3D Ultrastructural Study of Synapses in the Human Entorhinal Cortex.

The entorhinal cortex (EC) is a brain region that has been shown to be essential for memory functions and spatial navigation. However, detailed three-dimensional (3D) synaptic morphology analysis and identification of postsynaptic targets at the ultrastructural level have not been performed before in the human EC. In the present study, we used Focused Ion Beam/Scanning Electron Microscopy to perform a 3D analysis of the synapses in the neuropil of medial EC in layers II and III from human brain autopsies. Specifically, we studied synaptic structural parameters of 3561 synapses, which were fully reconstructed in 3D. We analyzed the synaptic density, 3D spatial distribution, and type (excitatory and inhibitory), as well as the shape and size of each synaptic junction. Moreover, the postsynaptic targets of synapses could be clearly determined. The present work constitutes a detailed description of the synaptic organization of the human EC, which is a necessary step to better understand the functional organization of this region in both health and disease.

3D Electron Microscopy Study of Synaptic Organization of the Normal Human Transentorhinal Cortex and Its Possible Alterations in Alzheimer's Disease.

The transentorhinal cortex (TEC) is an obliquely oriented cortex located in the medial temporal lobe and, together with the entorhinal cortex, is one of the first affected areas in Alzheimer's disease (AD). One of the most widely accepted hypotheses is that synaptopathy (synaptic alterations and loss) represents the major structural correlate of the cognitive decline observed in AD. However, very few electron microscope (EM) studies are available; the most common method to estimate synaptic density indirectly is by counting, at the light microscopic level, immunoreactive puncta using synaptic markers. To investigate synaptic morphology and possible alterations related to AD, a detailed three-dimensional (3D) ultrastructural analysis using focused ion beam/scanning EM (FIB/SEM) was performed in the neuropil of Layer II of the TEC in human brain samples from non-demented subjects and AD patients. Evaluation of the proportion and shape of asymmetric synapses (AS) and symmetric synapses (SS) targeting spines or dendritic shafts was performed using 3D reconstructions of every synapse. The 3D analysis of 4722 synapses revealed that the preferable targets were spine heads for AS and dendritic shafts for SS, both in control and AD cases. However, in AD patients, we observed a reduction in the percentage of synapses targeting spine heads. Regarding the shape of synapses, in both control cases and AD samples, the vast majority of synapses had a macular shape, followed by perforated or horseshoe-shaped synapses, with fragmented synapses being the least frequent type. Moreover, comparisons showed an increased number of fragmented AS in AD patients.

Axonal Anatomy Optimizes Spatial Encoding in the Rat Entorhinal-Dentate System: A Computational Study.

OBJECTIVE: The network architecture connecting neural regions is defined by the organization and anatomical properties of the projecting axons, but its contributions to neural encoding and system function are difficult to study experimentally. METHODS: Using a large-scale, spiking neuronal network model of rat dentate gyrus, the role of the anatomy of the entorhinal-dentate axonal projection was evaluated in the context of spatial encoding by incorporating grid cell activity to provide physiological, spatially-correlated input. The dorso-ventral extents of the entorhinal axon terminal fields were varied to generate different feedforward architectures, and the resulting spatial representations and spatial information scores of the network were evaluated. Position was decoded from the population activity using a point process filter to investigate the contributions of network architecture on spatial encoding. RESULTS: The model predicted the emergence of anatomical gradients within the dentate gyrus for place field size and spatial information along its dorso-ventral axis, which were dependent on the extents of the entorhinal axon terminal fields. The decoding results revealed an optimal performance at an axon terminal field extent of 2 mm that lies within the biological range. CONCLUSION: The axonal anatomy mediates a tradeoff between encoding multiple place field sizes or achieving a high spatial information score, and the combination of both properties is necessary to maximize spatial encoding by a network. SIGNIFICANCE: In total, this paper establishes a mechanistic neuronal network model that, in concert with information-theoretic and statistical methods, can be used to investigate how lower level properties contribute to higher level function.

Development of Parvalbumin-Expressing Basket Terminals in Layer II of the Rat Medial Entorhinal Cortex.

