Entorhinal cortex of the human, monkey, and rat: metabolic map as revealed by cytochrome oxidase.

THE JOURNAL OF COMPARATIVE NEUROLOGY 326:451-469 (1992)

Entorhinal Cortex of the Human, Monkey, and Rat: Metabolic Map as Revealed by Cytochrome Oxidase ROBERT F. HEVNER AND MARGARET T.T. WONG-RILEY Department of Cellular Biology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

ABSTRACT The entorhinal cortex (EC) is a medial temporal lobe area involved in memory consolidation. Results from previous studies suggest that the upper layers of the EC may be organized into anatomical-neurochemical modules associated with pathways through the neuron clusters in layers I1 and 111. To study metabolic patterns in the EC and to look for correlates of the proposed modules, we examined the distribution of cytochrome oxidase (CO) in the human, monkey, and rat EC. CO is a mitochondrial enzyme that has been used to study modules in other cortical areas. In all three species, the neuron clusters in layers 11-111 were darkly GO-reactive, whereas most of the neuropil between clusters was lightly or moderately CO-reactive. However, some neuropil regions directly adjacent to the neuron clusters were also darkly CO-reactive, especially in the human; these neuropil areas included portions of layers I and 11. In tangential sections through layers 1-11, the areas of dark staining formed a consistent pattern, comprised of partially interconnected islands and stripes associated with the neuron clusters. In the EC from one human hemisphere, 200-250 CO-reactive layer I1 islands were present. EC layers other than 1-111 also showed characteristic CO staining intensities, but no evidence of modularity. Our results indicate that CO staining labels distinct compartments related to the neuron clusters in the upper EC layers. We propose that these compartments may represent modules for cortical processing, analogous to the CO-labeled modules in some other areas of Cortex. o 1992 Wiley-Liss, Inc.

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Key words: hippocampus, temporal lobe, cerebral cortex, neural pathways, brain mapping

Modularity is increasingly being recognized as an important organizing principle in many brain areas. The identification of physiological modules in primary and higher order areas of the primate visual system, e.g., has provided evidence for hierarchical and parallel processing in that system. Neurons in the visual modules respond selectively to particular aspects of the visual scene, such as color, motion, or shape, and different modules at distinct processing levels are specifically connected in parallel pathways (Livingstone and Hubel, '88; Van Essen et al., '90, '92). Modules are also found in the rodent somatosensory cortex, where information from individual vibrissae is processed in the anatomical barrels (Welker, '71) and in the olfactory bulb, where olfactory inputs are processed in the glomeruli (Purves and LaMantia, '90). In several areas, including the olfactory bulb, the barrel field, and visual areas V1 and V2, the modular organization is associated with unique metabolic patterns revealed by staining for the mitochondrial enzyme cytochrome oxidase

o 1992 WILEY-LISS. INC.

(CO). Indeed, the modules in V1 and V2 were noted originally in CO-stained tissue; their distinct physiological properties, anatomical connections, and molecular markers were identified later (Vl: Horton and Hubel, '81; Livingstone and Hubel, '83, '84, '87; Carroll and Wong-Riley, '84; Horton, '84; V2: Livingstone and Hubel, '82; Tootell et al., '83; Wong-Riley and Carroll, '84; DeYoe and Van Essen, '85; Shipp and Zeki, '85; Hubel and Livingstone, '87; DeYoe et al., '90). Thus, CO staining may be a useful complement to other anatomical and physiological methods for studying modules. Previous studies of the entorhinal cortex (EC), a component of the medial temporal lobe memory system (Squire and Zola-Morgan, '911, have suggested that this area may Accepted August 14,1992. I Address reprint requests to Dr. Robert Hevner, who is now at the Department of Pathology, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115.

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have a modular organization. Structurally, the upper layers staining is that the level of oxidative metabolic activity in of the EC contain prominent clusters of neurons that are cortical layers and regions may be related to the types of highly suggestive of modules. The verrucae, or slight inputs and neurons in each location. We applied CO histochemistry and immunohistochemisbumps on the human EC surface, which are related to the neuron clusters, have likewise been cited as evidence of try to the human, monkey, and rat EC. We found that CO modularity (Klingler, '48; Amaral and Insausti, '90; Solod- was distributed in a compartmental pattern, consistent kin et al., '91; Van Hoesen et al., '91). Results from recent with modules, in the upper EC layers. We characterized myeloarchitectonic and neurochemical studies have added these compartments anatomically and found that they were to the evidence favoring modularity in the human EC (Beall related to the neuron clusters. We hypothesize that the CO-labeled compartments represent EC modules related to and Lewis, '91, '92; Solodkin et al., '91). Tract-tracing experiments have identified two separate specific neuronal pathways. pathways through the EC to the hippocampus: one that arises mainly in EC layer I1 and terminates in the dentate MATERIALS AND METHODS gyrus and hippocampal field CA3, and a second that passes mainly from EC layer I11 to CAI and subiculum (Steward Animal tissues. All animal experiments were done in and Scoville, '76; Witter and Amaral, '91). The dendritic accordance with NIH and Medical College of Wisconsin fields of the neurons giving rise to these two projections are regulations. Seven adult male Sprague-Dawley rats (300apparently segregated: studies of monkey EC have shown 400 g) were anesthetized with 4% chloral hydrate (10 mlikg that the apical dendrites of layer I11 neurons pass between i.p.) and perfused transcardially with 30 ml warm phosphatelayer I1 neuron clusters by bending into and through the buffered saline, followed by 100 ml cold buffered fixative interstices (Amaral et al., '87; Carboni et al., '90). Furthercontaining 4% paraformaldehyde. The brains were remore, anterograde transport studies have shown that the moved, postfixed by immersion in cold buffered fixative for layer I1 and I11 neurons, and their dendritic fields, receive 1hour, and cryoprotected with increasing concentrations of separate projections from different brain areas. Within sucrose (4-30%) in buffer. layer 11, the neuron clusters and interstices receive distinct Monkey EC specimens were obtained from six adult male inputs (Insausti et al., '87a,b; Saunders and Rosene, '88). and female macaques (two Macaca fascicularis, two M. Together, these data suggest that two segregated neural radiata, and two M . mulatta; 2.7-6.7 kg) used primarily for pathways pass through the upper layers of the EC and that another study; details of their treatment were given previthe neuron clusters are structural modules in these pathously (Hevner and Wong-Riley, '91). For anesthesia, the ways. monkeys received ketamine (20 mgikg i.m.) and sodium Currently, the concept of modularity in the EC is based pentobarbital (30 mgikgi.v. plus 30 mg/kgi.p., or 65 mgikg mainly on anatomical and neurochemical evidence; as yet, i.v.1. The animals were killed by high transection of the there have been no studies showing metabolic or physiologcervical cord followed by immediate removal of the brain. ical modules in the EC. Since CO has been useful as a Blocks containing the EC were dissected from the fresh metabolic probe for studying modules in other brain remonkey brains, fixed by immersion for 4-6 hours in cold gions, we hypothesized that CO staining might reveal buffered fixative containing 4% paraformaldehyde, and modular patterns in the EC. If so, CO staining could then cryoprotected in sucrose. be used to obtain further information about the EC modHuman tissue. Human EC specimens were obtained at ules regarding their shape, size, number, organization, and autopsy (12-24 hours postmortem) from normal individurelation to neuron clusters. An additional benefit of CO als without evidence of neurological disease. Specimens were used only if CO reactivity was intact in both EC and striate cortex, which was taken as a control because its CO Abbreviations pattern is well characterized in the human (Horton and Hedley-Whyte, '84). Specimens from six hemispheres from angular bundle ab four individuals (ages 3 days to 80 years) were found AChE acetylcholinesterase Alzheimer's disease AD acceptable. The human specimens were processed by immeramygdala Am€! sion fixation exactly as described above for monkey tissue. of amygdala basal nucleus Amg, The EC and striate cortex were removed and processed in of amygdala lateral nucleus Amg, parallel, using the same solutions, and identical fixation CA1, CA2, fields of hippocampus CA3 and CO reaction times. cytochrome oxidase co Histology. Cryoprotected specimens were sectioned at dentate gyrus DG 30 km (rat), 30-40 pm (monkey), or 40-60 pm (human) in entnrhinal cortex EC the coronal, sagittal, and horizontal planes. Some blocks caudal field of EC (monkey and human) E c, caudal limiting field of EC (monkey and human) Eri. from monkey and human were unfolded and flattened, then intermediate field of EC (monkey and human) EI sectioned tangential to the pial surface. This procedure was lateral entorhinal cortex (rat) Ei attempted with rat EC, but did not work well due to the lateral caudal field of EC (monkey and human) EIX lateral rostral field of EC (monkey and human) high curvature of the area. Alternate sections were stained El r medial entorhinal cort,ex (rat) EM for Nissl and/or CO (Wong-Riley, '791, mounted, and olfactory field of EC (monkey and human) E0 coverslipped. Immunohistochemistry was performed using rostral field of EC (monkey and human) EN antibodies and procedures that were described previously periamygdaloid cortex PAC (Hevner and Wong-Riley, '89, '90). For immunohistochemparasubiculum PaS piriform cortex PIR istry, antibodies were used at dilutions of 1:1,000 to 1:4,000 PrS presubiculum and were detected by the indirect immunoperoxidase rhinal sulcus rs method. Controls were incubated with the same concentrasubiculum SUB TH tion of preimmune serum substituted for anti-CO. field TH of parahippocampal gyrus (monkey)

