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Previously we showed that the vitamin A metabolite all-trans retinoic acid (atRA) induces synaptic plasticity in acute brain slices prepared from the mouse and human neocortex (Lenz et al., 2021). Depending on the brain region studied, distinct effects of atRA on excitatory and inhibitory neurotransmission have been reported. Here, we used intraperitoneal injections of atRA (10 mg/kg) in adult C57BL/6J mice to study the effects of atRA on excitatory and inhibitory neurotransmission in the mouse fascia dentata—a brain region implicated in memory acquisition. No major changes in synaptic transmission were observed in the ventral hippocampus while a significant increase in both spontaneous excitatory postsynaptic current frequencies and synapse numbers were evident in the dorsal hippocampus 6 hr after atRA administration. The intrinsic properties of hippocampal dentate granule cells were not significantly different and hippocampal transcriptome analysis revealed no essential neuronal changes upon atRA treatment. In light of these findings, we tested for the metaplastic effects of atRA, that is, for its ability to modulate synaptic plasticity expression in the absence of major changes in baseline synaptic strength. Indeed, in vivo long-term potentiation (LTP) experiments demonstrated that systemic atRA treatment improves the ability of dentate granule cells to express LTP. The plasticity-promoting effects of atRA were not observed in synaptopodin-deficient mice, therefore, extending our previous results regarding the relevance of synaptopodin in atRA-mediated synaptic strengthening in the mouse prefrontal cortex. Taken together, our data show that atRA mediates synaptopodin-dependent metaplasticity in mouse dentate granule cells.

Lewy body disorders are characterized by the emergence of α-synucleinopathy in many parts of the central and peripheral nervous systems, including in the telencephalon. Dense α-synuclein+ pathology appears in regio inferior of the hippocampus in both Parkinson’s disease and dementia with Lewy bodies and may disturb cognitive function. The preformed α-synuclein fibril model of Parkinson’s disease is growing in use, given its potential for seeding the self-propagating spread of α-synucleinopathy throughout the mammalian brain. Although it is often assumed that the spread occurs through neuroanatomical connections, this is generally not examined vis-à-vis the uptake and transport of tract-tracers infused at precisely the same stereotaxic coordinates. As the neuronal connections of the hippocampus are historically well defined, we examined the first-order spread of α-synucleinopathy three months following fibril infusions centered in the mouse regio inferior (CA2 + CA3), and contrasted this to retrograde and anterograde transport of the established tract-tracers FluoroGold and biotinylated dextran amines (BDA). Massive hippocampal α-synucleinopathy was insufficient to elicit memory deficits or loss of cells and synaptic markers in this model of early disease processes. However, dense α-synuclein+ inclusions in the fascia dentata were negatively correlated with memory capacity. A modest compensatory increase in synaptophysin was evident in the stratum radiatum of cornu Ammonis in fibril-infused animals, and synaptophysin expression correlated inversely with memory function in fibril but not PBS-infused mice. No changes in synapsin I/II expression were observed. The spread of α-synucleinopathy was somewhat, but not entirely consistent with FluoroGold and BDA axonal transport, suggesting that variables other than innervation density also contribute to the materialization of α-synucleinopathy. For example, layer II entorhinal neurons of the perforant pathway exhibited somal α-synuclein+ inclusions as well as retrogradely labeled FluoroGold+ somata. However, some afferent brain regions displayed dense retrograde FluoroGold label and no α-synuclein+ inclusions (e.g. medial septum/diagonal band), supporting the selective vulnerability hypothesis. The pattern of inclusions on the contralateral side was consistent with specific spread through commissural connections (e.g. stratum pyramidale of CA3), but again, not all commissural projections exhibited α-synucleinopathy (e.g. hilar mossy cells). The topographical extent of inclusions is displayed here in high-resolution images that afford viewers a rich opportunity to dissect the potential spread of pathology through neural circuitry. Finally, the results of this expository study were leveraged to highlight the challenges and limitations of working with preformed α-synuclein fibrils.

Because of the central role of the hippocampus in representing spatial and temporal details of experience, comparative studies of its volume and structure are relevant to understanding the evolution of representational memory across species. The hippocampal formation, however, is organized into separate anatomical subregions with distinct functions, and little is known about the evolutionary diversification of these subregions. We investigate relative volumetric changes in hippocampal subregions across a large sample of primate species. We then compare the evolution of the hippocampal formation to the neocortex. Results across hippocampal subregions indicate that, compared to strepsirrhines, the anthropoid lineage displays a decrease in relative CA3, fascia dentata, subiculum, and rhinal cortex volume in tandem with an increase in relative neocortical volume. These findings indicate that hippocampal function in anthropoids might be substantially augmented by the executive decision-making functions of the neocortex. Humans are found to have a unique cerebral organization combining increased relative CA3, subiculum, and rhinal cortex with increased relative neocortical volumes, suggesting that these regions may play a role in behaviors that are uniquely specialized in humans.

We studied the role of neurotransmitter signaling mediated by synaptic vesicles in the formation of aberrant functional connections between fascia dentata grafts and the somatosensory neocortex in adult rats. Quantitative analysis of the different populations of synaptic vesicles in the ectopic giant axonal endings of granular neurons was performed and the results were compared with the normal. Two pools of small clear vesicles (rapidly releasable pool and pool of reserve vesicles circulating in the active zone) and one pool of large dense-core vesicles were analyzed. Significant differences from the control suggest that synaptic integration of the transplants into the recipient brain is coordinated by transsynaptic signaling and mediated by different populations of synaptic vesicles.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the X-linked gene MECP2. Girls with RTT show dramatic changes in brain function, but relatively few studies have explored the structure of neural circuits. Examining two mouse models of RTT (Mecp2B and Mecp2J), we previously documented changes in brain anatomy. Herein, we use confocal microscopy to study the effects of MeCP2 deficiency on the morphology of dendrites and axons in the fascia dentata (FD), CA1 area of hippocampus, and motor cortex following Lucifer yellow microinjection or carbocyanine dye tracing. At 3 weeks of age, most (33 of 41) morphological parameters were significantly altered in Mecp2B mice; fewer (23 of 39) were abnormal in Mecp2J mice. There were striking changes in the density and size of the dendritic spines and density and orientation of axons. In Mecp2B mice, dendritic spine density was decreased in the FD (approximately 11%), CA1 (14-22%), and motor cortex (approximately 16%). A decreased spine head size (approximately 9%) and an increased spine neck length (approximately 12%) were found in Mecp2B FD. In addition, axons in the motor cortex were disorganized. In Mecp2J mice, spine density was significantly decreased in CA1 (14-26%). In both models, dendritic swelling and elongated spine necks were seen in all areas studied. Marked variation in the type and extent of changes was noted in dendrites of adjacent neurons. Electron microscopy confirmed abnormalities in dendrites and axons and showed abnormal mitochondria. Our findings document widespread abnormalities of dendrites and axons that recapitulate those seen in RTT.

– This article deals with the efferent connections of the hippocampal region, and considers the functional implications of these projections. The hippocampus receives indirect sensory information from many structures in the brain. Most of these afferents terminate in the superficial layers (I-III) of the entorhinal area. From cells in layers II and III of entorhinal area projections arise which terminate in hippocampus and fascia dentata, the perforant paths. Internal hippocampal projections constitute a unidirectional system of connections which project back to the deep layers (IV-VI) of the entorhinal area. The efferents from these layers may be divided into cortically terminating efferents, which originate from layer IV, and subcortically terminating efferents, which originate from layers V and VI. The cortically terminating projections end in regions of association cortex, and in limbic cortex. The subcortical projections terminate in the caudate, the putamen, and the accumbens nucleus. A hippocampal influence on these subcortical structures may seem surprising, but is logical when one takes into consideration reports on the functional associations between the striatum and the hippocampus. The findings suggest that the role of the hippocampus may be to gather input from all sensory modalities, to assign priorities continuously between these inputs, and as a result, to modify behavior via its influence on subcortical motor centres.

