Outer Molecular Layer Of Dentate Gyrus Research Articles

Re-innervation of the Denervated Dentate Gyrus by Sprouting Associational and Commissural Mossy Cell Axons in Organotypic Tissue Cultures of Entorhinal Cortex and Hippocampus.

Collateral sprouting of surviving axons contributes to the synaptic reorganization after brain injury. To study this clinically relevant phenomenon, we used complex organotypic tissue cultures of mouse entorhinal cortex (EC) and hippocampus (H). Single EC-H cultures were generated to analyze associational sprouting, and double EC-H cultures were used to evaluate commissural sprouting of mossy cells in the dentate gyrus (DG) following entorhinal denervation. Entorhinal denervation (transection of the perforant path) was performed at 14 days in vitro (DIV) and associational/commissural sprouting was assessed at 28 DIV. First, associational sprouting was studied in genetically hybrid EC-H cultures of beta-actin-GFPtg and wild-type mice. Using calretinin as a marker, associational axons were found to re-innervate almost the entire entorhinal target zone. Denervation experiments performed with EC-H cultures of Thy1-YFPtg mice, in which mossy cells are YFP-positive, confirmed that the overwhelming majority of sprouting associational calretinin-positive axons are mossy cell axons. Second, we analyzed associational/commissural sprouting by combining wild-type EC-H cultures with calretinin-deficient EC-H cultures. In these cultures, only wild-type mossy cells contain calretinin, and associational and commissural mossy cell collaterals can be distinguished using calretinin as a marker. Nearly the entire DG entorhinal target zone was re-innervated by sprouting of associational and commissural mossy cell axons. Finally, viral labeling of newly formed associational/commissural axons revealed a rapid post-lesional sprouting response. These findings demonstrate extensive and rapid re-innervation of the denervated DG outer molecular layer by associational and commissural mossy cell axons, similar to what has been reported to occur in juvenile rodent DG in vivo.

The mTOR Inhibitor Rapamycin Mitigates Perforant Pathway Neurodegeneration and Synapse Loss in a Mouse Model of Early-Stage Alzheimer-Type Tauopathy.

The perforant pathway projection from layer II of the entorhinal cortex to the hippocampal dentate gyrus is especially important for long-term memory formation, and is preferentially vulnerable to developing a degenerative tauopathy early in Alzheimer’s disease (AD) that may spread over time trans-synaptically. Despite the importance of the perforant pathway to the clinical onset and progression of AD, a therapeutic has not been identified yet that protects it from tau-mediated toxicity. Here, we used an adeno-associated viral vector-based mouse model of early-stage AD-type tauopathy to investigate effects of the mTOR inhibitor and autophagy stimulator rapamycin on the tau-driven loss of perforant pathway neurons and synapses. Focal expression of human tau carrying a P301L mutation but not eGFP as a control in layer II of the lateral entorhinal cortex triggered rapid degeneration of these neurons, loss of lateral perforant pathway synapses in the dentate gyrus outer molecular layer, and activation of neuroinflammatory microglia and astroglia in the two locations. Chronic systemic rapamycin treatment partially inhibited phosphorylation of a mechanistic target of rapamycin substrate in brain and stimulated LC3 cleavage, a marker of autophagic flux. Compared with vehicle-treated controls, rapamycin protected against the tau-induced neuronal loss, synaptotoxicity, reactive microgliosis and astrogliosis, and activation of innate neuroimmunity. It did not alter human tau mRNA or total protein levels. Finally, rapamycin inhibited trans-synaptic transfer of human tau expression to the dentate granule neuron targets for the perforant pathway, likely by preventing the synaptic spread of the AAV vector in response to pathway degeneration. These results identify systemic rapamycin as a treatment that protects the entorhinal cortex and perforant pathway projection from tau-mediated neurodegeneration, axonal and synapse loss, and neuroinflammatory reactive gliosis. The findings support the potential for slowing the progression of AD by abrogating tau-mediated neurotoxicity at its earliest neuropathological stages.