Grid cells in layer II of the medial entorhinal cortex (MEC LII) generate multiple regular firing fields in response to the position and speed of an individual within the environment. They exhibit a protracted postnatal development and, in the adult, show activity differences along the dorsoventral axis (DVA). Evidence suggests parvalbumin-positive (PV+) interneurons, most of which are perisomatic-targeting cells, play a crucial role in generation of the hexagonal grid cell activity pattern. We therefore hypothesized that the development and organization of PV+ perisomatic terminals in MEC LII reflect the postnatal emergence of the hexagonal firing pattern and dorsoventral differences seen in grid cell activity. We used immuno-electron microscopy to examine the development of PV+ perisomatic terminals and their target somata within dorsal and ventral MEC LII in rats of postnatal day (P)10, P15, and P30. We demonstrate that in dorsal and ventral MEC LII, the cross-sectional area of somata and number and density of perisomatic PV+ terminals increase between P10 and P15. A simultaneous decrease was observed in cross-sectional area of PV+ terminals. Between P15 and P30, both MEC regions showed an increase in PV+ terminal size and percentage of PV+ terminals containing mitochondria, which may enable grid cell activity to emerge and stabilize. We also report that dorsal somata are larger and apposed by more PV+ terminals than ventral somata at all stages, suggesting a protracted maturation in the ventral portion and a possible gradient in soma size and PV+ basket innervation along the DVA in the adult.

Peculiarities of Cyto- and Chemoarchitectonics of Human Entorhinal Cortex during the Fetal Period.

We studied peculiarities of the structure of human entorhinal cortex at weeks 20-26 of gestation (10 hemispheres). The samples were Nissl-stained and immunohistochemically treated with antibodies to parvalbumin, calretinin, calbindin, and cytoskeleton proteins (MAP2 and N200). 3D-reconstruction of the entorhinal cortex from serial sections was performed, caudomedial and rostrolateral areas were isolated. Parvalbumin+ cells in layer I, discrete distribution of layer II cells with colocalization of MAP2 and calretinin at the border with layer I, and two sublayers Va and Vb with MAP2+ neurons were typical for the caudomedial area. Rostrolateral area was characterized by the homogenous layer II with big amount of cells, high density of MAP2+ neurons only in layer III, and the unique layer V. Reelin+ Cajal-Retzius cells and N200+ fiber plexus in layer I were observed in the caudomedial and rostrolateral areas of the entorhial cortex. Layer IV was represented by a cell-free desiccant.

Axonal synapse sorting in medial entorhinal cortex.

Research on neuronal connectivity in the cerebral cortex has focused on the existence and strength of synapses between neurons, and their location on the cell bodies and dendrites of postsynaptic neurons. The synaptic architecture of individual presynaptic axonal trees, however, remains largely unknown. Here we used dense reconstructions from three-dimensional electron microscopy in rats to study the synaptic organization of local presynaptic axons in layer 2 of the medial entorhinal cortex, the site of grid-like spatial representations. We observe path-length-dependent axonal synapse sorting, such that axons of excitatory neurons sequentially target inhibitory neurons followed by excitatory neurons. Connectivity analysis revealed a cellular feedforward inhibition circuit involving wide, myelinated inhibitory axons and dendritic synapse clustering. Simulations show that this high-precision circuit can control the propagation of synchronized activity in the medial entorhinal cortex, which is known for temporally precise discharges.

Reduced Mitochondrial Activity is Early and Steady in the Entorhinal Cortex but it is Mainly Unmodified in the Frontal Cortex in Alzheimer's Disease.

BACKGROUND: It is well established that mitochondrial damage plays a role in the pathophysiology of Alzheimer's disease (AD). However, studies carried out in humans barely contemplate regional differences with disease progression. OBJECTIVE: To study the expression of selected nuclear genes encoding subunits of the mitochondrial complexes and the activity of mitochondrial complexes in AD, in two regions: the entorhinal cortex (EC) and frontal cortex area 8 (FC). METHODS: Frozen samples from 148 cases processed for gene expression by qRT-PCR and determination of individual activities of mitochondrial complexes I, II, IV and V using commercial kits and home-made assays. RESULTS: Decreased expression of NDUFA2, NDUFB3, UQCR11, COX7C, ATPD, ATP5L and ATP50, covering subunits of complex I, II, IV and V, occurs in total homogenates of the EC in AD stages V-VI when compared with stages I-II. However reduced activity of complexes I, II and V of isolated mitochondria occurs as early as stages I-II when compared with middle-aged individuals in the EC. In contrast, no alterations in the expression of the same genes and no alterations in the activity of mitochondrial complexes are found in the FC in the same series. CONCLUSION: Different mechanisms of impaired energy metabolism may occur in AD, one of them, represented by the EC, is the result of primary and early alteration of mitochondria; the other one is probably the result, at least in part, of decreased functional input and is represented by hypometabolism in the FC in AD patients aged 86 or younger.

