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Distribution of perihippocampo-hippocampal projection neurons in the lesser hedgehog tenrec
Abstract
The entorhinal cortex in the Madagascan lesser hedgehog tenrec is thought to be part of the three-layered subrhinal paleocortex (PCx) but cyto- and chemoarchitectural studies have failed so far to identify the area. To reach this goal tracer injections were made into the tenrec's hippocampus. Retrogradely labeled cells were found in dorsal portion of the posterior PCx, the adjacent rhinal cortex (RCx) and the so-called area XCx. The main paleocortical portion in the ventral PCx, however, remained unlabeled with the exception of a caudal region possibly equivalent to the amygdalo-piriform transition area. The labeled neurons showed a bilaminar distribution with the cells in the layer 2A giving rise to fibers to predominantly the dentate area and the cells in the layer 3A mainly projecting to the cornu ammonis and the subiculum. The latter regions, in addition, gave rise to a feedback projection to the layer 3B of especially the caudal RCx and the XCx. The analysis of the terminal projections, however, was hampered by the fact, that under certain conditions retrogradely transported biotinylated dextran was also transported in anterograde direction via collaterals of the entorhino-dentate fibers. The findings are compared with equivalent regions in more differentiated mammals particularly with regard to the perirhinal area showing little if any connections with the dentate gyrus.
... The majority of the Afrotheria are endemic to the African continent, but they are also found on the island of Madagascar, with the sirens showing a far broader distribution across the Indian, Pacific, and Atlantic oceans. The organization of the brain of the lesser hedgehog tenrec, Echinops telfairi, has been the subject of many studies (Alp ar et al., 2010;Kosaka et al., 2005;Krubitzer et al., 1997;Künzle, 1988Künzle, , 1992Künzle, , 1993Künzle, , 1994Künzle, , 1995aKünzle, ,1995bKünzle, , 1996Künzle, , 1998Künzle, , 2002Künzle, , 2003Künzle, , 2005aKünzle, , 2005bKünzle, , 2006Künzle, , 2009Künzle, , 2012Künzle et al., 2002;Künzle & Radtke-Schuller, 2000a, 2000b, 2001Künzle & Rehkämper, 1992;Künzle & Unger, 1992;Morawski et al., 2010;Radtke-Schuller & Künzle, 2000;Schmolke & Künzle, 1997;Stephan et al., 1981;Wolff & Künzle, 1997), making it one of the most intensely studied Afrotherian brains. To the authors' knowledge, no comprehensive mapping studies of the nuclei forming the cholinergic, catecholaminergic, serotonergic, or orexinergic systems in the lesser hedgehog tenrec have been undertaken, although the dopaminergic neurons of the olfactory bulb have been described (Kosaka et al., 2005). ...
The current study provides an analysis of the cholinergic, catecholaminergic, serotonergic, and orexinergic neuronal populations, or nuclei, in the brain of the lesser hedgehog tenrec, as revealed with immunohistochemical techniques. For all four of these neuromodulatory systems, the nuclear organization was very similar to that observed in other Afrotherian species and is broadly similar to that observed in other mammals. The cholinergic system shows the most variation, with the lesser hedgehog tenrec exhibiting palely immunopositive cholinergic neurons in the ventral portion of the lateral septal nucleus, and the possible absence of cholinergic neurons in the parabigeminal nucleus and the medullary tegmental field. The nuclear complement of the catecholaminergic, serotonergic and orexinergic systems showed no specific variances in the lesser hedgehog tenrec when compared to other Afrotherians, or broadly with other mammals. A striking feature of the lesser hedgehog tenrec brain is a significant mesencephalic flexure that is observed in most members of the Tenrecoidea, as well as the closely related Chrysochlorinae (golden moles), but is not present in the greater otter shrew, a species of the Potomogalidae lineage currently incorporated into the Tenrecoidea. In addition, the cholinergic neurons of the ventral portion of the lateral septal nucleus are observed in the golden moles, but not in the greater otter shrew. This indicates that either complex parallel evolution of these features occurred in the Tenrecoidea and Chrysochlorinae lineages, or that the placement of the Potomogalidae within the Tenrecoidea needs to be re‐examined.
... Hippocampus formation in the guinea pig and domestic pig is composed of the subiculum, hippocampus proper, and dentate gyrus, although only the dentate gyrus and hippocampus proper were investigated. Its general anatomical organization and three-layered structure appeared to be similar in primates: human [2], rhesus [25], and macaque [23], as well as non-primates mammals such as the pig [4], cat [31], hedgehog [15, 20], shrew [9], rat [17], and mouse [29]. The study showed that distribution of CART 61–102 and rhCART 28–116 -immunoreactivity in the guinea pig and domestic pig was similar in this respect, i.e. that both peptides were shown in the same layers of hippocampal formation. ...
This study provides a detailed description concerning the distribution of cocaineand amphetamine-regulated transcript (CART) subunits - CART(61-102) and rhCART(28-116) - in the hippocampal formation (HF) of the guinea pig and domestic pig, focussing on the dentate gyrus (DG) and hippocampus proper (HP). Although in both studied species CART-immunoreactive (CART-IR) neuronal somata and processes were present generally in the same layers, some species-specific differences were still found. In the granular layer (GL) of both species, the ovalshaped neurons and some thick varicose fibres were encountered. In the guinea pig there was an immunoreactive "band of dots", probably representing crosssectioned terminals within the DG molecular layer (MOL), whereas in the domestic pig, some varicose fibres were detected, thus suggesting a different orientation of, at least, some nerve terminals. Furthermore, some CART-positive cells and fibres were observed in the hilus (HL) of the guinea pig, whereas in the analogical part of the domestic pig only nerve terminals were labelled. In both species, in the pyramidal layer (PL) of the hippocampus proper, CART-IR triangular somata were observed in the CA3 sector, as well as some positive processes in MOL; however, a few immunoreactive perikarya were found only in the CA1 sector of the guinea pig. As regards the localization patterns of two isoforms of CART in the guinea pig, both peptide fragments were present simultaneously in each of the labelled neurons or fibres, whereas in the domestic pig three types of fibres may be distinguished within the area of the DG. In the hilus and MOL of the dentate gyrus, there were fibres expressing both isoforms of CART in their whole length (fibres of the first type). Fibres of the second type (in GL) coexpressed both peptides only on their short segments, and the last ones (in MOL) expressed solely rhCART(28-116). These results indicate that the distribution of the two CART isoforms are specifically related, thus the relationship between the two CART isoforms may imply different metabolic profiles of CART-expressing neurons.
... Wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP; Sigma; n = 7) and biotinylated dextran amine (BDA; Sigma; n = 5) were used as tracer substances. Most cases have been described previously with regard to other connections [26,28,82]. The additional tracer injections (Et01-47W, Et03-58W) were done in the same fashion: The WGA-HRP (1.5–8 nl of a 2–5% solution in distilled water) was pressure injected through a glass micropipette (tip diameter 8–15 μm) attached to a Hamilton syringe driven by a micromanipulator. ...
The dentate gyrus is well known for its mossy fiber projection to the hippocampal field 3 (CA3) and its extensive associational and commissural connections. The dentate gyrus, on the other hand, has only few projections to the CA1 and the subiculum, and none have clearly been shown to extrahippocampal target regions.
Using anterograde and retrograde tracer techniques in the Madagascan lesser hedgehog tenrec (Afrosoricidae, Afrotheria) it was shown in this study that the dentate hilar region gave rise to a faint, but distinct, bilateral projection to the most rostromedial portion of the olfactory tubercle, particularly its molecular layer. Unlike the CA1 and the subiculum the dentate gyrus did not project to the accumbens nucleus. A control injection into the medial septum-diagonal band complex also retrogradely labeled cells in the dentate hilus, but these neurons were found immediately adjacent to the heavily labeled CA3, while the tracer injections into the rostromedial tubercle did not reveal any labeling in CA3.
The dentate hilar neurons projecting to the olfactory tubercle cannot be considered displaced cells of CA3 but represent true dentato-tubercular projection neurons. This projection supplements the subiculo-tubercular projection. Both terminal fields overlap among one another as well as with the fiber terminations arising in the anteromedial frontal cortex. The rostromedial olfactory tubercle might represent a distinct ventral striatal target area worth investigating in studies of the parallel processing of cortico-limbic information in tenrec as well as in cat and monkey.