CYTOCHROME OXIDASE IN THE ENTORHINAL CORTEX

Nomenclature. EC layers were identified according to Ram6n y Cajal ('01-'02); in this system, the lamina dissecans is equal to layer IV.Fields (subdivisions within the EC) were recognized based on heterogeneities in the laminar pattern observed by Nissl staining. The rat EC was subdivided into medial (EM) and lateral (EL) fields as described by Blackstad ('56); further differentiation of the atypical EC was recognized as described by Haug ('76). Fields in the monkey and human EC were identified using the system ofAmaral et al.('87). Our rationale for using the Amaral et al. ('87) nomenclature rather than other maps developed specifically for the human EC (Rose, '27; Sgonina, '38; Filimonoff, '47; Macchi, '51; Braak, '72) is explicated in Results.

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in the human (Amaral and Insausti, '90; Beall and Lewis, '91, '92). The monkey nomenclature has the additional major advantage that it is much better defined than the previous human EC nomenclature systems. In our experience, the EC subdivisions described for the human (Rose, '27; Sgonina, '38; Filimonoff, '47; Macchi, '51; Braak, '72) were not as clearly recognized as those described for the monkey (Amaral et al., '87). Some of the previous human maps also seemed unnecessarily complex (recognizing up to 23 EC fields), and most could not be readily correlated with our CO and Nissl staining results. The criteria set forth by Amaral et al. ('87), in contrast, were more consistent and better defined. Whereas it is clear that the human and monkey EC are not identical in all respects, we feel that the relative simplicity and clarity of the monkey EC nomenclaRESULTS ture justify its use for human. Laminar and field CO reactivity. Figure 1 shows low Different laminar and intralaminar patterns of CO reactivity were observed in each species and within each EC magnification views of parasagittal sections through medial field. Thus, CO reactivity, like Nissl cytoarchitecture, was and lateral portions of the human EC. These sections heterogeneous in the EC. For this reason the CO staining illustrate the heterogeneity of the human EC along both the rostrocaudal and the mediolateral axes, evident by both results are presented according to species and EC field. Nissl (Fig. lA,C) and CO (Fig. lB,D) staining. This heterogeneity was the basis for subdividing the EC into seven Cytochrome oxidase in the human distinct fields (Amaral et al., '87). Five of the fields are entorhinal cortex visible in the low magnification sections shown in Figure I: The human EC was located on the exposed surface of the Eo, ER,El, Ec, and ECL.Higher magnification views of three parahippocampal gyrus, lateral to the uncus. The extent of fields-EI, Ec, and EcL-are shown in Figure 2. The the EC was approximated by examining the intact tissue for boundaries between fields were gradual rather than sharp. Some features of the CO staining pattern were fairly the surface "verrucae" (Klingler, '48; Amaral and Insausti, '90; Solodkin et al., '91; Van Hoesen et al., '911, and for the general in the human EC. Most significantly, the neuron dark spots that characterize this area (present study). The clusters in layers I1 and I11 were darkly CO-reactive and essential features of the EC seen by Nissl and CO staining were clearly associated with vertically and horizontally were the same in the 3-day-old as in the adult specimens we adjacent regions of neuropil that were likewise darkly examined; however, only adult tissue is shown in the stained. The remainder of the areas between clusters (the interstices) was composed primarily of less CO-reactive figures. Zntegrity of human specimens. In human specimens, it neuropil. In cross section, the darkly CO-reactive regions in was considered that tissue autolysis in the interval between the upper EC layers had the shape of short columns or death and fixation might significantly inhibit or alter CO bulbs; tangential sections, however, revealed a unique and reactivity. To assess tissue integrity and enzyme preserva- remarkable horizontal arrangement of these regions. The tion, we processed blocks of striate cortex in parallel with specific patterns observed in each EC field, and in tangenEC from the same human hemispheres. Previous studies tial sections, are described in full below. Eo lolfactoryl. This field was characterized by a very thin have shown that in human striate cortex, CO staining reveals a characteristic pattern with specific features, such layer I1 and poorly developed layers IV-VI (Fig. 1A).Layer I as the darkly-reactive puffs or blobs in the supragranular was lightly CO-reactive; layer I1 was darkly reactive; layer layers (Horton and Hedley-Whyte, '84). These features I11 was darkly reactive in its outermost portion, and were clearly visible in striate cortex by both histochemistry moderately reactive in its inner portion; and the indistinct and immunohistochemistry in the present study (not layers IV-VI were lightly-to-moderately stained (Fig. 1B). shown), and robust CO reactivity was found in the EC as ER lrostrall. In Nissl stains, this field was distinguished by well. Furthermore, the same pattern of CO reactivity in EC the presence of cell clusters in layer I1 and outer layer I11 was consistently observed by both histochemistry and and by the absence of distinct layers IV and Vc except at the immunohistochemistry. The specificityof the immunohisto- transition with EI (Fig. 1A). Layer I of ERwas mostly lightly chemical reaction was tested by substituting preimmune stained for CO; however, islands of moderate-to-darkreacserum for CO antibody; these controls showed only faint tivity were observed extending up into layer I from some layer I1 neuron clusters (Fig. IB). The neuron clusters in background staining (Hevner and Wong-Riley, '89, '90). Human EC field nomenclature. We subdivided the hu- layers I1 and I11 were darkly CO-reactive, whereas the man EC according to the field designations of Amaral et al. cell-sparse interstices between the neuron clusters were ('87). This nomenclature was originally developed for the somewhat less reactive. The inner portion of layer 111, and monkey EC, but we found it easily adaptable to the human layers V and VI of ERwere moderately reactive for CO. This appeared to be the largest field in the EC, as there are many structural similarities between EC El from the two species. For example, at the rostra1 tip of the human EC, as it is in the monkey (Amaral et al., '87). The human EC (Fig. lA,B), layer I1 became very thin and layers distinguishing Nissl features of EI were the prominent IV-VI were poorly developed, as in macaque field E, neuron clusters in layer I1 and upper layer I11 and the (Amaral et al., '87). Other groups have also determined that relatively acellular layers IV and Vc (Figs. lA,C, 2E). The the EC fields as defined for the monkey can be distinguished neuron clusters in upper layer I11 were largest and most

