Afferent connections of the entorhinal area in the rat as demonstrated by retrograde cell-labeling with horseradish peroxidase

Brain Research, 152 (1978)249~64

249

© Elsevier/North-HollandBiomedicalPress

Research Reports

AFFERENT CONNECTIONS OF THE ENTORHINAL AREA IN THE RAT AS DEMONSTRATED BY RETROGRADE CELL-LABELING WITH HORSERADISH PEROXIDASE

ROBERT M. BECKSTEAD* Department of Psychology, Massachusetts Institute of Technology, Cambridge, Mass, 02139 (U.S.A.)

(Accepted December22nd, 1977)

SUMMARY The entorhinal cortex (EC) of the rat has been divided into medial (MEA) and lateral (LEA) subdivisions. In order to analyze its afferent connections, small deposits of horseradish peroxidase (HRP) were placed at various loci within EC. The patterns of retrograde cell-labeling charted in 18 such cases suggested that EC is projected upon by several allocortical and subcortical structures and that there are differences in the afferent connections of the two subdivisions. Thus, although HRP injection of either division of EC led to cell-labeling in the hippocampal formation, most in ammonic field CA1 and the subiculum, several cells of the presubiculum were preferentially labeled by injection of MEA. Injections of LEA, but not those in MEA, resulted in substantial cell-labeling in the anterior piriform cortex of both hemispheres. Regardless of the location of its injection site within EC, the enzyme labeled cells in the diagonal band nucleus of Broca, amygdala and claustrum. The pattern of cell-labeling in the diagonal band nucleus extended into the ventrolaterally contiguous nucleus basalis after injection of LEA and into the dorsally contiguous medial septal nucleus after injection of MEA Whereas HRP deposits in either division of EC resulted in cell-labeling in the cortical and medial nuclei of the amygdala, only those deposits which involved LEA led to cell-labeling in the posterior part of the lateral nucleus. In the thalamus, labeled cells were found in the rostral part of the paratenial, periventricular and reuniens nuclei. Finally, at midbrain levels, numerous labeled cells appeared in the dorsal raphe nucleus, a few in the median raphe and locus coeruleus, and, only after rostral EC injection, in the ventral tegmental area.

* Present address: The RockefellerUniversity, 1230York Ave.,New York, N.Y. 10021,U.S.A.

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251 INTRODUCTION As first demonstrated in normal Golgi material by Cajal 7 and Lorente de N637, the entorhinal cortex supplies the hippocampus with one of its major inputs via the socalled perforant path. Understandably, this close association of the entorhinal area with the hippocampus prompted extensive experimental investigations of the terminal distribution of the perforant path4, 5,23,24,39,44,49,52, A reciprocating innervation from hippocampal subfield CA3 to layer IV of the medial entorhinal cortex has been described in the rat ~2 and from subfield CA 1 to the rostral part of the entorhinal cortex in the monkey 47. Beyond that concerning hippocampal connections, information on axons in the entorhinal area is scant. Most of our present knowledge of entorhinal afferents comes from anterograde tracing studies of fibers originating in other brain structures which have as one of their targets the entorhinal area. The recently developed horseradish peroxidase (HRP) retrograde tracing methodZ~, 3z provides a means of identifying neurons with processes projecting to, or through, the brain tissue into which it is injected. Since the H R P method has been used with encouraging results in previous studies of cortical afferents2,26, 2s, its application to a study of entorhinal afferents seemed promising. MATERIALS AND METHODS Each of 18 adult, female albino rats (Charles River Laboratories) received a single, microelectrophoretic is injection of H R P in various loci within the entorhinal cortex (EC) or, in control cases, in cortical areas adjacent to EC. The surgical technique used to expose the rhinal sulcus and subadjacent cortex was a slight modification of the one described in detail by Powell et al. 42. Injections were made from stereotaxically 29 guided glass micropipettes (internal tip diameter: 15-25/~m) filled with a 13 ~ solution of H R P (Sigma, type VI) in Tris.HC1 buffer at pH 8.6. The pipette was connected to a