The present study analyses the overall extrinsic connectivity of the non-olfactory amygdala (Ay) in the lesser hedgehog tenrec. The data were obtained from tracer injections into the lateral and intermediate portions of the Ay as well as several non-amygdalar brain regions. Both the solitary and the parabrachial nucleus receive descending projections from the central nucleus of the Ay, but only the parabrachial nucleus appears to project to the Ay. There is one prominent region in the ventromedial hypothalamus connected reciprocally with the medial and central Ay. Amygdalar afferents clearly arise from the dorsomedial thalamus, the subparafascicular nuclei and the medial geniculate complex (GM). Similar to other subprimate species, the latter projections originate in the dorsal and most caudal geniculate portions and terminate in the dorsolateral Ay. Unusual is the presence of amygdalo-projecting cells in the marginal geniculate zone and their virtual absence in the medial GM. As in other species, amygdalo-striatal projections mainly originate in the basolateral Ay and terminate predominantly in the ventral striatum. Given the poor differentiation of the tenrec's neocortex, there is a remarkable similarity with regard to the amygdalo-cortical connectivity between tenrec and rat, particularly as to prefrontal, limbic and somatosensorimotor areas as well as the rhinal cortex throughout its length. The tenrec's isocortex dorsomedial to the caudal rhinal cortex, on the other hand, may not be connected with the Ay. An absence of such connections is expected for primary auditory and visual fields, but it is unusual for their secondary fields.
The perihippocampal fields represent the most important regions connecting the neocortex and the hippocampus in rat, cat and monkey but little is known about their presence and connectivity in species with poorly differentiated brain. Using axonal tracer substances we have recently studied the distribution of cortical cells projecting to the hippocampus in the hedgehog tenrec. In the present study we determined the regions of the paleocortex and the rhinal cortex connected with the neocortex, and provide a tentative view of the site and the extent of the tenrec's entorhinal, perirhinal and postrhinal/parahippocampal fields. It is shown that only the dorsal portions of the posterior rhinal cortex may be considered equivalent to the perirhinal and postrhinal fields of higher mammals, while a considerable expanse of the ventral rhinal cortex may be part of the entorhinal area (its so-called dorsal portion) connected with both the dentate gyrus and the neocortex. A few cells projecting to the neocortex were also noted in the dorsal-most portion of the three-layered paleocortex (ventral entorhinal portion). These cells were linearly arranged and reminiscent of the neocortical projecting cells in the entorhinal layer 4/5 in more differentiated mammals. The main portion of the paleocortex caudal to the corpus callosum remained unlabeled following neocortical and hippocampal tracer injections. Unexpectedly, the area in the most ventral paleocortex adjacent to the amygdala also projected to the neocortex, particularly the tenrec's somatosensorimotor cortex.
The indusium griseum and its precallosal extension are usually considered poorly differentiated portions of the hippocampus. The connections of this so-called 'hippocampal continuation' (HCt) have only been analyzed so far in rodents, which show one of the least-developed HCt among mammals. In this study we have investigated the relatively well differentiated HCt of the small Madagascan hedgehog tenrec (Afrotheria) using histochemical and axonal transport techniques. The tenrec's HCt shows associative and commissural connections. It receives laminar specific afferents from the entorhinal cortex (collaterals from neurons projecting to the dentate area), the anterior and posterior piriform cortices as well as the supramammillary region. A few fibers also originate in the olfactory bulb and the dentate hilus. Among these input areas only the dentate hilus receives a significant reciprocal projection from the HCt. Additional HCt efferents are directed to the subcallosal septum (presumed septohippocampal nucleus), the olfactory tubercle and the islands of Calleja. With the exception of the supramammillary afferents and possible efferents to the supraoptic nucleus we failed, however, to demonstrate distinct thalamic and hypothalamic connections. A comparison of the connections of the HCt with those of the hippocampal subdivisions reveal some similarity between the HCt and the dentate area, but the overall pattern of connectivity does not permit a correlation of the HCt with the dentate area, let alone the cornu ammonis and the subiculum. This view is supported by histochemical findings in the tenrec (immunoreactivity to calcium binding proteins) as well as the rat (data taken from the literature). The HCt is therefore considered a region in its own right within the hippocampal formation. It may be tentatively correlated with the medial cortex of reptiles, while the dentate area and the cornu ammonis may have evolved de novo in mammals.
This paper provides a comprehensive description of the organization of projections from the entorhinal cortex to the dentate gyrus, which together with projections to other subfields of the hippocampal formation form the so-called perforant pathway. To this end, data that are primarily from anatomical studies in the rat will be summarized, complimented with comparative data from other species. The analysis of the organization of any of the connections of the hippocampus, including that of the entorhinal cortex to the dentate gyrus, is severely hampered because of the complex three-dimensional shape of the hippocampus. In particular in rodents, but to a lesser extent also in primates, all traditional planes of sectioning will result in sections that at some point or another do not cut through the hippocampus at an angle that is perpendicular to its long axis. To amend this, we will describe own unpublished tracing data obtained in the rat with the use of the so-called extended preparation. A number of issues will be addressed. First, data will be summarized which will clarify the laminar origin of the perforant pathway within the entorhinal cortex. Second, we will discuss whether or not a radial organization, along the proximo-distal dendritic axis of granule cells, characterizes the entorhinal-dentate projection. Third, we will discuss whether this projection is governed by any transverse organization, and fourth, we will focus on the organization along the longitudinal axis. Finally, the synaptic organization and the contralateral entorhinal-dentate projection will be described briefly. Taken together, the available data suggest that the projection from the entorhinal cortex to the dentate gyrus is a fairly well conserved connection, present in all species studied, exhibiting a grossly similar organization.
The precise hierarchy of ancient divergence events that led to the present assemblage of modern placental mammals has been an area of controversy among morphologists, palaeontologists and molecular evolutionists. Here we address the potential weaknesses of limited character and taxon sampling in a comprehensive molecular phylogenetic analysis of 64 species sampled across all extant orders of placental mammals. We examined sequence variation in 18 homologous gene segments (including nearly 10,000 base pairs) that were selected for maximal phylogenetic informativeness in resolving the hierarchy of early mammalian divergence. Phylogenetic analyses identify four primary superordinal clades: (I) Afrotheria (elephants, manatees, hyraxes, tenrecs, aardvark and elephant shrews); (II) Xenarthra (sloths, anteaters and armadillos); (III) Glires (rodents and lagomorphs), as a sister taxon to primates, flying lemurs and tree shrews; and (IV) the remaining orders of placental mammals (cetaceans, artiodactyls, perissodactyls, carnivores, pangolins, bats and core insectivores). Our results provide new insight into the pattern of the early placental mammal radiation.
The retrosplenial cortex is situated at the crossroads between the hippocampal formation and many areas of the neocortex, but few studies have examined the connections between the hippocampal formation and the retrosplenial cortex in detail. Each subdivision of the retrosplenial cortex projects to a discrete terminal field in the hippocampal formation. The retrosplenial dysgranular cortex (Rdg) projects to the postsubiculum, caudal parts of parasubiculum, caudal and lateral parts of the entorhinal cortex, and the perirhinal cortex. The retrosplenial granular b cortex (Rgb) projects only to the postsubiculum, but the retrosplenial granular a cortex (Rga) projects to the postsubiculu, rostral presubiculum, parasubiculum, and caudal medial entorhinal cortex. Reciprocating projections from the hippocampal formation to Rdg originate in septal parts of CA1, postsubiculum, and caudal parts of the entorhinal cortex, but these are only sparse projections. In contrast, Rgb and Rga receive dense projections from the hippocampal formation. The hippocampal projection to Rgb originates in area CA1, dorsal (septal) subiculum, and post-subiculum. Conversely, Rga is innervated by ventral (temporal) subiculum and postsubiculum. Further, the connections between the retrosplenial cortex and the hippocampal formation are topographically organized. Rostral retrosplenial cortex is connected primarily to the septal (rostrodorsal) hippocampal formation, while caudal parts of the retrosplenial cortex are connected with temporal (caudoventral) areas of the hippocampal formation. Together, the elaborate connections between the retrosplenial cortex and the hippocampal formation suggest that this projection provides an important pathway by which the hippocampus affects learning, memory, and emotional behavior.
Hippocampal area CA1 provides the major cortical output of the hippocampus, but only its projections to the subiculum and lateral septal nucleus are well characterized. The present study reexamines these extrinsic projections by using anterograde and retrograde tracing techniques. Injections of the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) in the septal one-third of CA1 label axons and terminals in subicular, postsubicular, retrosplenial, perirhinal, and entorhinal cortices, lateral septal nucleus, and diagonal band of Broca. The septal CA1 injections also label terminal fields in contralateral CA1, and in contralateral subicular, postsubicular, perirhinal, and entorhinal cortices. Injections into the splenial one-third of CA1 label axons and terminals in subiculum, postsubiculum, ventral area infraradiata, and lateral septal nucleus, but they do not label axons and terminals on the contralateral side of the brain. Injections in the temporal one-third of CA1 label axons and terminals in subicular, parasubicular, entorhinal, and infraradiata cortices, anterior olfactory nucleus, olfactory bulb, lateral septal nucleus, nucleus accumbens, amygdala, and hypothalamus. The temporal CA1 injections label no axons on the contralateral side of the brain. These data demonstrate that CA1 has more widespread projections than previously appreciated, and they provide the first clear evidence that CA1 projects to the contralateral cortex and to the ipsilateral olfactory bulb, amygdala, and hypothalamus. The results also demonstrate a heterogeneity in the efferent projections originating in different septotemporal levels of CA1.