The chondroitin sulfate proteoglycan NG2 is a component of the glial scar following brain injury. Because of its growth inhibiting properties, it has been suggested to impede axonal regeneration. To study whether NG2 could also regulate axonal growth in denervated brain areas, changes in NG2 were studied in the rat fascia dentata following entorhinal deafferentation and were correlated with the post-lesional sprouting response. Laser microdissection was employed to selectively harvest the denervated molecular layer and combined with quantitative RT-PCR to measure changes in NG2 mRNA (6 h, 12 h, 2 days, 4 days, 7 days post-lesion). This revealed increases of NG2 mRNA at day 2 (2.5-fold) and day 4 (2-fold) post-lesion. Immunocytochemistry was used to detect changes in NG2 protein (1 days, 4 days, 7 days, 10 days, 14 days, 30 days, 6 months post-lesion). NG2 staining was increased in the denervated outer molecular layer at day 1 post-lesion, reached a maximum 10 days post-lesion, and returned to control levels thereafter. Electron microscopy revealed NG2 immunoprecipitate on glial surfaces and in the extracellular matrix around neuronal profiles, indicating that NG2 is secreted following denervation. Double labeling of NG2-immunopositive cells with markers for astrocytes, microglia/macrophages, and mature oligodendrocytes suggested that NG2 cells are a distinct glial subpopulation before and after entorhinal deafferentation. BrdU labeling revealed that some of the NG2-positive cells are generated post-lesion. Taken together, our data revealed a layer-specific upregulation of NG2 in the denervated fascia dentata that coincides with the sprouting response. This suggests that NG2 could regulate lesion-induced axonal growth in denervated areas of the brain.

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.

Entorhinal cortex lesion (ECL) partially denervates the fascia dentata of the hippocampus. This is said to induce the sprouting of intact fibers from neighboring layers that invade the zone of the degenerating axons. However, recent studies using anterograde tracing failed to demonstrate sprouting across laminar boundaries. Sprouting does occur, but it mainly involves unlesioned fiber systems terminating within the layer of fiber degeneration. It is now of interest to identify the cues that could underlie this layer-specific sprouting response. Since extracellular matrix (ECM) molecules delineate boundaries of axonal growth during development, it was tested whether these molecules play a similar role during the sprouting process following ECL. After ECL, reactive astrocytes rapidly synthesize and secrete growth-inhibiting ECM molecules, such as tenascin-C and the chondroitin sulfate proteoglycan neurocan, into the ECM of the outer molecular layer. These molecules form a sharp border against the nondenervated inner molecular layer. This pattern of ECM molecule expression may contribute to the layer-specific sprouting response of surviving afferents after ECL: axons trying to grow into the denervated outer molecular layer, for example, from the inner molecular layer, would be deflected by a growth-inhibiting ECM barrier.

The Ts65Dn mouse is a genetic model for Down syndrome. Although this mouse shows abnormalities in cognitive function that implicate hippocampus as well as marked deficits in hippocampal long-term potentiation, the structure of the hippocampus has been little studied. We characterized synaptic structure in Ts65Dn and control (2N) mice, studying the hippocampus (fascia dentata, CA1) as well as the motor and somatosensory cortex, entorhinal cortex, and medial septum. Confocal microscopy was used to examine immunostained presynaptic boutons and to detail the structure of dendrites after Lucifer yellow microinjection. Both presynaptic and postsynaptic elements were significantly enlarged in Ts65Dn in all regions examined. The changes were detected at the youngest age examined (postnatal day 21) and in adults. In studies detailing the changes in fascia dentata and motor cortex, the enlargement of spines affected the entire population, resulting in the presence of spines whose volume was greatly increased. Electron microscopy confirmed that boutons and spines were enlarged and demonstrated abnormalities in the internal membranes of both. In addition, spine density was decreased on the dendrites of dentate granule cells, and there was reorganization of inhibitory inputs, with a relative decrease in inputs to dendrite shafts and an increase in inputs to the necks of spines. Taken together, the findings document widespread abnormalities of synaptic structure that recapitulate important features seen in Down syndrome. They establish the Ts65Dn mouse as a model for abnormal synapse structure and function in Down syndrome and point to the importance of studies to elucidate the mechanisms responsible for synapse enlargement. J. Comp. Neurol. 480:281–298, 2004. © 2004 Wiley-Liss, Inc.

Reactive axonal sprouting occurs in the fascia dentata after entorhinal cortex lesion. This sprouting process has been described extensively in the rat, and plasticity-associated molecules have been identified that might be involved in its regulation. To demonstrate causal relationships between these candidate molecules and the axonal reorganization process, it is reasonable to analyze knockout and transgenic animals after entorhinal cortex lesion, and because gene knockouts are primarily generated in mice, it is necessary to characterize the sprouting response after entorhinal cortex lesion in this species. In the present study, Phaseolus vulgaris-leucoagglutinin (PHAL) tracing was used to analyze the commissural projection to the inner molecular layer in mice with longstanding entorhinal lesions. Because the commissural projection to the fascia dentata is neurochemically heterogeneous, PHAL tracing was combined with immunocytochemistry for calretinin, a marker for commissural/associational mossy cell axons. Using both techniques singly as well as in combination (double-immunofluorescence) at the light or electron microscopic level, it could be shown that in response to entorhinal lesion mossy cell axons leave the main commissural fiber plexus, invade the denervated middle molecular layer, and form asymmetric synapses within the denervated zone. Thus, the commissural sprouting response in mice has a considerable translaminar component. This is in contrast to the layer-specific commissural sprouting observed in rats, in which the overwhelming majority of mossy cell axons remain within their home territory. These data demonstrate an important species difference in the commissural/associational sprouting response between rats and mice that needs to be taken into account in future studies.

Muscimol-stimulated radiotracer 36 Cl - uptake in synaptoneurosomes was used to investigate the function of the GABA A receptor complex in the CA1-3 area and fascia dentata (granular and molecular layers and hilus) of rats kindled by stimulation, twice a day, of the Schaffer collateral fibers. Two kindled groups were studied: (a) 24 h after the last generalized tonic-clonic seizure [fully kindled (FK) stage] and (b) 28 days after the last generalized seizure (long-term stage). Synaptoneurosomes were prepared in parallel from subslices of the CA1-3 area and fascia dentata. In FK animals, the muscimol-stimulated 36 Cl - uptake was significantly reduced by 21% in the CA1-3 area in comparison with nonstimulated controls, whereas a significant increase of 29% was found in the fascia dentata. Significant changes were no longer present at 4 weeks after the last generalized seizure. The observed changes in muscimol-stimulated 36 Cl - uptake at the FK stage closely parallel the recently observed changes in [ 3 H]muscimol binding in the CA1 area and fascia dentata. These results indicate that kindling causes a transiently decreased GABA A receptor-mediated function in the CA1-3, in contrast to an increased GABA A receptor-mediated function in the fascia dentata

The medial cerebral cortex of lizards, an area homologous to the hippocampal fascia dentata, shows delayed postnatal neurogenesis, i.e., cells in the medial cortex ependyma proliferate and give rise to immature neurons, which migrate to the cell layer. There, recruited neurons differentiate and give rise to zinc containing axons directed to the rest of cortical areas, thus resulting in a continuous growth of the medial cortex and its zinc-enriched axonal projection. This happens along the lizard life span, even in adult lizards, thus allowing one of their most important characteristics: neuronal regeneration. Experiments in our laboratory have shown that chemical lesion of the medial cortex (affecting up to 95% of its neurons) results in a cascade of events: first, massive neuronal death and axonal-dendritic retraction and, secondly, triggered ependymal-neuroblast proliferation and subsequent neo-histogenesis and regeneration of an almost new medial cortex, indistinguishable from a normal undamaged one. This is the only case to our knowledge of the regeneration of an amniote central nervous centre by new neuron production and neo-histogenesis. Thus the lizard cerebral cortex is a good model to study neuronal regeneration and the complex factors that regulate its neurogenetic, migratory and neo-synaptogenetic events.O córtex cerebral de lagartos, uma área homóloga à fascia dentata hipocampal, exibe neurogênese pós-natal prolongada, isto é, o epêndima do córtex medial prolifera e dá origem a neurônios imaturos, que migram para a camada celular. Nesta camada, neurônios recrutados se diferenciam e dão origem a axônios, ricos em zinco, que se projetam para as demais áreas corticais, do que resulta um crescimento contínuo do córtex medial e sua projeção axonal. Isto acontece por toda a vida do lagarto, mesmo em animais adultos, o que permite uma de suas características mais importantes: a regeneração neuronal. Experimentos em nosso laboratório demonstraram que uma lesão química do córtex medial (afetando até 95% de seus neurônios) resulta em uma cascata de eventos: inicialmente, morte celular e retração dendro-dendrítica maciças, seguidas de proliferação neuroblasto-ependimária e subseqüente neo-histogênese e regeneração de um novo córtex medial, indistingüível de um normal, não danificado. Ao que saibamos, este é o único caso de regeneração de uma estrutura nervosa central por produção de novos neurônios e neo-histogênese em amniotas. Portanto, o córtex cerebral do lagarto é um bom modelo para estudar a regeneração de neurônios e os fatores complexos que regulam os seus eventos neurogenéticos, migratórios e de neo-sinaptogênese.

