THE JOURNAL OF COMPARATIVE NEUROLOGY 291:583-608 (1990)

Neurons of the Lateral Entorhinal Cortex of the Rhesus Monkey: A Golgi, Histochemical, and Immunocytochemical Characterization A.A. CARBONI, W.G. IAVELLE, C.L. BARNES, ANDP.B.CIPOLLONI Department of SurgeryiDivision of Otorhinolaryngology, University of Massachusetts Medical Center, Worcester, Massachusetts 01655 (A.A.C., W.G.L.); Departments of Anatomy (C.L.B. and P.B.C.) Neurology (P.B.C.), Boston University School of Medicine, Boston, Massachusetts 021 18; Edith Nourse Rogers Memorial Veterans Administration Medical Center (A.A.C., C.L.B., P.B.C.), Bedford, Massachusetts 01730

ABSTRACT This study identifies the neuronal types of the rhesus monkey lateral entorhinal cortex (LEC) and discusses the importance of these data in the context of the connectional patterns of the LEC and the possible role of these cells in neurodegenerative diseases. These neuronal types were characterized with the aid of Golgi impregnation techniques. These characterizations were based upon their spine densities, dendritic arrays, and, where possible, axonal arborizations. The cells could be segregated into only spinous and sparsely spinous types. The most numerous spinous types were pyramidal neurons. Other spinous types included multipolar, vertical bipolar and bitufted, and vertical tripolar neurons. The sparsely spinous neuronal types consisted of multipolar, horizontal bipolar and bitufted, and neurogliaform cells. These cells were further classified with the aid of histochemical stains and immunocytochemical markers. Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry stained multipolar, bipolar, and bitufted neurons. Stain for cytochrome oxidase (CO) was found in pyramidal and nonpyramidal cell types. Immunocytochemical techniques revealed several nonpyramidal neurons that contain somatostatin (Som) or substance P (SP). This study complements previous analyses of the neuronal components described in the LEC and adds further information about the distribution of selected neurochemicals within this cortex. Key words: cellular morphology, NADPH-diaphorase, cytochrome oxidase, somatostatin, substance P, Alzheimer's disease

Several recent reports have illustrated that the entorhinal cortex (EC) has significant pathology in neurodegenerative diseases, such as Alzheimer's disease (AD), which have been shown to affect this cortical area (e.g., Hooper and Vogel, '76; Corwin et al., '85; Reyes e t al., '85). This cortical region, known to play a critical role in the formation of memories, has been shown to be altered by the pathological loss of specific neuronal subpopulations as demonstrated by the laminar specific presence of neurofibrillary plaques and tangles in AD (Hyman et al., '84; Braak and Braak, '85). In addition, pathology of the neuronal elements of the lateral entorhinal cortex (LEC) have been implicated, for example, in hyposmia associated with AD (e.g., Fleming and Rogers, '86). 0 1990 WILEY-LISS, INC.

Moreover, decreased levels of several neurochemicals have been noted in this cortical area in neurodegenerative diseases (Whitehouse et al., '82; Simpson e t al., '84; Beal et al., '85; Beal et al., '88). These reported cellular and chemical alterations may contribute to dysfunctions such as memory deficits or hyposmia, thereby indicating a role for the pathology of the cortical circuitry of the LEG in AD (e.g., McLean et al., '89). Characterization of the normal cortical circuitry ofthe EC, including the cellular and chemical comAccepted 29 August 1989 A preliminary report of this study waq presented a t the Annual Meeting of the Society for Neuroscience, 1987.

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A Fig. 3. Camera lucida drawings of layer I sparsely spinous multipolar neurons identified by silver impregnation with their s o n s (arrows) indicated. Scale bar: 100 pm.

ponents, is needed to better understand the changes observed in this cortical region in AD. Brodmann ('09) described the entorhinal cortex in several species of mammals, including man, and gave it the numerical designation of area 28 and subdivided this cortex into areas 28a and 28b. This cortex in the monkey is located ventromedially and within the rostral part of the temporal lobe, extending from the depths of the rhinal sulcus dorsomedially to the amygdaloid sulcus. Cortical area 51, consisting of prepiriform and periamygdaloid cortices, forms its rostral border and the proisocortical and the isocortical portions of the parahippocampal area constitute its caudal border (Van Hoesen and Pandya, '75a). The entorhinal cortex, a periallocortex, is an intermediate type of cortex for which Rambn y Cajal ('01) initially proposed the cytoarchitectonic structure of seven layers. Other investigators have subsequently subdivided this area into six layers in rodents and nonhuman primates (Lorente de No', '33; Blackstad, '56; Storm-Mathisen and Blackstad, '64; Mellgren and Blackstad, '67; Geneser-Jensen et al., '74; Saunders and Rosene, '88). We have followed the cytoarchitectonic model of six layers in this cortex. The connectional patterns of the entorhinal cortex have been studied extensively in nonprimates. These studies demonstrated that entorhinal cortical afferents originate from various cortical and subcortical regions (Krettek and Price, '74; Cragg, '61; Powell et al., '65; Price, '73; Van Hoesen and Pandya, '75a; Krettek and Price, '77a,b; Kosel

Fig. 1. A medial surface view of the rhesus monkey right hemisphere. Outlined is the approximate position of the entorhinal cortex (EC) on the ventromedial surface of the temporal lobe. Arrow indicates the rhinal sulcus (rs). Scale bar: 1cm.

Fig. 2. A Photomicrograph of Nissl-stained coronal section indicating the three subdivisions of the entorhinal cortex. The dotted lines outline the approximate boundaries. (M), medial; (I), intermediate; (L) lateral; Amg, amygdala; rs, rhinal sulcus. B: Photomicrograph of the laminar pattern of the lateral entorhinal cortex (L). Scale bar: 500 pm.

et al., '81; Wouterlood and Nederlof, '83; Ottersen, '82; Finch et al., '86; Room and Groenewegen, '86). The entorhinal cortex projects to the molecular layers of the hippocampal subfields and to the dentate gyrus via the perforant pathway (Blackstad, '56; Fink and Heimer, '67; HjorthSimonsen and Jeune, '72; Steward, '76; Steward and SCOville, '76; Wyss, '81; Witter et al., '86), as well as t o nonhippocampal areas (Kosel et al., '82). Relatively few studies have demonstrated these same connectional patterns in primates. These studies, as in the studies of nonprimates, indicated similar afferent and efferent connectivity in the primate entorhinal cortex (Van Hoesen and Pandya, '75a,b; Van Hoesen et al., '75; Van Hoesen, '82; Kosel et al., '82; Amaral et al., '83; Insausti et al., '87a,b). In spite of these data on the connectivity of the entorhinal cortex, a detailed description of the neuronal types found in this cortex has not been reported. Knowledge of the specific neuronal types and their connections in the LEC, therefore, becomes important to the study of human neurodegenerative diseases such as AD. This study describes the cellular morphology and their chemical reactivity in the monkey LEC and may provide functional insight into these neurons and their connections.

MATERIALS AND METHODS Golgi procedures While under deep sodium pentobarbital anesthesia (80 mg/kg of body weight, IV), a series of 15 monkeys were perfused by vascular perfusion with either a 10% neutral phosphate-buffered formalin solution or a combined 0.5 % paraformaldehyde-2 % glutaraldehyde fixative in a neutralbuffered phosphate solution. The entorhinal cortex was removed from each brain, and some blocks were silver impregnated by the rapid-Golgi method (Valverde, '70), and the Braitenberg (Braitenberg e t al., '67) or Golgi-Kopsch (Lee, '37) variations. Following silver impregnation, t h e blocks were dehydrated through aqueous solutions of increasing concentrations of glycerin to anhydrous glycerol and cut with a vibratome into 100 pm-thick sections (Fairen et al., '77).

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Fig. 4. A,B: Photomicrographs of rapid Golgi-silver impregnated layer 11 spinous multipolar neurons. C The same neuronal type identified in tissue prepared by the Braitenberg variation of the Golgi technique. Note: Throughout the micrographs of silver-impregnated tissue,

it is common to see amorphous densities (small white arrow) and impregnated glial processes (large white arrow) in the neuropil. The axon (bold arrow) is indicated. Scale bar: 100 bm.

Alternate intervening sections of 50 pm were cut and Nissl stained with thionin for cytoarchitectonic localization of somata. The 100 pm sections containing silver impregnated neurons from the brains that had been Derfused with the paraformaldehyde/glutaraldehyde fixative were examined with a light microscope and selected sections were goldtoned (Fairen et al., '77). These sections were then postfixed in buffered aqueous osmium tetroxide for 1 hour, dehydrated, stained "en bloc" with uranyl acetate, and embedded in thin sheets of plastic. This enables subsequent ultrastructural analysis of these neuronal types for more definitive characterizations (Peters et al., '76). Selected neurons were photographed and then drawn with the aid of a camera lucida drawing tube. The blocks from the brains perfused with the formalin fixative were embedded in celloidin, cut into 100 pm sections with a vibratome, and mounted onto slides for subsequent examination. The contralateral blocks from brains fixed by both methods were washed in 0.1 M phosphate buffer and sectioned with a vibratome in a consecutive alternate series of 100,50, and 30 pm-thick sections. The 100 pm sections were prepared by a slice Golgiimpregnation method (Harris, '78). Selected sections from this method were also gold-toned, dehydrated, and plasticembedded. The 50 wm sections were processed for immunocytochemical characterization of neuronal elements (see below), and the 30 Km sections were Nissl stained with thionin for cytoarchitectonic localization of the lateral entorhinal cortex (LEC) and those neurons within the LEC.

tinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) enzyme (Vincent et al., '83) or cytochrome oxidase (CO) activity (e.g., Wong-Riley, '79; Wong-Riley and Carroll, '84; DiFiglia et al., '87). Immunocytochemistry using polyclonal antisomatostatin (courtesy of L. Chun, Dept. of Neurology, MGH, Boston, MA) and anti-substance P (Tmmunonuclear, Stillwater, MN) was performed on the other sections (Polack and VanNoorden, '86). Light micrographs and camera lucida drawings were made of labeled cells. Representative distribution patterns of histochemically or immunocytochemically reactive cells were drawn and analyzed. This study was confined to the description of cortical neurons and their laminar organization in the rhesus monkey LEC. The characterization of these neuronal types was based upon density of dendritic spines, dendritic array, axonal arborization, histochemical staining, immunocytochemical labeling, and laminar localization of somata. Descriptions of nonpyramidal neurons in the LEC followed the nomenclature for neocortical neuronal types in the rat (Feldman and Peters, '78; Vogt and Peters, '81) and in the cat (Peters and Regidor, '81).

