Deafferented Dentate Gyrus Research Articles

Enhanced Microglial Clearance of Myelin Debris in T Cell-Infiltrated Central Nervous System

Acute multiple sclerosis lesions are characterized by accumulation of T cells and macrophages, destruction of myelin and oligodendrocytes, and axonal damage. There is, however, limited information on neuroimmune interactions distal to sites of axonal damage in the T cell-infiltrated central nervous system. We investigated T-cell infiltration, myelin clearance, microglial activation, and phagocytic activity distal to sites of axonal transection through analysis of the perforant pathway deafferented dentate gyrus in SJL mice that had received T cells specific for myelin basic protein (TMBP) or ovalbumin (TOVA). The axonal lesion of TMBP-recipient mice resulted in lesion-specific recruitment of large numbers of T cells in contrast to very limited T-cell infiltration in TOVA-recipient and -naïve perforant pathway-deafferented mice. By double immunofluorescence and confocal microscopy, infiltration with TMBP but not TOVA enhanced the microglial response to axonal transection and microglial phagocytosis of myelin debris associated with the degenerating axons. Because myelin antigen-specific immune responses may provoke protective immunity, increased phagocytosis of myelin debris might enhance regeneration after a neural antigen-specific T cell-mediated immune response in multiple sclerosis.

The β-amyloid-related proteins presenilin 1 and BACE1 are axonally transported to nerve terminals in the brain

In this study, we show that removal of entorhinal cortex (ERC) afferents to hippocampus reduces levels of presenilin 1 (PS1) in the dentate gyrus of APPswe/PS1ΔE9 transgenic (Tg) mice. PS1 immunoreactivity on the deafferented dentate gyrus decreases by approximately 25% and 50%, 2 and 4 weeks post-lesion compared to the contralateral side; by Western blotting, there is an approximately 40% decrease of the 43 kDa (full length) PS1 and an approximately 80% decrease of the 28 kDa (N-terminal fragment) PS1 on the lesioned dentate gyrus. Levels of β-site APP Cleavage Enzyme 1 (BACE1) immunoreactivity also decrease by approximately 50% and 65% 2 and 4 weeks post-lesion. Together, these data demonstrate that PS1 and BACE1 are transported from the entorhinal cortex to the hippocampus via axons of the perforant pathway.

Differential regulation of Ca 2+-calmodulin stimulated and Ca 2+-insensitive adenylyl cyclase messenger RNA in intact and denervated mouse hippocampus

The Ca 2+-calmodulin stimulated AC1 and Ca 2+-insensitive AC2 are major isoforms of adenylyl cyclase, playing an important role in synaptic plasticity in the mammalian brain. We studied the pattern of expression of AC1 and AC2 genes in the hippocampus of C57BL/6 mice. We found that there were differences in their patterns of distribution in the dentate gyrus. AC1 messenger RNA was detected both in the dentate granule cell bodies and the corresponding molecular field whereas AC2 messenger RNA was preferentially distributed in the dentate granule cell layer, suggesting that AC1 and AC2 messenger RNA are differentially regulated in the dentate gyrus. In order to examine the regulation of AC1 and AC2 expression in response to synaptic deafferentation and reinnervation, the distribution patterns of the two AC messenger RNA in the hippocampal fields and the parietal cortex were analysed 2, 5, 9 and 30 days following an unilateral entorhinal cortex lesion. Interestingly, we found significantly reduced levels of AC1 hybridization signal following the lesion whereas the level of AC2 messenger RNA remained unaffected in all lesioned groups. The changes in AC1 messenger RNA were transient, with a maximal reduction at five days postlesion, and were restricted to the granule cell bodies and stratum moleculare of the deafferented dentate gyrus. No significant change in AC1 messenger RNA levels was detected in other hippocampal fields nor for any other postlesion times studied. These findings suggest that, at least in the dentate gyrus, messenger RNA for AC1 and AC2 might be differentially compartmentalized in cell bodies and dendritic fields. The activity-dependent regulation of AC1 messenger RNA levels by afferent synapses may provide an elegant mechanism for achieving a selective local regulation of AC1 protein, close to its site of action.

