Entorhinal cortex of the rat: Topographic organization of the cells of origin of the perforant path projection to the dentate gyrus

Entorhinal Cortex of the Rat:Topographic Organization of the Cells

of Origin of the Perforant Path Projectionto the Dentate Gyrus

CYNTHIA L. DOLORFO1,3 AND DAVID G. AMARAL2*1Group in Neuroscience, University of California at San Diego, La Jolla, California 92193

2Department of Psychiatry, Center for Neuroscience and California Regional PrimateResearch Center, University of California, Davis, Davis, California 95616

3Institute of Experimental Medicine, Hungarian Academy of Science,Department of Functional Anatomy, H-1450 Budapest, Hungary

ABSTRACTBy using three-dimensional computer reconstruction techniques and the production of

two-dimensional unfolded maps, we analyzed the topographic organization of projections fromthe entorhinal cortex of the rat to the dentate gyrus. The retrograde tracers, Fast blue andDiamidino yellow, were injected at all septotemporal levels of the dentate gyrus, and thedistribution of retrogradely labeled layer II cells in the entorhinal cortex was plotted by usingcomputer-aided microscopy systems. Discrete injections of fluorescent dyes into the dentategyrus labeled bands of layer II neurons in the entorhinal cortex that covered approximately45% of its surface area. Injections confined to the septal half of the dentate gyrus resulted in aband that occupied the most lateral and caudomedial portions of the entorhinal cortex.Although there were subtle changes in the density of labeled cells in this region, essentiallythe same region of cells was labeled after any injection into the septal half of the dentategyrus. Injections into mid-septotemporal levels of the dentate gyrus (50–75% of the distancefrom the septal pole) led to a distinctly different pattern of retrograde labeling. A more medialportion of the lateral entorhinal cortex and a more rostral portion of the medial entorhinalarea were labeled in these cases. Another change in entorhinal labeling occurred when theinjection involved the most temporal quarter of the dentate gyrus. Injections into this area ledto a constrained region of entorhinal labeling that included the most medial portion of thelateral entorhinal area and the most rostral portion of the medial entorhinal area. Althoughthe domains of cells projecting to septal, mid-septotemporal, and temporal levels of thedentate gyrus were not entirely segregated, there was relatively little overlap of the threepopulations of neurons. These data raise the possibility that different portions of theentorhinal-hippocampal circuit are capable of semiautonomous information processing, atleast at the stage of input to the dentate gyrus. J. Comp. Neurol. 398:25–48, 1998.r 1998 Wiley-Liss, Inc.

Indexing terms: hippocampus; Fast blue; diamidino yellow; parvalbumin; three-dimensional

reconstruction

The entorhinal cortex is a prominent component of thehippocampal formation. Much of the cortical informationthat enters the hippocampal formation enters through theentorhinal cortex, and much of the return projection to theneocortex arises from the entorhinal cortex. It has beenknown for more than a century that the superficial layersof the entorhinal cortex give rise to a dense projection tothe dentate gyrus and hippocampus (Ramon y Cajal,1901). It was not until the 1970s, however, that the preciselaminar origins and general features of the topographical

organization of this projection were experimentally demon-strated. By studying the anterograde degeneration of

Grant sponsor: NIH; Grant numbers: NS 16980 and NS 16980-S1; Grantsponsor: Human Frontiers Science Program.

*Correspondence to: David G. Amaral, Ph.D., Center for Neuroscience,University of California, Davis, 1544 Newton Court, Davis, CA 95616.E-mail: [email protected]

Received 6 December 1996; Revised 25 March 1998; Accepted 30 March1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 398:25–48 (1998)

r 1998 WILEY-LISS, INC.

lesioned entorhinal projection axons, Hjorth-Simonsenand Jeune (1972) verified that the entorhinal cortex pro-jected to the dentate gyrus, hippocampus, and subiculum.Steward and Scoville (1976) established with retrogradetransport of horseradish peroxidase (HRP) that the projec-tions to the dentate gyrus arose from neurons in layer II ofthe entorhinal cortex. Both studies concluded that projec-tions originating laterally in the entorhinal cortex termi-nated in septal levels of the dentate gyrus, whereasprojections originating medially in the entorhinal cortexterminated temporally in the dentate gyrus. This lateral-to-medial topography of entorhinal-to-dentate projectionsshown in the rat (Hjorth-Simonsen, 1972; Wyss, 1981) wasalso observed in the cat (Witter and Groenewegen, 1984)and in the monkey (Witter et al., 1989). The studies ofRuth et al. (1982, 1988) were the first to study systemati-cally the topographic organization of the entorhinal-to-dentate gyrus projections. By using the HRP retrogradetracing technique, they demonstrated that largely nonover-lapping strips of layer II entorhinal neurons project todifferent septotemporal levels of the dentate gyrus. Theinjections of retrograde tracer, however, were widely spacedalong the septotemporal axis, and Ruth et al. indicatedthat a more gradientlike pattern of organization may havebeen observed had the injection sites been more closelyspaced. Although these studies provided important infor-mation on the organization of entorhinal projections to thedentate gyrus, several questions remained regarding thethree-dimensional organization of populations of entorhi-nal neurons that project to the dentate gyrus. It remainedunclear, for example, how spatially segregated are thepopulations of neurons that project to septal and temporalportions of the dentate gyrus. Nor, despite years of study,are the locations of neurons from the lateral and medialentorhinal cortices that project to a single level of thedentate gyrus well understood.

Such questions prompted us to carry out a comprehen-sive reanalysis of the three-dimensional organization ofthe origins of the perforant path projections to the dentategyrus. We have capitalized on the use of new visualizationand analysis techniques that are available for the analysisof neural connections. For these studies, we have combinedthe sensitive retrograde tracers Fast blue and Diamidinoyellow with computer-aided three-dimensional reconstruc-tion and two-dimensional unfolded mapping techniques.These techniques allowed us to determine precisely thethree-dimensional location of entorhinal neurons project-

ing to different septotemporal levels of the dentate gyrus.These data also provided helpful indications as to theprecise locations of the lateral and medial borders of theentorhinal cortex, an issue that has remained controver-sial.

MATERIALS AND METHODS

Surgery

Forty-two adult male Sprague-Dawley rats (280–350 g;Harlan Sprague-Dawley, Inc., Houston, TX) were used inthese experiments. All procedures were approved by theAnimal Use and Care Committee of the Salk Institute forBiological Studies and were performed in accordance withguidelines outlined by the U.S. Public Health ServicePolicy on Humane Care and Use of Laboratory Animals(Office for Protection from Research Risks, 1986). Allinjections were done in a single survival surgery. Animalswere first anesthetized with an anesthetic cocktail ofketamine (100 mg/ml), acepromazine (10 mg/ml), andrompun (20 mg/ml) at a surgical dose of 0.5 ml/100 g (i.p.).The anesthetic was supplemented when needed withNembutal (50 mg/ml) at a dose of 0.05 ml/100 g (i.p.).Atropine sulfate (0.1 ml/100 g, i.p.) was administered priorto the Nembutal injections. The animal was placed in aKopf stereotaxic instrument, and a midline incision wasmade. The location of bregma was determined, and twosmall (approximately 1 mm in diameter) burr holes weredrilled at locations depending on the placement of the twotracer injections. Injections were aimed at various septo-temporal levels of the dentate gyrus by using the atlas ofPaxinos and Watson (1986). After injection of tracers, thewound was sutured, and the animal monitored for recov-ery from anesthesia and returned to its home cage.