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CYTOCHROME OXIDASE IN THE ENTORHINAL CORTEX developed in the anteromedial regions of this field (Figs. lA, 2E); layer I11 was thinner and more nearly continuous posterolaterally (Fig. IC). In CO-stained sections (Figs. 1B,D, 2F), layer I was generally lightly reactive, except in some places overlying layer I1 neuron clusters, where dark CO reactivity extended up almost to the pial surface (Fig. 1D). The cell clusters were darkly CO-reactive in both layers I1 and 111. The hypocellular interstices in layers I1 and 111 were generally less CO-reactive than the neuron clusters; however, “bridges” of dark CO staining between neuron clusters in layer 11 were sometimes seen in the interstices (Fig. 2F). (These “bridges” were also seen in tangential sections; see below.) Layers IV and Vc were marked by light reactivity and in fact were distinctive markers of EI in CO-stained sections. Layer W was somewhat more distinct medially than laterally (Fig. 1). The upper, cellular portion of layer V appeared thinner and less laminated in the human than in the monkey (Amaral et al., ’87);thus we designated the entire cellular layer Vab rather than distinguishing between sublaminae Va and Vb. Layers Vab and VI were both moderately CO-reactive (Figs. 1B,D, 2F). Field Ec characteristically had neuron clusEc ters in layer I1 but not in layer 111, and had no distinct layer IV by Nissl staining (Figs. lC, 2 0 . CO reactivity in layer I was mostly light, but dark reactivity was found extending into layer I from layer I1 neuron clusters (arrowheads in Fig. 2D). As Figure 2D shows, the dark reactivity sometimes followed a slight curve through layer I, rather than ascending vertically straight to the pial surface. Darkly CO-reactive neuron clusters in layer I1 were separated by moderately CO-reactive interstices. Layer I11 was more cellular in its outer half than in its inner half, but was uniformly dark by CO staining (Figs. lC,D, 2C,D). Layer Vab was moderately-to-darkly reactive. Layer Vc was barely discernible by CO staining, since it was only slightly paler than layer VI, which was moderately CO-reactive. ECL(caudal ltmttcngl. This most caudal field was marked by its lack of distinct fibrous layers IV and Vc and by its relatively cell-poor layer V in the human (Figs. lC, 2A). This is in contrast to monkey ECL, where at least part of layer V is prominently cellular (Amaral et al., ’87). (Despite this difference in cellularity, monkey and human ECL appeared quite similar by CO staining; compare Figs. 2B and 5D). Layer I was lightly reactive, except above neuron clusters in layer I1 (arrowheads in Fig. 2B). The layer I1 cell clusters were darkly CO-reactive, whereas the interstices were lightly-to-moderately reactive. Layer 111 was darkly reactive, except for its outermost one-third, which was moderately reactive. The rather poorly cellular layer V was

Fig. 1. Nissl-stained (A and C) and CO histochemically stained (B and D) parasagittal sections through the human EC. A and B show adjacent sections from the anterior half of the medial portion of the EC, and C and D show adjacent sections from the lateral portion of the EC. The relations between the EC, the amygdala, and the hippocampus are clearly seen. The boundaries between the fields of the EC labeled in A and C are indicated by arrowheads. In C and D, the anterior portion of the EC was cut obliquely, so the most anterior fields could not be identified. The posterior boundary of the EC is seen at the far right in C and D. Heterogeneity in the human EC is observed by both CO and Nissl staining, and several parallels with monkey EC structure are observed by comparison with Figure 3. The asterisks in A and B indicate isolated CO-reactive neuron clusters in the presubiculum. Orientation: A, I, P, and S indicate anterior, inferior, posterior, and superior. Scale bar: 2 mm.

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darkly reactive in its upper portion and moderately reactive in its lower portion. Layer VI was lightly-to-moderately reactive. EL^ (lateral rostra[) and EL^ (lateral caudali. These small lateral fields were identified along the medial bank of the collateral sulcus (not shown). Like their monkey counterparts, human ELr and ELc lacked a distinct layer IV; however, layer Vc did not appear to be thickened in the human as it is in the monkey (Amaral et al., ’87). Other laminae were generally similar in their CO staining intensity to the medially adjacent fields ERand EI. Tangential sections. The horizontal distribution of CO reactivity and neuron clusters in EC layers 1-111 was studied in tangential sections. We also used these sections to determine if “dark spots,” visible on the surface of the intact parahippocampal gyrus without staining (Fig. 3A), might be related to the neuron clusters or CO-rich areas in the EC. Although we have found no mention of these dark spots in the literature, we consistently observed them over the EC territory and thought they might be useful EC landmarks. They were located over the same distribution as the surface irregularities, or verrucae, that have been described previously (Klingler, ’48; Amaral and Insausti, ’90; Solodkin et al., ’91; Van Hoesen et al., ’91), but it was unclear if the spots and verrucae were precisely related. In tangential sections, the darkly CO-reactive regions in the upper EC layers appeared as discrete, irregularly contoured islands, surrounded by much lighter areas associated mainly with interstices (Figs. 3B, 4).The CO-labeled islands were often aligned in short rows or connected in bands. The diameter of the CO islands was variable, but ranged mostly from 200-800 km in layers 1-11; a few CO islands were smaller or larger than these limits. In serial sections, the CO-reactive islands in layer I were continuous with those in layer 11, where CO-reactive neuronal somata became visible. Layer I11 also contained dark CO islands (except in fields Ec and ECL), but these were generally not in register with the layer 1-11 islands. The layer 1-11 CO islands were quite discrete and consistently organized (Figs. 3B, 41, but the layer I11 islands had less definite margins and were less coherently organized (not shown). The layer 1-11 CO islands seemed to form local groups based on the contrast, size, and shape of the islands (Figs. 3B, 4). Two main groups of CO islands were recognized consistently: an anteromedial group and a posterolateral group. The anteromedial islands varied less in size, formed more interconnecting stripes, and were more evenly spaced than the posterolateral islands. The stripes formed by the anteromedial islands were oriented mainly perpendicular to the anteromedial border of the EC (Figs. 3B, 4). A transition zone joined the anteromedial and posterolateral island groups. As the lateral border of the EC was approached, the posterolateral islands came closer together and finally merged into a continuous sheet (Figs. 3B, 4).The anteromedial CO islands were probably located in Eo, EE, and EI, whereas the posterolateral CO islands were probably in Ec, ECL,ELr, and ELc. However, field boundaries could not be determined with confidence in the tangential sections; the gradualness of field transitions was quite evident in this plane. A close relation was found between CO-dark islands and neuron clusters in serial tangential sections (Fig. 3C,D), confirming our impressions from cross sections. Double CO- and Nissl-stained sections further demonstrated the neuron clusters within CO islands in single sections (not

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Fig. 3. “Dark spots” on the surface of the unstained and uncut human parahippocampal gyrus (A), and adjacent CO histochemically stained (B,D) and Nissl-stained (C) tangential sections from the same specimen. Individual dark spots on the cortical surface (arrows in A) have the same size, shape, and position as darkly CO-reactive islands in layers 1-11 (arrows in B), indicating that the dark spots represent the CO islands or the closely associated neuron clusters. The CO islands come closer together and fuse into a continuous sheet at the posterolat-

era1 edge of the EC (B). Anteromedially, the CO islands are more striplike, with their long axis oriented perpendicular to the anteromedial edge of the EC (B). The boxed region in B is shown at higher magnification in D. The association of dark CO staining with neuron clusters is shown in C and D. Bridges of dark CO reactivity connecting neuron clusters are seen in the tangential plane (arrowheads in C,D), as they were in cross sections. Orientation: A, L, M, and P indicated anterior, lateral, medial, and posterior. Scale bar: 1mm.

