Subcortical connections of the perirhinal, postrhinal, and entorhinal cortices of the rat. II. efferents.

HHS Public Access Author manuscript Author Manuscript

Hippocampus. Author manuscript; available in PMC 2016 October 19. Published in final edited form as: Hippocampus. 2016 September ; 26(9): 1213–1230. doi:10.1002/hipo.22600.

SUBCORTICAL CONNECTIONS OF THE PERIRHINAL, POSTRHINAL, AND ENTORHINAL CORTICES OF THE RAT. II. EFFERENTS Kara L. Agster1,#,*, Inês Tomás Pereira2,#, Michael P. Saddoris2,*, and Rebecca D. Burwell1,2

Author Manuscript

1Department

of Neuroscience, Brown University, Providence RI, 02912

2Department

of Cognitive, Linguistic, and Psychological Sciences, Brown University, Providence

RI, 02912

Abstract

Author Manuscript Author Manuscript

This is the second of two studies detailing the subcortical connections of the perirhinal (PER), the postrhinal (POR) and entorhinal (EC) cortices of the rat. In the present study, we analyzed the subcortical efferents of the rat PER areas 35 and 36, POR, and the lateral and medial entorhinal areas (LEA and MEA). Anterograde tracers were injected into these five regions, and the resulting density of fiber labeling was quantified in an extensive set of subcortical structures. Density and topography of fiber labeling were quantitatively assessed in 36 subcortical areas, including olfactory structures, claustrum, amygdala nuclei, septal nuclei, basal ganglia, thalamic nuclei, and hypothalamic structures. In addition to reporting the density of labeled fibers, we incorporated a new method for quantifying the size of anterograde projections that takes into account the volume of the target subcortical structure as well as the density of fiber labeling. The PER, POR and EC displayed unique patterns of projections to subcortical areas. Interestingly, all regions examined provided strong input to the basal ganglia, although the projections arising in the PER and LEA were stronger and more widespread. PER areas 35 and 36 exhibited similar pattern of projections with some differences. PER area 36 projects more heavily to the lateral amygdala and much more heavily to thalamic nuclei including the lateral posterior nucleus, the posterior complex, and the nucleus reuniens. Area 35 projects more heavily to olfactory structures. The LEA provides the strongest and most widespread projections to subcortical structures including all those targeted by the PER as well as the medial and posterior septal nuclei. POR shows fewer subcortical projections overall, but contributes substantial input to the lateral posterior nucleus of the thalamus. The MEA projections are even weaker. Our results suggest that the PER and LEA have greater influence over olfactory, amygdala, and septal nuclei, whereas PER area 36 and the POR have greater influence over thalamic nuclei.

Please direct correspondence and reprint requests to: Rebecca D. Burwell, Ph.D., Department of Cognitive, Linguistic and Psychological Services, Brown University, Box 1821, Providence, RI 02912, Phone: (401) 863-9208, Fax: (401) 863-1300, [email protected]. #These authors contributed equally to this work. *KLA and MPS are currently located in the Department of Psychology and Neuroscience at the University of Colorado Boulder

Agster et al.

Page 2

Author Manuscript

Keywords parahippocampal; anatomy; connectivity; anterograde; memory

Introduction

Author Manuscript

Neuroanatomical studies suggest that the perirhinal (PER) and postrhinal (POR) cortices are the gateway for neocortical input to the hippocampal formation through their connections with the entorhinal cortices (EC). Historically, regions comprising the medial temporal lobe were believed to make similar functional contributions to episodic memory. By this view, the functions of the PER, POR, and EC were not differentiated from that of the hippocampus (HPC, Eacott et al., 1994; Squire et al., 2004). An historical neuroanatomical view posited that the PER and POR provided a gateway for neocortical input to the hippocampal formation through their connections with the EC. A more recent framework, however, assigns unique cognitive roles for the individual cortices (Eichenbaum and Lipton, 2008; Jarrard et al., 2004; Murray et al., 2007). Within this conceptual framework the PER is necessary for object recognition (Albasser et al., 2011; Bussey et al., 2003; Kealy and Commins, 2011; Meunier et al., 1993; Tu et al., 2011), the POR monitors the environment or context (Burwell and Hafeman, 2003; Furtak et al., 2012; Norman and Eacott, 2005), and the EC is involved in spatial/working memory (Brun et al., 2008; McGaughy et al., 2005; Steffenach et al., 2005; Stensola et al., 2012). This framework is complemented by findings that the PER, POR, and EC exhibit distinctly different connections with neocortical regions (Agster and Burwell, 2009; Burwell and Amaral, 1998a; Burwell and Amaral, 1998b; Lavenex et al., 2004; Witter et al., 1989).


The question of whether the PER, POR, and EC also exhibit different patterns of subcortical connectivity has not been fully investigated. Some studies focused on connections of a subset of the PER, POR, or EC regions with specific subcortical structures, for example, the relative strength of the amygdala connections with parahippocampal structures (Kemppainen et al., 2002; Majak and Pitkanen, 2003; Pikkarainen and Pitkänen, 2001; Pitkänen et al., 2000; Shi and Cassell, 1997). Those studies, however, do not permit an assessment of a more complete set of subcortical connections across structures in the parahippocampal region. The aim of this study was to provide a detailed analysis of the subcortical efferents of the major cortical structures in the parahippocampal region including the PER areas 35 and 36, POR, and the lateral and medial entorhinal areas (LEA and MEA) of the EC. The companion paper describes in detail, the subcortical afferents of the same structures (Tomás Pereira et al., submitted). Taken together, the two studies provide the capability to compare patterns of subcortical connectivity across the major cortical regions of the medial temporal lobe. These data will provide greater insight to the unique functional contributions of the PER, POR, and EC.

Hippocampus. Author manuscript; available in PMC 2016 October 19.

Agster et al.

Page 3

Author Manuscript

Materials and Methods Nomenclature A detailed rationale and description of the analyzed regions and associated nomenclature is presented in the companion paper (Tomás Pereira et al., submitted). Nomenclature is briefly described here. Originating regions included PER areas 35 and 36 and the POR according to Burwell (2001). For the EC, we used boundaries and nomenclature that subdivide the region into the lateral and medial entorhinal areas (LEA and MEA, respectively) (Blackstad, 1956; Brodmann, 1909). The borders and nomenclature for the subcortical regions in which we quantified labeled fibers are adapted from Swanson (1992; 1998). A subset of coronal sections illustrating those structures is shown in Figure 2 of the companion paper (Tomás Pereira et al., submitted).