Dexamethasone selectively suppresses microglial trophic responses to hippocampal deafferentation

Hippocampal deafferentation increases the expression of insulin-like growth factor-1 by microglia, and of ciliary neurotrophic factor and basic fibroblast growth factor by astroglia in fields and periods of reactive axonal growth. Glucocorticoids attenuate lesion-induced hippocampal sprouting, possibly by reducing trophic signals that stimulate growth. With an interest in this hypothesis, the present studies evaluated the influence of systemic treatment with the synthetic glucocorticoid dexamethasone on entorhinal lesion-induced increases in neurotrophic factor expression in young adult rat hippocampus. Daily dexamethasone injections almost completely blocked increases in insulin-like growth factor-1 messenger RNA content, but did not perturb increases in ciliary neurotrophic factor or basic fibroblast growth factor messenger RNA content, in the deafferented dentate gyrus molecular layer. To determine if the suppression of insulin-like growth factor-1 expression was secondary to a general inhibition of microglial responses, and to identify the time period of glucocorticoid sensitivity, additional rats were prepared to evaluate the effects of semi-chronic (i.e. daily) and single dexamethasone injections on microglial proliferation, ED-1 immunoreactivity (a marker of microglial reactivity) and insulin-like growth factor-1 messenger RNA expression. Semi-chronic dexamethasone treatment attenuated all three measures of deafferentation-induced microglial reactivity. However, a single dexamethasone injection given two (but not one or three) days postlesion inhibited deafferentation-induced increases in insulin-like growth factor-1 messenger RNA content, without having significant effects on other measures. These results demonstrate that dexamethasone treatment preferentially suppresses microglial, as opposed to astroglial, trophic responses to deafferentation, and suggest that glucocorticoids attenuate reactive axonal sprouting by inhibiting the microglial production of insulin-like growth factor-1.

Endogenous synaptogenesis in the deafferented dentate gyrus does not exclude synapse formation by embryonic entorhinal transplants

After partial deafferentation postsynaptic sites are reinnervated by local sprouting of remaining axons. We have investigated whether this process is sufficient to prevent new synapses being formed by transplanted embryonic tissue. We find that after unilateral entorhinal ablation endogenous sprouting by local axons is unable to reinnervate all the postsynaptic sites in the denervated outer dentate molecular layer. Axons from embryonic entorhinal tissue transplanted adjacent to the denervated area are able to reclaim a further proportion of the denervated postsynaptic sites. Thus, after a large lesion, endogenous sprouting is insufficient to preclude reinnervation by axons from embryonic transplants. ©1977 Elsevier Science B.V. All rights reserved.

Adhesion molecule expression on phagocytic microglial cells following anterograde degeneration of perforant path axons.

Entorhinal cortex lesion (ECL) leads to anterograde degeneration of perforant path axons and is known to induce a rapid and intense reaction of astrocytes and microglial cells in the deafferented dentate gyrus. Phagocytosis of degenerating axons involves the establishment and maintenance of cell-matrix and cell-cell interactions by activated glial cells. It was thus our aim to investigate whether the process of axon phagocytosis is accompanied by the expression of adhesion molecules on activated microglial cells or reactive astrocytes, as such molecules mediate bot cell-matrix and cell-cell interactions. We found that the integrin adhesion molecules leukocyte function antigen-1 (LFA-1), very late antigen-4 (VLA-4), and the ligand for LFA-1, intercellular adhesion molecule-1 (ICAM-1), were expressed on microglial cells accumulating in the outer molecular layer of the deafferented dentate gyrus. This upregulation of adhesion molecule expression on microglial cells showing morphological criteria of activation occurred rapidly following ECL, reached its peak at 3 days post lesion (dpl), and gradually returned to control levels after 9 dpl. Astrocytes were never labeled by antibodies directed against these adhesion molecules. Prelabeling of the perforant path with a fluorescent tracer and subsequent ECL led to phagocytosis of fluorescent-labeled axonal debris by cells that were located in the outer molecular layer and showed typical microglial morphology. Double-fluorescence labeling demonstrated that microglial cells engaged in the phagocytosis of axonal debris expressed LFA-1, VLA-4, and the LFA-1-ligand ICAM-1. In conclusion, our results demonstrate that anterograde degeneration of perforant path axons results in adhesion molecule expression on activated microglial cells engaged in axon phagocytosis. The expression of such molecules could represent a mechanism that retains activated microglia in areas of axonal degeneration and perhaps enables the interaction of microglial cells with each other or with other immunocompetent cells.