Fig. 2. Nissl-stained (A,C,E)and CO histochemically stained (B,D,F) parasagittal sections through the human EC. Adjacent sections through ECL(A,B), Ec (C,D), and EI (E,F) are shown. The cortical layers are indicated in A, C, E. Note that layers 11and 111are darkly CO-reactive in each of the fields. The clusters of large neurons in layer I1 are stained darker than the interstices, and dark reactivity extends up from the neuron clusters into layer I (arrowheads in A,B,C,D). A “bridge” of dark CO reactivity connects 2 neuron clusters in E and F (arrowheads). Layer 111 also contains darkly CO-reactive clusters, but only in anterior fields such as EI, and not in Ec or ECL.The posterior border of the EC, where ECL meets parahippocampal cortex, is marked by a sharp decrease in CO reactivity, especially in layer 111 (open arrows in A and B). Scale bar: 1mm.

shown). Tangential sections also revealed that small, presumably mostly glial cells were concentrated outside the CO-reactive islands in layer I (not shown), a Point that was not appreciated in cross sections. The horizontal “bridges” of dark CO reactivity between layer I1 neuron clusters, previous~ynoted in cross sections, were likewise visible in serial tangential sections (arrowheads in Fig. 3C,D)3 and in double co- and Nissl-stained sections (not shown). We compared the distribution of ‘‘dark spots” on the surface of the intact EC with the distribution of CO islands by photographing the intact parahippocampal mrus’ and then staining tangentid sections from the Same specimen for CO. The dark spots were clearly related to the CO

Fig. 4. Comparison of CO island patterns in right (A) and left (B) hemispheres from the same brain, and comparison of CO immunohistochemistry (A and B) and histochemistry (C) in tangential sections through layers 1-11 of the human EC. The section in A contained only the posterior two-thirds of the EC, including the posterior border, where the islands fuse into a continuous sheet. The lateral border is aiso clear in A, since the rhinal sulcus (rs) was unfolded and flattened. The sections in B and C contained almost the entire extent of the EC;

however, the boundaries of the EC are not quite visible, except posterolaterally where the CO islands fuse. The right (A) and left (B) hemispheres appeared to have no precise symmetry of the CO island pattern. Identical patterns were shown by CO immunohistochemistry (B) and histochemistry (C), except that slightly more contrast was obtained by immunohistochemistry. Orientation: A, L, M, and P indicate anterior, lateral, medial, and posterior. Scale bar: 2 mm.

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CYTOCHROME OXIDASE IN THE ENTORHINAL CORTEX islands (Fig. 3A,B); in some examples, individual dark spots and CO islands were matched in position, size and shape (arrows in Fig. 3A,B). We counted 247 dark spots and 224 CO islands in the photographs shown in Figure 3A,B. These counts probably underestimated the actual numbers of spots and islands, as the EC surface was not entirely exposed. The symmetry of the CO patterns was studied in left and right hemispheres from the same brain (Fig. 4A,B). The general features of the CO pattern, such as island size and shape, were similar in both hemispheres, but no precise right-left symmetry was observed. The results obtained by CO histochemistry and immunohistochemistry were also compared (Fig. 4B,C). Identical patterns were seen by both methods, indicating that the CO islands were enriched in both enzyme activity and enzyme protein. At the cellular level, however, neuronal cell bodies were more intensely labeled by immunohistochemistry than by histochemistry (not shown). Boundaries o f the human EC. The posterior boundary of the human EC was sharply demarcated in CO-stained cross sections (Figs. 1C,D, 2A,B). The anterior EC boundary was more gradual than the posterior boundary, but was nevertheless clearly seen by both CO and Nissl staining (Fig. 1A,B). The medial and lateral borders of the EC were also easily discerned in CO-stained cross sections (not shown). In tangential sections, the posterior and lateral limits of the EC were marked by fusion of the CO islands into a continuous sheet (Figs. 3B, 4). The anterior and medial limits, however, could not be defined by this criterion, since both periamygdaloid cortex (anteriorly) and parasubiculum (medially) contained CO-reactive cell clusters (not shown; see also Amaral and Insausti, '90).

Cytochrome oxidase in the monkey entorhinal cortex Our results are based on studies of three different species of macaques. The main features of the EC, observed by Nissl and CO staining, appeared to be essentially the same in these species. Witter et al. ('89) have also reported that the Nissl cytoarchitecture of the EC does not differ in major respects between the fascicularis and rhesus macaques. However, we did not examine all the planes of section necessary for a complete analysis in each species, and it is possible that some differences between species were present, but did not affect the structure or CO reactivity of the major EC fields and layers. Laminar and field CO reactivity. The EC and other major structures of the medial temporal lobe are shown in parasagittal sections at low magnification in Figure 5. In addition to the amygdala and hippocampus, five of the seven EC fields are visible in this figure. Heterogeneity was seen in both the Nissl and CO staining patterns (Fig. 5) and was the basis for marking the field boundaries (Amaral et al., '87). Generally, CO reactivity was dark in the neuron clusters and lighter in the interstices. The neuron clusters and CO-stained regions were, however, less distinct than they were in the human (Fig. 5). Also, layer I in the monkey did not contain distinct regions of dark CO staining above the layer I1 neuron clusters. Finally, tangential sections revealed that the organization of layer I1 neuron clusters and CO reactivity in monkey did not follow the island and stripe pattern that was observed in the human EC. More detailed

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descriptions of each field, and of the tangential sections, are given below. Eo. The most rostra1 of the EC fields, Eo was recognized by its very thin layer I1 and poorly developed layers IV-VI. Layer I was lightly CO-reactive;layer I1 was darkly reactive; and layer I11 was moderately-to-darkly reactive, with its outer portion being darker than its inner portion (Fig. 5). The rudimentary layers IV-VI were lightly-to-moderately reactive. ER. This field was recognized by the poorly developed layers IV and Vc and by the thick but nonlaminated layer VI. Layer I was lightly CO-reactive, and layers 11-111 were darkly reactive (Fig. 5). Many of the neurons in layers I1 and I11 of ER were grouped into clusters. The neuron clusters were stained slightly darker for CO than were the cell-sparse interstices. Layers IV-VI were moderately reactive. Layers IV and Vc were barely visible, as bands of somewhat paler CO staining compared to the adjacent layers Vab and VI. El. The largest EC field was characterized by the welldeveloped layers IV and Vc and by the coiled appearance of layer VI in Nissl stains. Since CO staining in EI was in many ways typical of the monkey EC, we show this field at higher magnification in Figure 6A,B. Layer I of EI was lightly reactive for CO (Fig. 6B). In layers I1 and 111, staining was darker over the neuron clusters than over the interstices between clusters. This point was sometimes difficult to appreciate in cross sections, because the neuron clusters sometimes ended or ran obliquely to the plane of section. However, tangential sections established the relation between dark CO staining and layer I1 neuron clusters more convincingly (Fig. 7). Layers IV and Vc in EI were lightly reactive for CO, consistent with their high contents of myelinated fibers (Amaral et al., '87). Layers Va, Vb, and VI were moderately CO-reactive. The radially oriented bundles of myelinated fibers that traverse EI (Amaral et al., '87) appeared as CO-pale lines streaming vertically through darker stained tissue in layers V and VI (Fig. 6B). Ec. In this field, layer IV characteristically lost its distinct appearance in Nissl stains, whereas layer Vc remained prominent. The neurons of layers I1 and I11 were mostly continuously distributed with rare cluster formation in layer 11. The CO reactivity of most layers was similar to that of the corresponding layers in El, except in layer VI, which appeared slightly lighter than in other EC fields (Fig. 5).Also, layer IV became indistinct by CO, as well as Nissl staining. EcL. In the most caudal EC field, layer V thinned and lost its laminar appearance, so that layer Vc no longer was visible. Higher magnification views of EcL are shown in Figure 6C,D. Layer I was lightly CO-reactive. Layer I1 was described by Amaral and coworkers ('87) as mostly continuous in ECL,but we observed a few small neuron clusters in this field (Fig. 6C), which were associated with dark CO staining (Fig. 6D). The remainder of layer I1 was slightly less reactive than the neuron clusters. The wide layer I11 stained darkly for CO, except for the uppermost portion, which was moderately CO-reactive. Layer V was slightly darker in its upper portion compared to its moderately reactive lower portion. Layer VI was lightly-to-moderately reactive. EL,.and Ek. In these small fields located on the medial bank of the rhinal sulcus, the wide layer Vc was lightly reactive for CO, and layer IV could not be distinguished by Nissl or CO staining (not shown). In other layers, staining


