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 (3) H-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.

Impaired in vivo synaptic plasticity in dentate gyrus and spatial memory in juvenile rats induced by prenatal morphine exposure.

Prenatal morphine exposure induces neurobiological changes, including deficits in learning and memory, in juvenile rat offspring. However the effects of this exposure on hippocampal plasticity, which is critical for learning and memory processes, are not well understood. The present study investigates the alterations of spatial memory and in vivo hippocampal synaptic plasticity in juvenile rats prenatally exposed to morphine. On gestation days 11-18, pregnant rats were randomly chosen to be injected twice daily with morphine or saline. Each juvenile offspring (postnatal day 22-31) performed one two-trial Y-maze task to evaluate spatial memory. Afterwards, the in vivo field excitatory postsynaptic potential (fEPSP) and population spike (PS) were recorded in the perforant path dentate gyrus (DG) pathway in the hippocampus. Prenatal morphine exposure reduced depotentiation (DP), but not long-term potentiation (LTP), of the EPSP slope. However, both LTP and DP of the EPSP slope were depressed in prenatal morphine-exposed juvenile offspring. The morphine group also showed poorer performance for the Y-maze task than the control group. Depressed PS LTP, but not EPSP LTP, in the morphine group suggested that prenatal morphine exposure changed GABAergic inhibition, which mediates EPSP-spike potentiation. Then a loss of GABA-containing neurons in the DG area of the morphine group was observed using immunohistochemistry. Taken together, our results suggest that prenatal morphine exposure impairs the juvenile offspring's dentate synaptic plasticity and spatial memory, and that decreased GABAergic inhibition may play a role in these effects. These findings might contribute to an explanation for the cognitive deficits in children whose mothers abuse opiates during pregnancy.

MR features of the developing perianterior horn structure including subcallosal fasciculus in infants and children.

INTRODUCTION: To describe the changes in the magnetic resonance (MR) signal of the perianterior horn structure (PAS) with increasing age, we studied 69 infants and children aged between 3 days and 9.4 years (average: 2.8 years) without any neurological deficits. METHODS: T1- and T2-weighted images and FLAIR (fluid attenuation inversion recovery) images were obtained in the axial plane. Based on a comparison of the intensity of the PAS with that of the cortex in each sequence (T1-WI/FLAIR/T2-WI), we classified the signal-intensity patterns into four types: I, low/low/high; II, low/high/high; III, iso/high/high; IV, high/low/low. RESULTS: Signal-intensity types I, II, III and IV were seen in 22, 8, 17, and 22 subjects, respectively, with younger subjects showing type I or II intensity patterns and older subjects showing type III or IV. In addition, T1-weighted and FLAIR images of subjects with a type I intensity pattern showed a rim of an isointensity component around the PAS that histologically coincided with migrating glial cells. The low-intensity area on FLAIR and T2-WI images of subjects with a type IV intensity pattern may represent myelinated fibers of the subcallosal fasciculus (ScF). CONCLUSION: The intensity of the MR signals of the PAS changes with increasing age, and this change may reflect histological features. A better understanding of these characteristics may help us to clarify myelination abnormalities, particularly those related to the ScF in the frontal lobe in infants and children.

Localization of HCN1 channels to presynaptic compartments: novel plasticity that may contribute to hippocampal maturation.

Increasing evidence supports roles for the current mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, I(h), in hippocampal maturation and specifically in the evolving changes of intrinsic properties as well as network responses of hippocampal neurons. Here, we describe a novel developmental plasticity of HCN channel expression in axonal and presynaptic compartments: HCN1 channels were localized to axon terminals of the perforant path (the major hippocampal afferent pathway) of immature rats, where they modulated synaptic efficacy. However, presynaptic expression and functions of the channels disappeared with maturation. This was a result of altered channel transport to the axons, because HCN1 mRNA and protein levels in entorhinal cortex neurons, where the perforant path axons originate, were stable through adulthood. Blocking action potential firing in vitro increased presynaptic expression of HCN1 channels in the perforant path, suggesting that network activity contributed to regulating this expression. These findings support a novel developmentally regulated axonal transport of functional ion channels and suggest a role for HCN1 channel-mediated presynaptic I(h) in hippocampal maturation.

Long-term depression in identified stellate neurons of juvenile rat entorhinal cortex.