Fig. 1. Chartings of frontal sections through the entorhinal area showing the HRP deposits at their largest diameter in various cases. The central portion of each injection is jet black and the area of visible diffusion of the enzyme is represented by shading. Abbreviations: abl, basal amygdaloid nucleus, pars lateralis; abm, basal amygdaloid nucleus, pars medialis; AC, anterior commissure; ac, central amygdaloid nucleus; aco, cortical amygaloid nucleus; ala, lateral amygdaloid nucleus, pars anterior; alp, lateral amygdaloid nucleus, pars posterior; am, medial amygdaloid nucleus; avt, ventral tegmental area (Tsai); CA1, cornu ammonis field CA1; CA3, cornu ammonis field CA3 ; cg, central gray substance; cl, claustrum; db, diagonal band nucleus (Broca); dr, dorsal raph6 nucleus; F, fornix; ip, interpeduncular nucleus; lea, lateral entorhinal area; Is, lateral septal nucleus; mea, medial entorhinal area; ML, medial lemniscus; ms, medial septal nucleus; nb, nucleus basalis (Meynert); obm, mitral cell layer of olfactory bulb; OT, optic tract; pac, periamygdaloid cortex; pas, parasubiculum; pc, piriform cortex; prh, perirhinal cortex; prs, presubiculum; pt, paratenial thalamic nucleus; pv, periventricular thalamic nucleus; r, thalamic reticular nucleus; re, thalamic reuniens nucleus; rn, red nucleus; s, subiculum; SM, stria medullaris; snc, substantia nigra pars compacta; snr, substantia nigra pars reticularis; ST, stria terminalis; tam, anteromedial thalamic nucleus; tav, anteroventral thalamic nucleus; v, ventral thalamic complex.

252 TABLE I

Average numbers o f labeled neurons distributed in various cell groups Jb[lowing H R P injections o f the diff'erent divisions o f the entorhinal area S y m b o l s : 5, 100 or m o r e cells; 4, 50-100 cells; 3, 20 50 cells; 2, 10-20 cells; 1, less t h a n 10 cells; 0, n o labeled cells. F o r abbreviation list o f cell g r o u p s see Fig. 1.

Injection site

Medial E C (n -- 2) Lateral E C (n -- 4) Anterior E C (n=3) Perirhinal Ctx. (n = 2) Subiculum (n -- 1)

Cell Group P C CA1 CA3 S P R S A L P ABL A M A C O C L

DB M S R E P T P V A V T D R

0

2

0

3

3

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1

1

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5

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3

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current source (Midgard Electronics) by a short segment of silver wire in contact with the H R P solution, and a positive D C current of 1.8 #A was passed through the pipette for 5-10 min. Twenty-four hours postoperatively the rats were re-anaesthetized and perfused transcardially with a solution of 1.5 ~ paraformaldehyde, 2 ~ glutaraldehyde and 5 ~o sucrose in 0.1 M phosphate buffer (pH 7.4) cooled to 4 °C. The brains were removed immediately, placed in fresh cold fixative for 6 h, stored overnight in pH 7.4 phosphate buffer containing 15 ~ sucrose, and cut on the freezing microtome into frontal sections of 50 #m thickness, Alternate tissue sections were incubated with either a diaminobenzidine (DAB) substrate according to the LaVail and LaVai133 modification of the Graham and Karnovsky 17 method, or with a benzidine dihydrochloride (BDH) substrate according to the method of Mesulam 3s. All tissue sections were mounted on gelatin-coated slides, lightly counterstained with either thionin (DAB reacted sections) or neutral red (BDH reacted sections), and scanned for the presence of HRP-positive perikarya. Retrograde cell-labeling due to H R P deposits confined to the hippocampus, subiculum, perirhinal cortex and neocortex dorsal to the rhinal sulcus were compared to the cell-labeling resulting from injections of EC as a means of controlling for undetectable diffusion of the enzyme into these adjacent cortical areas. The locations of HRP-labeled cells were charted onto tracings of projected sections. RESULTS