Author Manuscript Author Manuscript Author Manuscript

The structures, groupings, and abbreviations of subcortical structures are shown in Table 1. The olfactory areas analyzed were the anterior olfactory nucleus (AON), the olfactory tubercle (OT), the piriform transition area (PTA), the endopiriform nucleus (EP), and the taenia tecta (TT, Table 1). The claustrum (CLA) was analyzed as a single structure. Amygdala nuclei analyzed included the lateral nucleus (LA), the basolateral nucleus (BLA), the basomedial nucleus (BMA), the central nucleus (CEA), and the olfactory amygdala (OA). We analyzed four septal regions, including the lateral septal nucleus (LS), the medial septal complex (MS), the posterior septal complex (PS), and the bed nuclei of the stria terminalis (BST). For the basal ganglia, we included the caudate putamen (CP), the nucleus accumbens and the fundus of the striatum (ACB), the lateral and medial segments of the globus pallidus (GP), and the substantia innominata including the magnocellular preoptic nucleus (SI). We also included the dopaminergic cell groups in the substantia nigra pars compacta and pars reticulata with the ventral tegmental area (SN-VTA). Thalamic structures were grouped into dorsal structures and ventrolateral structures. For the dorsal thalamus, the anterior dorsal (DTHan) included anteroventral, anteromedial, anterodorsal, interanteromedial, interanterodorsal, and lateral dorsal nuclei. The medial group (DTHme) included the mediodorsal and submedial thalamic nuclei, and the perireuniens nucleus. The lateral group (DTHla) included the suprageniculate nucleus and the lateral posterior nucleus, the posterior limiting nucleus, and the posterior complex of the thalamus. The ventral group (DTHve) included the ventral anterior-lateral complex, the ventral medial nucleus and the ventral posterior complex of the dorsal thalamus. We analyzed the intralaminar nucleus of the dorsal thalamus (ILM) as a single structure. The ventrolateral thalamus included the medial geniculate (MG), the lateral geniculate (LG), the reticular nucleus (RT), the zona incerta (ZI) and the ventrolateral group (VLTH). The VLTH included the subthalamic nucleus, the perifascicular nucleus, and the peripeduncular nucleus. In the hypothalamus, the periventricular zone (PVZ) included the paraventricular, anteroventral, anterior, intermediate, and posterior paraventricular hypothalamic nuclei. The mammillary bodies (MBO) included the dorsal, medial, and lateral mammillary nuclei, the tuberomammillary nucleus, and the supramammillary nucleus. The medial zone (MEZ) included the medial, anterodorsal, anteroventral, and posterodorsal preoptic nuclei, the parastrial nucleus, the suprachiasmatic nucleus, the retrochiasmatic area, the subparaventriclar zone, the anterior


Agster et al.

Page 4

Author Manuscript

hypothalamic area, and the tuberal area of the hypothalamus. The lateral zone (LZ) included the lateral preoptic area and the lateral hypothalamic area. Subjects Twenty-nine male Sprague-Dawley rats weighing between 300–400g at the time of surgery served as subjects for these experiments. Animals were housed as described in the companion paper (Tomás Pereira et al., submitted). All methods involving the use of live subjects conform to NIH guidelines and were approved by the appropriate Institutional Animal Care and Use Committee. Data from these subjects were used in analyses of the interconnections and/or the cortical efferents and hippocampal connections of the PER, POR, and EC (Agster and Burwell, 2009; Agster and Burwell, 2013; Burwell and Amaral, 1998b). In addition, a version of these data were previously reviewed in highly summarized form (Furtak et al., 2007; Kerr et al., 2007).

Author Manuscript

Surgery

Author Manuscript

Surgical procedures were performed as previously reported (Burwell and Amaral, 1998b; Tomás Pereira et al., submitted). Briefly, animals were anaesthetized and secured in a stereotaxic apparatus (Kopf, Tujunga, CA). An incision was made in the scalp, and the skin and connective tissue retracted. A small hole was drilled in the region of skull overlying the intended injection site. Animals received injections of biotinylated dextran amine (BDA) or Phaseolus vulgaris-leuccoagglutinin (PHA-L). Tracer injections were located in the PER (n=10), POR (n=5), or EC (n=14). BDA was prepared in a 10% solution in 0.1 M phosphate buffered saline. PHA-L was prepared in a 2.5% solution in 0.1 M phosphate buffered saline. These anterograde tracers were injected via iontophoresis with positive DC current (4 μamps; 8 sec on and 8 sec off) for 8 min through glass micropipettes. Micropipettes had an average tip diameter of 4–5 μm. Following the injection, the wound was sutured and the animal was monitored for several hours before returning to the colony. Tissue Processing Animals were perfused as previously described (Tomás Pereira et al., submitted). Brains were coronally sectioned at 30 μ on a freezing microtome. Sections were collected in a 1:5 series for processing or storage. Two series were collected in potassium phosphate buffered saline (KPBS) for immunohistochemical processing and one series was collected and mounted for Nissl staining using thionin. The remaining two series were collected and stored at −80°C in a cryoprotectant tissue collecting solution of 30% ethylene glycol and 20% glycerol in 0.1M phosphate buffered sodium.

Author Manuscript

Visualization of PHA-L fibers was accomplished using a biotinylated secondary antibody with avidin-biotin incubation (method adapted from Gerfen and Sawchenko, 1984). Sections were incubated for 2–3 h in 5% normal goat serum (NGS) and 0.5% Triton-X 100 (TX) in KPBS to minimize non-specific binding. Sections were then incubated in the primary antiserum solution of rabbit anti-PHA-L (1:12,000 dilution; Dako, Carpenteria, CA) in 0.3% TX and 2% NGS in KPBS for 42–48 h. Following two 10 min washes in 2% NGS in KPBS, sections were incubated in the biotinylated secondary antibody solution containing goat antirabbit IgG (1:277 dilution; Vector Laboratories, Burlingame, CA), 0.3% TX, and 2% NGS


Agster et al.

Page 5

Author Manuscript

in KPBS for 1 h. Sections were then washed twice in 2% NGS in KPBS and incubated in a solution of avidin reagent and stabilizer (1:100 and 1:200 dilutions, respectively; Super ABC Kit; Biomeda Corporation, Foster City, CA) in KPBS for 45 min. Following another two 10 min washes in 2% NGS in KPBS, tissue was re-incubated into the biotinylated secondary solution for 45 min. Sections were then placed into two 10 min washes of KPBS and recycled into the avidin solution for 30 min. After three 10 min washes in KPBS, the sections were processed for visualization by incubation in 0.05% diaminobenzidine (DAB; Pierce, Tacoma, WA) and 0.04% hydrogen peroxide in KPBS for 5–10 min.

Author Manuscript

Visualization of BDA fibers was accomplished using an avidin-biotin reaction. Sections were initially pre-treated in a solution of 1% TX in KPBS for 1 h, in order to facilitate penetration of the reagents. The sections were then incubated overnight at 4°C in a solution of avidin reagent and stabilizer (1:25 and 1:50 dilutions, respectively; Super ABC Kit; Biomeda Corporation) in KPBS plus 0.1% TX. Following three 10 min washes in KPBS, sections were processed for visualization by incubation in 0.05% DAB and 0.04% hydrogen peroxide in KPBS for 10–30 min. Subsequent to immunohistochemical processing all sections were washed, mounted on gelatin coated slides, dried, defatted, and intensified with gold chloride and thiocarbohydrazide (Lewis et al., 1986). Data Analysis


Sections were examined using dark field microscopy (Leica MZ6 stereomicroscope with darkfield capability). Fibers within 36 subregions or nuclei were qualitatively rated for density of fibers on a scale of 0–6. A score of zero indicated no fibers were present, whereas a score of 6 denoted dense fiber labeling within a region. Due to the large number of nuclei within some subcortical structures, subsets of individual nuclei were condensed in some cases to facilitate analysis (Tomás Pereira et al., submitted). The ratings were adjusted for each animal such that the full scoring range was used, therefore, differences across injection sites were normalized across animals. Using a methodology developed previously (Burwell and Amaral, 1998b), the ratings from each case were recorded in a 2D unfolded map allowing for analysis of the rostro-caudal patterns of density. Two measures were derived from the density scores, comparable to what was done in previous studies (Agster and Burwell, 2009). First, the scores were averaged across each subcortical region for each animal. This measure represents the relative influence of each parahippocampal region onto one subcortical structure (e.g. does the LA receive stronger input from PER area 35 or from POR?). The second measure used takes the volume of the subcortical regions into account, allowing for the evaluation of the overall strength of the projection. We used the standard volume calculated for each subcortical structure from 20 Sprague-Dawley male rats analyzed in the companion afferent study (Tomás Pereira et al., submitted). The average density scores for each subcortical region was multiplied by its standardized volume. This measure allows for comparison of the strength of the projection from one parahippocampal region to different subcortical regions (e.g. is the subcortical output from LEA stronger to the CP or to the BLA?).


