Dentate Gyrus Of Aged Rats Research Articles

Isolation and Culture of Neural Stem/Progenitor Cells from the Hippocampal Dentate Gyrus of Young Adult and Aged Rats.

Adult neural stem/progenitor cells (NSPCs) in two neurogenic areas of the brain, the dentate gyrus and the subventricular zone, are major players in adult neurogenesis. Addressing specific questions regarding NSPCs outside of their niche entails in vitro studies through isolation and culture of these cells. As there is heterogeneity in their morphology, proliferation, and differentiation capacity between these two neurogenic areas, NSPCs should be isolated from each area through specific procedures and media. Identifying region-specific NPSCs provides an accurate pathway for assessing the effects of extrinsic factors and drugs on these cells and investigating the mechanisms of neurogenesis in both healthy and pathologic conditions. A great number of isolation and expansion techniques for NSPCs have been reported. The growth and expansion of NSPCs obtained from the dentate gyrus of aged rats are generally difficult. There are relatively limited data and protocols about NSPCs isolation and their culture from aged rats. Our approach is an efficient and reliable strategy to isolate and expand NSPCs obtained from young adult and aged rats. NSPCs isolated by this method maintain their self-renewal and multipotency. Key features • NSPCs isolated from the hippocampal dentate gyrus of young adult and aged rats, based on Kempermann et al. (2014) and Aligholi et al. (2014). • Maintenance of NSPCs isolated from the dentate gyrus of aged rats (20-24 months) in our culture condition is feasible. • According to our protocol, maximum growth of primary neurospheres obtained from isolated NSPCs of young and aged rats took 15 and 35 days, respectively.

Neuroprotective effects of a Smoothened receptor agonist against postoperative cognitive dysfunction by promoting autophagy in the dentate gyrus of aged rats

ABSTRACTObjectives: To investigate the effect of purmorphamine (PUR), a Shh co-receptor Smoothened (Smo) agonist, on postoperative cognitive dysfunction (POCD) rat models.Methods: Eighteen-month-old male Sprague–Dawley rats were subjected to intramedullary fixation of a tibial fracture with 7% chloral hydrate anesthesia to mimic human clinical surgery. PUR was administered via an intraperitoneal injection at a dose of 15mg/kg/day for 3 consecutive days at 6 h after surgery. The aged rats were sacrificed after performing a Morris water maze test 1, 3, and 7 days postoperatively to evaluate the expression of related proteins at the appointed time.Results: Compared to the POCD + vehicle group and sham + PUR group, the POCD + PUR group restored neurological deficit (P = 0.01). PUR administration induced upregulation of Shh expression on postoperative day 1 (P = 0.02), which continued on the third day (P = 0.008) but dropped by the 7th day (P = 0.03). Immunofluorescent analysis, similar to western blot analysis, showed a significant increase in the autophagy-marker LC3 (P = 0.006) as well as p62 degradation (P = 0.000) in the dentate gyrus of the aged rats (P = 0.000) after PUR treatment. Importantly, LC3 was mainly found in the presynaptic and postsynaptic membranes of the hippocampus.Conclusions: These results indicate a link between Shh and autophagy in the rat model of POCD, providing new insights into Shh signaling pathway-mediated mechanisms of neuroprotection and cognitive repair after POCD. It also provides a potential entry point for the development of clinical drugs.

Is Vulnerability of the Dentate Gyrus to Aging and Amyloid-β1-42 Neurotoxicity Linked with Modified Extracellular Zn2+ Dynamics?