RESULTS Cytoarchitecture

The nrimate entorhinal cortex. located in the medial uortion of the temporal lobe (Fig. l ) , has been previously subdiHistochemistry and immunocflochemis*vided by using cytoarchitectonic characteristics into three Eight postmortem whole brains were removed fresh from areas: the classical subareas defined as areas 28a (medial) adult rhesus monkeys perfused by overdose injections of and 28b (lateral) by Brodmann ('09), with area 2% (intermesodium pentobarbital. These brains were immersion fixed in diate) more recently defined by Van Hoesen and Pandya ('75a). T o parcellate the entorhinal cortex (EC) in the pres2 cc, PLP solution (periodate, lysine, and paraformaldehyde) in a neutral phosphate buffer (McIAeanand Nakane, '74) ent study, we used the three main subdivisions described in and cut with a vibratome into 50 pm sections. Histochemis- nonprimates (Lorente de No', '34; Krieg, '46; Blackstad, '56; try was performed on some sections for either the nico- Haug, '76) and the modified nomenclature of lateral (LEC),

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Fig. 5. Photomicrographs of (A) a rapid Golgi-impregnated layer I1 typical pyramidal neuron. B A rapid Golgi-impregnated layer I1 biapical pyramid. C: A layer I1 biapical pyramid from tissue prepared with

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Braitenberg variation. D A rapid Golgi-impregnated layer I1 oblique pyramidal neuron. Bold arrows indicate their axons. Scale bar: 100 wm.

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Fig. 6. A-D Photomicrographs of layer I1 spinous multipolar neurons with triangular somata identified in tissue prepared with the Brai-

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tenberg variation. B:2, A layer I1 pyramid; arrows indicate s o n s . Scale bar: 100 pm.

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Fig. 7. A$: Photomicrographs of typical pyramidal neurons in layer I11 of LEC impregnated by the Braitenberg variation. b, Blood vessel. Scale bar: 100 grn.

medial (MEC), and intermediate (IEC) entorhinal cortex for the three subdivisions 28L, 28M, and 281, respectively. The area we have designated LEC (28L), has been previously called area 28b in the rhesus monkey (Van Hoesen and Yandya, ’75a; Saunders and Rosene, ’88) and E R , E L R , plus ELCin the fascicularis monkey (Amaral and Price, ’84; Amaral et al., ’87) (Fig. 2A). Although previous investigators have divided the entorhinal cortex (EC) into varying numbers of cortical laminae (e.g., Lorente de No’, ’33; Kosel et al., ’82), we subdivided this cortex, as did Brodmann (’09), into six fundamental layers. The most noted differences among investigators is in the nomenclature of layer IV. The cortical lamina we have designated layer IV in the LEC was called layer IIIa by Lorente de No’ (’38); however, other investigators also enumerated this lamina as layer IV (e.g., Van Hoesen and Pandya, ’75a; Saunders and Rosene, ’88). The presence of a distinct layer 1V in this study was corroborated by the silver impregnation methods and by the distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) enzyme. The laminar pattern of six layers that can be followed throughout the three subdivisions of entorhinal cortex was easily defined in coronal sections stained by the Nissl method and thus made it possible for us to be precise in our definition of the lamination of the LEC (Fig. 2B).

Layer I was a wide, neuron sparse, superficial lamina of loosely packed small, round cell bodies. Layer I1 o f t h e LEC was a thin lamina of compact cells that stained darker than did adjacent layers. The large round cell bodies of this layer were arranged in prominent small clusters or “islands.” This “island” effect seen in layer I1 distinguished the LEC from the IEC and MEC and continued through layer I11 as a columnar arrangement of somata within these laminae. Based on cell packing density, layer 111 was a broad layer with two subdivisions: a narrow cell sparse area immediately deep to layer 11, whereas below this was a wider, more densely packed area of medium-sized cells. Layer IV (lamina dissecans), although not as clearly distinct in the LEC as it is in the MEC and IEC, did appear as a relatively cell sparse lamina. Layer V was densely populated with medium-sized cells. Layer VI was composed of medium-sized cells densely packed in variegated bands.

SILVER IMPREGNATION The following description of the neuronal types found in the silver-impregnated tissue came from the separate investigation of each lamina of the lateral entorhinal cortex (LEC) beginning in layer I and continuing through layer VI. Although other studies have included axonal arborizations

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Fig. 8. A,B,C Photomicrographs of upper layer 111 biapical p y x mids. A: Golgi-Kopsch;B: Braitenberg variation; C: rapid Golgi. D Pho-

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tomicrograph of a Golgi-Kopsch layer I11 oblique pyramid. Arrows indicate axons. Scale bars: 100wn.

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Fig. 9. A , B Photomicrographs of large spinous multipolar neurons of layer I11 impregnated by the Braitenberg variation. Scale bars: 100

w as criteria for characterizing neurons in the neocortex (Ramon-Moliner, '61, '67; Valverde, '76; Peters and Jones, '84), we generally limited our characterizations to cell bodies and dendritic arrays due to the partial axonal impregnation that usually occurs with silver impregnation methods in the monkey (Jones, '75; Sloper and Powell, '79; Lund, '87). In the neocortex, neurons have been segregated into spinous (spiny), sparsely spinous, or smooth (spine free or aspiny) types. The spinous types include both pyramidal and nonpyramidal neurons with the nonpyramidal neurons having a dendritic spine density approaching and, in some cases equal to, the pyramidal neurons (Feldman and Peters, '78). Smooth or aspiny neurons rarely have completely spine-free dendrites (Lund, '73; Jones, '75; Lund et al., '81). Contrary to similar studies in the neocortex, the neurons found in the monkey LEC could be segregated in this study into only spinous and sparsely spinous types. The relative sizes of nonpyramidal neurons described here were based on the extent of the dendritic arrays, the size of the somata, or both. Individual pyramidal neuronal size was based on the linear extent of their apical trees.

Layer I Layer I was clearly distinguished as a wide layer containing many vertically as well as horizontally oriented impregnated segments of cells but contained very few completely

impregnated neurons. The impregnated neurons were small, sparsely spinous, multipolar cells (Figs. 3, 21A,B). The cell bodies were round, 13-15 pm in diameter, with eccentric nuclei. Neurons of this type had between 4 and 12 primary dendrites in radial array, all of approximately the same diameter, with few spines and were generally confined within layer I. In spite of the paucity of impregnation of the thin axons originating from the somata of these cells, they appeared to ramify locally.

Layer 11 Layer I1 had several morphologically distinct cell types. The "islands" of layer I1 were composed mainly of large spinous multipolar neurons that had cell bodies of 25-30 Wm in diameter with large centric nuclei (Figs. 4,21C,D,E). They had three to eight primary dendrites that were free of spines, approximately equal in diameter, and bifurcated very near the cell body. Second- and third-order dendrites branched in radial arrays and were heavily populated along their lengths with a constant density of spines nearly equal to the spine density on the dendrites of a pyramidal neuron. These dendrites did not taper appreciably as they coursed in all directions from the soma. Dendritic branches directed horizontally remained predominantly in layer 11, creating a layer of dendritic bundles between the islands of cell bodies. Other dendrites directed superficially had terminal seg-

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c

Fig. 10. A Photomicrograph of a layer I11 spinous multipolar neuron with inverted triangular soma impregnated by the Braitenberg vari-

ation. B: Camera lucida drawing of a similar neuron of this type identified in Golgi-impregnated tissue. Scale bars: 100 Fm.

ments in layer I, whereas deeper branches coursed into layer 111. The axons originated from the soma or a primary den-

Layer III

dritic segment and apparently ramified locally. Pyramidal neurons of layer I1 were relatively small, having somata, of 15-20 pm, located within deeper portions of layer I1 (Fig. 5A). These spinous neurons had one thick, stunted apical dendritic shaft that bifurcated a short distance from the cell body and had terminal segments in layer I. The basal dendritic skirt branched almost horizontally, but had some terminal segments in upper layer 111. A variation on this appearance was also found with some of these neurons having biapical shafts (Figs. 5B,C). Other variants were oriented obliquely with pear-shaped somata and basal skirts of a lesser complexity than of the typical pyramids (Fig. 5D). All the pyramidal neurons exhibited a single, long, thick, tapering axon originating from the deep surface of their somata. Another spinous neuronal type found in layer I1 was multipolar with triangular cell bodies, the base toward the pia, and a diameter of 25-30 pm (Figs. 6, 21F). The somata of these multipolar neurons were not located within the “islands” of layer 11. The primary dendritic segments were radially arrayed but not of equal diameter. There were often two or three predominant dendrites that coursed obliquely toward the pia from the superficial side of the soma, having some of their terminal segments extending into layer I, whereas other dendrites were directed horizontally within layer 11, having some terminal segments in superficial portions of layer 111. These latter dendrites were longer and more extensive than were those directed toward the pia. These cells exhibited single, long, thick, tapering axons originating from the deep surface of their somata.

