Neurophysiology and neuropharmacology of projections from entorhinal cortex to striatum in the rat

We studied projections from the entorhinal cortex (Ent) to the striatum in anesthetized rats using extra- and intracellular recording and multibarrel iontophoresis. The majority of recordings were from the caudate-putamen (CPu) and core of the nucleus accumbens (AcbC). Electrical stimulation of the Ent evoked synaptic responses in 77% of tests with AcbC neurons and 48% of tests with CPu neurons. In the case of AcbC neurons, 61% of these tests proved to be excitatory and were often followed by inhibitory phases. In contrast to this, only 18% of tests from CPu neurons were excitatory. Intracellular HRP labeling showed that responsive cells were medium spiny neurons.

During iontophoretic experiments, application of the glutamatergic AMPA antagonist DNQX could selectively decrease or block excitatory responses. The GABA_A antagonist bicuculline methiodide increased cellular firing rates and could reveal excitatory responses, suggesting block of a short-latency, short-duration inhibitory component. Ejection of the GABA_B antagonist CGP-35348 could attenuate a later, longer-duration component of inhibition. The results indicate that the Ent excites striatal neurons at least in part by glutamatergic receptors and suggest that this excitation is followed by secondary prolonged GABAergic inhibition.     

GABAergic neurons in the entorhinal cortex project to 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.     

GABAergic projections from the hippocampus to the retrosplenial cortex in the rat

The retrosplenial cortex (RS) in rats has been implicated in a wide range of behaviors, including spatial navigation and memory. Relevant to this, the RS is closely interconnected with the hippocampus by multiple direct and indirect routes. Here, by injecting the retrograde tracer cholera toxin subunit B conjugated with Alexa488 (CTB-Alexa488) in the granular retrosplenial cortex (GRS), we demonstrate a moderately dense non-pyramidal projection from CA1. Neurons are in several layers, but mainly (about 65%) at the border of the stratum radiatum (SR) and stratum lacunosum moleculare (SLM). In particular, by double-labeling with GAD67 or gamma-aminobutyric acid (GABA), we establish that these neurons are GABAergic. Further immunocytochemical screening for calcium-binding proteins, somatostatin (SS) or cholecystokinin (CCK) failed to identify additional neurochemical subgroups; but a small subset (about 14%) is positive for the m2 muscarinic acetylcholine receptor (M2R). Terminations target layer 1 of the GRS, as shown by biotinylated dextran amine (BDA) injections into CA1 and confirmed by a very superficial injection of CTB-Alexa488 in GRS. The superficial injection shows that there is a sparse GABAergic projection from the subiculum to layer 1 of the GRS, in addition to the dense excitatory connections to layer 3. The role of these dual inhibitory-excitatory pathways - within the subiculum, and in parallel from CA1 and the subiculum - remains to be determined, but may be related to synchronized oscillatory activity in the hippocampal complex and GRS, or to the generation of rhythmic activity within the GRS.     

Non-hippocampal cortical projections from the entorhinal cortex in the rat and rhesus monkey

The entorhinal cortices are known to give rise to powerful projections that terminate in the hippocampus and dentate gyrus. Collectively, these link the hippocampal formation to many parts of the cortex and to subcortical structures like the amygdala. Non-hippocampal projections from the entorhinal cortices are understood poorly. Such projections to neighboring temporal areas in the rat and rhesus monkey have been investigated using the autoradiographic and horseradish peroxidase (HRP) tracing procedures. In the rat, HRP-labeled neurons were observed in the intermediate and lateral fields of the entorhinal cortices after injections of temporal cortical areas 20, 35, 36 and 41. They were located predominantly in layers II, III and IV. In the monkey , HRP-labeled neurons were observed in the entorhinal cortices after injections of the rostral superior temporal gyrus (area TA or 22); the temporal polar cortex (area TG or 38); the inferior temporal cortex (area TE or 20); the perirhinal cortex (area 35) and the posterior parahippocampal cortices (areas TF and TH). Unlike the rat, labeled entorhinal neurons in the monkey were located in layer IV. Autoradiographic experiments in the monkey yielded complimentary results. In view of the fact that layer IV of the entorhinal cortex in both the rat and monkey receives a powerful projection from the subicular-CA1 fields of the hippocampal formation, the results imply that this layer mediates an indirect non-fornical connection between the hippocampal formation and the temporal cortex.     