The injection sites were easily identified as a heavy deposition of the H R P reaction product and the number of labeled cells were essentially the same with either the DAB or BDH substrate. In some of the initial cases, the injection was deliberately made large in order to maximize the probability of retrograde transport of the enzyme

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Fig. 2. Chartings of selected frontal sections from case EC-14 showing the distribution of HRPpositive perikarya (black dots) resulting from an H R P deposit in the anteroventral part of the entorhinal cortex. For abbreviations see Fig. 1.

254 to as many cell groups as might provide afferents to EC. In several other cases, however, more discrete H R P deposits were obtained that did not involve adjacent cortical areas nor underlying white matter (Fig. 1). Considered together, the injection sites composed a closely spaced series extending from the anteroventral transition of EC with the periamygdaloid cortex to its posterodorsal transition with the subiculum. Additionally, injections were obtained that appeared to be restricted to either the medial or lateral subdivision of EC as defined on cytoarchitectonic grounds by Blackstad 4. Table I allows one to compare the retrograde cell-labeling in various nuclei after injections of lateral or medial EC, the perirhinal cortex and the subiculum. Localized intracortical injections of H R P were found to label cells in many allocortical areas and in cell groups of the basal forebrain, thalamus and midbrain tegmentum. However, some notable differences were found in cell-labeling patterns depending upon the location of the injection site within EC. For this reason, the results of representative anterior, lateral and medial injections are shown in Figs. 2, 3 and 4, respectively. A l l o c o r t i c a l areas

Labeled cells appeared ipsilaterally in hippocampal and subicular cortices in all cases of EC injection, but only in cases involving the lateral subdivision of EC were large numbers of labeled cells found in the ipsilateral piriform cortex and, in lesser numbers, in the contralateral piriform cortex. HRP-positive cells appeared exclusively in the pyramidal cell layer (layer II) of the piriform cortex (Figs. 2B and 3A) and tended to be more numerous in the anterior part of that cortex regardless of the anteroposterior position of the H R P deposit in the lateral EC. However, whereas injection of any part of the lateral EC resulted in bilateral cell-labeling of the piriform cortex, injections of the extreme anterior tip of EC additionally led to retrograde transport of the enzyme to a few neurons in the mitral cell layer of the ipsilateral olfactory bulb (Fig. 2A). Regardless of the location of its injection site in EC, the H R P also labeled numerous cells in the hippocampal formation. After injection of the lateral EC, HRPpositive cells were found mostly in the stratum pyramidale of the ventral two-thirds of ammonic field CA1, in somewhat lesser numbers in the same stratum of field CA3, and scattered throughout all cell strata of the subiculum (Figs. 2G and 3F). Similarly, field CA1 and the subiculum contained labeled neurons after medial EC injection. However, compared to injections of lateral EC, H R P injections of the medial EC led to no cell-labeling in field CA3 but did result in the labeling of cells in the ventral part of the presubiculum (Fig. 4D-F). It should be stressed that the cortical area found to be most abundantly labeled was the anterior piriform cortex after lateral EC injection and that even then the number of HRP-positive cells was moderate and fell within a range of about 120-200 cells per case. Cell-labeling in the hippocampal formation was always substantially less, with ammonic field CA1 and the subiculum invariably containing the most labeled neurons. In no case of EC injection was any neocortical area found to contain HRP-positive cells. Control injections in the subiculum labeled cells only in ammonic

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Fig. 3. Chartings of selected frontal sections from case EC-16 showing the cell-labeling resulting from an HRP deposit in the lateral subdivision of the entorhinal cortex. For abbreviations see Fig. 1.

256 field CA1 and the presubiculum, and a few labeled cells could be found in the subiculum after injection of the perirhinal cortex.

