pm for Figures 10 and 11. CONNECTIONS OF THE NUCLEUS ACCUMBENS 189 190 R. NJ3WMAN AND S.S. WINANS jecting to the caudal part of the nucleus organization of these projections. While the through the fimbria. substantia nigra was found to receive efferents Figure 6C shows that the afferents to nucle- from the rostral and caudolateral portions of us accumbens from the basolateral nucleus of nucleus accumbens, a terminal field was not the amygdala terminate most extensively in observed in the nigra after an application of the caudal portion of accumbens; labeling in HRP in the caudomedial or intermediate por- this nucleus was sparse when HRP was de- tions of the nucleus. However, the apparent posited in the rostral pole of accumbens (brain absence of nigral projections from these parts 7714; Fig. 21, but was considerably more ex- of nucleus accumbens may have been due to tensive in brains having more caudal appli- technical factors as discussed above. There- cation sites (experiments 7711, 7715, 7814; fore, the possibility of topographical organi- Figs. 3-5). Furthermore, labeling in the an- zation in this projection cannot be evaluated. terior division of the basolateral nucleus was The efferent projection from nucleus accum- heaviest when HRP was deposited in caudo- bens to the ventral pallidum was also not lateral nucleus accumbens (brain 7715; Fig. found to be topographically organized in the 4), while HRP applications in caudomedial present study, in contrast to the finding in the accumbens labeled the posterior division most rat of Swanson and Cowan ('75) that medial heavily (brain 7814; Fig. 5). These latter re- portions of the nucleus project medially within sults confirm the findings of Krettek and Price the pallidum, and efferents from lateral parts ('78a) in the rat and cat. of nucleus accumbens terminate laterally. Intralaminar thalamic efferents to accum- The experimental results described in this bens arise from the rostral portions of the paper substantiate the classification of nucle- central medial and paracentral nuclei and us accumbens as a striatal structure and pro- from rostral parafascicular neurons ventro- vide experimental evidence supporting the medial to the fasciculus retroflexus. The origin concept of the ventral striatum. Since experi- of these projections in restricted portions of ments described in the second paper in this the intralaminar nuclei is in contrast to the series on the connections of the olfactory tu- midline thalamic projections to nucleus ac- bercle (Newman and Winans, this issue) pro- cumbens, which arise throughout their re- vide additional information concerning the spective nuclei. The pattern of termination of organization and possible function of the ven- the intralaminar and midline thalamic projec- tral striatal system, the evidence for the above tions in nucleus accumbens is such that all statements will be discussed in that paper, parts of nucleus accumbens are innervated by together with the results of the olfactory tu- the parafascicular, parataenial, and reuniens bercle experiments. nuclei, while the central medial and parav- entricular nuclei project to all but the caudo- ACKNOWLEDGMENTS medial portion of the nucleus (Fig. 5; brain We gratefully acknowledge the fine artistic 7814). contributions of our colleague William L. Bru- The projection to nucleus accumbens from don, and the thoughtful criticism of the man- the ventral tegmental area displays a rostro- uscript by Dr. Lennart Heimer. caudal topography, such that the ventral teg- This research was supported in part by mental area efferents terminate most heavily NIMH grant MH29375. Dr. Newman was the in rostral accumbens. This can be seen by recipient of a Student Research Fellowship examining the amount of labeling in the ven- from the University of Michigan Medical tral tegmental area in experiments having School. successively more caudal HRP application LITERATURE CITED sites (brains 7714,7711,7715, and 7814; Figs. Beckstead, R.M. (1976) Convergent thalamic and mes- 2-51. In contrast to the findings of Fallon and encephalic projections to the anterior medial cortex in Moore ('78) in the rat, this projection was not the rat. J. Camp. Neurol., 166:403-416. found to display point-to-point topography, Beckstead, R.M. (1979) An autoradiographic examina- tion of corticocortid and subcortical projections of the and a medial-lateral component was not noted medicdorsal projection (prefrontal) cortex in the rat. J. in the topography of these fibers. Comp. Neurol., 184:43-62. The data obtained here with HRP on the Bjorklund, A, and 0. Lindvall (1978) The mesotelence- efferents of nucleus accumbens support the phalic dopamine system: A review of its anatomy. In. Limbic Mechanisms: The Continuing Evolution of the findings most consistently reported with au- Limbic System Concept. K. E. Livingston and 0. Hor- toradiographic and degeneration techniques, nykiewicz, eds. Plenum Press, New York, pp. 307-332. but do not provide evidence for a topographical 307-332. CONNECTIONS OF THE NUCLEUS ACCUMBENS 191