The topographic organization of the projections from the entorhinal cortex to the dentate gyrus in the macaque monkey was studied with anterograde and retrograde tracing methods. Injections of WGA-HRP or the fluorescent retrograde tracers, Fast blue and Diamidino yellow, were placed at various levels along the rostrocaudal axis of the dentate gyrus and hippocampus. In 5 experiments the fluorescent dyes were injected at 2 rostrocaudal levels of the same dentate gyrus. Labeled neurons were observed mainly in layers II and III of the entorhinal cortex, though some were also seen in layers V and VI. The labeled layer II cells resulting from each of the tracer injections were located throughout much of the rostrocaudal extent of the entorhinal cortex, though they tended to have a more limited distribution in the transverse or mediolateral axis. Injections of retrograde tracers located caudally in the dentate gyrus resulted in a rostrocaudally oriented zone of labeled cells that was situated laterally in the entorhinal cortex adjacent to the rhinal sulcus. The zone of labeled cells was not oriented strictly parallel to the rhinal sulcus since at caudal levels it extended medially to encompass the full transverse extent of the most caudal portion of the entorhinal cortex. When injections were placed more rostrally in the dentate gyrus and hippocampus, the rostrocaudally oriented zone of labeled cells was situated more medially in the entorhinal cortex. Anterograde tracing experiments using 3H-amino acid injections into different rostrocaudal and mediolateral positions of the entorhinal cortex confirmed the organization demonstrated by the retrograde tracers and further indicated that the entorhinal fibers terminate in the outer two-thirds of the molecular layer of the dentate gyrus. Unlike in the rat, where the entorhinal termination zone in the molecular layer is clearly bilaminate, projections from all portions of the entorhinal cortex appeared to terminate more diffusely throughout the outer two-thirds of the molecular layer. The results of the present study indicate that rostrocaudally oriented zones of cells that cut across several cytoarchitectonic subdivisions of the entorhinal cortex give rise to topographically organized projections to the dentate gyrus. Cells located laterally in the entorhinal cortex project to caudal levels of the dentate gyrus, whereas progressively more medially situated cells project to progressively more rostral parts of the dentate gyrus.
The longer connections of the entorhinal cortex have been studied autoradiographically in a series of rats, each of which received a small injection of 3H-amino acids in one of the various cytoarchitectonic subfields of the entorhinal cortex. The major findings can be summarized as follows. Whereas the projection of the lateral entorhinal area (LEA) to the dentate gyrus is broad in its longitudinal extent, the medial entorhinal area (MEA), and especially the ventral portion of this zone, projects in a more lamellar fashion. In the transverse plane the LEA preferentially projects to the inner (dorsal) blade of the dentate gyrus, while the MEA innervates both blades equally. Within the radial dimension, the entorhinal cortex projects to the dentate gyrus according to a medial to lateral gradient, with lateral portions of the LEA projecting along the pial surface and successively more medial portions of the entorhinal projecting closer to the granule cells. The commissural entorhinal to dentate projections are similar to the ipsilateral projections in location; however, they are considerably reduced in septotemporal extent and do not arise from cells in the ventral half of either LEA or the intermediate entorhinal area (IEA). The projection of the entorhinal cortex to Ammon's horn reflects the same longitudinal characteristics as the dentate projections. An alvear input which extends only to the pyramidal cells at the CA1-subicular junction was most noticeable at ventral hippocampal levels. Finally the extrahippocampal projections have been analyzed. These arise predominantly from cells in the LEA and project forward along the angular bundle to the piriform and periamygdaloid cortices, as well as the endopiriform nucleus, the lateral, basolateral, and cortical amygdaloid nuclei, the nucleus of the lateral olfactory tract, the olfactory tubercle, the anterior olfactory nucleus, the taenia tecta, and the indusium griseum. These extrinsic projections are to a large extent reciprocal to the major extrinsic inputs to the LEA.
Entorhinal fibers to the fascia dentata originating from layer II stellate neurons are known to terminate exclusively in the outer two thirds of the molecular layer, where they innervate distal dendritic portions of dentate neurons. Using anterograde tracing with Phaseolus vulgaris leucoagglutinin, we unraveled a previously unknown entorhinal projection that directly innervates proximal dendritic portions and somata of granule cells and GABAergic neurons. This projection originates from neurons located in entorhinal layers IV-VI of the medial entorhinal area. These fibers enter the outer two thirds of the molecular layer, traverse the inner molecular layer (IML) and granule cell layer, where they form numerous boutons, and finally arborize subjacent to the granule cells. Correlated light and electron microscopy revealed that the boutons formed by these fibers establish asymmetric synapses on dendrites in the IML, on spines and somata of granule cells, and on spineless dendrites subjacent to the granule cell layer. Postembedding immunogold staining indicates that this entorhino-dentate projection is not GABAergic and that it also terminates on GABAergic inhibitory neurons. These data demonstrate that not all entorhino-dentate fibers display a similar high laminar specificity for the outer molecular layer (OML). Although fibers from the superficial layers of the entorhinal cortex terminate exclusively in the OML, entorhinal fibers arising from deeper layers are not confined to laminar boundaries. Finally, the possibility that these supposedly excitatory entorhinal afferents may monosynaptically activate proximal dendrites and somata of dentate neurons needs to be incorporated into contemporary concepts of the hippocampal network.
We characterized presubicular neurons giving rise to bilateral projections to the medial entorhinal cortex (MEA) of the rat. Retrograde labeling of presubiculo-entorhinal projections with horseradish peroxidase and subsequent GABA immunocytochemistry revealed that 20-30% of the ipsilaterally projecting neurons are GABAergic. No GABAergic projections to the contralateral MEA were observed. GABAergic projection neurons were observed only in the dorsal part of the presubiculum, which, when taking into account the topography of presubicular projections to MEA, indicates that only the dorsal part of MEA receives GABAergic input. The GABAergic projection neurons constitute approximately 30-40% of all GABAergic neurons present in the superficial layers of the dorsal presubiculum. Using double-label fluorescent retrograde tracing, we found that the ipsilateral and contralateral presubiculo-entorhinal projections originate from different populations of neurons. Anterograde labeling of presubiculo-entorhinal projections and electron microscopical analysis of labeled terminals substantiated the presence of a restricted GABAergic presubiculo-entorhinal projection. A small fraction of afferents to only ipsilateral dorsal MEA formed symmetrical synapses with dendritic shafts. No symmetrical synapses on spines were noted. Most afferents to the dorsal part of ipsilateral MEA, as well as all afferents to the remaining ipsilateral and contralateral MEA, formed asymmetrical synapses with both spines and dendritic shafts in an almost equal ratio. Thus, we conclude that the majority of the presubiculo-entorhinal projections exert an excitatory effect on both principal neurons and interneurons. The projections from the dorsal part of the presubiculum comprise a small inhibitory component that originates from GABAergic neurons and targets entorhinal interneurons.
This study investigates direct hippocampal efferent projections to the temporal lobe of the rhesus monkey. Tritiated amino acid injections were placed into the hippocampal formation to identify terminal fields, and complementary fluorescent retrograde tracer injections were placed into the cortex to identify the cells of origin. Tritiated amino acid injections into CA1, prosubicular or subicular subfields produced anterograde label over parts of the parahippocampal gyrus and temporal pole Injections of fluorescent retrograde tracers demonstrated that these projections originate from longitudinal strips of neurons that occupy part of the CA1 subfield as well as from strips of neurons in adjacent prosubicular and subicular subfields. Thus, an injection into area TH of the posterior parahippocampal gyrus labeled neurons in a longitudinal strip of proximal CA1 (i.e., near CA2) as well as a strip in the subiculum; injections into areas TF, TL, 35, or Pro labeled neurons in a longitudinal strip of distal CA1 (i.e., near the prosubiculum) as well as one in the prosubiculum; and an injection into al ea TFO labeled neurons in a longitudinal strip in the middle of CA1. These strips of neurons extended longitudinally throughout the entire rostrocaudal length of the hippocampus. These results demonstrate that, in the monkey, CA1 projections to cortex arise topographically from longitudinally oriented strips of neurons that occupy only a part of the transverse extent of CA1 but that cover most of the anteroposterior extent of the hippocampus. Thus, in the monkey, CA1 is not a single uniform entity and may have a unique role as a source of direct hippocampal projections to the cerebral cortex. (C) 1998 Wiley-Liss, Inc.
