Projections from the hippocampal region to the mammillary bodies in macaque monkeys

Transcription

1 European Journal of Neuroscience, Vol. 22, pp , 2005 ª Federation of European Neuroscience Societies Projections from the hippocampal region to the mammillary bodies in macaque monkeys John P. Aggleton, 1 Seralynne D. Vann 1 and Richard C. Saunders 2 1 School of Psychology, Cardiff University, Tower Building, Park Place, Cardiff, Wales CF10 3AT UK 2 Laboratory of Neuropsychology, National Institute of Mental Health, Bethesda, MD USA Keywords: entorhinal cortex, fornix, hippocampus, hypothalamus, memory, subiculum, temporal lobe Abstract A combination of anterograde and retrograde tracers mapped the direct hippocampal and parahippocampal inputs to the mammillary bodies in two species of macaque monkey. Dense projections arose from pyramidal cells in layer III of the subiculum and prosubiculum, and terminated in the medial mammillary nucleus. While there was no evidence of an input from the dentate gyrus or fields CA1 3, a small contribution arose from the presubiculum and entorhinal cortices. All of the hippocampal and parahippocampal projections to the mammillary bodies appeared to use the fornix as a route. The caudal portions of the subiculum and prosubiculum contained the greatest numbers of cells projecting to the mammillary bodies. A light contralateral projection to the medial mammillary nucleus was also observed, although this appeared to arise primarily from the more rostral portions of the subiculum and prosubiculum. There was a crude topography within the medial mammillary nucleus, with the caudal subicular projections terminating in the mid and dorsal portions of the nucleus while the rostral subicular and entorhinal projections terminated in the ventral and lateral portions of the medial nucleus. Light ipsilateral projections throughout the lateral mammillary nucleus were sometimes observed. Comparisons with related studies of the macaque brain showed that the dense hippocampal projections to the mammillary bodies arise from a population of subicular cells separate from those that project to the anterior thalamic nuclei, even though the major output from the mammillary bodies is to the anterior thalamic nuclei. Other comparisons revealed underlying similarities with the corresponding projections in the rat brain. Introduction Tracts containing direct projections from the hippocampal formation to the mammillary bodies can readily be followed in the human brain. More detailed descriptions have come from other primate species, including the rhesus macaque (Simpson, 1952; Rosene & Van Hoesen, 1977), squirrel monkey (Poletti & Cresswell, 1977; Krayniak et al., 1979) and the African green monkey (Simpson, 1952). These projections have attracted considerable speculation concerning their function as they form one of the major subcortical outputs from the hippocampal formation. Papez (1937), for example, placed these projections at the centre of his putative brain circuit for emotion. Replacing that view, Delay & Brion (1969) regarded the same circuit as crucial for normal memory, drawing support from the neuroanatomy of amnesia. In spite of their probable importance for memory (Gaffan, 1992; Aggleton & Brown, 1999), the hippocampal projections to the mammillary bodies are poorly understood in the primate brain. The majority of evidence comes from lesion degeneration studies (Simpson, 1952; Valenstein & Nauta, 1959; Poletti & Cresswell, 1977), but this information is incomplete as not all efferents show degeneration and it is impossible to confirm the precise origin of these projections. It is, nevertheless, assumed that hippocampal fibres pass through the fornix and terminate bilaterally in the medial mammillary nucleus and nucleus intercalatus (Valenstein & Nauta, 1959; Poletti & Correspondence: Professor John Aggleton, as above. aggleton@cf.ac.uk Received 11 July 2005, revised 6 September 2005, accepted 19 September 2005 Cresswell, 1977), with inconclusive evidence of a projection to the lateral mammillary nucleus (Simpson, 1952). All other information about these mammillary body inputs comes from two studies that used axonal transport methods (Rosene & Van Hoesen, 1977; Krayniak et al., 1979). Both studies found that the subiculum is the principal source of the hippocampal efferents. Rosene & Van Hoesen (1977) reported that injections of radioactive amino acids into the subiculum, but not into fields CA1 3 or the dentate gyrus, resulted in anterograde label in the mammillary bodies. No other information was provided. Krayniak et al. (1979) injected large volumes of the tracer horseradish peroxidase (HRP) into the mammillary body region of the squirrel monkey and observed retrograde label throughout the rostrocaudal extent of the subiculum and parts of the presubiculum. Many issues concerning the hippocampal mammillary body projections therefore remain unresolved in the macaque brain. These issues include whether, as is found in the rat, these projections arise from a particular cell layer within the subiculum and if their terminations are topographically arranged (Meibach & Siegel, 1975; Allen & Hopkins, 1989; Kishi et al., 2000; Ishizuka, 2001). Other unresolved issues include whether the hippocampal formation projects to the lateral mammillary nucleus, and whether adjacent parahippocampal regions (the entorhinal, perirhinal and parahippocampal cortices) also project to the mammillary bodies. A further issue is whether all of the inputs from the hippocampal region use the fornix. Electrophysiological evidence from the squirrel monkey suggests that there is a nonfornical efferent pathway from the hippocampus to the hypothalamus (Poletti & Sujatanond, 1980), although this has not doi: /j x