The neuronal types found in layer I11 were generally of the spinous type and included both multipolar and pyramidal neurons. Typical pyramidal neurons (Figs. 7; 21N) found in this layer had pyramidal-shaped somata with large centric nuclei, a prominent apical shaft that coursed toward the pia, and dendrites radiating from the base of the cell body to form a basal skirt. The cell bodies were approximately 20-30 pm at their widest diameter. The large primary dendritic segments of these neurons had few spines. In some cases, the primary segments gave rise to a few horizontal branches prior to their apical tufts. These spinous secondary apical branches were thin with little branching. The tufts of their apical dendrites had many branches with some terminal segments in layer I (Fig. 21L). The axons, originating from the base of the cell bodies, were considerably larger in diameter than were those of the large spinous multipolar neurons of the “islands” of layer 11. These axons were smooth, except for the presence of local “bleb-like” thickenings and had several thin collateral branches arising at right angles to the parent process. In addition to the typical pyramidal neurons, layer I11 also had four populations of atypical pyramids. One of these types, the “biapical pyramid,” characteristically had two “apical” dendrites (Figs. 8A,B,C, 21G). Each of these apical dendrites had an apical tuft that branched as prolifically as, and had a spine density approaching or equal to, the typical pyramidal neuron. The cell bodies of the “biapical pyramids” were mainly in the middle to upper portion of the more densely packed band of layer I11 with their primary

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Fig. 11. A: Photomicrograph of a rapid Golgi-impregnated upper layer 111spinous multipolar neuron similar to a “star pyramid.” B: Camera lucida drawing of the same cell. Arrows indicate axons. Scale bars: 100 rrm.

segments in the less dense band of upper layer III and subsequent branches extending into layer 11. The second type of atypical pyramid, usually found in superficial layer 111,was obliquely oriented (Figs. 8D, 21M). Their somata gave rise to a basal skirt, many small dendrites, and one apical dendritic shaft that branched into the apical tree. Several of the primary dendrites of these neurons had spines. A third type of pyramidal neuron found in this layer exhibited characteristics previously considered atypical by Globus and Scheibel (’67) in the rabbit cortex. Very near their somata, the apical shafts gave rise to single, short, thin, secondary horizontal branches (Fig. 21P). Finally, inverted pyramids were also found in small numbers in this layer. Their characteristics were the same as the typical pyramid with the exception of their orientation. We also found a population of large spinous multipolar neurons in layer III (Figs. 9, 21s). In general, this neuronal type exhibited morphological characteristics similar to those of the large spinous multipolars in the “islands” of layer 11, although their somata were not as round and their dendrites branched more profusely. Their somata were approximately 30 hm in diameter and gave rise to a radial array of dendrites, each having a spine density comparable to that of the pyramidal neurons. Some spinous multipolar neurons exhibited additional morphological characteristics. The somata of one of these variant spinous multipolar neurons were sparsely populated with spines and had a distinctive inverted triangular shape

(Figs. 10, 210). Although their overall dendritic arrays had radial appearances, the primary segments originated from the three “corners” of the somata with the two thickest dendrites coursing obliquely toward the pia. These primary segments were moderately spinous. Subsequent dendritic segments of these cells tapered and were heavily populated with spines. Another spinous neuronal type was a large multipolar neuron located in superficial layer 111 (Figs. 11, 21Q). A dense population of spines on apical and basal dendrites gave this neuron an appearance similar to that of a pyramidal cell; however, the axon was thin and apparently locally ramifying. This cell type is very similar to the neuron described as the “star pyramid” by Lorente de No’ (’38). Large spinous vertical bipolar neurons were also found in layer I11 (Fig. 12, ZlR). These neurons had elliptical cell bodies that bore occasional spines. Primary dendritic segments emerged from both the superior and inferior poles of the somata. Each primary segment gave rise to several tapering dendritic branches densely populated with spines. Some of these spinous vertical bipolar neurons also had a short, thin spinous dendrite that originated from one side of the cell body and extended horizontally in layer 111. The superior primary dendritic segment was longer than was the inferior primary segment that bifurcated closer to the soma. The ascending dendrites were confined to layer I11 and were grouped in a much narrower array than was the descending skirt. The descending dendrites extended into deeper

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Figure 12

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Fig. 13. Photomicrographs of (A) a layer 111sparsely spinous vertical bipolar neuron and (B) a layer I11 sparsely spinous horizontal bipolar neuron. Scale bars: 100 qm.

layers. The smooth, thin axons, which appeared to ramify locally, arose from the inferior pole of the cell body or a descending dendrite or both. In layer 111, there were also vertical and horizontal sparsely spinous bipolar and bitufted neurons (Fig. 13). These were usually smaller than was the spinous vertical bipolar cell type described above. These smaller bipolar and bitufted cell types had ovoid somata that gave rise to one primary dendrite from each pole. The dendrites extended up to 300 fim in either direction but did not branch profusely. The silver methods also impregnated some layer I11 sparsely spinous multipolar neurons having somata that were pear-shaped (Figs. 14, 21K). Their dendrites were radially arrayed and confined predominantly to layer 111. Their axons originated from the cell body and/or from a primary dendrit,e to ramify locally. The columnar arrangement of neurons through layers I1 and 111 of the LEC as suggested in Nissl preparations was corroborated in the silver-impregnated tissues (Fig. 15). Typically, apical dendrites of pyramidal neurons in layer TI1 Fig. 12. A Camera lucida drawing of a large layer I11 vertical spinous bipolar neuron identified with rapid Golgi-impregnation. B: Photomicrograph of a similar cell identified with Golg-Kopsch impregnation. C: Photomicrograph of a similar cell t y p e identified with the Braitenberg variation of the Golgi technique. Arrows indicate axons. Scale bars: 100 pm.

were arranged in narrow bundles that coursed through the cell sparse areas between the cellular columns of layer 111, the islands of layer I1 neurons, and into layer I.

Layer IV Layer IV, as defined in the Nissl preparations, contained in the silver-impregnated preparations (Fig. 14). It had, however, a distinctive reticulation of axonal and dendritic processes from neurons with somata in the more superficial and deep layers.

no somata

Layer V-VI Since the laminar delimitations between layers V and VI were unclear in the tissue prepared by the rapid-Golgi method, neurons in layers V and VI will be described as being in the superficial, mid, or deep portions of the layer V-VI band. Very superficial in the layer V-VI band we found large spinous multipolar neurons with variously shaped cell bodies. One such type was a spinous horizontal tripolar neuron (Fig. 21W). Neurons of this type had large irregularly shaped, triangular cell bodies with eccentric nuclei. Their somata gave rise to three predominantly horizontal dendritic segments that were moderately to heavily populated with spines. In the mid-layer V-VI band, there were large spinous multipolar neurons with irregularly shaped somata

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Fig. 14. Photomicrographs of layer I11 sparsely spinous multipolar neurons. Dotted lines indicate the approximate borders of layer IV with

its distinctive fiber reticulation found in both Golgi (A) and Braitenberg (B) preparations. Arrows indicate axons. Scale bar: 100 pm.

(Fig. 21U). Their three primary dendritic segments were radially oriented and heavily populated with spines. Large typical pyramids were also found in the layer V-VI band (Fig. 21V,Z). Their cell bodies were generally smaller, more elongated, and narrower than those found in the other layers. The basal skirts of this cell type had many branches extending into the white matter, and their axons also coursed directly into the white matter. The layer V-VI band also contained spinous vertical bitufted and bipolar neurons of varying sizes (Figs. 16, 21X,Y,ZZ). These neurons had spindle-shaped cell bodies with dendrites originating from each pole and were oriented either vertically or horizontally. Their short primary dendritic segments had relatively few spines, whereas their subsequent dendritic branches had a higher density of spines. The deeper portion of the LEC V-VI band contained spinous vertical tripolar neurons (Fig. 17, 21T). These had an ovoid cell body with centric nuclei. Each soma gave rise to three primary dendritic segments; one directed toward the pia, one toward the white matter, and the third horizontally. Subsequent dendritic segments were equal in diameter, nontapering, and heavily populated with spines. With the rapid-Golgi, the Braitenberg, and Golgi-Kopsch methods, sparsely spinous neurogliaform cells were found in layers 11,111,V, and VI. These cells had small round somata that gave rise to as many as 12 very thin dendrites. Their spherical array extended not more than 75-80 pm from the soma. Many dendrites branched back toward the soma, cre-

ating the “clewed” or “spiderweb” effect described in other cortices (Valverde, ’71; Jones, ’75).