The Madagascan lesser hedgehog tenrec was investigated to get insight into the areal evolution of the hippocampal formation in mammals with poorly differentiated brains. The hippocampal subdivisions were analyzed using cyto- and chemoarchitectural criteria; long associational and commissural connections were demonstrated with tracer techniques. The hedgehog tenrec shows a well differentiated dentate gyrus, CA3 and CA1. Their major intrinsic connections lie within the band of variations known from other species. The dentate hilar region shows calretinin-positive mossy cells with extensive projections to the molecular layer. The calbindin- and enkephalin-positive granule mossy fibers form a distinct endbulb and do not invade the CA1 as reported in the erinaceous hedgehog. Isolated granule cells with basal dendrites were also noted. A CA2 region is hard to identify architecturally; its presence is suggested due to its contralateral connections. Subicular and perisubicular regions are clearly present along the dorsal aspects of the hemisphere, but we failed to identify them unequivocally along the caudal and ventral tip of the hippocampus. A temporal portion of the subiculum, if present, differs in its chemoarchitecture from its dorsal counterpart. The perisubicular region, located medially adjacent to the dorsal subiculum may be equivalent to the rat's presubiculum; evidence for the presence of a parasubiculum was rather weak.
2Department of Anatomy and Embryology, Research Institute of Neurosciences, Vrije Universiteit, Graduate School of Neurosciences Amsterdam, Amsterdam, The Netherlands
Retrograde tracing experiments were performed to clarify the topographic projection from medial (area 28m) and intermediate (area 28i) divisions of the entorhinal cortex to the dentate gyrus. Pipets filled with horseradish peroxidase (HRP) were positioned by electrophysiologic guidance at one of several septotemporal (S‐T) levels in the dentate molecular layer of anesthetized rats; the tracer was expelled iontophoretically to minimize its spread. Retrograde labeling of neurons within areas 28m and 28i was analyzed in relation to cytoarchitectonic as well as spatial features of the region (obtained by histologic reconstruction).
Regardless of the S‐T level, ejections of HRP which were confined to the dentate gyrus labeled only layer II neurons of each area. Following septal pole ejections, labeled neurons were located in the posterolateral, extreme posterior, and postermedial parts of both areas 28m and 28i. Mid S‐T ejections produced not only a ventral, but also an anteromedial, shift in the location of entorhinal projection cells; no cells were labeled posterolaterally. After temporal dentate ejections labeled neurons occupied the most anteromedial part of these entorhinal areas. For both areas, but especially for area 28i, convergence of entorhinal efferents upon a single S‐T level in the dentate gyrus occurred from neurons which lay in a dorsoventral (i. e., frontal), and to a lesser extent a rostrocaudal, plane.
The efferent axes of both areas 28m and 28i thus appear to be curved and are therefore best described in three dimensions. The entorhinal axes begin in a posterodorsolateral location, wrap around the posterior cortical convexity, and end in an anteroventromedial position. The results provide a useful map for in situ exploration of entrohinodentate connections in the rat, emphasize the parallel innervation of the dentate gyrus by distinct entorhinal fiber systems, and reflect the importance of the S‐T axis as a framework for interpreting hippocampal organization.
The Projections o the entorhinal and perirhinal cortices to the hippocampus in the cat have been studied with retrograde and anterograde tracing techniques. Retarogradely transported tracers, which were injected at different levels along the septotemporal longitudinal hippocampal axis, result in labeled neurons in superficial entorhinal cortical layers II and III. Occasionally, labeled cells were also observed in the deepest entorhinal layer as well as in the superficial layers of the perirhinal area 35. It could further be shown that labeled neurons located superficially in the entorhinal cortex corresponds to a septotemporal gradient along the longitudinal axis of the hippocampus. This topographical organization of the entorhinal-hippocampal projection system could be substantiated by the use of anterograde tracing of radioactively labeled amino acids. Injections in the entorhinal cortex produce labeled fibers in the hippocampus. Injections in the perirhinal area 35 result also in labeling over the hippocampus, whereas area 36 does not seem to distribute fibers to the hippocampus. As anticipated from the results of the retrograde tracing experiments, injections located laterally, in or close to the posterior rhinal sulcus, produce prominent labeling over the septal pole of the hippocampus, whereas progressively more medially located injections result in progressively more temporally located labeling. This topographical distribution of perforant path fibers along the septotemporal axis of the hippocampus, which is related to a lateromedial axis in the entorhinal cortex, has been observed following injections in the lateral entorhinal area (LEA) as well as in the medial entorhinal area (MEA). The present observations are discussed in regard of other connectional and putative functional differences between the septal and temporal hippocampus.
The present study re-examines, with autoradiographic methods, the pattern of termination of fibers originating from various medio-lateral divisions of the entorhinal cortex on dentate granule cells and on hippocampa pyramidal cells of the rat. Entorhinal fibers were found to distribute in a proximodistal gradient along the dendrites of dentate granule cells, with afferents from the medial entorhinal area terminating in the innermost portion of the entorhinal synaptic field, afferents from the lateral entorhinal area terminating in the most superficial portions of the entorhinal synaptic field, and intermediate medio-lateral locations in the entorhinal area terminating in intermediate loca tions in the entorhinal synaptic zone. A similar graded pattern of termination of medial and lateral entorhinal fibers was apparent in the very slight crossed projection of the entorhinal area to the contralateral dentate gyrus. In addition, a comparable gradient in the pattern of termination of entorhinal fibers was evident in the entorhinal projection field in the distal dendritic regions of the pyramidal cells of regio inferior of the hippocampus proper.
In order to examine whether the entorhinal-hippocampal-entorhinal circuit is reciprocal and topographic, the connections between the subiculum, the CA1 field, and the entorhinal cortex were studied with the carbocyanine dye (Dil), which moves in both retrograde and anterograde directions. We investigated the organization of reciprocal connections revealed by injections of Dil in the entorhinal cortex along the rhinal sulcus. Anterograde fluorescent labeling showed the same pattern reported in previous studies of the dorsal hippocampus. When the injection site of DiI extended into the deep layers (IV–VI) of the same cortical column, the anterograde labeling of the perforant path was accompanied by retrograde labeling of the subicular neurons and the CA1 neurons. The distribution of labeled cells overlapped the distribution of labeled fibers, and the distribution of labeled cells paralleled that of the labeled fibers in the CA1 field. DiI injection into the medial entorhinal cortex revealed fewer retrogradely labeled subicular neurons than injection into the lateral entorhinal cortex, whereas the number of labeled CA1 neurons was not dependent on the injection site. The number of labeled CA1 neurons was always several times greater than the number of subicular neurons. Thus, the amount of information conveyed by the CA1 projection might be higher than that conveyed by the subicular projection. These results indicate that the entorhinal cortex, CA1, and the subiculum are connected reciprocally and topographically. We believe that the framework of the major hippocampal circuit proposed in previous studies should be reconsidered. We propose that the CA1 projection, rather than the subicular projection, is the main projection that feeds back information from the hippocampus to the entorhinal cortex. © 1995 Wiley-Liss, Inc.
The topology of the connections between the entorhinal cortex (EC), area CA1, and the subiculum is characterized by selective and restricted origin and termination along the transverse or proximodistal axis of CA1 and the subiculum. In the present study, we analyzed whether neurons in CA1 and the subiculum that receive EC projections are interconnected and give rise to return projections to EC, such that they terminate deep in the area of origin of the EC-to-CA1/subiculum projections. Both for the lateral and medial subdivision of EC, the projections to CA1/subiculum, as well as the projections from CA1 to the subiculum and back to EC, are rather divergent. Interestingly, we only rarely observed evidence for the presence of “reentry loops,” i.e., cells in layer III of EC giving rise to projections to interconnected neurons in CA1 and the subiculum, while the targeted CA1 neurons also projected back to the deep layers of the area of origin of the pathway in EC. We conclude that although fibers originating from a restricted part of EC distribute extensively in a divergent way along the longitudinal axis of CA1 and the subiculum, only restricted portions of the latter two areas, receiving inputs from the same entorhinal area, are interconnected. Moreover, only a small percentage of the CA1 neurons that project to the correspondingly innervated subicular neurons give rise to projections that return to the deep layers of the originating part of EC. The present findings are taken to indicate that the EC-hippocampal circuitry functionally comprises many parallel-organized specific “reentry loops.” Hippocampus 2001;11:99–104. © 2001 Wiley-Liss, Inc.