The effects of hippocampal treatment with a phosphorothioate oligodeoxynucleotide (ODN) antisense to the γ-aminobutyric acid (GABA) A receptor γ2 subunit on neuropeptide Y (NPY) were studied. Adult male Wistar rats were treated with unilateral intrahippocampal infusion of γ2 subunit antisense ODN for 5 days. Rats infused with mismatch ODN and naive rats served as controls. Brain sections were analysed for levels of NPY mRNA by in situ hybridisation, NPY-immunoreactivity (NPY-ir) by means of immunocytochemistry, and specific NPY binding sites by in vitro receptor autoradiography. Following infusion of antisense ODN, a marked increase in cytoplasmic NPY-ir was observed in hilar neurones of the fascia dentata. Further, intense NPY-ir was visualised in the mossy fibres and in cell bodies of the entorhinal cortex and throughout the neocortex. High levels of NPY mRNA were detected in the same cortical areas of antisense treated rats. A very large increase was observed in the piriform and parietal areas. NPY gene expression also occurred in the granular cell layer, in which no NPY mRNA could be detected in normal animals. The level and distribution of cells displaying high levels of NPY mRNA differed among animals, perhaps as a result of the distinct anatomical location of ODN infusion. Finally, hippocampal levels of NPY specific binding increased, suggesting that NPY neurotransmission is markedly increased. These findings are reminiscent of reported changes in the expression of NPY mRNA and immunoreactivity in conditions of increased neuronal excitation and support the usefulness of the present animal model for the study of epileptic phenomena.

A modulatory role for the hippocampal theta rhythm in synaptic plasticity is suggested by the observations that theta occurs during exploratory behaviors, spatial learning is impaired when the theta rhythm is disrupted, and excitation of hippocampal principal cells is phase-coupled to the theta wave. The theta phase affects the nature of the plasticity induced in urethane-anesthetized rats and in the carbachol-treated in vitro slice preparation, but these oscillations are phenomenologically different from natural theta, and the effects of theta phase on plasticity under natural conditions have not been reported. We therefore examined the effects of theta phase on the magnitude of long-term potentiation (LTP) in awake rats running on a linear track for a food reward. Twelve adult and 10 aged F344 male rats were implanted with a stimulating electrode in the perforant path and a recording electrode in the hilus of the fascia dentata. Stimuli were delivered at the peak or trough of the hilar theta rhythm. In both adult and aged, memory-impaired rats, LTP lasting at least 48 h was induced when stimuli were delivered at the positive theta peak, whereas LTP was not induced when stimuli were delivered at the negative troughs. Consistent with the finding that the threshold for LTP induction is increased at this synapse in old rats, the magnitude of LTP induced at the peak of theta rhythm was significantly lower in old animals. These data confirm that LTP can be modulated by locomotion-induced theta, and that this modulation is at least qualitatively preserved across age.

By using slice cultures of hippocampus as a model, we have studied the development of dendritic spines in fascia dentata granule cells. We raised the question as to what extent spine development is dependent on a major afferent input to these neurons, the fibers from the entorhinal cortex and neuronal activity mediated by these axons. Our results can be summarized as follows: (i) the entorhino-hippocampal projection develops in an organotypic manner in co-cultures of entorhinal cortex and hippocampus. Like in vivo, entorhinal fibers, labeled by anterograde tracing with biocytin, terminate in the outer molecular layer of the fascia dentata. (ii) The layer-specific termination of entorhinal fibers is not altered by the blockade of neuronal activity with tetrodotoxin. Likewise, the differentiation of the dendritic arbor of postsynaptic granule cells does not require neuronal activity. Blockade of neuronal activity did not affect the mean spine number of granule cell dendrites in entorhino-hippocampal co-cultures, but led to a relative increase in thin, long filiform spines that are characteristic of immature neurons. (iii) The maturation of the granule cell dendritic arbor is, however, controlled by the afferent fibers from the entorhinal cortex in an activity-independent manner. In single slice cultures of hippocampus lacking entorhinal input, Golgi-impregnated granule cells have much shorter, less branched dendrites when compared with granule cells in entorhino-hippocampal co-cultures. This reduction in dendritic length in granule cells lacking entorhinal input results in a lower mean total number of spines per neuron, but the mean number of spines per microm is not reduced in the absence of entorhinal innervation. These results indicate that innervation by fibers from the entorhinal cortex, but not neuronal activity mediated via these axons, is essential for the normal development of the granule cell dendritic arbor. Neuronal activity is required, however, for the maturation of dendritic spines.

The extracellular matrix protein reelin plays an important role in neuronal pattern formation and axonal collateralization during the development of the central nervous system. With the concept that reelin might also be important for axonal growth in the injured nervous system we investigated whether reelin is re-expressed in areas of collateral sprouting after brain injury. The expression of reelin messenger RNA was studied in the denervated fascia dentata of adult rats one, four, seven and 14 days following entorhinal cortex lesion. In adult control animals, in situ hybridization histochemistry with digoxigenin-labeled reelin riboprobes revealed reelin messenger RNA expression in neurons located in the outer molecular layer and beneath the granule cell layer of the dentate gyrus. After entorhinal cortex lesion, this expression pattern did not change during the whole post-lesional time period investigated despite a strong glial activation and reactive sprouting in the outer molecular layer of the dentate gyrus as visualized by immunohistochemistry for glial fibrillary acidic protein and acetylcholinesterase histochemistry, respectively. The expression of reelin messenger RNA was also unaffected by entorhinal cortex lesion in the dentate gyrus of young animals (postnatal day seven), where an even stronger sprouting response occurs.

Intracellular filamentous inclusions containing abnormally phosphorylated tau protein are hallmarks of several human neurodegenerative disorders. This study reveals tau-positive cytoskeletal abnormalities in neurons and glial cells of aged baboons. The brains of four baboons (Papio hamadryas, 20-30 yr of age) were examined using the Gallyas silver technique for neurofibrillary changes and phosphorylation-dependent anti-tau antibodies (AT8, AT100, AT270, PHF-1, TG-3). Conspicuous changes were noted in two animals, 26 and 30 yr of age. In both animals, a combination of neuronal and glial cytoskeletal pathology was seen preferentially affecting limbic brain areas, including the hippocampal formation. In the 30-yr-old animal, numerous tau-positive inclusions were seen in the granule cells of the fascia dentata. These cells even exhibited an accumulation of argyrophilic neurofibrillary tangles. The glial changes affected both astrocytes and oligodendrocytes. Tau-positive astrocytes were seen in perivascular, subpial, and subependymal locations. Tau-positive oligodendrocytes preferentially occurred in limbic fiber tracts including the entorhinal perforant path. Ultrastructurally, tau-positive straight filaments (10-14 nm) in both neurons and glial cells were revealed by anti-tau immunoelectron microscopy. This study thus indicates the potential usefulness of aged baboons for experimental investigation of neuronal and glial filamentous tau pathology. This nonhuman primate species may provide valuable information pertinent to the broad spectrum of human tauopathies.

The elicitation of repeated focal seizures (kindling) induces mossy fiber sprouting in the hippocampus of the rat. The present study investigated whether repeated generalized seizures also induce mossy fiber sprouting. Human psychiatric patients receive repeated generalized seizures during electroconvulsive therapy (ECT). Male Long-Evans rats received a course of eight electroconvulsive shock (ECS) seizures administered on a 48-h schedule over a course of 2 1/2 weeks. Control subjects received matched handling, but no stimulation. Fourteen days after the last ECS trial, all subjects were sacrificed and their brains subjected to Timm staining. Cell counts and area measures were also taken in the hilus. Significant sprouting, but not significant cell loss, was seen in the fascia dentata of the subjects that had received ECS.

