morphology and kainate-receptor immunoreactivity of identified neurons within the entorhinal cortex...

THE JOUKNAL OF COMPARATIVE NEUROLOGY 35795-35 (1995)

Morphology and Kainate-Receptor Immunoreactivity of Identified Neurons

Within the Entorhinal Cortex Projecting to Superior Temporal Sulcus in the

Cynomolgus Monkey

PAUL F. GOOD AND JOHN H. MORRISON Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine,

New York. New York 10029

ABSTRACT Projections of the entorhinal cortex to the hippocampus are well known from the classical

studies of Cajal (Ramon y Cajal, 1904) and Lorente de NO (1933). Projections from the entorhinal cortex to neocortical areas are less well understood. Such connectivity is likely to underlie the consolidation of long-term declarative memory in neocortical sites. In the present study, a projection arising in layer V of the entorhinal cortex and terminating in a polymodal association area of the superior temporal gyrus has been identified with the use of retrograde tracing. The dendritic arbors of neurons gving rise to this projection were further investigated by cell filling and confocal microscopy with computer reconstruction. This analysis demonstrated that the dendritic arbor of identified projection neurons was largely confined to layer V, with the exception of a solitary, simple apical dendrite occasionally ascending to superficial laminae but often confined to the lamina dissecans (layer IV). Finally, immunoreactivity for glutamate-receptor subunit proteins GluR 5/61 7 of the dendritic arbor of identified entorhinal projection neurons was examined. The solitary apical dendrite of identified entorhinal projection neurons was prominently immunolabeled for GluR 5/6/7, as was the dendritic arbor of basilar dendrites of these neurons. The restriction of the large bulk of the dendritic arbor of identified entorhinal projection neurons to layer V implies that these neurons are likely to be heavily influenced by hippocampal output arriving in the deep layers of the entorhinal cortex. Immunoreactivity for GluR 51617 throughout the dendritic arbor of such neurons indicates that this class of glutamate receptor is in a position to play a prominent role in mediating excitatory neurotransmission within hippocampal-entorhinal circuits. o 1995 WiIey-Liss, Inc.

Indexing terms: hippocampal formation, memory, glutamate receptor, confocal microscopy

The entorhinal cortex (ERC), which is located medial to the rhinal sulcus in the monkey cerebral cortex, is an allocortical region providing a key route of connectivity between neocortical association areas and the hippocampus. Storage of memories of specific objects or events such as faces or places, often called declarative memory, is believed to be dependent on processing of neocortical information by the hippocampus (Squire and Zola-Morgan, 1991). Although such processing of sensory information is necessary in the early phase of memory consolidation, later phases of memory become independent of the hippocampus (Zola-Morgan and Squire, 1990). Furthermore, studies by Damasio et al. (1982) and Squire and Zola-Morgan (1991) have demonstrated that the hippocampus is not the reposi- tory of declarative memory. To the extent that memory can

be localized, these studies have shown that lesions of specific cortical sites known to underlie processing of specific sensory submodalities (e.g., faces) result in the loss of the ability to recognize or remember specific features associated with that submodality. These findings imply that information flow both into the hippocampal formation and from the hippocampal formation returning to such neocortical sites is essential for the storage and function of declarative memory.

Accepted November 17, 1994. Address reprint requests to John H. Morrison, Fishberg Research Center

for Neurobiology, Box 1065, Mount Sinai School of Medicine, New York, NY 10029.

O 1995 WILEY-LISS, INC.

26 P.F. GOOD AND J.H. MORRISON

Previous studies with anterograde and retrograde tracers (Van Hoesen et al., 1972, 1975; Van Hoesen and Pandya, 1975a; Turner et al., 1980; Insausti et al., 1987; Suzuki and Amaral, 1994) have clarified the areal and laminar organization of af€erents to the ERG, yet little is known about reciprocal connections of many identified afferents. The study of Insausti et al. (1987) demonstrated regional and laminar organization of widespread afferents to ERC all arising from periallocortical areas with the exception of a projection to ERG from a polymodal sensory association area located within the superior temporal cortex that is coincident with areas TPO and TAa of Seltzer and Pandya (1978). Full reports of ERC projections reciprocating identified afferents have been provided only for those to parahippocampal (Kosel et al., 1982) and cingulate cortices (Baley- dier and Mauguiere, 1980), whereas reciprocal projections to other limbic and sensory association cortices have been confirmed but have not been presented in detail (Arnold et al., 1988; Tourtellotte et al., 1988; Good et al., 1991). Of particular interest for the present study was the projection to ERG from a subdivision of the superior temporal gyrus lying on the dorsal bank of the superior temporal sulcus (STS), including areas TPO and TAa, and shown to have distinct multimodal sensory properties (Bruce et al., 1981; Baylis et al., 1987). These studies demonstrated that areas forming the roof and depth of the STS represent a conflu- ence of high-order sensory processing of visual, auditory, and somatosensory information.