Basal forebrain cell groups Although HRP deposited in any part of EC was transported retrogradely to label cells in the ipsilateral nucleus of the diagonal band of Broca, by far the most abundant cell-labeling in that nucleus occurred after injections that extensivelyinvolved the lateral subdivision of EC (Figs. 2C and 3B). The number of labeled neurons in the diagonal band nucleus appeared to be proportional to the extent of the HRP deposit in lateral EC, but the greatest density of cell-labeling tended always to be situated in the genu of the nucleus and could extend from there in a more or less decremental scattering into both the vertical and horizontal limbs of the nucleus. The pattern of randomly distributed HRP-positive cells present in the horizontal limb of the diagonal band nucleus after lateral EC injection appeared to continue into the caudolaterally adjacent nucleus basalis (magnocellular preoptic nucleus) without interruption (Figs. 2D and 3C). On the other hand, injections of medial EC resulted in retrograde labeling of large multipolar neurons in the medial septal nucleus which is dorsally contiguous with the vertical limb of the diagonal band nucleus (Fig. 4A). A few septal cells would invariably be labeled after such medial EC injections that appeared to lie in the medialmost fringe of the lateral septal nucleus (Fig. 4A). Control injections of the subiculum resulted in more abundant cell-labeling in both the medial and lateral septal nuclei but less celllabeling in the diagonal band nucleus. No labeled cells were found in the nucleus basalis after subiculum injection. Although less endowed with HRP-positive cells than the diagonal band nucleus, the ipsilateral amygdaloid complex was found to contain a significant number of labeled neurons after HRP deposits in any part of EC. In cases of lateral EC injection, the posterior part of the lateral amygdaloid nucleus contained numerous labeled cells and cell-labeling in the cortical nucleus and lateral part of the basal nucleus was found to be only very sparse although consistent across injections (Figs. 3E, 4D and E). The medial and cortical amygdaloid nuclei and the lateral part of the basal nucleus were found to contain a few HRP-positive cells after medial EC injection (Fig. 4C). Unlike cases of lateral EC injection, very little cell-labeling occurred in the lateral nucleus of the amygdala when the HRP deposit was restricted to the medial EC. Regardless of its site of injection within EC, HRP also labeled a few cells in the periamygdaloid cortex (Fig. 4C). Deposits of HRP in the subiculum labeled moderate numbers of neurons in the lateral part of the basal nucleus and a few cells in the posterior part of the lateral nucleus; deposits in the perirhinal cortex labeled cells in the posterior part of the lateral nucleus and the cortical nucleus. Other cells labeled by HRP deposited in lateral EC were found randomly distributed throughout the anteroposterior extent of the ipsilateral claustrum, whereas HRP injection of the medial EC again elicited only a minimum of such labeling (Figs. 2C and D, 3B-E, 4A and C). Thalamic and mesencephalic cell groups In the thalamus, HRP-positive cells were present in the nucleus reuniens,

257

Fig. 4. Chartings of selected frontal sections from case EC-4 showing cell-labeling resulting from ar H R P deposit in the medial subdivision of the entorhinal cortex. For abbreviations see Fig. 1.

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Fig. 5. Schematic diagram summarizing the major afference sources of the entorhinal area shown in this study. For abbreviations see Fig. 1.