Expression of the neural adhesion molecule L1 in the deafferented dentate gyrus.

Expression of the neural adhesion molecule L1 and its potential involvement in axonal sprouting were examined in the deafferented rat dentate gyrus. We focused on the dentate gyrus because of its well-defined cytoarchitecture and well-characterized neuronal degeneration and sprouting response following entorhinal cortex lesions. In the molecular layer of the dentate gyrus, a trilaminar staining pattern was observed, with the middle molecular layer exhibiting slightly denser immunolabeling compared to both inner and outer molecular layers. Two to 12 days after a unilateral entorhinal cortex lesion, a progressive loss of L1 immunolabeling was noted in the ipsilateral middle and outer molecular layers, followed by a substantial reappearance of immunostaining 65 days after lesion incidence. The width of the immunostained ipsilateral inner molecular layer revealed a progressive widening and by postlesion day 65 occupied about 50% of the total width of the molecular layer. Immunoelectron microscopy localized L1 to the surface of unmyelinated axons in both normal and deafferented dentate gyrus. In situ hybridization revealed L1 messenger RNA confined to neurons throughout the hippocampal formation, but did not indicate changes in L1 messenger RNA levels in the hippocampus, dentate gyrus, entorhinal cortex or basal forebrain in response to unilateral entorhinal cortex lesions. Changes in L1 immunolabeling in the deafferented dentate gyrus corresponded in a spatial and temporal manner to changes of the synaptic marker synaptophysin and axonal marker phosphorylated tau. Results of the present study are most consistent with the view that L1 is expressed on reinnervating fibers after they make synaptic contacts with other structures. Thus, L1 appears to be involved in the maturation and stabilization of reinnervating fibers and consequently may play an important role in the repair process of the lesioned adult CNS.

Expression of Rat Cathepsin S in Phagocytic Cells

Cysteine lysosomal proteases are essential for turnover of intracellular and extracellular proteins. These enzymes are strongly implicated in normal and pathological processes involving tissue remodeling. Among the cysteine proteases, cathepsin S seems to be best suited for such a process since it retains most of its enzymatic activity at neutral pH. In situ hybridization analyses of the adult rat brain, spleen, and lung reveal that cathepsin S mRNA is preferentially expressed in cells of mononuclear-phagocytic origin. After entorhinal cortex lesion of adult rat brain (a paradigm for neuronal degeneration and reactive synaptogenesis), cathepsin S mRNA is dramatically increased in activated microglia in the deafferented dentate gyrus and in macrophages at the wound site, suggesting a role in lesion-induced tissue remodeling. This possibility is further supported by the finding that cathepsin S degrades a number of extracellular matrix molecules at neutral pH and by the finding that inflammatory mediators stimulate its secretion from the microglia and macrophages. These data suggest that cathepsin S is an important player in degenerative disorders associated with the cells of the mononuclear phagocytic system.