The phylogenetic position of the lesser hedgehog tenrec, Echinops telfairi, was studied on the basis of analysis of the concatenated sequences of 12 mitochondrial protein-coding genes. In addition to the tenrec, the analysis included two other representatives of the insectivore order Lipotyphla, the hedgehog and the mole. The eutherian tree was rooted with three non-eutherian mammalian taxa. The analysis joined the tenrec, the African elephant (order Proboscidea) and the aardvark (order Tubulidentata) on a common branch. The three lipotyphlan taxa, the tenrec (Tenrecidae), the mole (Talpidae) and the hedgehog (Erinaceidae) were dispersed in the eutherian tree, demonstrating lipotyphlan polyphyly.
The Madagascan lesser hedgehog tenrec was investigated to get insight into the areal evolution of the hippocampal formation in mammals with poorly differentiated brains. The hippocampal subdivisions were analyzed using cyto- and chemoarchitectural criteria; long associational and commissural connections were demonstrated with tracer techniques. The hedgehog tenrec shows a well differentiated dentate gyrus, CA3 and CA1. Their major intrinsic connections lie within the band of variations known from other species. The dentate hilar region shows calretinin-positive mossy cells with extensive projections to the molecular layer. The calbindin- and enkephalin-positive granule mossy fibers form a distinct endbulb and do not invade the CA1 as reported in the erinaceous hedgehog. Isolated granule cells with basal dendrites were also noted. A CA2 region is hard to identify architecturally; its presence is suggested due to its contralateral connections. Subicular and perisubicular regions are clearly present along the dorsal aspects of the hemisphere, but we failed to identify them unequivocally along the caudal and ventral tip of the hippocampus. A temporal portion of the subiculum, if present, differs in its chemoarchitecture from its dorsal counterpart. The perisubicular region, located medially adjacent to the dorsal subiculum may be equivalent to the rat's presubiculum; evidence for the presence of a parasubiculum was rather weak.
We determined the cortical regions that project directly to the CA1 field of the monkey hippocampus by injecting the retrograde tracers Fast blue, Diamidino yellow or WGA-HRP into CA1 and examining the distribution of labeled cells. In the temporal lobe, large numbers of retrogradely labeled cells were observed in the perirhinal and parahippocampal cortices. Only an occasional labeled cell, however, was observed in the unimodal visual area TE. Additional projections to CA1 arose in the dorsal bank of the superior temporal sulcus, in the rostral and retrosplenial portions of the cingulate cortex, in the agranular insular cortex, and in the caudal orbitofrontal cortex.
Neurons of origin of the perforant path were labeled in entorhinal cortex after exposure of their injured axons to horseradish peroxidase. Almost all neurons in layer II of portions of the entorhinal cortex ipsilateral to the injury site contained diffuse and/or granular label. They included stellates, pyramids, fusiforms, and two cell types whose dendrites arose from two or three characteristic sites and divided into branches which were largely parallel or oblique to the pial surface. These two types predominated in the area lateralis, and stellates with dendrites mainly oriented perpendicular to the pia were the most numerous cells in the area medialis.
Tetramethyl benzidine (TMB) is a presumptively non-carcinogenic chromogen which yields a blue reaction-product at sites of horseradish peroxidase activity. Sixty-six distinct procedures were performed in rats and monkeys in order to determine the optimal incubation parameters for TMB. As a result, a procedure is recommended whose sensitivity greatly surpasses that of a previously described benzidine dihydrochloride method. Indeed, the sensitivity of this new method in demonstrating retrograde transport is markedly superior to that of the previously described benzidine dihydrochloride method. Furthermore, as a consequence of this enhanced sensitivity, many efferent connections of the injection site are also visualized. The injection site demonstrated by this TMB procedure is significantly larger than the one demonstrated when benzidine dihydrochloride or diaminobenzidine is used as a chromogen. Finally, this TMB procedure has been compared to two other TMB procedures and found to provide superior morphology and sensitivity.
The pathway from the entorhinal cortical region to the hippocampal formation has previously been shown to be comprised of two sub‐systems, one of which projects predominantly to the ipsilateral fascia dentata and regio inferior of the hippocampus proper, and a second which projects bilaterally to regio superior. The goal of the present investigation was to determine if these two pathways might originate from different cell populations within the entorhinal area. The cells of origin of these entorhinal pathways were identified by retrograde labeling with horseradish peroxidase (HRP).
Injections which labeled the entorhinal terminal fields in both the fascia dentata and regio superior resulted in the retrograde labeling of two populations of cells in the entorhinal area. Ipsilateral to the injection, HRP reaction product was found in the cells of layer II (predominantly stellate cells) and the cells of layer III (predominantly pyramidal cells). Contralateral to the injections, however, the reaction product was found almost exclusively in the cells of layer III.
With selective injections of the entorhinal terminal field in regio superior, only the cells of layer III were labeled, but these were labeled bilaterally. Selective injection of the entorhinal terminal field in the fascia dentata, however, resulted in the labeling of cells of layer II, but not of layer III, and these cells of layer II were labeled almost exclusively ipsilaterally. A very small number of labeled cells in layer II were, however, found contralateral to the injection as well.
No labeled cells were found either in the presubiculum or parasubiculum following injections of the hippocampal formation. These cell populations were found capable of retrograde transport of HRP, however, since cells in both presubiculum and parasubiculum were labeled following HRP injections into the contralateral entorhinal area.
These results suggest that the projections to the fascia dentata originate from the cells of layer II, while the projections to regio superior originate from the cells of layer III of the entorhinal region proper. The very slight crossed projection from the entorhinal area to the contralateral area dentata probably originates from the small population of cells in layer II which are labeled following HRP injections in the contralateral area dentata.
In this investigation the efferent projections of the entorhinal and prorhinal cortices relative to their sites of termination in the hippocampus and fascia dentata were investigated in the rhesus monkey using experimental silver impregnation methods. Contrary to the often cited observations of Lorente de No, all entorhinal areas, including the laterally lying prorhinal cortex, were found to give rise to the perforant pathway, and furthermore, each cytoarchitectonically defined subarea was found to contribute a unique component. These perforant pathway components terminate in distinct regions of the dendritic zones of the fascia dentata granule cell and the hippocampal pyramidal cell. A previously undescribed projection to the prosubiculum and hippocampus has been found to originate from the prorhinal cortex which forms the medial wall of the rhinal sulcus along the lateral-most portion of the entorhinal cortex in the rhesus monkey. These results, in conjunction with our previous observations regarding differential afferents to the entorhinal cortex, indicate that specific afferent and efferent connections characterize each cytoarchitectonically definable subareas of this periallocortical region. Additionally, they indicate that the perforant pathway might be conceptualized as the final link in a multisynaptic series of connections instrumental in providing the hippocampus with potential modality specific and multimodal input.
The topographic and laminar organization of entorhinal projections to the dentate gyrus, hippocampus, and subicular complex was investigated in the Macaca fascicularis monkey. Injections of 3H-amino acids were placed at various positions within the entorhinal cortex and the distribution of anterogradely labeled fibers and terminals within the other fields of the hippocampal formation was determined. Injections of the retrograde tracers Fast blue, Diamidino yellow, and wheat germ agglutinin-horseradish peroxidase (WGA-HRP) were also placed into the dentate gyrus, hippocampus, and subicular complex, and the distribution of retrogradely labeled cells in the entorhinal cortex was plotted using a computer-aided digitizing system. The entorhinal cortex gave rise to projections that terminated in the subiculum, in the CA1, CA2, and CA3 fields of the hippocampus, and in the dentate gyrus. Projections to the dentate gyrus, and fields CA3 and CA2 of the hippocampus, originated preferentially in layers II and VI of the entorhinal cortex whereas projections to CA1 and to the subiculum originated mainly in layers III and V. Anterograde tracing experiments demonstrated that all regions of the entorhinal cortex project to the outer two-thirds of the molecular layer of the dentate gyrus and to much of the radial extent of the stratum lacunosum-moleculare of CA3 and CA2. While the terminal distributions of entorhinal projections to the dentate gyrus, CA3, and CA2 were not as clearly laminated as in the rat, projections from rostral levels of the entorhinal cortex preferentially innervated the outer portion of the molecular layer and stratum lacunosum-moleculare, whereas more caudal levels of the entorhinal cortex projected relatively more heavily to the deeper portions of the entorhinal terminal zones. The entorhinal projection to the CA1 field of the hippocampus and to the subiculum followed a transverse rather than radial gradient of distribution. Rostral levels of the entorhinal cortex terminated most heavily at the border of CA1 and the subiculum. More caudal levels of the entorhinal cortex projected to progressively more distal portions of the subiculum (towards the presubiculum) and more proximal portions of CA1 (towards CA2). Lateral portions of the entorhinal cortex projected to caudal levels of the recipient fields and more medial parts of the entorhinal cortex projected to progressively more rostral portions of the fields.