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Figure 5


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intensity was similar to that in the medially adjacent fields ERand EI. Tangential sections. As mentioned above, the tangential sections confirmed that dark CO staining was associated with neuron clusters in layers 11-111 throughout most of the monkey EC. The neuron clusters were most prominent, however, in ERand EI (Fig. 7). The tangential sections also revealed that the neuron clusters formed a few discrete islands (these being most common in anterior fields), but were more often interconnected with each other by cellular bridges (Fig. 7A). Bakst and Amaral ('84)have also observed this interlocking arrangement in Nissl-stained tangential sections. Boundaries of the monkey EC. The posterior boundary with neocortical area TH was sharply delimited by the higher CO reactivity in the EC, especially in layer I11 (Figs. 5B, 6D). The anterior border, where EC meets periamygdaloid cortex, was comparatively gradual by both CO and Nissl staining (Fig. 5). The medial and lateral borders of the EC were distinct in both CO- and Nissl-stained sections (not shown).

islands. Details of the CO distribution in each field are given below. EMimedLal,. The distinguishing Nissl features of EMwere the thick and continuous layer 11, and the distinct lamina dissecans, which thinned ventrally (Figs. 8A, 9A,C,E). CO staining showed consistent laminar reactivity patterns, which differed between the dorsal and ventral portions of EM (Figs. 8B, 9B,D,F). Layer I was lightly-to-moderately reactive, and layer I1 was darkly reactive, throughout EM. The dark reactivity in layer I1 extended slightly above and below this layer, into adjacent portions of layers I and 111. Layer I11 was moderately CO-reactive, except along the border with layer 11, where dark reactivity was seen. Layer IV was lightly CO-reactive in dorsal EM,but darkened to become moderately reactive in ventral EM.Layer V was only moderately reactive in dorsal EM, but was quite darkly stained in ventral EM (Figs. 8, 9). Examination at higher magnifications indicated that the dark reactivity was located in both cell bodies and neuropil of layer V (not shown). Layer VI was lightly CO-reactive dorsally, but moderately reactive in ventral EM. The inner portion of layer VI was slightly darker than the more superficial Cytochrome oxidase in the rat portion at all dorsoventral levels. entorhinal cortex EI, (lateral,. This field was seen best in horizontal sections The rat EC curves sharply around the posterolateral (Fig. 9). As originally noted by Blackstad ('561, most of EL cerebral surface. For this reason, our attempts to flatmount was located ventrally; EL narrowed dorsally, so that only EM and tangentially section the rat EC were unsuccessful. Our remained at the most dorsal levels (Fig. 9). The neuron analysis was thus restricted to parasagittal and horizontal clusters in layer I1 of ELwere also most prominent ventrally planes, which cut the EC approximately in cross section (arrowheads in Fig. 9E). CO reactivity in EL was similar to (coronal sections were also obtained, but they intersected that in EM, with a few exceptions (Fig. 9B,D,F). The most the EC obliquely). noticeable difference was that CO staining was heterogeLaminar and field CO reactivity. Figure 8 shows neous within layer I1 of EL: dark CO reactivity marked the parasagittal sections through the entire dorsoventral ex- neuron clusters, while lighter staining was seen in the gaps tent of the medial EC (field EM). Figure 9 shows horizontal between clusters (Fig. 9E,F). Also, layers 111-VI in EL sections through both EM and the lateral EC (field EL). appeared somewhat less reactive than the same layers in These figures demonstrate heterogeneity in the Nissl and EM.The dark staining of layer V ventrally in EMdid not CO staining patterns, not only between the classical fields extend into EL, but faded in the transition zone between EMand EL (Blackstad, '561, but also to some extent within these fields (Fig. 9D,F). these fields, along the dorsoventral axis. Boundaries o f the rat EC. The dorsal boundary was Neuron clusters in the rat EC were found only in layer 11, clearly marked by sharp differences of CO activity in the and only in field EL (Figs. 8, 9). In general, the neuron clusters were darkly reactive in CO-stained sections. There adjacent neocortex (Fig. 8B). The lateral and medial borwas no evidence of CO-reactive bridges between neuron ders of the EC were likewise easily distinguished by CO clusters, nor of CO-rich columnar extensions into layer I. staining (Fig. 9B,D,F). Ventrally, the EC met a thinner The curvature of the rat EC prevented us from obtaining cortex with fewer laminae, which Haug ('76) interpreted as the tangential sections necessary for studying the horizon- the atypical EC (Fig. 8).CO staining supported this interpretal organization of the neuron clusters and CO-reactive tation, since CO reactivity was fairly continuous from the classical EC into the ventral atypical region. In particular, the thin ribbon of dark CO reactivity marking layer V in ventral EMcontinued into the atypical EC (arrow in Fig. 8B). Farther ventrally, where the atypical EC met the periamygdaloid cortex and amygdala, a fairly sharp border Fig. 5. Adjacent Nissl-stained (A) and CO histochemically stained (B) parasagittal sections through the macaque monkey medial tempo- was seen by CO staining (not shown). ral lobe. The relation of the EC to the amygdala and hippocampus is clearly seen. The boundaries between EC fields, labeled in A, are indicated by arrowheads. The heterogeneity of the EC is evident by both Nissl and CO staining. Note the sharp decrease in CO reactivity at the posterior boundary of the EC, where Eel, meets neocortical area TH. The other structures of the medial temporal lobe are also well demarcated by GO staining. For example, the basal nucleus of the amygdala is stained darker than the lateral nucleus. (These 2 nuclei are seen together because the plane of section was tilted slightly from the parasagittal, to intersect the EC more perpendicularly.) The structures of the hippocampus are also recognized by their characteristic CO staining patterns (Kageyama and Wong-Riley, 1982). Orientation: A, I, P, and S indicate anterior, inferior, posterior, and superior. Scale bar: 1 mm.

DISCUSSION In the present study we used cytochrome oxidase (CO) as a marker to analyze the metabolic organization of the entorhinal cortex (EC), with particular attention to modular patterns. We studied the distribution of CO as it relates to layers, fields, and neuron clusters in the EC. The distribution of darkly CO-reactive neurons, although not a focus of the present study, has been examined by Carboni et al. ('90).