Agster et al.

Page 6

Author Manuscript

Results Description of Injection Sites Twenty-nine experiments were analyzed for strength of subcortical efferents. The locations of individual injection sites are shown in Figure 1. Injection sites are represented on a twodimensional unfolded map of the EC, PER, and POR. An injection site was defined as the region containing labeled cell bodies, and a cell count was performed to obtain an approximation of the size of each tracer injection. Cell counts were performed in the section where the spread of the injection site appeared largest. Additionally, the spread of the injection was estimated by quantifying the area of cortex containing labeled cells. The number of labeled cells visible at the injection site, the approximate size of the injection, and laminar location of the injection sites in each region are listed in Table 2.

Author Manuscript

Pathways The axonal trajectories from the PER, POR, and EC to the subcortical nuclei are similar across regions. In all cases, most fibers arising from injection sites in the PER, POR, and EC were observed to enter the external capsule. After traveling various distances through the external capsule, fibers exited and innervated subcortical nuclei within the basal forebrain. Fibers of passage from each of the three cortical regions also traveled through the stria terminalis, though this route was utilized more frequently by PER and POR fibers. Fibers emerging from the EC injection sites were more commonly observed in the fornix.

Author Manuscript

Fiber pathways of projections to the midbrain nuclei were more variable. Fibers of passage from all cortices to the thalamic and hypothalamic nuclei were found to travel dorsally adjacent to the optic tract. POR fibers directed to these structures were additionally observed in the external medullary lamina of the thalamus. EC projections to midbrain structures were very weak, and accordingly, fibers directed to midbrain were not obvious. Density of Fiber Labeling For each target region, two measures of fiber density are presented. In this section we describe the average density of fiber labeling (Table 3). This measure reflects the varying strengths of projections from the originating parahippocampal structures onto specific subcortical nuclei.

Author Manuscript

Perirhinal Cortex Area 35—Injections of anterograde tracer into area 35 of the perirhinal cortex lead to greatest density of fibers in the claustrum, amygdala and olfactory nuclei (Table 3). More specifically, within the olfactory nuclei, dense fibers were observed in the PTA (Figure 2B), and the TT (particularly the more caudal portion). Moderate density was also observed in the OT and the EP. For the olfactory nuclei, most of the input arose from rostral area 35. In the amygdala, the LA, BLA and BMA had the densest fibers (Figure 3B). The LA projection was most dense to the rostral portion and originated from rostral area 35 as well. The basal ganglia presented moderate density of fibers, but analysis of the different nuclei reveals that fibers terminated most densely in the CP and the ACB (Figure 4B). The rostral injection site (but not the caudal one) projects more strongly to the caudal region of ACB, whereas the CP is targeted evenly across the rostrocaudal extent. The septal nuclei


Agster et al.

Page 7

Author Manuscript

exhibited moderate to low density of fibers, whereas the thalamic and hypothalamic nuclei showed little or no fiber labeling.

Author Manuscript

Perirhinal Cortex Area 36—Perirhinal area 36 shows a pattern of efferent projections similar to area 35. Injections in area 36 lead to greatest density of fibers in the claustrum, amygdala, olfactory nuclei, and basal ganglia (Table 3). In the olfactory nuclei, PTA has the greatest density of fibers, particularly in the rostral portion of the region (Figure 2C). The injections that generated the densest fibers originated in both deep and superficial layers of the mid rostro-caudal region of area 36. The projection from area 36 to PTA is comparatively weaker than the projection originating in area 35. In the amygdala, the strongest projection from area 36 terminated in the LA (Figure 3C). This was the highest density of fibers observed for any projection analyzed in the study. The projection targeted mostly the caudal portion of the LA and originated from everywhere in area 36, but most intensely from the more caudal injection sites. In the BLA, dense fibers were also observed, particularly in the midpoint of the rostrocaudal extent. In the basal ganglia, the CP (particularly the caudal portion) and ACB (evenly across the rostrocaudal axis) also showed high density of fibers following injections of anterograde tracer into area 36 (Figure 4C). The septal nuclei had overall modest density of fibers, with the exception of the PS, where fiber density was moderately high. The projection to the BST targets the bed nucleus of the stria terminalis exclusively. The hypothalamus, similarly to the pattern of results observed in area 35, received few projections from area 36.

Author Manuscript

One of the biggest differences between the projections from area 35 and 36 was observed in the subnuclei of the dorsal and ventrolateral thalamus. Area 35 projections were overall weak, whereas projections from area 36 were considerably stronger to specific nuclei, including DTHla in the dorsal thalamus and the VLTH in the ventral thalamus. In the DTHla, there was some segregation of projections, with rostral injections leading to fiber labeling only in the posterior complex of the thalamus (Figure 6B), whereas the remaining cases showed labeled fibers in both the suprageniculate and the lateral posterior nuclei of the thalamus. Fiber labeling in the DTHmi was moderate, but restricted to the nucleus reuniens. The density of fibers terminating in VLTH was stronger in the caudal portion and targeted primarily the subparafascicular nucleus. Finally, the MG showed moderately dense fibers, mostly located in the dorsal region (Figure 6C).

Author Manuscript

Postrhinal Cortex—The pattern of subcortical projections from the POR stands in stark contrast to the projections from PER. On the whole, the projections are much weaker. In addition to the weak projections to the hypothalamus (also observed in PER), projections to the olfactory nuclei and septal nuclei were also very weak. The claustrum, amygdala and specific subnuclei of the basal ganglia and thalamus show highest density of fibers. Most of the projections arise from the deep layers of POR, with very few fibers observed following injections in the superficial layers. In the amygdala, LA and CEA (the caudal portion particularly) showed the highest density of fibers, with the BLA showing moderate density. Most basal ganglia nuclei showed low density of fibers with the exception of the CP which showed high levels of fiber density, with the caudal portion being particularly targeted (Figures 5B–C). For the thalamic regions, the output pattern appears generally similar Hippocampus. Author manuscript; available in PMC 2016 October 19.

Agster et al.

Page 8

Author Manuscript

between POR and area 36, however, the specific target nuclei are distinct. Similarly to area 36, the DTHla showed the highest density of fibers following anterograde tracer injections in the POR. Fiber labeling was observed only in the lateral posterior nucleus (Figure 6D), as opposed to the different nuclei targeted by area 36 (Figure 6B). The DTHan showed moderate density of fibers, found mostly in the lateral dorsal nucleus. As for PER area 36, the only DTHmi structure in which fibers were observed following POR injections was the nucleus reuniens. In the ventrolateral thalamus, the LG also presented moderately high density of fibers (Figure 6D).

Author Manuscript

Lateral Entorhinal Area—The EC displayed a pattern of projections distinct from both PER and POR. Within the entorhinal cortex, LEA and MEA also showed differing strength of projections, with the LEA in general directing much stronger input to the subcortical nuclei. The highest density of fiber labeling following injections in the LEA was observed in the olfactory nuclei, the claustrum and the amygdala. In the olfactory subcortical region, the PTA showed the highest density of fibers (Figure 2D), but all other nuclei (AON, OT, EP and TT) also presented dense fiber labeling. These fibers were most dense following injections of the lateral and intermediate bands of LEA. The claustrum also received strong projections from LEA, particularly to the mid-rostrocaudal and caudal portions. These projections were also more likely to arise from the lateral and intermediate bands of LEA. In the amygdala, the densest fiber labeling was observed in the BLA, but LA, BMA and OA also showed dense fibers (Figure 3D). These projections originated primarily from the lateral and intermediate LEA bands and targeted the whole rostrocaudal extent of the amygdala nuclei.