The entorhinal cortex (EC) serves as a gateway to the hippocampus and plays a pivotal role in memory processing in the brain. Superficial layers of the EC convey the cortical input projections to the hippocampus, whereas deep layers of the EC relay hippocampal output projections back to the superficial layers of the EC or to other cortical regions. Whereas the EC expresses long-term potentiation (LTP) and depression (LTD), the underlying cellular and molecular mechanisms have not been determined. Because the axons of the stellate neurons in layer II of the EC form the perforant path that innervates the dentate gyrus granule cells of the hippocampus, we studied the mechanisms underlying the long-term plasticity in identified stellate neurons. Application of high-frequency stimulation (100 Hz for 1 s, repeated 3 times at an interval of 10 s) or forskolin (50 microM) failed to induce significant changes in synaptic strength, whereas application of pairing (presynaptic stimulation at 0.33 Hz paired with postsynaptic depolarization from -60 to -10 mV for 5 min) or low-frequency stimulation (LFS, 1 Hz for 15 min) paradigm-induced LTD. Pairing- or LFS-induced LTDs were N-methyl-D-aspartate receptor-dependent and occluded each other suggesting that they have the similar cellular mechanism. Pairing-induced LTD required the activity of calcineurin and involved AMPA receptor endocytosis that required the function of ubiquitin-proteasome system. Our study provides a cellular mechanism that might in part explain the role of the EC in memory.

Synaptic fatigue at the naive perforant path-dentate granule cell synapse in the rat.

Synaptic activation at low frequency is often used to probe synaptic function and synaptic plasticity, but little is known about how such low-frequency activation itself affects synaptic transmission. In the present study, we have examined how the perforant path-dentate granule cell (PP-GC) synapse adapts to low-frequency activation from a previously non-activated (naive) state. Stimulation at 0.2 Hz in acute slices from developing rats (7-12 days old) caused a gradual depression of the AMPA EPSC (at -80 mV) to about half within 50 stimuli. This synaptic fatigue was unaffected by the NMDA and metabotropic glutamate (mGlu) receptor antagonists d-AP5 and LY-341495. A smaller component of this synaptic fatigue was readily reversible when switching to very low-frequency stimulation (0.033-0.017 Hz) and is attributed to a reversible decrease in release probability, which is probably due to depletion of readily releasable vesicles. Thus, it was expressed to the same extent by AMPA and NMDA EPSCs, and was associated with a decrease in quantal content (measured as 1/CV(2)) with no change in the paired-pulse ratio. The larger component of the synaptic fatigue was not readily reversible, was selective for AMPA EPSCs and was associated with a decrease in 1/CV(2), thus probably representing silencing of AMPA signalling in a subset of synapses. In adult rats (> 30 days old), the AMPA silencing had disappeared while the low-frequency depression remained unaltered. The present study has thus identified two forms of synaptic plasticity that contribute to fatigue of synaptic transmission at low frequencies at the developing PP-GC synapse; AMPA silencing and a low-frequency depression of release probability.

Bcl-2 overexpression does not promote axonal regeneration of the entorhino-hippocampal connections in vitro after axotomy.

CNS lesions trigger cell death in injured neurons and glia. Genes of the bcl-2 family play crucial roles in the control of apoptosis and cell survival in the CNS. Recently, it has been suggested that overexpression of bcl-2 induces axonal elongation and regeneration in vitro and in vivo. Here, we analyze the regenerative potential of bcl-2 overexpression in the axotomized entorhino-hippocampal connection in organotypic slice cocultures. Our results show that in slice cocultures from bcl-2-overexpressing mice, there is a decrease in the number of dead neurons in the entorhinal cortex. In addition, axonal regeneration is not enhanced after axotomy. Thus, in the entorhino-hippocampal formation in vitro, bcl-2 overexpression rescues neurons from axotomy-induced cell death but fails to enhance the regeneration of the entorhino-hippocampal connection.

Projections of the pontine nuclei to the cochlear nucleus in rats.