2 2520 J. P. Aggleton et al. been tested using anatomical techniques. For these reasons the present study examined mammillary body inputs from the hippocampal region using axonal transport techniques. A subset of animals had the fornix transected prior to injection of a tracer (anterograde and retrograde) in order to determine whether there were any direct, nonfornical routes. While fornix section results in a decreased volume of the medial mammillary nucleus this shrinkage is primarily due to the loss of neuropil as there is little or no reduction in cell numbers (Loftus et al., 2000). For this reason, the subsequent injection of retrograde tracers into the mammillary bodies is still informative. Materials and methods Two different techniques were used to trace the connections from the hippocampal region to the mammillary bodies. The source of these projections was examined by injecting the retrograde tracer HRP into the mammillary bodies. The anterograde transport of amino acids was used to identify those nuclei within the mammillary bodies that receive projections from the hippocampal region. Animals were pooled from related studies in order to obtain the maximum amount of information concerning temporal lobe efferents or diencephalic afferents. It is for this reason that data are presented from more than one species of macaque monkey and there are a number of minor variations in the experimental procedures. The monkeys with fornix lesions had previously been used for behavioural studies into the effects of this surgery (e.g. Bachevalier et al., 1985). The cases with amino acid injections and previous fornix lesions had also been used to determine the routes taken by hippocampal efferents to a range of sites, including the thalamus, nucleus accumbens and amygdala (Aggleton et al., 1986; Aggleton, 1986; Friedman et al., 2002; Saunders et al., 2005). All experimental procedures were carried out under strict adherence to the NIH Guide for Care and Use of Laboratory Animals or current Home Office (UK) guidelines. Retrograde transport Intact animals The subjects were three male cynomolgus monkeys (Macaca fascicularis) ranging in weight from 4.0 to 5.6 kg at the time of surgery (cases MB1 3). In all three cases a 0.15-lL injection of 35% HRP (Boehringer, Mannheim) in 2% dimethyl sulphoxide solution was aimed at the region of the left mammillary body. Following the induction of anaesthesia with intravenous pentothal, the monkey was placed in a stereotaxic apparatus. A bone flap was made to the left of the dorsal midline and the tip of a 1-lL Hamilton syringe (Bonaduz, Switzerland) was lowered into the mammillary region. The location of the mammillary bodies was determined using X-ray guidance procedures (Aggleton, 1985). Monkeys survived 48 h and were deeply anaesthetized with pentobarbitone sodium and then perfused intracardially with physiological saline followed by 1 L of a solution of 2.5% paraformaldehyde and 1.5% glutaraldehyde in phosphate buffer (ph 7.2). The brains were blocked in the coronal plane and then cryoprotected in cold 30% sucrose in (0.1 m) phosphate buffer solution for 3 days. Frozen sections were cut at 50 lm and a one-infive series collected in phosphate buffer. Two series of coronal sections were processed for each monkey. One series was processed using tetramethyl benzidine following the procedure of Mesulam (1978). The other series was processed according to a modified Hanker Yates technique (Hanker et al., 1977; Perry & Linden, 1982). This second series was used for the cell counts in the hippocampal region. The Hanker Yates procedure was as follows: the brain sections were incubated in 700 ml of a 0.1 m sodium cacodylate buffer solution (ph 5.1, containing 2.4 g cobalt chloride, 1.6 g ammonium nickel sulphate, 700 mg catechol and 350 mg p-phenylenediamine) for 15 min and then washed in phosphate buffer for 3 5 min. The sections were then transferred into a fresh solution of 700 mg catechol, 350 mg p-phenylenediamine and one drop of hydrogen peroxide (H 2 O 2 ), and incubated for 15 min. Sections were mounted on glass slides, counter-stained with Cresyl Violet and coverslipped. Cells labelled with HRP were charted and counted on coronal sections at 1-mm intervals. Animals with fornix transection Two male cynomolgus monkeys (MBfx 4 and 5), weighing 4.0 and 5.6 kg at the time of surgery, received a complete transection of the fornix up to 1 year before the HRP injections. Both operated animals were first sedated with an intramuscular injection of Ketalar (10 mg kg) and anaesthetized intravenously with pentothal. A midline craniotomy and a unilateral dura flap exposed the midline. One hemisphere was gently retracted to access the corpus callosum. A small slit was made in the corpus callosum through which the anterior fornix was transected with a small metal sucker and cautery. The fornix section was made just caudal to the descending column. The details concerning the HRP injections are the same as those for the intact animals. The craniotomy for the HRP injections was made in the opposite (right) hemisphere to that previously used for the approach to the fornix. Anterograde transport Intact monkeys A total of five rhesus monkeys (Macaca mulatta: cases PRh1, ERh1, ERh2, ERh3 and PSub) weighing from 1.7 to 5.5 kg, and five cynomolgus monkeys (Macaca fascicularis: ACy9, ACy12, ACy14, ACy25 and ACy28) weighing from 2.7 to 7.0 kg, received tritiated amino acid injections in different parts of the hippocampus or parahippocampal region. The animals were tranquilized with ketamine hydrochloride (10 15 mg kg, intramuscular injection) and deeply anaesthetized with intravenous sodium pentobarbital before being placed in a stereotaxic apparatus or other head-holding device. Under aseptic conditions, dural and bone flaps were opened to permit access to the cortex of the temporal lobe. The tip of a 1- or 5-lL Hamilton syringe was then positioned in the temporal region and an equal-parts cocktail of either