In a comparative approach, the anatomical organization of the hippocampus was investigated in two species of megachiropteran bats, the grey-headed flying fox, Pteropus poliocephalus, and the little red flying fox,Pteropus scapulatus. In general, the cytoarchitectonic appearance of the flying fox hippocampus corresponded well with that of other mammals, revealing all major subdivisions. While the dentate fascia was trilaminated with a molecular layer, a granule cell layer, and a distinct polymorphic layer, the ammonic subfields were subdivided into stratum lacunosum moleculare, stratum radiatum, stratum lucidum or mossy fiber layer (restricted to the CA3 region), pyramidal cell layer, and stratum oriens. In Ammon's horn, only subfields CA1, CA3, and CA3c were clearly discernible, whereas the CA2 region remained indistinct. In some cytoarchitectonic features, such as the dispersion of the pyramidal layer in CA1, the megachiropteran hippocampus resembled the corresponding region in primates.
Five characteristic neuronal cell types of the megachiropteran hippocampus were studied in fixed slice preparations after intracellular injection with Lucifer Yellow. While the morphological appearance of CA3 pyramidal cells, horizontal stratum oriens cells, aspiny stellate cells, and mossy cells strongly resembled their counterparts in rodents, primates, and carnivores, granule cells showed an interesting variation from the nonprimate pattern. Like a subset of granule cells in the primate dentate gyrus, 75% of flying fox granule cells revealed 1–2 basal dendrites that ramified in the polymorphic layer. These processes are presumed to form the morphological substrate for recurrent excitation.
Entorhinal afferents to Ammon's horn and the dentate fascia were revealed by employing the method of tract tracing in fixed tissue with the carbocyanine dye Dil. Similar to the rat and cat, but unlike the monkey, the entorhino-dentate projection in the flying fox is bilaminate, with medial entorhinal afferents occupying the middle third of the molecular layer and lateral entorhinal axons ramifying closer to the hippocampal fissure. The remaining inner third of the molecular layer was free from entorhinal input. In contrast to the radial organization of the projection to dentate gyrus and subfield CA3, entorhinal afferents to region CA1 followed a proximo-distal gradient, with medial entorhinal afferents terminating closer to the CA3/CA1 border. Photoconverted preparations were used to determine the trajectory of individual axons. The majority of entorhino-dentate axons traversed the hippocampal fissure, usually close to the crest region, and gave rise to several terminal branches with numerous en passant varicosities. Individual fibers coursed for considerable distances parallel to the granule cell layer, thus presumably activating a large number of postsynaptic granule cells.
It is concluded that in several features of its anatomical organization, the flying fox hippocampus corresponds with that of primates. Therefore, these findings corroborate previous evidence that megachiropteran bats have evolved from an early branch of the primate lineage.
The methods of retrograde fluorescent tracing and anterograde transport of the lectin Phaseolus vulgaris leucoagglutinin (PHA-L) were used to demonstrate the existence of projections from layers IV and VI of the entorhinal area to the hippocampal formation in the rat brain. These two layers of the medial and lateral entorhinal area innervate the molecular layer of Ammon's horn and the area dentata. In the area dentata the projection from layer IV follows that of the perforant path, while that from layer VI innervates the outer two-thirds of the molecular layer, the subgranular zone and the deep part of the hilus of the area dentata.
This is the first in a series of papers investigating the neuroanatomical basis for the interaction of the amygdala and the hippocampal formation in the rhesus monkey. The present report focuses on the complementary and convergent projections of the amygdala and hippocampal formation to the entorhinal and perirhinal cortices. These results were obtained from complementary experiments using injections of radioactively labeled amino acids to identify the anterograde projection patterns and injections of horseradish peroxidase and fluorescent retrograde tracers to confirm the cytoarchitectonic location of the neurons of origin for each projection.
The results of this investigation demonstrate that both the hippocampal formation and the amygdala project to the entorhinal and perirhinal cortices where, with a few exceptions, the major projections of each structure generally are found in different layers of the same cytoarchitecture subdivisions of the entorhinal cortex but overlap in the same layers of the perirhinal cortex. Thus, the lateral and accessory basal nuclei of the amygdala project to layer 3 of areas Pr1, 28I, 28L, and 28S, and the accessory basal nucleus projects strongly to layer 1 of these same areas. In contrast, the subiculum, prosubiculum, and subfield CA1 of the of the hippocampal formation all have a projection to layer 5 of these same areas. In area 28M, the accessory basal nucleus of the amygdala projects to layer 1, while the subiculum, prosubiculum, and subfield CA1 of the hippocampal formation all project to layer 5, and the presubiculum projects to layer 3.
In addition to these complementary laminar projectios, there are a few areas of laminar overlap. Thus in area 28S, both the presubiculum and the CA1 subfield project to layer 3, where the lateral and accessory basal amygdaloid nuclei also project. Similarly, in 28I there is a major projection from the presubiculum and a lighter projection from the subiculum and CA1 to layer 3, where the lateral and accessory basal nuclei also project. There is also extensive laminar overlap in the perirhinal cortex. From the amygdala, the accessory basal nucleus projects to layers 1 and 3 and the lateral basal nucleus to layers 3, 5, and 6, while from the hippocampal formation, the prosubiculum projects to layers 3, 5, and 6, and the CA1 subfield projects to layer 5. This pattern of hippocampal and amygdaloid projections to the entorhinal and perirhinal cortices indicates that these cortices constitute a region of potentially extensive interaction between the amygdala and the hippocampus.
Among the entorhinal neurons that give rise to the perforant path, a small population is sparsely spinous and displays either a multipolar or a horizontal-bipolar dendritic tree. By application of post-embedding immunocytochemistry to neurons of these types with previously identified projections to the hippocampus we found immunoreactivity for gamma-aminobutyric acid (GABA). Thus, it appears that the perforant path not only contains an excitatory but also a small inhibitory component.
Projections from the rat lateral entorhinal cortex (area 28‐l) to the dentate gyrus were traced and then interpreted according to a parcellation scheme that recognized four cytoarchitectonic subdivisions of area 28‐1: areas dorsolateral (dl), ventrolateral (vl), ventromedial (vm), and TR. Following lesions of area 28‐l, anterograde degeneration was traced with the Fink‐Heimer method. In parallel experiments iontophoretic injections of horseradish peroxidase (HRP) were made in the lateral perforant path terminal zone of the dentate molecular layer. Retrograde neuronal labeling patterns within area 28‐l were charted following dorsal, midseptotemporal (mid ST), and ventral dentate injections. In two additional cases HRP was deposited in the ventral subiculum.
Lesions of area dl (which lies entirely on the posterolateral cortex) produced terminal degeneration that was confined to the dorsal one‐half of the dentate gyrus. Lesions involving primarily areas vl and vm (which lie on the posteroinferior cortex) caused a complementary pattern of degeneration; silver grains predominated in the ventral dentate gyrus.
Injections of HRP into the outer dentate molecular layer labeled layer II neurons within area 28‐l. Deposits of HRP in the dorsal one‐third of the dentate gyrus labeled a rostrocaudal strip of neurons within the dorsal one‐third of area dl; no other subdivisions of area 28vl contained labeling. After mid‐ST deposits of HRP, a rostrocaudally oriented strip of labeled cells appeared in the ventral one‐third of area dl. Mid‐ST injections also labeled neurons in the caudolateral quadrant of area vl. Injection of HRP into the ventral dentate gyrus labeled neurons in the caudomedial quadrant of area vl as well as a few neurons in caudal area vm. No labeled cells were ever found in area dl following ventral dentate HRP deposits. Neurons within area TR were never retrogradely labeled from injections of HRP into the dentate perforant path zone. However, ventral subicular injections of HRP labeled a few cells in the posterior part of area TR, as well as hundreds of neurons throughout the rostrocaudal extent of area vl.
The results indicate a highly organized innervation of the dentate gyrus by several subdivisions of area 28‐l. In area dl, rostrocaudal strips of layer II neurons innervate distinct segments of the dorsal ST axis. The posterior half of areas vl and vm innervates the ventral half of the ST axis; a lateromedial gradient there corresponds to increasingly ventral terminations along the dentate ST axis. Finally, in contrast to the other lateral entorhinal subdivisions area TR does not appear to innervate the distal dendrites of dentate granule cells. The implications of this organization for the study of functional diversity along the ST axis are considered.
This paper describes the retrohippocampal projections of individual layers of the lateral entorhinal area as studied by the method of anterograde transport of the lectin Phaseolus vulgaris leucoagglutinin (PHA‐L) in the rat. As in the medial entorhinal area (EA), (Köhler, '86a) PHA‐L injections restricted to individual layers of the lateral EA resulted in labeling of sparse projections to the subicular complex (e.g., subiculum, pre‐ and parasubiculum), whereas projections to the perirhinal area and piriform cortex were prominent. All PHA‐L injections resulted in the labeling of axons projecting longitudinally within the entorhinal area, in both dorsal and ventral directions, albeit the ventral projections were the most prominent ones.