Transection of the entorhino-dentate perforant path is a well known model for lesion-induced axonal sprouting and glial reactions in the rat. In this study, we have characterized the microglial reaction in the dentate molecular layer of the SJL/J and C57Bl/6 mouse. The morphological transformation of the microglial cells and their densitometrically measured Mac-1 immunoreactivity were correlated with the density of silver-impregnated axonal and terminal degeneration and the myelination of the degenerating medial and lateral perforant pathways. Anterograde axonal and terminal degeneration leads to: (i) altered myelin basic protein immunoreactivity with the appearance of discrete myelin deposits preferentially in the denervated medial and significantly less so in the lateral perforant path zone from day 2 after lesioning; (ii) an increase in number and Mac-1 immunoreactivity of morphologically-changed microglial cells in the denervated perforant path zones with more pronounced morphological transformation of microglia in the medial than in the lateral perforant path zones at day 2 but not day 5 after lesioning; and (iii) a linear correlation between the density of microglial Mac-1 reactivity and axonal degeneration in the medial but not in the lateral perforant path zone at two days postlesion, and a linear correlation in both zones at five days postlesion. We propose that the differentiated microglial response is due to the different densities of axonal and terminal degeneration, as observed in the individual cases. The finding of a potentiated or accelerated microglial activation in the medial as compared to the lateral perforant path zone suggests different kinetics of microglial activation in areas with degenerating myelinated and unmyelinated fibers.

The complete absence of handling of male rats during neonatal development (from birth to postnatal day 21) correlates with an impairment of latent inhibition [J. Feldon, I. Weiner, From an animal model of an attentional deficit towards new insights into the pathophysiology of schizophrenia, J. Psychiatr. Res. 26 (1992) 345–366.]. Such nonhandling of rats reportedly also correlates with a decreased expression of reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd) reactivity in the hippocampus in adult rats (6 months of age) when compared with rats of the same age that were handled during the same neonatal period [R.R. Vaid, B.K. Yee, U. Shalev, J.N. Rawlins, I. Weiner, J. Feldon, S. Totterdell, Neonatal nonhandling and in utero prenatal stress reduce the density of NADPH-diaphorase-reactive neurons in the fascia dentata and Ammon's horn of rats, J. Neurosci. 17 (1997) 5599–5609.]. The present study investigated whether such a decrease in NADPHd activity would be detectable at earlier ages. Therefore, the present study assessed the density of nitric oxide (NO) producing neurons in the fascia dentata and Ammon's horn in 28-, 54-, and 118-day-old nonhandled and handled male rats using NADPHd histochemistry and immunohistochemical localization of neuronal isoform of nitric oxide synthase (nNOS), a NADPHd. This showed that in these three age groups, the numbers of NADPHd positive neurons per unit area throughout the hippocampus of rats that received no handling during neonatal development did not differ significantly from those of rats that received regular daily handling. In addition, we found in the rats of 118 days of age that the areal density of nNOS immunopositive neurons in the hippocampus also did not differ significantly between nonhandled and handled rats. Nevertheless, in a parallel study, rats from the same experimental group receiving identical treatments showed the expected impairment of latent inhibition at 4 months of age [R. Weizman, J. Lehmann, S. Leschiner, I. Allmann, T. Stoehr, C. Heidbreder, A. Domeney, J. Feldon, M. Gavish, Long-lasting effect of early handling on the peripheral-type benzodiazepine receptor, Pharmacol. Biochem. Behav. in press.]. These results suggest that nonhandling of rats during the early neonatal period, that does result in impairment in latent inhibition, does not affect the numbers of NO producing neurons in the hippocampus in rats of young ages, including the age of observed impairment of latent inhibition.

Hilar mossy cells are the main cells of origin of the commissural/associational projection to the inner molecular layer of the rat fascia dentata. In order to analyze the cholinergic innervation of hilar mossy cells, a light and electron microscopic double-labeling technique was used. Immunolabeling for calcitonin gene-related peptide (CGRP) was employed to identify mossy cells and immunocytochemistry for choline acetyltransferase (ChAT) was used to label cholinergic septohippocampal fibers. Cholinergic boutons were abundant around mossy cell somata and on their proximal dendrites. Electron microscopy confirmed that many of these boutons formed synapses with the CGRP-positive mossy cells. These data demonstrate a direct innervation of hilar mossy cells by cholinergic septohippocampal afferents. This connectivity could contribute to the electrophysiological behavior of mossy cells during theta oscillations.

Neurofilament proteins (NFPs), the cytoskeletal proteins that are essential for axogenesis and maintenance of neuron shape in the nervous system, were studied for their spatial distributions at nine postnatal days (PN 3, 5, 7, 10, 14, 17, 21, 28, and 120). Simultaneously non-phosphorylated (SMI-32; 150/200 kDa; Sternberger) and phosphorylated (SMI-31; 200 kDa) NFP immunoreactivity in the entire developing rat hippocampus was studied, quantified, and compared to that of mossy fiber (MF) axons and terminals using Neo-Timm's histochemistry, the most selective, sensitive, and reproducible technique. Differential developmental expressions were observed between the two NFP states. SMI-32 was initially expressed on PN 3 only in the perikarya of pyramidal neurons in CA3. As early as PN 5, SMI-31 appeared in the MF pathway, in parallel to the growth of MF axons. By contrast, SMI-32 did not appear at any age in the MF pathway, including the MF terminal zone of stratum lucidum. At PN 14, the distribution of both NFPs in the MF system (MFs and their target neurons, i.e., CA3/CA4 pyramidal neurons and hilar neurons) was nearly complete; however, the peak densities of SMI-32 and SMI-31 were later at PN 21 and statistically equal to the most adult level (PN 120). The temporal regulation and maximal levels of SMI-32 and SMI-31 expressions on MF target neurons (CA3: SMI-32) and in the MF terminal zone (stratum lucidum: SMI-31) were nearly parallel to the progressive and rapid PN growth of the MF axons and terminals occurring between PN 14 and PN 17, suggesting that the mechanisms for maturation of MF synaptogenesis occur after PN 17.

This study determined whether hippocampal kainate (KA) receptor mRNA levels were increased or decreased in temporal lobe epilepsy patients compared with nonseizure autopsies. Hippocampal sclerosis (HS; n = 17), nonsclerosis (non-HS; n = 11), and autopsy hippocampi (n = 9) were studied for KA1-2 and GluR5-7 mRNA levels using semiquantitative in situ hybridization techniques, along with neuron densities. Compared with autopsy hippocampi, HS and non-HS cases showed decreased GluR5 and GluR6 hybridization densities per CA2 and/or CA3 pyramid. Furthermore, HS patients demonstrated increased KA2 and GluR5 hybridization densities per granule cell compared with autopsy hippocampi. These findings indicate that chronic temporal lobe seizures were associated with differential changes in hippocampal KA1-2 and GluR5-7 hybridization densities that vary by subfield and pathology group. In temporal lobe epilepsy patients, these results support the hypothesis that pyramidal cell GluR5 and GluR6 mRNA levels are decreased as a consequence of seizures, and in HS patients granule cell KA2 and GluR5 mRNA levels are increased in association with aberrant fascia dentata mossy fiber sprouting and/or hippocampal neuronal loss.

This study compared temporal lobe epilepsy patients, along with kindled animals and self sustained limbic status epilepticus (SSLSE) rats for parallels in hippocampal AMPA and NMDA receptor subunit expression. Hippocampal sclerosis patients (HS), non-HS cases, and autopsies were studied for: hippocampal AMPA GluR1-3 and NMDAR12b mRNA levels using in situ hybridization: GluR1, GluR2/3, NMDAR1, and NMDAR2(ab) immunoreactivity (IR); and neuron densities. Similarly, spontaneously seizing rats after SSLSE, kindled rats, and control animals were studied for: fascia dentata neuron densities: GluR1 and NMDAR2(ab) IR; and neo-Timm's staining. In HS and non-HS cases, the mRNA hybridization densities per granule cell, as well as molecular layer IR, showed increased GluR1 (relative to GluR2/3) and increased NMDAR2b (relative to NMDAR1) compared to autopsies. Likewise, the molecular layer of SSLSE rats with spontaneous seizures demonstrated more neo-Timm's staining, and higher levels of GluR1 and NMDAR2(ab) IR compared to kindled animals and controls. These results indicate that hippocampal AMPA and NMDA receptor subunit mRNAs and their proteins are differentially increased in association with spontaneous, but not kindled, seizures. Furthermore, there appears to be parallels in fascia dentata AMPA and NMDA receptor subunit expression between HS (and non-HS) epileptic patients and SSLSE rats. This finding supports the hypothesis that spontaneous seizures in humans and SSLSE rats involve differential alterations in hippocampal ionotrophic glutamate receptor subunits. Moreover, non-HS hippocampi were more like HS cases than hippocampi from kindled animals with respect to glutamate receptors; therefore, hippocampi from kindled rats do not accurately model human non-HS cases, despite some similarities in neuron densities and mossy fiber axon sprouting.