tritiated proline and leucine (final concentration of 50 lci ml; New England Nuclear) or tritiated proline, leucine, lysine and an amino acid mixture derived from algal protein hydrosylate (final concentration of 100 lci ml; New England Nuclear) was then injected into the target region. In six cases there was a single injection of between 6 and 30 lci. In one of these cases (PSub), both hemispheres received a single injection of amino acids. In three cases (ACy9, ACy25 and ACy28) there were multiple injections in a single hemisphere totalling between 12 and 22 lci. Appreciably larger injections were made in one additional case (ERh3), which received multiple injections in different sites within the entorhinal region totalling 120 lci. The dura and skin were then sutured, and prophylactic doses of antibiotics were administered to prevent infection. Analgesics were administered during the postoperative period. After a postoperative survival period of 5 10 days each monkey was deeply anaesthetized with sodium pentobarbital and perfused intracardially with 0.9% saline followed by 10% formol saline. The brain was then removed and in six cases (ACy9, ACy12, ACy14, ACy25, ACy28 and PRh1) cryoprotected with 30% sucrose solution before being cut in

3 Hippocampal projections to mammillary bodies lm coronal sections on a freezing microtome. The sections were then mounted from phosphate buffer. In four other cases (ERh1, ERh2, ERh3 and PSub) the brains were embedded in paraffin and cut in 10-lm coronal sections. The sections of all cases were then coated with emulsion and exposed at 4 C for between 6 and 30 weeks. The sections were then developed and counterstained with thionine. Monkeys with fornix transection In five cases the fornix was surgically transected 2 12 months prior to the injection of the amino acids. These cases consist of two rhesus monkeys (ARhF23 and ARhF24) and three cynomolgus macaques (ACyF15, ACyF19 and ACyF27). As described above, a small slit was made in the corpus callosum exposing the fornix just caudal to the descending columns. The fornix was then severed bilaterally by coagulation and aspiration. The completeness of this surgery, details of which have been described (Bachevalier et al., 1985), was confirmed histologically. Three of the cases (ACyF15, ACyF19 and ARhF23) received a single amino acid injection of between 5.5 and 10 lci. The two other animals (ARhF24 and ACyF27) received multiple injections totalling between 12 and 22 lci. In case ARhF24 the injections were in one hemisphere but in ACyF27 injections were placed in both hemispheres. In all other respects the methods were the same as those for the intact animals. mammillary nucleus. The lateral nucleus is distinguished by its relatively large cells, which stain intensely in Nissl-stained sections. Finally, some authors have described a third mammillary nucleus, the nucleus intercalatus. This nucleus lies lateral to pars lateralis of the medial nucleus and is skirted by fornix fibres (Veazey et al., 1982a). There is, however, disagreement over the existence of a distinct nucleus intercalatus (Crosby & Showers, 1969; Nauta & Haymaker, 1969) and, while the present study uses the nuclear subdivisions proposed by Veazey et al. (1982a), at many levels a separate nucleus intercalatus is not readily distinguished. Anatomical designations Rose (1939) provided a detailed anatomical description of the mammillary bodies in a range of mammalian species, including the rhesus monkey and human. The mammillary bodies can be divided into two principal nuclear groups, the medial and lateral nuclei (Fig. 1). The medial nucleus, which comprises > 90% of the volume of the mammillary bodies, can be readily distinguished as it is surrounded by a dense fibre capsule (Fig. 1). At many levels the medial nucleus can be further subdivided. Rose (1939) described three nuclei (medial, basal and intermediate) within the medial mamillary complex of Macaca mulatta. A later analysis of the macaque posterior hypothalamus (Veazey et al., 1982a) also subdivided the medial mammillary nucleus into three similar components: pars medialis, pars basalis and pars lateralis (Fig. 1). The present study uses these later designations (Veazey et al., 1982a). While the cells in pars medialis are the largest in the medial mammillary nucleus, the cells in pars basalis are among the smallest in the posterior hypothalamus (Fig. 1). Pars lateralis occupies the lateral half of the medial nucleus but there is no distinct boundary with pars medialis. Pars lateralis is distinguished from pars medialis by the lower density of cells, mainly as a consequence of the large number of fibres in this lateral part of the medial nucleus. Lateral and ventral to the medial nucleus is the lateral Fig. 1. Photomicrographs of coronal sections from the macaque brain showing the appearance of the mammillary bodies and hippocampal formation. (Upper) Nissl-stained section showing nuclei within the mammillary bodies of the rhesus monkey. (Middle) Silver-stained (Gallyas) section showing the arrangement of tracts at the mid-ap level of the mammillary bodies. (Lower) Nissl-stained section showing the cytoarchitectonic fields and subfields within the hippocampus and parahippocampal region. The subiculum is divided into subregions a and b and the location of the lamina prinicipalis externa is indicated. The dotted line shows the location of layer III within the subiculum and prosubiculum. Abbreviations: b, medial mammillary nucleus, pars basalis; CA1, hippocampal field CA1; Ent, entorhinal cortex; ic, nucleus intercalatus; l, medial mammillary nucleus pars lateralis; LMN, lateral mammillary nucleus, LPE, lamina principalis externa; PaS, parasubiculum; Prh, perirhinal cortex; PrS, presubiculum; PS, prosubiculum; m, medial mammillary nucleus pars medialis; MTT, mammillothalamic tract; V, ventricle. Scale bars, 1 mm.