HISTOCHEMISTRY AND IMMUNOCYTOCHEMISTRY We have further characterized neurons of the lateral entorhinal cortex (LEC) according to their histochemical and immunocytochemical reactivity. LJtilizing techniques that stain for the nicotinamide adenine dinucleotide phosphate-diaphorase enzyme (NADPH-d) and cytochrome oxidase (CO), we identified subpopulations of neuronal types in the monkey LEC. Other subpopulations were identified by their immunoreactivity for somatostatin (Som) and substance P (SP) (Polak and VanNoorden, ’86). A high density of cells stained for NADPH-d enzyme were in layers 11,111,V, and VI, whereas layers I and IV had only a few NADPH-d-positive cells (Fig. HA). The thinner dendritic segments appeared more mottled in the NADPH-dpositive tissue the more distal they were to the cell body. There was a lower density of visible dendritic spines on the neurons stained for the diaphorase enzyme as compared to the Golgi preparations. This is possibly due to the variability in staining techniques, section thickness, or the inability of enough formazan to deposit in the spine to be visualized (Ferrante and Kowall, ’87). The spine densities did appear, however, to be qualitatively higher than those found on the

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found in the “islands” of layer I1 in the silver-impregnated tissue, were NADPH-d-positive (Fig. ZlC). These cells had round somata, with, at least, three nontapering dendrites in a radial array. Their axons ramified locally. The neuronal types stained with this method in layers 111, V, and VE were sparsely spinous and spinous multipolar neurons (Fig. 19B,C). The sparsely spinous types included multipolar neurons of different sizes and complexity of dendritic arrays, in addition to many bipolar and bitufted neurons oriented either vertically or horizontally. The spinous neurons were multipolar cells with somata of varied sizes and dendritic arrays. Thin and smooth horizontal and vertical fiber processes in all layers, particularly in layers I and IV, appeared to be axonal elements. The CO method stained only the somata and proximal portions of primary dendritic segments. Spines were not labeled with this technique. Staining was adequate enough, however, to identify pyramidal and nonpyramidal neurons. They were seen only in layer 111 and the V-VI band (Fig. 18B). The highest density of CO-positive neurons was in the upper portion of the layer V-VI band. Most of these cells throughout layers 111, V, and VI were lightly stained; however, some were darkly stained with the highest density of these darkly stained neurons being in layer 111. I t is unclear whether this is due to the variability of staining or an indication of differences in enzymatic activity. The majority of cells that were heavily stained for CO were vertical or horizontal bipolar and bitufted neurons (Fig. 19D). A few multipolar and horizontal tripolar as well as pyramidal neurons also stained for CO (Fig. 19E,F). Somatostatin antibodies labeled neurons mainly in layers I1 and I11 with a few in the upper portion of the layer V-VI band (Fig. 18C). Large spinous and sparsely spinous multipolar and sparsely spinous bipolar and bitufted cells were labeled by Som antibodies. Spinous multipolar, horizontal, and vertical bipolar and bitufted neurons were labeled in layers I1 and 111. In the layer V-VI band, there were more Som-immunoreactive vertical bipolar and bitufted cells than there were multipolar cells (Fig. 20A.B). Substance P antibodies labeled a few cells in layer 111and the upper portion of the layer V-VI band (Fig. l8D). They were vertical and horizontal bipolar and bitufted cells (Fig. 20C,D). Although no somata were labeled with the aid of Som or SP antibodies in layers I and IV, fiber plexuses were labeled in both laminae. We were not able to identify any pyramidal neurons that were Som-or SP-positive.

DISCUSSION Fig. 15. Photomicrographs of (A) a Nissl-stained coronal section of rhesus monkey LEC illustrating the columnar arrangement of somata, from the “islands” of layers 11-111. B A rapid Golgi-silver impregnated coronal section from the same level of the monkey LEC illustrating the arrangement of narrow bundles of dendrites that course through the cell sparse areas between the columns of layer I11 and the “islands” (dotted lines) of layer 11. Scale bar: 1 mm.

NADPH-d-positive cells of the neocortex (Tagaki e t al., ’83; Vincent and Johansson, ’83; Vincent et al., ’83). Labeled cells in layer I were small, sparsely spinous multipolar neurons (Fig. 19A). Although our previous silver stains had not impregnated neurons in layer IV, the NADPH-d enzyme method stained horizontal bipolar and bitufted cells. Spinous multipolar neurons of layer 11, similar to those

Several investigations have shown that the entorhinal cortex (EC) has marked neuropathological and neurochemical changes in neurodegenerative diseases known to affect memory, such as Alzheimer’s disease (AD) (Hooper and Vogel, ’76; Davies et al., ’80; Rossor et al., ’80; Delfs and Dichter, ’83; Kemper, ’84; Morrison et al., ’85, ’86; Reyes et al., ’87). Detailed neuropathological studies of AD on the distribution of neurofibrillary plaques and tangles have shown a specific laminar distribution within the EC that has been postulated as effectively isolating the hippocampal formation by interrupting its connections, thereby providing an anatomical explanation for the memory losses associated with this disease (Hyman et al., ’84; Hyman et al., ’86). Additional studies have identified alterations of peptidergic markers in this disease (Beal et al., ’85).

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Fig. 16. Photomicrographs of layer V-VI vertical sparsely spinous bitufted neurons; Golgi-Kopsch impregnated (A, B). C A layer V-VI vertical sparsely spinous bipolar neuron. D: A horizontal sparsely spi-

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nous bipolar neuron of layer V-VI. Arrows indicate axons. Scale bars: 100pm.

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A

Fig. 17. Camera lucida drawing of (A) a layer V-VI spinous vertical tripolar neuron. B: Photomicrograph of the same neuron, Golgi-impregnated and gold-toned. Arrow indicates the soma. Scale bar: 100 pm.

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C Fig. 18. Camera lucida drawings of representative distribution pattern of (A) NADPH-diaphorase stained neurons; (B) cytochrome oxi-

dase-stained neurons; (C) somatostatin reactive neurons: (D) Substance P reactive neurons. Amg, amygdala;RS, rhinal sulcus.

MONKEY ENTORHINAL CORTICAL NEURONS Investigations of the nonhuman primate have established a proposed connectional scheme in which the EC plays a role in the input and output of the hippocampal formation. The functional circuitry of any neuronal system is, however, more completely defined by its cellular and chemical constituents. In the present study, we have, therefore, expanded on previous descriptions of the neuronal types found in the nonhuman primate lateral entorhinal cortex (LEC). In addition, we have correlated this detailed morphological information with the reactivity for specific neuroenzymes and neuropeptides that are found in the intrinsic neuronal subpopulations of this cortical area. Several of the cell types in the monkey LEC, a paleocortex, were very similar to cell types described in the neocortex by other investigators. The pyramidal neurons and several of their variants described here have been reported in other primate and nonprimate cortices (Globus and Scheibel, ’67; Kaiserman-Abramof and Peters, ’72; Braak, ’80; Feldman, ’84). Except for the spinous stellate in the visual cortex (Lund, ’73), pyramidal neurons are thought to be the sole source of output in the primate neocortex (Gray, ’59; Feldman, ’84; Jones, ’84). In view of this concept and the morphological similarities between pyramidal neurons of the monkey LEC and the neocortex, we postulate that t,he efferent pathways of the LEC arise, in part, from these cell types found in layers 11, 111, V, and VI. Projections of the LEC, for example, studied with retrograde and anterograde labeling methods illustrating cells of origin in layer I1 that terminate selectively in the fascia dentata (HjorthSimonsen and Jeune, ’72; Steward and Scoville, ’76) probably arise, in part, from the typical, biapical, and oblique pyramids of layer 11. The pyramidal cells did not label with the chemical markers we applied in this study with the exception of cytochrome oxidase (CO). Previous studies, however, have shown that pyramidal cells use the excitatory amino acids, glutamate and aspartate as neurotransmitters (Streit and Cuenod, ’79; Baughman and Gilbert, ’81; Cuenod et al., ’82; Streit, ’84; Conti et al., ’87; Kaneko and Misuno, ’88). Several of the neuronal types in the monkey LEC are nonpyramidal cells, including the sparsely spinous and spinous multipolar, bitufted, and bipolar types. These types are commonly seen in other cortices. This “transitional cortex” (Nauta and Karten, ’70) also included cell types not routinely described in the neocortex such as spinous tripolar and spinous projection multipolar neurons. The tripolar neuron was most commonly found in the V-VI band and was oriented either horizontally or vertically. The projection spinous multipolar type was found in layer I1 outside of the “cellular islands” (Fig. 6). The spine densities present on this cell type coupled with projection-like axons, seen entering the white matter by other investigators, lead us to believe that these neurons are the previously described “intermediate neurons” (Lund et al., ’77; Lund, ’84), “modified pyramids” (Braak et al., ’76; Braak, ’80; Braak and Braak, ’85), or “extraverted nerve cells” (Sanides and Sanides, ’72). This neuronal type is also probably the same neuronal type as the projection “stellates” found in layer I1 of the human parahippocampal gyrus that have been proposed as an example of the transition between the nonpyramidal and the pyramidal neuronal morphologies (Braak et al., ’76). In addition to the pyramidal neurons, these projection spinous multipolar cells of layer I1 apparently also contribute to the extrinsic projections of the LEC. In this cortex, the intrinsic neurons, including the most sparsely spinous ones, were far more spinous than were simi-