Bobillier, P., S. Seguin, F. Petitjean, D. Salvert, M. Touret, Hardy, H., and L. Heimer (1977) A safer and more sen- and M. Jouvent (1976) The of the brain- sitive substitute for diaminohnzidine in the light micro- stem: A topographical atlas of their efferent projections scopic demonstration of retrograde and anteropade ax- as revealed by autaradicgraphy. Brain Rm.,113,449-486. onal transport of HRP.Neumsci. Lett., 5235-240. Colman, D., F. Scalia, and E. Cabrales (1976) Light and Haug, F.-M.S. (1976) Sulphide silver pattern and cy- electron microscopic observations on the anterograde toarchitectonics of parahippocampal areas in the rat. transport of horseradish peroxidase in the optic pathway Adv. Anat. Embryol. Cell Biol., 52:l-73. in the mouse and rat. Brain Res.. 102:15%163. Hedreen, J.C., and S. McGrath (1978) Observations on Conrad, L.C.A., and D.W. Pfaf€ (1976) Autoradiographic labeling of neuronal cell' bodies, axons, and terminals tracing of nucleus aaumbens efferents in the rat. Brain aRer injection of horseradish peroxidase into rat brain. Res., 113:589-596. J. Comp. Neurol., 176:225-246. Cowan, W.M., and T.P.S. Powell (1955) The projections Heimer, L. (1978) The olfactory cortex and the ventral of the midline and intralaminar nuclei of the thalamus striatum. In: Limbic Mechanism%The Continuing Evo- of th &bit J. M. v.M, 18:2€6-279. lution of the Limbic System Concept. K. E. Livingston Cowan, W.M., G. Raisman, and T.P.S. Powell (1965) The and 0. Hornykiewia, eds. Plenum Press, New York, pp. connexions of the amygdala. J. Neurol. Neurusurg. Psy- 95-187. chiat., 28:137-151. Heimer, L., and R.D. Wilson (1975) The subcortical pre Dahlstrom, A., and K. Fuxe (1964) Evidence for the jections of the : Similarities in the neural con- existence of monamine containing neurons in the central nections of the hippocampus, the , and the nervous system. I. Demonstration of monamines in the . In: Gulgi Centennial Symposium: Perspectives cell bodies of brainstem neurons. Acta Physiol. Seand., in Neurobiology. M. Santini, ed. Raven, New York, pp. 62 (Suppl. 232):l-55. 177-193. de Olmos, J.S. (1977) An improved HRF' method for the Herkenham, M. (1978) The connections of nucleus reu- study of central nervous connections. Exp. Brain Res., niens thalami:Evidence for a direct thalamo-hippocam- 29:54-551. pal pathway in the rat. J. Comp. Neurol., 177:589-610. de Olmos, J., H. Hardy, and L. Heimer (1978) The affer- Jones, E.G., and R.Y. Leavitt (1974) Retrograde axonal ent connections of the main and accessory olfactory bulb transport and the demonstration of nonspecific projec- formations in the rat An experimental HRP-study. J. tions to the and striatum from thalamic Comp. Neurol., 181:213-244. intrahninar nuclei in the rat, cat, and monkey. J. Comp. de Olmos, J.S., and W.R. Ingram (1972) The projection Neurol., 154:349-378. field of the in the rat brain. An experi- Knmk, H.L. (1966) The Fiber Connections of the Fore- mental study. J. Comp. Neurol., 146:303-334. brain. F.A. Davis, Philadelphia, p. 148. Dill, R.E., and E. Costa (1977) Behavioral dissociation Koikegami, H., Y. Hirata, and J. Ogrema (1967) Stud& of the enkephalinergic system of accumbens and the on the paralimbic brain structures and some experiments nucleus caudatus. Neuropharmawlogy, 16:323-326. on the nucleus accumbens. Folia Psyehiat. Neurol. Jpn., Domesick, V.B., G.P. Smith, and W.J.H. 21: 151-180. Nauta (1976) Contrasting projections of the nucleus Krettek, J.E., and J.L. Price (1977a) The cortical projec- accumbens and caudatoputamen in the rat. Abstract, tions of the mediodorsal nucleus and adjacent thalamic Cajal Club Proceedings. nuclei in the rat. J. Comp. Neurol., 171:157-192. Fallon, J.H., and R.Y. Moore (1978) Catecholamlne in- Krettek, J.E., and J.L. Price (1977b) Projections from nervation of the . lV. Topography of the the amygialoid complex to the cerebral cortex and thal- dopamine projection to the basal forebrain and neostria- amus in the rat and cat. J. Comp. Neurol., 172:687-722. tum. J. Comp. Neurol., 180:545-580. Krettek, J.E., and J.L. Price (1978a) Amygdaloid projec- FaUon, JH., JN. Riley, J.C. Sip, and RY. Moore (1978) The tions to subcortical structures within the basal forebrain : Organization and connections. J. and brainstem in the rat and cat. J. Comp. Neurol., Comp. Neurol., 181:375-396. 