Retrograde tracer injections. The retrograde fluores-cent dyes Fast blue (FB; Bentivoglio et al., 1980) andDiamidino yellow (DY; Keizer et al., 1983) were depositedinto two different septotemporal locations of each animal.A glass micropipette (60–120 µm outer diameter) contain-ing either FB or DY (Dr. Illing GmbH & Co., Germany) waslowered into the dentate gyrus. To avoid contamination ofadjacent hippocampal fields, the dye was deposited in themolecular layer of the infrapyramidal blade of the dentategyrus. After the pipette remained in place for 5 minutes,an interval timed air pressure-injection system (Amaraland Price, 1983) was used to inject 50–70 nl of 3% FB or150–250 nl of 2% DY. Before slowly removing the pipettefrom the brain, it was first raised 100 µm above theinjection site, where it remained for 5–10 minutes. Thiswas done to minimize diffusion of dye along the pipettetract. The second dye was then injected in the same wayinto the ipsilateral dentate gyrus at a second location.

Tissue processing

After a postinjection survival period of 6–8 days, theanimals were deeply anesthetized with 35% chloral hy-drate and transcardially perfused at a rate of 37 ml/minutewith 0.9% saline (room temperature) for 1 minute, fol-lowed by one of the following fixatives: (A) 4% paraformal-dehyde in 0.1 M sodium phosphate buffer (PB; pH 7.4) for20 minutes or (B) 4% paraformaldehyde in 0.1 M sodiumacetate buffer (pH 6.5) for 10 minutes followed by 4%paraformaldehyde in 0.1 M sodium borate buffer (pH 9.5)for 15 minutes. The animal’s head was covered with icethroughout. The brain was carefully removed and then

Abbreviations

ab angular bundleaf amygdaloid fissureAPir amygdala-piriform transition areaDG dentate gyrusDY Diamidino yellowFB Fast blueIV layer IV of the entorhinal cortex (lamina dissecans)LEA lateral entorhinal areaMEA medial entorhinal areaPAC periamygdaloid cortexPaS parasubiculumPC piriform cortexPOR postrhinal cortexPR perirhinal cortexrs rhinal sulcus

26 C.L. DOLORFO AND D.G. AMARAL

postfixed and cryoprotected in either 4% paraformalde-hyde–20% glycerol in PB for brains perfused with fixativeA or in 4% paraformaldehyde–20% glycerol in 0.1 Msodium borate buffer for brains perfused with fixative B.Postfixation and cryoprotection was carried out for at least24 hours at 4°C. Thirty-two of the brains were blocked inthe coronal plane at levels through the anterior commis-sure and midway through the cerebellum, frozen in pulver-ized dry ice, and stored at 270°C until sectioned. Tomaintain the structural integrity of the most caudalportions of the cortex during sectioning, the remaining 10brains were individually embedded in a solution contain-ing 13% gelatin and 26% sucrose. The gelatin blockscontaining the brains were cured in the 4% paraformalde-hyde fixative for 4 hours at 4°C until the blocks hardenedand were then cryoprotected in a solution of 2% dimethylsulfoxide and 20% glycerol in PB for 48–72 hours at 4°C.These blocks were frozen in cooled isopentane (,270°C)for 30–45 minutes prior to sectioning.

The brains were sectioned on a freezing microtome inthe coronal plane (30 µm). For analysis of FB- andDY-labeled cells, a 1-in-5 series of sections through thehippocampus and entorhinal cortex was immediatelymounted on gelatin-coated slides. The slides were quicklyair dried and then placed in slide boxes containing desic-cant and stored at 220°C until the sections were analyzed.After analysis, these sections were stained with 0.25%thionin to aid in cytoarchitectonic identification of entorhi-nal borders. For parvalbumin immunocytochemistry, anadjacent 1-in-5 series of sections was collected in a cryopro-tectant solution (TCS; 25% glycerin, 30% ethylene glycol in0.05 M sodium phosphate buffer). These sections werestored in TCS at 220°C until reacted for parvalbuminimmunoreactivity.

Parvalbumin immunostaining. Tissue from four ani-mals perfused with fixative B was removed from TCS andrinsed in three changes of 0.02 M potassium phosphatebuffered saline (pH 7.4). Parvalbumin immunoreactivitywas localized by using the peroxidase–antiperoxidasemethod described by Pitkanen and Amaral (1993).

Data analysis

A 1-in-5 series of sections through the entire rostrocau-dal extent of the entorhinal cortex from each case wasanalyzed by using a Leitz Dialux 20 fluorescent micro-scope. Sections stained with thionin and for parvalbuminimmunoreactivity were analyzed with brightfield and dark-field optics on a Leitz Dialux 20 microscope and docu-mented with a Leitz DMRD photomicrography system andwith a Nikon Multiphot 4- 3 5-inch camera system.Photographs were scanned with a UMAX UTA840 flatbedscanner linked to a Macintosh Power PC for high-resolution hardcopy representation. Illustrations were con-structed by using the Adobe Photoshop software packageon a Macintosh Power PC computer. To better appreciatethe overall distribution of FB- and DY-labeled cells in theentorhinal cortex, two-dimensional unfolded maps andthree-dimensional computer-generated models were madeof the distribution of retrogradely labeled cells from thecoronal plots (see below).

Construction of two-dimensional density maps of

retrograde labeling. The method for making straight-line unfolded maps is a modification of the method de-scribed by Van Essen and Maunsell (1980) and Suzuki andAmaral (1996). The distribution of FB- and DY-labeled

cells in the entorhinal cortex (ipsilateral to the injectionsites) and the outlines of the cerebral hemisphere wereplotted at a 150-µm rostrocaudal sampling interval byusing a computer-aided digitizing system (MD-2; Minne-sota Datametrics, Minneapolis, MN) linked to a LeitzDialux 20 microscope. The FB and DY injection sites werealso plotted. The plots were printed at a magnification of27.53 to allow counting of individual plotted cells. Byusing a Nikon-SMZ-10 stereomicroscope and attacheddrawing tube, each plot was aligned with the image of thecounterstained Nissl section, and prominent features suchas the rhinal sulcus, the classically defined entorhinalareal and laminar boundaries were drawn onto the plots.The FB and DY dye injection cores and surroundingnecrosis were also drawn on the plots.

The procedure for making a straight-line unfolded mapof the rat entorhinal cortex is schematically illustrated inFigure 1. Starting at the fundus of the rhinal sulcus, thepial surface of each plot was divided into 6-mm-widecolumnar bins (i.e., 250 µm-wide bin magnified 27.53),each extending from the pial surface to the layer II–IIIborder. The number of FB- and DY-labeled neurons inlayer II of each bin was counted and noted. The unfoldedmap was constructed by ‘‘straightening’’ the cortical sur-face of each plot and then ‘‘stacking’’ the straightenedsections to produce a flattened representation of thesurface of the entorhinal cortex. The fundus of the rhinalsulcus was used to align the sections. The number ofretrogradely labeled cells in each bin was entered into aMicrosoft Excel database, in which the elements of thedatabase corresponded to the sampling bins on the coronalplots (Suzuki and Amaral, 1996). An Excel macro was usedto convert the cell count data into four distinguishableshades of gray representing four density ranges of labeledcells (see below).

Density ranges for maps of retrograde labeling.

Determining the density ranges or cutoff values arithmeti-cally (i.e., dividing the highest observed cell count by 4)overemphasized low densities in the shading scheme andunderemphasized moderately high densities. To adjust forthis, the FB and DY cell count data of each case werenormalized by using a natural log transformation of cellcounts. Following log transformation of the cell count data,the density ranges were calculated by dividing the distribu-tion of log values by 4. The anti-log of the three cutoffvalues in this transformed range were used for determin-ing the four ranges of the raw densities. An Excel macrowas used to apply a different gray level to each of theranges calculated for each density of labeling of theindividual cases. Low densities were shaded light gray,high densities were shaded black, and bins in which nolabeled neurons were observed were left unshaded.