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in postsynaptic cell bodies and dendrites (Kageyama and Wong-Riley, '82; Mjaatvedt and Wong-Riley, '88, '91). At the tissue level, CO activity has been correlated with capillary density and glucose utilization (Borowsky and Collins, '89b). Ion pumping appears to be the major biochemical process related to neural functional activity (Erecinska and Silver, '89), and in this regard, we have found that the distribution of CO in brain is correlated with that of Na+,K+-ATPase,the ion pump responsible for most neuronal ATP consumption (Hevner et al., '92). On the basis of such findings, we have proposed that CO may be a marker for functional activity in neurons and neural pathways (Wong-Riley, '89). According to this proposition, we would suggest that the areas of dark CO reactivity in the EC-such as the islands in layers II-IIIare sites of intense neuronal activity and that the CO distribution represents a functional (as well as metabolic) map of EC. Further studies will be needed to assess the accuracy of this interpretation.

Comparative laminar and field analysis In this section, we relate the intensity of CO staining in each layer to relevant anatomical and physiological data and interpret differences in CO staining between species to anatomical and physiological differences. Layer I. CO staining intensity was mostly low-tomoderate throughout layer I, except over neuron clusters in Fig. 7. Adjacent Nissl-stained (A) and CO histochemically stained human EC. The significance of this dark staining over the (B) tangential sections through layers 1-11 of the monkey EC. In these neuron clusters is discussed with layer I1 (see below). sections, the CO-reactive neuron clusters (arrows) are most prominent Layer I is unique in being the only layer to receive direct in the middle portion of the EC, but they were also prominent projections from the olfactory bulb in the rat and monkey anteriorly in neighboring sections. Note how most of the cell clusters (Turner et al., '78; Kosel et al., '81; Amaral et al., '87), and interlock and surround the less cellular interstices. The shallow medial curve of the rhinal sulcus (rs), marking the posterior boundary of the presumably human. These projections end in the outer EC, is visible in these sections. Orientation: A, L, M, and P indicate portion of layer I ("layer Ia") throughout most of the rat EC, but only in Eo in the monkey (Turner et al., '78; Kosel anterior, lateral, medial, and posterior. Scale bar: 2 mm. et al., '81; Amaral et al., '87). No difference between outer and inner layer I was discerned by CO staining in any of the species; edge effects caused apparent dark staining of outer Cytochrome oxidase as a functional marker layer I in some sections, but only low or moderate staining Previous studies have established that CO activity is was observed in areas where sections lay flattest on the often closely related to the level of functional activity in slide. Thus the direct olfactory bulb projection to the EC neural pathways: stimulation of a given pathway generally may not be robust compared to other layer I inputs. In the leads to increased CO activity, whereas deprivation of input rat, other layer I inputs include intrinsic connections from leads to decreased CO activity. This has been demonstrated the lower cortical layers (Kohler, '86, '88) and extrinsic in many systems including the visual, auditory, somatosenconnections from the periamygdaloid and piriform cortices, sory, and limbic pathways (reviewed by Wong-Riley, '89). predominantly to EL (Krettek and Price, '77). In the The neurophysiologic events underlying the increased monkey, projections to layer I have been traced from functional and metabolic activity remain to be identified neocortex and from amygdala, among other structures exactly, although several correlations have been observed. (Insausti et al., '87a,b; Saunders and Rosene, '88). In studies involving neuronal physiological recordings, high Layer ZZ. Dark CO staining was observed in much of CO levels were associated with strong synaptic inputs layer I1 in all three species. The cellular portions of this (Mawe et al., '90) and with high spontaneous activity layer, and in particular the neuron clusters, were the sites (Livingstone and Hubel, '84). In particular, putative strong of darkest CO staining. This suggests that the perforant excitatory projections have been linked to high CO activity pathway, which projects from the EC layer I1 cell clusters to the ipsilateral dentate gyrus and CA2-3 (Steward and Scoville, '76; Witter and Amaral, '911, is highly active Fig. 6 . Nissl-stained (A$) and CO histochemically stained (B,D) metabolically (and perhaps functionally). This conclusion is supported by the findings that (1) CO staining is dark in parasagittal sections through the monkey EC. Adjacent sections through El (A,B)and EcL(C,D) are shown. The cortical layers are indicated in A dendrites postsynaptic to the perforant pathway in the and C. Layers I1 and I11 were stained darkly for CO in both EI and ECL. outer two-thirds of the dentate molecular layer (Kageyama Layer I1 was broken into clusters associated with dark CO staining and Wong-Riley, '82), and (2) CO reactivity in the outer (arrowheads in A,B,C,D). The clusters were larger in ELthan in ECL. two-thirds of the dentate molecular layer declines following Note the CO-pale fibers running vertically through layers Va-VI in EI. The posterior boundary of the EC with TH is indicated by open arrows ablation of the ipsilateral EC (Borowsky and Collins, '89a). Furthermore, it has been known for many years that in C and D; it coincides with a shallow sulcus, which is the medial stimulation of the EC causes massive excitation of the continuation of the rhinal sulcus. Scale bar: 0.5 mm.

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Fig. 8. Adjacent Nissl-stained (A) and CO histochemically stained (B)parasagittal sections through the rat medial EC (EM).The cortical layers (I-VI) are labeled in A. Note the dark CO reactivity in layer 11, and in the ventral half of layer V (arrow in B). The dark reactivity in

layer V was observed only in EM.The superior boundary of EMis clearly visible by both staining methods (arrowheads in A,B). The thinner cortex seen inferiorly is the atypical EC. Orientation: C, D, R, and V indicate caudal, dorsal, rostral, and ventral. Scale har: 0.5 mm.

dentate granule cells (Andersen et al., '66). In the rat, the smaller projection from layer I1 in EL to neocortex may likewise be highly active (Kosel et al., '82; Kohler, '86). The dark CO reactivity could also be a consequence of strong excitatory synaptic input to the layer I1 islands, necessitating active ion pumping. In the rat, layer I1 receives inuut from uarasubiculum and uresubiculum (to EM),from periamygdaloid cortex (to EL), and from lower layers via intrinsic projections (Krettek and Price, '77; Kohler, '85, '86, '88). In the monkey, layer I1 inputs come from many structures, including a strong projection from parahippocampal cortical area TF (Insausti et al., '87a,b; Saunders and Rosene, '88). Alternatively, the intrinsic electrical properties of the layer I1 neurons may account for the high energy requirements of these cells. Alonso and coworkers have detected intrinsic Na+-dependent membrane potential oscillations in rat layer I1 stellate neurons, which may be involved in generating the theta rhythm (Alonso and Garcia-Austt, '87a,b; Alonso and Llinas, '89).

The layer ZZ interstices. CO activity was lower in the layer I1 interstices than in the neuron clusters. It might be supposed that this difference could reflect variations in cell density, but no relation between cell density and CO reactivity has generally been found in brain. In primate V1

Fig. 9. Nissl-stained (A,C,E)and CO histochemically stained (B,D,F) horizontal sections through the rat EC. Each pair of adjacent sections and came from a different dorsoventral level. A and B were dorsal; C and D were intermediate; and E and F were ventral. The cortical layers (I-VI) are indicated in A. The boundary between the medial (EM)and lateral (EL) fields of the EC is indicated by an open arrow in the Nissl-stained section at each level, except in A where only EMis visible. Note the dark CO reactivity of layer I1 throughout the EC, and of layer V in ventral EM(arrows in D,F). In ventral EL,layer I1 is broken up into neuron clusters (arrowheads in E) that are darkly CO-reactive (arrowheads in F), and are separated by less reactive interstices. Orientation: C, L, M, and R indicate caudal, lateral, medial, and rostral. Scale bar: 0.5 mm.


