Topographical organization of projections from the entorhinal cortex to the striatum of the rat

Pergamon

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Neuroscience Vol. 78, No. 3, pp. 715–729, 1997 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/97 $17.00+0.00 S0306-4522(96)00592-1

TOPOGRAPHICAL ORGANIZATION OF PROJECTIONS FROM THE ENTORHINAL CORTEX TO THE STRIATUM OF THE RAT S. TOTTERDELL* and G. E. MEREDITH† *University Department of Pharmacology, Oxford OX1 3QT, U.K. †Department of Anatomy, Royal College of Surgeons in Ireland, Dublin 2, Ireland Abstract––The efferent projections of the entorhinal cortex to the striatum were studied with retrograde (horseradish peroxidase–wheat germ agglutinin) and anterograde (biocytin and biotinylated dextran amine) tracing methods. The bulk of the entorhinal cortical fibres were found to project to the nucleus accumbens in the ventral striatum, but the caudate–putamen is only sparsely and diffusely innervated, rostrally, along its dorsal and medial borders. Fibres arising from neurons in the lateral entorhinal cortex project throughout the rostrocaudal extent of the nucleus accumbens but are most abundant in the core and lateral shell of that nucleus. The rostral neurons of the medial entorhinal cortex were found to project sparsely to the striatum, whereas caudal neurons provide a dense input to the rostral one-third of the nucleus accumbens, especially to the rostral pole, where they concentrate more in the core than in the shell. Contralateral entorhinal projections, which are very sparse, were found in the same parts of the nucleus accumbens and the caudate–putamen as the ipsilateral terminal fields. The present observations that entorhinal inputs to the nucleus accumbens are regionally aligned suggest that disruption of these connections could produce site-specific deficits with, presumably, specific behavioural consequences. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: shell, core, rostral pole, nucleus accumbens, limbic system, anterograde tracing.

Much recent work has demonstrated pathological changes in the entorhinal cortex (EC) and hippocampal formation in subjects who have suffered from schizophrenia.3,51 Post mortem findings include decreases in the volume of both structures and changes in their cyto- and chemo-architecture,11,51 alterations that can be correlated with the level of cognitive impairment and pre-morbid functioning of schizophrenic patients.50 Certainly, such structural alterations could be expected to affect significantly the integrative capacity of both the EC and the hippocampus, and presumably disrupt their connections with other parts of the brain. The hippocampal formation is reciprocally linked with the EC and projects to subcortical targets, including a major projection to the nucleus accumbens in the ventral striatum.15,56 The EC is thought to process convergent information from all major association cortices in order to send it on to the hippocampal formation, but it also projects to subcortical targets, the most important of which may be the nucleus accumbens.13,28,54,55,61,62 In contrast to the hippocampal inputs to the nucleus accumbens, which are dense and topographically organized, Abbreviations: BDA, biotinylated dextran amine; DAB, 3,3*-diaminobenzidine tetrahydrochloride; EC, entorhinal cortex; HRP–WGA, horseradish peroxidase–wheat germ agglutinin; LEC, lateral entorhinal cortex; MEC, medial entorhinal cortex.

terminal fields arising from neurons in the EC seem to be diffuse and encompass much of the caudate– putamen.54,55,61,62 Reports on the origin, pathway and extent of these EC projections6,28,48,54,55,61,62 are conflicting and although recent physiological work has confirmed the projection and established its excitatory nature, providing evidence for glutamate as the neurotransmitter,13 that work provides little insight into the topography or the extent of the terminal fields. It was proposed in the early eighties36,39 that the nucleus accumbens functions as a limbic–motor interface because its excitation resulting from fornix or amygdalar stimulation can be selectively gated by activation of midbrain dopaminergic systems.36,39,65 Since that time, these observations have been confirmed and extended by other studies.33,45–47,52,56 These and other data from behavioural studies37,38,47,52 provide evidence in the nucleus accumbens for limbic–dopamine interactions that mediate motivational and reward-based conduct, behaviour presumably disturbed in psychotic illness. Moreover, recent work in rats has shown that certain exploratory and stereotypic behaviours are mediated by regionally specific circuitry in the nucleus accumbens.27,30 In the mid 1980s, Za`borszky et al.66 identified two parts of the nucleus accumbens, an outer shell and an inner core. These subdivisions, which are best identified with immunostaining for Calbindin-D28k,25,35,67