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lar neurons found in neocortical regions (Peters, ’84). Although similar neuronal types have been classified as “nonspinous” or “aspiny” in other cortices, we classified none of the neurons in this study of the monkey LEC as nonspinous. The cell type with the least number of spines, for example, was a small sparsely spinous multipolar neuron found only in layer I. Similar cells have been described in layer I of the neocortex as small “stellates” having short dendrites and few spine-like structures, although fewer spines than seen on this cell type in the LEC (Ramon y Cajal, 1899; Meller et al., ’68; Colonnier, ’68; Colonnier and Rossignol, ‘69; Lund and Lund, ’70; Sousa-Pinto et al., ’75; Baron, ’76; MarinPadilla and Marin-Padilla, ’82). Our findings of a richness of dendritic spines and an abundance of varied nonpyramidal dendritic arrays, in particular, on cells whose dendrites predominantly extended horizontally and obliquely within their own layer, may be a characteristic of this phylugenetically older cortex. These observations made from Golgi preparations were corroborated with the aid of the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) enzyme staining technique that also qualitatively demonstrated a greater number of spines on labeled intrinsic neurons of the LEC as compared to similar cells positive for NADPH-d in other systems (Kowall et al., ’85; Ferrante et al., ’87). The NADPH-d method was employed in order to expand on our Golgi results of the morphologies of the nonpyramidal neurons, since it is known to provide complete staining of neuronal elements of intrinsic circuit cells in reproducible numbers, including axons (DiFiglia et al., ’82 a,b; Gray, ’83; Tagaki et al., ’83; Vincent and Johansson, ’83; Vincent et al., ’83; Johansson e t al., ’84; Kowall et al., ’85; Ferrante and Kowall, ’87; Ferrante et al., ’87). I t has also been shown that some somatostatin (Som) and substance P (SP) immunoreactive cells colocalize with the enzyme NADPH-d; therefore, i t was possible to morphologically characterize subsets of these cells with the NADPH-d technique. Indeed, all the nonpyramidal neuronal types seen with the silver impregnation techniques, except for the large projection spinous multipolar cells of layer 11, were stained with this method. Although colocalization studies were not done here, we believe, in view of previous reports, at least some of the NADPH-d-positive nonpyramidal cells are those neurons involved in the local circuitry of the LEC that contain the peptides Som and SP. The functional significance of the histo- and immunocytochemical markers used in these studies is not fully understood. Cytochrome oxidase is, however, known to be involved in the electron transport system and has been used as an indicator of cellular activity (Wong-Riley, ’79; WongRiley and Carroll, ’84; DiFiglia et al., ’87). Cells containing NADPH-diaphorase enzyme are well known to be local circuit cells in both the cortex and the striatum. Although SP and Som immunoreactive cells and their tissue levels have been shown to be affected by human diseases (e.g., Beal et al., ’85), the mechanism of action of these neuropeptides has yet to be completely defined, although they have been proposed as neuromodulators (Siggins and Gruol, ’86). Since NADPH-d and neuropeptides are contained in intrinsic neurons and the majority of intrinsic neurons have been shown to be GABAergic (Meinecke and Peters, ’87), it follows logically that these substances are clearly contained in cells that are known to provide inhibitory influences within cortical circuitry. The role of these interneurons in the LEC may be assumed to be the same as that proposed for other cortices.

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Figure 19

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Fig. 20. Photomicrographs of (A) a somatostatin (Som) positive field of vertical bipolar and bitufted layer V-VI neurons; (B)a layer VVI horizontal bitufted neuron having Sam-like immunoreactivity; (C) a

layer I11 vertical bipolar neuron found to have substance P (SP) immunoreactivity; (D) layer I11 SP immunoreactive neurons: a horizontal bipolar cell and a lightly labeled multipolar neuron. Scale bar: 100 pm.

In terms of the circuitry of the LEC of the monkey, it is known that the input from the neocortex is received across layers I, 11, and IT1 and it is in these layers that the cells of origin for the perforant pathway are located (Powell et al., '65; Scalia, '66; Heimer, '68; Jones and Powell, '70; Price and Powell, '71; Van Hoesen et al., '72; Van Hoesen and Pandya, '75a; Krettek and Price, '77b; Seltzer and Pandya, '78;

Braak, '80; Brodal, '81; Pandya et al., '81; Schwartz and Coleman, '81; Amaral et al., '83; Insausti et al., '87a; Saunders and Rosene, '88). The input to layers V and V1 is from the subicular cortices and the CA1 subfields of the hippocampal formation (Heimer, '68; Jones and Powell, '70; Anderson et al., '71; Price and Powell, '71; Hjorth-Simonsen, '72; Hjorth-Simonsen and Jeune, '72; Segal and Landis, '74; Van Hoesen and Pandya, '75b; Steward, '76; Steward and Scoville, '76; Kosel et al., '82; Insausti et al., '87a,b; Saunders and Rosene, '88). It is from layers V and VI that projections from the LEC back to the neocortical regions arise. If true in the LEC as in the neocortex, all extrinsic input into the LEC, such as that from the neocortex and the hippocampus, may contact any element in the cortical projection fields capable of forming asymmetrical, presumably excitatory, synapses (White, '89). These synapses may be made with

Fig. 19. Photomicrographs of (A)a layer I small sparsely spinous multipolar neuron positive for NADPH-diaphorase (NADPH-d) (arrow indicates axon); (B) a layer I1 sparsely spinous bitufted neuron positive for NADPH-d a lightly stained multipolar neuron, (open arrow); (C) a spinous layer 111neuron positive for NADPH-d; (D) a deep layer V-VI vertical bitufted neuron stained for cytochrome oxidme; (E)a cytochrome oxidase-stained horizontal tripolar cell of layer 111; (F)a cytochrome oxidase-stained layer 111pyramidal neuron. Scale bars: 100 pm.

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I

II

IV

Fig. 21. Collage of camera lucida drawings of neuronal types identified in the monkey LEC and their representative laminar arrangement. A,B: Layer I multipolar; C,D,E,F layer I1 spinous multipolar; G: layer I11 biapical pyramidal; H:layer I1 sparsely spinous horizontal bipolar; I: layer I11 sparsely spinous horizontal bipolar; J,O,Q,S layer I11 spinous multipolar; K layer 111 sparsely spinous multipolar; L,N,P: layer I11 pyramidal; M: layer 111oblique pyramidal; B:layer I11 large spinous vertical bipolar, and in the layer V-VI band; T spinous vertical tripolar; U: spinous multipolar; V,Z pyramidal; W horizontal tripolar; X sparsely spinous vertical bipolar; Y,ZZ spinous vertical bitufted neurons.

the spines and dendrites of pyramidal cells and presumably some of the subclasses of spinous nonpyramidal neurons, such as the projection spinous multipolar cells found between the “cellular islands” of layer 11. They may also be formed with the somata as well as the dendrites and spines of intrinsic neurons. As noted above, the extrinsic pathways of the LEC presumably arise from pyramidal cells, as in the neocortex, as

well as large spinous multipolar projection cells. The cells of origin of each extrinsic pathway reside in specific laminae. In addition to extrinsic axons, these cells of origin also have recurrent collateral axons that contribute to local intrinsic circuitry. Thus, by virtue of their axonal and dendritic ramifications, both pyramidal as well as nonpyramidal cells have intracortical connections with neurons of, and the afferents to, several laminae (Peters and Proskauer, ’80;

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*B

i

'

I

,

IV

V

VI

Figure 21 continued

Winfield et a]., '81; Jones et al., '87; White '89). As previously speculated by Lorente de No' ('38),it would follow from this concept that the intrinsic neurons with only local axons influence both local cortical circuitry as well as extrinsic pathways. The information processing via the complex-

ity of dendritic and axonal arrays of both the pyramidal and nonpyramidal cells, therefore, allows for many cortical layers to influence the output of one specific cortical lamina. Cognizance of the intracortical distributions of all the neuronal processes of the LEC (both dendritic and axonal) is

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obviously important in attempting to understand further how this cortex normally processes information (see, White, '89). Although these suppositions are from observations made in the neocortex, they remain to be defined for the LEC. This study does, however, provide information needed for identification of labeled axonal pathways onto identified cells of the monkey LEC with ultrastructural techniques. Similar techniques used in conjunction with on-grid immunocytochemistry, utilized in implying neocortical functional anatomy (Cipolloni and Keller, '89), will aid in clarifying further and more precisely the role of these various neuronal types in the LEC. In human disease studies, such as Alzheimer's disease, there are apparently laminar and chemically specific neuronal losses. These neuronal losses (Hyman et al., '84; Joynt and McNeill, '84; Kemper, '84; Roberts et al., '85; Morrison et al., '86) or changes in the levels of neurotransmitters or neuromodulators (Davies et al., '80; Rossor et al., '80; Delfs and Dichter, '83; Beal et al., '85; Morrison et al., '85; Hyman et al., '86) imply alterations in the normal connectivity of the brain. The morphological data on the neurons of the LEC of the monkey and their associated neurochemicals described in this report may provide further insight into the pathological substrate of Alzheimer's disease.

ACKNOWLEDGMENTS The authors are grateful to Ms. Valerie Knowlton, Ms. Elena Yotides, Mr. Robert Ferrante, and Mr. Robert Landrigan for their technical assistance. We also thank Dr. Neil W. Kowall for providing some tissue prepared for immunocytochemical analysis, and Drs. Christine Nelsen, M. Flint Beal, Massinio Fiandaca, James Hamos, Deepak Pandya, and Edward Yeterian for providing us with useful comments on the manuscript. This study was supported by BRSG #6-32724, University of Massachusetts Medical Center, Worcester, MA 01655; NIH Grant #20967; the Veterans Administration; the Edith Nourse Rogers Memorial Veterans Administration Medical Center; the Institute for Neurologic Research, Bedford, MA 01730; and Scottish Rite Schizophrenia Program, NMJ, U.S.A.