Although data on corticocortical connectivity provide understanding of information flow between cortical areas, more precise knowledge of the cellular and molecular characteristics of identified efferent neurons correlated with the laminar pattern of afferents to identified projection neurons has rarely been generated in the context of a specific corticocortical circuit. Morphological analysis of neurons of the lateral ERG has been reported (Lorente de NO, 1933; Carboni et al., 1990); however, such analyses of neurons of the ERC did not correlate dendritic morpholoa or laminar organization of dendritic arbor with known efferent targets or afferent inputs of such neurons. In the present study, the identification of neurons by retrograde transport of the fluorescent dye fast blue has been com- bined with the intracellular injection of identified neurons with the fluorescent dye Lucifer yellow (LY). This procedure allows the determination of the dendritic morphology of ERG neurons projecting to known neocortical regions. The dendritic arbor of identified projection neurons was further investigated by immunolabeling for kainate-class glutamate-receptor subunit proteins. Along with N-methyl- D-aspartate (NMDA) and a-amino-3-hydroxy-5-methyl-4- isoxazole-propionic acid (AMPA) receptors, kainate receptors form a major class of ligand-gated ion channel receptors responsive to glutamate, a major excitatory neurotransmit- ter of the cortex. With the use of a monoclonal antibody of the IgM class (mAb-4F5) that specifically recognizes glutamate receptor subunits GluR 5, 6, and 7 (Huntley et al., 1993), sections of ERG containing LY-filled neurons were immunolabeled in order to determine the distribution of kainate receptors in excitatory circuits of identified ERC efferent neurons.

MATERIALS AND METHODS Eight adult male cynomolgus monkeys weighing 4-7 kg

were used in this study. Animals were housed and handled

in accordance with NIH and Mt. Sinai Institutional Animal Care and Use Committee guidelines. Animals were anesthetized with ketamine (25 mgikg), intubated, and maintained on an oxygenihalothane mixture. The animals were placed in a Kopf large-animal stereotaxic apparatus (David Kopf Instruments, Tujunga, CA), and a vertical incision was made 2-3 cm rostral to the ear bar and extending 5-8 cm dorsally from the zygomatic arch. A large bone flap was removed and the dura reflected, exposing the lateral and superior temporal sulci. A 5 p1 Hamilton syringe (Reno, NV) with a 24 gauge needle was positioned 2-3 mm dorsal to the STS and inclined at an angle perpendicular to the cortical surface, approximately 5" above horizontal. The needle was inserted 7 mm deep into the cortex in the dorsal bank of the STS, creating a penetration parallel to the cortical surface of the sulcus. At this site, 400 nl of 5%) aqueous fast blue (Sigma Chemical Co., St. Louis, MO) was injected over 4 minutes, the needle was allowed to remain in place for an additional 5 minutes and was then withdrawn to a point 3.5 mm from the surface, and an additional 400 nl was injected in the same manner. The needle was then moved 1-2 mm along the external surface of the sulcus to the next injection site, and another pair of injections was made. Six to eight total injections were made in this manner. The bone flap was closed with Gelfoam (Upjohn, Kalamazoo, MI) and bone wax (Ethicon, Somerville, NJ) and covered with acrylic dental cement (Duralay Dental Mfg. Co., Worth; IL).

After a survival period of 3 weeks, animals were deeply anesthetized with ketamine (25 mgikg) and Nembutal (30 mgikg), intubated, and manually ventilated. Animals were perfused transcardially with 1% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) for 45 seconds, followed by 4% paraformaldehyde in PUS for 9 minutes. The brain was removed, and the temporal lobe was dissected from the brain, blocked coronally, and postfixed for 2 hours in the same fixative. Blocks were washed in PBS, and ERC was dissected from the remaining temporal lobe.

From seven animals, Vibratome sections 200 pm thick were cut in the coronal plane, washed, and held in cold PBS. Sections were then placed on a nitrocellulose membrane in a dish containing PBS, and fast blue-labeled cells were identified with a Nikon microscope equipped with UV (330-380 nm excitation, 400 nm dichroic mirror, 420 nm barrier) filter block for observation of fast blue, B2 (450- 490 nm excitation! 510 nm dichroic mirror, 520 nm barrier) filter block for observation of LY, and long-working- distance neofluor objectives. Labeled cells were impaled with a glass micropipette electrode containing 5% aqueous LY (Sigma), and the dye was iontophoretically (World Precision Instruments Inc., New Haven, CT) injected by passing a 5-10 nA current for 5-15 minutes. Fast blue- containing neurons were filled across the ERC with enough spacing to avoid dendritic overlap. Sections for morphological analysis were then mounted with Permafluor (Lipshaw Immunon, Pittsburgh, PA). From one animal, sections 100 pm thick were cut in the coronal plane and mounted directly on slides for mapping of retrograde transport. From three animals, sections with filled neurons processed for immunolabeling were equilibrated in 30% sucrose and mounted on frozen OCT (Lab-Tek Products, Naperville, IL), resectioned at 30 p m on a cryostat (Reichert, Vienna, Austria), and incubated for 3 days in 0.01 M PBS (PBSb) containing 5% nonfat dry milk and primary antibody mAb-4F5 (available from Pharmingen, San Diego, CA)