Author Manuscript

Considering all the parahippocampal regions analyzed, the septal nuclei (and particularly the MS), received their strongest input from LEA. These projections arose from all bands of the LEA and targeted especially the rostral portion of all septal nuclei. The projection to BST arose mainly from the lateral band and targeted mainly the septohippocampal nucleus, but also the bed nucleus of the stria terminalis. In the basal ganglia, the CP, ACB and SI showed dense fiber labeling (Figure 4D). The ACB showed increased density of fibers in the caudal portion, the SI showed higher density in the rostral portion and the CP showed equally distributed density across its rostrocaudal extent. Injections in the lateral and intermediate bands of LEA induced greater density than the medial band. Overall, the dorsal thalamus, ventrolateral thalamus, and hypothalamus showed weak to negligible density of fiber labeling.

Author Manuscript

Medial Entorhinal Area—The density of fibers in the subcortical nuclei observed after injections of anterograde tracer in MEA are the weakest of the parahippocampal regions. Fiber density levels ranged from not present to moderate. Still, differences among the subcortical structures are apparent. The olfactory nuclei, claustrum, amygdala, septal nuclei, and basal nuclei overall show more densely labeled fibers than the dorsal thalamus, ventrolateral thalamus, and hypothalamus. The highest density was observed after injections to the medial band of MEA. In the olfactory nuclei, OT and TT show the greatest density of fibers, whereas in the septal nuclei, the density of fibers is greatest in MS. In the basal ganglia, similarly to the other parahippocampal projections, CP and ACB show the most dense fiber labeling (Figure 5D).


Agster et al.

Page 9

Volume-Normalized Density of Fibers

Author Manuscript

The average density measure allows for comparing the amount of input to a particular subcortical structure across projecting regions. One can imagine, however, that when projections to a larger and a smaller structure result in the same average density of fiber labeling, that more fibers are labeled in the larger structures. This measure reflects the relative strength of projections from each parahippocampal region onto all subcortical structures. For that reason, we constructed a second measure in which the density of fiber labeling is normalized to the volume of the efferent structure (Table 4). In other words, the average density is multiplied by the volume of a structure. For composite structures, this number is then summed. This second measure allows for comparison of the size of the output projections emerging from the PER, POR, and EC.


For all parahippocampal structures, the largest projection targets the basal ganglia, and more specifically the CP. This effect is certainly due to the large volume of this region, however, the ACB and SI are also the targets of very strong projections from PER areas 35 and 36 and from LEA. Aside from the basal ganglia, area 35 has large projections to almost all olfactory nuclei, the claustrum and the BMA. For area 36, the strongest projections target OA, OT, the claustrum and DTHla, including the posterior complex and the suprageniculate and lateral posterior nuclei of the thalamus. The POR also projects strongly to DTHla, but in this case targeting exclusively the lateral posterior nucleus. The third strongest output from POR projects to the DTHan, mostly the lateral dorsal nucleus. For the LEA, after the basal ganglia projection, the strongest outputs targets several olfactory nuclei, the OA in the amygdala, and the claustrum. The output to other structures was weak except for a moderately strong projection to LS and MS in the septal nuclei. As described previously, the MEA shows overall very weak output projections to the subcortical regions. Aside from the strong output to the CP, the only other noteworthy projections include the OT, OA and, similarly to LEA, LS and MS. Dentate gyrus-projecting entorhinal bands of the entorhinal cortex For the LEA, there were five injections in the lateral band, two in the intermediate band, and one in the medial band. For the MEA, there were four injections in the lateral band, one in the intermediate band, and one in the medial band. The injections in the LEA and MEA medial bands included both deep and superficial layers. The intermediate MEA band injection largely involved deep layers (Figure 1). Although there were different numbers of injection sites in each band, taken together the cases represented input from both deep and superficial layers.

Author Manuscript

The lateral and intermediate bands of the LEA exhibited similar patterns and strengths of projections to subcortical structures. Injections in both these bands produced dense fiber labeling in olfactory structures, the caudate, amygdala structures, CP, ACB, and SI. Structures in the thalamus and hypothalamus were weakly labeled or not at all. The one injection in the medial band of the LEA resulted in much less fiber labeling. The strongest labeling was observed in the BLA and BMA and the MS (Tables 5 and 6).


Agster et al.

Page 10

Author Manuscript

For the MEA, overall, injections produced much less labeling in subcortical structures. Interestingly, the strongest labeling arose from the injection in the medial band. The fact that this injection site (654B) involved all layers does not account for the stronger resultant fiber labeling. This site produced much stronger labeling compared with a site in the lateral band that also involved all layers (652B). The pattern of labeling resulting from the MEA medial band was comparable to that of the LEA lateral band with the most labeling observed in olfactory structures, the claustrum, septal nuclei, and the basal ganglia (Tables 5 and 6). There was little or no fiber labeling in the thalamus.

Author Manuscript

Although there were similar overall patterns of labeling resulting from the LEA lateral/ intermediate band and the MEA medial band, there were some differences in the details. Labeling in the olfactory structures was comparable. Labeling in the claustrum was not as strong arising from the MEA medial band as that arising from sites in the LEA lateral and intermediate bands. The amygdala labeling was weaker overall and the pattern was different. Sites in the LEA lateral and intermediate bands caused strongest labeling in the BLA followed by the BMA and the OA. The MEA medial band produced the strongest labeling in the OA, followed by the BMA. Septal fiber labeling was similar in strength, but the patterns were different. LEA lateral and intermediate band sites produced moderate fiber labeling that was similar across all septal structures. In contrast, the MEA medial band produced strong labeling in MS, moderate labeling in the LST and BST and little to no labeling in the PS. Basal ganglia labeling was not as strong following the MEA medial band injection, but the pattern was similar to that of the LEA lateral band sites such that heavier labeling was observed in the ACB followed by the CP. Regarding the hypothalamus, the MEA intermediate site produced moderate labeling in the MBO.

Author Manuscript

Discussion The purpose of this study was to provide a comprehensive and semiquantitative analysis of the subcortical efferents of the PER, POR, and EC. Data were analyzed in two ways. A summary of the subcortical input from parahippocampal regions to the subcortical nuclei assessed by the density of labeled fibers is presented in Figure 7. A summary that reflects the overall size of the projections emerging from parahippocampal regions is presented in Figure 8.

Author Manuscript

The first method assessed the overall density of fiber labeling in the efferent structures to provide an average (Table 3 and Figure 7). This allows for comparing the amount of input to a particular subcortical structure across projecting regions. For example, if one examines the projections to the LA depicted in Figure 7, one can see that injections in PER area 36 resulted in denser labeling that injection sites in PER area 35, LEA, or POR. The second measure normalizes the density of fiber labeling by the volume of the efferent structure (Table 4 and Figure 8). This allows for comparison of the size of projections emerging from the origin structures. For example, in Figure 8, one can see that the strongest projection emerging from the MEA targets the CP, whereas smaller projections target the ACB and the olfactory OT.


Agster et al.

Page 11

Author Manuscript

Our findings reveal that PER areas 35 and 36, POR, LEA and MEA each display a unique complement of subcortical efferents. In general, the LEA provides the strongest and most robust input to subcortical structures, closely followed by areas 36 and 35. The POR provides more restricted input targeting mainly the thalamus, but also providing weak inputs to a few basal ganglia and amygdala structures. With the exception of the MEA site located in the medial band, projections originating in the MEA were very weak and limited.