In the cochlear nucleus, there is a magnocellular core of neurons whose axons form the ascending auditory pathways. Surrounding this core is a thin shell of microneurons called the granule cell domain (GCD). The GCD receives auditory and nonauditory inputs and projects in turn to the dorsal cochlear nucleus, thus appearing to serve as a central locus for integrating polysensory information and descending feedback. Nevertheless, the source of many of these inputs and the nature of the synaptic connections are relatively unknown. We used the retrograde tracer Fast Blue to demonstrate that a major projection arises from the contralateral pontine nuclei (PN) to the GCD. The projecting cells are more densely located in the ventral and rostral parts of the PN. They also are clustered into a lateral and a medial group. Injections of anterograde tracers into the PN labeled mossy fibers in the contralateral GCD. The terminals are confined to those parts of the GCD immediately surrounding the ventral cochlear nucleus. There is no PN projection to the dorsal cochlear nucleus. These endings have the form of bouton and mossy fiber endings as revealed by light and electron microscopy. The PN represent a key station between the cerebral and cerebellar cortices, so the pontocochlear nucleus projection emerges as a significant source of highly processed information that is introduced into the early stages of the auditory pathway. The cerebropontocerebellar pathway may impart coordination and timing cues to the motor system. In an analogous way, perhaps the cerebropontocochlear nucleus projection endows the auditory system with a timing mechanism for extracting temporal information.

Selective cholinergic denervation inhibits expression of long-term potentiation in the adult but not infant rat hippocampus.

The present study assessed the role of the cholinergic systems on the expression of perforant path long-term potentiation (LTP) in rat hippocampal slices from the infant and adult brain. To denervate the cholinergic systems, 192 IgG--saporin was injected into the lateral ventricle of the infant (2-weeks-old) and adult (6-weeks-old) rat brain. There, choline acetyltransferase-immunoreactive fibers were barely detectable 2 weeks and 2 months after injection for both the groups. For the infant rats, perforant path LTP was not affected by selective cholinergic denervation; the probability of LTP development was 0.83 (five out of six slices) and 0.78 (seven out of nine slices) at 2 weeks and 2 months later in 192 IgG--saporin-treated slices, as compared with 0.83 at each period in control saline-treated slices. In contrast, the expression of the LTP was blocked by selective cholinergic denervation for the adult rats; the probability of LTP development was 0 (zero out of 10 slices) and 0.38 (three out of eight slices) at 2 weeks and 2 months later in 192 IgG--saporin-treated slices, as compared with 0.8 (eight out of 10 slices) and 0.83 (five out of six slices) at each period in control saline-treated slices. The results of the present study thus suggest that the cholinergic systems play a crucial role in the expression of LTP in the adult brain and that the denervated systems in the infant brain could be compensated by the sprouting of non-cholinergic fibers.

Development of the entorhino-hippocampal projection: guidance by Cajal-Retzius cell axons.

The entorhinal cortex gives rise to a massive projection to the hippocampus and fascia dentata. In the rat, this projection forms early in development with first entorhinal axons reaching the hippocampus around embryonic day (E) 17. From the very beginning, the entorhinal axons recognize their appropriate termination zones in the hippocampus proper and fascia dentata, i.e., stratum lacunosum-moleculare and the outer molecular layer of the dentate. This is remarkable, because at the time of entorhinal fiber ingrowth, the definitive target cells of entorhinal axons, pyramidal cells and granule cells, are not yet fully developed, and the majority of their distal dendritic tips have not yet reached these layers. This raises the question as to the cellular and molecular signals guiding the entorhinal axons to and keeping them in their target layers. Here we hypothesize that early generated Cajal-Retzius (CR) cells located in stratum lacunosum-moleculare and the outer molecular layer of the dentate, and in particular their axons projecting to the entorhinal cortex, provide a template that is used by the entorhinal axons to find their target layers in the hippocampus.

Outgrowth-promoting molecules in the adult hippocampus after perforant path lesion.