ERC-STS PROJECTION NEUKONS 27

diluted at 1: 1,000. Additional sections were processed in parallel, with the omission of primary antibody. Sections were further processed for 2 hours in biotinylated anti- mouse IgM (Vector Laboratories, Burlingame, CA) and 2 hours in avidin-Texas red (Amersham, Arlington Heights, IL), with three washes of PBSb intervening. and mounted with Permaffuor. Contralateral temporal lobes from three animals were sectioned at 30 pm on a cryostat or 50 pm on a Vibratome and were incubated in primary antibody at 1: 1,000 as described above with diaminobenzidine (DAB) as a chromogen.

Fast blue-containing neurons were mapped with an Axiophot microscope (Zeiss), and the dendritic arbors of LY-filled neurons were reconstructed with a confocal microscope (Zeiss LSM), both supported by a custom-designed Electronic Mapping and Morphology Analysis (EMMA) system, which was developed through a collaborative effort of Mt. Sinai and Scripps Research Institute. Retrogradely labeled neurons were mapped in a 1 of 8 series of the 100-pm-thick sections. A total of 140 LY-filled neurons were examined by fluorescence (Zeiss filter set Blue H 485) and confocal (488 nm argon laser) microscopy, and 26 LY-filled neurons were reconstructed with confocal microscopy; images were captured at 3 or 4 pm through the z axis and over the entire lateral extent of their dendritic arbor and were collapsed in the z axis. The somata and dendritic arbor of LY-filled neurons were then mapped into their location within the cortical slice. The dendritic arbors of resectioned, glutamate-receptor 51 6 / 7-immunolabeled, LY- filled neurons were reconstructed by aligning each subsection with adjacent sections and mapping superimposed images of LY-filled neurons. Immunolabeling of LY-filled dendrites by mAb-4F5-Texas red was observed using Zeiss filter set green 530-585 and was recorded on the map of the LY-filled neuron. Immunolabeling by mAb-4F5 was only mapped onto the dendritic arbor of LY-filled cells where immunolabeling of surrounding neuronal processes could be clearly identified within the plane of focus. Nomencla- ture of entorhinal cortex follows that ofAmaral et al. (1987) and recognizes the lamina dissecans of ERC as layer IV and the layer under investigation in the present study as layer V.

RESULTS Injections into the superior temporal sulcus in all eight

monkeys included area TPO (Fig. 1). In the mediolateral axis in one case, there was significant encroachment into the fundus of the STS, and, in all cases, there was extension into the lateral superior temporal fields TAa and TS. Because the injection needle was inserted parallel to the gray matteriwhite matter border, the white matter was not disrupted by this procedure. Although minor diffusion of fast blue into the white matter could not be ruled out, in all sections examined the apparent injection site excluded the underlying white matter. Retrograde transport in all cases labeled somata in layer V of ERC (Figs. 2, 5A), with labeled cells occasionally appearing in deep layer 111. Fast blue- labeled neurons were found at times in clusters, especially at the depth of the rhinal sulcus and at the center of the mediolateral aspect of the ERC. Retrogradely labeled cells were also seen in layers I11 and V of area TE (von Bonin and Bailey, 1947) but not in areas 35/36 (Brodmann, 1909) and were not observed in CA1 or subiculum. Labeling was present across the mediolateral extent of the ERC, but

- A

rs

Fig. 1. Fast blue injection site in superior Lemporal sulcus. Map of a representative fast blue injection site in the superior temporal sulcus of a cynomolgus monkey. Sections are taken at levels of the entorhinal cortex (dissected for morphologic analysis: A). hippocampal genu 031, lateral geniculate nucleus (C) , and posterior hippocampus (D). sts, Superior temporal sulcus; inj (stippled areas), maximum spread of injected tracer; rs, rhindl sulcus; lgn, lateral geniculate nucleus. Scale bar = 5 rnm.

some cases demonstrated a concentration of labeled neurons in the lateral portion of the ERC, and all labeling terminated at the rhinal sulcus. Retrograde labeling was concentrated to the greatest extent in intermediate, caudal, and caudal-limiting subfields (Ei, Ec, and Ecl) of the ERC, as described by Amaral et al. (1987).