Author Manuscript

The PER areas 35 and 36 show a pattern of subcortical projections that is generally similar across the two regions. The main targets of these projections are the olfactory group, the claustrum, the amygdala and the basal ganglia. The connection with the amygdala has been documented previously in different species (Herzog and Van Hoesen, 1976; Shi and Cassell, 1999; Witter and Groenewegen, 1986b). The present study identified primary projections to the LA, BLA and BMA. Together with the accompanying study (Tomás Pereira et al., submitted) this indicates strong reciprocal connections between these regions. The strong connectivity reinforces the role both the amygdala and the PER have in emotional processing (Janak and Tye, 2015; Kent and Brown, 2012; LeDoux, 1992). The PER connection to olfactory subcortical nuclei is consistent with the findings from the cortical connectivity (Agster and Burwell, 2009) and highlight the multisensory nature of stimulus processing that occurs in the PER (Albasser et al., 2011; Burwell, 2001).

Author Manuscript

The greatest distinction in subcortical projections between PER areas 35 and 36 are those that target the thalamic nuclei. Whereas area 35 shows overall week projections to all thalamic nuclei, area 36 projects strongly to a few specific nuclei. The pattern of projections from area 36 to the thalamus, instead appears similar to that of the POR. For example, both structures very strongly project to the DTHla. However, closer examination reveals that the projections from rostral PER area 36 target the posterior complex (mid and caudal injections target both the suprageniculate and the lateral posterior nucleus), whereas the POR projections exclusively and strongly targets the lateral posterior nucleus. It should be noted that the dense projections from the PER to the amygdala, as well as the thalamic nuclei, including the posterior thalamic nuclei projection from the rostral regions have been previously observed (McIntyre et al., 1996; Witter and Groenewegen, 1986a; Witter and Groenewegen, 1986b). However, the present study allows for comparisons across different parahippocampal regions, like the one highlighted above. PER area 36 also projects to the VLTH, including the subparafascicular nucleus which is involved in sexual behavior (Veening and Coolen, 1998), and to MG, which has been implicated in auditory fear conditioning (LeDoux et al., 1984). This pattern of connectivity emphasizes the role of PER area 36, in emotional processing, particularly when it relates to learning and memory paradigms.

Author Manuscript

The POR subcortical projections are much weaker than the ones originating in PER. However, a very strong projection was found, as was described above, to the lateral posterior nucleus of the thalamus. Interestingly, this structure is associated with visual attention (Petersen et al., 1987; Robinson et al., 1991) and it projects strongly to the posterior parietal cortex (Chandler et al., 1992). The POR is also strongly interconnected with the posterior parietal cortex (Agster and Burwell, 2009; Burwell and Amaral, 1998a), an area implicated in spatial attention (Corbetta et al., 1993; Posner et al., 1984; Reep and Corwin, 2009). Also


Agster et al.

Page 12

Author Manuscript

in the dorsal thalamus a moderate projection was found in the nucleus reuniens, a structure increasingly implicated in studies of spatial navigation and as a relay between prefrontal cortex and the hippocampus (Ito et al., 2015; Jankowski et al., 2014). In the ventrolateral thalamus, moderately dense fibers were found in the LG, a major target of visual input (Hughes and Chi, 1981). This pattern of connectivity, coupled with the very low levels of POR output to olfactory subcortical regions, complement what was previously known in regards to the cortical connectivity of this region. Studies examining the cortical afferents of the parahippocampal region revealed that the individual cortices differed greatly in the type of sensory input each received (Burwell and Amaral, 1998), with the POR being overwhelmingly skewed toward visuo-spatial information. In contrast, PER receives input more evenly distributed between olfactory, auditory, visual, and visuospatial cortices.

Author Manuscript

The subcortical projections arising in the LEA and MEA were different in both strength and targets. Subcortical projections of the MEA were limited to the basal ganglia and olfactory structures. The MEA medial band, however, does provide stronger inputs to the amygdala and medial septal nuclei. The LEA, on the other hand, projects strongly to the olfactory regions, claustrum, amygdala, and septal nuclei. The LEA projections overlap with the projections from PER areas 35 and 36, but also include other projections. For example, the LEA projects more extensively to olfactory and amygdala structures. Previous characterization of the interconnections of the parahippocampal regions has described a pattern of connectivity compatible with these results.

Author Manuscript

For all parahippocampal regions, the CP and ACB of the basal ganglia were major efferent targets. In fact, when taking the volume of the target regions into consideration, the projection to CP was the largest for all regions. Given the variety of cognitive functions these regions contribute to, it is not surprising that these regions are heavily interconnected with the medial temporal lobe. While some of these projections have been described before (Krayniak et al., 1981; McIntyre et al., 1996; Witter and Groenewegen, 1986b), it is interesting to compare across parahippocampal regions. Specifically, the PER and LEA showed stronger output to basal ganglia than POR and MEA. However, for the rest of the basal ganglia, there is some segregation of parahippocampal input. For example, SI receives much stronger input from LEA than the other regions and it will be interesting to further investigate the significance of this anatomical connection.

Author Manuscript

One last subcortical structure was strongly targeted by all parahippocampal structures (and more so by the PER and LEA): the claustrum. This strong connectivity is unsurprising given the vast cortical connections of the claustrum itself. Because its function is still unknown, with suggestions ranging from the simple facilitation of cortical function (Baizer et al., 2014), to the basis of consciousness (Crick and Koch, 2005), it is difficult to speculate as to the role this anatomical connection might have. The data from this study taken together with the companion study allows an assessment of the reciprocity of the subcortical inputs and outputs of PER areas 35 and 36, POR, LEA, and MEA. The claustrum projects to all five regions, with the strongest projection targeting the LEA and the weakest targeting POR and MEA. Only the inputs from areas 35 and 36 and the LEA are reciprocated. Regarding olfactory structures, the strongest inputs are to the LEA


Agster et al.

Page 13

Author Manuscript

and the MEA medial band, arising largely from the PTA and the EP. Interestingly, the LEA and PER area 35 project back to all olfactory structures, although the projections to the PTA are the strongest. All structures in the amygdala project to PER areas 35 and 36, LEA, and MEA. The strongest projection is from LA to area 36. These projections are reciprocal. In addition, the POR provides noteworthy projections to the LA and the CEA. All parahippocampal regions project to the CP and the ACB, although MEA projections arise only in the medial band. The heaviest projections arise in PER areas 35 and 36. The projections to structures in the basal ganglia are not reciprocated. The medial septal nucleus projects to both the LEA and the MEA. This projection is reciprocal, though the MEA projection arises only in the medial band. Structures in the midline thalamic nuclei project to all five target regions, but these are largely not reciprocated. There is a robust reciprocal connection between the POR and the lateral posterior nucleus of the thalamus.

Author Manuscript

We have found that the subcortical targets of the PER areas 35 and 36, POR, LEA, and MEA differ substantially. The LEA provides the strongest and most widespread input to subcortical structures, particularly olfactory, amygdala, and basal ganglia structures. PER areas 35 and 36 also provided strong, widespread input to most of the structures targeted by the LEA. In contrast, the POR and MEA provided more limited and weaker inputs to subcortical structures. The primary target of the POR is the lateral posterior nucleus of the thalamus. The overall primary target of the MEA are basal ganglia structures, although the MEA medial band appears to also target the amygdala and medial septal nucleus. Given that the PER regions show stronger connectivity with the LEA and the POR shows stronger connectivity with MEA (Burwell and Amaral, 1998b), our findings suggest that the PER/LEA complex may exert substantially more influence over subcortical functions than the POR/MEA.

Author Manuscript

Acknowledgments Grant sponsor: NSF; Grant number: IBN9875792 to RDB. Grant sponsor: NIMH; Grant number: MH072144 to KLA. Grant sponsor: NSF; Grant number: IOB0522220 to RDB. The authors thank Sanford Brown for his extensive technical support.

