RSPERIAIEI\‘T~~L

Afferents

NEWMXX

57,

750-765

(1977)

to the Entorhinal Cortex of the Rat by the Method of Retrograde Transport of Horseradish Peroxidase MENAHEM

Isotope

Dcpartiifmt, Rccrivcd

?‘Ac l~l’&rirm~~t Mag

2. 1977;

Studied

SEGAL 1 Imfitufc

revision

of Scirilcr,

vccrizjcd July

Kckovot,

Israrl

5, 1977

The afferents to the parahippocampal area of the rat were studied with retrograde transport of horseradish peroxidase injected into the medial entorhinal cortex, lateral entorhinal cortex, parasubiculum, presubiculum, or a large injection which stained all these structures as well as the ventral hippocampus. Control rats were injected with horseradish peroxidase into the overlying visual cortex. Labeled neurcns in brains with injections into the medial entorhinal cortex and the adjacent parasubicular region were found in the ipsilateral and contralateral presubicular region, the medial septal nucleus, the thalamic nucleus reuniens, the dorsal part of the lateral nucleus of thalamus, the anterior periventricular nucleus of the thalamus, and the dorsal raphe nucleus. Brains with injections into the lateral entorhinal cortex yielded labeled neurons in the medial septal nucleus, nucleus reuniens, dorsal raphe nucleus, and nucleus locus ceruleus. Injections into the presubiculum resulted, in addition, in labeling of neurons in the lateral nucleus of the thalamus. Control injections aimed at the sensory cortex overlying the parahippocampal area yielded labeled neurons in the medial septal nucleus, the dorsal lateral geniculate nucleus, and the nucleus locus ceruleus.

INTRODUCTION The entorhinal cortex (Brodnlann’s area 28) constitutes the nrajor cortical conq>onent of the liil~l~ocainpal region. Its cytoarcliitecture is siiiipler than that of the neocortex (14). Its efferents forni the l>erforant path which

innervates the l~ipl~ocanqmsand were analyzed anatomically (7, 20) and physiologically ( 1) . I n contrast, the knowledge of its afferents is scarce. Lorente

de Nb

(14)

found

three

nlain

inlnrt

fibers

of unknown

origin

in

Abbreviations : HRP-horseradish peroxidase. 1 This study was supported by Grant l/75 from the Israeli Center for Psychobiology, Charles E. Smith Foundation. I would like to acknowledge the excellent assistance of V. Greenberger. 750 Copyright All rights

0 1977 by Academic Press, of reproduction in any form

Inc. reserved.

ISSN

0014-4886

the entorhinal cortex. Detailetl tle~cril)tions ol IJIII~ s0111cof the entorhinal at&rents have been lmblislied in recent years. Altogether, the a&rents to the entorhinal cortex form three main groups: the cortical, local, and subcortical afferents. The cortical afferents were studied in detail by several investigators. Whitlock and Nauta (27) described, in the monkey. a connection between the inferior temporal cortex and the entorhinal cortex. Powell ct al. (18) suggestedthat the pyriforim tortes projects to the lateral entorhinal cortex, Krettek and Price (13 j found that the an~ygdaloitl nuclei project to the entorhinal cortex, and Kerr and Dennis (11) added that even the lateral olfactory tract may arrive at the ventral entorhinal cortex. Van Hiiesen and Pandya (25. 26), in an extensive study in the monkey, described several cortical afferents to the entorhinal cortex, the caudal orbitofrontal cortex and, in the temporal lobe, areas 51 and 27. Some of the local afferents were clescribed in recent years as well; Karten (10) found that the presubiculurm and parasubicuhun project to the entorhinal cortex of the cat. Shipley (24) extended these observations to the guinea pig and described a pathway from the presubiculunl both ipsi- and contmlateral to dorsal and nledial entorhinal areas. The hippocaml)us proper. and niorc specifically, its posterior CA3 segment appears to project to the entorhinal cortex as well (7 j. Compared to the aforenientioned structures much less is known about subcortical afferents to the entorhinal region. The nleclial sepal nucleus nlq contribute afferents to the entorhinal region which are probably cholinergic. The nionoamine-containing brain stem nuclei, the locus ceruleus? and the raphe nuclei which innervate nlost of the forebrain niay also innervate the entorhinal cortex. In addition, Hiikfelt ct al. (9) suggestedthat clopaminecontaining fibers of brain stem origin may also be present in the limbic cortex. The discovery that horseradish peroxidasc (HRP) is taken up by asonal endings and transported soniatopetally made this enzyme an ideal tool for studying connections in the brain, and indeed it was used successfully in a nuniber of laboratories. The present report sunin7arizesa study of afferents to the entorhinal cortex of the rat with the method of retrograde transport of HRP. MATERIALS