The basal levels of extracellular Zn2+ are in the range of low nanomolar concentrations in the hippocampus and perhaps increase age-dependently. Extracellular Zn2+ dynamics is critical for cognitive activity and excess influx of extracellular Zn2+ into hippocampal neurons is a known cause of cognitive decline. The dentate gyrus is vulnerable to aging in the hippocampus and affected in the early stage of Alzheimer's disease (AD). The reasons remain unclear. Neurogenesis-related apoptosis may induce non-specific neuronal depolarization by efflux of intracellular K+ in the dentate gyrus and be markedly increased along with aging. Extracellular Zn2+ influx into dentate granule cells via high K+-induced perforant pathway excitation leads to cognitive decline. Modified extracellular Zn2+ dynamics in the dentate gyrus of aged rats is linked with vulnerability to cognitive decline. Amyloid-β1-42 (Aβ1-42) is a causative candidate for AD pathogenesis. When Aβ1-42 concentration reaches picomolar in the extracellular compartment in the dentate gyrus, Zn-Aβ1-42 is formed in the extracellular compartment and rapidly taken up into dentate granule cells, followed by Aβ1-42-induced cognitive decline that is due to Zn2+ released from Aβ1-42, suggesting that dentate granule cells are sensitive to extracellular Zn2+-dependent Aβ1-42 toxicity. This paper deals with proposed vulnerability of the dentate gyrus to aging and Aβ1-42 neurotoxicity.

Weakened Intracellular Zn2+-Buffering in the Aged Dentate Gyrus and Its Involvement in Erasure of Maintained LTP.

Memory is lost by the increased influx of extracellular Zn2+ into neurons. It is possible that intracellular Zn2+ dynamics is modified even at non-zincergic medial perforant pathway-dentate granule cell synapses along with aging and that vulnerability to the modification is linked to age-related cognitive decline. To examine these possibilities, vulnerability of long-term potentiation (LTP) maintenance, which underlies memory retention, to modification of synaptic Zn2+ dynamics was compared between young and aged rats. The influx of extracellular Zn2+ into dentate granule cells was increased in aged rats after injection of high K+ into the dentate gyrus, but not in young rats. This increase impaired maintained LTP in aged rats. However, the impairment was rescued by co-injection of CaEDTA, an extracellular Zn2+ chelator, or CNQX, an AMPA receptor antagonist, which suppressed the Zn2+ influx. Maintained LTP was also impaired in aged rats after injection of ZnAF-2DA into the dentate gyrus that chelates intracellular Zn2+, but not in young rats. Interestingly, the capacity of chelating intracellular Zn2+ with intracellular ZnAF-2 was almost lost in the aged dentate gyrus 2h after injection of ZnAF-2DA into the dentate gyrus, suggesting that intracellular Zn2+-buffering is weakened in the aged dentate gyrus, compared to the young dentate gyrus. In the dentate gyrus of aged rats, maintained LTP is more vulnerable to modification of intracellular Zn2+ dynamics than in young rats, probably due to weakened intracellular Zn2+-buffering.

Surface L‐type Ca2+ channel expression levels are increased in aged hippocampus

Age-related increase in L-type Ca2+ channel (LTCC) expression in hippocampal pyramidal neurons has been hypothesized to underlie the increased Ca2+ influx and subsequent reduced intrinsic neuronal excitability of these neurons that lead to age-related cognitive deficits. Here, using specific antibodies against Cav1.2 and Cav1.3 subunits of LTCCs, we systematically re-examined the expression of these proteins in the hippocampus from young (3 to 4 month old) and aged (30 to 32 month old) F344xBN rats. Western blot analysis of the total expression levels revealed significant reductions in both Cav1.2 and Cav1.3 subunits from all three major hippocampal regions of aged rats. Despite the decreases in total expression levels, surface biotinylation experiments revealed significantly higher proportion of expression on the plasma membrane of Cav1.2 in the CA1 and CA3 regions and of Cav1.3 in the CA3 region from aged rats. Furthermore, the surface biotinylation results were supported by immunohistochemical analysis that revealed significant increases in Cav1.2 immunoreactivity in the CA1 and CA3 regions of aged hippocampal pyramidal neurons. In addition, we found a significant increase in the level of phosphorylated Cav1.2 on the plasma membrane in the dentate gyrus of aged rats. Taken together, our present findings strongly suggest that age-related cognitive deficits cannot be attributed to a global change in L-type channel expression nor to the level of phosphorylation of Cav1.2 on the plasma membrane of hippocampal neurons. Rather, increased expression and density of LTCCs on the plasma membrane may underlie the age-related increase in L-type Ca2+ channel activity in CA1 pyramidal neurons.