Distinct increased metabotropic glutamate receptor type 5 (mGluR5) in temporal lobe epilepsy with and without hippocampal sclerosis

Metabotropic glutamate receptor type 5 (mGluR5) upregulation in temporal lobe epilepsy (TLE) and the correlation of its expression with features of hippocampal sclerosis (HS) remains unclear. Here we characterized mGluR5 immunoreactivity in hippocampus, entorhinal cortex (EC), and subiculum of TLE specimens with confirmed HS, with neocortical TLE (non-HS) and necropsy controls. We correlated mGluR5 immunoreactivity with neuronal density, mossy fiber sprouting, astrogliosis (GFAP), and dendritic alterations (MAP2). TLE specimens showed increased mGluR5 expression, which was most pronounced in the EC, subiculum, CA2, and dentate gyrus outer molecular layer. Increased mGluR5 expression was seen in hippocampal head and body segments and was independent of neuronal density, astrogliosis, or dendritic alterations. Positive correlation between mGluR5 expression with mossy fiber sprouting and with MAP2 in CA3 and CA1 was found only in HS specimens. Negative correlation between mGluR5 expression with seizure frequency and epilepsy duration was found only in non-HS cases. Specimens from HS patients without previous history of febrile seizure (FS) showed higher mGluR5 and MAP2 expression in CA2. Our study suggests that mGluR5 upregulation is part of a repertoire of post-synaptic adaptations that might control overexcitation and excessive glutamate release rather than a dysfunction that leads to seizure facilitation. That would explain why non-HS cases, on which seizures are likely to originate outside the hippocampal formation, also exhibit upregulated mGluR5. On the other hand, lower mGluR5 expression was related to increased seizure frequency. In addition to its role in hyperexcitability, mGluR5 upregulation could play a role in counterbalance mechanisms along the hyperexcitable circuitry uniquely altered in sclerotic hippocampal formation. Inefficient post-synaptic compensatory morphological (dendritic branching) and glutamatergic (mGluR5 expression) mechanisms in CA2 subfield could potentially underlie the association of FS with HS and TLE. © 2013 Wiley Periodicals, Inc.

Testing Mitochondrial Metabolic Competence by Cytochrome Oxidase Preferential Cytochemistry Versus Immunoreactivity of Subunits I and IV

Cytochemically evidenced COX activity was compared with levels of immunohistochemically stained mitochondrial- and nuclear-encoded subunits (CO I and CO IV) in the dentate gyrus outer molecular layer (OML) and cerebellar granular layer (GL) of adult and old rats. COX activity decreased significantly in aging, whereas CO I and CO IV levels were significantly increased both in GL and OML of old animals. These findings suggest that the age-related decay of the mitochondrial metabolic competence is not caused by a reduction of COX subunits levels, but causal events affecting mitochondria as discrete morphofunctional units of the cellular bioenergetic machinery.

Estrogen increases the number of spinophilin-immunoreactive spines in the hippocampus of young and aged female rhesus monkeys.

It is well documented that estrogen increases dendritic spine density in CA1 pyramidal cells of young female rats. However, this effect is attenuated in aged rats. We report here a quantitative analysis of estrogen effects on hippocampal spine number as visualized with antispinophilin in young (6-8 years old) and aged (19-23 years old) female rhesus monkeys, a species with a pattern of female endocrine senescence comparable to that of humans. Monkeys were ovariectomized and administered either vehicle or estradiol cypionate 3 months postovariectomy, followed by an additional dose 3 weeks later, with perfusion 24 hours after the last estrogen treatment. Immunolocalization of spinophilin, a spine-associated protein, was used for quantitative stereologic analyses of total spinophilin-immunoreactive spine numbers in CA1 stratum radiatum and the inner and outer molecular layers of dentate gyrus. In both young and aged female monkeys, the estrogen-treated groups had an increase in spinophilin-immunoreactive spines (37% in young, P <.005; 35% in aged, P <.05) compared with the untreated groups that amounted to more than 1 billion additional immunoreactive spines. The young group also showed a trend toward an estrogen-induced increase in immunoreactive spines in the dentate gyrus outer molecular layer, but this effect was not statistically significant (P =.097). We conclude that spine number in the rhesus monkey hippocampus is highly responsive to estrogen, yet, unlike the female rat, aged female rhesus monkeys retain the capacity for spine induction in response to estrogen. These data have important implications for cognitive effects of estrogen replacement in postmenopausal women and demonstrate that an estrogen replacement protocol that mimics normal physiological cycles with timed, intermittent peaks can have profound neurobiological effects.