Template map of dentate gyrus. To summarize thesize and location of the retrograde injection sites, all FBand DY injection sites (the core of the dye injection plus thesurrounding necrotic zone) were transferred from thecoronal plots onto an unfolded template map (Fig. 2) of thedentate gyrus (adapted from Swanson et al., 1978). Byusing the crest of the dentate gyrus as an alignment point,the width of the injection at each rostrocaudal level was‘‘unfolded’’ onto the dentate map.

Three-dimensional reconstructions

Computer files of selected cases were generated bytracing features of each retrograde plot by using the 3DED

ENTORHINAL PROJECTION TO THE DENTATE GYRUS 27

software package (Young et al., 1987) and a Hitachi digitiz-ing tablet linked to a PC. Outlines of the cortical hemi-sphere, granule cell layer of the dentate gyrus, FB and DYinjection sites, and the position of FB- and DY-labeledlayer II entorhinal neurons were digitized. Cross-sectionalprofiles of blood vessels were used as fiducial marks forregistration of adjacent sections. Because of unavoidablelocal physical stretching and compressing of sections dur-ing the processing of the tissue, adjacent hemisphere

outlines were occasionally out of register. These outlineswere digitally warped to produce a smoother reconstruc-tion of the cortical surface. The data files containing thecontours and cell positions were used as input to athree-dimensional surface modeling program (SyntheticUniverses [SYNU]; Hessler et al., 1992) on a SiliconGraphics Iris workstation. Rendered three-dimensionalcomposite models of the FB- and DY-labeled cells, granulecell layer, and hemisphere surfaces were visualized and

Fig. 1. Location of the entorhinal cortex and summary of themethods for making straight-line unfolded density maps of retro-gradely labeled cells in the entorhinal cortex. A–C: Photomicrographsof a rat brain showing the approximate position of the lateral (LEA,stripes) and medial (MEA, dots) subdivisions of the entorhinal cortexof the rat. A: Lateral view. Numbered lines indicate approximatedistance (mm) from bregma according to the stereotaxic atlas ofPaxinos and Watson (1986). B: Ventral view. C: Caudal view. D: Un-folded surface map of the entorhinal cortex. The positions of fourrostrocaudal coronal sections are shown. Starting at the rostral end ofthe entorhinal cortex, the mediolateral length of the entorhinal cortex(patterned strips) from each coronal plot is ‘‘straightened’’ and ‘‘stacked’’

along the rostrocaudal length of the entorhinal cortex by using therhinal sulcus (rs) as an alignment point. In section AP 28.7, where theplane of sectioning is tangential to layer II of the entorhinal cortex, a‘‘cut’’ was made (indicated by the dotted line) and the cell layer wasunfolded away from the cut. To map the distribution of retrogradelylabeled cells, the pial surface of the entorhinal cortex was divided into250-µm bins (see asterisk at AP 25.2). The number of labeled layer IIcells per bin was entered into an Excel database template. Thequantity was graphically converted into a two-dimensional map. Theentorhinal cortical map has been rotated 90° relative to the view in A;the rostral tip of the unfolded entorhinal cortex points upward. Forabbreviations, see list. Scale bars 5 1 mm in A–C.


rotated into informative views with SYNU. Digital ‘‘snap-shots’’ of these views were exported to a Macintosh PowerPC for hardcopy preparation.

RESULTS

Nomenclature

In the present report, we follow the nomenclature basedon the classical descriptions of the entorhinal cortex(Brodmann, 1909; Krieg, 1946a,b; Blackstad, 1956). Theapproximate positions of the entorhinal cortex and of itstwo main subdivisions, the lateral (LEA) and medial(MEA) entorhinal areas, are illustrated in Figure 1.

The LEA is located rostrolateral to the MEA, and itsdorsolateral border lies nearest to the rhinal sulcus.Throughout its rostrocaudal extent, the LEA is boundeddorsally by the perirhinal and postrhinal cortices. Atrostral levels, the LEA lies dorsolateral to piriform cortex;at mid-rostrocaudal levels it lies dorsal to periamygdaloidcortex; and at caudal levels, it lies between medial andlateral components of the MEA.

The MEA occupies a caudomedial position in the entorhi-nal cortex. At rostral levels, it is limited laterally byventral portions of the LEAand medially by the parasubicu-lum. At caudal levels, the MEA occupies approximately theventral one-third of the occipital pole and is boundeddorsolaterally by the postrhinal cortex and dorsomediallyby the parasubiculum.

Description of injection sites

The 39 injections analyzed in the present study encom-passed the entire septotemporal extent of the dentategyrus. The sizes and locations of injections from sixrepresentative cases are illustrated in Figure 2, whichdemonstrates their position both on a two-dimensionaltemplate of the dentate gyrus and on standard coronalsections. Injection sites are also illustrated in Figures 3and 7.

The FB and DY injection sites consisted of a central coreof dye surrounded by a necrotic zone. We defined the FBand DY injection sites according to the criteria of Huisman

Fig. 2. Two-dimensional unfolded map of the dentate gyrus show-ing the septotemporal positions and sizes of Fast blue and Diamidinoyellow injection sites of six representative cases. Standard coronalsections at six rostrocaudal levels are also illustrated to show the same

six injection sites (black profiles) that are represented on the unfoldedmap. Dashed line in map represents the crest of the dentate gyrus.Map adapted from Swanson et al. (1978).


et al. (1980; see also Conde, 1987) in which the central coreplus adjacent necrotic zone are considered to constitute thearea of effective dye uptake. Injections were generallycentered in the molecular and granule cell layers of theinfrapyramidal blade of the dentate gyrus. In none of theanalyzed cases did the injection involve the extrahilar CA3field, the CA1 field, the subiculum, or the presubiculum.With the exception of four cases in which only the outerhalf of the molecular layer was involved by the injection,the remaining injection sites covered the entire radialwidth of the molecular layer and usually some of thegranule cell layer. On average, the FB injections wereapproximately 245 µm in diameter in the coronal planeand extended for approximately 540 µm in rostrocaudallength; the DY injections averaged 273 µm in diameter and510 µm in rostrocaudal length.

Topographic organizationof entorhinal-to-dentate projections

Of the 39 cases containing either FB or DY injections, 31cases, with injections covering virtually all of the septotem-poral extent of the dentate gyrus, were selected to demon-strate the organization of entorhinal-to-dentate-projec-tions. The cells of origin of the layer II dentate-projectingneurons displayed an orderly distribution pattern thatwas systematically related to the location of the retrogradetracer injection along the septotemporal axis of the den-tate gyrus. In general, we observed that cells locatedlaterally and caudally in the entorhinal cortex were la-beled after injections into the septal half of the dentategyrus, whereas cells located in more medial and rostralportions of the entorhinal cortex were labeled after injec-tions into the temporal half of the dentate gyrus.

The patterns of retrogradely labeled entorhinal layer IIneurons are illustrated in the unfolded maps of Figure 3.In this multipart figure, the maps showing the distributionof retrogradely labeled cells are ordered according to thelocation of the fluorescent dye injections. Cases withseptally placed injections are shown first, followed by caseswith progressively more temporally placed injections. Theunfolded maps of the dentate gyrus on which the injectionsites are located are divided into 10 percentile divisions. InFigure 3A–G, all injections are located within the septal25% of the dentate gyrus. In Figure 3H–N, injections arelocated in the next 25% of the dentate gyrus. In Figure

3O–V, the first case (DG-36FB) is located on the septal sideof the 50th percentile location, and the remaining injec-tions are located in the next 25%. In Figure 3W–CC, thefirst injection (DG-35DY) was located on the septal side ofthe 75th percentile location, and the remaining injectionswere in the temporal 25% of the dentate gyrus.