AND

hlETHODS

Adult (200 to 300-g) male Wistar rats of a local breeding colony were used. Rats were anesthetized with halothane (2 to 3%), chloral hydrate (350 mg/kg), or Netmbutal (40 nig/kg). During a S- to lo-min interval. 0.1 to 0.2 pl SOS HRP in saline was injected stereotaxically into their brains through a 30- to 50-q glass pipet which was glued to a lo-p1 Hanlilton syringe. The volume injected was controlled by observing an air

752

MENAHEM

SECAL

bubble in the syringe. The syringe was tested before each rat by injecting a minute quantity of HRP into saline. HPR was purchased from Sigma (Type VI), BGehringer Manheim GmbH, or received as a gift from Miles Yeda, Israel. Most experiments were done with Sigma HRP which yielded the best results. Twenty four hours after the injection the rats were anesthetized with urethane (1.2 g/kg) and perfused through the heart with 50 ml saline followed by 200 to 250 ml of a mixture of 1% glutaraldehyde and 1.5% paraformaldehyde prepared in 0.1 M phosphate buffer, $H 7.4, and applied with a pressure of 125 mm Hg. The brains were removed, kept 2 h in fixative, and then washed overnight in several changes of cold 5% sucrose in 0.1 M phosphate buffer, pH 7.4 (21), to remove excess aldehydes. Frozen sections were cut at 60 to 70 pm in the coronal plane for most brains, and in the horizontal plane for some. The sections were preincubated 15 min in a saturated solution of 3,3’-diaminobenzidine tetrahydrochloride (Sigma) in phosphate-buffered sucrose, followed by incubation 30 min in the same medium which now contained 0.01% HZOZ. The sections were mounted on glass slides and examined in a Zeiss microscope equipped with dark-field condenser (N.A. 0.7/0.85). Selected brains were counterstained with cresyl violet to facilitate anatomic localization of the labeled neurons. No~r~enclat~cre. The hippocampal region is here divided into the “hippocampus” which includes the dentate gyrus area, CA4-CA1 and the subiculum, and the “parahippocampal area ” which includes the lateral entorhinal cortex (area 28b), medial entorhinal cortex (area 28a), parasubiculum, retrosplenial area (29e), and presubiculum (Fig. 1). The structures were identified on cresyl violet sections and named according to Blackstad (3) and Lorente de N6 (14). Comparisons were made between horizontal and coronal sections (Fig. 1) and localization of the various areas in the different planes was done according to horizontal sections of the entorhinal cortex (8). Identification of other structures was facilitated by reports of the work of Sherwood and Timiras (23), Kijnig and Klippel (12), and Nauta and Haymaker ( 17). The term “stain” is used throughout to describe the extent of spread of HRP in the injection site. The term “label” refers to neurons containing HRP which was taken up and transported somatopetally. There is a clear distinction between the two conditions; the stained cells are homogeneously covered with brown precipitate of the HRP reaction product whereas transported HRP appears in small characteristic grains. RESULTS One hundred and eighty-nine rats were injected with HRP. An initial screening, based on injection site and quality of staining, reduced the num-