References

Author Manuscript

Agster KL, Burwell RD. Cortical efferents of the perirhinal, postrhinal, and entorhinal cortices of the rat. Hippocampus. 2009; 19(12):1159–1186. [PubMed: 19360714] Agster KL, Burwell RD. Hippocampal and subicular efferents and afferents of the perirhinal, postrhinal, and entorhinal cortices of the rat. Behavioral Brain Research. 2013; 254:50–64. Albasser MM, Amin E, Iordanova MD, Brown MW, Pearce JM, Aggleton JP. Separate but interacting recognition memory systems for different senses: the role of the rat perirhinal cortex. Learn Mem. 2011; 18(7):435–43. [PubMed: 21685150] Baizer JS, Sherwood CC, Noonan M, Hof PR. Comparative organization of the claustrum: what does structure tell us about function? Front Syst Neurosci. 2014; 8:117. [PubMed: 25071474] Blackstad TW. Commissural connections of the hippocampal region in the rat, with special reference to their mode of termination. J Comp Neurol. 1956; 105:417–537. [PubMed: 13385382]


Agster et al.

Page 14

Author Manuscript Author Manuscript Author Manuscript Author Manuscript

Brodmann, K. Vergleichende lokalisationslehre der grosshirnrinde in ihren prinzipien dargestellt auf grund des zellenbaues. Liepzig, Barth: 1909. Brun VH, Leutgeb S, Wu HQ, Schwarcz R, Witter MP, Moser EI, Moser MB. Impaired spatial representation in CA1 after lesion of direct input from entorhinal cortex. Neuron. 2008; 57(2):290– 302. [PubMed: 18215625] Burwell RD. Borders and cytoarchitecture of the perirhinal and postrhinal cortices in the rat. The Journal of Comparative Neurology. 2001; 437(1):17–41. [PubMed: 11477594] Burwell RD, Amaral DG. Cortical afferents of the perirhinal, postrhinal, and entorhinal cortices of the rat. The Journal of Comparative Neurology. 1998a; 398(2):179–205. [PubMed: 9700566] Burwell RD, Amaral DG. Perirhinal and postrhinal cortices of the rat: interconnectivity and connections with the entorhinal cortex. The Journal of Comparative Neurology. 1998b; 391(3): 293–321. [PubMed: 9492202] Burwell RD, Hafeman DM. Positional firing properties of postrhinal cortex neurons. Neuroscience. 2003; 119(2):577–588. [PubMed: 12770570] Bussey TJ, Saksida LM, Murray EA. Impairments in visual discrimination after perirhinal cortex lesions: testing ‘declarative’ vs. ‘perceptual-mnemonic’ views of perirhinal cortex function. The European Journal of Neuroscience. 2003; 17(3):649–660. [PubMed: 12581183] Chandler HC, King V, Corwin JV, Reep RL. Thalamocortical connections of rat posterior parietal cortex. Neuroscience Letters. 1992; 143(1–2):237–242. [PubMed: 1436673] Corbetta M, Miezin FM, Shulman GL, Petersen SE. A PET study of visuospatial attention. J Neurosci. 1993; 13(3):1202–26. [PubMed: 8441008] Crick FC, Koch C. What is the function of the claustrum? Philos Trans R Soc Lond B Biol Sci. 2005; 360(1458):1271–9. [PubMed: 16147522] Eacott MJ, Gaffan D, Murray EA. Preserved recognition memory for small sets, and impaired stimulus identification for large sets, following rhinal cortex ablations in monkeys. Eur J Neurosci. 1994; 6(9):1466–78. [PubMed: 8000570] Eichenbaum H, Lipton PA. Towards a functional organization of the medial temporal lobe memory system: role of the parahippocampal and medial entorhinal cortical areas. Hippocampus. 2008; 18(12):1314–24. [PubMed: 19021265] Furtak SC, Ahmed OJ, Burwell RD. Single neuron activity and theta modulation in postrhinal cortex during performance on a visual discrimination task. Neuron. 2012; 76(5):976–988. [PubMed: 23217745] Furtak SC, Wei SM, Agster KL, Burwell RD. Functional neuroanatomy of the parahippocampal region in the rat: the perirhinal and postrhinal cortices. Hippocampus. 2007; 17(9):709–22. [PubMed: 17604355] Gerfen CR, Sawchenko PE. An anterograde neuroanatomical tracing method that shows the detailed morphology of neurons, their axons and terminals: immunohistochemical localization of an axonally transported plant lectin, Phaseolus vulgaris-leucoagglutinin (PHA-L). Brain Research. 1984; 290(2):219–238. [PubMed: 6198041] Herzog AG, Van Hoesen GW. Temporal neocortical afferent connections to the amygdala in the rhesus monkey. Brain Res. 1976; 115(1):57–69. [PubMed: 824015] Hughes CP, Chi DY. Afferent projections to the ventral lateral geniculate nucleus in the cat. Brain Res. 1981; 207(2):445–8. [PubMed: 7470913] Insausti R, Herrero MT, Witter MP. Entorhinal cortex of the rat: cytoarchitectonic subdivisions and the origin and distribution of cortical efferents. Hippocampus. 1997; 7(2):146–183. [PubMed: 9136047] Ito HT, Zhang SJ, Witter MP, Moser EI, Moser MB. A prefrontal-thalamo-hippocampal circuit for goal-directed spatial navigation. Nature. 2015 Janak PH, Tye KM. From circuits to behaviour in the amygdala. Nature. 2015; 517(7534):284–92. [PubMed: 25592533] Jankowski MM, Islam MN, Wright NF, Vann SD, Erichsen JT, Aggleton JP, O’Mara SM. Nucleus reuniens of the thalamus contains head direction cells. Elife. 2014:3. Jarrard LE, Davidson TL, Bowring B. Functional differentiation within the medial temporal lobe in the rat. Hippocampus. 2004; 14(4):434–449. [PubMed: 15224981] Hippocampus. Author manuscript; available in PMC 2016 October 19.

Agster et al.