Morphology of ERC projection neurons The extent of the dendritic arbor of identified ERC-

projection neurons was assessed by following the filling of LY in a given dendrite to its termination. By inspection, it could be determined whether filling was entirely within a section or terminated at the cut surface of a section (de Lima et al., 1990). Dendrites that could be followed within a section were seen to taper continuously until they no longer could be resolved with the LSM and were judged to be completely filled. Other dendrites that were observed end- ing abruptly at the surface of a section were determined to be cut dendrites. Dendritic arbors of identified layer V projection neurons were classified as typical pyramidal, asymmetric pyramidal, vertical fusiform, and multipolar (Table 1). The criteria employed in these definitions are similar to those employed by de Lima et al. (1990). In addition, there were rare inverted-pyramidal neurons seen in deep layer V or layer VI, accounting for 3 of 140 neurons analyzed for dendritic morphology. LY-filled neurons all had moderately spiny dendrites (Fig. 5E). Both their apical dendrite and their basal dendritic arbor appeared to have the same spine density, and the spine density was similar among all morphological types.

Pyramidal cells were defined as those cells having a large, unbranched apical dendrite extending toward superficial laminae. A second major category of identified projection neurons was the asymmetrical pyramid; the majority of


Fig. 2. Maps of' retrogradely labeled neurons in entorhinal cortex. Sections 100 Km thick are mapped at ROO bm intervals through the entorhinal cortex and are displayed from most rostra1 (upper left) to most caudal (lower right). Each point represents one labeled neuron. Outer solid linc rcprcscnts the layer IIiIII border; inner solid line

represents the layer IIIiIV border. amyg, Amygdala; dg, dentate gyrus; hf, hippocampal fissure; hip, hippocampus; Iv, lateral ventricle; pac, periamygdaloid cortex; rs, rhinal sulcus;. D, dorsal; V, ventral; M, medial; L, lateral. Scale bar = 2 mm.

these neurons had an apical dendrite terminating at the lamina dissecans (Table 1) and a second large primary dendrite traveling laterally or obliquely in layer V (Fig. 3).

The majority of identified neurons in four animals were of the pyramidal type (1111140; 78%). The basilar dendritic arbor of pyramidal neurons consisted of uniformly or,

ERC-STS PR0,JECTION NEURONS 29

TABLE 1. Distribution of Neuronal Types of ERC-STS Projection Neurons

Cell type Number Percent

Pyramidal Apical dendrite’

Superficial to LD Deep to LD nla2 Total

Asymmetric pyramidal Apical dendrite

Superficial tn LD Deep to LD Total

‘Total pyramidal neurons Multipolar Vertical fusifiirm Inverted pyrdmld Total

29 25 28 82

8 2 1 29 111 15 11 3

140

58

2n 78 11

R 2

’Apical dendrite indicates the extent of the apical dendrite, terminating superficially to the lamina dissecans (layer IV) or terminating deep to the lamina dissecans in thosc neurons that had complete apical dendrites. ’Cut dendrites that could not be analyzed.

occasionally, laterally distributed dendrites that were confined almost entirely to layer V of the ERC (Figs. 3, 5B). Together, pyramidal and asymmetrical pyramidal cells accounted for 78% of all identified neurons observed (Table 1). A striking feature of identified pyramidal-projection neurons was the simplified nature of their apical dendrite. These dendrites arose as an unbranched process, often displaying a few delicate lateral processes in layers IV and V. In about half of the pyramidal neurons analyzed. the apical dendrite did not extend above layer IV and gave these neurons the appearance of multipolar neurons (Table 1). The apical dendrite of the remainder of pyramidal cells extended to layer I11 and, often, to layer I1 but showed no arborization in superficial layers, remaining as a single shaft to its termination.

The remaining morphological types represented a minor- ity of the neurons analyzed. Multipolar cells possessed a dendritic arbor distributed uniformly around their somata. Vertical fusiform cells possessed vertically oriented somata, vertical apical and basal primary dendrites. and multiple lateral dendrites, the majority of which were confined to layer V. Finally, a few cells with the appearance of typical pyramids were found inverted deep in layer V with their “apical” dendrite extending through layer VI to the white matter. Mean, minimum, and maximum spread of dendrites of 25 fully reconstructed neurons along with the branching patterns of their basilar dendritic arbor are given in Table 2. In the coronal plane, the maximum spread of all neuronal types was similar, at approximately 550 pm. Because measurements were made on collapsed, two- dimensional images of neurons in a 200-km-thick section, actual dendritic spread could be as much as 35 pm greater.