4 2522 J. P. Aggleton et al. The terminology used for the hippocampus and subicular cortices (subiculum, prosubiculum, presubiculum and parasubiculum) largely follows that established by Lorente de Nó (1934). For this reason the region often called the subiculum is divided into two areas, the prosubiculum and the subiculum (Fig. 1). The prosubiculum lies between the subiculum and hippocampal field CA1, and is distinguished from the subiculum by an additional layer II, which contains modified Ammon s horn pyramidal cells. In accordance with standard practice (Swanson & Cowan, 1977), the border of the subiculum with the presubiculum is principally defined by the emergence of a new layer of small pyramidal cells (II), the lamina principalis externa. At the same time, layers III V of the subiculum [the subiculum a of Lorente de Nó (1934)] extend for a short way under the lamina principalis externa (Fig. 1). The terminology used in delineating the entorhinal cortex follows that described by Saunders & Rosene (1988). The perirhinal cortical designations, areas 35 and 36 (Brodmann, 1909), are taken from Amaral et al. (1987) and match those of Saleem & Tanaka (1996). Results Retrograde tracers (HRP injections into the mammillary bodies) Representative examples of the HRP injection site are shown in Fig. 2, which shows an injection in an intact animal (MB2) and an animal with a fornix transection (MBfx5). All the HRP injection sites were centred in the medial nucleus of the mammillary bodies but appeared to include the lateral nucleus. While all of the injections filled most of the mammillary bodies none of them was restricted to the medial and lateral mammillary nuclei, but spread into the immediately surrounding areas of the hypothalamus, including parts of the ventral medial hypothalamus, the posterior hypothalamus, the lateral hypothalamus, the tuberomammillary area and the supramammillary area. In all cases the injection spread into the medial mammillary nucleus in the contralateral hemisphere but did not appear to reach the lateral mammillary nucleus. The patterns of labelled cells in the hippocampal region were strikingly similar in the three intact cases (MB1 3), although there were differences in the absolute numbers of retrogradely labelled cells. In contrast, no labelled cells were observed in the hippocampal formation or parahippocampal region of the three cases in which the fornix had been transected. For these reasons the pattern of hippocampal label is depicted on a series of coronal sections (Fig. 3) from just one of the intact cases (MB2; Fig. 2). Large numbers of labelled cells were found in the subicular cortices, with the large majority in the subiculum (Figs 3 and 4). The cells were, for the most part, in the mid and deeper parts of layer III of the subiculum (Lorente de Nó, 1934) and were primarily pyramidal-type cells (Fig. 4). These cells were found in similar numbers across the distal proximal extent of the subiculum and so there were equivalent numbers in subiculum a and subiculum b (Lorente de Nó, 1934). The prosubiculum also contained labelled cells. Once again, nearly all of these prosubicular cells were in layer III and were of pyramidal type. These labelled prosubicular cells formed a continuous group with those in the subiculum (Fig. 4), although there was typically less than half of the number found in the prosubiculum as in the subiculum. While the overwhelming majority of cells were in layer III, very occasional labelled cells were observed in the deeper layers of the prosubiculum and subiculum. The presubiculum contained < 2% of the total number of labelled cells in the hippocampal formation. These presubicular cells were scattered in the layers below the lamina principalis externa, and were Fig. 2. Brightfield photomicrographs of coronal sections showing the placement of the HRP injections in two representative cases (MB2, MBfx5). In both cases there was some spread of the HRP in the tract made by the injection. Abbreviations: CP, cerebral peduncle; V, ventricle. Scale bar, 1 mm. smaller than those in the subiculum. The presubicular cells consisted of a mix of pyramidal cells and smaller stellate type cells. No labelled cells were observed in the lamina principalis externa. In the large majority of sections no labelled cells were found in the parasubiculum; however, one or two labelled cells were observed in each animal. No labelled cells were found in the dentate gyrus or fields CA1 3. Evidence was found of a very light projection from the entorhinal cortex (Fig. 3). A scattering of labelled cells was found in the entorhinal cortex (area 28S, Saunders & Rosene, 1988), often close to the perirhinal region (area 35). These entorhinal cells were primarily in layer III with some in layers II and V. The perirhinal cortex (areas 35 and 36) typically contained no labelled cells, although in a small number of sections an occasional cell was present in the middle layers of area 35.

5 Hippocampal projections to mammillary bodies 2523 Fig. 3. Series of coronal sections showing the distribution of HRP-positive cells in the medial temporal lobe in case MB2. The numbers correspond to the approximate AP level of the section with respect to ear-bar 0. Abbreviations: Amy, amygdala; Ent, entrohinal cortex; PaS, parasubiculum; PS, prosubiculum; Prh, perirhinal cortex; PrS, presubiculum, RhS, rhinal sulcus; S, subiculum; TH, parahippocampal area TH. There was clear evidence of a rostrocaudal gradient in the source of the mammillary body inputs from the subicular region (Fig. 5). In all three cases the greatest numbers of labelled cells were in the most caudal portion of the prosubiculum and subiculum. From the rostral to the middle portions of the prosubiculum and subiculum there was little change in the numbers of labelled cells, but in the caudal hippocampal formation there was a marked increase in the number of HRP-positive cells in the prosubiculum, subiculum and, to a lesser extent, the presubiculum (Fig. 5). As a consequence there was a doubling or more in the numbers of labelled cells at the very posterior 2 mm of the hippocampus (Figs 3 and 5). At this very caudal level, just posterior to the end of the splenium, the presubiculum extends towards the retrosplenial cortex and begins to curve around the splenium. Large numbers of labelled cells were present in layer III of this most caudal part of the subiculum (Figs 4 and 5). The findings in the two animals with fornix lesions prior to the HRP injection (MBfx4 and 5) were all consistent. In no case were labelled cells identified in the hippocampal formation or the parahippocampal gyrus. Anterograde tracers (amino acid injections into medial temporal lobe structures) The location and extent of the various injections sites are shown in Fig. 6. A series of standard coronal sections depicts the locations of the amino acid injections in the hippocampus and