LITERATURE CITED Amaral, D.G., and J.L. Price (1984) Amygdalo-cortical projections in the J . Comp. Neurol. 230r463496. monkey ( M Q C ~ C fascicularis). U Amaral, D.G., R. Insausti, and W.M. Cowan (1983) Evidence for a direct projection from the superior temporal gyrus to the entorhinal cortex in the monkey. Brain Res. 275:263-277. Amaral, D.G., R. Insausti, and W.M. Cowan (1987) The entorhinal cortex of the monkey: I. Cytoarchitectonic organization. J. Comp. Neurol. 2641326 355. Andersen, P., T.V.P. Bliss, and K.K. Skrede (1971) Lamellar organization of hippocampal excitatory pathways. Exp. Brain Res. 13:222-238. Baron, M. (1976) Organizacion functional de la capa I de la corteza cerebral: Cellulas de Cajal. [Doctoral thesis]. Ann. Ins. Farm. Esp. 22r23-240. Baughman, R.W., and C.D. Gilbert (1981) Aspartate and glutamate as possible neurotransmitters of cells in layer 6 of the visual cortex. Nature 287:84%849. Beal, M.F., M.F. Mazurek,V.T. Tran, G. Chattha, E.D. Bird, and J.B. Martin (1985) Reduced numbers of Somatostatin receptors in the cerebral cortex in Alzheimer's disease. Science 229:289-291. Beal, M.F., N.W. Kowall, K.J. Schwartz, R.J. Ferrante, and J.B. Martin (1988) Chronic quinolinic acid lesions in rats closely mimic Huntington's disease. Neurosci. Abstr. 14r299.1. Blackstad, T.W. (1956) Commissural connections of the hippocampal region in the rat, with special reference to their mode of termination. J. Comp.

Neurol. 105.417-538, Braak, H. (1980) Types of nerve cells forming the telencephalic cortex. In H. Braak (ed.): Architectonics of the Human Telencephalic Cortex. New York: Springer-Verlag. Braak, H., and E. Braak (1985) On areas of transition between entorhinal allocortex and temporal isocnrtex in the human brain. Normal morphology and lamina-specific pathology in Alzheimer's disease. Acta Neuropathol. (Berl.) 68:325-332. Braak, H., E. Braak, and H. Strenge (1976) Gehoren die Inselneurone der Regio entorhinalis zur Klasse der Pyramiden-oder der Sternzellen? 2. Mikrosk. Anat. Forsch. W.%,S. 1017-1031. Braitenberg, V., V. Guglielmotti, and E. Sada (1967) Correlation of crystal growth with the staining of axons by the Golgi procedure. Stain Technol. 42t277-282. Brodal, A. (1981) The olfactory pathways. In A. Brodal (ed) Neurological Anatomy in Relation to Clinical Medicine, Ed. 3. New York, Oxford: University Press, Chap. 10, pp. 640494. Brodmann, K. (1909) Vergleichende Lokalisationslehre der Grosshirnrinde in ihren Prinzipien dargestellt auf Grind des Zellenbaues. Z. Zellforsch. Leipzig: J.A. Barth, 324 pp. Cipolloni, P.B., and A. Keller (1989) Thalamocortical synapses with identified neurons in monkey primary auditory cortex: A combined Golgi/EM and GABA/peptide immunochemistry study. Brain Res. 4921347-355, Colonnier, M. (1968) Synaptic patterns on different cell types in the different laminae of the cat visual cortex: An electron microscope study. Brain Res. 9:268-287. Colonnier, M., and S. Rossignol (1969) On the heterogeneity of the cerebral cortex. In H. Jasper, A. Pope, and A. Ward (eds): Basic Mechanisms of the Epilepsies. Boston: Little Brown. Conti, F., M. Fabri, and T. Manzoni (1987) An immunocytochemical study on glutaminergic cortico-cortical neurons in the somatic sensory areas of monkeys. SOC.Neurosci. Ahstr. 13:73.16. Corwin, J., M. Serby, and P. Conrad (1985) Olfactory recognition deficits in Alzheimer's and Parkinsonian patients. IRCS Med. Sci. 131260. Cragg, B.G. (1961)Olfactory and other afferent connections ofthe hippocampus in the rabbit, rat and cat. Exp. Neurol. 3t588-600. Cuenod, M., P. Bagnoli, A. Beaudet A. Rustiono, L. Wiklund, and P. Streit (1982) Transmitter-specific retrograde labeling of neurons. In V. ChanPalay and S.L. Palay (eds): Cytochemical Methods in Neuroanatomy, vol. 1 New York: Alan R. Liss, Inc., pp. 17-44. Davies, P., R. Katzman, and R.D. Terry (1980) Reduced somatostatin-like immunoreactivity in cerebral cortex from cases of Alzheimer disease and Alzheimer senile dementia. Nature 288:27%280. Delfs, J.R., and M.A. Dichter (1983) Effects of somatostatin on mammalian cortical neurons in culture: Physiological actions and unusual dose response characteristics. J. Neurosci. 3r1176-1188. DiFiglia, M., N. Aronin, and J.B. Martin (1982a) The light and electron microscopic localization of immunoreactive somatostatin in the rat caudate nucleus. J. Neurosci. 9rl267-1274. DiFiglia, M., N. Aronin, and J.B. Martin (1982h) Light and electron microscopic localization of imrnunoreactive leuenkephalin in the monkey basal ganglia. J. Neurosci. 2(3):303-320. DiFiglia, M., G.A. Graveland, and L. Schiff (1987) Cytochrome oxidase activity in the rat caudate nucleus: Light and electron microscopic observations. J. Comp. Neurol. 255:137-145. Fairen, A,, A. Peters, and J. Saldanha (1977) A new procedure for examining Golgi impregnated neurons by light and electron microscopy. J. Neurocytol. 6:311-337. Feldman, M. (1984) Morphology of the neocortical pyramidal neuron. In A. Peters and E.G. Jones (eds): Cerebral Cortex. I. Cellular Components of the Cerebral Cortex. New York Plenum Press, pp. 123-200. Feldman, M.L., and A. Peters (1978) The forms of non-pyramidal neurons in the visual cortex of the rat. J. Comp. Neurol. 1791761-794. Ferrante, R.J., and N.W. Kowall(l987) Nicotinamide adenine dineucleotide phosphate-diaphorase (NADPH-d) histochemistry of the human caudate nucleus. SOC.Neurosci. Abstr. 13:205.19. Ferrante, R.J., N.W. Kowall, M.F. Beal, J.B. Martin, E.D. Bird, and E.P. Richardson (1987) Morphologic and histochemical characteristics of a spared subset of striatial neurons in Huntington's disease. J. Neuropathol. Exp. Neurol. 46:1227. Finch, D.M., E.E. Wong, E.L. Derian, X.Chen, N.L. Nowlin-Finch, and L.A. Brothers (1986) Neurophysiology of limbic system pathways in the rat: Projections from the amygdala to the entorhinal cortex. Brain Res. 370:273-~284. Fink, R.P., and L. Heimer (1967) Two methods for selective silver impregna-