paratenial nucleus and periventricular nucleus. However, whereas anterior EC injections labeled but a few cells in each of the 3 nuclei (Fig. 2F), H R P deposits placed more posteriorly in either the medial or lateral subdivision of EC resulted in a marked increase of HRP-positive cells in the nucleus reuniens with no similar increase in the number of labeled cells in the paratenial and periventricular nuclei (Figs. 3B and 4B). Furthermore, only those H R P injections which involved lateral EC resulted in celllabeling in the periventricular thalamic nucleus. Cell-labeling in these 3 thalamic nuclei was always confined to their rostral segments and, in the nucleus reuniens, were also conspicuously confined to the ventrolateral corner of the nucleus. None of the 3 thalamic nuclei were found to contain labeled cells after injections of the perirhinal or subicular cortex. Other HRP-positive cells were found in cell groups of the midbrain tegmentum. Injections of H R P involving the anterior tip of EC, but not more posteriorly placed injections, resulted in the labeling of a few cells in the ipsilateral ventral tegmental area of Tsai (Fig. 2G). The labeled cells were located at an anteroposterior level corresponding approximately to the level at which the oculomotor nerve exits the brain stem, where they were situated dorsal and lateral to the anterior one-half of the interpeduncular nucleus and interspersed among the outgoing fibers of the third nerve roots. Labeled cells, lacking in obvious lateralization, were present in large numbers in the dorsal raphe nucleus after H R P injections involving the lateral EC but not when the injection was confined to the medial EC (Figs. 2H and 3H). Finally, one or two labeled neurons were found in the median raphe nucleus, nucleus locus coeruleus and nucleus tegmenti dorsalis lateralis as a result of most, but not all, H R P deposits in EC (not illustrated).

259 DISCUSSION The structures exhibiting the greatest quantities of HRP-positive neurons after injections of EC and, therefore, possibly the major afference sources of the entorhinal area are summarized schematically in Fig. 5. These structures are the piriform and hippocampal cortices, the diagonal band nucleus, amygdala, nucleus reuniens thalami and the dorsal raphe nucleus. Although apparently not a quantitatively important afference source, the ventral tegmental area is included among the significant cell groups because it is a previously undescribed source of afferents to EC and may be the location of the parent cells of the dopamine axons found to be present in the outer 3 layers of the (rostro-)ventral EC 35. The present finding of an olfactory input from the piriform cortex is consistent with earlier reports based on degeneration methodsg, 42, and in the present study was found to be provided in part from the contralateral piriform cortex as well, the axons from which, in all likelihood, cross in the anterior commissure. However, whereas earlier reports suggested a direct input to the ventrolateral part of the lateral EC from the olfactory bulb20,4~,58, only the extreme anteroventral margin of EC could be shown to receive such a projection in the present study. If the lateral EC is defined, as suggested by Blackstad 4, as a lateral zone characterized by a condensation of the cells in the outer part of layer II into a thin, tightly-packed layer which is broken into cell islands, by a lower packing density in layer III and by the presence of the lamina dissecans, then there is a cortical area ventrolateral to it which does not have these characteristics but instead appears to be transitional with the piriform cortex 19 and, in the present study, was found to receive fibers from the olfactory bulb. Completely compatible with the present finding is the recent autoradiographic evidence provided by Rosene and Heimer 46 in the monkey. They report that no autoradiographically detectable fibers could be followed posterior to this transitional area after injections of tritiated amino acids confined to the olfactory bulb. It is obvious, however, that the lateral EC is not structurally homogeneous throughout and, indeed, several different parcellations have been proposed19, ~6,37,45. It is possible, therefore, that the authors of previous studies in which a projection was noted from the bulb to this ventrolateral transition cortex chose to classify it as part of the lateral EC. Even so, it would seem likely that the largest part of the lateral EC, at least, receives its olfactory input via tertiary sensory neurons in the piriform cortex. Hjorth-Simonsen 22 has described a projection from hippocampal field CA3 to the entorhinal area which is confined in its distribution to the medial EC. According to Swanson and Cowan 53, however, fibers from CA3 distribute to both the medial and lateral subdivisions of EC. Unfortunately, the present findings do not clarify this point since cell-labeling in CA3 was elicited only by H R P deposits in the lateral EC. It m u s t be noted, however, that the extreme posterodorsal portion of EC, which contains a substantial part of the medial subdivision was not involved by any of the H R P deposits. The present results also suggest that both subdivisions receive fibers from field CA1 and the subiculum. Since Swanson and Cowan 5z have observed a projection from both CA1 and the subiculum to the perirhinal cortex but not to EC, the possibility can not be excluded that the cell-labeling in these hippocampal areas is the