Long‐term treadmill exposure protects against age‐related neurodegenerative change in the rat hippocampus

The potential of exercise or environmental enrichment to prevent or reverse age-related cognitive decline in rats has been widely investigated. The data suggest that the efficacy of these interventions as neuroprotectants may depend upon the duration and nature of the protocols and age of onset. Investigations of the mechanisms underlying these neuroprotective strategies indicate a potential role for the neurotrophin family of proteins, including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). In this study, we have assessed the effects of 8 months of forced exercise, begun in middle-age, on the expression of long-term potentiation (LTP) and on spatial learning in the Morris water maze in aged Wistar rats. We also assessed these measures in a cage control group and in a group of rats exposed to the stationary treadmill for the same duration as the exercised rats. Our data confirm an age-related decline in expression of LTP and in spatial learning concomitant with decreased expression of NGF and BDNF mRNA in dentate gyrus (DG). The age-related impairments in both plasticity and growth factor expression were prevented in the long-term exercised group and, surprisingly, the treadmill control group. Given the extensive handling that the treadmill control group received and their regular exposure to an environment outside the home cage, this group can be considered to have experienced environmentally enriched conditions when compared with the cage control group. Significant correlations were observed between both learning and LTP and the expression of NGF and BDNF mRNA in the dentate gyrus. We conclude that decreased expression of NGF and BDNF in the dentate gyrus of aged rats is associated with impaired LTP and spatial learning. We suggest that the reversal of these age-related impairments by enrichment and exercise may be linked with prevention of the age-related decline in expression of these growth factors and, furthermore, that enrichment is as efficacious as exercise in preventing this age-related decline.

Cannabinoids attenuate the effects of aging upon neuroinflammation and neurogenesis

WIN-55,212-2 (WIN-2) can elicit anti-inflammatory and cognitive-enhancing effect in aged rats. The current study was designed to determine the differential role of the endocannabinoid receptor sub-types 1 (CB1) and 2 (CB2) and transient receptor potential vanilloid 1 receptor (TRPV1) in the reduction of age-associated brain inflammation and their effects on neurogenesis in the dentate gyrus of aged rats. Our results demonstrate that 1) the antagonist actions of WIN-2 at the TRPV1 receptor are responsible for the reduction in microglial activation and 2) the agonist actions of WIN-2 at CB1/2 receptors can trigger neurogenesis in the hippocampus of aged rats. Chronic treatment with WIN-2 established an anti-inflammatory cytokine profile within the hippocampus. Our results provide insight into the role of the endocannabinoid and vanilloid systems upon two different and detrimental aspects of normal and pathological aging, chronic neuroinflammation and decline in neurogenesis.

Genome-wide analysis of aging and learning-related genes in the hippocampal dentate gyrus

We have previously described the transcriptional changes that occur in the hippocampal CA1 field of aged rats following a Morris Water Maze (MWM) training paradigm. In this report we proceed with the analysis of the dentate region from the same animals. Animals were first identified as age learning-impaired or age-superior learners when compared to young rats based on their performance in the MWM. Messenger RNA was isolated from the dentate gyrus of each animal to interrogate Affymetrix RAE 230A rat genome microarrays. Microarray profiling identified 1129 genes that were differentially expressed between aged and young rats as a result of aging, and independent of their behavioral training ( p < 0.005). We applied Ingenuity Pathway Analysis (IPA) algorithms to identify the significant biological processes underlying age-related changes in the dentate gyrus. The most significant functions, as calculated by IPA, included cell movement, cell growth and proliferation, nervous system development and function, cellular assembly and organization, cell morphology and cell death. These significant processes are consistent with age-related changes in neurogenesis, and the neurogenic markers were generally found to be downregulated in senescent animals. In addition, statistical analysis of the different experimental groups of aged animals recognized 85 genes ( p < 0.005) that were different in the dentate gyrus of aged rats that had learned the MWM when compared to learning impaired and a number of controls for stress, exercise and non-spatial learning. The list of learning-related genes expressed in the dentate adds to the set of genes we previously described in the CA1 region. This long list of genes constitutes a starting tool to elucidating the molecular pathways involved in learning and memory formation.