Single-labeled immunoreactivity for GluR 5/6/7 in ERC

Throughout the ERC, many neurons were GluR 51617 immunoreactive (GluR 5/6/7-ir; Fig. 4A-Cj. In layer I of both rostral and caudal ERC, a network of GluR 5/6/7-ir neuronal processes was present through the layer and was continuous with processes of layer 11, where the intensity of immunolabeling increased. Neurons of layer 11, particularly the islands characteristic of rostral ERC, were densely immunoreactive and showed thick initial dendrites amid finer immunoreactive processes At rostral levels of layer 11, a narrow region of reduced immunoreactivity was present, forming a cap deep to the layer I1 islands. In layer 111, overall intensity of immunoreactivity was lower than in

layer 11, especially in the deeper part, and was present in both thick and thin apical dendrites and in a lightly labeled network of neuronal processes. Layer IV was a cell-sparse lamina and contained immunolabeling of bundled apical dendrites arising in deeper layers and few fine neuronal processes. Layer V contained heavily immunolabeled somata and initial apical-dendritic segments among labeled bundles of apical dendrites and a densely immunoreactive plexus of dendritic processes. The densely labeled dendritic plexus of layer V ended abruptly at the boundary of layer IV, forming a clear demarcation between the two layers. Layer VI was similar in overall somatic labeling intensity to layer V with a lower intensity of immunolabeled fine dendritic processes.

Dendritic distribution of GluR 5/6/7 immunoreactivity in identified

projection neurons Three animals were analyzed for triple labeling by retro-

grade transport, LY filling, and GluR 51617 immunocyto- chemistry for a total of 53 reconstructed, immunolabeled neurons. Dendritic segments of all identified entorhinal cortex-superior temporal sulcus (ERC-STS) projection neurons were immunoreactive for GluR 5/6/7. Identified projection neurons in layer V, as demonstrated above, possess an apical dendrite that does not ramify in superficial layers and often does not extend above layer 111. The apical dendrites of identified layer V pyramidal-projection neurons were strongly immunoreactive in all neurons analyzed (Figs. 5. 6). The basilar arbor of these neurons was GluR 5/6/7-ir as well, demonstrating immunolabeling in tertiary and higher order dendrites. Antibody 4F5 is an IgM class of immunoglobulin, and, given the incomplete penetration of the IgM primary antibody into 30-km-thick subsections, it was not possible under this limitation in the present study to determine definitively whether irnmunolabeling was excluded from limited portions of the dendritic arbor of identified entorhinal projection neurons. However, the wide distribution of GluR 5i6/7-immunopositive segments throughout all components of the dendritic tree suggests that there is no terminal zone from which GluR 5 / 6 / 7 subunits are excluded. Neuronal processes in sections processed without primary antibody were not seen when viewed with the Texas red-filter combination.

DISCUSSION The principal findings of the present report are the

following: 1) A significant projection exists from ERC to the dorsal bank of the STS that reciprocates afferents described by Insausti et al. (1987); 2) the dendritic arbor of such neurons is virtually exclusively confined to layer V of ERC; and 3) kainate-receptor immunoreactivity is found throughout the dendritic arbor of all identified neurons analyzed, including solitary apical dendrites extending to superficial lamina.

Regional organization of entorhinal-neocortical projections

In describing projections to the ERG from neocortical areas, Insausti et al. (1987) made the observation that only multimodal-association areas provided direct projections to the ERC, and their observations demonstrated that the perirhinal/parahippocampal belt, which is composed of areas 35, 36, TF, and TH, formed one such multimodal sensory area, whereas the dorsal bank of the superior


B

Fig. 3. Morphological maps of entorhinal cortex-superior temporal sulcus (ERC-STS) projection neurons. Low-magnification map of ERC (A) demonstrates dendritic arbor of five identified ERC-STS projection neurons at their in situ location in layer V. All are of simple pyramidal type with unbranched apical dendrites. Solid line indicates layer IV/V border. B: Illustration of the neuron indicated at arrow in A at higher magnification. A photomicrograph of the neuron in B is presented in

temporal sulcus formed another. A number of studies have focused on connectivity of ERG with the perirhinali parahippocampal belt and have linked such connectivity with declarative memory (Van Hoesen and Pandya, 1975a; Kosel et al., 1982; Van Hoesen, 1982; Insausti et al., 1987; Suzuki and Amaral, 1994). However, Jones and Powell (1970) observed that the depth of the superior temporal sulcus contained a region of convergence of the three major sensory systems, where input to limbic regions commenced, and that this region might be expected to project directly or indirectly to the hippocampus. Reciprocal connectivity between the ERC and STS demonstrated here indicates that the dorsal bank of the STS is in a position to provide multimodal sensory-association information to the ERC and to receive information back that has been processed by