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are currently recognized as cytoarchitecturally, hodologically, physiologically and pharmacologically distinct,7–10,16,31–34,40,41,43–45,67 and presumably subserve quite separate functions.10 The nucleus accumbens also has a rostral pole, which comprises approximately the rostral quarter of the nucleus,67,68 and although this sector can be divided immunohistochemically into a shell and core,25 other features distinguish it from the rest of the nucleus.21,24,60,68 In light of the role that the EC plays in cognitive and memory functions and the importance of the ventral striatum in reward-based behaviour, it now seems expedient to examine entorhinal–striatal connections in some detail, especially in relationship to the shell, core and rostral pole of the nucleus accumbens. EXPERIMENTAL PROCEDURES

In this study, female Wistar rats (Olac, U.K. and Trinity College Dublin; 160–350 g) were anaesthetized either with Equithesin (0.25 M chloral hydrate: 9.7 mg/ml pentobarbitone sodium: 0.3 ml/100 g, i.p.) or a 4:3 mixture (1 ml/kg, i.m.) of 1% ketamine hydrochloride (Vetelar, U.K.) and 2% xylazine (Rompun, Bayer, Belgium). Each animal was placed in a stereotaxic apparatus and the skull opened. Using coordinates derived from the atlas of Paxinos and Watson,42 microinjections of biocytin (n=12) or biotinylated dextran amine (BDA; n=20) were made in the entorhinal area or of horseradish peroxidase conjugated to wheat germ agglutinin (HRP–WGA; n=7) in the ventral striatum. Biocytin and HRP–WGA were purchased from Sigma (U.K.) and BDA from Molecular Probes (U.S.A.). Small volumes (250–300 nl) of biocytin were injected with pressure (Nanoliter Injection System, W.P.I., U.K.) using pipettes broken to a tip diameter of 30–35 µm. Iontophoretic deposits of BDA were made from pipettes (tip diameter 20–30 µm) with a current of 6 µA, 7 s on/5 s off, for a total of 30 min. Forty to fifty nanolitres of HRP–WGA were introduced into target areas from pipettes (tip diameter 30 µm) with pressure over a period of 15 min. Following surgery, animals were returned to their cages and provided with food and water ad libitum. Survival times varied depending upon the tracer: biocytin, 24–36 h; HRP–WGA, 48 h; BDA, seven days. The optimal survival time for biocytin was found to be 36 h, considerably longer than is usually used in studies with this tracer. Each animal was then reanaesthetized with an overdose of pentobarbitol (1 ml/kg Sagatal, Rhoˆne Me´rieux, U.K.) and perfused through the aorta with physiological saline, followed by a fixative mixture (500 ml) of 3% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3–7.4). Each brain was removed, postfixed in the same solution for 30–45 min at room temperature and the forebrain cut into either coronal or horizontal sections (70–100 µm thick) with a vibrating microtome. Sections cut coronally or horizontally at 100 µm from brains injected with biocytin or BDA were rinsed thoroughly with phosphate-buffered saline and incubated overnight in avidin–biotin–peroxidase complex (Vector Laboratories, U.K.) with gentle agitation at 4)C. Bound peroxidase was then revealed using either 3,3*diaminobenzidine tetrahydrochloride (DAB) or a nickelenhanced DAB protocol. In the former, sections were washed in Tris–HCl buffer (pH 7.6) and reacted in 0.05% DAB and 0.01% H2O2 for 10–30 min, while in the latter sections were washed in Tris–HCl buffer (pH 8.0) and reacted in 0.002% DAB with 0.05% nickel ammonium sulphate and 0.0015% H2O2 for 15–30 min. Following the nickel–DAB procedure, some sections were incubated for a second time in freshly made avidin–biotin–peroxidase

complex, overnight at 4)C, rinsed and reacted in 0.05% DAB and 0.01% H2O2 (pH 7.6) for 5–10 min. Sections from brains in which HRP–WGA was deposited were reacted in 0.05% DAB and 0.01% H2O2, in Tris–HCl buffer (pH 7.6), for 5–10 min. Following these reactions, sections were either mounted serially on gelatin-coated slides, dehydrated and coverslipped, or immersed in 1% osmium tetroxide in 0.1 M phosphate buffer for 30 min, washed in distilled water, dehydrated and embedded in Durcupan resin (Fluka, U.K.). The material was studied with a light microscope (Leica Dialux or Nikon Labophot) and injection sites, fibre projection pathways, terminal fields and retrogradely labelled neuronal populations were mapped with the aid of a drawing tube on to a series of diagrams prepared from the atlas of Paxinos and Watson,42 and a reference series of sections through the rat striatum and EC stained for Nissl substance. A further series through the striatum was immunohistochemically reacted with antibodies directed against the calcium binding protein, Calbindin-D28k (see Meredith et al.35 for details). Shell and core boundaries, based on the calbindin-immunoreacted material, were drawn on to the reference diagrams (Fig. 4b–e, Fig. 5b–e), where anterogradely labelled fibres and varicosities from biocytin or BDA injections into the EC were plotted. Calbindin immunoreactivity in the caudate–putamen produced the typical patch/striosome and matrix regions14 in all immunoreacted sections, but in the interest of clarity, this pattern was not transferred to the diagrams. Representative sections of tracer-injected material were photographed. For electron microscopy, 100-µm-thick sections containing anterogradely labelled fibres were first identified in the light microscope. The coverslip was then removed from the slide, and the area of interest cut out and mounted on a pre-formed resin block with cyanoacrylate glue. Serial ultrathin sections were cut using a Leica Ultracut E, collected on Pioloform-coated, single-slot, copper grids, stained with lead citrate49 and examined in a Philips 201C electron microscope. RESULTS