MONKEY ENTORHINAL CORTICAL NEURONS tion of degenerating axons and their synaptic endings in the central nervous system. Brain Res. 4:369-374. Fleming, M.D., and J. Rogers (1986) Neuropathology of the olfactory system in Alzheimer’s disease and normal aging. Sac. Neurosci. Abstr. 12356.5. Geneser-Jensen, F.A., F.M.S. Haug, and G. Danscher (1974) Distribution of heavy metals in the hippocampal region of the guinea pig. A light microscope study with Timm’s sulphide silver method. Z. Zellforsch. 147r441478. Globus, A., and A.B. Scheibel (1967) Pattern and field in cortical structure: The rabhit. d. Comp. Neurol. 231.3:155-172. Gray, E.G. (1959) Axo-somatic and axo-dendritic synapses of the cerebral cortex. An electron microscope study. J. Anat. 93~420-433. Gray, T.S. (1983) The morphology of somatostatin-immunoreactive neurons in the lateral nucleus of the rat amygdala. Peptides 4r663-668. Harris, K.M. (1978) A rapid Golgi technique for brain tissue less than 0.5 mm thick. Soc. Neurosci. Abstr. 4:1053. Haug, F.-M.S. (1976) Sulphide silver pattern and cytoarchitectonics of parahippocampal areas in the rat. Adv. Anat. Embryol. Cell Biol. 52(4):1-73. Heimer, L. (1968) Synaptic distributions of centripetal and centrifugal nerve fibers in the olfactory system of the rat. An experimental anatomical study. J. Anat. IU3r413-432. Hjorth-Simonsen, A. (1972) Projections of the lateral part of the entorhinal area to the hippocampus and fascia dentata. J. Comp. Neural. 146r219232. Hjorth-Simonsen, A., and B. Jeune (1972) Origin and termination of the hippocampal perforant path in the rat studied by silver impregnation. J. Comp. Neurol. 144:21.5-232. Hooper, M.W., and F.S. Vogel (1976) The limbic system in Alzheimer’s disease: A neuropathologic investigation. Am. J. Pathol. 85:l-13. Hyman, B.T., G.W. Van Hoesen, A.R. Damasio, and C.L. Barnes (1984) Alzheimer’s disease: Cell-specific pathology isolates the hippocampal forrnation. Science 225:1168-1170. Hyman, B.T., G.W. Van Hoesen, and A.R. Damasio (1986) Glutamate depletion of the perforant pathway terminal zone in Alzheimer’s disease. Soc. Neurosci. Abstr. 12944. Insausti, R., D.G. Amaral, and W.M. Cowan (1987a) The entorhinal cortex of the monkey: 11. Cortical d e r e n t s . d. Comp. Neurol. 264356-395. Insausti, R.: D.G. Amaral, and W.M. Cowan (1987b) The entorhinal cortex of the monkey: 111. Subcortical afferents. J. Comp. Neurol. 264:396-408. Johansson, O., T. Hokfelt, and R. Elde (1984) Immunohist,ochemical distribution of somatostatin-like immiinoreactivity in the central nervous system of the rat. Neuroscience 13265-339. Jones, E.G. (1975) Varieties and distribution of non-pyramidal cells in the somatic sensory cortex of the squirrel monkey. J. Comp. Neurol. 16Ur205268. Jones, E.G. (1984) Laminar distribution of cortical efferent cells. In A. Peters and E.G. Jones (eds): Cerebral Cortex. I. Cellular Components of the Cerebral Cortex. New York Plenum Press, pp. 521-553. Jones, E.G., and T.P.S. Powell (1970) An anatomical study of converging sensory pathways within the cerebral cortex of the monkey. Brain 935’9% 820. Jones, E.G., S.H.C. Hendry, and J. DeFelipe (1987) GABA-peptide neurons of the primate cerebral cortex: A limited cell class. In E.G. Jones and A. Peters (eds): Cerebral Cortex. VI. Further Aspects of Cortical Function, Including Hippocampus. New York Plenum Press, pp. 237-266. Joynt, R., and T.H. McNeill (1984) Neuropeptides in aging and dementia. Peptides 5 (Suppl. 1):269-274. Kaiserman-Abramof, LR., and A. Peters (1972) Some aspects of the morphology of Betz cells in the cerebral cortex of the cat. Brain Res. 43:527-546. Kaneko, T., and N. Misuno (1988) Immunohistochemical study of glutaminase-containing neurons in the cerebral cortex and thalamus of the rat. J. Comp. Neurol. 267:59&602. Kemper, T. (1984) Neuroanatomical and neuropathological changes in normal aging and dementia. In M.L. Albert (ed): Clinical Neurology of Aging. New York Oxford University Press, pp. 9-52. Kosel K.C., G.W. Van Hoesen, and J.R. West (1981) Olfactory bulb projections to the parahippocampal area of the rat. J. Comp. Neural. 198:467482. Kosel K.C., G.W. Van Hoesen, and D.W. Rosene (1982) Non-hippocampal cortical projections from the entorhinal cortex in the rat and the rhesus monkey. Brain Res. 244~201-213. Kowall, N.W., M.F. Beal, R.d. Ferrante, and J.B. Martin (1985) Topography of nicotinamide adenine dinucleotide phosphate-diaphorase staining neurons in rat striatum. Neurosci. Lett. 593561-66.

607 Krettek, J.E., and J.L. Price (1974) Projections from the amygdala to the perirhinal and entorhinal cortices and the suhiculum. Brain Res. 71r150154. Krettek, J.E., and J.L. Price (1977a) The cortical projections of the mediodorsal nucleus and adjacent thalamic nuclei in the rat. J. Comp. Neurol. 171:157-192. Krettek, J.E., and J.L. Price (1977b) Projections of the amygdaloid complex and adjacent olfactory structures to the entorhinal cortex and suhiculum in the rat and cat. J. Comp. Neurol. 172323-752. Krieg, W.J.S. (1946) Connections of the cerebral cortex. I. The albino rat. A. Topography of the cortical areas. J. Comp. Neurol. 84:221-275. Lee, A.B. (1937) Axis-cylinder and dendrite stains (Golgi and others). In J.B. Gatenby and T.S. Painter (eds): Lee’s Microtomist’s Vademecum. London: Churchill, pp. 496-520. Lorente de No’, R. (1933) Studies on the structure of the cerebral cortex. I. The area entorhinalis. J. Psychol. Neurol. 45381-438. Lorente de No’, R. (1934) Studies on the structure of the cerebral cortex. 11. Cont,inuation of the study of the ammonic system. J. Psychol. Neurol. 4fil13-177. Lorente de No’, R. (1938) Cerebral cortex: Architecture, intracortical connections, motor projections. In d.F. Fulton (ed): Physiology of the Nervous System, Ed. 2. London: Oxford University Press, pp. 288-213. Lund, .J.S. (1973) Organization of neurons in the visual cortex, area 17, of the monkey (Macaca mulatta).J. Comp. Neurol. 147r455-496. Lund, J.S. (1984) Spiny stellate neurons. In A. Peters and E.G. Jones (eds): Cerebral Cortex, vol. 1:Cellular Components of the cerebral cortex. New York: Plenum Press, pp. 225-308. Lund, J.S. (1987) Local circuit neurons of macaque monkey striate cortex: Neurons of laminae 4c and 5a. J. Comp. Neural. 257:60-92. Lund, J.S. and R.D. Lund (1970) The termination of callosal fibers in the paravisual cortex of the rat. Brain Res. 17r25-45. Lund, J.S., R.G. Boothe, and R.D. Lund (1977) Development of neurons in the visual cortex (area 17) of the monkey (Macaca nemestrina): A Golgi study from fetal day 127 to post natal maturity. J. Comp. Neurol. 176:149-188. Lund, J.S., A.E. Hendrickson, M.P. Ogren, and E.A. Tobin (1981) Anatomical organization of the primate visual cortex area VII. J. Comp. Neurol. 202: 19-45. Marin-Padilla, M., and M.T. Marin-Padilla (1982) Origin, prenatal development and structural organization of layer I of the human cerebral (motor) cortex: A Golgi study. Anat. Embryol. 164r161-206. McLean, I.W., and P.K. Nakane (1974) Periodate-lysine-paraformaldehyde fixative: A new fixative for immunoelectron microscopy. J. Histochem. Cytochem. 22(12):1077-1083. McLean, J.H., M. Shipley, and D.I. Bernstein (1989) A possible role of the olfactory system in Alzheimer’s disease. AHAF Alzheimer’s Disease Conference Abstract, p. 4. Meinecke, D.L. and A. Peters (1987) Gaba immunoreactive neurons in rat visual cortex. J. Comp. Neurol. 261 (3):38&404. Meller, K., W. Breipohl, and P. Glees (1968) The cytology of the developing molecular layer of mouse motor cortex. Z. Zellforsch. Mikrosk. Anat. 96:171-183. Mellgren, S.I., and T.W. Blackstad (1967) Oxidative enzymes (tetrazolium reductases) in the hippocampal region of the rat. Distribution and relation to architectonics. Z. Zellforsch. 78:167-207. Morrison, d.H., J. Rogers, S. Scherr, R. Benoit, and F.E. Bloom (1985) Somatostatin immunoreactivity in neuritic plaques of Alzheimer’s patients. Nature 31490-92. Morrison, J.H., S. Scherr, D.A. Lewis, M.J. Campbell, F.E. Bloom, J. Rogers, and R. Benoit (1986) The laminar and regional distribution of neocortical somatostatin and neuritic plaques: Implications for Alzheimer’s disease as a global neocortical disconnection syndrome. In A.B. Scheibel and A.F. Weschler (eds): The Biological Substrates of Alzheimer’s Disease, New York Academic Press, pp. 115-131. Nauta, W.J.H., and H.J. Karten (1970) A general profile of the vertebrate brain, with sidelights on the ancestry of cerebral cortex. In F.O. Schmitt (ed): The Neurosciences: Second Study Program. New York Rockefeller, IJniversity Press pp. 7-25. Ottersen, O.P. (1982) Connections of the amygdala of the rat. I V Corticoamygdaloid and intraamygdaloid connections as studied with axonal transport of horseradish peroxidase. d. Comp. Neurol. 20530-48. Pandya, D.N., G.W. Van Hoesen, and M.M. Mesulam (1981) Efferent connections of the cingulate gyrus in the rhesus monkey. Exp. Brain Res. 42r319-330.