F G

Figure 5B. Note that neurons in situ (Fig. 5A) are presented with pia down, whereas subsequent maps and photomicrographs are presented with pia up. Other morphological types of identified layer V projection neurons: asymmetrical pyramids (C,D), vertical fusiform (E), and multipolar (F). G: Inverted pyramid of layer VI. Abbreviations as in Figure 2. Scale bars = 2 mm in A, 200 bm in G (also applies to B-F).

the hippocampal formation. The injection site employed in the present study was purposely made quite large in order to ensure that the largest population of ERC projection neurons was available for analysis. Therefore, a detailed point-to-point analysis of the density of connections could not be made. Thus. it is possible that a particular subfield of STS may receive the bulk of the projection of ERC or that there is an organized topographic projection from ERC to STS, but such a pattern would not have been determined in this study.

Suzuki et al. (1993) have recently observed that the perirhinal iparahippocampal belt plays a role in mediation of declarative memory similar to that proposed for the function of ERC and hippocampus. The present results suggest that polymodal areas of STS play a role in anatomic

ERC-STS PROJECTION NEURONS 31

TABLE 2. Morphometric Data for ERC-STS Projection Neurons

Tvpe

Basal arbor diameter

(wm)'

Apical dendritic

length (u,mIz

Pyramid Range Mean

Range Mean

Range Mean

Multipolar Range Mean

Asymmetric pyramid

Vertical fusiform

260580 448

420-520 450

480-550 510

380-580 490

300440 492

210-240 223

240-520 420

- -

Basal dendritic branchin2

First-degree Second-degree Third-degree Fourth-degree Fifth-degree

5-16 2~.15 0-16 0-6 0 4 9 8 6 2 1

6-10 6 1 0 2-14 0-5 0 4 8 8 6 3 2

11-16 1&13 2-9 2-5 1-2 14 11 5 3 2

5-13 5-13 &13 0-4 wl 8 R 4 1 0

'Basal arbor diam~ter indicates the maximum extent of the basal dendritic arbor confined to layer V and deep lamina dissecans 2Apical dendritic length indicates length of the dendrite measured frnm the snnia "he hnsal dendritic hranching pattern is given by the number of primary first-degree, etc., dendrites.

connectivity parallel to that of pcrirhinal and parahippocampal cortices, possibly subserving a similar function in relaying polymodal sensory information to and from the hippocampus. The functional necessity of parallel circuits mediating sensory input to the ERC is unknown. Studies of response properties of STS neurons (Bruce et al., 1981; Perrett et al., 1982, 1985a,b, 1987; Baylis et al., 1987) have demonstrated neuronal populations in the superior temporal sulcus that are responsive to faces as well as to other discrete complex stimuli. Perrett et al. (1985a,b) and Rolls (1992) described neurons of the STS having responses invariant for certain facial gestures and movements rather than the responses to the identity of faces found more frequently in inferior temporal visual areas. Such properties may have evolved to generate an immediate response to a presenting social or threatening interaction and, therefore, may require a memory circuit separate from that for identity of an individual. Studies similar to those of Suzuki et al. (1993) involving lesions of the dorsal bank of the STS along with testing of discrete memory modalities would help to illuminate the role of the STS in memory function.

Morphology of ERC projection neurons A prominent feature of neurons projecting from ERC to

STS was the limited extent and negligible branching of their apical dendrites. In addition, the basilar arbor of these neurons was confined to layer V and deep lamina dissecans. Such a configuration effectively limits the majority of synaptic input of ERC-STS projection neurons to afferents terminating in the deep layers of the ERC. Of the two main groups of ERC afferents, neocortical and allocortical, it is known that those from the hippocampus largely terminate in the deep layers of the ERC (Rosene and Van Hoesen, 1977), whereas those from other cortical regions, although less well described, appear to terminate prominently in superficial and, more diffusely, in deep layers of the ERC (Insausti et al., 1987; Suzuki and Amaral, 1994). Accord- ingly, it may be suggested that processing of information from ERC to STS does not bypass the hippocampus and is, in fact, likely to be heavily influenced by output from the hippocampus.

Single, unbranched apical dendrites have been noted in analyses of dendritic morphology of other corticocortical projections. In an anterograde-transport study in the cynomolgus monkey, Rockland and Virga (1989) noted a neuron found in layer VI of area V2, projecting to V1, and possess- ing a delicate, apparently unbranched dendrite, while Lund et al. (19811, in describing Golgi-impregnated neu-

rons in area V2 of the nemestrine monkey, noted a similar group of layer VI neurons with some degree of apical dendritic arborization in more superficial layers.