A high-resolution study of hippocampal and medial temporal lobe correlates of spatial context and prospective overlapping route memory.

When navigating our world we often first plan or retrieve an ideal route to our goal, avoiding alternative paths that lead to other destinations. The medial temporal lobe (MTL) has been implicated in processing contextual information, sequence memory, and uniquely retrieving routes that overlap or "cross paths." However, the identity of subregions of the hippocampus and neighboring cortex that support these functions in humans remains unclear. The present study used high-resolution functional magnetic resonance imaging (hr-fMRI) in humans to test whether the CA3/DG hippocampal subfield and parahippocampal cortex are important for processing spatial context and route retrieval, and whether the CA1 subfield facilitates prospective planning of mazes that must be distinguished from alternative overlapping routes. During hr-fMRI scanning, participants navigated virtual mazes that were well-learned from prior training while also learning new mazes. Some routes learned during scanning shared hallways with those learned during pre-scan training, requiring participants to select between alternative paths. Critically, each maze began with a distinct spatial contextual Cue period. Our analysis targeted activity from the Cue period, during which participants identified the current navigational episode, facilitating retrieval of upcoming route components and distinguishing mazes that overlap. Results demonstrated that multiple MTL regions were predominantly active for the contextual Cue period of the task, with specific regions of CA3/DG, parahippocampal cortex, and perirhinal cortex being consistently recruited across trials for Cue periods of both novel and familiar mazes. During early trials of the task, both CA3/DG and CA1 were more active for overlapping than non-overlapping Cue periods. Trial-by-trial Cue period responses in CA1 tracked subsequent overlapping maze performance across runs. Together, our findings provide novel insight into the contributions of MTL subfields to processing spatial context and route retrieval, and support a prominent role for CA1 in distinguishing overlapping episodes during navigational "look-ahead" periods.

The role of topography in the transformation of spatiotemporal patterns by a large-scale, biologically realistic model of the rat dentate gyrus.

A large-scale, biologically realistic, computational model of the rat hippocampus is being constructed to study the input-output transformation that the hippocampus performs. In the initial implementation, the layer II entorhinal cortex neurons, which provide the major input to the hippocampus, and the granule cells of the dentate gyrus, which receive the majority of the input, are modeled. In a previous work, the topography, or the wiring diagram, connecting these two populations had been derived and implemented. This paper explores the consequences of two features of the topography, the distribution of the axons and the size of the neurons' axon terminal fields. The topography converts streams of independently generated random Poisson trains into structured spatiotemporal patterns through spatiotemporal convergence achievable by overlapping axon terminal fields. Increasing the axon terminal field lengths allowed input to converge over larger regions of space resulting in granule activation across a greater area but did not increase the total activity as a function of time as the number of targets per input remained constant. Additional simulations demonstrated that the total distribution of spikes in space depends not on the distribution of the presynaptic axons but the distribution of the postsynaptic population. Analyzing spike counts emphasizes the importance of the postsynaptic distribution, but it ignores the fact that each individual input may be carrying unique information. Therefore, a metric should be created that relates and tracks individual inputs as they are propagated and integrated through hippocampus.

Superficially projecting principal neurons in layer V of medial entorhinal cortex in the rat receive excitatory retrosplenial input.

Principal cells in layer V of the medial entorhinal cortex (MEC) have a nodal position in the cortical-hippocampal network. They are the main recipients of hippocampal output and receive inputs from several cortical areas, including a prominent one from the retrosplenial cortex (RSC), likely targeting basal dendrites of layer V neurons. The latter project to extrahippocampal structures but also relay information to the superficial layers of MEC, closing the hippocampal-entorhinal loop. In the rat, we electrophysiologically and morphologically characterized RSC input into MEC and conclude that RSC provides an excitatory input to layer V pyramidal cells. Ultrastructural analyses of anterogradely labeled RSC projections showed that RSC axons in layer V of MEC form predominantly asymmetrical, likely excitatory, synapses on dendritic spines (90%) or shafts (8%), with 2% symmetrical, likely inhibitory, synapses on shafts and spines. The overall excitatory nature of the RSC input was confirmed by an optogenetic approach. Patterned laser stimulation of channelrhodopsin-expressing presynaptic RSC axons evoked exclusively EPSPs in recorded postsynaptic layer V cells. All responding layer V pyramidal cells had an axon extending toward the white matter. Half of these neurons also sent an axon to superficial layers. Confocal imaging of RSC synapses onto MEC layer V neurons shown to project superficially by way of retrogradely labeling from superficial layers confirmed that proximal dendrites of superficially projecting cells are among the targets of inputs from RSC. The excitatory RSC input thus interacts with both entorhinal-cortical and entorhinal-hippocampal circuits.