Functional Alterations in Alzheimerʼs Disease

Positron emission tomography (PET) studies measuring glucose utilization have demonstrated cerebral hypometabolism in Alzheimer's disease (AD). The anatomic and biochemical basis for this observation remains unknown. We have examined the distribution in the hippocampal formation of the neuron-specific glucose transporter 3 (Glut3) protein. Using quantitative immunohistochemistry, we find a large reduction (49.5%) in Glut3 immunoreactivity in the outer portion of the molecular layer of the dentate gyrus in AD brains. This region corresponds to the terminal zone of the perforant pathway, whose cells of origin in layer II of the entorhinal cortex are selectively destroyed in AD. Because glucose uptake reflects metabolic demand, these results suggest a decrement of functional activity in the deafferented dentate gyrus granule cells. Generalizing from this observation, decreased glucose uptake seen on PET studies may reflect, in part, decreased glucose transport and utilization in functionally deafferented cortical fields.

Glial beta-amyloid precursor protein: expression in the dentate gyrus after entorhinal cortex lesion.

Stereotactic lesioning of the rat entorhinal cortex leads to an induction of beta-amyloid precursor protein (APP) immunoreactivity in non-neuronal cells of the deafferented dentate gyrus. Double immunofluorescence against APP and the microglia-binding isolectin B4 from Griffonia simplicifolia revealed that APP immunoreactivity was confined to activated microglia. The microglial APP expression became detectable 3 days after lesioning, reached its peak after 7 days and disappeared after 10 days. The early accumulation of APP in microglia supports the view that microglia play an important role in the initial stages of amyloid plaque formation. Such a glial APP accumulation occurs rapidly in distant but anatomically connected areas. This is in line with the preferential localization of amyloid plaques in the dentate gyrus, which is the projection field of the degenerating neurones of the entorhinal cortex in patients with developing dementia.

Lesion of the rat entorhinal cortex leads to a rapid microglial reaction in the dentate gyrus

Stereotaxic lesioning of the entorhinal cortex leads to an anterograde axonal degeneration in the molecular layer of the dentate gyrus. As revealed by immunocytochemical and histochemical methods, lesion of the entorhinal cortex induced a proliferation of microglia and an increased expression of established microglial activation markers within the deafferented zone. Reactive microglial cells were detected as early as 24 h after the lesion. The microglial reaction showed a maximum around day 3 post-lesion and disappeared by day 8 post-lesion. Reactive microglia were strongly positive for the B4-isolectin from Griffonia simplicifolia (GSI-B4), expressed high levels of CR3 complement receptor and 5'-nucleotidase, but lacked CD4 and MHC class I and II antigens. In addition, microglial cells were identified using MUC 102, a new monoclonal antibody against rat microglia. At the ultrastructural level, reactive microglial cells were consistently seen to phagocytose degenerating terminals. Our data suggest that (1) axonal degeneration represents a sufficient stimulus for inducing microglial activation and proliferation in the deafferented dentate gyrus; (2) these activated microglial cells are characterized by immunophenotypes different from those observed in other types of CNS injury; (3) the early microglial reaction precedes the well-documented astrocyte reaction in the dentate gyrus; and (4) the timed interaction of microglia and astrocytes could be important for regulating regenerative sprouting processes in the mature CNS.

Astrocytosis in the molecular layer of the dentate gyrus: A study in Alzheimer's disease and schizophrenia

Recently, several authors have claimed prominent abnormalities in the entorhinal cortex of both patients with Alzheimer's disease (AD) and schizophrenia. The entorhinal cortex is the origin of the perforant pathway, a major input to granule cells of the dentate gyrus of the hippocampus. The present study explored the possibility of a lesion in the entorhinal cortex of both AD and schizophrenic patients by quantitating astrocytic markers within the terminal fields of the perforant pathway. An increase in fibrillary astrocytes was found in half ( 3 6 ) of the AD patients while none of the schizophrenic ( n = 6) or control ( n = 7) brains exhibited gliosis. Since the redistribution and hyperplasia of astrocytes within the molecular layer of the partially deafferented dentate gyrus depend on the chronicity of the entorhinal lesion, the abnormalities observed in AD patients are consistent with the progressive course of the illness. Furthermore, the presence of gliosis in the subiculum of three out of six AD patients suggested pathology secondary to projections from the entorhinal region, amygdala, or prepyriform cortex. The absence of similar changes in schizophrenic patients does not disprove previous claims of entorhinal pathology but suggests that the lesion, if it exists, is either static in nature or occurred long before death.