FIG. 1. Coronal (top) and horizontal (bottom) sections through the parahippocampal region of the rat. The horizontal sections were redrawn from Hjorth-Simonsen and Jeune (8) and represent three levels from dorsal (left) to ventral (right). The letters to the left of the horizontal sections represent the approximate locations of the corresponding coronal sections. Structures were identified on cresyl violet-stained sections, and named according to Lorente de N’6 (14). Abbreviations here and in the following figures : AH-anterior nucleus of the hypothalamus ; AbiT-anterior medial nucleus of the thalamus; AMY-amygdala; AVT-anterior ventral nucleus of the thalamus : CB -cerebellum ; CAl-area C-41 of the hippocampus; CC-corpus callosum; CIA-claustrum ; CN-caudate nucleus; DB-nucleus of the diagonal band; DG-dentate gyrus ; DH-dorsal hypothalamic area ; DLG-dorsal lateral geniculate nucleus ; D?vIH -dorsomedial nucleus of the hypothalamus ; DRAorsal raphe nucleus ; F-fornix ; H-hippocampus ; LC-nucleus locus ceruleus ; LE-lateral entorhinal cortex ; LTlateral nucleus of the thalamus ; ME-medial entorhinal cortex ; MLF-medial longitudinal fasciculus ; MR-median raphe nucleus ; MS-medial septal nucleus ; MTmedial thalamus group ; NRT-nucleus reuniens thalamus ; PAS-parasubicular cortex ; PC-pyriform cortex ; PR-prerhinal cortex ; PRS-presubiculum; PV-paraventricular nucleus of the thalamus ; RS-retrosplenial area ; RT-nucleus reticularis thalami ; S-subiculurn ; Sal-supramammillary nucleus; V-ventricle; VMH-ventromedial nucleus of the hypothalamus ; VT-ventral tier nuclei of the thalamus; ZIzona incerta.

her to about 60 brains. Of these, 18 brains ysis. The selection of brains for analysis injection and the general appearance of regions. All selected brains except those frontal plane. The sections were projected of a camera lucida. The injection site and the diagrams. The analysis consisted of:

were selected for a detailed anaIwas based on the size and site of labeled neurons in several brain mentioned were sectioned in the and traced on paper by means labeled neurons were marked on (i) two brains with a large injec-

tic111

site (Fig.

3. lmin

117)

wlwe

tlie l-1 RF- tlif‘fmetl

211~1stnincd

the medial

latcml el~torl~itlal a~;ts. 1~:~r;~5~~lJi~ulu1il~ pTstl~Jicdlllll. sul~ic~il~ui~, ait1 tile posterior anti ventral l~i~~pocanipis.(ii) In two hills tlic iiijccticm was placed in the posterior tip of the hemispliere, staining mainly the nietlial entorkinal cortex but also the posterior lateral entorhinal tortes (Fig. ZC, brain 232). (iii) In two rats the staining was confined to the lateral entorhinal cortex (Fig. 2D. brain 188) with someof the stain spreading into the overlying neocortex. (iv) The medial entorhinal group (six rats) was difficult to analyze becausethe stain usually spread into the parasubiculun~ and presubiculun~ rostra1 to the medial entorhinal cortex. The amount of :uxl

FIG. 2. Representative sections of the maximum spread of HRP in several brains. The sections were unstained. A-Horizontal section with an injection localized in the lateral entorhinal cortex. X 7. B-Injection site into the presubiculum, case 234. X 7. C-Injection site into the medial entorhinal cortex (case 232) with spread into the posterior lateral entorhinal cortex. X 7. D-Injection site into the lateral entorhinal cortex (case 188). X 12. Note the presence of extensive blood vessel labeling, resulting from cndogenous red blood cell peroxidasc staining.