Summary The objective of this study was to demonstrate the effects of prolonged exposure to 6-ANA at low dose-levels in dogs. A male and a female Beagle dog received daily oral repetitive doses of 1 mg/kg or less for 20 weeks. Both dogs showed lacrimation, conjunctivitis, reduced motility and anemia since the second week of treatment. The female dog was more affected than the male and at the end of treatment period it had tremor, hanging lower jaw, stepping gait of the hind limbs, hunched posture, and general debilitation. Postmortem examination of the female dog revealed prominent brain edema with pressure atrophy of the dorsal cranial bones. Microscopic examination of the nervous system revealed spongiform neuropathy in both animals mainly affecting the telencephalic cortex and hippocampal fascia dentata, the substantia gelatinosa in the spinal cord and the dorsal root and autonomic ganglia. The changes were produced by vacuolation of astrocytes in the central nervous system and perineuronal satellite cells in the ganglia. Examination of the other organs revealed thymic atrophy and high hematopoietic activity of the bone marrow in both dogs. The male had severe interstitial edema and vacuolar degeneration of the testicular seminiferous tubules and the female had marked chronic pyelonephritis. This chemically induced spongiform neuropathy in dogs obviously represents a sub-chronic form of the “energy deprivation syndrome” induced by impaired glucose utilization. Vacuolar degeneration of the testicular seminiferous epithelium may have the same pathogenesis.

To elucidate the role of aspartate as a signal molecule in the brain, its localization and those of related amino acids were examined by light and electron microscopic quantitative immunocytochemistry using antibodies specifically recognizing the aldehyde-fixed amino acids. Rat hippocampal slices were incubated at physiological and depolarizing [K(+)] before glutaraldehyde fixation. At normal [K(+)], aspartate-like and glutamate-like immunoreactivities were colocalized in nerve terminals forming asymmetrical synapses on spines in stratum radiatum of CA1 and the inner molecular layer of fascia dentata (i.e., excitatory afferents from CA3 and hilus, respectively). During K(+)depolarization there was a loss of aspartate and glutamate from these terminals. Simultaneously the immunoreactivities strongly increased in glial cells. These changes were Ca(2+)-dependent and tetanus toxin-sensitive and did not comprise taurine-like immunoreactivity. Adding glutamine at CSF concentration prevented the loss of aspartate and glutamate and revealed an enhancement of aspartate in the terminals at moderate depolarization. In hippocampi from animals perfused with glutaraldehyde during insulin-induced hypoglycemia (to combine a strong aspartate signal with good ultrastructure) aspartate was colocalized with glutamate in excitatory terminals in stratum radiatum of CA1. The synaptic vesicle-to-cytoplasmic matrix ratios of immunogold particle density were similar for aspartate and glutamate, significantly higher than those observed for glutamine or taurine. Similar results were obtained in normoglycemic animals, although the nerve terminal contents of aspartate were lower. The results indicate that aspartate can be concentrated in synaptic vesicles and subject to sustained exocytotic release from the same nerve endings that contain and release glutamate.

Patterns of neuronal activity recorded in CA1 of the hippocampus and in neocortex during waking-behavior, are reactivated during subsequent slow-wave sleep (SWS). It has been suggested that this reactivation may originate in the hippocampal CA3 region, where modifiable excitatory recurrent connections are abundant and where sharp-waves, in which the reactivation is most robust, appear to arise. The present experiment investigated whether ensemble firing patterns of granule cells in the fascia dentata (FD), an area ‘upstream’ from CA3, are also reactivated during sleep. Populations of FD granule cells were recorded from during spatial behavior and during prior and subsequent SWS. Firing rate correlations between cell-pairs with overlapping place fields were significantly enhanced during post behavioral sleep compared to pre behavioral sleep. Correlations between cells with non-overlapping place fields or which were silent during maze behavior, were not changed. Thus, reactivation of experience-specific correlation states also occurs in granule cells during sleep. Because these cells do not have excitatory interconnections, but form a major input to CA3 pyramidal cells, current models predicted that sleep reactivation would appear first in CA3. There are, however, both extensive polysynaptic excitatory interactions among granule cells and feedback from CA3 pyramidal cells. Granule cells also receive indirect input from neocortical regions known to undergo trace reactivation. Although a simple model for a CA3 origin of the reactivation phenomenon cannot be confirmed, the present results extend our understanding of the generality of this phenomenon.

The present study aimed to assess the developmental progress of the hippocampal nitric oxide (NO) system within adulthood by comparing the density of NO-producing neurons in the fascia dentata and Ammon's horn in two groups of adult male rats using NADPH-diaphorase (NADPH-d) histochemistry. One group comprised 6-month-old rats (early adulthood), and the other 12-month-old rats (middle-adulthood). Areal density (number of neurons per unit area) of NADPH-d positive neurons along the three hippocampal axes (septo-temporal, transverse and radial axes) were subjected to quantitative analyses. There were significant variations in the density of NADPH-d-reactive neurons along the transverse and radial axes of the hippocampus, similar to what have been described previously. Comparison between 6-month and 12-month-old rats indicated a substantial reduction in the density of NADPH-d-reactive neurons in the fascia dentata (69%) and Ammon's horn (54%) of the latter group. This reduction was relatively uniform along the septotemporal and radial axes, but appeared to be more pronounced in the fascia dentata and in the proximal region of Ammon's horn. Our finding showed that the hippocampal NO system can undergo significant changes within adulthood. It further highlighted the possibility that an age-related reduction in the capacity to produce NO may not be directly responsible for the cognitive decline associated with senescence, but rather predisposes neuronal degeneration in later life.

Lübke, Joachim, Michael Frotscher, and Nelson Spruston. Specialized electrophysiological properties of anatomically identified neurons in the hilar region of the rat fascia dentata. J. Neurophysiol. 79: 1518–1534, 1998. Because of their strategic position between the granule cell and pyramidal cell layers, neurons of the hilar region of the hippocampal formation are likely to play an important role in the information processing between the entorhinal cortex and the hippocampus proper. Here we present an electrophysiological characterization of anatomically identified neurons in the fascia dentata as studied using patch-pipette recordings and subsequent biocytin-staining of neurons in slices. The resting potential, input resistance ( RN), membrane time constant (τm), “sag” in hyperpolarizing responses, maximum firing rate during a 1-s current pulse, spike width, and fast and slow afterhyperpolarizations (AHPs) were determined for several different types of hilar neurons. Basket cells had a dense axonal plexus almost exclusively within the granule cell layer and were distinguishable by their low RN, short τm, lack of sag, and rapid firing rates. Dentate granule cells also lacked sag and were identifiable by their higher RN, longer τm, and lower firing rates than basket cells. Mossy cells had extensive axon collaterals within the hilus and a few long-range collaterals to the inner molecular layer and CA3c and were characterized physiologically by small fast and slow AHPs. Spiny and aspiny hilar interneurons projected primarily either to the inner or outer segment of the molecular layer and had a dense intrahilar axonal plexus, terminating onto somata within the hilus and CA3c. Physiologically, spiny hilar interneurons generally had higher RNvalues than mossy cells and a smaller slow AHP than aspiny interneurons. The specialized physiological properties of different classes of hilar neurons are likely to be important determinants of their functional operation within the hippocampal circuitry.

The rat fascia dentata is characterized by a simple cytoarchitecture and characteristic lamination of afferents. Entorhinal afferents are believed to terminate exclusively in the outer two thirds of the molecular layer, whereas commissural fibers are believed to terminate exclusively in the inner molecular layer of the fascia dentata. A sharp border divides these two major afferent fiber systems and is regarded as the main boundary of the fascia dentata. This concept of a highly laminated brain structure has made the fascia dentata attractive for studies analyzing normal or pathological processes of the brain. Recently, entorhinal as well as commissural fibers have been identified which do not follow the classical lamination of the fascia dentata. Using anterograde tracing with Phaseolus vulgaris-Leucoagglutinin, an entorhino-dentate projection to the molecular layer, granule cell layer, and hilus of the fascia dentata was described. With the same technique, GABAergic commissural fibers to the outer molecular layer of the fascia dentata were revealed and a previously unknown heterogeneity of the commissural projection was demonstrated. These previously unknown fiber systems complicate the interpretation of lesion effects in this brain region and have to be taken into account as possible sources of sprouting fibers following the partial denervation of the fascia dentata.