Astrocytosis in the molecular layer of the dentate gyrus: A study in Alzheimer's disease and schizophrenia

Recently, several authors have claimed prominent abnormalities in the entorhinal cortex of both patients with Alzheimer's disease (AD) and schizophrenia. The entorhinal cortex is the origin of the perforant pathway, a major input to granule cells of the dentate gyrus of the hippocampus. The present study explored the possibility of a lesion in the entorhinal cortex of both AD and schizophrenic patients by quantitating astrocytic markers within the terminal fields of the perforant pathway. An increase in fibrillary astrocytes was found in half ( 3 6 ) of the AD patients while none of the schizophrenic ( n = 6) or control ( n = 7) brains exhibited gliosis. Since the redistribution and hyperplasia of astrocytes within the molecular layer of the partially deafferented dentate gyrus depend on the chronicity of the entorhinal lesion, the abnormalities observed in AD patients are consistent with the progressive course of the illness. Furthermore, the presence of gliosis in the subiculum of three out of six AD patients suggested pathology secondary to projections from the entorhinal region, amygdala, or prepyriform cortex. The absence of similar changes in schizophrenic patients does not disprove previous claims of entorhinal pathology but suggests that the lesion, if it exists, is either static in nature or occurred long before death.

Absence of long-term potentiation in the subcortically deafferented dentate gyrus

All subcortical afferents to the dorsal hippocampus, running in the fimbria-fornix and supracallosal path, were removed by aspiration. Three to 5 months later the rats were implanted with chronic recording electrodes in the dentate gyrus and CA1 region, and stimulating electrodes in the angular bundle. In non-lesioned rats, high-frequency trains delivered to the angular bundle gave rise to a sustained increase of the evoked population spike in the dentate gyrus. In lesioned animals, high-frequency stimulation resulted in only short-lasting changes, and by 15 min after the conditioning trains the amplitude of both the population spike and field postsynaptic potentials returned to baseline. In lesioned rats large amplitude interictal spikes (< 40 ms, 3–8 mV) occurred spontaneously. These findings suggest that either (1) coactivation of entorhinal and subcortical inputs is essential for the induction of long-lasting plastic changes in the dentate gyrus, or (2) the long-term potentiation mechanism is saturated by the chronically occurring interictal discharges in the subcortically denervated dentate gyrus.

Glial contribution to amino acid content and metabolism of the deafferented dentate gyrus.

The time course of tissue content and evoked release of endogenous amino acids was analyzed in the partially deafferented dentate gyrus of the rat hippocampus 2-24 days following unilateral lesion of the perforant path. Amino acids in tissue extracts and perfusates were determined after precolumn derivatization and hplc separation. The astrocytic glial cell reaction was monitored with immunohistochemistry of S-100. The tissue content of glutamate decreased significantly on the lesioned side, whereas only a moderate reduction in taurine, aspartate, and alanine occurred. Glutamine was significantly elevated at 7 days. The evoked efflux of glutamate was reduced at 2 and 7 days, whereas no change was seen at longer survival periods. The evoked release of GABA and aspartate increased on the denervated side after 12 and 24 days. The rate of carbon utilization into amino acid pools was followed with 14C-glucose and 14C-acetate. The incorporation of acetate showed a peak 2-9 days following lesion, which paralleled in time the hypertrophic glial cells. The incorporation of glucose decreased during this period. The metabolic events are discussed in relation to the morphological changes in synapses and glial cells.

Terminal proliferation in the partially deafferented dentate gyrus: time courses for the appearance and removal of degeneration and the replacement of lost terminals.