parahippocampal regions. In all cases with fornix lesions the tract transections were complete (Fig. 6). Occasionally, the fornix surgery resulted in minor unilateral damage to the cingulate cortex. Subiculum Mammillary body label could be seen in those intact cases in which the amino acid injection involved the subiculum at either rostral (ACy12, ACy14), mid (ACy25) or caudal (ACy28) levels (Fig. 6). In one case (ACy12) the injection was placed in the rostral portion of the prosubiculum and adjacent parts of the subiculum. All cell layers were involved, and the injection did not spread to the presubiculum. The injection in ACy14 was centred in the rostral subiculum and so was slightly more distal to CA1 than that in ACy12 (i.e. closer to the presubiculum). All layers of the subiculum were involved in Case ACy14, with the limit of the injection site just reaching the prosubiculum (laterally) and the presubiculum (medially). For these two cases with rostral subiculum injections (ACy12 and ACy14) the pattern of mammillary body label was very similar, although it was appreciably denser and more widespread in ACy12 (the case with the injection more proximal to CA1). In both cases ipsilateral fibres could be traced in the fornix, which approached the rostral mammillary bodies lateral to the medial mammillary nucleus. In case ACy12 silver grains were present across the dorsal two-thirds of the rostral part of the medial nucleus (Fig. 7 upper), spreading

6 2524 J. P. Aggleton et al. Fig. 5. Numbers of HRP-labelled cells in the subicular cortices at different AP levels in cases MB1, MB2 and MB3. Cell counts are taken from the subiculum, prosubiculum and presubiculum. A rostrocaudal gradient can be seen, with the clearest increase at the most caudal hippocampus. Fig. 4. Photomicrographs of HRP-positive cells in the mid (Upper) and caudal (Lower) levels of the hippocampus following an injection of HRP in case MB2. A region of the subiculum and prosubiculum from the upper photomicrograph is enlarged in the middle figure. These coronal sections show how the label is restricted to the subicular cortices. Abbreviations: Ent, entorhinal cortex; DG, dentate gyrus; PaS, parasubiculum; Prh, perirhinal cortex; PS, prosubiculum; PrS, presubiculum, RS, rhinal sulcus; Rspl, retrosplenial cortex; S, subiculum; TH, parahippocampal area TH. Scale bars, 1 mm. throughout almost the entire medial mammillary nucleus just a little more caudally. As a consequence, label was found in pars medialis, pars lateralis and pars basalis (Fig. 7). While some of this label was caused by fibres passing caudally, much appeared to reflect termination in the medial mammillary nucleus. Label was also present in nucleus intercalatus. The termination pattern in ACy12 changed appreciably in the caudal half as label was now largely restricted to an arc from the most ventral and medial part of pars medialis, through pars basalis, to the ventral half of pars lateralis (Fig. 7, lower). This dense label then formed a distinctive crescent around the lateral and ventral margins of the medial nucleus in ACy12 (Fig. 7). This crescent of label, which included fibres, involved all of pars basalis. The amount of label in this crescent decreased markedly near the caudal limit of the medial mammillary nucleus. While there was also label in the region of nucleus intercalatus, some of this reflected the continuation of those fornical fibres that passed caudally beyond the mammillary bodies. In case ACy14 the injection was also centred in the rostral subiculum but it was located nearer the presubiculum. A more restricted pattern of label was found in the medial mammillary nucleus; it was concentrated in the lateral and ventral parts of the medial nucleus (Fig. 8). Once again, the densest label was in pars basalis. A much lighter scattering of label was found in the contralateral medial mammillary nucleus in both ACy12 and ACy14. This label formed a much fainter copy of that present in the ipsilateral hemisphere (Figs 7 and 8), and so involved all three regions within the medial nucleus in ACy12. Fibres were visible crossing directly between the most medial portions of pars medialis. This appeared to be the sole route for these contralateral projections as no label could be discerned in the fornix in the contralateral hemisphere. In both ACy12 and ACy14 there was evidence of a light increase in label across the ipsilateral lateral mammillary nucleus. This label was, however, diffuse and very light. The lack of this label caudal to the lateral nucleus indicated that some of this represented terminals. An injection centred in the mid AP levels of the dentate gyrus but involving the dorsal layers of the subiculum (ACy25) resulted in a slightly different pattern of label to that observed with the more rostral subicular injections. At the more rostral levels, label was present across a central horizontal band in the medial mammillary nucleus, involving pars medialis and pars lateralis. More caudally, the label in the medial mammilary nucleus was concentrated in a continuous arc from the most ventral to the more lateral parts of the medial mammillary nucleus. The majority of this label was in the most ventral part of pars medialis, but the label extended into pars lateralis and pars

7 Hippocampal projections to mammillary bodies 2525 Fig. 7. Distribution of anterograde label in the mammillary bodies after an injection involving the rostral subiculum (case ACy12). The upper (darkfield) and middle (brightfield) show the same coronal section from the more rostral portion of the mammillary bodies. Considerable label is visible in the ipsilateral medial mammillary nucleus, with much lighter label in the contralateral medial nucleus. (Lower) Coronal darkfield and brightfield sections in case ACy12, showing how the distribution of label changes in the more caudal portion of the mammillary bodies. Abbreviations: LMN, lateral mammillary nucleus; TB, tuberomammillary area. Scale bar, 1 mm. Fig. 6. Schematic illustrations and photomicrograph showing the placement of the amino acid injections (Upper and Mid) and the appearance of the fornix transections (Lower). The upper series of coronal sections depict the injection sites in the 10 normal cases. The middle coronal sections depict the six injection sites in the five animals in which the fornix was sectioned prior to amino acid injection. The lower panel shows a Nissl-stained coronal section in case ARhF24. The fornix has been removed via a dorsal approach. Abbreviations: Amyg, amygdala; CC, corpus callosum; ATN, the anterior thalamic nuclei; PaS, parasubiculum; PrS, presubiculum, RhS, rhinal sulcus; S, subiculum. Scale bar, 1 mm. basalis. No label was visible in the lateral mammillary nucleus or in the contralateral mammillary bodies. The mammillary body label in ACy25 was appreciably lighter than that observed in ACy12 and ACy14, and this presumably reflects the greater involvement of the subiculum in the latter two cases. In case ACy28 the amino acid injection into the caudal hippocampus filled the subiculum, prosubiculum, fields CA1 3 and dentate gyrus, and included the medial edge of the presubiculum. This large injection resulted in very dense label throughout the rostrocaudal extent of the mammillary bodies. Labelled fibres could be seen entering the lateral margin of the medial mammillary nucleus and, for all but the very caudal limit of the medial nucleus, there was a very dense region of label filling the dorsal two-thirds of the medial nucleus (Fig. 8). This region of label involved part of the dorsal pars medialis and all of pars lateralis. In contrast the ventral parts of the medial nucleus, including pars basalis, contained considerably less label. The only exception was at the most caudal part of the medial nucleus where the densest label was in the ventral pars medialis. A very dense