The term entorhinal area, as used in this study, includes the medial (MEC) and lateral (LEC) entorhinal cortices and the perirhinal cortex.29 MEC and LEC divisions, as determined by others,2,22 were identified in a series of Nissl-stained sections of our material (Fig. 1A). The MEC and rostral part of the EC have a less distinct lamination, due to a thinner layer II and clumping of neurons in layer III. The lamina dissecans is either small or absent. The LEC at the same level has a wider layer II, a more columnar layer III, wider lamina dissecans and better differentiated layers V and VI. More caudally, the EC takes on more isocortical features and the lamina dissecans is absent. Retrograde tracing studies The seven injections of HRP–WGA were centred on the nucleus accumbens, with the bulk of the tracer deposited being rostral and medial in that nucleus (Fig. 1F). All injection sites involved the shell of the nucleus accumbens, but the core and rostral pole were labelled in four of the rats. In one case, tracer spread to the caudolateral parts of the nucleus accumbens. Although there was some spread of

Fig. 1. (A) A Nissl-stained coronal section through the rat entorhinal area, showing the medial (MEC) and lateral (LEC) divisions of the entorhinal cortex. The asterisks in A and B mark the rhinal fissure. (B) Neurons in the LEC, at a similar rostrocaudal level to A, retrogradely labelled following injection of HRP–WGA into the nucleus accumbens. The boxed areas in deep, mid and superficial levels appear in C, D, and E respectively. (C–E) Neurons in the LEC showing the characteristic granular DAB reaction product (arrows). In the deep and mid layers the labelled cells contain many pronounced granules, but more superficially (E, sup) there are fewer labelled neurons and some contain very few granules (double arrows). (F) A representative HRP–WGA injection site (hatched area) in the medial part of the nucleus accumbens. In the EC, black dots mark regions containing retrogradely labelled neurons. Arrowheads in A and F indicate the approximate boundary between the MEC and LEC, and LEC and perirhinal cortex. Scale bars=100 µm (B), 50 µm (C–E).

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HRP–WGA to the overlying medial part of the caudate–putamen, there was little involvement of the septal area and none of the overlying cortex. The results from these seven injection sites were compared with those from a further 15 injections of HRP– WGA into the rostromedial nucleus accumbens that formed part of another study. All injections resulted in retrograde labelling, ipsilateral to the injection site, in the subiculum and to varying degrees in the CA1 area of the hippocampus. In addition, there was considerable retrograde labelling of neurons in the EC and perirhinal cortex from all injections, although generally the intensity of labelling was considerably less in these cells than in the subiculum and CA1 area. All injections retrogradely labelled neurons in the LEC (Fig. 1B–E) and the perirhinal cortex and most labelled some neurons in the MEC, with these lying mainly ventrally at the mid anterior–posterior level. Labelled neurons in the LEC are present mainly in the mid to deep cell layers5 (Fig. 1C, D) and occasionally in the more superficial layers (Fig. 1E); medially, cells are most common, only in the deepest layers, close to the alveus (Fig. 1F). In addition, in horizontal sections, the more caudally and ventrolaterally labelled cells appear to occupy the entire depth below the lamina dissecans. Retrogradely labelled neurons in the contralateral entorhinal area were found only rarely. Anterograde tracing studies Injections of BDA or biocytin into the EC and perirhinal cortex (Fig. 2A, B) resulted in fibre labelling in the nucleus accumbens and, to a lesser extent, the caudate–putamen (Figs 3–6). At the site of injection, the tracer was taken up into cell bodies and dendrites (Fig. 3A, D). Fibres originating from the injection sites could be followed through the series of coronal or horizontal sections by virtue of the dark brown appearance of the DAB reaction product or the dark blue deposit following a nickel–DAB reaction. The combination of a nickel–DAB procedure followed by the DAB protocol was very effective in revealing the fibres, which appeared black. Although injections that did not involve the deep layers of the EC resulted in the labelling of fibres, these mainly innervate the hippocampal formation (not illustrated). From superficial LEC injections, no fibres reach the nucleus accumbens, although occasionally some could be seen in the olfactory tubercle. No injection sites involved the subiculum of the hippocampus or cortices dorsal to the perirhinal cortex. All injections labelled very fine and varicose fibres that are diffusely dispersed in the nucleus accumbens and caudate–putamen (Fig. 6A). Both small and large varicosities are present in the nucleus accumbens (Fig. 6C, D). Projection routes from the lateral entorhinal cortex. Injections in the LEC gave rise to labelled fibres that reach the rostral parts of the brain via a number of