stain in the ~);~r;~s~~l)icuI~u~~was relatively siiiall iii three of these hraiiis which were characterizrtl as a scpratr sul)group. (,v) 111 two additional rats tht~ l)ipet tips were fount1 to lx short of tlie nietlial elitorlriilal cortex aiitl the stain spread only iii the parasul~iculunl and adjacent presubiculuni (Fig. 2B, brain 234). (vij. In two other brains the injection was placed in the lateral entorhinal cortex (Fig. 2A, one rat) or in both medial and lateral entorliinal cortex (one rat). These brains were sectioned in the horizontal plane. (vii) One of eight control rats in which the injection was aimed at the lateral posterior neocortes was analyzed. In this brain the injection stained the neocortes and the corpus callosuni and there was also some HRP spread into the retroq)lenial region and the subiculm~, sparing the entorhinal cortex. The strategy of the experiments was to first search for labeled neurons in brains with a large injection spreading throughout the posterior ventral cerebrum and then attempt to localize labeled neurons in the structures previously verified with cresyl violet stain in brains where small injections stained only restricted regions of the ~~arahip~~ocaii~l~alregion. The rl~rrygdula. An example of a large injection spread was brain 117 (Fig. 3). In this brain which was counterstained with cresyl violet, labeled neurons were found in the ipsilateral amygdala, mainly in the posterior part of the lateral amygdaloid nucleus, but some cells were also seen in the anterior component of the nucleus. Many labeled neurons were also found in the adjacent claustrum lateral to the esternal capsule throughout its anterior-posterior extent. In none of the brains with the restricted injection sites were there clearly labeled neurons in either the amygdala or the claustrum. The hJcdial Srptar rlrca. This area constitutes a major cholinergic affere!lt pathway of the hippocamps and the neocortex (5, 15? 22). All the brains contained some labeled neurons in this region (Figs. 3, S). In the brains with a large injection site there was a continuum of labeled neurons extending from the dorsal coniponent of the medial septum ventrally toward the horizontal and vertical limbs of the nucleus of the diagonal band, including neurons in the base of the medial preoptic regions. This cell population is known to project to wide regions of the neocortex (5). Neurons in the medial sepal region were large (20- to 25pii] multipolar cells and alinost all of them were ipsilateral to the injection site (Fig. SB). No labeling was found in neurons in the lateral sepal nuclear groups or in other sepal nuclei. The number of labeled neurons in the septum of each brain appeared to correlate with the size of the injection site ; the larger the injection area, the larger the number of labeled neurons. There were no apparent differencesin intensity of labeling per neuron with different injection sizes. There was no clear anatomical difference between neurons projecting to the lateral

FIG. 3. The injection site and labeled neurons in a brain with a large injection (case 117). Heavy injection staining is marked with dark black, light spread of injected material with light gray. Labeled neurons are marked with stars. Sections are arranged from rostra1 to caudal levels from top left to bottom right.

and medial entorhinal cortex, pre- and parasubicultun7, or the overlying visual cortex. Local am! Co~r~n~iss~~ml .-1flcuents. Detection of local afferents was dependent on the size of the injection. Because the labeletl neurons were localized in close proximity (1 to 3 mm) to the injection site, the smaller the spread of HRP, the clearer the distinction between stained and labeled cells. To avoid erroneous identification of neurons which were stained through somatic or dendritic transport as retrogradely labeled neurons, only neurons completely outside the area stained with diffuse HRP reaction product were considered for the analysis. In agreement with a previous report (lo), labeled neurons were found in the dorsal presubicular and parasubicular region both ipsilateral and contralateral to the injection site. (Figs. 4, 5, brain 166). These labeled neurons were found in brains injected in the medial entorhinal cortex or the parasubicular region. Labeled presubicular cells were not found in rats injected in the lateral entorhinal cortex. Labeled neurons were not found in the presubiculum after a control injection into the overlying visual cortex. The size of the area containing labeled neurons was not the same in the ipsi- aid contralateral hcniispheres.

FIG. 4. X-Labeled neurtm in dorsal rrtid~e nucIws (brain 117). Note the granular appearance of labeling in the neurons ( rbc = red blocrd cell ). B, C--T.aheling of neurons in the prcsubiculun~. ipsilateral ( R ) mtl contralateral CC) to the injection site. See brain 166 (Fig. 5) for complete diagram. Sotc the differences it1 the extent of labeling between the ipsilntcral :mtl clmtralateral ~~r~*~ubiculum. x 75.

758

MENAIIEM

SEGAL

FIG. 5. A detailed description of ipsilateral (top row) and contralateral labeling of local neurons after injection in the medial entorhinal cortex with some spread into the parasubiculum.