8 2526 J. P. Aggleton et al. patch of labelled fibres was present in the area between the lateral mammillary nucleus and the medial mammillary nucleus, and for this reason it was not possible to determine whether there were projections to nucleus intercalatus. Label of terminal appearance was, however, found throughout the lateral mammillary nucleus. This label, which was not as dense as that observed in the medial mammillary nucleus, was distributed uniformly across the lateral nucleus. In spite of the density of the label in the ipsilateral hemisphere, only very few labelled fibres and very light termination were observed in the contralateral medial mammillary bodies. The location of this label corresponded to the densest label in the ipsilateral medial nucleus. Presubiculum and parasubiculum In case PSub an amino acid injection was placed in the caudal presubiculum and parasubiculum. Label could be seen in the fornix and followed to the region of the mammillary bodies. The only label was light and was limited to the mid and caudal levels of the mammillary bodies. At these levels the silver grains formed a pale continuous band between pars medialis and pars lateralis of the medial nucleus. The label was confined to a curved horizontal strip that was between a half and two-thirds up the dorsal axis of the nucleus. Light terminal label appeared to be scattered throughout the lateral mammillary nucleus. There was no evidence of contralateral label or label in nucleus intercalatus. Entorhinal cortex (area 28) A large amino acid injection involving most of the area 28 (case ERh3) resulted in a distinct projection to the medial mammillary bodies similar in some respects to that seen in case ACy14 (rostral subiculum). A mixture of fibres and probable terminal label was found across the most rostral portion of the medial mammillary nucleus. For the remaining rostrocaudal levels the label was concentrated in the ventrolateral part of the medial nucleus (Fig. 9). This more ventral label was centred in and around pars basalis. No evidence was found of a projection to the lateral mammillary nucleus, to nucleus intercalatus or to the contralateral mammillary bodies. A more selective injection centred in area 28m within the entorhinal cortex (case ERh2) resulted in a similar locus of label except that it was more discrete, being restricted to the mid rostrocaudal level of pars basalis. Perirhinal cortex (areas 35 and 36) Data from two cases indicate that there is a barely discernable input from the perirhinal cortex to the medial mammillary nucleus. In case PRh1 the amino acid injection involved the mid and caudal levels of areas 35 and 36. For most of the mammillary bodies there was no evidence of a projection, although a small increase in silver grains was found in the dorsal two-thirds of pars medialis and pars lateralis on one section. A very similar result was found for ACy9, where the injection was placed into caudal area 35 with some spread into area 36 and rostral TF. Once again, there was no evidence of an input for most of the mammillary bodies, with only one section showing a slight increase in silver grains in the dorsolateral portion of the medial mammillary nucleus. Fig. 8. The different distributions of anterograde label in the mid-ap level of the mammillary bodies after an amino acid injection involving either the rostral subiculum (ACy14; upper two photomicrographs) or the caudal subiculum (ACy28; lower two photomicrographs). (Upper panels) The darkfield (lower) photomicrograph shows the presence of label in the basal and lateral parts of the medial mammillary nucleus, with lighter label in the corresponding areas in the contralateral hemisphere. (Lower panels) The darkfield (lower) photomicrograph shows a region of dense label across the dorsal and middle portions of the medial nucleus. Label is also visible in the ipsilateral lateral mammillary nucleus (LMN) but not in the mammillary bodies in the contralateral hemisphere. Abbreviations: b, medial mammillary nucleus, pars basalis; LMN, lateral mammillary nucleus. Scale bars, 1 mm.

9 Hippocampal projections to mammillary bodies 2527 Fig. 9. Photomicrographs showing distribution of anterograde label in the mammillary bodies after an injection involving much of the entorhinal cortex (ERh3). The darkfield (lower) photomicrograph shows a region of label centred in the basal region of the medial nucleus. Abbreviation: LMN, lateral mammillary nucleus. Scale bar, 500 lm. Cases with prior fornix transaction Amino acid injections were placed in the subiculum (ACyF15, ACyF27L and ARhF24), dentate gyrus (ACyF19), the caudal part of the rhinal sulcus (ARhF23) and the deep layers of areas 35 and 36 (ACyF27R). In none of these cases was there evidence of any label in the mammillary bodies. It is noteworthy that this failure to find mammillary body label included cases (ACyF15, ACyF27L and ARhF24) in which the injections involved the subiculum, and so included the same areas as those injected in the intact cases. Discussion The present study is the first to describe in detail both the source and termination patterns of the direct projections from the hippocampal formation to the mammillary bodies in the monkey brain. The principal projection in the macaque brain arises from pyramidal cells in layer III of the subiculum and prosubiculum. These regions provide the overwhelming majority of the input, with much smaller contributions from the lamina principalis interna of the presubiculum and from the entorhinal cortex. These medial temporal projections were focused on the medial mammillary nucleus, and evidence was found that different parts of the subicular complex terminate in different parts of the medial nucleus. A much lighter projection to the lateral mammillary nucleus was noted although, when label was observed in this nucleus, it was distributed uniformly throughout, unlike the inputs to the medial mammillary nucleus. Some