A simplified apical dendrite and the modified pyramidal nature of a subset of ERC-STS projection neurons present a pattern that contrasts with that of the extensive branching of apical dendritic tufts seen more commonly in neocortical pyramidal neurons (de Lima et al., 1990; Einstein and Fitzpatrick, 1991). Although neocortical neurons furnish- ing corticocortical output are all pyramidal, the efferent neurons described in the present study are pyramidal with simplified apical dendrite or modified pyramidal neurons. However, there are other examples of nonpyramidal projection neurons in the ERC; specifically, the spiny stellate neurons of layer 11, which give rise to the portion of the perforant pathway that projects to the dentate gyrus in rat (Steward and Scoville, 1976) and monkey (Van Hoesen and Pandya, 1975b; Witter and Amaral, 1991).

The only portion of the dendritic arbor of identified ERC projection neurons that was not confined to layer V and the lamina dissecans was the single, unbranched apical dendrite that often extended to layer I l l or 11. A recent study of neurons of the lateral ERC (Carhoni et al., 1990) identified neurons similar to those described in the present study, although it did not demonstrate the simplified apical dendrites shown here. However, in his classical study of the mouse entorhinal cortex, Lorente de NO (1933) demonstrated neurons of ERC layer V (his layer VI) with single unbranched apical dendrites. Therefore, the simplified dendrites of identified ERC projection neurons described here may represent a subset of ERC layer V neurons not detected in a general analysis of layer V neurons. Further analysis of layer V neurons that project to other neocortical sites will reveal whether simplified apical dendrites are a common property of such neurons or are restricted to STS-projecting neurons.

Implications for connectivity of ERC projection neurons

Given that the majority of neocortical input to the ERC is thought to be directed to layers 1-111 (Insausti et al., 1987; Schwerdtfeger et al., 1990; Suzuki and Amaral, 1994) and that hippocampal output is thought to be directed to layers V and VI (Rosene and Van Hoesen, 1977), it is possible that apical dendrites of identified ERC projection neurons receive inputs differing from those to basilar dendrites. The preponderance in the dendritic arbor of identified ERC


projection neurons in layer V makes it likely that neocortical input to such neurons is small relative to the hippocampal input. Determining whether there are fundamental differences between inputs to apical dendrites of these neurons compared to the basal dendritic arbors in deep layers of ERC must await tracing experiments that will allow the precise determination of synaptic sites of hippocampal and neocortical afferents to ERC projection neurons.

Kainate-receptor immunoreactivity The ERC receives information as a final link in sensory

processing that proceeds from primary sensory cortices through higher order unimodal-association and polymodal- association areas. The projection from polymodal areas to ERC is believed to be necessary for the storage of sensory phenomena as memory (Squire and Zola-Morgan, 1991). It has been shown by Huntley et al. (1993) that the densities of immunolabeling for kainate-class receptors vary across primary sensory and association cortical areas. Their study demonstrated quantitatively that Brodmann area 9, a prefrontal association area, contained a high density of GluR 5/61 7-immunolabeled neurons; area 18, the second visual area, contained an intermediate density; and area 17, primary visual cortex, contained the lowest density of the three areas analyzed. The present investigation has shown that kainate-receptor immunolabeling in ERC is extremely dense, which is consistent with the correlation between high-order association cortex and density of kainate- receptor immunoreactivity.

The ERC, hippocampus, and other high-order association cortices are areas of substantial convergence of cortical processing in which complex constructs of sensory information are represented. The particularly high density of kainate-class receptor immunolabeling in the ERC suggests that kainate receptors may play an important role in mediating the extensive convergent corticocortical input to

Fig. 5. Fluorescence photomicrographs of LY-filled fast blue-labeled and 4F5-immunolabeled neurons (top). A: Neurons in layer V retrogradely labeled by fast blue. Nonspecific fluorescence is lipofuscin. B: Micrograph of cell reconstructed in Figure 3B; note that, in this micrograph, pia is presented up. C,D and E,F are pairs of micrographs of double-labeled dendrites: C,E, LY fluorescence; D,F, 4F5-Texas red immunofluorescence. C and D are from the neuron mapped in Figure 6A. and E and F are from the neuron mappcd in Figure 6C. Arrows in C and D indicate the dendritic segment indicated by arrow in Figure 6A. Arrows in E and F indicate the dendritic segment indicated by arrow in Figure 6C. Only a portion of the dendritic arbor of a single 30 pm subsection of the originally complete 200 pm section is shown in the photomicrograph, whereas the complete dendritic arbor of a neuron is mappcd in Figure 6. Scale bars = 100 pm in A, 60 pm in B, 50 pm in E (also applies to C,D,F).