different routes (Figs 3B, C, 4). The bulk of the projection is associated with the ventral external capsule (Figs 3C, 4i), at approximately the same dorsal/ventral level as the original injection, moving forward to the point where it becomes continuous with the posterior part of the anterior commissure; here, the fibres turn sharply medially (Fig. 4g). Thereafter, they travel either in the anterior commissure itself (Figs 3B, 4g) or immediately adjacent to it, medially and rostrally, until the commissure enters the nucleus accumbens, where the fibres then emerge and become varicose (Fig. 4a–f ). A few fibres also reach the nucleus accumbens via the route taken by subicular efferents,15 moving first dorsally and rostrally in the alveus to the fimbria (Fig. 4h, i) and then passing down the fornix through the septum to the medial nucleus accumbens (Fig. 4e, f ). Other fibres were traced in the ventral external capsule to the amygdala, where varicose fibres are present in the lateral and basolateral nuclei (Fig. 4h, i). A few then pass into the stria terminalis (Fig. 4h, i) and, via this route, move with the amygdalar efferent fibres rostrally to the nucleus accumbens. Fibres also pass caudally from the injection sites to enter the external capsule, before travelling dorsally, where they turn rostrally again and could then be seen in the dorsal fornix (Fig. 4h, i). Here they travel forward, until some fibres pass into the corpus callosum (Fig. 4e–g) and from there into the medial and dorsal caudate– putamen (Fig. 4e–g). Some also course more medially and cross into the septum, where they appear to be associated with the fibres from the fornix (Fig. 4g). Projection routes from the medial entorhinal cortex. Injections of tracer in the MEC labelled some fibres that join the alveus and then follow the route that has already been described for subicular inputs to the nucleus accumbens15 (Fig. 5h, i). On route, many fibres leave the dorsal fornix to enter the corpus callosum and others continue on to the septum (Figs 3E, 5e–g), but unlike subicular efferents, few labelled fibres enter the septal pole of the medial nucleus accumbens (Fig. 5d, e). Other fibres were traced to the lateral and basolateral nuclei in the amygdala (Fig. 5h, i). From there, a few then pass into the stria terminalis and, via this route, move rostrally to the nucleus accumbens. Fibres were also seen caudal to the nucleus accumbens at the base of the lateral ventricle (Figs 3F, 5g). It is possible that these reach the striatum via the bed nucleus of the stria terminalis (Fig. 5g). Labelled fibres from the corpus callosum, presumably those entering from the dorsal fornix, enter the dorsal border of the striatum, along much of its rostrocaudal extent (Fig. 5c–h). However, labelled callosal fibres are always sparse except rostrally, where dense bundles are present in the ventral part of the forceps minor (Fig. 5a, b, Fig. 6A). Many of those fibres are continuous with varicose fibres in the rostral pole of the nucleus accumbens (Fig. 6C) and the rostromedial caudate–putamen (Fig. 5b, c).

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Fig. 2. Injection sites of anterograde tracers into the EC mapped on to diagrams prepared from tracings of Nissl stained sections. Injection sites from horizontally sectioned brains are not included. (A) Eleven injection sites (a–k) into the LEC. (B) Seventeen injection sites (a–r) into the MEC. Arrowheads indicate the approximate LEC/MEC boundary, double arrowheads the border between the LEC and the perirhinal cortex.

Topography of the entorhinal projection to the nucleus accumbens. Anterogradely labelled fibres resulting from tracer injections into the LEC were found widely distributed throughout the nucleus accumbens, in the core, shell and rostral pole (Fig.