of this lateral mammillary nucleus label appeared to reflect the passage of axons. It was also possible to confirm that the inputs from the hippocampal formation and the parahippocampal region are completely reliant on the fornix. In this regard they differ from the direct medial temporal inputs to the thalamus, some of which have both fornical and nonfornical routes (Saunders et al., 2005). Perhaps the most important finding is the confirmation that the principal source of the hippocampal projection is from pyramidal cells in layers III of the prosubiculum and subiculum. Krayniak et al. (1979) also described how subicular cells in the squirrel monkey project to the mammillary bodies, but these retrogradely labelled cells were present throughout the total depth of the subiculum. In the present study the labelled cells were confined to layer III, but were distributed across its depth. As the tracer injections made by Krayniak et al. (1979) involved an unusually large volume of HRP (the same concentration as the present study but over six times the volume) there is some question as to how much spread occurred beyond the mammillary bodies. Other differences arise from the finding that in the macaque brain the prosubiculum contributes appreciably to the mammillary body projections, while in the squirrel monkey only a few scattered cells in the prosubiculum appear to innervate the mammillary bodies (Krayniak et al., 1979). A further difference was that Krayniak et al. (1979) did not include anterograde tracers. In the present study, the complementary anterograde tracer experiments showed that the projections from the hippocampal formation to the posterior hypothalamus were largely confined to the medial and lateral mammillary bodies. This finding makes it less probable that the HRP-labelled cells in the subicular cortices in the present study are the result of retrograde transport from regions immediately adjacent to the mammillary bodies. A novel finding for the primate brain was the rostrocaudal gradient in the density of the subicular projections to the mammillary bodies (Fig. 5). Although inputs arose along the length of the structure there was an increase from the more caudal subiculum, prosubiculum and presubiculum regions. Other hippocampal and subicular efferents in the macaque brain also show rostrocaudal gradients. While the inputs to nucleus accumbens, the orbitofrontal cortex and the anterior thalamus arise primarily from the rostral subiculum (Aggleton et al., 1986; Carmichael & Price, 1995; Friedman et al., 2002), the subicular projections to the retrosplenial cortex arise predominantly from the caudal subicular cortices (Kobayashi & Amaral, 2003). In fact, the projections to the retrosplenial area 29 show further similarities with those to the mammillary bodies. Not only do both sets of projections have a rostrocaudal gradient, but both arise primarily from layer III pyramidal cells in the subiculum and prosubiculum, with the majority from the subiculum (Kobayashi & Amaral, 2003). There is, in addition, a much smaller retrosplenial input from deeper cells in the presubiculum, as is found for the mammillary body inputs. These various rostrocaudal anatomical gradients suggest that there should be corresponding functional correlates, especially as electrophysiological and functional imaging evidence also point to rostrocaudal differences in hippocampal function (Columbo et al., 1998; Schacter & Wagner, 1999). Of especial relevance is evidence that activity in the caudal subiculum increases during retrieval of arbitrary face name combinations (Zeineh et al., 2003). As the hippocampal links with the

10 2528 J. P. Aggleton et al. mammillary bodies and the retrosplenial cortex have frequently been implicated in episodic memory (Maguire, 2001; Vann & Aggleton, 2004) this caudal activation may, in part, reflect these connectional patterns. Clear evidence was found that the projections to the medial mammillary nucleus were topographically organized as several different termination patterns were observed. These patterns did not usually correspond to the cytoarchitectonic divisions within the medial mammillary nucleus, although it is possible that much more discrete injections could have revealed such an arrangement. One repeated pattern comprised a concentration of label in the most ventral and lateral parts of the medial mammillary nucleus, forming a continuous arc that involved pars basalis. While some of this label appeared to be fibres, much was consistent with terminal label. This pattern was associated with injections in the rostral subiculum and entorhinal cortex. An injection involving the mid rostrocaudal level of the subiculum terminated in a central band in the medial mammillary nucleus. The injection into the presubiculum also labelled the central axis of the medial mammillary nucleus. Finally, injections into the caudal hippocampus filled the central and dorsal parts of the medial nucleus. The areas of terminal label from these various injections did, however, overlap so that all parts of the medial nucleus appear to receive subicular inputs. This coarse topography closely matches that described by Simpson (1952) for Macaca mulatta, even though he could only use lesion degeneration data. Simpson (1952) reported that the rostal hippocampus appears to terminate in the ventral and rostral medial mammillary nucleus and the mid rostrocaudal levels of the hippocampus terminate in an axial zone, while the caudal hippocampus terminates in the caudal and dorsal medial mammillary nucleus. Although it was not possible for Simpson (1952) to distinguish the importance of the subiculum, and the lesion approach is confounded by damage to fibres of passage, this general pattern of rostrocaudal projections is in surprisingly good agreement with the present study. Subicular projections reach the contralateral medial nucleus but not the contralateral lateral mammillary nucleus (Simpson, 1952; Poletti & Cresswell, 1977). As a consequence, the medial mammillary nucleus receives bilateral subicular inputs, but principally has ipsilateral Fig. 10. Photomicrographs of coronal sections of the subiculum showing the different sources of the inputs from the hippocampal formation to the mammillary bodies and the thalamus. (Upper) Following an injection in the mammillary bodies (Case MB2) the large majority of label is found in pyramidal cells scattered across layer III. (Lower) Darkfield (left) and brightfield (right) photomicrographs of coronal sections through the subiculum in an animal in which HRP was injected into the anterior thalamic nuclei (case A1, Aggleton et al., 1986). The HRP-positive cells, which are visible in the darkfield photomicrograph (lower left), are concentrated in layers IV and V in the polymorphic cell layer (outlined by dashed lines). Abbreviations: AL, alveus, PS, prosubiculum; S, subiculum. Scale bar, 250 lm.