Page 15

Author Manuscript Author Manuscript Author Manuscript Author Manuscript

Kealy J, Commins S. The rat perirhinal cortex: A review of anatomy, physiology, plasticity, and function. Prog Neurobiol. 2011; 93(4):522–48. [PubMed: 21420466] Kemppainen S, Jolkkonen E, Pitkanen A. Projections from the posterior cortical nucleus of the amygdala to the hippocampal formation and parahippocampal region in rat. Hippocampus. 2002; 12(6):735–55. [PubMed: 12542226] Kent BA, Brown TH. Dual functions of perirhinal cortex in fear conditioning. Hippocampus. 2012; 22(10):2068–79. [PubMed: 22903623] Kerr KM, Agster KL, Furtak SC, Burwell RD. Functional neuroanatomy of the parahippocampal region: the lateral and medial entorhinal areas. Hippocampus. 2007; 17(9):697–708. [PubMed: 17607757] Krayniak PF, Meibach RC, Siegel A. A projection from the entorhinal cortex to the nucleus accumbens in the rat. Brain Res. 1981; 209(2):427–31. [PubMed: 7225802] Lavenex P, Suzuki WA, Amaral DG. Perirhinal and parahippocampal cortices of the macaque monkey: Intrinsic projections and interconnections. The Journal of Comparative Neurology. 2004; 472(3): 371–394. [PubMed: 15065131] LeDoux JE. Brain mechanisms of emotion and emotional learning. Current Opinion in Neurobiology. 1992; 2:191–197. [PubMed: 1638153] LeDoux JE, Sakaguchi A, Reis DJ. Subcortical efferent projections of the medial geniculate nucleus mediate emotional responses conditioned to acoustic stimuli. J Neurosci. 1984; 4(3):683–98. [PubMed: 6707732] Lewis DA, Campbell MJ, Morrison JH. An immunohistochemical characterization of somatostatin-28 and somatostatin-28-1-12 in monkey prefrontal cortex. The Journal of Comparative Neurology. 1986; 248(1):1–18. [PubMed: 2873154] Majak K, Pitkanen A. Projections from the periamygdaloid cortex to the amygdaloid complex, the hippocampal formation, and the parahippocampal region: a PHA-L study in the rat. Hippocampus. 2003; 13(8):922–942. [PubMed: 14750655] McGaughy J, Koene RA, Eichenbaum H, Hasselmo ME. Cholinergic deafferentation of the entorhinal cortex in rats impairs encoding of novel but not familiar stimuli in a delayed nonmatch-to-sample task. The Journal of Neuroscience. 2005; 25(44):10273–10281. [PubMed: 16267235] McIntyre DC, Kelly ME, Staines WA. Efferent projections of the anterior perirhinal cortex in the rat. The Journal of Comparative Neurology. 1996; 369(2):302–318. [PubMed: 8727002] Meunier M, Bachevalier J, Mishkin M, Murray EA. Effects on visual recognition of combined and separate ablations of the entorhinal and perirhinal cortex in rhesus monkeys. The Journal of Neuroscience. 1993; 13(12):5418–5432. [PubMed: 8254384] Murray EA, Bussey TJ, Saksida LM. Visual perception and memory: a new view of medial temporal lobe function in primates and rodents. Annu Rev Neurosci. 2007; 30:99–122. [PubMed: 17417938] Norman G, Eacott MJ. Dissociable effects of lesions to the perirhinal cortex and the postrhinal cortex on memory for context and objects in rats. Behavioral Neuroscience. 2005; 119(2):557–566. [PubMed: 15839802] Paxinos, G.; Watson, C. The Rat Brain in Stereotaxic Coordinates. San Diego: Academic Press; 1998. Petersen SE, Robinson DL, Morris JD. The contribution of the pulvinar to visual spatial attention. Neuropsychologia. 1987; 25(1):97–105. [PubMed: 3574654] Pikkarainen M, Pitkänen A. Projections from the lateral, basal and accessory basal nuclei of the amygdala to the perirhinal and postrhinal cortices in rat. Cerebral Cortex. 2001; 11(11):1064– 1082. [PubMed: 11590116] Pitkänen A, Pikkarainen M, Nurminen N, Ylinen A. Reciprocal connections between the amygdala and the hippocampal formation, perirhinal cortex, and postrhinal cortex in rat. A review. Annals of the New York Academy of Sciences. 2000; 911:369–391. [PubMed: 10911886] Posner MI, Walker JA, Friedrich FJ, Rafal RD. Effects of parietal injury on covert orienting of attention. The Journal of Neuroscience. 1984; 4(7):1863–1874. [PubMed: 6737043] Reep RL, Corwin JV. Posterior parietal cortex as part of a neural network for directed attention in rats. Neurobiol Learn Mem. 2009; 91(2):104–13. [PubMed: 18824116]


Agster et al.

Page 16


Robinson DL, McClurkin JW, Kertzman C, Petersen SE. Visual responses of pulvinar and collicular neurons during eye movements of awake, trained macaques. Journal of Neurophysiology. 1991; 66(2):485–496. [PubMed: 1774583] Shi CJ, Cassell MD. Cortical, thalamic, and amygdaloid projections of rat temporal cortex. The Journal of Comparative Neurology. 1997; 382(2):153–175. [PubMed: 9183686] Shi CJ, Cassell MD. Perirhinal cortex projections to the amygdaloid complex and hippocampal formation in the rat. The Journal of Comparative Neurology. 1999; 406(3):299–328. [PubMed: 10102498] Squire LR, Stark CE, Clark RE. The medial temporal lobe. Annu Rev Neurosci. 2004; 27:279–306. [PubMed: 15217334] Steffenach HA, Witter M, Moser MB, Moser EI. Spatial memory in the rat requires the dorsolateral band of the entorhinal cortex. Neuron. 2005; 45(2):301–313. [PubMed: 15664181] Stensola H, Stensola T, Solstad T, Froland K, Moser MB, Moser EI. The entorhinal grid map is discretized. Nature. 2012; 492(7427):72–8. [PubMed: 23222610] Swanson, LW. Brain Maps: Structure of the Rat Brain. Amsterdam: Elsevier; 1992. Swanson, LW. Brain Maps: Structure of the Rat Brain. Amsterdam: Elsevier; 1998. Tomás Pereira I, Agster KL, Burwell RD. Subcortical connections of the perirhinal, postrhinal, and entorhinal cortices of the rat. I. Afferents. submitted. Tu HW, Hampton RR, Murray EA. Perirhinal cortex removal dissociates two memory systems in matching-to-sample performance in rhesus monkeys. J Neurosci. 2011; 31(45):16336–43. [PubMed: 22072685] Veening JG, Coolen LM. Neural activation following sexual behavior in the male and female rat brain. Behav Brain Res. 1998; 92(2):181–93. [PubMed: 9638960] Witter MP, Groenewegen HJ. Connections of the parahippocampal cortex in the cat. III. Cortical and thalamic efferents. The Journal of Comparative Neurology. 1986a; 252(1):1–31. [PubMed: 3793972] Witter MP, Groenewegen HJ. Connections of the parahippocampal cortex in the cat. IV. Subcortical efferents. The Journal of Comparative Neurology. 1986b; 252(1):51–77. [PubMed: 3793975] Witter MP, Groenewegen HJ, Lopes da Silva FH, Lohman AH. Functional organization of the extrinsic and intrinsic circuitry of the parahippocampal region. Progress in Neurobiology. 1989; 33(3):161– 253. [PubMed: 2682783]

Author Manuscript Hippocampus. Author manuscript; available in PMC 2016 October 19.

Agster et al.

Page 17

Author Manuscript Author Manuscript Figure 1.

Author Manuscript

Location of injection sites within the parahippocampal regions. Injection sites are represented on an unfolded map of the parahippocampal region. Dark grey shading represents injection sites to deep layers of cortex, medium grey represents tracer injections to deep and superficial layers of cortex, and light grey shading represent tracer injections to superficial layers of cortex. Solid lines separate the parahippocampal regions (PER area 35, PER area 36, POR, LEA and MEA), the wide-dash line represents the rhinal sulcus, and the small dashed lines separate projecting bands from the EC to the HPC into lateral, intermediate and medial.

Author Manuscript Hippocampus. Author manuscript; available in PMC 2016 October 19.

Agster et al.

Page 18


Figure 2.

Author Manuscript

Darkfield photomicrographs showing the distribution of fiber labeling in the PTA (one of the olfactory subcortical nuclei) following anterograde tracer injections in parahippocampal regions. A. Schematic showing the approximate level of the photomicrographs. B. Fiber labeling following anterograde tracer injection to PER area 35 (Case 24P). Very dense fiber labeling was observed. C. Fiber labeling as a result of tracer injection to PER area 36 (Case 62B). Dense fibers were observed but less than in area 35 or LEA. D. Fiber labeling as a result of tracer injection to LEA (Case 57P). Very dense fibers were observed in PTA. Other dense labeling is observed at the injection site in LEA. For abbreviations, see Table 1. Scale bar: 250μm.


Agster et al.

Page 19


Figure 3.

Author Manuscript

Darkfield photomicrographs showing the distribution of fiber labeling in the amygdala nuclei. A. Schematic illustrating the approximate level of the photomicrographs. B. Fiber labeling following anterograde tracer injection to PER area 35 (Case 24P). Moderate fiber labeling is observed in the LA, BLA, and BMA. Within BLA, the posterior region shows stronger labeling than the anterior region. C. Fiber labeling as a result of anterograde tracer injection to PER area 36 (Case 128B). Very dense fiber labeling is observed in the LA. Moderate fiber labeling is observed in the BLA. D. Fiber labeling as a result of anterograde tracer injection to the LEA (Case 57P). Strong fiber labeling is observed in the BLA, particularly the posterior region. LA and BMA shown moderate fiber labeling. For abbreviations, see Table 1. Other abbreviations: BLAa – basolateral nucleus of the amygdala (anterior region); BLAp – basolateral nucleus of the amygdala (posterior region). Scale bar: 250 μm.