A cell adhesion molecule mimetic, FGL peptide, induces alterations in synapse and dendritic spine structure in the dentate gyrus of aged rats: a three‐dimensional ultrastructural study

The FGL peptide is a neural cell adhesion molecule (NCAM) mimetic comprising a 15-amino-acid-long sequence of the FG loop region of the second fibronectin type III module of NCAM. It corresponds to the binding site of NCAM for the fibroblast growth factor receptor 1. FGL improves cognitive function through enhancement of synaptic function. We examined the effect of FGL on synaptic and dendritic structure in the brains of aged (22-month-old) rats that were injected subcutaneously (8 mg/kg) at 2-day intervals until 19 days after the start of the experiment. Animals were perfused with fixative, brains removed and coronal sections cut at 50 microm. The hippocampal volume was measured, tissue embedded and ultrathin sections viewed in a JEOL 1010 electron microscope. Analyses were made of synaptic and dendritic parameters following three-dimensional reconstruction via images from a series of approximately 100 serial ultrathin sections. FGL affected neither hippocampal volume nor spine or synaptic density in the middle molecular layer of the dentate gyrus. However, it increased the ratio of mushroom to thin spines, number of multivesicular bodies and also increased the frequency of appearance of coated pits. Three-dimensional analysis showed a significant decrease in both post-synaptic density and apposition zone curvature of mushroom spines following FGL treatment, whereas for thin spines the convexity of the apposition zone increased. These data indicate that FGL induces large changes in the fine structure of synapses and dendritic spines in hippocampus of aged rats, complementing data showing its effect on cognitive processes.

Age-related changes in CREB binding protein immunoreactivity in the cerebral cortex and hippocampus of rats

Although the role of cAMP-response-element-binding protein (CREB) binding protein (CBP) in the neuroprotective mechanisms has been the focus of many studies, very little is known about the expression or function of CBP in aged brains. We have therefore examined age-related changes in CBP expression in the cerebral cortex and hippocampus with an immunohistochemical technique. In the cerebral cortex, the distribution patterns were not different between adult and aged groups, but the staining intensity of CBP was significantly decreased in aged rats. In the hippocampus, a distinct immunoreactivity pattern was observed in the CA1–3 areas and dentate gyrus. CBP immunoreactivity was significantly deceased in the pyramidal layer of CA1–3 regions in aged hippocampus. In the dentate gyrus of aged rats, significant decreases were also found in the granule cell layer and polymorphic layer. The first demonstration of age-related decreases in CBP expression in the cerebral cortex and hippocampus may provide useful data for investigating the pathogenesis of age-related neurodegenerative diseases and depression.

Exacerbation of Apoptosis in the Dentate Gyrus of the Aged Rat by Dexamethasone and the Protective Role of Corticosterone

Glucocorticoid-induced cell loss in the dentate gyrus of rats of various ages was studied using the TUNEL procedure to detect apoptotic cells. A highly significant increase in the incidence of apoptosis was observed within the dentate hilus and granule cell layer within 24 h of a single injection of dexamethasone (DEX) in rats aged between 1 and 36 months; DEX-induced apoptosis was more pronounced with increasing age. Corticosterone (CORT) did not cause an increase in the rate of apoptosis above that found in age-matched controls. However, CORT pretreatment (3 h) resulted in a significantly attenuated DEX-induced apoptosis in both areas of the dentate gyrus. Serum CORT levels in saline-treated rats peaked at 6 months of age and reached a nadir at 36 months of age. The results indicate that (i) aged subjects are more susceptible to DEX in terms of dentate gyrus cell loss by apoptosis, (ii) CORT, which binds to Type I corticosteroid receptors with a high affinity, might serve to protect against the damaging effects of DEX which is a ligand of the Type II glucocorticoid receptor, and (iii) declining endogenous levels of CORT may increase the vulnerability of the dentate gyrus of aged rats to insult by DEX.