Recombinant DNA vaccine against neurite outgrowth inhibitors attenuates behavioral deficits and decreases Abeta in an Alzheimer's disease mouse model.

Alzheimer's disease (AD) is a chronic neurodegenerative disease that causes a progressive loss in learning and memory capabilities and eventually results in dementia. The non-renewable nature of neurons in the central nervous system leads to the basic pathological changes that are related to the various behavioral and psychological symptoms of AD. Oligodendrocyte- and myelin-related neurite outgrowth inhibitors (NOIs) tend to hinder the regeneration of neurons. We designed a recombinant DNA vaccine composed of multiple specific inhibitory domains of NOIs. Vaccination induced effective antibodies against the specific domains in the sera of mice treated with a DNA primed-vaccinia virus boost regimen. The vaccine attenuated neuronal degeneration in the mouse brain and protected the model mice from behavioral deficits. Vaccination also decreased the formation of soluble Aß oligomer and amyloid plaques in the co-transgenic mice brain. What's more, astrocytosis in brains of APP/PS1 co-transgenic mice was also relieved. The results suggested that immunotherapy with multiple specific domains of myelin- and oligodendrocyte-related NOIs may be a promising approach for Alzheimer's disease and other degenerative central nervous system diseases.

Presynaptic HCN1 channels regulate Cav3.2 activity and neurotransmission at select cortical synapses.

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are subthreshold, voltage-gated ion channels that are highly expressed in hippocampal and cortical pyramidal cell dendrites, where they are important for regulating synaptic potential integration and plasticity. We found that HCN1 subunits are also localized to the active zone of mature asymmetric synaptic terminals targeting mouse entorhinal cortical layer III pyramidal neurons. HCN channels inhibited glutamate synaptic release by suppressing the activity of low-threshold voltage-gated T-type (Ca(V)3.2) Ca²(+) channels. Consistent with this, electron microscopy revealed colocalization of presynaptic HCN1 and Ca(V)3.2 subunit. This represents a previously unknown mechanism by which HCN channels regulate synaptic strength and thereby neural information processing and network excitability.

Proepileptic influence of a focal vascular lesion affecting entorhinal cortex-CA3 connections after status epilepticus.

In limbic seizures, neuronal excitation is conveyed from the entorhinal cortex directly to CA1 and subicular regions. This phenomenon is associated with a reduced ability of CA3 to respond to entorhinal cortex inputs. Here, we describe a lesion that destroys the perforant path in CA3 after status epilepticus (SE) induced by pilocarpine injection in 8-week-old rats. Using magnetic resonance imaging, immunohistochemical, and ultrastructural analyses, we determined that this lesion develops after 30 minutes of SE and is characterized by microhemorrhages and ischemia. After a longer period of SE, the lesion invariably involves the upper blade of the dentate gyrus. Adult rats treated with subcutaneous diazepam (20 mg kg for 3 days) did not develop the dentate gyrus lesion and had less frequent spontaneous recurrent seizures (p < 0.01). Young (3-week-old) rats rarely (20%) developed the CA3 lesion, and their spontaneous seizures were delayed (p < 0.01). To investigate the role of the damaged CA3 in seizure activity, we reinduced SE in adult and young epileptic rats. Using FosB/DeltaFosB markers, we found induction of FosB/DeltaFosB immunopositivity in CA3 neurons of young but not in adult rats. These experiments indicate that SE can produce a focal lesion in the perforant path that may affect the roles of the hippocampus in epileptic rats.

Agrin expression during synaptogenesis induced by traumatic brain injury.