Injections placed into the septal half of the dentate gyrus(0–50th percentiles) retrogradely labeled a rostrocaudallyoriented band of cells located in the dorsolateral andcaudomedial portions of the entorhinal cortex (Fig. 3A–O).Injections placed into the 50th–75th percentile levelslabeled a population of cells located in mid-mediolateralportions of the entorhinal cortex (Fig. 3P–W). Within thisband of labeled cells, there were both rostrocaudally andmediolaterally oriented components, which gave the over-all band a ‘‘boomerang’’ or arclike appearance in theunfolded maps. Injections placed into the 75th–100thpercentile levels of the dentate gyrus labeled a mediolater-ally oriented band of cells located in the extreme rostrome-dial portions of the entorhinal cortex (Fig. 3X–CC). Inter-estingly, there was a fairly discrete change in the domainsof labeled cells at the three levels described such thatlargely nonoverlapping populations of entorhinal neuronsappeared to project to septal, mid-septotemporal, andtemporal levels of the dentate gyrus. Thus, the distribu-tion of labeled cells in the unfolded maps shown in Figure3A-G and 3H–O were very similar. In Figure 3P–W, therewas an abrupt change in the pattern when the injectionsite moved across the 50th percentile marker. And therewas another abrupt change in distribution in Figure3X–CC when the injection site moved across the 75thpercentile marker.

This ‘‘bandlike’’ organization of the entorhinal-to-dentate-projecting neurons can be best appreciated in three-dimensional computer reconstructions of retrogradely la-beled neurons in the entorhinal cortex (Fig. 4). Figure4A,B illustrates data from one experiment (DG-35) inwhich the blue band of retrogradely labeled cells resultedfrom an FB injection involving the septal portion of thedentate gyrus (20th percentile). The yellow band of cellsresulted from a DY injection located at a mid-septotempo-ral level of the dentate gyrus (70th percentile). The distri-bution of these retrogradely labeled cells is also illustratedon the two-dimensional map of the entorhinal cortex (Fig.4C) and in coronal sections (Fig. 5). The results from a

Fig. 3. A–CC: Twenty-nine representative two-dimensional den-sity maps showing the patterns of retrogradely labeled cells in theentorhinal cortex following Diamidino yellow or Fast blue injectionsinto different septotemporal levels of the dentate gyrus. The lateralentorhinal is the larger area at the left in each map, and the medialentorhinal area is the region at the right of each map. Insets at topright of each panel show the percentile level along the septotemporalaxis of the various injection sites, with the zero percentile levelrepresenting the septal tip of the dentate gyrus, and the 100 percentilelevel representing the temporal tip. Maps are arranged from mostseptal (A) to most temporal (CC) injection sites. Pixels shaded blackrepresent the densest areas of labeled cells and progressively grayerpixels indicate sparser labeling. The algorithm for determining thegray level for these pixel values is described in the Materials andMethods section. White areas indicate areas where no labeled cellswere observed. Injections in the septal 0–25th percentiles of thedentate gyrus resulted in retrograde labeling in essentially the sameregion of the entorhinal cortex, i.e., the same regions of the lateral andmedial entorhinal areas have indications of labeling in each map. Thesame regions of the unfolded maps are also labeled when the injections

involve the 25–50th percentile levels of the dentate gyrus (H–O). Achange takes place, however, in the third panel of this illustration. Orepresents an injection located just septal to the 50th percentile markof the dentate gyrus. The distribution of labeled cells is essentially thesame as all previous maps, but P–W represent injections in the50th–75th percentile levels of the dentate gyrus. There has been ashift in the pattern of retrograde labeling to a region more medial inthe lateral entorhinal area and more rostral in the medial entorhinalarea. It is perhaps easiest to appreciate this shift by looking at theregion of the rhinal sulcus that is marked by the vertical bar at the leftside of each map. In O, there are numerous labeled cells indicated inthis region. However, in P–W, the rhinal sulcus and lateral portion ofthe lateral entorhinal area are devoid of labeled cells. A similar shift inthe pattern of retrograde labeling is shown in W–CC. W represents aninjection located septal to the 75th percentile and has a labelingpattern similar to that shown in O–V. The remainder of the mapsillustrate labeling from injections in the temporal quartile of thedentate gyrus. There has been a shift such that labeled cells arelocated even more medial in the lateral entorhinal area and morerostral in the medial entorhinal area. Scale bar 5 1 mm.


Figure 3


Figure 3 (Continued)






second experiment (DG-26) are illustrated in Figure 4D–F.In this figure, the blue band of cells resulted from an FBinjection located at mid-septotemporal levels of the den-tate gyrus (60th percentile). The blue band is comparablein location to the yellow band of cells in Figure 4A,B. Theyellow band of cells resulted from a DY injection located intemporal levels of the dentate gyrus (80th percentile). Thedistribution of these retrogradely labeled cells is alsoillustrated on a two-dimensional map of the entorhinalcortex (Fig. 4F) and in coronal sections (Fig. 6).

It is particularly clear in the three-dimensional recon-structions that there was minimal overlap between theseptally projecting band of cells (blue spheres) and themid-septotemporally projecting band (yellow spheres; Fig.4A,B). The minimal overlap between septally and mid-septotemporally projecting bands is also apparent in thecoronal sections (Fig. 5). Similarly, there is minimal over-lap between mid-septotemporally projecting cells (bluespheres) and temporally projecting cells (yellow spheres;Fig. 4D,E). The minimal overlap can also be seen byexamining representative coronal sections (Fig. 6). Takentogether, these data suggest that three relatively discreteand largely nonoverlapping domains of entorhinal layer IIneurons innervate septal (0–50th percentiles), mid-septotemporal (50th–75th percentiles), and temporal(75th–100th percentiles) portions of the dentate gyrus.

Gradient organization of bands ofentorhinal-to-dentate-projecting neurons

Whereas the general organization of entorhinal bandsprojecting to septal, mid-septotemporal, and temporallevels of the dentate gyrus appeared to be largely nonover-lapping, the distribution of the density of labeled cellswithin each of the bands appeared to shift in a gradedfashion related to the septotemporal location of the injec-tion site. These graded shifts in the density of retrogradelylabeled layer II neurons can be best appreciated by compar-ing the locations of the pixels indicating the most denselylabeled areas in the unfolded maps of Figure 3.

Two gradients were observed. Within each of the threebands, as injections were placed at progressively moretemporal locations, the highest density of labeled cellsshifted medially in the lateral entorhinal component androstrally in the medial entorhinal component.

Lateral and medial components ofthe entorhinal projection to different

septotemporal levels of the dentate gyrus

Septally, mid-septotemporally, and temporally placedFB and DY injections, covering the entire outer two-thirdsof the dentate molecular layer, produced nonoverlappingbands of retrogradely labeled entorhinal layer II neurons.These bands include cells located in the LEA and MEA.However, in the rare occasions when the dye injectionswere limited to the middle third of the dentate molecularlayer, retrogradely labeled entorhinal layer II neuronswere located only in the MEA. In the two clearest cases ofthis illustrated in Figure 7, the topography outlined abovewas confirmed for the MEA projection alone. Thus, for aninjection located in the middle molecular layer of theseptal dentate gyrus (DGEC-12FB), the band of retro-gradely labeled cells was located in the caudal pole of theMEA. When the injection was located at a mid-septotempo-ral level (DG-23 DY), in contrast, the band of MEA labeledcells was located more rostrally.