Delayed peripheral nerve regeneration and central nervous system collateral sprouting in leucocyte common antigen-related protein tyrosine phosphatase-deficient mice.

Cell adhesion molecule-like receptor-type protein tyrosine phosphatases have been shown to be important for neurite outgrowth and neural development in several animal models. We have previously reported that in leucocyte common antigen-related (LAR) phosphatase deficient (LAR-deltaP) mice the number and size of basal forebrain cholinergic neurons, and their innervation of the hippocampal area, is reduced. In this study we compared the sprouting response of LAR-deficient and wildtype neurons in a peripheral and a central nervous system lesion model. Following sciatic nerve crush lesion, LAR-deltaP mice showed a delayed recovery of sensory, but not of motor, nerve function. In line with this, neurofilament-200 immunostaining revealed a significant reduction in the number of newly outgrowing nerve sprouts in LAR-deltaP animals. Morphometric analysis indicated decreased axonal areas in regenerating LAR-deltaP nerves when compared to wildtypes. Nonlesioned nerves in wildtype and LAR-deltaP mice did not differ regarding myelin and axon areas. Entorhinal cortex lesion resulted in collateral sprouting of septohippocampal cholinergic fibres into the dentate gyrus outer molecular layer in both genotype groups. However, LAR-deltaP mice demonstrated less increase in acetylcholinesterase density and fibre number at several time points following the lesion, indicating a delayed collateral sprouting response. Interestingly, a lesion-induced reduction in number of (septo-entorhinal) basal forebrain choline acetyltransferase-positive neurons occurred in both groups, whereas in LAR-deltaP mice the average cell body size was reduced as well. Thus, regenerative and collateral sprouting is significantly delayed in LAR-deficient mice, reflecting an important facilitative role for LAR in peripheral and central nervous system axonal outgrowth.

Analysing metabotropic glutamate group III receptor mediated modulation of synaptic transmission in the amygdala-kindled dentate gyrus of the rat

Metabotropic glutamate receptors (mGluRs) provide a powerful control of synaptic transmission in the hippocampus and may serve as a target for drug development in human temporal lobe epilepsies. Agonists and antagonists at these receptors influence the development and propagation of seizures in some animal models of epilepsy. Experimental seizures can change the level of expression of mGluRs in the rat hippocampus. In the human dentate gyrus of patients suffering from temporal lobe epilepsy (TLE), group III mGluR mediated inhibition of synaptic transmission is almost lost in the sub-group with Ammon's horn sclerosis. We tested the modulation of synaptic transmission by the group III mGluR specific agonist l(+)-2-amino-4-phosphonobutyric acid ( l-AP4) in the dentate gyrus outer molecular layer in control and amygdala-kindled rats, a common model for TLE. Extracellular field potential recordings upon subthreshold stimulation of lateral perforant path fibers were measured simultaneously in the outer molecular layer and granule cell layer. Analysis of `paired-pulse' characteristics in the absence and presence of l-AP4 and group III mGluR mediated inhibition of synaptic transmission in the lateral perforant path revealed no significant alterations in fully kindled rats. Since there is no evidence of altered l-AP4 responses, a loss of group III mGluR function, particularly that of subtype mGluR8, seems not necessary for the kindling epilepsy.

Astroglial ciliary neurotrophic factor mRNA expression is increased in fields of axonal sprouting in deafferented hippocampus

Evidence that ciliary neurotrophic factor promotes axonal sprouting and regeneration in the periphery raises the possibility that this factor is involved in reactive axonal growth in the brain. In situ hybridization was used in the present study to determine whether ciliary neurotrophic factor mRNA expression is increased in association with axonal sprouting in deafferented adult rat hippocampus. In untreated rats, ciliary neurotrophic factor cRNA labeling density was high in the olfactory nerve, pia mater, and aspects of the ventricular ependyma and was relatively low within areas of white matter (fimbria, internal capsule) and select neuronal fields (hippocampal cell layers, habenula). After an entorhinal cortex lesion, hybridization was markedly increased in fields of anterograde degeneration, including most prominently the ipsilateral dentate gyrus outer molecular layer and hippocampal stratum lacunosum moleculare. Labeling in these fields was increased by 3 days postlesion, was maximal at 5 days, and returned to normal levels by 14 days. Double labeling demonstrated that, in both control and experimental tissue, ciliary neurotrophic factor mRNA was colocalized with glial fibrillary acidic protein immunoreactivity in astroglia, but it was not colocalized with markers for oligodendrocytes or microglia. These results demonstrate that astroglial ciliary neurotrophic factor expression is increased in fields of axonal and terminal degeneration and that increased expression is coincident with 1) increased insulin-like growth factor-1 and basic fibroblast growth factor expression and 2) the onset of reactive axonal growth. The synchronous expression of these glial factors in fields of deafferentation suggests the possibility of additive or synergistic interactions in the coordination of central axonal growth. J. Comp. Neurol. 386:137–148, 1997. © 1997 Wiley-Liss, Inc.