Septotemporal distribution of entorhinal projections to the hippocampus in the cat: electrophysiological evidence

The projection of the entorhinal cortex (EA) to the hippocampus in the cat has been studied by electrophysiological methods. Field potentials elicited by EA stimulation sites were measured in the hippocampus (fascia dentata). Different topographic distributions of the amplitude and of the onset latency of average evoked potentials (AEPs) were obtained depending on the place of the stimulation along a lateromedial axis in the Ea. The lateral EA elicited the largest AEPs in the septal part of the hippocampus and the medial EA evoked maximal responses in the temporal part of the hippocampus, while the intermediate part of the EA evoked the largest AEPs in the splenial (intermediate) part of the hippocampus. Unit activity elicited by hippocampal stimulation was measured in the EA. Analysis of the antidromic unit activity showed that the pathways analysed were monosynaptic. Different conduction velocities to the septal part of the hippocampus were found; the pathway from the lateral EA was the fastest and the pathway from the medial EA the slowest. Assuming that the sites of maximal AEP amplitude correspond to the location of the major synaptic inputs, it can be concluded that the active synaptic inputs arising along a latero-medial axis in the EA are distributed within the hippocampus according to a septotemporal axis, although with some overlap between the different projections. Therefore it may be concluded that the hippocampus is not homogeneous with respect to the inputs from the EA. The present observations are discussed regarding anatomical data and putative functional differences between septal and temporal hippocampus.     

An analysis of entorhinal cortex projections to the dentate gyrus,hippocampus, and subiculum of the neonatal macaque monkey

The entorhinal cortex is the primary interface between the hippocampal formation and neocortical sources of sensory information. Although much is known about the cells of origin, termination patterns, and topography of the entorhinal projections to other fields of the adult hippocampal formation, very little is known about the development of these pathways, particularly in the human or nonhuman primate. We have carried out experiments in which the anterograde tracers ³H‐amino acids, biotinylated dextran amine, and Phaseolus vulgaris leucoagglutinin were injected into the entorhinal cortex in 2‐week‐old rhesus monkeys (Macaca mulatta). We found that the three fiber bundles originating from the entorhinal cortex (the perforant path, the alvear pathway, and the commissural connection) are all established by 2 weeks of age. Fundamental features of the laminar and topographic distribution of these pathways are also similar to those in adults. There is evidence, however, that some of these projections may be more extensive in the neonate than in the mature brain. The homotopic commissural projections from the entorhinal cortex, for example, originate from a larger region within the entorhinal cortex and terminate much more densely in layer I of the contralateral entorhinal cortex than in the adult. These findings indicate that the overall topographical organization of the main cortical afferent pathways to the dentate gyrus and hippocampus are established by birth. These findings add to the growing body of literature on the development of the primate hippocampal formation and will facilitate further investigations on the development of episodic memory. J. Comp. Neurol. 522:1485–1505, 2014. © 2013 Wiley Periodicals, Inc.     

Species differences in mechanosensory projections from the mouth to the ventrobasal thalamus.

To determine whether the largely ipsilateral, inverted representation of mouth parts in the ventrobasal thalamus of sheep was unique to that species or an expansion of a general mammalian pattern, the corresponding thalamic projections were mapped electrophysiologically in a selected series of mammals (oppossums, agoutis, squirrel monkeys, cats, raccoons, and sheep) representing major branches of evolution among therian mammals. In mapping, tungsten microelectrodes were used to record multi-unit discharges in the thalamus in response to mechanical stimulation of oral surfaces. The pattern of projections seen in sheep is not a general mammalian pattern; there is extensive variability among mammals in the laterality and internal orgainzation of the projections from the mouth. In spite of the great variability, the results suggest an hypothesis concerning phylogenetic trends: descendants of palaeoryctoid insectivores (cats, raccoons, and sheep in our sample) have extensive ipsilateral projections from the mouth, in other therian mammals (opossums, agoutis, and squirrel monkeys in our sample) the ipsilateral component is small or absent.     