For each experiment, we calculated the total surfacearea of the entorhinal cortex, the surface area occupied bythe band of retrogradely labeled cells, and the proportionof the band located within the LEA and the MEA (Table 1).The total surface area of the rat entorhinal cortex wasapproximately 19.6 mm2. The LEA occupies more of thesurface area of the entorhinal cortex (68%) than does theMEA (32%). Of this surface, the area occupied by the bandsof retrogradely labeled cells averaged 43%, with a range of19.9–59%. We found that the proportion of the surface areaof labeled bands in the LEA and MEA corresponded almostexactly to the overall proportions of these divisions to theentire surface area of the entorhinal cortex. Thus, theproportion of the surface area occupied by the labeledbands that was located in the LEA was about 67.5%, andthe part that was in the MEA was about 32.5% (Table 1).

Retrograde labeling as a marker forthe borders of the entorhinal cortex

One controversial issue concerning the rat entorhinalcortex is the exact location of its borders, in particular itslateral border with perirhinal cortex. Different stereotaxicatlases of the rat brain place the lateral border anywherefrom the fundus of the rhinal sulcus to a few millimetersbelow the rhinal sulcus (Zilles, 1985; Paxinos and Watson,1986; Swanson, 1992). One reason for this variability isthat the region immediately around the rhinal sulcus iscytoarchitectonically complex. For example, layer II andIII cells in this dorsolateral region of the entorhinal cortex,especially at caudal levels, are loosely organized andmerge with equally diffuse cells of the dorsally adjacentperirhinal areas. Thus, it is often very difficult to differen-tiate the lateral entorhinal cortex from the perirhinalcortex solely on the basis of cytoarchitectonic characteris-tics. The medial entorhinal border with the parasubiculumis also difficult to determine by using cytoarchitectoniccriteria alone, particularly at rostral levels. One definingfeature of the entorhinal cortex is that it projects to thedentate gyrus, whereas adjacent cortical areas do not.Thus, the border between retrogradely labeled layer IIneurons and nonlabeled neurons provides an excellentmarker for the limit of the entorhinal cortex. In addition,recent studies in the monkey and rat have shown denseimmunoreactivity for parvalbumin, a calcium-binding pro-tein, in lateral portions of the entorhinal cortex. Thisstaining also defines a distinct border with the perirhinalcortex (Pitkanen and Amaral, 1993; Dolorfo and Amaral,1993; Wouterlood, 1994; Burwell et al., 1995; Insausti etal., 1997). To clarify the location of the lateral border of theentorhinal cortex, we compared the distribution of retro-gradely labeled cells with Nissl counterstained sectionsand with adjacent sections stained immunohistochemi-cally for the distribution of parvalbumin (Fig. 8).

The lateral entorhinal border was best demarcated bythe band of retrogradely labeled layer II cells resultingfrom retrograde tracer injections located at the septal poleof the dentate gyrus (Figs. 4A,B, 8). At rostral levels,FB-labeled cells corresponded to clusters of medium-sized,oval-shaped layer II neurons approximately 250–500 µmventral to the fundus of the rhinal sulcus. The location ofthese septally projecting labeled cells coincided with aregion of dense parvalbumin immunoreactivity that alsoprovided a distinct border with the dorsally adjacentperirhinal cortex. At rostral levels, the Nissl image of theperirhinal cortex is also different from the entorhinal


cortex, with the merged layers II and III of the perirhinalcortex populated with smaller and more densely packedneurons.

At progressively more caudal levels (Fig. 8), however, itbecomes increasingly difficult to distinguish the borderbetween the entorhinal and postrhinal cortices purely onthe basis of the Nissl-stained material. And here both thedistribution of retrogradely labeled cells and the parvalbu-min labeling provided clear borders. At these more caudallevels, the border is located more dorsally, first in thefundus of the rhinal sulcus (Fig. 8A–C) and then dorsal tothe sulcus (Fig. 8D–F).

The medial border of the entorhinal cortex was demar-cated by the medial edge of the bands of retrogradelylabeled cells as shown in the three-dimensional reconstruc-tions (Fig. 4). At rostral and mid-rostrocaudal levels, themedial entorhinal border with the parasubiculum was bestdemarcated by retrogradely labeled cells resulting fromdye injections located in temporal and mid-septotemporalportions of the dentate gyrus, respectively (Fig. 4D–F).The retrogradely labeled cells formed a sharp border at themedial extreme of the entorhinal cortex (Fig. 6B–E). Atcaudal levels, the medial border was delimited by retro-gradely labeled cells resulting from injections into theseptal portions of the dentate gyrus (Fig. 4B).

DISCUSSION

Although it has been known for more than a centurythat the entorhinal cortex gives rise to a powerful, unidirec-tional projection to the dentate gyrus, the so-called perfo-rant pathway (Ramon y Cajal, 1901), several generaltopographical features of this projection remain unclear.By using three-dimensional computer reconstruction tech-niques and the production of two-dimensional unfoldedmaps, we have analyzed the three-dimensional topo-graphic organization of projections from the entorhinalcortex to the dentate gyrus in the rat. Our findingsconfirmed the observations of Ramon y Cajal and othersthat all septotemporal levels of the dentate gyrus receive aprominent input from the entorhinal cortex. We alsoconfirmed the findings of previous studies (Hjorth-Simon-sen and Jeune, 1972; Steward and Scoville, 1976; Ruth etal., 1982, 1988; Pohl and Ott, 1984; Witter and Groenwe-gen, 1984) indicating that the perforant pathway displaysa general lateral-to-medial topography that is related tothe septotemporal level of the dentate gyrus which isinnervated. Cells located laterally and caudally in theentorhinal cortex project to septal levels of the dentategyrus, whereas cells located medially and rostrally projectto more temporal levels. Importantly, our data extend andclarify these findings by showing that, with respect to theMEA, entorhinal-to-dentate projecting neurons display acaudal-to-rostral topography related to the septotemporalaxis of the dentate gyrus. Our data also indicate that thereis substantial convergence and divergence of the entorhi-nal projection to the dentate gyrus. Thus, even a discreteinjection into a particular septotemporal level of thedentate gyrus led to retrogradely labeled cells that weredistributed throughout, on average, 45% of the surfacearea of the entorhinal cortex, i.e., 45% of the surface areaof the entorhinal cortex converged onto a relatively smallpoint within the dentate gyrus. Conversely, injections atany point throughout the septal half of the dentate gyruslabeled cells in the same general region of the entorhinal

cortex, indicating substantial divergence of entorhinal-to-dentate terminations. We also found that the distributionof retrogradely labeled cells at the rhinal sulcus providesan accurate border between the entorhinal and perirhinaland postrhinal cortices. When injections were clearlyconfined to the molecular layer of the dentate gyrus, we didnot observe labeled cells in the perirhinal or postrhinalcortices. The border indicated by the retrograde labelingcorresponded to a change from high (in the entorhinalcortex) to low (in the perirhinal and postrhinal cortices)immunoreactivity for the calcium-binding protein parval-bumin, which thus also provides a reliable boundary forthese regions.