608 Peters, A. (1984) Bipolar cells. In A. Peters and E.G. Jones (eds): Cerebral Cortex, vol. 1: Cellular Components of the Cerebral Cortex. New York: Plenum Press, pp. 381-407. Peters, A., and E.G. Jones (1984) In A. Peters and E.G. Jones (eds):Cerebral Cortex, vol. 1: Cellular Components of the Cerebral Cortex. New York Plenum Press, chap. 4. Peters, A,, and C.C. Proskauer (1980) Synaptic relations between a multipolar stellate cell and a pyramidal neuron in the rat visual cortex. A combined Golgi-electron microscope study. J. Neurocytol. 9:1&183. Peters, A., and d. Regidor (1981) A reassessment of the forms of nonpyramidal neurons in area 17 of cat visual cortex. J. Comp. Neurol. 203.685716. Peters, A., M. Feldman, and J. Saldanha (1976) The projection of the lateral geniculate nucleus to area 17 of the rat cerebral cortex. 11. Terminations upon neuronal perikarya and dendritic shafts. J. Neurocytol. 5235-107. Polack, J.M., and S. VanNoorden (eds) (1986) Immunoeytochemistry. Modern Methods and Applications, Ed. 2. Winston-Salem: Wright Publishing. Powell, T.P.S., W.M. Cowan, and G. Raisman (1965) The central olfactory connections. J. Anat. 99391-813. Price, J.L. (1973) An autoradiographic study of complementary laminar patterns of termination of afferent fibers to the olfactory cortex. J. Comp. Neurol. 150:87-108. Price, J.L., and T.P.S. Powell (1971) Certain observations in the olfactory pathway. J. Anat. 110:105-126. R a m h y Cajal, S. (1899) Histologie du Systhme nerveux de 1’Homme et des Vertkhres, vol. 11. Paris: Maloine. Ram6n y Cajal, S. (1901) Studies on the Cerebral Cortex [Limbic Structures]. Trans. L.M. Kraft. London: Lloyd-Luke, 1955. Ramon-Moliner, E. (1961) The histology of the postcruciate gyrus of the cat. 111. Further observations. J. Comp. Neurol. 117:229-249. Ramon-Moliner, E. (1967) La differentiation morpbologique des neurones. Arch. Ital. Biol. 105t149-188. Reyes, P.F., G.T. Golden, P.L. Fagel, R.G. Fariello, L. Katz, and E. Carner (1985) Olfactory pathways in Alzheimer’s disease. Soc. Neurosci. Ahstr. 2(1):168. Reyes, P.F., G.T. Golden, P.L. Fagel, R.G. Fariello, L. Katz, and E. Carner (1985) Olfactory pathways in Alzheimer’s disease. Neurosci. Ahstr. 2(1):168. Roberts,G.W.,T.J. Crow, and J.M.Polak (1985) Locationof neuronal tangles in somatostatin neurones in Alzheimer’s disease. Nature 314.92-94. Room, P., and H.J. Groenewegen (1986) Connections of the parahippocampal cortex. I. Cortical afferents. J. Comp. Neurol. 251r415-450. Rossor, M.N., P.C. Emson, Q.Mountjoy, M. Roth, and L.I. Iverson (1980) Reduced amounts of immunoreactive somatostatin in the temporal cortex in senile dementia of the Alzheimer type. Neurosci. Lett. 2Or373-377. Sanides. F., and D. Sanides (1972) The “extraverted neurons” of the mammalian cerebral cortex. Z. Anat. EntwickL-Gesch. 136t272-293. Saunders, R.C., and D.L. Rosene (1988) A comparison of the amygdala and the hippocampal formation in the rhesus monkey. I. Convergence in the entorhinal, prorhinal and perirhinal cortices. J. Comp. Neurol. 271(2):153-184. Scalia, F. (1966) Some olfactory pathways in the rabbit brain. J. Comp. Neurol. I26:285-310. Schwartz, S.P.,and P.D. Coleman (1981) Neurons of the perforant path. Exp. Neurol. 741305-312. Segal, M., and S. Landis (1974) Afferents to the hippocampus of the rat studied with the method of retrograde transport of horseradish peroxidase. Brain Res. 7R.1-15. Seltzer, B., and D.N. Pandya (1978) M e r e n t cortical connections and architectonics of the superior temporal sulcus and surrounding cortex in the rhesus monkey. Brain Res. 149:l-24. Siggins, G.R., and D.L. Gruol(l986) Mechanisms of transmitter action in the vertebrate central nervous system. In V.B. Mountcastle, F.E. Bloom, and S.R. Geiger (eds): Handbook of Physiology, Sect. I: The Nervous System. Bethseda, Md: American Physiological Society, pp. 1-114. Simpson, J., C.M. Yates, and A. Gordon (1984) Olfactory tubercle choline acetyltransferase activity in Alzheimer-type dementia, Down’s syndrome and Huntington’s disease. J. Neurol. Neurosurg. Psychiatry 47t1136 1139. Sloper, J.J., and T.P.S. Powell (1979) An electron microscopic study of d e r ent connections to the primate motor and somatic sensory cortices. Phil. Trans. R. SOC. Lond. [Biol.] 285:19%226. Sousa-Pinto, A,, M. Paula-Barhosa, and M. Carmo Matos (1975) A Golgi and electron microscopical study of nerve cells in layer I of the cat auditory cortex. Brain Res. 9S:443458.

A.A. CARBON1 ET AL. Steward, 0. (1976) Topographic organization of the projections from the entorhinal area to the hippocampal formation of the rat. J. Comp. Neurol. 167:285-314. Steward, O., and S.A. Scoville (1976) Cells of origin of entorhinal cortical afferents to the hippocampus and fascia dentata of the rat. J. Camp. Neurol. 169:347-370. Storm-Mathisen, J., and T.W. Blackstad (1964) Cholinesterase in the hippocampal region. Distribution and relation to architectonics and afferent systems. Acta Anat. (Basel)56:21&253. Streit, P. (1984) Glutamate and aspartate as transmitter eandidates for systems of the cerehral cortex. In E.G. Jones and A. Peters (eds): Cerebral Cortex, vol. 2. New York Plenum Press, pp. 119-143. Streit, P., and M. Cuenod (1979) Transmitter specificity and connectivity revealed by differential retrograde labeling of neuronal pathways. Neurosci. Lett. Suppl. 3:340. Tagaki, H., P. Somogyi, J. Somogyi, and A.D. Smith (1983) Fine structural studies on a type of somatostatin-immunoreactive neuron and its synaptic connections in the rat neostriatum: A correlated light and electron microscopic study. J. Comp. Neurol. 214:l-16. Valverde, F. (1970) The Golgi method. A tool for comparative structural analyses. In W.J.H. Nauta and S.O.E. Ehbesson (eds): Contemporary Research Methods in Neuroanatomy. New York Springer-Verlag, pp. 11-31. Valverde, F. (1971) Short axon neuronal subsystems in the visual cortex of the monkey. Int. J. Neurosci. Ir181-197. Valverde, F. (1976) Aspects of cortical organization related to the geometry of neurons with intracortical axons. J. Neurocytol. 5509-529. Van Hoesen, G.W. (1982) The parahippocampal gyrus. New observations regarding its cortical connections in the monkey. TINS 5345-350. Van Hoesen, G.W., and D.N. Pandya (1975a) Some connections of the entorhinal (area 28) and perirhinal (area 35) cortices of the rhesus monkey. I. Temporal lobe afferents. Brain Re.95:1-24. Van Hoesen, G.W., and D.N. Pandya (1975b) Some connections of the entorhinal (area 28) and the perirhinal (area 35) cortices of the rhesus monkey. 111. Efferent connections. Brain Res. 95:39-59. Van Hoesen, G.W., D.N. Pandya, and N. Butters (1972) Cortical d e r e n t s to the entorhinal cortex of the rhesus monkey. Science 175:1471-1473. Van Hoesen, G.W., D.N. Pandya, and N. Butters (1975) Some connections of the entorhinal (area 28) and perirhinal (area 35) cortices of the rhesus monkey. 11. Frontal lohe afferents. Brain Res. 95:25-38. Vincent, S.R., and 0. Johansson (1983) Striatal neurons containing both somatostatin- and avian pancreatic polypeptide (APP)-like immunoreactivities and NADPH-diaphorase activity: A light and electron microscopic study. J. Comp. Neurol. 217:26&270. Vincent, S.R., 0. Johansson, T. Hokfelt, L. Skirboll, R.P. Elde, L. Terenius, J. Kimmel, and M. Goldstein (1983) NADPH-diaphorase: A selective histochemical marker for ntriatal neurons containing both somatostatin- and avian pancreatic polypeptide (APP)-like immunoreactivities. J. Comp. Neurol. 21 7r252-263. Vogt, B.A., and A. Peters (1981) Form and distribution of neurons in rat cingulate cortex: Areas 32,24 and 29. J. Comp. Neurol. 195;603425. White, E.L. (1989) Synaptic Connections Between Identified Elements. In E.B. White (ed): Cortical Circuit Synaptic Organization of the Cerebral Cortex-Structure, Function and Theory. Boston: Birkhauser. Whitehouse, P.J., D.L. Price, R.G. Struble, A.W. Clark, J.T. Coyle,and M.R. Delong (1982) Alzheimer’s disease and senile dementia: Loss of neurons in the basal forebrain. Science 215:1237-1239. Winfield, D.A., R.N.L. Brook, J.J. Sloper, and T.P.S. Powell (1981)A combined Golgi-electron microscopic study of the synapses made hy the proximal axon and recurrent collaterals of a pyramidal cell in the somatic sensory cortex of the monkey. Neuroscience 6:1217 -1230. Witter, M.P., P. Room, H.J. Groenewegen, and A.H.M. Lohman (1986) The connections of the parahippocampal cortex in the cat. V. Intrinsic connections; Comments on the input/output connections with the hippocampus. J. Comp. Neurol. 2.52:7694. Wong-Riley, M. (1979) Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry. Brain Res. I71:I 1-28. Wong-Riley, M.T., and E.W. Carroll (1984) Quantitative light and electron microscopic analysis of cytochrome oxidase-rich zones in V I1 prestriate cortex of the squirrel monkey. J. Comp. Neurol. 222:1%37. W o u t e r l d , F.G., and J. Nederlof (1983) Terminations of olfactory afferents on layer I1 and 111 neurons in the entorhinal area: Degeneration-Golgielectron microscopic study in the rat. Neurosci. Lett. .366:105-110. Wyss, J.M. (1981) An autoradiographic study of the efferent connections of the entorhinal cortex in the rat. J. Comp. Neurol. 199t495-512.