6C–E). Labelled fibres and varicosities were also seen as far caudolaterally as the fundus striati (Fig. 4f). Injections into the rostral and mid levels of the LEC result in more labelled fibres in the rostral pole than injections into the caudal LEC. The densest terminal

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fields are present in the caudal and lateral parts of the nucleus, where they do not respect the core/shell boundary and are spread between the core and lateral shell (Figs 4c, d, 6D, E). Injections of tracer into the MEC also result in labelled fibres in the core, shell and rostral pole, with labelling concentrated medially. Injections in the rostral MEC (Fig. 2B, sites a and b) result in little or no labelling in the nucleus accumbens. Mid to caudal level injection sites in the MEC lead to intense labelling of fibres and varicosities in the rostral half of the nucleus accumbens (Figs 5a–c, 6A, C). The projections are relatively intense at the rostral pole (Fig. 6C), where fibres and varicosities are denser in the core than in the shell. More caudally in the nucleus, terminal fields and fibres are very sparse (Fig. 5e, f). Topography of the entorhinal projection to the caudate–putamen. Following tracer injections into the LEC, varicose labelled fibres were seen along the dorsal and medial borders of the caudate–putamen (Fig. 4e–g) just adjacent to the lateral ventricle. These fibres do not penetrate far into the caudate–putamen from the corpus callosum (Fig. 6B). Caudally, the fibres extend down the medial edge of the caudate– putamen, but more rostrally they are most concentrated dorsally as well as medially, juxtaposed to the lateral ventricle. Fibres originating in the MEC are more densely distributed in the caudate–putamen than are fibres from the LEC. Also located primarily at the dorsomedial border, the fibres from the MEC are most dense in the rostral sections, where they are continuous with the terminal fields in the rostral nucleus accumbens (Fig. 5a–c). The entorhinal cortical fibres in the caudate–putamen are diffusely distributed and are not restricted to either patches/ striosomes or matrix. Contralateral projections of the entorhinal area Tracer injections into both the LEC and MEC labelled terminal fields in the contralateral hemisphere. In the striatum, these fields coincide with those containing varicose fibres in the ipsilateral hemisphere, but are more sparsely innervated. Major routes between the hemispheres are the dorsal hippocampal commissure (Figs 4h, i, 5h, i), which contains labelled fibres following injections into either the

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LEC or the MEC, and the anterior commissure (Fig. 4g), which contains labelling only after injections into the LEC. Electron microscopy Boutons in the lateral shell (Fig. 7A) or core (Fig. 7B) of the nucleus accumbens, labelled anterogradely from the lateral entorhinal area, were examined in the electron microscope. Occasionally, reaction product was detected in fine fibres that presumably represent the very fine axons seen with the light microscope. A number of anterogradely labelled structures, containing at most one mitochondrion and pleiomorphic and small, round vesicles and occasional large vesicles without dense cores, were followed through serial ultrathin sections. In a sample of 20 vesicle-containing profiles, nine were found in asymmetrical synaptic contact with their postsynaptic targets (Fig. 7A, B), while in a further five, a synapse was identified but could not be classified, either due to an incomplete series of sections or a suboptimal plane of section. Most synaptic boutons are in contact with structures with small profiles and, in six, the spine apparatus was present (Fig. 7A). In four cases the postsynaptic structure had a concave profile and in three the synapse was perforated (Fig. 7A). DISCUSSION

The main goal of this study was to examine, in detail, the fibre trajectories and topography of the terminal fields of entorhinal neurons in relation to the neostriatum and compartmental subdivisions of the nucleus accumbens in the ventral striatum. Anatomical evidence that the EC projects to the striatum has been presented in the past for rat,6,28,48,55,62 cat61 and guinea-pig.54 The existence of these projections is also supported by physiological investigations.13 All studies agree that the bulk of this projection is made to the nucleus accumbens and that the input to the caudate–putamen is relatively sparse. Nevertheless, there have been differing claims regarding the extent and topography of the terminal fields and the trajectory of projecting fibres. For example, Sørensen54 and Swanson and Ko¨hler55 describe entorhinal projections to the entire rostrocaudal extent of the caudate–putamen and nucleus accumbens, whereas

Fig. 3. Photomicrographs depicting MEC and LEC injection sites and fibre projections. Representative sites of BDA injections into the LEC (A), seen in horizontal section (the edge of the section lies lateral to the site) or biocytin into the MEC (D), seen in coronal section (the edge of the section lies medial to the site). Note the appearance of individual cells (arrows) and extensive dendritic labelling. From the LEC injection site, fibres (arrowheads) were found in the lateral limb of the anterior commissure (B, coronal section) and the external capsule (C, horizontal section). In E, labelled fibres (arrowheads) from the MEC injection were seen ipsilaterally in the medial septum (ms) and fornix (fx), and in F running close to the medial edge of the lateral ventricle (lv). These fibres course further laterally to enter the caudomedial striatum, as seen in Fig. 5f. In F, orientation is indicated at the top of the micrograph. Scale bar in A (applies to all photographs)=500 µm (A, D), 200 µm (B, F), 40 µm (C), 900 µm (E).