Technical considerations

A potential problem to consider when using FB and DYin neural tracing studies is whether uptake by fibers ofpassage could confound the results. There is relativelylittle information concerning the trajectory of individualperforant path fibers as they innervate the dentate gyrus.The prevailing view is that entorhinal fibers travel withinthe angular bundle to approximately the septotemporallevel that they will innervate and then perforate thesubiculum to enter the dentate gyrus. Presumably thefibers travel for some distance in the molecular layerbecause anterograde tracing studies indicate that a particu-lar point within the entorhinal cortex innervates 30% ormore of the septotemporal extent of the dentate gyrus.Although we cannot dismiss the possibility that somefibers of passage were labeled in our studies, the dataactually provide evidence against this possibility. First,the density of retrogradely labeled cells projecting to theseptal half of the dentate gyrus demonstrated a subtlelateral-to-medial shift (in the lateral entorhinal area) andcaudal-to-rostral shift (in the medial entorhinal cortex) asthe injections were placed progressively temporally in thedentate gyrus. If fibers of passage were being substantiallyinvolved by the more temporal injections, one would expecta more homogeneous pattern of labeling. Second, the moreconvincing argument is that when the injections wereplaced temporal to the 50% percentile point of the dentategyrus, there were only a few labeled cells in the lateral andcaudal bands that were labeled after septal injections. Iffibers were passing through temporal levels to reach septallevels of the dentate gyrus and if the fibers were beinglabeled by the dye injections, one would expect the lateraland intermediate bands to show retrogradely labeled cells.

Fig. 4. Three-dimensional computer reconstructions of the caudalhalf of the left cerebral hemisphere and two-dimensional unfoldedmaps of case DG-35 (A–C) and case DG-26 (D–F). A: Lateral view ofcase DG-35 showing the position of Fast blue (FB; blue) and Diamidinoyellow (DY; yellow)-labeled cells. The band of FB-labeled cells demar-cates the dorsolateral entorhinal border with the dorsally adjacentperirhinal and postrhinal cortices. At progressively caudal levels, thisborder extends into and then dorsal to the rhinal sulcus. Inset: Medialview of the dentate gyrus showing the septotemporal positions of theFB and DY dye injections for case DG-35. C: Two-dimensional map ofthe distribution of FB- (blue) and DY- (yellow) labeled cells in theentorhinal cortex of case DG-35. B: Medial view of case DG-35. D:Medial view of case DG-26 showing the position of FB- (blue) and DY-(yellow) labeled cells. These bands demarcate the rostral and mid-rostrocaudal entorhinal borders with the parasubiculum. E: Ventralview of case DG-26. Each dot represents one labeled cell. F: Two-dimensional map of the distribution of FB- (blue) and DY- (yellow)labeled cells in the entorhinal cortex of case DG-26.


Figure 4


Because this was not observed, we conclude that theresults were not heavily influenced by the fiber-of-passageproblem.

Comparison with previous studies

Although our findings are consistent with those ofprevious studies on the organization of the entorhinal-to-dentate projection, the use of three-dimensional computerreconstruction, two-dimensional unfolded mapping tech-niques, and the systematic sampling of all septotemporallevels of the dentate gyrus using sensitive retrograde dyesenabled the three-dimensional organization of entorhinal-to-dentate projections to be appreciated. The major findingfrom this new analysis is that the entorhinal-to-dentateprojection can be divided into three largely nonoverlappingdomains. The septal half of the dentate gyrus receivesinput from the laterocaudal portion of the entorhinalcortex, a region occupying approximately the third quarterof the dentate gyrus is innervated by the mid-mediolateralportion of the entorhinal cortex, and the temporal quarterof the dentate gyrus receives input from the most rostrome-dial portion of the entorhinal cortex.

Ramon y Cajal (1901) showed that the entorhinal projec-tion to the dentate gyrus originated in the superficiallayers. However, it was not until the retrograde tracerstudies of Steward and Scoville (1976) that the entorhinalprojection to the dentate gyrus was shown to originateprimarily from layer II neurons. These retrograde experi-ments confirmed previous anterograde degeneration experi-ments (Hjorth-Simonsen and Jeune, 1972) showing thatlateral parts of the entorhinal cortex project to septal partsof the dentate gyrus. However, Steward and Scoville (1976)did not study the septotemporal topography of the perfo-rant path projection.

The most extensive study to date of the origins of theentorhinal projection to the dentate gyrus is that of Ruthet al. (1982, 1988). They used the HRP technique andevaluated the distribution of retrogradely labeled cells inseries of coronal sections. From their two-dimensionalanalysis, they proposed a three-dimensional organizationfor the entorhinal-to-dentate projection. The nomencla-ture used by Ruth et al. (1982, 1988) differs substantiallyfrom the one used in the present paper; they had lateral,intermediate, and medial divisions of the entorhinal cor-tex, and the lateral entorhinal cortex was divided intothree subdivisions and a transitional zone. One complicat-ing feature to the three-dimensional organization thatthey described was that the projections originating fromthe lateral and medial entorhinal areas (Ruth et al., 1982,1988) were covered in separate papers and the organiza-tion of the consolidated projection was not discussed ineither paper.

Judging from their illustrations of retrogradely labeledcells, it appears that the general topographic patternobserved by Ruth et al. (1982, 1988) is consistent with thepattern portrayed in the present paper. Perhaps the mostclear-cut difference is in their description of the projectionsfrom the lateral entorhinal area. As summarized in theirFigure 10 (Ruth et al., 1988), they concluded that lateralentorhinal projections to different foci in the septal half ofthe dentate gyrus arose from distinctly different mediolat-erally situated strips within the lateral entorhinal area.Our data suggest that the entire septal half of the dentategyrus receives input from the same region of the lateralentorhinal area, although the strength of the projection

(based on the density of retrogradely labeled cells) doesvary in a consistent fashion. We found that the location ofthe highest density of labeled cells moved from mostlaterally in the lateral entorhinal cortex with extremeseptal injections to more medially in the lateral entorhinalarea with more temporally situated injections. One expla-nation for this apparent difference may relate to thesensitivity of the tracers used in the two studies. If, whenusing the less sensitive HRP, Ruth et al. (1988) onlyobserved the regions of densest labeling, then they wouldobtain the pattern of discontinuous bands that they re-ported. With the more sensitive fluorescent dyes that weused, we observed a much larger region of retrogradelabeling. This was the case despite the fact that ourinjection sites were generally smaller than their HRPsites. This may also explain why Ruth et al. (1982)reported that at most 30% of the lateral entorhinal cortexprojected to a particular septotemporal level of the dentategyrus, whereas we found, on average, that 45% of theentire entorhinal cortex projects to a discrete location inthe dentate gyrus. Our data are consistent with themeasures of axonal spread along the septotemporal axis ofthe dentate gyrus resulting from bulk anterograde tracerinjections in the entorhinal cortex (Tamamaki, 1997), fromintracellularly labeled entorhinal layer II neurons (Tama-maki and Nojyo, 1993), and with electrophysiologicalrecordings of dentate granule cells (Pare and Llinas, 1994)following entorhinal stimulation. Tamamaki (1997), forexample, placed injections of carbocyanine dye into themost lateral aspect of the rat entorhinal cortex andobserved anterograde labeling throughout the entire sep-tal half of the dentate gyrus.

Topographic organizationof entorhinal–dentate connections:

Gradient versus segmented

Based on previously published anterograde tracing stud-ies of the perforant pathway (Wyss, 1981), we expected toobserve a gradient organization to the location of cellsprojecting to different septotemporal levels of the dentategyrus. Had we simply examined the distribution of labeledcells after a small set of injections placed at septal, middle,and temporal levels of the dentate gyrus, the patternwould have been consistent with a gradient organization.The advantage we had in the current series of experimentswas that there was a large number of injections (39) that

Fig. 5. Retrograde tracer injections that define the extreme lateral,caudomedial, and mid-rostrocaudal medial borders of the entorhinalcortex. Unfolded density maps of the distribution of Fast blue (FB; topleft) and Diamidino yellow (DY; top right) labeling and correspondingcoronal sections of case DG-35. Density maps show the distribution ofretrogradely labeled cells that resulted from FB and DY injections intoseptal and mid-septotemporal dentate gyrus, respectively. A–F: Plotsof coronal sections from six rostrocaudal levels through the rat brainshow the mediolateral distribution of retrogradely labeled FB (black)and DY (gray) neurons. A88–F88: Photomicrographs of Nissl-counter-stained sections of the plots in A–F. The FB-labeled cells demarcatethe entire rostrocaudal extent of the dorsolateral entorhinal borderwith the dorsally adjacent perirhinal cortex. The FB-labeled cells alsodemarcate the medial entorhinal border with the parasubiculum atcaudal levels (E,F). The DY-labeled cells demarcate the medial entorhi-nal border with the parasubiculum at mid-rostrocaudal levels (D).Filled arrowheads indicate the lateral and medial extents of theentorhinal cortex derived from the pattern of retrograde labeling.Scale bar 5 1 mm.