PHA‐L injections into layers 2a and 2b resulted in labeling of axons that could be followed into layers 2a, 2b, and layer 1 on both sides of the injection site. Whereas numerous axons appeared to terminate in layer 2, most fibers ascended into layer 1, where they ran in a medial direction, passing the medial EA, around the parasubiculum to the presubiculum. Numerous axons were found to take a lateral route running past the lateral aspect of the lateral EA to the piriform cortex. The axons running medial in layer 2 did not enter the medial EA.
After PHA‐L injections into layer 3, a large number of axons left the labeled cells on both sides of the injection site, in addition to massive projections that ascended into layers 2b, 2a and 1, just above the injection. Few axons entered layers 2‐6 of the medial EA, but numerous axons innervated layer 1, where they were found to run in the outer half of this layer. The axons running in a medial direction reached layer 1 of the presubiculum, whereas the laterally oriented ones innervated the molecular layer of the piriform cortex. PHA‐L injections into layer 4 resulted in massive labeling of projections to all superficially located layers. Layers 1, and 2b through 5 were innervated lateral to, and layer 4 medial to, the injection site.
After a PHA‐L injection into layer 5, ascending projections were found innervating layers 1 through 4. The terminal fields were found to be particularly dense in the deep parts of layer 3 and in layer 1. This projection expanded laterally, but few projections reached into the medial sector of the lateral EA or into the medial EA.
PHA‐L injections into layer 6 resulted in massive projections to layers 1 through 6 of the lateral EA. Layer 4 was among the most densely innervated layers of the lateral EA after this injection, and the innervation of layers 4 through 6 expanded into the medial EA after layer 6 injections.
Taken together, these findings have shown that: (1) most of the projections from individual layers of the lateral EA are confined within this cortical area or run to extrahippocampal areas by way of the piriform cortex, and (2) with the exception of layers 4 and 6, the lateral EA sends few projections to layers 2 through 6 of the media EA or to the deep layers of the subicular complex.
The connections between the subiculum (SUB) and the entorhinal cortex (EC) were studied in the cat with retrograde and anterograde tracing techniques.
Injections of the retrogradely transported tracer WGA-HRP at different levels along the septotemporal axis of the subiculum result in labeled neurons predominantly in the medial entorhinal cortex (MEA) in the superficial layers II and III. In the deep layers labeled cells are found more widespread over the EC. The superficially located labeled EC neurons are topographically distributed in a lateromedial gradient, which corresponds to a septotemporal gradient along the longitudinal axis of the subiculum. This organization of the EC-SUB projection system could be substantiated by the use of injections anterogradely transported radioactively labeled amino acids in EC.
The SUB to EC projections were investigated with the anterograde transport of WGA-HRP and with radioactively labeled amino acids that were injected at different levels along the septotemporal axis of the subiculum. This results in a patch of anterogradely labeled fibers and terminals in MEA, predominantly in layers II and III, with a wider band of label in the deep layers. Again, a topographical distribution along the lateromedial axis of the EC corresponding to the septotemporal axis of the SUB was observed.
Contralateral reciprocal connections between EC and SUB are also present, and exhibit a similar topographical organization.
The intrahippocampal projections of the subicular complex were studied in the rat with the aid of the anterogradely transported lectin Phaseolus vulgaris leucoagglutinin (PHA‐L). After iontophoretic injections of the lectin into the subiculum proper, presubiculum, or the parasubiculum, axons and terminal processes immunoreactive for PHA‐L were traced to their respective terminal fields within the hippocampal region. After subicular injections PHA‐L‐stained axons could be followed both in a caudal and a rostral direction. The caudally directed fibers course around or within the angular bundle to enter layers VI and V of the medial entorhinal area (MEA). Many fibers penetrate through these layers to terminate in layer IV of the medial and the lateral entorhinal area, which contains a major terminal field of this projection. At more ventral levels, all layers of the entorhinal area are innervated by cells located in the subiculum. Other retrohippocampal projections of the subiculum proper include the deep and the outer two layers of the presubiculum and the medial sector of the parasubiculum, in addition to a massive projection which terminates in the retrosplenial cortex. The rostrally directed projections from the subiculum form a dense innervation of strata lacunosum, radiatum, oriens, and of individual pyramidal cells in the regio superior of the Ammon's horn. All these projections of the subiculum are exclusively ipsilateral.
After injections of PHA‐L into layers n and III of the presubiculum, both ipsi‐ and contralateral projections were traced to the outer three layers of the medial entorhinal area; the lateral entorhinal area apparently receives no innervation from the presubiculum. The innervation of layer III is very dense while in layer II and deep layer I, restricted zones of innervation are found. The fibers reach these layers via the deep layers of the MEA and through the molecular layer after first coursing around the parasubiculum. In addition, a minor projection from the presubiculum to the pyramidal cell layer of the subiculum and to the molecular layer of the hippocampal formation was found.
PHA‐L injections into the parasubiculum labeled fibers that form a dense innervation of layer II in the MEA and the medial part of the lateral EA, and of the most medial sector of layer III in the MEA. Layer I and the superficial part of layer II of the contralateral MEA also contain a dense terminal network after PHA‐L injections into the parasubiculum. The commissural fibers reach the contralateral side via the dorsal hippocampal commissural system, the angular bundle, and finally through all layers of the contralateral EA. With the exception of a small projection to the molecular layer of the hippocampal formation, the parasubiculum appears not to innervate any of the other hippocampal subfields.
Taken together, these studies have shown that each field of the subicular complex has projections restricted to separate layers of the entorhinal area, and thus, that each field participates in its own unique way in the control of entorhinal function.
A detailed study of the origin and termination of the so-called perforant path of the hippocampal region has been made in the rat. This tract connects the entorhinal area with the hippocampus and the fascia dentata. At all dorso-basal levels of the hippocampus, the terminal field has been shown to occupy the middle part of the molecular layer of the fascia dentata and that level of the stratum lacunosum-moleculare of the hippocampal subfield CA3 which is close to the stratum radiatum. The outer and inner parts of the dentate molecular layer, the stratum lacunosum-moleculare of subfield CA1, and the superficial part of the latter layer in CA3 contained fibers en passage only. The origin of the perforant path has been found to be in the medial part of the entorhinal area, from the most dorsal to the most ventral levels. The fibers arise in part at least from cells in layers I—III. Lesions of the lateral part of the entorhinal area leave the perforant path unaffected. A topical organization has been demonstrated: Lesions dorsal in the entorhinal area evoke terminal degeneration in antero-rostral (septal) parts of the hippocampus only. More ventral lesions produce degeneration in increasingly caudal (temporal) segments of the hippocampus. Earlier descriptions of the route traversed by the perforant path have been confirmed. In addition, the axons to the most rostral parts of the hippocampus and fascia dentata have been shown to course superficially in the dorsal and rostral parts of the subiculum and CA1 which form an exposed part of the medial aspect of the hemisphere.
Horseradish peroxidase (HRP) injected into rat hippocampus was transported to the perikarya of neurons which project to the hippocampus. HRP-labeled cells were present in both medial and lateral entorhinal cortex; cells of the medial entorhinal cortex appeared to be topographically organized. The mediaal septal nucleus contained stained cells; its mediaal aspect was labeled after dorsal hippocampal injections, while ventral hippocampal injections resulted in the labeling of more laterally located cells. Stained cells were also observed in the ipsilateral nucleus locus coeruleus, dorsal and median raphe nuclei and areas CA3–4 of the contralateral hippocampus. In additions, cells in the supramammillary region, an area not previously recognized to project to the hippocampus, were labeled. Finally, the mossy fiber terminal zone and the CA3–4 terminal zone in the dentate molecular layer of the ipsilateral hippocampus demonstrated HRP activity, presumably the result of orthograde axonal transport from the injection site.
The hippocampus and fascia dentata receive their major extrinsic input from the entorhinal area through the so-called perforant path. This pathway is now shown to be composed of at least two distinct fiber systems: (1) A medial perforant path coming from the medial part of the entorhinal area and terminating in the middle of the dentate molecular layer and in the deep half of the stratum lacunosum-moleculare of the hippocampal subfield CA3. (2) A lateral perforant path from the lateral part of the entorhinal area to a superficial zone in the dentate molecular layer and to the superfcial part of the stratum lacunosum-moleculare of CA3. This paper deals specifically with the lateral perforant path. A third group of perforant fibers, bing intermediate to the others with regard to both origin and termination has been noticed in one animal.