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Fig. 4. A representative series of coronal diagrams that show the anterograde labelling following an injection (f in Fig. 2A) into the LEC. In a–e, the boundary between the core and shell of the nucleus accumbens is delineated by a solid line. Fibres are represented by short wavy lines and terminal fields by small dots. Abbreviations: ac, anterior commissure; bn, bed nucleus of the stria terminalis; c, core of the nucleus accumbens; cp, caudate–putamen; dhc, dorsal hippocampal commissure; ex, external capsule; f, fimbria–fornix; fm, forceps minor of the corpus callosum; fs, fundus striati; lv, lateral ventricle; ms, medial septum; sh, shell of the nucleus accumbens; st, stria terminalis.

we show, in agreement with others,48,61 that the EC projects predominantly to the rostral nucleus accumbens and rostromedial caudate–putamen. In addition, studies that employed retrograde tracers alone6,48 found little or no topography in the

entorhinal input to the striatum. However, we know from our own retrograde work that injections into the medial nucleus accumbens invariably label neurons in the LEC, MEC and perirhinal cortex, while anterogradely labelled terminal fields roughly

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Fig. 5. A representative series of coronal diagrams that show the anterograde labelling following an injection into the MEC (tracing based on c and m in Fig. 2B). Fibres are plotted as short wavy lines and terminal fields as small dots. In a–e, the boundary between the core and shell of the nucleus accumbens is delineated by a dotted line. Abbreviations are the same as in the legend for Fig. 4.

correspond topographically to the mediolateral axis of the EC (present results and Refs 28 and 61, but see Ref. 55). Further, in contrast to the findings of Krayniak et al.28 for rat and Witter and Groenewegen61 for cat, our tracings show that the fornix is not the principal route for entorhinal fibres. Moreover, fibres carried in the fornix to the nucleus

accumbens originate primarily in the hippocampal formation15 and not in the EC as described by Krayniak et al.28 Finally, the present study extends the physiological results of Finch et al.13 and provides, for the first time, the topographical arrangement of entorhinal terminal fields in the core, shell and rostral pole of the nucleus accumbens.

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Fig. 6. Anterograde labelling in the striatum following tracer injections into the MEC (A–C) or LEC (D, E). A–C are from tissue reacted with nickel–DAB+DAB, D is from material processed with DAB alone, and E is from material reacted with DAB and processed for electron microscopy. (A) Intense fibre labelling (arrowheads) in the forceps minor of the corpus callosum at about the level of section b in Figs 4 and 5. Orientation is indicated with medial to the left: note positions of the prefrontal cortex (pfc) and rostral pole of the nucleus accumbens (rp) in relationship to the marked fibres. (B) Very fine varicosities (arrows) in the corpus callosum (cc) and along the dorsal edge of the caudate–putamen, which is recognized by characteristic fibre bundles. (C) Fibres with large (double arrowheads) and small (single arrowheads) varicosities in the rostral pole of the nucleus accumbens. (D) Fibres with large (double arrowheads) and small (single arrowheads) varicosities in a horizontal section through the shell of the nucleus accumbens. (E) Varicose fibres (arrows) in a horizontal section through the core of the nucleus accumbens. Scale bar in A (applies to all photographs)=25 µm (A, C), 40 µm (B), 30 µm (D, E).

Technical considerations In the present study, we employed the retrograde tracer, HRP–WGA, and the anterograde tracers biocytin and BDA, all of which were selected for their sensitivity and selectivity.17,53,57 Nevertheless, they

have their limitations. For example, HRP–WGA is transported bidirectionally and is taken up by fibres of passage.17 Presumably, the interruption of passing fibres is one explanation for the lack of orderly topography seen in retrograde studies of entorhinal projections, as discussed above. Further, the two

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Fig. 7. Electron micrographs of boutons in the nucleus accumbens anterogradely labelled following injection of BDA into the LEC. (A) A bouton in the lateral shell of the nucleus accumbens in synaptic contact (arrowheads) with a spine (sa, spine apparatus). The synapse in this section is perforated (star) and, in an adjacent section, it was identified as asymmetric. (B) A bouton in the nucleus accumbens core in synaptic contact (arrowheads) with a small profile (which may be a spine). Note the large vesicles without dense cores (stars). Scale bar in A (applies to both photographs)=0.2 µm.