Alterations in opiate receptor binding in the hippocampus of aged Long-Evans rats

Quantitative in vitro autoradiography was used to examine [ 3H] d-Ala 2, McPhe 4, Gly-01 5 enkephalin (DAGO) (μ-agonist) and [ 3H]diprenorphine (general opiate antagonist) binding sites in the hippocampal formation of young (6–8 months) and aged (25–28 months) Long-Evans rats. Age-related changes in these binding sites were restricted to specific regions but were not generally dependent on the ligand used. No reliable age-related changes in opiate binding were observed in the CA1 field or subicular region. In contrast, a decrease in the density of binding was found in both dorsal and ventral hippocampus within the CA3 field of aged brains. An age-related decrease in opiate binding within the dentate gyrus differed in its topography at dorsal and ventral levels of the hippocampus. A uniform decrease of opiate receptor binding was found throughout the dorsal dentate gyrus, while a more localized decrease of these sites occurred in hilar and granular layers of the ventral dentate gyros. These results indicate that a decrease of opiate binding in the hippocampal formation is largely localized to the CA3 region and dentate gyrus of aged rats. These findings are discussed with reference to age-related changes in hippocampal pathways containing opioid peptides. The implications for hippocampal opioid function in learning and age-related cognitive decline are also considered.

Effect of 5-hydroxytryptophane on behavior and hippocampal physiology in young and old rats

Spatial memory ability, tested in a water maze, was severely impaired in control, 24-month-old hooded rats. A daily injection of the serotonin precursor, 5-hydroxytryptophane (5-HTP), prior to training sessions, had no effect on the behavior of the young rats but improved considerably the performance of the old rats in the watermaze. In the same groups of young and aged rats, the response properties of the hippocampal dentate gyrus to perforant path stimulation was assessed before and after parenteral administration of 5-HTP. The dentate gyrus of aged rats produces a smaller EPSPs in response to perforant path stimulation but a larger population spike for a given EPSP than that produced in young rat brains. These differences are not affected by 5-HTP. In young brains, priming commissural stimulation suppresses subsequent reactivity to perforant path stimulation. This priming effect is nearly absent in aged rat hippocampus but reappears when the rat is injected with 5-HTP. It is suggested that the serotonergic innervation of the rat hippocampus plays a major role in regulation of the excitability of the hippocampus and in behavioral functions associated with this structure.

Synaptic plasticity in the dentate gyrus of aged rats is altered after chronic nimodipine application

We examined ultrastructural correlates of synaptic plasticity in the hippocampus of young (3 months) vs aged (30 months) Wistar rats and established the effects of the calcium antagonist nimodipine in animals chronically treated from 24 to 30 months. The effects of nimodipine was studied since this compound improves hippocampal neuronal physiology and enhances cognitive function during aging. In the supragranular layer of the dentate gyrus we found a 24% decrease in synaptic density (Nv) in aged animals, while synaptic size (S)_was not significantly alatered. After nimodipine treatment Nv in aged rats was not significantly different from young adults, thus being significantly increased conpared to age-matched controls. The size of synapses was not significantly altered after nimodipine administration. Total synaptic surface area (Sv) in nimodipine-treated animals was significantly increased compared to aged controls, however, Sv remained significantly lower than in young adults. These data indicate that chronic administration of nimodipine enables granular cells in the dentate gyrus to maintain its number of synaptic contacts during the aging process. Furthermore, the presented influence of nimodipine on synaptic plasticity processes may underlie previously reported improved cognitive functioning of aged animals treated similarly with nimodipine.

Aged-Related Changes in Learning and Memory, Choline Acetyltransferase Activity and Number of Neuronal Cells in Rats.

In a water maze task, the goal latency and distance of swimming onto the platform of aged rats (24 months old) were slowly shortened by repeated training compared with those of young rats (8 weeks old). A significant decrease in choline acetyltransferase activity in the frontal cortex, parietal cortex and striatum was observed in aged rats. Moreover, the number of neuronal cells in the hippocampal CA1 subfield and dentate gyrus of aged rats was smaller than that of young rats. The atrophy of striatal cells was observed. These results suggest that age-related delay of acquisition is due to the above-mentioned biochemical and histological changes, and that rates of aging in biochemical and morphological parameters are different in the discrete brain areas.

The effect of chronic hydergine treatment on the plasticity of synaptic junctions in the dentate gyrus of aged rats.