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and V2, CO reactivity varies independently of cell density (Horton and Hubel, '81; Livingstone and Hubel, '82; Tootell et al., '83; Carroll and Wong-Riley, '84; Horton, '84; Wong-Riley and Carroll, '84). In the rodent somatosensory cortex, CO staining is actually lower in the cell-dense barrel walls than in the neuropil-filled barrel hollows (Wong-Riley and Welt, '80). It seems more likely that the CO staining differences between interstices and neuron clusters reflected differences in neuronal anatomical and physiological properties, as would be consistent with most results concerning CO. This concept is intriguing in view of recent anatomical work indicating that the interstices and layer I1 neuron clusters may be associated with different neural pathways. Golgi studies of monkey EC have shown that the apical dendrites of layer I11 cells bend away from the layer I1 neuron clusters and curve upward through the interstices (Amaral et al., '87; Carboni et al., 'go), suggesting that the dendritic fields of cells in these layers are segregated. Anterograde tracing studies in monkey have demonstrated that many afferents to the EC end with a patchy distribution localized mainly over either the neuron clusters, e.g., from parahippocampal area TF, or over the interstices, e.g., from the lateral amygdaloid nucleus (Insausti et al., '87a,b; Saunders and Rosene, '88). Patchy inputs to the rat EC also seem to end preferentially over the neuron clusters or interstices, although the correlation is less clear in this species (Krettek and Price, '77; Kohler, '85, '86, '88). These data suggest that the layer I1 interstices are associated with the pathway through layer 111, which is separate from the pathway through layer I1 (Steward and Scoville, '76; Witter and Amaral, '91). This segregation of the layer I1 and layer I11 pathways probably extends vertically into the neuropil of layer I; this idea is strengthened by our observation that layer I regions above layer I1 neuron clusters in human were more darkly reactive than layer I regions above interstices. Presumably, metabolic (and possibly functional) activity differ between the apical dendritic fields of layer I1 neurons and the apical dendritic fields of layer I11 neurons. Electron microscopic cytochemistry could be used to determine exactly which neuronal elements in layers 1-11 are responsible for these staining differences. Electrophysiological studies could also provide insight into the functional differences between the different pathways through layers I1 and 111 and the layer I subregions associated with each. Layer ZfZ. In the rat, CO staining was lighter in layer I11 than in layer 11. In the monkey and human, however, cellular portions of layers I1 and 111 were both quite darkly reactive for CO. The comparison between layers I1 and I11 is important because these layers give rise to the two main components of the projection from EC to hippocampus and dentate gyrus (Steward and Scoville, '76; Witter and Amaral, '91). Our results suggest that the pathway from layer 111 to CA1 and subiculum has different properties (and is perhaps less functionally active) than the pathway from layer I1 to dentate gyrus and CA3, especially in the rat. The pattern of CO reactivity in monkey and human layers 1-11 was consistent with this idea (see discussion of layer 11, above). For both pathways, the hippocampal regions postsynaptic to their efferent projections are darkly COreactive, in the rat as well as the monkey (Kageyama and Wong-Riley, '82). Inputs to the layer 1-11 apical dendritic fields of layer 111 neurons were discussed with layer I1 (see above). Other

R.F. HEVNER AND M.T.T. WONG-RILEY inputs, ending within layer 111,have also been described. In the rat, layer 111 receives major projections from olfactoryrelated cortices, and the strength of these inputs increases ventrolaterally within the EC (Krettek and Price, '77; Van Groen et al., '87). This input may be a basis for the increasing CO reactivity that was noticed ventrolaterally in layer I11 of rat EC, especially in EL. However, the size of the efferent projection from layer I11 to CA1 also increases ventrolaterally (Witter et al., '881, and this could also influence CO activity in this layer. In the monkey, many inputs to layer I11 have been identified, including a prominent afferent projection from perirhinal cortical area 35 (Insausti et al., '87a,b; Saunders and Rosene, '88). The formation of CO-reactive neuron clusters in layer 111 in some fields of the primate EC may indicate that layer 111 is partially organized into modules, as appears to be true throughout layer 11. The layer 111 neuron clusters, like those in layer 11, were darkly CO-reactive. Layer ZV. This layer, the lamina dissecans, had lowmoderate CO activity. Layer IV contains a plexus of basal dendrites of layer I11 neurons (Ramon y Cajal, '01-'02; Lorente de No, '331, as well as many myelinated axons, at least in monkeys (Amaral et al., '87). Myelinated axons are low in CO and tend to decrease the overall metabolic activity of regions with high myelin contents (Wong-Riley, '89). No specific afferents to this layer have been identified. Layer V. CO activity was low-to-moderate in most of layer V, with a few exceptions. Most strikingly, CO activity was specifically heightened within this la,yer in the ventral half of EMin the rat. Layer V receives a major afferent input from the subiculum (Shipley and Sgirensen, '75; Rosene and Van Hoesen, '77; Beckstead, '78; Sgirensen and Shipley, '79; Saunders and Rosene, '88), which in the rat is densest ventromedially (Kohler, '85). This suggests that the projection from subiculum may be a major excitatory input to layer V. This would be consistent with the important role ascribed to this projection as a link in the feedback pathway from the hippocampus, through EC, t o the neocortex (Rosene and Van Hoesen, '77; Kosel et al., '82; Van Hoesen, '82; Sgirensen, '85; Swanson and Kohler, '86; Witter and Groenewegen, '86; Amaral, '87). Other inputs that may influence CO levels in rat are the intrinsic projection to layer V in EMfrom the same layer in EL (Kohler, '881, and the projection from CA3 to this layer (Swanson and Cowan, '77; Swanson et al., '78). The pyramidal cells in layer V, especially in EL, send outputs to many areas of the rat neocortex (Kosel et al., '82; Swanson and Kohler, '86). In the monkey and human, layers Va-b had CO levels consistent with moderate functional activity, but layer Vc had lower CO levels, in accordance with its higher content of myelinated fibers (Amaral et al., '87). Layer V in the monkey receives input from many sources, including prominent projections from the amygdala and subiculum (Rosene and Van Hoesen, '77; Insausti et al., '87a,b; Saunders and Rosene, '88). In turn, layer V in the monkey projects to neocortex, CA1, and back to subiculum, as it does in the rat (Kosel et al., '82; Witter and Amaral, '91). Layer V f . CO staining was generally moderate in this layer, except dorsally in the rat and caudally in the monkey, where CO reactivity fell somewhat. The significance of these variations in CO activity is unclear. In the rat, layer VI receives afferent input from CA1 (Swanson et al., '78) and probably from other extrinsic sources. In the monkey, layer VI receives a strong afferent projection from the orbitofrontal cortex (area 13),as well as several inputs from