Fig. 6. A-D: Maps of dendritic morphology and double labeling for GluR 5/6/7 immunoreactivity in representative identified projection neurons of cntvrhindl corlex (bottom). The neurons in B and C are simple pyramidal cells with portions of apical dendrites missing, whereas neurons in A and D contain entire apical dendrites. The portion of dendritic arbor with only LY label is represented in green, whereas LY-filled dendritic segments with identifiable GluR 5 /6 /7 immunoreactivity are represented in red. Under the limitations of penetration of mAb-4F5 into 30 pm subsections, it is not possible to conclude definitively that GluR 5/6/7 epitopc is abscnt from unlabeled portions of dendrites (see t,ext). A illustrates the apical dendrite of an identified neuron traversing five subsections, which demonstrates immunolabeling at the sudace of each subsection and not within the depths of cach subsection. Arrows in A and C correspond to arrows in Figure 5C,D and Figure 5E,F, respectively. In B-D, outlines of somata and axon (a) are black. Scale bar = 100 km.

Fig. 4. GluR 5!6/7 immunoreactivity in enturhinal cortex. Photomi- crographs of GluR 5itii7 immunoreactivity in caudal (A) and rostra1 (B) entorhinal cortex. Asterisks mark laminar boundaries. Roman numerals mark the lamina. Note the distinct plexus of irnmunolabelcd dendrites in layer V and the discrete boundary of immunolabeling hetween layers IV and V. Scale bar = 40 pm.


this region. In addition, density of kainate-receptor immunoreactivity in the ERC correlates well with the distribution of kainate receptors demonstrated in kainate-binding studies in rat, monkey, and human (Monaghan et al., 1983; Cross et al., 1987; Jansen et al., 1989). These studies have revealed that the densest binding is located in layers 1/11 and deep layers VIVI. which is the same distribution seen via single-label immunoreactivity for kainate receptors in the monkey ERC.

Finally, kainate-receptor immunoreactivity is present throughout the dendritic arbor of ERC projection neurons, which indicates that neurotransmission from afferents synapsing on all parts of these neurons may be mediated by kainate-class receptors. Apical dendrites in particular dis- play kainate-receptor immunoreactivity throughout their extent and may provide an important kainate receptor- mediated input to ERC projection neurons. The apical dendrite mapped in Figure 6A demonstrates that, although antibody did not penetrate the complete depth of each of the five subsections traversed by the dendrite, immunolabeling was present within the same dendrite in the superficial portion of each section.

An earlier study (Good et al., 1993) demonstrated that axodendritic synapses were common on apical dendritic shafts found in layers IVIV of ERC and were likely to represent the synaptic substrate of apical dendritic immunoreactivity. However, the presence of intradendritic glutamate-receptor immunoreactivity does not necessarily imply that all spines arising from immunolabeled dendrites or all postsynaptic densities on such spines are immunolabeled (Siegel et al., 1994). Further studies aimed at determining the relationship between immunolabeled dendrites and the localization of immunolabeling to axospinous receptors are presently in progress.

Implications for Alzheimer’s disease Reports describing the pattern of neurodegeneration in

Alzheimer’s disease have noted that layer V neurons in ERC are heavily affected by neurofibrillary degeneration (Van Hoesen and Solodkin, 1993), although they are affected somewhat later in the progression of the disease than layer I1 neurons. Other areas of the hippocampal formation and ERC that are noted to undergo early neurofibrillary degeneration in Alzheimer’s disease are field CA1 of the hippocampus and entorhinal layer I1 neurons. The latter two fields share a dense immunoreactivity for GluR 5/6/7 (Good et al., 1993; this report) that may be related to their heightened vulnerability to neurofibrillary degeneration. Alzheimer’s disease has been noted to progress among cortical areas that are related by high-order associational connectivity (Braak and Braak, 1991; Price et al., 1991). The polymodal region of the superior temporal sulcus is one such region that is affected by Alzheimer’s disease (Van Hoesen and Solodkin, 1993) in contrast to relatively unaf- fected surrounding areas of unimodal auditory or visual processing. The present results demonstrate that the STS is closely linked neuroanatomically with the ERC. Further investigations of markers of entorhinal-superior temporal projection neurons in the monkey, which uniquely describe such cells and differentiate them from neurons projecting to other areas, may allow the identification of such a subpopulation of entorhinal projection neurons in the human. With these data, a population of neurons that is vulnerable to early degeneration in Alzheimer’s disease may be identified, which can lead to greater understanding

of cellular characteristics associated with a known projection that might underlie neuronal degeneration.

ACKNOWLEDGMENTS The authors thank Drs. George Huntley and Steven

Siegel for critical comments on the manuscript and Mr. Bob Woolley for photographic assistance. This study was supported by NIH grant AG06647; the Human Brain Project funded jointly by NIMH, KIDA, and NASA, and The Charles A. Dana Foundation.

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morphology and kainate-receptor immunoreactivity of identified neurons within the entorhinal cortex...

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