178:225-254. Fox, C.A. (1943) The stria terminalis, longitudinal as- Krettek, J.E.,and J.L. Price (1978b) A description of the sociation bundle, and precommissural fornix fibers in the amygdaloid complex in the rat and cat with observations cat. J. Comp. Neurol., 79:277-295. on intraamygdaloid axonal connections. J. Comp. Neu- Graham, R.C., and M.J. Karnovsky (1966) The early rol., 178:255-280. stages of absorption of injected horseradish peroxidase in Lauer, E.W. (1945) The nuclear pattern and fiber con- the proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique. J. Histochem. Cyto- nections of certain basal telencephalic centers in the macaque. J. Comp. Neurol., 82:215-245. chem., 14:291-303. Leonard, C.M. (1969) The of the rat. I. Guillery, R.W. (1959) Merent fibers to the dorso-medial Cortical projections of the mediodorsal nucleus. 11. Effer- thalamic nucleus in the cat. J. Anat., 93:403-419. ent connections. Brain Res., 12:321-343. Gdian,E.S. (1927) The diencephalon of the albino rat. Nauta, H.J.W., M.B. Pritz, and RJ. kk(1974) Afferents J. Comp. Neurol., 43:l-114. to the rat caudatoputamen studied with horseradish Gdian, E.S. (1928) The corpus striatum of the rat. J. peroxidase. An evaluation of a retrograde neuroanatom- Comp. Neurol., 45:249-281. ical reaearch method. Brain Res., 67:219-238. Haberly, L.B., and J.L. Price (1978) Association and Nauta, W.J.H., G.P. Smith, R.L.M. Faull, and V.B. Dome- commissural fiber systems of the olfactory cortex of the sick (1978) Efferent connections and nigral afferent8 rat. I. Systems originating in the piriform cortex and of the nucleus accumbens in the rat. adjacent areas. J. Comp. Neurol., 78:711-740. 3:385-401. Halperin, J.J., and J.H. LaVail (1975) A study of the Newman, R., and S.S. Winans (1980) An experimental dynamics of retrograde transport and accumulation of study of the ventral striatum of the golden hamster. 11. horseradish peroxidase in injured neurons. Brain h., The neuronal connections of the olfactory tubercle. (this 100:253-270. issue). 192 R. NEWMAN AND S.S. wI"S

Phillipson, O.T. (1979) Afferent projections to the ven- Smith, O.C. (1930) The corpus striatum, amygdala, and tral tegmental area of 'bai and interfascicular nucleus: stria terminalis of Tarnandua tetrudactyla. J. Comp. Neu- A horseradish peroxidase study in the rat. J. Comp. rol., 51:65-127. Neurol., 187: 117-143. Swanson, L.W. (1976) An autoradiographic study of the Pijenburg, A.J.J., G.N. Woodruff, and J.M. van efferent connections of the preoptic area in the rat. J. Rossum (1973) Ergometrine induced locomotor activ- Comp. Neurol., 167:227-256. ity following intracerebral injection into the nucleus Swanson, L.W., and W.M. Cowan (1975) A note on the accumbens. Brain Res., 59:289-302. connections and development of the nucleus accumbens. Powell, T.P.S., and W.M. Cowan (1956) A study of thal- Brain Res., 92:324-330. amo-striate relations in the monkey. Brain, 79:364-390. Swanson, L.W., and W.M. Cowan (1977) An autoradi- Raisman, G., W.M. Cowan, and T.P.S. Powell (1966) An ographic study of the organization of the efferent connec- experimental analysis of the efferent projections of the tions of the hippocampal formation in the rat. J. Comp. hippocampus. Brain, 89: 83-108. Neurol., 172:49-84. Rioch, D.M. (1931) Certain myelinated fiber connections Swanson, L.W., and W.M. Cowan (1979) The connections of the diencephalon of the dog, cat, and aevisa. J. Comp. of the septal region in the rat. J. Comp. Neurol., Neurol., 53:319-388. 186: 621- 655. Siegel, A,, S. Ohgami, and H. Edinger (1975) Projections Ungerstedt, U. (1971) Stereotaxic mapping of the mon- of the hippocampus to the in the squirrel amine pathways in rat brain. Acta Physiol. Scand. 197 monkey. Brain Res., 99:247-260. (Suppl. 367):l-48. Siegel, A., and J.P. Tassoni (1971a) Differential efferent van Alphen, H.A.M. (1969) The anterior mmmissure of projections from the ventral and dorsal hippocampus of the rabbit. Acta Anat. (Baself, 74 (Suppl. 57):l-112. the cat. Brain Behav. Evol., 4:185-200. Williams, D.J., A.R. Crossman, and P. Slater (1977) The Siegel, A,, and J.P. Tassoni (1971b) Differential efferent efferent projections of the nucleus accumbens in the rat. projections of the lateral and medial septal nuclei to the Brain Res., 130:217-227. hippocampus in the cat. Brain Behav. Evol., 4:201-219. Smith, M.O., and R. Holland (1975) Effects of lesions of the nucleus accumbens on lactation and postpartum be- havior. Physiol. Psychol., 3:331-350.