The rat fascia dentata is frequently used as a model system to analyze normal as well as pathological processes of the brain. The normal anatomy of the fascia dentata is the basis for a meaningful interpretation of experimentally induced changes in this brain region. Using anterograde tracing with Phaseolus vulgaris-leucoagglutinin (PHAL) previously unknown commissural as well as entorhinal fiber systems to the fascia dentata were described. These fiber systems need to be incorporated into current concepts of the hippocampal network since they have profound implications for studies of lesion effects in this brain region.

The extracellular matrix protein tenascin-C has been implicated in the regulation of axonal growth. Using unilateral entorhinal cortex lesions, which induce a massive sprouting response in the denervated outer molecular layer of the rat fascia dentata, the role of tenascin-C for axonal growth was investigated in vivo. Monoclonal antibodies against the neurite outgrowth and anti-adhesive domains of the molecule were employed. Immunostaining was increased throughout the denervated outer molecular layer by day 2, reached a maximum around day 10, and was back to control levels by four weeks post lesion. Growth cone deflecting as well as neurite outgrowth promoting isoforms of tenascin-C were up-regulated after the lesion. Using electron microscopy, single intensely tenascin-C immunoreactive cells were identified as reactive astrocytes that phagocytose degenerated terminals. In situ hybridization histochemistry for tenascin-C messenger RNA revealed numerous cellular profiles in the denervated outer molecular layer of the ipsilateral and contralateral dentate gyrus two days post lesion. Tenascin-C messenger RNA-positive cells in the outer molecular layer were identified as astrocytes using double-labelling for tenascin-C messenger RNA and glial fibrillary acidic protein immunohistochemistry. Thus, a tenascin-C-rich substrate is present in the outer molecular layer during the time of sprouting and a sharp boundary is formed against the inner molecular layer. This pattern may contribute to the layer-specific sprouting response of surviving afferents after entorhinal lesion. Neurite outgrowth may be promoted within the denervated zone, whereas axons trying to grow into the denervated outer molecular layer, for example from the inner molecular layer, would be deflected by a tenascin-C-rich barrier.

The expression of the transcription factor c-JUN was investigated in the rat fascia dentata under normal conditions and after entorhinal cortex lesion. As shown by immunocytochemistry and in situ hybridization histochemistry c-JUN and its messenger RNA are present in the principal cell layers of the dentate gyrus and Ammon's horn (except hippocampal region CA2). Pre-embedding immunogold electron microscopy revealed an almost exclusive nuclear localization of c-JUN, where it is associated with chromatin. In addition, double immunolabelling for c-JUN and parvalbumin demonstrated that c-JUN immunoreactivity is primarily found in principal neurons since GABAergic parvalbumin-positive interneurons did not express c-JUN. After unilateral electrolytic lesion of the entorhinal cortex c-JUN was strongly up-regulated in the ipsilateral dentate gyrus within 2 h postlesion. This up-regulation was also present in the contralateral fascia dentata 12 h after entorhinal cortex lesion and returned to control levels on both sides 24 h postlesion. The cellular distribution of c-JUN did not change after entorhinal cortex lesion: parvalbumin-positive interneurons never contained c-JUN. These results point to a specific role of c-JUN in the granule cells of the fascia dentata in the normal animal and in rats with entorhinal cortex lesions. The selective induction of c-JUN after entorhinal lesion could be one of the first molecular steps that regulate transneuronal changes within granule cells after their denervation. A different mechanism has to be assumed for GABAergic interneurons known to receive an entorhinal innervation as well.

Kainic acid (KA) lesions of the rat hilus model hippocampal sclerosis and temporal lobe epilepsy. Unilateral hilar cell loss denervates the associational afferents normally projecting to the inner molecular layer (IML) granule cell dendrites, followed by ipsilateral mossy fiber (MF) sprouting. Hilar neurons also project through the hippocampal commissure to the contralateral IML. This study compared densities of IML MF sprouting following unilateral versus bilateral low dose KA lesions, using Neo-Timm stain 30 days later. Unilateral KA (0.4 microg) caused only dense ipsilateral MF sprouting. Bilateral lesions with lower doses of KA (0.1 with 0.2 or 0.3 microg) induced dense bilateral MF sprouting. However, the same low doses of KA injected unilaterally did not induce significant sprouting ipsilaterally or contralaterally. These data show that denervations of both associational and commissural afferents to the same IML dendritic zones of granule cells induce non-linear, additive bilateral MF neoinnervations.

The most frequently used model of neonatal cerebral hypoxia–ischemia consists of a 7-day postnatal rat model with combined common carotid artery ligation and hypoxemia. Neuropathologic studies have shown major differences between this 7-day postnatal rat model and a similar adult model in regard to overall cerebral vulnerability, type and distribution of lesions. It is not clear how and when during animals' development these changes in cerebral vulnerability take place. To determine this we studied groups of rats of 2 to 30 postnatal days. The animals underwent unilateral common carotid artery ligation followed by breathing in 8% oxygen for 30, 60, 90, or 120 min and their brains were examined at 24- or 72-h recovery intervals. Due to resistance of 2–3-day-old rats to develop cerebral hypoxic–ischemic damage, 5% O2 was used instead of 8% O2. The results indicate that: (i) There is an overall increase in severity of cerebral lesions on the side of common carotid artery ligation between 2 and 7 postnatal days. There is also an increase in the frequency of cerebral lesions in developing animals with increasing age. (ii) Hippocampus is remarkably resistant to hypoxic–ischemic insult at 2–3 postnatal days but becomes progressively vulnerable, and by age 13 postnatal days hippocampal vulnerability far exceeds that of cortex. (iii) Cortical lesions change from predominantly columnar cell death to laminar selective neuronal death at age 13 postnatal days. (iv) Also significant changes occur in relative vulnerability of various hippocampal regions during development. During the first 5 postnatal days relative vulnerability of hippocampal regions is similar, but as the animals' development proceeds and hippocampal vulnerability increases lesions tend to involve specific regions while sparing others. By age 13 postnatal days CA1 and lateral CA3 develop increased vulnerability while medial CA3 and fascia dentata become relatively resistant and by 21 postnatal days adult pattern of CA1 selective vulnerability is approached. The underlying mechanisms for these changes in regional vulnerability to cerebral hypoxia-ischemia during development should be sought in complex regional anatomic, functional, and metabolic alterations that take place as brain matures.

Mice lacking prion protein (PrP-null) are resistant to transmissible spongiform encephalopathies. However, the normal functions of this highly conserved protein remain controversial. This study examines whether PrP-null mice develop normal neuronal pathways, specifically the mossy fibre pathway, within the hippocampus. Timm stained hippocampal sections from the PrP-null group had more granules than the controls in: the granule cell layer, the inner molecular layer of the dentate gyrus, and the infrapyramidal region of CA3. This resembles the mossy fibre collateral and terminal sprouting seen in certain epilepsies. The abnormal connectivity might be predicted to promote epileptiform activity, but extracellular electrophysiological recordings from the granule cell layer revealed a reduced excitability in the PrP-null group, both with and without blockade of GABAA receptor-mediated inhibition. We propose that reorganization of neuronal circuity is a feature of PrP-null mice.