Figure 5



involved essentially all septotemporal levels of the dentategyrus. And, in each case, a two-dimensional unfolded mapof the distribution of retrogradely labeled cells was pre-pared, allowing for easy comparisons of the three-dimensional distribution of cells. Upon examination of allof these unfolded maps, three distinct patterns of labelingemerged. Septally projecting entorhinal cells were locatedin the most lateral and caudomedial portions of theentorhinal cortex. Mid-septotemporally projecting cellswere located at mid-mediolateral portions of the entorhi-nal cortex, and temporally projecting cells were located inrostromedial portions. Although there were subtle shifts inthe density of cells within each of these zones, it becameincreasingly clear that all of the injections located withinapproximately the septal half of the dentate gyrus (0th–50th percentiles) lead to generally the same pattern oflabeling in the entorhinal cortex. Injections located withinthe 50th–75th levels, however, lead to a distinctly differentpattern of labeling. And injections located within the75th–100th percentile levels resulted in an entirely dis-tinct pattern of labeling. Thus, there appeared to be threesegregated and perhaps functionally distinct componentsof the perforant path system. This organization is all themore interesting given that the associational connectionswithin the entorhinal cortex appear to respect this tripar-tite subdivision (Dolorfo and Amaral, 1994, 1998).

Entorhinal connections andthe ‘‘lamellar hypothesis’’

We have previously argued (Amaral and Witter, 1989)that the intrinsic circuitry of the hippocampal formation isnot consistent with the notion that it is organized intorelatively thin, functionally isolated slices. This was atenet of the ‘‘lamellar hypothesis’’ initially advanced byAndersen et al. (1971). In fact, it is now clear that all, orvirtually all, of the neurons in a typical 350-µm in vitrohippocampal slice have at least a portion of their axonalplexus transected. The current findings indicate that theperforant path projections are also not organized in alamellar fashion. Because the rat hippocampus has aseptotemporal extent of approximately 10 mm, our dataindicate that neurons located in the lateral portion of theentorhinal cortex may distribute collaterals over a dis-tance of 5 mm in the dentate gyrus. Even the moreconstrained projections arising from mid and medial bandsof the entorhinal cortex are distributed for distances ofapproximately 2.5 mm. Our data suggest that the conver-gent–divergent organization of the perforant path projec-tion predicts a very widespread rather than a point-to-point distribution of information from the entorhinal cortexto the dentate gyrus.

What other evidence is therefor septotemporal segmentation

of hippocampal function?

A clear prediction from the organization of entorhinalprojections is that the septal half of the dentate gyrus maybe involved in different functions than the mid and tempo-ral quarters. Although there is currently relatively littlefunctional information to substantiate this prediction,there are additional neuroanatomical data that supportseptotemporal differences in the functional organization ofthe hippocampal formation. For example, cholinergic pro-jections are much stronger to temporal portions of thehippocampus than to septal levels (Hoover et al., 1978;

Amaral and Kurtz, 1985; Wainer et al., 1985). Similarly,monoaminergic projections arising from the ventral teg-mental area and the locus coeruleus (Verney et al., 1985;Haring and Davis, 1985) terminate most heavily withintemporal levels of the hippocampus. Other inputs to thehippocampal formation also demonstrate a septotemporalpreference. Thus, the projection from the amygdala to theCA1 field of the hippocampus preferentially terminates inthe temporal third of the field (Krettek and Price, 1977).The pattern of associational and commissural connectionsalso distinguishes septal and temporal levels of the den-tate gyrus. Hilar projections arising from the septal half ofthe hilus distribute widely and heavily innervate theseptal two-thirds of the dentate gyrus. Projections arisingfrom the temporal portions of the hilus, in contrast, have amuch more limited distribution and terminate mainly inthe temporal one-third of the dentate gyrus (Fricke andCowan, 1978). The associational projections arising fromCA3 of the hippocampus have a similar distribution. Thoseoriginating in the septal half of the hippocampus extendthroughout as much as two-thirds of the septotemporallength of the hippocampus, whereas projections originat-ing from temporal portions terminate most heavily withinthe temporal one-third of the hippocampus (Ishizuka et al.,1990; Li et al., 1994).

Recently, Risold and Swanson (1996) also divided theseptotemporal length of the hippocampus into ‘‘three broaddomains’’ on the basis of its projections to the lateral septalnucleus. The septal half of the hippocampus and subicu-lum give rise to only meager projections to the mostdorsomedial portion of the lateral septal nucleus. Progres-sively heavier projections originate from more ventrallevels of CA1 and the subiculum. Projections from theseregions are topographically organized such that moretemporal levels project to more ventral levels of the lateralseptal nucleus. Similarly, the septal two-thirds of thesubiculum project heavily to the mammillary nuclei,whereas the temporal one-third does not; the temporalone-third, in contrast, projects heavily to the ventromedialnucleus of the hypothalamus, whereas the septal two-thirds does not (Swanson and Cowan, 1977; Witter andGroenwegen, 1990; Canteras and Swanson, 1992). Thus,the organization of both intrinsic and extrinsic connectionsindicates that temporal levels of the hippocampal forma-tion have a different circuitry from septal levels. It isdifficult with available neuroanatomical data to determine

Fig. 6. Retrograde tracer injections that define the rostral andmid-rostrocaudal medial borders of the entorhinal cortex. Unfoldeddensity maps of the distribution of Fast blue (FB; top left) andDiamidino yellow (DY; top right) labeling and corresponding coronalsections of case DG-26. Density maps show the distribution of retro-gradely labeled cells that resulted from DY and FB injections intotemporal and mid-septotemporal dentate gyrus, respectively. A–F: Plotsof coronal sections from six rostrocaudal levels through the rat brainshow the mediolateral distribution of retrogradely labeled DY (gray)and FB (black) neurons. Large black and gray profiles in B (largearrows) coincide with the FB and DY dye cores shown in B8 (largearrows) at this level. A88–F88: Photomicrographs of Nissl-counterstainedsections of the plots in A–F. The DY-labeled cells demarcate the rostralmedial entorhinal border with the dorsally adjacent parasubiculum.The FB-labeled cells demarcate the medial entorhinal border with theparasubiculum at mid-rostrocaudal levels (D,E). Filled arrowheadsindicate the lateral and medial extents of the entorhinal cortex derivedfrom the pattern of retrograde labeling. Scale bars 5 1 mm.


Figure 6




whether the septotemporal axis should be divided into two,three, or more ‘‘domains.’’

That the septal portion of the hippocampus may func-tion differently from the temporal hippocampus is alsosupported by the organization of neocortical sensory andother inputs to the entorhinal cortex. The vast majority ofneocortical sensory information to the entorhinal cortexterminates in the lateral and caudal portions of theentorhinal cortex that project to the septal half of thedentate gyrus (Deacon et al., 1983; Burwell and Amaral,1996). Projections from the amygdala, in contrast, termi-nate primarily in the more rostromedially situated por-tions of the entorhinal cortex that terminate preferentiallyin the temporal half of the dentate gyrus (Krettek andPrice, 1977; Miettinen et al., 1996).