Astroglial ciliary neurotrophic factor mRNA expression is increased in fields of axonal sprouting in deafferented hippocampus

Evidence that ciliary neurotrophic factor promotes axonal sprouting and regeneration in the periphery raises the possibility that this factor is involved in reactive axonal growth in the brain. In situ hybridization was used in the present study to determine whether ciliary neurotrophic factor mRNA expression is increased in association with axonal sprouting in deafferented adult rat hippocampus. In untreated rats, ciliary neurotrophic factor cRNA labeling density was high in the olfactory nerve, pia mater, and aspects of the ventricular ependyma and was relatively low within areas of white matter (fimbria, internal capsule) and select neuronal fields (hippocampal cell layers, habenula). After an entorhinal cortex lesion, hybridization was markedly increased in fields of anterograde degeneration, including most prominently the ipsilateral dentate gyrus outer molecular layer and hippocampal stratum lacunosum moleculare. Labeling in these fields was increased by 3 days postlesion, was maximal at 5 days, and returned to normal levels by 14 days. Double labeling demonstrated that, in both control and experimental tissue, ciliary neurotrophic factor mRNA was colocalized with glial fibrillary acidic protein immunoreactivity in astroglia, but it was not colocalized with markers for oligodendrocytes or microglia. These results demonstrate that astroglial ciliary neurotrophic factor expression is increased in fields of axonal and terminal degeneration and that increased expression is coincident with 1) increased insulin-like growth factor-1 and basic fibroblast growth factor expression and 2) the onset of reactive axonal growth. The synchronous expression of these glial factors in fields of deafferentation suggests the possibility of additive or synergistic interactions in the coordination of central axonal growth.

Distribution of AMPA receptor subunits in the hippocampal formation of temporal lobe epilepsy patients

The immunocytochemical distribution of the AMPA-selective receptor subunits GluR1 and GluR2/3 were mapped in the human hippocampal formation obtained from surgery for medically intractable temporal lobe epilepsy. GluR2/3 immunoreactivity was detected in all principal cell types of the hippocampal formation, including hilar neurons, granule cells of the dentate gyrus, and pyramidal cells of the cornu ammonis fields and subiculum. GluR2/3 immunostaining typically filled the cell bodies and processes of neurons. A comparison of GluR2/3 immunoreactivity in a sclerotic specimen versus a non-sclerotic specimen demonstrated a profound loss of staining, specifically in the areas where neuronal dropout was occurring, including CA1, CA3 and the hilus. An analysis of GluR1 immunoreactivity in non-sclerotic specimens revealed that it was predominantly localized to cellular processes throughout the cornu ammonis fields, with a sparse staining of the dentate gyrus outer molecular layer and little to no staining of the dentate gyrus inner molecular layer. Similar to the GluR2/3-immunostained patterns, GluR1 immunoreactivity was lost in the cornu ammonis fields of sclerotic hippocampal specimens, corresponding to patterns of neuronal dropout. Our most compelling finding was a unique extensive pattern of GluR1 and Glu2/3 immunoreactivity throughout the molecular layers of the dentate gyrus of severely compromised hippocampi. The altered staining of GluR1 and GluR2/3 complements some of the patterns of axonal sprouting already described for the dentate gyrus, with a conjecture that their anatomy and distribution pattern underlies to some degree the reorganization of the sclerotic hippocampus. A combination of enhanced glutamatergic transmission and changes in neuropeptides that modulate hippocampal circuitry could greatly affect the degree of excitability in the hippocampal formation. The alterations of GluR1 and GluR2/3 immunoreactivity in the dentate gyrus add another component to the concept of reorganization in the epileptic sclerotic hippocampus.