(bottom) (brain 166)

were rather concentrated in one layer (layer 2), all with the same orientation (Fig. 4). There were fewer labeled cells in the contralateral hemisphere, No labeled neurons were found in the ipsilateral hippocampus proper, a proposed source of afferents to the entorhinal cortex (7). Two other types of local labeled neurons were detected. In the uiedial eutorhinal-parasubicular injections, labeled neurons were found in the far lateral entorhinal cortex (rat 141) on the border of the prerhinal region. Lateral entorhinal cortex injections (Fig. 2A) yielded in some cases labeled neurons in the parasubicular region, adjacent to the medial eutorhinal cortex. Diencephalic Afferents. Tlfc nzrcleusvcmiens: The nucleus reuuiens separates the medial hypothalamic paraventricular and dorsomeclialnuclei from the ventromedial nucleus of the thalamus, at the levels of the anterior and medial hypothalamic regions ( 17). The ipsilateral nucleus reuniens and the dorsal hypotlialainic area contained lahcletl neurous in brains injected iii the medial entorhinal cortex, lateral entorhiual cortex, and the parasubiculuul. There were no labeled neurosisin tllis region in Ijraills injcctetl in the overlying ncocortcs or the hilq~ocampus (21). The largest number of labeled

760

MENAIIEM

SEGAL

FIG. 7. Serial sections through the nucleus reuniens of brain 166 (see Fig. 6) to show the extent of labeling of neurons in the nucleus reuniens and the dorsal hypothalamic region. neurons was found in brain 117 (Figs. 3, 6, 7) but smaller injections also yielded substantial labeling of neurons in this region. The extent of labeling in the nucleus reuniens was analyzed in serial sections through the region (rat 166, Fig. 7). Labeled neurons were found as far anterior as the middle level of the anterior hypothalamus, where they were in the heaviest concentration. Fewer cells were found in progressive posterior sections and there were almost no cells in the middle level of the dorsomedial hypothalanuls. At its anterior division, this labeled cell group was just lateral and dorsal to the tip of the third ventricle (Fig. 6) aud appeared to extend into the dorsal hypothalamic region as identified on the cresyl violet-stained sections. The more posteriorly located cells were found farther away from the ventricle and appeared to be associated with the ventral aspect of the n. reuniens. No labeled neurons were associatedwith the posterior half of the ii. reuniens. Midline Thalamus. Cells in this region were labeled only in brains with large injection sites (i.e., brain 117). The labeled neurons were in the periventricular nucleus of the thalamus just underneath the ventricular lumen (Fig. 8). These labeled cells were restricted to the anterior thalamic area, rostra1 to the anterior thalamic nuclei and ipsilateral to the injection site. No cells were found at the level of and caudal to the anterior thalamic group. Lateral Tlzahrus. Cells in this region were labeled in brains with small injections localized in the medial entorhinal cortex and the I’arasubiculuiii. Injections placed in more dorsal locations. i.e.. the parasubiculum, the presuhiculuni, ant1 the overlying retrosplenial area. _ yielded labeled neurons situated as far anterior as the anterior ventral nucleus (Fig. 9, brain 234).

ErlwKIITxAL

(‘OKTICAL

EFFISKEXTS

761

RIore ventral injections, staining the medial entorhinal cortex. caused labeling of more caudally placed neurons. The labeled neurons were found in the dorsal part of the lateral thalatnus near the ventricle (Fig. 8, brain 152) and, similar to other subcortical affcrents, were ipsilateral to the injection site. Dorsal Latrral Gmiculatc. Labeling was found in the dorsal lateral gelliculate bodv after injection into either the lateral entorhinal cortex (brain 1%) or the medial entorhinal corks (braiu 131). The neurons were

FIG. 8. .%-Labeled neurons in the periventricular area of the thalamus (brain 117). Sate the presence of labeling mainly ipsilateral (left) to the injection site. Blurred tnarks are labeled neurons out of focus. X 75 B-A labeled neuron in the medial septal I-