The fiber-course of the lateral perforant path is found to be identical to that previously described for the medial path. The terminal field is present along the whole axial extent of the hippocampus and fascia dentata, i.e., from the temporal tip to the subsplenial portion.
No sings of degeneration corresponding to the so-called alvear path were observed following lesions of either the medial or the lateral part of the entorhinal cortex.
Terminal degeneration appeared in the molecular layer of the subiculum and CA1 and in the anterior continuation of the hippocampal formation subsequent to lesions including the prepyriform cortex.
After topical injection of horseradish peroxidase into the dorsal hippocampal formation, the distribution of retrogradely labelled neurons of the entorhinal cortex was investigated. The distribution of these cells, which are projecting to the dorsal hippocampal formation, is demonstrated by drawings representing series of frontal, sagittal and horizontal sections and including stereotaxic coordinates. These drawings can be used as a morphological and stereotaxic tool in neurobiological research. Controversial opinions as to the dividing of the entorhinal cortex into subfields are discussed.
An efferent projection from the perirhinal cortex (area 35) in the rat was studied using the anterograde transport of tritiated amino acids as well as horseradish peroxidase (HRP). Following injections of either tracer in either the dorsal or ventral parts of area 35, anterogradely transported label was observed in the molecular layer of the subiculum, adjacent prosubiculum and CAla. Regardless of the dorsoventral level of the injection, the label was most dense at mid-dorsoventral levels of the subiculum and decreased in density in both the septal and temporal directions. Small injections of the same tracers made into the surrounding entorhinal, ectorhinal or prepiriform cortices did not reproduce this pattern. While the entorhinal cortex is the main cortical source of afferent input to the molecular layer of the subiculum as well as the hippocampus and dentate gyrus, the perirhinal cortex appears to constitute a complementary cortical pathway for afferent input to the subiculum.
A peroxidase reaction product that can be easily distinguished from standard diaminobenzidine (DAB) reaction products is needed for pre-embedding electron microscopic double-antibody labelling studies. Benzidine dihydrochloride (BDHC) and gold-substituted silver peroxidase reactions are unsatisfactory for double labelling because they lack sensitivity and reliability and/or compromise ultrastructure. We show here that light and electron microscopic immunocytochemistry can be done with a modification of the tungstate-stabilized tetramethylbenzidine (TMB) reaction (Weinberg and Van Eyck 1991) which yields a crystalline reaction product. With this method, we have obtained excellent immunolabelling for a variety of antigens, including tyrosine hydroxylase, enkephalin, serotonin, Fos protein and retrogradely transported cholera toxin B subunit (CTB). The TMB-tungstate reaction is useful for ultrastructural double labelling because the crystals contrast well with the amorphous product of diaminobenzidine reactions. The TMB-tungstate reaction is more sensitive and reliable for immunocytochemistry than the benzidine dihydrochloride reaction and gives better ultrastructure than the gold-substituted silver peroxidase reaction. We also show that neurons filled with biocytin by intracellular injection can be visualized with TMB-tungstate for either light (LM) or electron (EM) microscopy.
Because convulsive seizures develop very rapidly from kindling sites in the anterior perirhinal cortex, we studied perirhinal efferents by using the anterograde tracer Phaseolus vulgaris leucoagglutinin (PhAL). PhAL injections into the anterior perirhinal cortex labelled a prominent network of fibers within the frontal cortex that was most dense within layers I and II and layer VI. As individual PhAL injection sites within the perirhinal cortex were restricted to one or two adjacent laminae, we were able to determine that layer V was the main source of the perirhinofrontal projection. This was confirmed by frontal cortex injections of the retrograde tracer Fluorogold (FG). Other cortical areas with densely labelled fibers following perirhinal PhAL injections included the agranular insular, infralimbic, orbital, parietal, and entorhinal cortices. Moderate to mild fiber labelling was also noted in the posterior piriform, temporal and occipital cortices, and the claustrum. Subcortical labelling was seen in the nucleus accumbens; fundus striati; basal and lateral amygdala nuclei; the "acoustic thalamus"; and the central grey. Several of these cortical and subcortical projections were bilateral. The different laminar origin of these perirhinal efferents is discussed. These results confirmed our prediction of extensive direct projections from the anterior perirhinal cortex to the frontal cortex in the rat. The significance of this projection is discussed with special reference to the anatomical basis of convulsive limbic seizures.
It has previously been shown that olfactory input to the hippocampus (HPC) is mediated polysynaptically via the lateral entorhinal cortex (LEC), the site of origin of the lateral perforant pathway (LPP). Because previous anatomical studies have shown that olfactory projections also terminate in perirhinal cortex and that this latter region projects directly to the hippocampus, we investigated the role of perirhinal cortex (PRC) in the mediation of the olfactory-hippocampal potential in the rat. Single-pulse stimulation of the lateral olfactory tract (LOT) resulted in a long onset latency (12-20 ms) evoked response in the dentate gyrus of the ipsilateral hippocampal formation. LOT-HPC potentials were rapidly and completely abolished following the microinfusion of procaine into the LPP, suggesting that they are ultimately mediated via this pathway. In support of this finding, current source density analysis indicated that the LOT-HPC response was generated by a current sink at the outer molecular layer of both dorsal and ventral blades of the dentate gurus. Electrolytic and ibotenic acid lesions of PRC produced a significant decrease in the amplitude of LOT-HPC potentials when testing was conducted 4-7 days postlesion. Lesions of LEC produced similar effects and combined lesions of LEC and PRC resulted in an almost complete eradication of the potential, suggesting that parallel entorhinal-hippocampal and perirhinal-hippocampal pathways are involved. These data suggest, therefore, that a portion of the olfactory input to the hippocampus is mediated via polysynaptic connections routed through perirhinal cortex. Because recent research has suggested that PRC plays an important role within the temporal lobe memory system, this connectivity may be important for olfactory memory processes.
It has been suggested that the entorhino-hippocampal circuit is involved in memory formation. To investigate the way that associative memory is elaborated in the circuit, the entorhino-dentate projection was studied with the fluorescent lipophilic tracer Dil. We investigated the projection originating in the dorsal part of the entorhinal cortex by injecting Dil along the rhinal sulcus. Anterograde fluorescent labeling allowed us to examine sections of the sample with a confocal microscope or in wholemount preparations with a fluorescence microscope. Quantitative analysis of the distribution of the Dil-labeled perforant path by confocal microscopy was performed in the septal one third level of the hippocampus. The analysis confirmed that the topographical map along the mediolateral dimension of the entorhinal cortex was transferred to the proximodistal level (from the inner one third to the edge of the molecular layer) of the granule cell dendrites in a gradually shifting manner. The fiber profile observed after lateral entorhinal injection was thick in the suprapyramidal blade and thin in the infrapyramidal blade. The fiber profile observed after medial entorhinal injection was thin in the suprapyramidal blade and thick in the infrapyramidal blade. Fluorescence microscopic observation of wholemount preparations showed that projections from the Dil injection site were distributed wider than half the dentate gyrus in the longitudinal direction. In transverse sections, the range of the labeled fiber distribution was confirmed to be more than two thirds of the dentate gyrus in the same direction regardless of the mediolateral level of the injection site. It has been suggested that the dorsoventral axis of the entorhinal cortex is represented in the septotemporal levels of the dentate gyrus, but that the topographical correspondence might be weak and vague. Although our investigation was limited to the projection from the dorsal entorhinal cortex to the dorsal part of the dentate gyrus, we conclude that the widely distributed projection covers the dentate gyrus in a nontopographic manner.
It is well established that some neuroanatomical tracers may be taken up by local axonal terminals and transported to distant axonal collaterals (e.g., transganglionic transport in dorsal root ganglion cells). However, such collateral-collateral transport of tracers has not been systematically examined in the central nervous system. We addressed this issue with four neuronal tracers--biocytin, biotinylated dextran amine, cholera toxin B subunit, and Phaseolus vulgaris-leucoagglutinin--in the cerebellar cortex. Labelling of distant axonal collaterals in the cerebellar cortex (indication of collateral-collateral transport) was seen after focal iontophoretic microinjections of each of the four tracers. However, collateral-collateral transport properties differed among these tracers. Injection of biocytin or Phaseolus vulgaris-leucoagglutinin in the cerebellar cortex yielded distant collateral labelling only in parallel fibres. In contrast, injection of biotinylated dextran amine or cholera toxin B subunit produced distant collateral labelling of climbing fibres and mossy fibres, as well as parallel fibres. The present study is the first systematic examination of collateral-collateral transport following injection of anterograde tracers in brain. Such collateral-collateral transport may produce false-positive conclusions regarding neural connections when using these tracers for anterograde transport. However, this property may also be used as a tool to determine areas that are innervated by common distant afferents. In addition, these results may indicate a novel mode of chemical communication in the nervous system.