Insulin‐like growth factor‐1 mRNA is increased in deafferented hippocampus: Spatiotemporal correspondence of a trophic event with axon sprouting

Deafferentation is known to induce axonal sprouting in adult brain, but the signals that direct this response are not understood. To evaluate the possible roles of insulin-like growth factor-1 (IGF-1) and basic fibroblast growth factor (bFGF) in central axonal sprouting, the present study used in situ hybridization to evaluate IGF-1 and bFGF mRNA expression in entorhinal deafferented rat hippocampus. Alternate tissue sections were processed for Fink-Heimer impregnation of axonal degeneration, Bandeiraea simplicifolia (BS-1) labeling of microglia, and glial fibrillary acidic protein immunocytochemistry. In control hippocampus, IGF-1 mRNA was localized to a few neurons, with no labeled cells in the dentate gyrus molecular layer; bFGF cRNA hybridization was diffuse in dendritic fields but was dense in CA2 stratum pyramidale. Both mRNA species were increased by deafferentation. The distribution of elevated IGF-1 mRNA corresponded precisely to fields of axonal degeneration and was greatest in the dentate gyrus outer molecular layer and stratum lacunosum moleculare. In these fields, IGF-1 mRNA was elevated by 2 days, reached maximal levels at 4 days, and declined by 10 days postlesion. Double labeling revealed that the majority of IGF-1 cRNA-labeled cells were microglia. In deafferented hippocampus, bFGF mRNA was broadly increased across fields both containing and lacking axonal degeneration. In the dentate, bFGF mRNA levels peaked at 5 days postlesion and remained elevated through 14 days. These results demonstrate that reactive microglia within deafferented hippocampal laminae express IGF-1 mRNA just prior to and during the period of reactive axonal growth and suggest that IGF-1 plays a role in directing the sprouting of spared afferents into these fields.

Deafferentation removes calretinin immunopositive terminals, but does not induce degeneration of calbindin D‐28k and parvalbumin expressing neurons in the hippocampus of adult rats

Unilateral combined transections of the fimbriafornix and angular bundle in adult Fischer 344 rats were used to study the effects of deafferentation on hippocampal expression of calretinin, calbindin D-28k, and parvalbumin. Reflecting the widespread degeneration of synaptic contacts, immunostaining for glial fibrillary acidic protein 6 days after the lesions was increased in lacunosum-molecular and oriens layers of CA1, 2, and 3 in ipsi- and contralateral hippocampus and in the ipsilateral dentate gyrus outer molecular layer. At 21 days the immunoreactivity had decreased to control levels except for a still slightly increased signal in the oriens layer of CA1-3. At 6 and 21 days after the combined lesions the numbers of hippocampal neurons containing calretinin, parvalbumin, and calbindin D-28k was unaltered. The combined lesions abolished calretinin containing terminals in the dentate gyrus inner molecular layer on the deafferentated side. This could be reproduced by single unilateral fimbria-fornix transections, suggesting that the axons of these calretinin positive terminals project to the hippocampus through the fimbria-fornix. The most likely origin of the calretinin positive terminals are neurons in the supramammillary hypothalamic nucleus. Our findings demonstrate that the extensive lesion-induced synaptic rearrangements in the adult hippocampus do not induce degeneration of hippocampal neurons expressing calretinin, calbindin D-28k, and parvalbumin, but do remove calretinin containing terminals which reach their targets in the hippocampus through the fimbria-fornix.

The effect of entorhinal cortical ablation on the distribution of muscarinic cholinergic receptors in the rat hippocampus

Removal of the entorhinal cortical projection to the hippocampus in adult rats decreased the density of muscarinic cholinergic receptors in the denervated dentate gyrus outer molecular layer at two days postlesion. Thirty days following the lesion (in adults and neonates) there is a small receptor density increase in the outer molecular layer (may be due to tissue shrinkage), and a larger increase in the lacunosum-moleculare. The receptor density decrease seen two days postlesion suggests the presence of presynaptic muscarinic receptors on the lost entorhinal cortical fibers. The distribution and extent of the receptor changes seen at 30 days postlesion are inconsistent with the cholinergic fiber reorganization which follows an entorhinal cortical lesion, but are consistent with a proposed model of non-cholinergic afferent mediated control of muscarinic receptor density in the rat hippocampus.