Lesion-induced neuronal plasticity in the adult central nervous system of higher vertebrates appears to be controlled by region- and layer-specific molecules. In this study we demonstrate that membrane-bound hippocampal outgrowth-promoting molecules, as present during the development of the entorhino-hippocampal system and absent or masked in the adult hippocampus, appear 10 days after transection of the perforant pathway. We used an outgrowth preference assay to analyse the outgrowth preference of axons from postnatal entorhinal explants on alternating membrane lanes obtained from hippocampus deafferented from its entorhinal input taken 4, 10, 20, 30 and 80 days post-lesion and from adult control hippocampus. Neurites from the entorhinal cortex preferred to extend axons on hippocampal membranes disconnected from their entorhinal input for 10 days in comparison with membranes obtained from unlesioned adult animals. Membranes obtained from hippocampi disconnected from their entorhinal input for 10 days were equally as attractive for growing entorhinal cortex (EC) axons as membranes from early postnatal hippocampi. Further analysis of membrane properties in an outgrowth length assay showed that entorhinal axons extended significantly longer on stripes of lesioned hippocampal membranes in comparison with unlesioned hippocampal membranes. This effect was most prominent 10 days after lesion, a time point at which axonal sprouting and reactive synaptogenesis are at their peak. Phospholipase treatment of membranes obtained from unlesioned hippocampi of adult animals strongly promoted the outgrowth length of entorhinal axons on these membranes but did not affect their outgrowth preference for deafferented hippocampal membranes. Our results indicate that membrane-bound outgrowth-promoting molecules are reactivated in the adult hippocampus following transection of the perforant pathway, and that neonatal entorhinal axons are able to respond to these molecules. These findings support the hypothesis of a temporal accessibility of membrane-bound factors governing the layer-specific sprouting of remaining axons following perforant path lesion in vivo.

A role for the Eph ligand ephrin-A3 in entorhino-hippocampal axon targeting.

Neurons of layers II and III of the entorhinal cortex constitute the major afferent connection of the hippocampus. The molecular mechanisms that target the entorhinal axons to specific layers in the hippocampus are not known. EphA5, a member of the Eph receptor family, which has been shown to play critical roles in axon guidance, is expressed in the entorhinal cortex, the origin of the perforant pathway. In addition, ligands that interact with EphA5 are expressed in distinct hippocampal regions during development of the entorhino-hippocampal projection. Of these ligands, ephrin-A3 mRNA is localized both in the granular cell layer of the dentate gyrus and in the pyramidal cell layer of the cornu ammonis, whereas ephrin-A5 mRNA is only expressed in the pyramidal cell layer of the cornu ammonis. In the dentate gyrus, the ligand protein is not present in the termination zone of the entorhinal efferents (the outer molecular layer of the dentate gyrus) but is concentrated in the inner molecular layer into which entorhinal efferents do not grow. We used outgrowth and stripe assays to test the effects of ephrin-A3 and ephrin-A5 on the outgrowth behavior of entorhinal axons. This functional analysis revealed that entorhinal neurites were repelled by ephrin-A3 but not by ephrin-A5. These observations suggest that ephrin-A3 plays an important role in the layer-specific termination of the perforant pathway and that this ligand may interact with the EphA5 receptor to restrict entorhinal axon terminals in the outer molecular layer of the dentate gyrus.

Development of afferent fiber lamination in the infrapyramidal blade of the rat dentate gyrus.

In the rat dentate gyrus, the lateral perforant path, the medial perforant path, and the major part of the hilar projection to the molecular layer share the lamination domain, mainly in the outer one-third of the molecular layer, the middle one-third, and the inner one-third, respectively. To reveal the order of the afferent fiber lamination and to have an indication of how the synaptic sites on dendrites are determined, we investigated the ontogeny of afferent fiber lamination in the dorsal hippocampus by injecting 1, 1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) into the entorhinal cortex and hippocampus in vivo. Fibers from the contralateral hilar region were found under the pia mater of the infrapyramidal blade at postnatal day 3 (P3), whereas the entorhinal afferent fibers were absent in the infrapyramidal blade. Then the medial and the lateral perforant path appeared under the pia mater in the infrapyramidal blade as riding on top of the preexisting laminae by P7 and by P11, respectively. Based on the established knowledge that most entorhinal layer II neurons simultaneously innervate both the suprapyramidal blade and infrapyramidal blade by branching, it is assumed that the medial and lateral perforant path in the suprapyramidal blade await an appropriate timing for sprouting of interstitial branches into the infrapyramidal blade. The granule cells in the infrapyramidal blade had dendritic growth cones by P11. Calretinin immunohistochemistry revealed Cajal-Retzius cells in the infrapyramidal blade even at P14. Under the pia mater, axon growth cones of ingrowing afferent fibers may interact with the dendritic growth cones or the Cajal-Retzius cells, and determines the synaptic sites on the granule cell dendrites.