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where input from the lateral geniculate nucleus is received. In normal animals, the layer IV columns subserving left and right eyes are equal in CO reactivity, and no differences between ocular dominance columns are seen. It could be that some type of modular organization is likewise masked in the deeper layers of EC, but it is equally possible that the deeper EC layers lack modularity entirely. In any case, functional modules are not always revealed by CO staining. Modular patterns in the entorhinal cortex Rather, physiological methods provide the most accurate The most distinct modular elements in EC were the information concerning functional organization. neuron clusters and CO islands in layer 11. The darkly Species differences. In the rat, layer I1 neuron clusters reactive islands defined by CO staining included more than and CO-reactive islands were found only in EL,suggesting just the neuron clusters, since dark CO reactivity extended that layer I1 is only partially modular in this species, like outside the neuron clusters and into some contiguous zones layer I11 in the primates. Layer I11 in rat appeared to lack of neuropil. The CO-reactive islands thus define a type of structural or metabolic modularity. The significance of metabolic compartment in the EC. They enclose not only these interspecies differences in EC modularity is uncertain the neuron clusters, which may be considered structural in the absence of physiological data. The primate EC modules, but also overlying zones in layer I and adjacent receives much more neocortical input than does the rat EC neuropil in layer 11. This leaves most portions of the (Jones and Powell, '70; Van Hoesen and Pandya, '75a,b; CO-poor layer I1 interstices, and presumably the CO-poor Van Hoesen et al., '75; Beckstead, '78; Amaral, '87; Insausti layer I regions above them, as separate and distinct modu- et al., '87a), but other differences could also account for the lar compartments. Although the interstices do not form greater modularity in the primate EC. discrete units like the neuron clusters, they may nevertheCorrelation with acetylcholinesterase (AChE). Like CO, less be considered separate modular compartments (com- AChE staining specifically labels zones associated with the pare with V1 interpuffs; see below). We hypothesize that layer I1 neuron clusters in human EC (Kelovic and Kosthe different compartments in EC layers 1-11 may represent tovic, '81; Green and Mesulam, '88). The AChE-labeled functional modules having distinct physiological properties, zones extend vertically into layer I neuropil and appear as as in the primate visual cortex, the rodent barrel cortex, and islands or bands in tangential sections (Kelovic and Kosthe olfactory bulb. Clearly, physiological studies would be tovic, '81). Thus the distribution ofAChE is probably quite needed to test this idea. similar to that of CO in the upper layers of the human EC. In the primates, layer I11 also contained some modular This suggests that cholinergic input is associated with the elements (both neuron clusters and CO-reactive islands), same modules as are labeled by CO staining. Both methods but they were less differentiated than in layer I1 and were may be useful for revealing the modular pattern, although found only in some EC fields. Also, their organization was they are based on different reactions and yield different poorly defined even in tangential sections. As in layer 11, types of information (metabolic vs. neurochemical). though, a close relation was observed between the modules AChE staining is also heterogeneous in the upper layers identified structurally (neuron clusters) and metabolically of monkey and rat EC. The spots of dark AChE reactivity in (CO staining). These data suggest that layer I11 is partially rat EC, although clearly visible, have not received much modular in primates, at least at the structural and metaattention in previous studies (Storm Mathisen and Blacksbolic levels. Physiological methods could be used to test for tad, '64; Paxinos and Watson, '86). Their relation to either functional modules in layer 111. We found no metabolic or the neuron clusters or the CO-rich islands is uncertain at structural evidence for modularity in the deeper layers present. In monkey EC, dark AChE reactivity labels neuron (IV-VI) of the EC. clusters in some fields, but mainly interstices in other fields Comparison with striate cortex. The CO-defined meta- (Bakst and Amaral, '84). This lack of strict correlation bolic organization of EC is in some ways reminiscent of that between CO and AChE in the monkey EC is consistent with in primate visual area V1. In V1, the puffs of dark CO previous work, which has generally shown no specific reactivity in layers 1-111 form discrete units surrounded by relation between CO and transmitter systems (Wong-Riley, a matrix of lower CO reactivity known as the interpuffs '89). Other methods that have previously been applied to (Horton and Hubel, '81; Carroll and Wong-Riley, '84; the EC, such as Timm's (Amaral et al., '87) and pigment Horton, '84). Thus the spatial relation between puffs and staining (Braak, '721, likewise seemed to have no consistent interpuffs resembles that between clusters and interstices relation with CO. in the EC. The V1 puffs are not, however, associated with any structural modules observable by Nissl staining. The Cytochrome oxidase, the entorhinal cortex, V1 puff and interpuff neurons have distinct physiological and Alzheimer's disease properties and anatomical connections (Livingstone and Hubel, '83, '84, '871, indicating that they are functional as The modular distribution of CO in the EC may have some well as metabolic modules. For example, neurons in the relevance to studies of human neurologic diseases, espepuffs are more responsive to specific color stimuli (Living- cially Alzheimer's disease (AD). Both the EC and CO have stone and Hubel, '84). An analogous modular physiological previously been implicated in AD. Parker et al. ('90) have organization could be present in EC, and the anatomical reported that platelet mitochondria from AD patients are data identifying two separate pathways through the EC deficient in CO, but not other respiratory enzymes. Our would be consistent with this idea. However, there is as yet own preliminary evidence indicates that AD patients may also have CO abnormalities in brain tissue as well (R. Egan no physiological evidence bearing on this point. In V1, the puffs are centered within ocular dominance and M. Wong-Riley,unpublished observations). Neuropathocolumns extending throughout the cortical thickness. The logical studies have found that the EC, and the adjacent ocular dominance columns are most discrete in layer IV, transentorhinal and perirhinal cortices, are the regions

other sources (Insausti et al., '87a,b; Saunders and Rosene, '88). Efferents from layer VI project to many neocortical areas and to intrinsic hippocampal formation targets in the rat (Swanson and Kohler, '86; Kohler, '86, '88). In the monkey, layer VI sends projections to the dentate gyrus, CA2, and CA3 (Witter and Amaral, '91).


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that are first and most severely affected by AD pathology (Hyman et al., '84, '86; Van Hoesen and Damasio, '87; Bra& and Braak, '91; Van Hoesen et al., '91). Within the EC, AD lesions are concentrated in the layer I1 neuron clusters, which were found to be CO-rich in the present study. These findings raise the intriguing possibility that primary or secondary deficits of neuronal CO activity may be related to cell death and lesion formation in the EC of AD patients. Our present results concerning CO in the normal EC may serve as a basis for future studies to investigate this possibility.

ACKNOWLEDGMENTS We thank Dr. K.-C. Ho for providing human brain tissue at autopsy, and Drs. C. Nguyen-minh and V.M. Haughton for providing brains from monkeys used in their studies. This work was supported by NIH Grant NS18122 (M.W.R.) and by the Medical Scientist Training Program at the Medical College of Wisconsin (R.F.H.).

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The compartmentalization of the monkey and rat cerebellar cortex: zebrin I and cytochrome oxidase.

Localization of cytochrome oxidase (COX) activity and COX mRNA in the hippocampus and entorhinal cortex of the monkey brain: correlation with specific neuronal pathways.

Functional topography of the human entorhinal cortex.

Relationships between dendritic morphology and cytochrome oxidase compartments in monkey striate cortex.

A map of abstract relational knowledge in the human hippocampal-entorhinal cortex.

Functional subregions of the human entorhinal cortex.

Entorhinal Cortex Thickness across the Human Lifespan.

Structure of cytochrome a3-Cua3 couple in cytochrome c oxidase as revealed by nitric oxide binding studies.

Neurons of the lateral entorhinal cortex of the rhesus monkey: a Golgi, histochemical, and immunocytochemical characterization.

Entorhinal cortex of the monkey: V. Projections to the dentate gyrus, hippocampus, and subicular complex.

Connections between the pulvinar nucleus and the prestriate cortex in the squirrel monkey as revealed by peroxidase histochemistry and autoradiography.

Repeating spatial activations in human entorhinal cortex.

Acceleration of the oxygen reaction in CuA-deficient Nitrosomonas europaea cytochrome c oxidase as revealed by the flow-flash measurement.

Subcortical projections to the prefrontal cortex in the rat as revealed by the horseradish peroxidase technique.

Laminar and dorsoventral molecular organization of the medial entorhinal cortex revealed by large-scale anatomical analysis of gene expression.

What Does Cytochrome Oxidase Histochemistry Represent in the Visual Cortex?

Maturation of prefrontal cortex in the monkey revealed by local reversible cryogenic depression.

Noradrenergic innervation of the human adrenal cortex as revealed by dopamine-beta-hydroxylase immunohistochemistry.

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

Context-dependent spatially periodic activity in the human entorhinal cortex.

Entorhinal cortex and consolidated memory.

A microcolumnar structure of monkey cerebral cortex revealed by immunocytochemical studies of double bouquet cell axons.

Development of synaptic transmission at autonomic synapses in vitro revealed by cytochrome oxidase histochemistry.

Heterogeneity of layer II neurons in human entorhinal cortex.