Disynaptic olfactory input to the hippocampus mediated by stellate cells in the entorhinal cortex

Electrophysiological and anatomical studies indicate functional relationships between the olfactory bulb and the hippocampus, mediated by the lateral olfactory tract and perforant path. Fibres from the lateral olfactory bulb terminate in the molecular layer of the lateral entorhinal cortex, which contains stellate and pyramidal cells that project to the hippocampus. Therefore this study was performed to analyze whether a trineuronal, disynaptic chain connects the olfactory bulb and the hippocampus. In adult rats, Fast Blue was unilaterally injected into the septal hippocampus to label cells of origin of the entorhinohippocampal pathway. Lesions of the ipsilateral olfactory bulb induced anterograde terminal degeneration in the entorhinal cortex of the same animals. Fast Blue labelled, and thus hippocampally projecting entorhinal neurones in fixed vibratome slices of the operated brains were injected with Lucifer Yellow. Most of these neurones were stellate layer II and pyramidal layer III cells; in addition there were some sparsely spinous multipolar cells in layers II and III and sparsely spinous horizontal cells at the layer I/II border. Injected cells were photoconverted and processed for electron microscopy. Olfactory bulb lesions resulted in electron-dense degeneration of abundant terminal boutons in the outer zone of entorhinal layer I. The relative frequency of degenerating boutons decreased towards deeper zones of the layer. In the outer zone, degenerated terminals predominantly contacted dendritic spines. These contacts could be seen on injected stellate cells but not on pyramidal cells. This study shows that the area dentata of the rat is reached by disynaptic afferent input from the olfactory bulb and thus is likely to process olfactory information. Oligosynaptic pathways might provide the hippocampus also with visual and auditory inputs; such fast transmitted polysensory information could be essential for the proposed participation of the hippocampus in attention-related mechanisms.     

Laminar origin and septotemporal distribution of entorhinal and perirhinal projections to the hippocampus in the cat

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.     

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.     

Topographic activation of the medial entorhinal cortex by presubicular commissural projections

Previous investigations have shown that presubicular commissural fibers traveling in the caudal part of the dorsal hippocampal commissure (PSD) selectively activated the dorsalmost portion of the entorhinal cortex (EC), where they discharged perforant path neurons to the dorsal dentate gyrus. The dentate activation was followed by that of the dorsal hippocampus. The aim of the present study was to ascertain whether presubiculum commissural projections traveling in the PSD can also activate ventral levels of the EC and, if so, whether this activation is followed by that of the dentate gyrus-hippocampal system in the ventral hippocampus. The experiments were carried out in adult, anesthetized guinea pigs by field potential analysis. The results showed that presubicular fibers traveling at different PSD loci selectively activated specific EC portions, with caudal fibers activating only the dorsal EC and more rostral fibers activating ventral EC points. The region activated by PSD projections corresponded to the medial EC. Current source-density (CSD) analysis revealed that at both dorsal and ventral EC levels excitatory synaptic potentials followed by neuron discharge were generated in layer II, site of origin of the perforant path to the dentate gyrus. Activation of either dorsal or ventral levels of the EC was followed by activation of the dentate gyrus-hippocampal system in corresponding hippocampal segments. The results provide physiological evidence that the commissural presubicular projections activate the EC in a topographic manner. The massive activation of perforant path neurons at all EC levels suggests that presubicular signals may strongly influence the functions played by the EC-dentate-hippocampal system.     

Age-dependent changes in projections from locus coeruleus to hippocampus dentate gyrus and frontal cortex

Age-dependent changes in noradrenergic innervations of the hippocampal dentate gyrus (DG) and the frontal cortex (FC) have been studied in male F344 rats. The projections from the nucleus locus coeruleus (LC) to DG or FC with advancing age (from 7 to 27 months) in rats have been quantified by electrophysiological and immunohistochemical methods. In the electrophysiological study, we observed that the percentage of LC neurons activated antidromically by electrical stimulation (P-index) of DG or FC decreased with age. We found that the percentage of LC neurons showing multiple antidromic latencies (M-index), which suggests axonal branching of individual LC neurons, increased markedly between 15 and 17 months in DG or FC. In DG, the M-index increased steadily between 15 and 24 months. In contrast, the increased M-index in FC was maintained until 24 months. The increased M-index in both targets declined at 27 months. These results suggest that LC neurons give rise to axonal branching following the loss of projections to DG or FC with age. In the immunohistochemical study, the density of dopamine-beta-hydroxylase-positive axonal varicosities was measured in molecular, granule cell and polymorphic layers of DG. The density in the polymorphic layer significantly decreased in the earlier stage of ageing (7-19 months), whilst the density in the molecular and granule cell layers decreased in the later stage (27 months). These findings suggested that a layer-specific decline occurred with age in the noradrenergic axon terminals in DG.     