This study determined whether there were differences in hippocampal neuron loss and synaptic plasticity by comparing rats with spontaneous epilepsy after limbic status epilepticus and animals with a similar frequency of kindled seizures. At the University of Virginia, Sprague-Dawley rats were implanted with bilateral ventral hippocampal electrodes and treated as follows; no stimulation (electrode controls; n=5): hippocampal stimulation without status (stimulation controls; n=5); and limbic status from continuous hippocampal stimulation (n=12). The limbic status group were electrographically monitored for a minimum of four weeks. Four rats had no recorded chronic seizures (status controls), and all three control groups showed no differences in hippocampal pathology and were therefore incorporated into a single group (controls). Eight limbic status animals eventually developed chronic epilepsy (spontaneous seizures) and an additional eight rats were kindled to a similar number and frequency of stage 5 seizures (kindled) as the spontaneous seizures group. At the University of California (UCLA) the hippocampi were processed for: (i) Niss1 stain for densitometric neuron counts; (ii) neo-Timm's histochemistry for mossy fiber sprouting; and (iii) immunocytochemical staining for glutamate decarboxylase, N-methyl-D-aspartate receptor subunit 2, AMPA receptor subunit 1 and the GABA(A) receptor. In the fascia dentata inner and outer molecular layers the neo-Timm's stain and immunoreactivity was quantified as gray values using computer image analysis techniques. Statistically significant results (P

The reactions of microglial and astroglial cells to anterograde axonal degeneration were studied in the fascia dentata of adult rats from 1 to 42 days after removal of the entorhinal perforant path projection. The observations focused on the kinetics of glial activation in terms of induction of immunomolecules on the glial cells and the possible correlation between these changes and lesion-induced extravasation of plasma constituents. Normal and activated microglial cells were identified by immunohistochemical visualization of the constitutively expressed complement type 3 receptor (CR3/CD11b). Activated microglial cells were stained immunohistochemically for the inducible major histocompatibility complex (MHC) antigen class I and class II and the leukocyte common antigen (LCA/CD45). Astroglial cells were identified by immunohistochemical staining for glial fibrillary acidic protein (GFAP). Blood–brain barrier (BBB) conditions were primarily evaluated by immunohistochemical staining for extravasated Immunoglobulin G (IgG), but also by intravenously injected horseradish peroxidase (HRP) and Evans Blue. Twenty-four hours after entorhinal cortex ablation, microglial cells in the perforant path terminal zones displayed an increase in CR3 immunoreactivity, changes of morphology and an induced expression of MHC antigen class I. At the same time there was a hitherto undescribed leakage of IgG through the BBB (albeit without detectable extravasation of HRP and Evans Blue). One day later microglial cells also expressed LCA, but MHC antigen class II was not induced under these degenerative conditions. The activation of microglial cells occurred prior to a noticeable hypertrophy of astroglial cells and increase in GFAP immunoreactivity, as this first became evident on Postlesional Day 2. From the results we conclude (1) that perforant path axonal degeneration induces an endothelial transcytosis of blood-borne IgG by mechanisms which cannot be envisioned by conventional HRP tracer methods and (2) that the early activation of both microglial cells and astroglial cells is likely to be initiated and later influenced by both axonal degeneration and extravasated plasma constituents. The demonstration of an early induction of immunomolecules on activated microglial cells and extravasation of blood borne molecules might moreover form the basis for a correspondingly early intervention aiming to regulate microglial immunomolecule, cytokine, and growth factor gene expression in the affected areas.

Background Aging is typically accompanied by memory decline and changes in hippocampal function. Among these changes is a decline in the activity of the dentate gyrus (DG) during behavior. Lasting memory, however, is thought to also require recapitulation of recent memory traces during subsequent rest – a phenomenon, termed memory trace reactivation, which is compromised in hippocampal CA1 with progressive age. This process has yet to be assessed in the aged DG, despite its prominent role in age-related memory impairment. Using zif268 transcription to measure granule cell recruitment, DG activity in adult and aged animals was assessed both during spatial exploration and as animals remained at rest in the home cage in order to detect potential memory-related replay. Results Consistent with the observation of memory trace reactivation in DG, the probability that an individual granule cell transcribes zif268 during rest in the animal’s home cage is increased by recent experience in a novel environment. Surprisingly, a comparable increase was observed in the probability of granule cells in the aged DG expressing zif268 during rest. Moreover, no significant age-related difference was observed in the number of granule cells expressing zif268 during rest. Thus, the number and pattern of granule cell expression of zif268 during rest is preserved in aged animals, despite a significant decline in exploration-related zif268 expression. Conclusions These data lead to the hypothesis that the input the aged DG receives from backprojections from CA3 (the region widely hypothesized to mediate reactivation) remains functionally intact despite loss of innervation from the perforant path.

The commissural and associational projections to the rat dentate gyrus are believed to be anatomically homologous fiber systems. They are often referred to as the so-called commissural/associational system of the dentate gyrus. However, whereas characteristic laminar termination patterns within the molecular layer of the dentate gyrus have been described for the different cells of origin of the associational projection, the axons of the different cell types of commissural neurons have long been believed to terminate exclusively within the inner molecular layer. Only recently, a previously unknown commissural projection to the outer molecular layer of the dentate gyrus was described and the question was raised whether the commissural fibers could exhibit a heterogeneity similar to that of the associational projections. Using the anterograde tracer Phaseolus vulgaris leucoagglutinin, which labels individual axons and their collaterals, we have studied the termination pattern of commissural axons in the dentate gyrus of the septal hippocampus. At least four different commissural fiber types could be revealed on the basis of their laminar termination pattern: fibers to the inner molecular layer (type 1), fibers to the outer molecular layer (type 2), fibers terminating throughout the molecular layer (type 3), and fibers terminating in both the granule cell layer and the molecular layer (type 4). These observations demonstrate a previously underestimated heterogeneity of the commissural projection. In addition, there is a great deal of parallelism between the different commissural and associational fibers, pointing to a coordinated action of the two systems in the two hippocampi.

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 withPhaseolus vulgarisleucoagglutinin, 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.

This study reports that lesion of the adult lizard medial cortex (lizard hipocampal fascia dentata) induces a short period of intensive neurogenesis which we have termed reactive neurogenesis; a cell proliferation event that occurs in the subjacent ependyma. Specific lesion of the medial cortex was achieved by intraperitoneal injection of the neurotoxin 3-acetylpyridine and proliferating cells were detected using tritiated thymidine or 5-bromodeoxiuridine pulse labelling. After lesion, granule neurons in the lizard medial cortex cell layer appeared pyknotic and died; they were then removed and progressively replaced by a set of new neurons. These neurons were mostly generated from the second to the seventh day post-lesion. A dramatic temporal increment of labelled ependymal cells was detected when either tritiated thymidine or 5-bromodeoxiuridine pulses were delivered in that period. The maximum of about five thousand labelled cells per hemisphere was reached by the fourth day after the lesion. Beyond the seventh day post-lesion, the numbers of labelled cells returned to a level of about four hundred per hemisphere, similar to that of the control specimens. Electron microscopy revealed that the recently generated cells were neuroblasts or immature neurons with a characteristic pattern of chromatin condensation and a high number of ribonucleic granules.

The calcium-binding protein calretinin is present in an intrinsic GABAergic and an extrinsic non-GABAergic system in the rat and monkey hippocampal formation. Important species differences have been noted in hippocampal cell types immunostained for calretinin and the termination pattern of calretinin containing hypothalamic afferents in the hippocampus. In the present study, calretinin-containing neurons were visualized using immunocytochemistry in the human hippocampal formation of individuals which showed no significant neuropathological alterations. Calretinin-immunoreactivity was present exclusively in non-granule cells of the dentate gyrus and in non-pyramidal cells of Ammon's horn. Calretinin-positive neurons were found most frequently in the hilus of the fascia dentata and in strata radiatum and lacunosum-moleculare of CA1, whereas neurons in CA2 and CA3 were rarely immunostained. The majority of calretinin-immunoreactive neurons were small, bipolar or fusiform neurons. The dendritic trees of the calretinin-positive neurons were, for the most part, parallel to the dendrites of the principal cells. In the hilus, however, we observed cells with dendrites restricted to the hilar area. These dendrites were parallel to the granule cell layer. In the stratum lacunosum-moleculare, neurons with dendrites oriented parallel to the hippocampal fissure were frequently detected. In general, dendrites were smooth or sparsely spiny, displaying small conventional spines. The axons usually emerged from the proximal dendrite and could be followed over long distances. Axons were thin, had small varicosities and displayed only few collaterals which branched relatively far away from the cell body. Distinct bands of darkly stained calretinin-positive fibers occupied the innermost portion of the dentate molecular layer and the pyramidal cell layer of CA2. This distribution of calretinin-immunoreactive structures in the human hippocampus is similar to that observed in other primates but differs from that described in lower mammals, i.e., the rat. Our findings suggest that primates may share a common hippocampal calretinin-containing system, presumably both the intrinsic GABAergic and the extrinsic hypothalamic non-GABAergic components.