Agster et al.

Page 20


Figure 4.

Author Manuscript

Darkfield photomicrograph showing the distribution of fiber labeling in a selection of basal ganglia structures following anterograde trace injections into PER and LEA. A. Schematic showing the approximate level of the photomicrographs. B. Fiber labeling as a result of anterograde tracer injection to PER area 35 (Case 24P). Dense fibers are observed in ACB with moderate density in CP. C. Fiber labeling following anterograde tracer injection to PER area 36 (Case 54P). Dense fiber labeling is observed in both CP and ACB. D. Fiber labeling as a result of anterograde tracer injection to the LEA (Case 127B). Dense fibers observed in ACB, CP and moderate fiber density in SI. For abbreviations, see Table 1. Scale bar: 250μm.


Agster et al.

Page 21


Figure 5.

Author Manuscript

Darkfield photomicrograph showing the distribution of fiber labeling in the CP following tracer injections to the POR and MEA. A. Schematic showing the approximate level of the photomicrographs in C and D. B. Fiber labeling in CP at the anterior level as a result of anterograde tracer injection the POR (Case 39P). Weak fiber labeling is observed clustered particularly in the medial region. C. Fiber labeling in posterior CP in the same case as B (Case 39P). Very dense labeling is observed in clusters along the lateral wall of the nucleus. D. Fiber labeling observed in the CP following anterograde tracer injection to the MEA (Case 28P). Few fibers are observed. For abbreviations, see Table 1. Scale bar: 250μm.


Agster et al.

Page 22


Figure 6.

Author Manuscript

Darkfield photomicrographs showing examples of fiber labeling in some nuclei of the dorsal and ventrolateral thalamus. A. Schematic showing the approximate level of the photomicrographs in B and D. B. Fiber labeling as a result of anterograde tracer injection to PER area 36 (Case 54B). Very dense labeling is observed in the posterior complex of the DTHla, but not in the lateral posterior nucleus of DTHla. C. Fiber labeling as a result of anterograde tracer injection to PER area 36 (Case 45P). Labeling is moderate considering the MG as a whole, but dense fibers are present in the dorsal region. D. Fiber labeling as a result of anterograde tracer injection to the POR (Case 83B). Fibers terminate strongly in the lateral posterior nucleus of the DTHla, but weakly in the posterior complex. Some fiber labeling is also observed in the lateral geniculate (dorsal region). For abbreviations, see Table 1. Other abbreviations: LGd – lateral geniculate (dorsal region); LGv – lateral geniculate (ventral region); LP – lateral posterior nucleus of the thalamus (part of DTHla); MGd – medial geniculate (dorsal region); MGv – medial geniculate (ventral region); PO – posterior complex of the thalamus (part of DTHla); VPL – ventral posterior complex of the


Agster et al.

Page 23

Author Manuscript

dorsal thalamus (lateral region; part of DTHve); VPM – ventral posterior complex of the dorsal thalamus (medial region; part of DTHve). Scale bar: 250μm.

Author Manuscript Author Manuscript Author Manuscript Hippocampus. Author manuscript; available in PMC 2016 October 19.

Agster et al.

Page 24


Figure 7.

Wiring diagram representing subcortical output of the parahippocampal regions based on the densities of labeled cells in each subcortical structure (Table 3). The density measure allows for comparisons of the strength of the parahippocampal input to a specific subcortical nucleus. Accordingly, the projections are color coded according to their region of origin. The thickness of the lines indicates the relative strength of the projection. Information for connections shown in black are from prior studies (Agster and Burwell, 2013; Burwell and Amaral, 1998b). For simplicity, the weakest connections (3.0. Note that only one injection was analyzed for the LEA medial band and the MEA lateral and medial bands.

+++++

Lateral

Olfactory

Efferent Regions

Author Manuscript

Density of Fiber Labeling from injections in Entorhinal Cortex

Author Manuscript

Table 5 Agster et al. Page 33


Author Manuscript

Author Manuscript 0.72 0.17 0.51

D. Thalamus

V.L. Thalamus

Hypothalamus

0.95

0.06

0.38

32.38

6.02

11.10

3.61

19.63

Intermediate

LEA

0.55

0.00

1.00

1.51

1.91

2.71

0.59

1.66

Medial

0.17

0.03

0.30

12.17

1.71

1.00

0.64

1.74

Lateral

1.70

0.00

0.00

6.50

1.87

0.49

0.21

2.33

Intermediate

MEA

2.00

0.13

0.67

26.14

5.47

9.43

2.27

14.53

Medial

Volume-normalized density ± standard error of the mean density of fibers following injections of anterograde tracers in the parahippocampal regions. Values for each animal were normalized by a standardized subcortical volume for each region. Note that only one injection was analyzed for the LEA medial band and the MEA lateral and medial bands.

5.00 38.68

Basal Ganglia

Amygdala

Septal Nuclei

3.60 11.41

Claustrum

16.83

Lateral

Olfactory

Efferent Regions

Author Manuscript

Volume-normalized Density of Fibers from injections in Entorhinal Cortex

Author Manuscript

Table 6 Agster et al. Page 34


Subcortical connections of the perirhinal, postrhinal, and entorhinal cortices of the rat. I. afferents.

Some connections of the entorhinal (area 28) and perirhinal (area 35) cortices of the rhesus monkey. II. Frontal lobe afferents.

Some connections of the entorhinal (area 28) and perirhinal (area 35) cortices of the rhesus monkey. III. Efferent connections.

Some connections of the entorhinal (area 28) and perirhinal (area 35) cortices of the rhesus monkey. I. Temporal lobe afferents.

Complementary Functional Organization of Neuronal Activity Patterns in the Perirhinal, Lateral Entorhinal, and Medial Entorhinal Cortices.

Topographic organization of the basal forebrain projections to the perirhinal, postrhinal, and entorhinal cortex in rats.

Commissural connections between the auditory cortices of the rat.

Connections of the magnocellular medial preoptic nucleus (MPN mag) in male Syrian hamsters. II. The efferents.

Cortical and subcortical connections of the pars compacta of the anterior pretectal nucleus in the rat.

Complementary roles of the orbital prefrontal cortex and the perirhinal-entorhinal cortices in an odor-guided delayed-nonmatching-to-sample task.

Cross connections among crayfish claw efferents.

Tracing of axonal connections by rhodamine-dextran-amine in the rat hippocampal-entorhinal cortex slice preparation.

Subcortical connections of area V4 in the macaque.

Distinct Spatiotemporal Activation Patterns of the Perirhinal-Entorhinal Network in Response to Cortical and Amygdala Input.

Physiological studies of the reciprocal connections between the hippocampus and entorhinal cortex.

Subcortical connections of visual areas MST and FST in macaques.

An experimental electron microscopic study of afferent connections to the primate motor and somatic sensory cortices.

Autoradiographic tracing of nucleus accumbens efferents in the rat.

The connections of presubiculum and parasubiculum in the rat.

Epileptiform synchronization and high-frequency oscillations in brain slices comprising piriform and entorhinal cortices.

Quantitative studies on the supraoptic nucleus in the rat. II. Afferent fiber connections.

The perirhinal cortex.

Claustrum in the hedgehog (Erinaceus europaeus) brain: cytoarchitecture and connections with cortical and subcortical structures.

Functional reciprocal connections of the rat entorhinal cortex and subicular complex with the medial frontal cortex: an in vivo intracellular study.