anterograde tracers can potentially be carried retrogradely. If this had occurred, it is possible that subicular neurons that also project to the nucleus accumbens might have been retrogradely labelled from the injection sites in the EC, an event that would have interfered with the analysis of our results. Biocytin, however, seems to be transported retrogradely in very few pathways and only after injections of large volumes.53 The injection system we used (W.P.I., U.K.) fully controlled injection volumes, which were kept small, and retrogradely labelled cell bodies were never seen in the hippocampal formation of our biocytin-injected material. BDA is also preferentially transported anterogradely.57 However, there are studies that show retrograde transport of this marker,63 although retrograde transport of BDA seems to be most efficient when low-molecular-weight BDA is used and the tissue is processed at a low pH.26 For the present study, injection volumes of BDA were kept small and the pH of all buffers was neutral. Following injection of BDA into the LEC, presumably retrogradely labelled cells in the ventral subiculum were found in only two animals, whereas anterogradely labelled fibres in the fimbria–fornix were found in an additional eight cases. Of methodological importance for the present study was the sequential reaction of the same tissue with two chomogens: nickel–DAB and DAB. The major advantage of this dual procedure was to enhance the visualization of the extremely fine fibres and small varicosities of entorhinal efferents (see,

e.g., Fig. 6A–C). Such structures reacted accordingly, appeared darker and more distinctive than those stained singly with nickel–DAB or DAB (unpublished observations), although it is possible that the staining would not be superior to a procedure where either of the chromogens was used twice in sequential reactions. Projections from the entorhinal area to the striatum Neurons giving rise to the projection to the nucleus accumbens lie in a relatively broad band that occupies the whole of the deeper half of the EC (present results and Refs 6 and 61). Our study and those of most others48,54,61,62 show that neurons in the deep layers of the LEC provide most of the input to the nucleus accumbens, but we found, along with others,28,48,61 that some retrogradely labelled neurons also lie in the more superficial layers. These more superficially located neurons appear to be less intensely labelled (observe the label in superficial cells in Fig. 1E), however, which may explain why they are not always described. The failure to confirm a projection to the striatum from the more superficially located neurons in the LEC by anterograde tracing methods is difficult to explain. However, it may be due to the relative lack of sensitivity of the methods employed to reveal an extremely sparse projection,17 but it is also possible that the superficial neurons project preferentially to the olfactory tubercle,62 which was sometimes involved in the injection sites. We found little evidence of neurons retrogradely

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labelled in the contralateral EC following our injections of tracer, made predominantly into the medial shell of nucleus accumbens. This is consistent with the report that the core has stronger bilateral connections than the shell.6 The subcortical projections of the EC appear to reach the striatum via a number of different routes. In contrast to other findings,28,61 our tracings show that the fornix is not the main route for these fibres. We find, as have others,54,55,61,62 that the more lateral neurons of the EC give rise to fibres which mainly pass in or just beside the external capsule, rostrally. Swanson and Ko¨hler55 suggest that these fibres reach the striatum directly from this lateral position, but we find that many fibres at the level of the posterior limb of the anterior commissure turn sharply in a medial direction and pass either within the anterior commissure or enter an area immediately adjacent to it that is recognized to be a part of the central division of the extended amygdala.19 These labelled axons then move rostrally into the nucleus accumbens, where they distribute in the core, lateral shell and fundus striati. Furthermore, most axons of MEC neurons reach the rostral pole, presumably by a dorsal route via the dorsal fornix, from which they enter the corpus callosum and eventually pass out of the forceps minor, a route not reported previously. Fibres innervating the caudate–putamen from the LEC may reach their destination via the corpus callosum, but were not found in the cingulate bundle as described for fibres from the dorsal EC in the guinea-pig.54 A few fibres, probably from the MEC, pass through the fornix to enter the nucleus accumbens at the septal pole28,54,61 and ascend dorsally into the caudate–putamen. The bundle subjacent to the corpus callosum, previously described as the fasciculus subcallosus in the cat,61 could give rise to fibres that enter the caudate–putamen, but our own work suggests that the labelled fibres along the ventral edge of the corpus callosum are not in a separate tract (see Fig. 6B). Most studies are in agreement that the EC distributes fibres diffusely to both dorsal and ventral parts of the striatum, but that there appears to be a mediolateral and a rostrocaudal topography in the terminal fields.54,55,61 Our investigation confirms both the diffuse nature of the projections and, to a certain extent, the mediolateral topography of their inputs. We also found, in contrast to what has been reported previously for rat28 and cat,61 that the projections from the LEC and adjacent perirhinal cortex reach the whole rostrocaudal extent of the nucleus accumbens, including the rostral pole, but largely exclude the septal pole of the shell. These differences may be related to the use of tritiated amino acid autoradiography for anterograde tracing in these earlier studies, a method that is not ideal for defining terminal fields.17 In addition, we are unable to confirm a clear rostrocaudal topography for terminal fields for, in agreement with Phillipson