260 result of involvement of axons passing to the perirhinal area. In agreement with Shipley 50, only the medial EC was found to receive fibers from the presubiculum which itself is projected upon by the anterior thalamus 13. In addition to its input from allocortex, the lateral EC has been reported to receive fibers from several multisensory neocortical areas in the primate2V,a4, 55-~7. However, in the present study, no neocortical cells were found to be marked by the H R P reaction product after any injections confined to EC. Furthermore, this inconsistency does not appear to be attributable to species differences since it has been observed in this laboratory that tritiated amino acids, when injected into either medial prefrontal or anterior cingulate cortex, are transported so as to label axons in the lateral EC in an autoradiographically detectable manner. Such a failure of injected H R P to label neocortical cells is reminiscent of a similar failure in the study by Nauta et al. 4° of the enzyme to label parent cells of the well-documented corticostriatal system after massive injections in the nucleus caudatoputamen. The reasons for such failure by certain cells to transport H R P in detectable quantities are largely unknown, but Jones 25 has suggested that the labeling or non-labeling of any particular neuronal soma is determined in part at least by the characteristics of the cell's terminal axon arbor. A recent study by autoradiography 1~ had led to the conclusion that the nucleus of the diagonal band of Broca projects to the entorhinal area. In that study, no reference was made to any differences in the projection to one or the other division of EC. From the present experiments, it would seem that the heaviest projection is to the lateral EC since H R P deposits in that subdivision elicited much more cell-labeling than comparably sized injections of medial EC, and, further, that the lateral EC is also the recipient of axons from cells in the nucleus basalis, whereas medial septal cells project selectively to the medial EC. A similar projection from the medial septal nucleus to the medial EC has been described in the catl0, 51. Krettek and Price al using the autoradiographic fiber-tracing method have shown amygdaloentorhinal projections having well-defined laminar patterns of termination from the lateral complex of nuclei to the lateral entorhinal area in both the cat and rat thus confirming their earlier degeneration studies a0. The results of the present experiments suggest that such a projection arises primarily from neurons of the posterior subdivision of the lateral amygdaloid nucleus and, to a lesser extent, from the lateral part of the basal nucleus. The present findings suggest that the medial and cortical nuclei of the amygdala also contribute to the amygdaloentorhinal projection terminating in the lateral EC and, further, that the medial EC is projected upon by neurons in these two amygdaloid nuclei. In their study, Krettek and Price 31 also describe a projection from the claustrum (their dorsal endopiriform nucleus) to the entorhinal area. The present findings support this observation and suggest that the neurons of the claustrum which project to EC are not distributed in any obvious topographical pattern within that nucleus. The initial indication of a thalamic input to EC was provided by Nauta and Whitlock 41 in the cat after large lesions involving nuclei along the midline of the anterior thalamus. Subsequently, Raisman et al. 44 described fibers in EC derived from