Kindling is an animal model of epilepsy which is accompanied by morphological and biochemical changes in the brain, including sprouting of fibers and increased transmitter release. Here we have examined the immunocytochemical expression of 1) GAP-43, a growth-associated protein, which is a neuron-specific PKC substrate, particularly expressed in development and regeneration and 2) glial fibrillary acidic protein (GFAP), part of the astrocytic cytoskeleton, after perforant path kindling. Subsequent to kindling, GAP-43 immunoreactivity was increased in CA1 stratum lacunosum-moleculare and the inner and outer molecular layer of the fascia dentata. Other hippocampal subregions showed a lower increase. GFAP immunoreactivity was increased in the entire hippocampus, but especially in stratum lacunosum-moleculare of the CA1 and the hilus of fascia dentata. The difference between the number of GFAP-positive profiles in the hippocampus of control rats and in fully kindled rats was found to be non-significant. We interpret these findings as being related to both plastic neuronal changes and possible neuronal degeneration.

We studied the binding of (35S)t-butylbicyclophosphorothionate to the GABA, receptor- mediated chloride channel in the CA1 area and fascia dentata of control and Schaffer collateral kindled rats, by means of semi-quantitative autoradiography. The (35S)t-butylbicyclophosphorothionate binding was determined at three stages during kindling aquisition: (i) after six afterdischarges, (ii) after 14 afterdischarges and (iii) after the induction of fully kindled seizures. Furthermore, the binding was studied at the long-term stage, 28 days after the last generalized tonic-clonic seizure (Racine R. J. (1972) Electroenceph. din. Neurophysiol. 32, 281-2941. The binding was investigated at three (35S)t-butyl- bicyclophosphorothionate concentrations, 4, 47.5 (&, value) and 180 nM (B,,,_ value). A significant decrease in (35S)t-butylbicyclophosphorothionate binding in the CA1 area ( - 6 to - 20%) and hilar formation (- 17 to - 37%), in one or more of the three (35S)t-butylbicyclophosphorothionate concentrations tested at the six and 14 afterdischarges and fully kindled stages was observed, but no significant changes at the long-term kindling stage were found. In contrast, the granular and molecular layers of the fascia dentata presented a significant increase in (35S)t-butylbicyclophosphorothionate binding (+ 15 to +38%) at the 14 afterdischarges, fully kindled and long-term kindled stages. These changes in r5S)t-butylbicyclophosphorothionate binding are in line with the previously observed changes in the (3H)muscimol lTitulaer M. N. G. et al. (1993) Neuroscience 59, 817-826 and ('Hlfluni- trazepam pitulaer M. N. G. et al. (submitted)) binding, indicating that Schaffer collateral kindling stimulation is accompanied by changes of all three main GABA, receptor binding sites: (i) the "cage" convulsant site associated with the chloride channel, (ii) the GABA, agonist binding site and (iii) the benzodiazepine receptor agonist binding site. We hypothesize that this modification of GABA,/ benzodiazepine receptor complex binding may underlie the changes in GABA-mediated inhibition in the kindled rat hippocampus, as previously indicated in el&rophysiological studies, (Kamphuis W. et al. (1988) Brain Rex 440,2052151, consequently contributing to the establishment of a kindling stimulation induced epileptogenic focus.

Numerous studies have shown a lamina-specific termination of commissural fibers to the dentate gyrus in the inner molecular layer. However, the exact course and arborization pattern of individual fibers remained unknown. In this study, the commissural fiber tract to the dentate gyrus of the rat has been studied using the anterograde tracer Phaseolus vulgaris -leucoagglutinin (PHA-L), which labels individual axons and their collaterals. Following iontophoretic application of the tracer, anterogradely labeled fibers were followed through the posterior basal fornix and medial fimbria where they formed a dense fiber bundle. Labeled fibers then entered the dentate gyrus close to the medial blade of the granule cell layer where they separated and traversed the hilus. Only in those cases where the injection also involved CA3 pyramidal cells could axons arborizing in the hilus be observed. Typically, fibers that continued into the molecular layer did not arborize in the hilus. Upon their entrance into the molecular layer, these fibers changed direction, gave off several collaterals, and followed a new path parallel to the granule cell layer where they preferentially formed en passant contacts. These commissural fibers to the inner molecular layer terminated in a wide septotemporal (longitudinal) extension. However, a considerable number of fibers reached the outer molecular layer where some of them formed extensive arborizations. Moreover, these commissural fibers to the outer molecular layer appeared to be restricted to the hippocampal lamella, corresponding to the level of the contralateral injection site. These data suggest the existence of three commissural projections to the rat dentate gyrus: (1) commissural fibers to the hilus arising from CA3 neurons, (2) commissural fibers to the inner molecular layer, and, (3) commissural fibers to the outer molecular layer. © 1995 Wiley-Liss, Inc.

The binding of the GABAA/benzodiazepine receptor agonist [3H]flunitrazepam was studied in the hippocampus of rats kindled by daily stimulation of the Schaffer collaterals, using semi-quantitative autoradiography. Two kindled stages were investigated: (i) 24 h after the last generalized tonic-clonic seizure (fully kindled) and (ii) 28 days after the last generalized seizure (long-term). The binding of [3H]flunitrazepam was determined at two concentrations, 3 and 16 nM. In the CA1 area, we found a small but significant decrease (ca. 10%), both in the 3 and 16 nM [3H]flunitrazepam binding at the fully kindled stage. In contrast, there was a significant increase in the 3 nM binding (c. 15%) at the long-term stage. The 16 nM binding was not significantly different from control binding at this stage. In the granular and molecular layers of the fascia dentata, we found at both kindled stages a significantly increased 3 nM (ca. 9 and 19%, respectively) and 16 nM (ca. 19 and 14%, respectively) binding. Furthermore, we found that muscimol was still able to enhance the [3H]flunitrazepam binding in kindled animals, indicating that the GABAA receptor agonist binding site and benzodiazepine agonist binding site are still functionally coupled. The changes in [3H]flunitrazepam binding at the fully kindled stage are in agreement with the recently observed kindling-induced changes in [3H]muscimol binding in the hippocampal formation of the same animals [Titulaer M. N. G. et al. (1994) Neuroscience 59, 817-826] and extend these observations to the benzodiazepine modulatory site of the GABAA receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

To investigate the changes underlying kindling epileptogenesis in the rat hippocampus, the levels of the messenger RNAs encoding for the subunits of the N-methyl-D-aspartate-receptor (1, 2A-D) and the kainate-receptor (1, -2, GluR-5, -6, -7) were determined in hippocampal principal neurons using in situ hybridization techniques and semi-quantitative analysis of the autoradiograms. Schaffer collateral-commissural pathway kindled rats were investigated at three different stages of kindling acquisition, always 24 h after the last stimulation. Furthermore, fully kindled rats were studied at long-term (28 days) after termination of kindling stimulations. NR1 messenger RNA levels were slightly decreased in CA1 area of fully kindled animals. In the fascia dentata region, a minor increase of NR2A and NR2B transcripts was found at all stages of kindling acquisition. KA-2 messenger RNA was enhanced in all hippocampal subfields during kindling development. However, none of these changes persisted at long-term after the last seizure and only the low-abundant GluR-7 expression was slightly depressed in the fascia dentata. From our observations we conclude that it is unlikely that alterations in N-methyl-Daspartate or kainate receptor gene expression play an important role in kindling acquisition or maintenance.

Nonpyramidal neurons containing the calcium-binding protein parvalbumin (PV) are one of the inhibitory elements of the hippocampal network. Previous studies have indicated that they are involved in septohippocampal disinhibitory circuits. This study analyzes the commissural and ipsilateral associational afferents of parvalbumin neurons. Injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) into the hilus of the fascia dentata labeled numerous axons in the molecular layer that established synaptic contacts with parvalbumin-immunoreactive neurons on both the injection and the contralateral side. Mossy fibers, labeled by injections into the granule cell layer, terminated on parvalbumin neurons in the hilus and in CA3. Injections of PHA-L into CA3 resulted in a dense labeling of fibers in the hilus and in CA3, CA2, and CA1 on both the injection and the contralateral side. In all these hippocampal fields, PHA-L-labeled fibers established asymmetric contacts with PV-immunoreactive, presumably GABAergic, inhibitory neurons. These observations indicate that parvalbumin-immunoreactive inhibitory neurons in the hippocampus are targets of presumably excitatory associational and commissural projections and suggest that they are involved in feed-forward and feed-back circuits. © 1994 Wiley-Liss, Inc.