Contributions of the hippocampus and entorhinal cortex to rapid visuomotor learning in rhesus monkeys

The hippocampus and adjacent structures in the medial temporal lobe are essential for establishing new associative memories. Despite this knowledge, it is not known whether the hippocampus proper is essential for establishing such memories, nor is it known whether adjacent regions like the entorhinal cortex might contribute. To test the contributions of these regions to the formation of new associative memories, we trained rhesus monkeys to rapidly acquire arbitrary visuomotor associations, i.e., associations between visual stimuli and spatially directed actions. We then assessed the effects of reversible inactivations of either the hippocampus (Experiment 1) or entorhinal cortex (Experiment 2) on the within‐session rate of learning. For comparison, we also evaluated the effects of the inactivations on performance of problems of the same type that had been well learned prior to any inactivations. We found that inactivation of the entorhinal cortex but not hippocampus produced impairments in acquiring novel arbitrary associations. The impairment did not extend to the familiar, previously established associations. These data indicate that the entorhinal cortex is causally involved in establishing new associations, as opposed to retrieving previously learned associations. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.     

Development and topographical organization of projections from the hippocampus and parahippocampus to the retrosplenial cortex

The rat hippocampal formation (HF), parahippocampal region (PHR), and retrosplenial cortex (RSC) play critical roles in spatial processing. These regions are interconnected, and functionally dependent. The neuronal networks mediating this reciprocal dependency are largely unknown. Establishing the developmental timing of network formation will help to understand the emergence of this dependency. We questioned whether the long‐range outputs from HF‐PHR to RSC in Long Evans rats develop during the same time periods as previously reported for the intrinsic HF‐PHR connectivity and the projections from RSC to HF‐PHR. The results of a series of retrograde and anterograde tracing experiments in rats of different postnatal ages show that the postnatal projections from HF‐PHR to RSC display low densities around birth, but develop during the first postnatal week, reaching adult‐like densities around the time of eye‐opening. Developing projections display a topographical organization similar to adult projections. We conclude that the long‐range projections from HF‐PHR to RSC develop in parallel with the intrinsic circuitry of HF‐PHR and the projections of RSC to HF‐PHR.     

MRI measures of entorhinal cortex vs hippocampus in preclinical AD

BACKGROUND: MRI measures of the entorhinal cortex and the hippocampus have been used to predict which nondemented individuals with memory problems will progress to meet criteria for AD on follow-up, but their relative accuracy remains controversial. OBJECTIVES: To compare MRI measures of the entorhinal cortex and the hippocampus for predicting who will develop AD. METHODS: MRI volumes of the entorhinal cortex and the hippocampus were obtained in 137 individuals comprising four groups: 1) individuals with normal cognition both at baseline and after 3 years of follow-up (n = 28), 2) subjects with memory difficulty but not dementia both at baseline and after 3 years of follow-up (n = 73), 3) subjects with memory difficulty at baseline who were diagnosed with probable AD within 3 years of follow-up (n = 21), and 4) patients with mild AD at baseline (n = 16). RESULTS: Measures of both the entorhinal cortex and the hippocampus were different for each of the pairwise comparisons between the groups (p < 0.001) and were correlated with tests of memory (p < 0.01). However, the volume of the entorhinal cortex differentiated the subjects from those destined to develop dementia with considerable accuracy (84%), whereas the measure of the hippocampus did not. CONCLUSION: These findings are consistent with neuropathologic data showing substantial involvement of the entorhinal cortex in the preclinical phase of AD and suggest that, as the disease spreads, atrophic change develops within the hippocampus, which is measurable on MRI.