261 the cingulum bundle which were possibly of thalamic origin. Herkenham 21 has recently shown, using autoradiography, that neurons in the nucleus reuniens thalami project over the fasciculus cinguli to EC. The present study supports this finding, showing that the cells of origin of such a projection are situated in a specific position in the ventrolateral corner of the rostral end of the nucleus reuniens. Other previously unknown thalamoentorhinal projections were also suggested by the present experiments from the paratenial nucleus and, to a lesser extent, the periventricular nucleus. Monoamine-containing axons have recently been identified in several corticalareas including EC. Noradrenergic axons in EC have been described in the histofluorescence studies of Fuxe et al. 16 and Ungerstedt 54, and Lindvall et al. 3~ using the highly sensitive glyoxylic acid method reported the presence of dopamine axons in the outer 3 layers of the ventral entorhinal area. The present findings of HRP-labeled cells in the locus coeruleus and ventral tegmental area are consistent with the notion that the catecholamine containing axons present in EC originate in these tegmental cell groups - - norepinephrin axons from the locus coeruleus and dopamine axons from the ventral tegmental area - - within the boundaries of which cell bodies of such a catecholaminergic nature are known to reside la. Axons containing the indoleamine, serotonin, have also been demonstrated in EC 1,16 and a direct projection to EC from the dorsal raphe nucleus has been shown autoradiographically6,S. It is not surprising, therefore, that H R P deposits in EC resulted in substantial labeling of cells in the dorsal raphe nucleus. However, whereas cell-labeling in the dorsal raphe and locus coeruleus appeared to be independent of the location of the H R P deposit in EC, it was notable that only the most anteroventrally placed injections resulted in cell-labeling in the ventral tegmental area, suggesting a more restricted distribution of the latter's fibers within EC. This finding is supported by autoradiographic chartings of a corticipetal projection from the ventral tegmental area to only the rostroventral segment of EC lz. The retrograde-labeling histochemical method using H R P proved to be very useful, with either the DAB or BDH substrate, in revealing neurons in several allocortical and subcortical cell groups from which axons appear to reach the entorhinal area. Although the numbers and locations of HRP-positive perikarya were comparable regardless of the substrate used for reaction, the BDH reacted material provided the following two advantages over the DAB reacted tissue. First, the labeled cells were so much more visible under the microscope with bright-field illumination that the need for dark-field examination was obviated and localization of the cells on cytoarchitectural grounds as seen in the Nissl-stained material was greatly facilitated. Second, the filling of the cell somata and proximal segments of their dendritic processes was more complete, imparting to them a Golgi-like appearance which further aided in their cytoarchitectural classification. For these reasons, the BDH, blue reaction for use with H R P seems preferable to the DAB, brown reaction. Neither method, of course, provides escape from the two major problems which confront the experimenter in using the H R P histochemical technique in its present application: the well-known fiber-of-passage problem and the extreme caution that must be adopted in the interpretation of negative results (for discussion see refs. 25, 40). An additional qualification is made necessary by the fact that while the retrograde cell-labeling

262 m e t h o d with H R P allows precise localization o f m a n y afference-sources o f a given area, it provides little c e r t a i n t y regarding the exact m o d e o f t e r m i n a t i o n o f the afferent p r o j e c t i o n system. F o r this reason, it is salutary to note t h a t the distinctions d r a w n between afferent c o n n e c t i o n s o f the medial a n d lateral subdivisions o f E C in the present r e p o r t , a l t h o u g h s u p p o r t i v e o f a previous n o t i o n o f differential afferent connections for the two areas, can only be stated tentatively a n d a detailed description o f the d i s t r i b u t i o n o f fibers in E C m u s t be p r o v i d e d by a n t e r o g r a d e tracing studies a n d studies o f greater resolving power. Even despite these constraints, it w o u l d seem significant t h a t the cell-labeling in the present experiments i n v a r i a b l y o c c u r r e d in such a diverse range o f cell groups a n d t h a t consistent differences o c c u r r e d in cell-labeling p a t t e r n s d e p e n d i n g u p o n the l o c a t i o n o f injected H R P in the medial or lateral EC. This finding suggests that, for the rat at least, the e n t o r h i n a l area which a p p e a r s to p l a y a n i m p o r t a n t role in h i p p o c a m p a l functionZ,15, 4s is subject to the influences o f a wide variety o f b r a i n activity. ACKNOWLEDGEMENTS I wish to express m y sincere a p p r e c i a t i o n to Drs. W a l l e J. H. N a u t a a n d Valerie B. D o m e s i c k for their e n c o u r a g e m e n t a n d advice. This study was s u p p o r t e d by U S P H S grants NS-06542, MH-25515 a n d N I H - 5 - T 0 1 - G M 0 1 6 4 - 1 3 .

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