Interaction between extracellular matrix proteins and regulatory proteinases can mediate synaptic integrity. Previously, we documented that matrix metalloproteinase 3 (MMP-3) expression and activity increase following traumatic brain injury (TBI). We now report protein and mRNA analysis of agrin, a MMP-3 substrate, over the time course of trauma-induced synaptogenesis. Agrin expression during the successful synaptic reorganization of unilateral entorhinal cortical lesion (UEC) was compared with expression when normal synaptogenesis fails (combined fluid percussion TBI and bilateral entorhinal lesion [BEC]). We observed that agrin protein was increased in both models at 2 and 7 days postinjury, and immuohistochemical (IHC) co-localization suggested reactive astrocytes contribute to that increase. Agrin formed defined boundaries for sprouting axons along deafferented dendrites in the UEC, but failed to do so after combined insult. Similarly, Western blot analysis revealed greater increase in UEC agrin protein relative to the combined TBI+BEC model. Both models showed increased agrin transcription at 7 days postinjury and mRNA normalization by 15 days. Attenuation of synaptic pathology with the NMDA antagonist MK-801 reduced 7-day UEC agrin transcript to a level not different from unlesioned controls. By contrast, MK-801 in the combined insult failed to significantly change 7-day agrin transcript, mRNA levels remaining elevated over uninjured sham cases. Together, these results suggest that agrin plays an important role in the sprouting phase of reactive synaptogenesis, and that both its expression and distribution are correlated with extent of successful recovery after TBI. Further, when pathogenic conditions which induce synaptic plasticity are reduced, increase in agrin mRNA is attenuated.

Running induces widespread structural alterations in the hippocampus and entorhinal cortex.

Physical activity enhances hippocampal function but its effects on neuronal structure remain relatively unexplored outside of the dentate gyrus. Using Golgi impregnation and the lipophilic tracer DiI, we show that long-term voluntary running increases the density of dendritic spines in the entorhinal cortex and hippocampus of adult rats. Exercise was associated with increased dendritic spine density not only in granule neurons of the dentate gyrus, but also in CA1 pyramidal neurons, and in layer III pyramidal neurons of the entorhinal cortex. In the CA1 region, changes in dendritic spine density are accompanied by changes in dendritic arborization and alterations in the morphology of individual spines. These findings suggest that physical activity exerts pervasive effects on neuronal morphology in the hippocampus and one of its afferent populations. These structural changes may contribute to running-induced changes in cognitive function.

Plasticity of synaptopodin and the spine apparatus organelle in the rat fascia dentata following entorhinal cortex lesion.

Synaptopodin is an actin-associated molecule essential for the formation of a spine apparatus in telencephalic spines. To study whether synaptopodin and the spine apparatus organelle are regulated under conditions of lesion-induced plasticity, synaptopodin and the spine apparatus were analyzed in granule cells of the rat fascia dentata following entorhinal denervation. Confocal microscopy was employed to quantify layer-specific changes in synaptopodin-immunoreactive puncta densities. Electron microscopy was used to quantify layer-specific changes in spine apparatus organelles. Within the denervated middle and outer molecular layers, the layers of deafferentation-induced spine loss, synaptogenesis, and spinogenesis, the density of synaptopodin puncta and the number of spine apparatuses decreased by 4 days postlesion and slowly recovered in parallel with spinogenesis by 180 days postlesion. Within the nondenervated inner molecular layer, the zone without deafferentation-induced spine loss, a rapid loss of synaptopodin puncta and spine apparatuses was also observed. In this layer, spine apparatus densities recovered by 14 days postlesion, in parallel with plastic remodeling at the synaptic level and the postlesional recovery of granule cell activity. These data demonstrate layer-specific changes in the distribution of synaptopodin and the spine apparatus organelle following partial denervation of granule cells: in the layer of spine loss, spine apparatus densities follow spine densities; in the layer of spine maintenance, however, spine apparatus densities appear to be regulated by other signals.

Feedforward inhibition regulates perirhinal transmission of neocortical inputs to the entorhinal cortex: ultrastructural study in guinea pigs.