The number of synapses (Nv), the surface density of contact zones (Sv) as well as the average size (S) of E-PTA stained synapses in the supragranular layer of the dentate gyrus from adult (12 months), old (30 month), and Hydergine-treated old (30 months) rats were measured by using quantitative morphometric techniques. In old animals, Nv and Sv were significantly reduced, whereas S was significantly increased as compared with the values in adult rats. Hydergine (Codergocrine mesylate) treatment of old animals (3 mg/Kg/day for 4 weeks) influenced these three parameters, differentially. The Sv in aged animals receiving Hydergine, relative to that in untreated old rats, was significantly increased; the number and size of synapses in the treated old rats were significantly higher and smaller, respectively, than that in old controls. We interpret the present findings to indicate a modulating effect of Hydergine on the morphological plasticity of synaptic junctions in the dentate gyrus of aged rats.

Stability of synaptic density and spine volume in dentate gyrus of aged rats

The number of synapses per unit volume and per granule cell and the size of dendritic spines were studied in the dentate gyrus of Sprague-Dawley rats 6, 24, and 30 months of age. Neither synaptic density nor mean spine volume showed any age-related trends. An increase in granule cell packing density at 24 months and concomitant stability of the height of the granule cell layer is consistent with the idea that postnatal generation of granule cells may continue late into life. Possible explanations for the discrepancies in the literature regarding synaptic loss in this area include differences in morphometric techniques, age of animals used, regional differences within dentate gyrus, and sampling variability. Generalized synapse loss in the senescent rodent brain remains to be established.

Lesion‐induced synaptogenesis in the dentate gyrus of aged rats: II. Demonstration of an impaired degeneration clearing response

Previously we reported that a delayed onset in the reinnervation of the outer two-thirds of the dentate molecular layer occurred in aged rats after an entorhinal lesion. Several factors associated with formation of new synaptic contacts and removal of degenerative debris may affect the reinnervation process. In this study the appearance and removal of degeneration was analyzed and evaluated with respect to the delayed reinnervation process in aged rats. After a complete lesion of the entorhinal cortex, 85-90% of the input to the outer two-thirds of the ipsilateral molecular layer is lost. Electron-dense and electron-lucent degeneration are present throughout the outer two-thirds of the denervated molecular layer. In both aged and young adult rats, the electron-lucent degeneration disappears by 10 days postlesion. The predominant electron dense degeneration, however, is removed at a different rate by young adult and aged rats. Young adults demonstrate a biphasic degeneration removal process, with almost half of this degeneration rapidly lost by 10 days postlesion, and nearly all by 60 days postlesion. Aged animals in contrast, have lost only 16% of the dense degeneration at 10 days postlesion, with about 30% of the degeneration remaining at 60 days postlesion. The impaired removal of the degeneration from the denervated zone appears to be reciprocally related to the reinnervation response in both age groups and may be related to the normal astrocyte hypertrophy and elevated corticosteroid levels in aged rats.

Lesion-induced synaptogenesis in the dentate gyrus of aged rats: I. Loss and reacquisition of normal synaptic density.

Quantitative electron microscopy was used to examine the ability of aged (2-year-old) and young adult (90-day-old) rats to replace those synapses lost (85-90%) in the outer two-thirds of the molecular layer of the dentate gyrus after a complete unilateral lesion of the entorhinal cortex. In aged rats the synaptic density is significantly lower than that of young adults at 10 days postlesion. Synaptic replacement begins between 2 and 4 days postlesion in young adults, whereas there is a delay until after 10 days postlesion in aged rats. Once synapse replacement begins in aged rats, the rate of synapse reappearance is about equal that of young adults. Thus the initial 10 days postlesion appears critical to growth of responding afferents and reformation of synaptic contacts. Analysis of synapses in terms of noncomplex and complex synaptic types shows that the noncomplex type accounts for the significant synaptic density difference between the two age groups. Replacement of complex synapses is nearly indistinguishable between age groups and is complete by 60 days postlesion. In contrast the initial replacement rate of noncomplex synapses in aged rats is much slower than young adults, though the control synaptic density is achieved by the end of the time course.

Loss of axon terminals contacting neuronal somata in the dentate gyrus of aged rats

Loss of axon terminals contacting neuronal somata in the dentate gyrus of aged rats