and Griffiths,48 the EC projects principally to the rostral parts of nucleus accumbens. Indeed, our work shows that the input from the caudal MEC is to the rostral pole. Probably, the most important findings of the present study are the dense entorhinal input to the rostral pole of the nucleus accumbens and the LEC terminal fields in the lateral shell of this nucleus. The rostralmost part of the nucleus accumbens is somewhat ill-defined anatomically,67 but can be separated from the rest of the nucleus by its connections,64,68 the coupling of its cells41 and by its cellular responses to the manipulation of dopamine.21,24,60 The dopaminergic innervation of this region is dense58,59 and when depleted, neurons here show very pronounced increases in mRNA for the peptide enkephalin, decreases in mRNA for dynorphin and substance P, and changes in the mRNA for both D1 and D2 dopaminergic receptors.24,60 Moreover, dopamine D2 receptor stimulation inhibits acetylcholine release in rostral but not caudal parts of the nucleus.21 Therefore, rostral pole neurons could be especially sensitive to changes in dopamine transmission. In the nucleus accumbens, dopaminergic function has been studied with interest ever since early research pointed to its role in mood, motivation and attention (see Phillips et al.47). The EC, which provides an excitatory, glutamatergic input to the striatum, may sort and consolidate information required for reinforcing behaviours through its connections with the nucleus accumbens.13 Although Finch et al. found no discernible differences in the cellular responses of shell and core neurons with entorhinal stimulation, their investigation examined few if any responses at the rostral pole. Much work points to the striatal, and therefore the ‘‘extrapyramidal’’, character of the core and the limbic nature of the shell of the nucleus accumbens.9,10,18,30,67 Neurons in the core project to ventral pallidal targets, whereas those in the shell, and especially its medial part, provide a major output to the lateral hypothalamus and mesencephalic reticular formation.20 Furthermore, there is evidence that extracellular levels of dopamine differ between the core and shell in response to externally applied stimuli.8 Moreover, dopamine-mediated behaviours are dependent upon the stimulation of glutamatergic receptors,4,27,30 the antagonism of which impairs spatial learning. While the physiological relevance of lateral entorhinal inputs to the lateral shell is not known, the glutamatergic nature of this innervation13 may provide, at least in part, the basis for such behavioural processes. If so, this EC input could be important in mediating some of the effects of neuroleptic drug treatment in psychotic illness. The entorhinal cortex in disease In schizophrenia, entorhinal cortical damage seems to be an important pathological feature.1,11 A

Entorhinal projections to the striatum

reduction in size of the parahippocampal gyrus has been related to disturbances in the normal migration or cell differentiation processes.1,23 In addition, significant differences in cell density in the EC of schizophrenic patients compared to controls, with trends for reduced densities in the deep cell layers and the superficial cell clusters, have been reported.11 Such deficits presumably alter the entorhinal input to the nucleus accumbens and therefore change the physiological function of regional circuits. Certainly, latent inhibition, proposed as a model of the attentional deficit of schizophrenia, is a behaviour apparently dependent upon the integrity of connections with the ventral striatum and is abolished by lesions involving the EC.12 Further work is needed to

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establish the functional importance of this regionally specific entorhinal input to the nucleus accumbens.

Acknowledgements—The authors wish to thank Peter Kelleghan, and Drs Hong Lin and Thomas Farrell, for technical assistance and discussion. We are also indebted to Dr Ben Yee for helpful suggestions and advice, and to Professor Barry Roberts and Dr Richard Greene for critical comments on the manuscript. Further, we are grateful to the Media Services Department of the Royal College of Surgeons in Ireland for their assistance with the photomicrographs. The work was funded by a Wellcome Trust project grant, no. 042767/94 (S.T.), a grant from the Research Committee of The Royal College of Surgeons in Ireland (G.E.M.) and a Forbairt/British Council Joint Research Scheme award for 1995, no. 005.R.

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