Both behavioral and electrophysiological studies are alsobeginning to demonstrate septotemporal differences in hippo-campal function. Rats with lesions restricted to the septalportions of the hippocampus exhibit longer escape latencies inthe Morris water maze than do rats with lesions restricted totemporal portions of the hippocampus (Moser et al., 1993),suggesting that septal portions of the hippocampus may bemore involved in spatial memory processing than temporalportions. Evidence for this conclusion was provided by Jung etal. (1994) who observed more place cells in septal portions ofthe hippocampus than in temporal levels. Moreover, placefields at septal levels of the hippocampus were more finelytuned than those recorded in progressively more temporalportions of the hippocampus.

Implications for functional analysesof the entorhinal-dentate projections

The accurate representation of the three-dimensionaltopography of the entorhinal projections to the dentate

DG-23 DY

LEA

MEA

DGEC-12 FB

LEAMEA

0

10

20

30

40

50

60

70

80

90

100

DGEC-12 FB

DG-23 DY

Fig. 7. Two representative two-dimensional density maps showingthe patterns of retrogradely labeled cells in the entorhinal cortex afterFast blue (FB) or Diamidino yellow (DY) injections limited to themiddle one-third of the molecular layer of septal (DGEC-12 FB) andmid-septotemporal (DG-23 DY) levels of the dentate gyrus. Inset

(right) shows the percentile level along the septotemporal axis of thelocations of these injections. Pixels shaded black represent the densestareas of labeled cells, and progressively grayer pixels indicate sparserlabeling. White areas indicate areas where no labeled cells wereobserved.

TABLE 1. Lateral and Medial Entorhinal Area Componentsof Entorhinal-to-Dentate Projecting Bands

Case

Septotemporalposition ofinjection

(percentilelevel)

EC1

(mm2)

% Areaof EC

occupiedby band

Percentageof bandin LEA

Percentageof bandin MEA

DG-39 DY 10 20.6 45.5 74.9 25.1DG-34 DY 10 17.9 40.5 79.0 21.0DG-36 DY 10 19.2 45.6 80.4 19.6DGEC-16 DY 20 19.1 33.2 66.1 33.9DG-35 FB 20 20.5 44.7 55.0 45.0DGEC-15 FB 20 18.5 32.5 49.6 50.4DGEC-1 FB 20 21.1 40.3 51.2 48.8DG-22 FB 20 19.3 42.3 49.0 51.0DG-24 FB 20 19.0 52.7 44.6 55.4DG-44 FB 30 19.0 28.7 61.0 39.0DG-44 DY 30 19.0 53.9 69.2 30.8DG-41 FB 40 15.3 54.8 76.8 23.2DG-43 DY 40 18.5 59.3 63.2 36.8DG-34 FB 50 17.9 59.0 68.9 31.1DG-36 FB 50 19.2 58.6 65.6 34.4DG-25 FB 60 20.3 52.0 63.5 36.5DGEC-1 DY 60 21.1 46.3 68.3 31.7DG-26 FB 60 21.1 53.7 38.1 61.9DGEC-15 DY 60 18.5 56.2 81.9 18.1DGEC-16 FB 60 19.1 46.7 67.0 33.0DG-39 FB 70 20.6 44.2 84.3 15.7DGEC-18 DY 70 19.2 30.7 86.8 13.2DG-35 DY 70 20.5 51.2 78.6 21.4DG-27 DY 70 22.3 35.1 90.4 9.6DGEC-18 FB 70 19.2 37.3 84.0 16.0DG-28 DY 70 23.0 37.0 73.2 26.8DGEC-12 DY 80 18.2 19.9 63.8 36.2DGEC-11 DY 80 19.8 29.6 69.8 30.2DG-26 DY 80 21.1 23.8 53.5 46.5Mean 19.6 43.3 67.5 32.5

1Areas of two-dimensional unfolded maps of the entorhinal cortex (EC), as determinedby the area measuring tool of the computer drawing program Canvas (see Materials andMethods in Dolorfo and Amaral, 1998). LEA, lateral entorhinal area; MEA, medialentorhinal area.


gyrus has both practical and theoretical implications forfuture research. One practical implication is that thisinformation can be used to determine appropriate posi-tions in which to place electrodes to stimulate both themedial and lateral perforant path projections to a particu-lar septotemporal level of the dentate gyrus. Figure 9illustrates eight coronal sections through the entorhinalcortex (arranged from rostral to caudal) that illustratewhich portions of the entorhinal cortex project to differentseptotemporal levels of the dentate gyrus. If one is record-ing from the septal dentate gyrus, for example (Fig. 9A),the appropriate portion of the lateral entorhinal cortex tostimulate is located laterally, subjacent to the rhinalsulcus (Fig. 9B). However, the corresponding portion of themedial entorhinal cortex to stimulate is actually locatedcaudally (Fig. 9H) rather than medially. This statementcan be confirmed by examining the band of blue-labeledneurons in Figure 4A that resulted from a FB injectioninto the septal dentate gyrus. If one wanted to record from

a mid-level of the dentate gyrus (Fig. 9D), then both medialand lateral perforant path fibers could be stimulated at thesame rostrocaudal level (e.g., Fig. 9F or G). And if one isinterested in stimulating perforant path fibers to the mosttemporal portion of the dentate gyrus, then stimulatingelectrodes would need to be situated rostromedially withinthe entorhinal cortex (Fig. 9E). However, the electrodeplacement for the lateral perforant path stimulation wouldneed to be placed rostral (Fig. 9C or D) to the medialperforant path stimulation (Fig. 9E).

On a more theoretical level, the topography described inthe present study suggests some autonomy of functionwithin different regions of the entorhinal cortex and atdifferent septotemporal levels of the hippocampus anddentate gyrus. Given the extensive associational connec-tions that occur at many stages of the intrinsic hippocam-pal circuitry (Amaral and Witter, 1989), it will be ofinterest to determine to what extent they preserve ordegrade this potential functional segregation.

Fig. 8. High magnification photomicrographs of case DG-35 show-ing the dorsolateral border of the entorhinal cortex at mid-rostrocau-dal (A–C) and caudal (D–F) levels. A,D: Nissl-stained sections show-ing the cytoarchitectonic characteristics of the portion of the entorhinalcortex adjacent to the rhinal sulcus. B,E: Brightfield photomicrograph

of adjacent sections immunostained for parvalbumin. C,F: Correspond-ing plots of the same sections illustrated in A and D, respectively,showing the distribution of layer II neurons labeled with Fast blue.Blood vessels are indicated by asterisks and serve as points ofreference. Scale bars 5 170 µm.


AP -7.64

LEA

MEA

AP -8.30

LEA

MEAMEA

MEA

AP -8.72

F

G

H

LEA

AP -3.8

A

AP -6.04

LEA

D

AP -4.52

LEA

AP -5.2

LEA

AP -6.72

LEA MEA

B

C

E

Figure 9

ACKNOWLEDGMENTS

We thank Janet Weber, Mary Ann Lawrence, and KaonTong for excellent histological assistance, Miriam Frankfor technical assistance, and Dr. Brian W. Leonard forcomments on an earlier version of the manuscript. We alsothank Dr. Paul Adams, Rick Avila, and David Hessler forassistance with the three-dimensional reconstructions.This work was supported in part by NIH grants NS 16980(D.G.A.) and NS 16980-S1 (C.L.D.).

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Entorhinal cortex of the rat: Topographic organization of the cells of origin of the perforant path projection to the dentate gyrus