The time courses for the appearance and removal of degenerating terminals and the loss and reappearance of intact terminals were investigated in the partially denervated inner molecular layer of the dentate gyrus of the adult rat. Dense degeneration was evident in the neuropil within 26 hours following contralateral hippocampectomy. These profiles increased rapidly in number until the maximal degree was reached at two to three days postlesion, after which the degenerating terminals were quickly removed from the neuropil. A more rapid rate of removal occurred during the 3-to 5-day survival period than from 6 to 50 days postlesion. The intact terminal population dropped 35% within two days of the lesion and remained at this level until six to eight days postlesion when the number began to steadily increase. The time course for this reappearance can be divided into two phases: a period of rapid terminal addition from 6 to 15 days followed by a phase of slower acquisition. This recovery continued until the normal synaptic density was regained by 50 to 65 days postlesion. These results indicate that a substantial proportion of degenerating endings are removed well in advance of the time at which terminal proliferation begins, suggesting that certain changes other than merely the removal of competitive inputs must take place prior to growth of new terminals. Possible explanation suggested by the present results for the delay in the onset of sprouting include: (1) an absence of appropriate postsynaptic targets during the 2-to 5-day postlesion period and (2) inhibition of axonal growth by the glial cells which are phagocytizing the degenerating products. Beyond the sixth postlesion day the rate at which new terminals appear does correlate with the rate at which degeneration is removed. This suggests that once underway the time course for sprouting may be determined by the avaiabliity of postsynaptic sites.

Ultrastructural evidence for bouton proliferation in the partially deafferented dentate gyrus of the adult rat.

A quantitative morphological study of the changes in the dentate gyrus molecular layer in response to the removal of perforant path afferents was made utilizing electron microscopic techniques. Alterations in 1. the population of remaining afferents, 2. glial cells, and 3. granule cell dendrites are reported. The major observation was an increase in intact bouton density in the region of denervation which began at 5 days post-lesion and continued through 11 days post-lesion, the longest post-lesion survival time studied.

Changes in the distribution of the dentate gyrus associational system following unilateral or bilateral entorhinal lesions in the adult rat

The distribution of the dentate gyrus associational system was analyzed in naive adult rats and in those with either unilateral or bilateral lesions of the entorhinal cortex. Horseradish peroxidase histochemistry was used to trace the origin and course of this intrinsic fiber system. The fibers originated in the CA3-4 pyramidal cell field, apparently medial to the origin of the Schaffer collateral system, and followed a trajectory which was essentially identical to that described for this system by Zimmer36. The associational terminal field occupied the inner 26% of the dentate gyrus molecular layer in normal rats and 35-38% of the normal width of that layer following either ipsilateral or bilateral entorhinal lesion. These measurements are quite similar to those previously obtained on the commissural system terminal field in the normal and partially deafferented dentate gyrus. These results are interpreted to reflect axon sprouting by the associational fibers into the adjacent deafferented dendritic field.

Hypertrophy and redistribution of astrocytes in the deafferented dentate gyrus

The response of the astroglial population of the dentate gyrus molecular layer to removal of that region's primary afferent was investigated using Cajal's gold sublimate method. Deafferentation caused the astrocytes to hypertrophy, an effect which was detectable at 24 hr and maximal at 72–96 hr post-lesion. Following this, the astroglia entered a lengthy period of gradual atrophy. Counts of the astrocytes in the various sublayers of the molecular layer led to the conclusion that these cells migrate into denervated dendritic areas from neighboring, nondeafferented zones.

Growth of a new fiber projection in the brain of adult rats: Re-innervation of the dentate gyrus by the contralateral entorhinal cortex following ipsilateral entorhinal lesions.

Ablation of the entorhinal cortex of the rat removes the major synaptic input to the granule cells of the ipsilateral dentate gyrus. Following unilateral entorhinal lesions in adult rats, we have examined the efferent projections of the remaining contralateral entorhinal cortex to determine if these might sprout to re-innervate the deafferented dentate gyrus. Autoradiographical tracing of the fiber projections of the remaining contralateral entorhinal cortex 60 days following lesions indicates that new fibers sprout and grow for several hundred microns into the denervated regions, to terminate on portions of the granule cell dendrites which would normally receive ipsilateral entorhinal afferents.