The rhinal cortices constitute the main route for impulse traffic to and from the hippocampus. Tracing studies have revealed that the perirhinal cortex forms strong reciprocal connections with the neo- and entorhinal cortex (EC). However, physiological investigations indicate that perirhinal transmission of neocortical and EC inputs occurs with a low probability. In search of an explanation for these contradictory findings, we have analyzed synaptic connections in this network by combining injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHAL) into the neocortex, area 36, or area 35 with gamma-aminobutyric acid (GABA) immunocytochemistry and electron microscopic observations. Within area 36, neocortical axon terminals formed only asymmetric synapses, usually with GABA-negative spines (87%), and less frequently with GABA-immunopositive (GABA+) dendrites (13%). A similar synaptic distribution was observed within area 35 except that asymmetric synapses onto GABA+ dendrites were more frequent (23% of synapses). Examination of the projections from area 36 to area 35 and from both regions to the EC revealed an even higher incidence of asymmetric synapses onto GABA+ dendrites (35 and 32%, respectively) than what was observed in the neocortical projection to areas 36 and 35. Furthermore, some of the neocortical and perirhinal terminals containing PHAL and GABA immunolabeling formed symmetric synapses onto GABA-negative dendrites in their projection sites (neocortex to area 35, 16%; area 36 to 35, 7%; areas 36-35 to EC, 12%). Taken together, these findings suggest that impulse transmission through the rhinal circuit is subjected to strong inhibitory influences, reconciling anatomical and physiological data about this network.

Status epilepticus in 12-day-old rats leads to temporal lobe neurodegeneration and volume reduction: a histologic and MRI study.

PURPOSE: Whether status epilepticus (SE) in early infancy, rather than the underlying illness, leads to temporal lobe neurodegeneration and volume reduction remains controversial. METHODS: SE was induced with LiCl-pilocarpine in P12 rats. To assess acute neuronal damage, brains (five controls, five with SE) were investigated at 8 h after SE by using silver and Fluoro-Jade B staining. Some brains from the early phase were processed for electron microscopy. To assess chronic changes, brains from nine controls and 13 rats with SE at P12 were analyzed after 3 months by using histology and magnetic resonance imaging (MRI). RESULTS: MRI analysis of the temporal lobe of adult animals with SE at P12 indicated that 23% of the rats had hippocampal, 15% had amygdaloid, and 31% had perirhinal volume reduction. Histologic analysis of sections from the MR-imaged brains correlated with the MRI data. Analysis of neurodegeneration 8 h after SE by using both silver and Fluoro-Jade B staining revealed degenerating neurons located in the same temporal lobe regions as the volume reduction in chronic samples. Electron microscopic analysis revealed irreversible ultrastructural alterations. As with the chronic histologic and MRI findings, interanimal variability was seen in the distribution and severity of acute damage. CONCLUSIONS: Our data indicate that SE at P12 can cause acute neurodegeneration in the hippocampus as well as in the adjacent temporal lobe. It is likely that acute neuronal death contributes to volume reduction in temporal lobe regions that is detected with MRI in a subpopulation of animals in adulthood.

Input from the presubiculum to dendrites of layer-V neurons of the medial entorhinal cortex of the rat.

The entorhinal cortex (EC) and the hippocampus are reciprocally connected. Neurons in the superficial layers of EC project to the hippocampus, whereas deep entorhinal layers receive return connections. In the deep layers of EC, pyramidal neurons in layer V possess apical dendrites that ascend towards the cortical surface through layers IIII and II. These dendrites ramify in layer I. By way of their apical dendrites, such layer-V pyramidal cells may be exposed to input destined for the superficial entorhinal neurons. A specific and dense fiber projection that typically ends in superficial entorhinal layers of the medial EC originates in the presubiculum. To investigate whether apical dendrites of deep entorhinal pyramidal neurons indeed receive input from this projection, we injected the anterograde tracer PHA-L in the presubiculum or we lesioned the presubiculum, and we applied in the same experiments the tracer Neurobiotin trade mark pericellularly in layer V of the medial EC of 17 rats. PHA-L labeled presubiculum axons in the superficial layers apposing apical segments of Neurobiotin labeled layer-V cell dendrites were studied with a confocal fluorescence laserscanning microscope. Axons and dendrites were 3D reconstructed from series of confocal images. In cases in which the presubiculum had been lesioned, material was investigated in the electron microscope. At the confocal fluorescence microscope level we found numerous close contacts, i.e. appositions of boutons on labeled presubiculum fibers with identified dendrites of layer-V neurons. In the electron microscope we observed synapses between degenerating axon terminals and spines on dendrites belonging to layer-V neurons. Hence we conclude that layer-V neurons receive synaptic contacts from presubiculum neurons. These findings indicate that entorhinal layer-V neurons have access to information destined for the superficial layers and eventually the hippocampal formation. At the same time, they have access to the hippocampally processed version of that information.