11 Hippocampal projections to mammillary bodies 2529 projections to the anterior thalamic nuclei (Veazey et al., 1982b). In contrast, the lateral mammillary nucleus only appears to receive ipsilateral subicular inputs, yet has bilateral projections to the anterior dorsal nucleus (Veazey et al., 1982b). Fibres reaching the contralateral mammillary bodies appeared to cut directly across the midline from the medial mammillary nucleus and, consistent with this observation, no evidence could be found of labelled fibres in the contralateral fornix. Some evidence was found that the rostral subiculum provides most of the contralateral input. This gradient is therefore in the opposite direction to that for the density of inputs to the ipsilateral medial mammillary nucleus (Fig. 5). All previous evidence of hippocampal projections to the lateral mammillary nucleus of the monkey has come from degeneration studies (Simpson, 1952; Poletti & Cresswell, 1977), making it impossible to confirm the source of the input. The present study found evidence of a light diffuse projection throughout this nucleus, arising from the length of the subiculum. The inputs to the lateral mammillary nucleus have been described in more detail in the rat brain, where they arise from the presubiculum, parasubiculum and postsubiculum (Allen & Hopkins, 1989; Shibata, 1989; van Groen & Wyss, 1990a,b). Interest in contrasts between the medial and lateral mammillary nuclei has grown with the discovery that the lateral mammillary nucleus contains head-direction cells (Blair et al., 1998) while the medial mammillary nucleus contains cells showing theta activity (Kocsis & Vertes, 1994). From such evidence it has been proposed that these different mammillary nuclei reflect stages in two parallel memory systems (Vann & Aggleton, 2004), but almost all of the relevant data have so far come from studies of the rodent brain. It is therefore of especial interest that head-direction cells have been recorded in the presubiculum of macaque monkeys (Robertson et al., 1999). Coupled with this finding are the present results that confirm the existence of direct subicular inputs to the lateral mammillary nuclei, with additional evidence that the presubiculum contributes to this projection. There have been many more studies describing the hippocampal and parahippocampal projections to the mammillary bodies in the rat brain than in the primate brain. It is therefore useful to see how these sets of connections compare. It should first be noted that for the relevant rat studies a separate prosubiculum is not distinguished, and is treated as part of the subiculum. In both the macaque and the rat, the subiculum is by far the most important source of direct connections, with no inputs from CA1 and much lighter inputs from the lamina prinicipalis interna of the presubiculum (Ishizuka, 2001). As in the macaque, the parasubiculum provides little if any input to the medial mammillary bodies (Allen & Hopkins, 1989; Ishizuka, 2001) while there is an entorhinal projection to the ventral and lateral part of the medial mammillary nucleus (Shibata, 1988). The rat subicular projections, like those in the monkey, arise from pyramidal cells distributed across the layer III as designated by Lorente de Nó (1934). This projection arises from the entire septotemporal length of the subiculum, and there does not seem to be a rostrocaudal gradient similar to that described in the present study. Greater precision in the placement of injections in rodents has made it possible to uncover more about the topography of terminations within the mammillary bodies. This topography arises from two axes. First, there is evidence that, along the transverse axis, the part of the subiculum which is closer to the presubiculum (the equivalent of subiculum a) projects preferentially to lateral and caudal parts of the medial mammillary nucleus while the part closer to CA1 projects more to the medial parts of the medial mammillary nucleus (Shibata, 1989; Ishizuka, 2001). The presbiculum, in contrast, projects to the posterior mammillary nucleus and the lateral mammillary nucleus (Shibata, 1989). Second, the dorsal, caudal and ventral subiculum cortices project to the dorsal, central and ventral portions of the medial mammillary nucleus, respectively. As in the macaque, these projections do not map onto the cytoarchitectonic divisions within the medial mammillary nucleus; rather, they are aligned in more horizontal tiers (Shibata, 1989). One of the most striking features of the subicular inputs to the mammillary bodies is that they arise in a quite different cell population from that which projects to the anterior thalamus. The hippocampal anterior thalamic projections, which are also largely confined to the prosubiculum and subiculum within the subicular complex, arise almost exclusively from polymorphic cells in the deepest cell layer (Krayniak et al., 1979; Aggleton et al., 1986). Using the nomenclature of Lorente de Nó (1934), the thalamic projections arise from layer IV of the prosubiculum and layers IV and V of the subiculum. This contrasts with the projections to the mammillary bodies, which originate from layer III of both the subiculum and the prosubiculum. This arrangement means that two parallel projections arise in exactly the same regions but from different lamina (Fig. 10). The same parallel arrangement has been reported for the hippocampal projections to the mammillary bodies and anterior thalamus in the rat brain (Meibach & Siegel, 1975; Sikes et al., 1977; Ishizuka, 2001). This very precise separation is all the more remarkable given that the principal output from the mammillary bodies is to the anterior thalamic nuclei, i.e. a major target of the subicular polymorphic cells. The conclusion is that the direct influences of the subicular cortices on the mammillary bodies and on the thalamus must differ in some way that has yet to be identified. At the same time, these different connections presumably converge within the anterior thalamic nuclei, as all three anterior thalamic nuclei receive dense inputs from the mammillary bodies as well as from the hippocampus (subiculum). As the mammillary bodies do not project directly back upon the hippocampus (Amaral & Cowan, 1980), understanding the relationship between the direct hippocampal inputs to the thalamus and the indirect thalamic inputs, via the mammillary bodies, remains a key goal in determining how these pathways contribute to memory processes. Acknowledgements The authors are grateful to M. Mishkin and L. Woods for their support and technical assistance. Part of this research was supported by the MRC. Abbreviation HRP, horseradish peroxidase. References Aggleton, J.P. (1985) X-ray localization of limbic structures in the cynomolgus monkey (Macaca fascicularis). J. Neurosci. Meth., 14, Aggleton, J.P. (1986) A description of the amygdalo-hippocampal interconnections in the macaque monkey. Exp. Brain Res., 64, Aggleton, J.P. & Brown, M.W. (1999) Episodic memory, amnesia, and the hippocampal anterior thalamic axis. Behav. Brain Sci., 22, Aggleton, J.P., Desimone, R. & Mishkin, M. 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