Kathy R. Magnusson

Isoflurane Differentially Affects Neurogenesis and Long-term Neurocognitive Function in 60-day-old and 7-day-old Rats

Background:Anesthetic agents cause cell death in the developing rodent brain and long-term, mostly hippocampal-dependent, neurocognitive dysfunction. However, a causal link between these findings has not been shown. Postnatal hippocampal neurogenesis affects hippocampal function into adulthood; therefore, the authors tested the hypothesis that isoflurane affects long-term neurocognitive function via an effect on dentate gyrus neurogenesis. Methods:The S-phase marker 5-bromodeoxyuridine was administered at various times before, during, and after 4 h of isoflurane given to postnatal day (P)60 and P7 rats to assess dentate gyrus progenitor proliferation, early neuronal lineage selection, and long-term survival of new granule cell neurons. Fear conditioning and spatial reference memory was tested at various intervals from 2 weeks until 8 months after anesthesia. Results:In P60 rats, isoflurane increased early neuronal differentiation as assessed by BrdU/NeuroD costaining, decreased progenitor proliferation for 1 day, and subsequently increased progenitor proliferation 5–10 days after anesthesia. In P7 rats, isoflurane did not induce neuronal lineage selection but decreased progenitor proliferation until at least 5 days after anesthesia. Isoflurane improved spatial reference memory of P60 rats long-term, but it caused a delayed-onset, progressive, persistent hippocampal deficit in P7 rats in fear conditioning and spatial reference memory tasks. Conclusion:The authors conclude that isoflurane differentially affects both neurogenesis and long-term neurocognitive function in P60 and P7 rats. Neurogenesis might mediate the long-term neurocognitive outcome after isoflurane at different ages.

Age-related changes in excitatory amino acid receptors in two mouse strains

The present study examined the binding of [3H]-L-glutamate to NMDA receptors, [3H]-kainate to kainate receptors, and [3H]-AMPA to AMPA/quisqualate receptors in the brains of C57Bl and BALB/c mice as a function of increasing age. Significant decreases in binding to NMDA receptors occurred with increasing age (3 to 30 months) in a majority of cortical and hippocampal brain regions from the C57Bl and BALB/c strains of mice. Significant decreases in binding to kainate and AMPA receptors were found in the inner frontal and parietal cortices and stratum lacunosum/moleculare of CA1 in both strains. These regions also exhibited the greatest percent decline in NMDA binding sites with aging. The loss of NMDA receptors in the stratum lacunosum/moleculare of CA1 was greater in the BALB/c mice than the C57Bl strain. These results demonstrate that a few brain regions have age-associated reductions in all three ionotropic EAA receptors; however, the NMDA receptor appears to be selectively vulnerable to the aging process throughout much of the cerebral cortex and hippocampus.

Aging of glutamate receptors: correlations between binding and spatial memory performance in mice.

C57B1/6 mice aged 3, 10, and 26 months were tested for spatial learning in the Morris water maze. Ten and 26 month old mice were ad libitum-fed or diet restricted (60% of ad libitum-fed calories). Diet restriction significantly improved memory performance among the 10 and 26 month olds. In age/diet group comparisons, aged ad libitum-fed mice had significantly higher average proximity scores, indicating poorer performance, in probe trials for place learning than the 3 month olds and diet restricted 10 month olds. Diet restricted 26 month olds did not differ significantly from 3 month olds or any other groups in probe trial measures. The group means for average proximity scores were significantly correlated with binding densities for the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors in the frontal cortex and CA1 region of the hippocampus. Alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA) binding correlated with group proximity scores in frontal and parietal cortices and within the CA1 and CA3 regions of the hippocampus. Kainate and metabotropic binding sites showed no significant correlations with behavior. These results suggest that there is a sparing of spatial memory with diet restriction in aging C57B1/6 mice and that the effects of aging on NMDA and AMPA receptors may be associated with age-related declines in spatial learning.

Age-related changes in the protein expression of subunits of the NMDA receptor.

C57Bl/6 mice show decreased expression of the mRNA for the epsilon2 and zeta1 subunits of the N-methyl-D-aspartate (NMDA) receptor in subregions of the cerebral cortex and hippocampus with increased age. The purpose of this study was to determine the effects of aging on the protein expression of the three major subunits of the NMDA receptor. Semi-quantitative Western blot techniques were applied with the use of antibodies that recognize either the epsilon1 (NR2A), epsilon2 (NR2B) or zeta1 (NR1) subunits of the NMDA receptor or a synaptic terminal protein, synaptophysin. In the cerebral cortex of 30-month-old mice, the level of protein expression of both the epsilon2 and zeta1 subunits were decreased significantly from levels found in the 3- and 10-month-old mice and the protein expression of the epsilon1 subunit showed a significant decline between 10 and 30 months of age. In the hippocampus, the epsilon2 subunit exhibited a higher protein expression level in the 10-month-old mice as compared to both the young and old mice and the zeta1 subunit showed a significant drop in expression in the old mice from both 3- and 10-month-olds. Synaptophysin showed significant declines in protein expression with increasing age. These results demonstrated that changes in the protein expression of the major subunits of the NMDA receptor occur during the aging process and, in some cases, were greater than changes seen previously in mRNA expression. These subunit alterations may explain some of the changes that are seen in NMDA receptor functions during aging.

The aging of the NMDA receptor complex.

N-methyl-D-aspartate (NMDA) receptors are present at high density in the cerebral cortex and hippocampus and play an important role in learning and memory. These receptors are negatively affected by the aging process, but this effect does not appear to be uniform throughout the cortex and hippocampus. This review discusses the age-associated changes that occur in the different binding sites of the NMDA receptor complex, in the expression of subunits that comprise the complex, in the electrophysiological properties of the receptor, and in the ability of NMDA to stimulate the release of other transmitters. Spatial memory and some types of passive avoidance memory tasks have been shown to involve NMDA receptors. Aged animals show deficiencies in the performance of these tasks, as compared to young, and some studies have identified an association between lower densities of NMDA receptor binding and poor memory performance. A number of drug and diet interventions have shown potential for reversing or slowing the effects of aging on the NMDA receptor. These studies suggest that the development of treatments that are aimed at preventing or reversing the effects of aging on the NMDA receptor will aid in preventing the memory declines that are associated with aging.

Relationships between diet-related changes in the gut microbiome and cognitive flexibility

Western diets are high in fat and sucrose and can influence behavior and gut microbiota. There is growing evidence that altering the microbiome can influence the brain and behavior. This study was designed to determine whether diet-induced changes in the gut microbiota could contribute to alterations in anxiety, memory or cognitive flexibility. Two-month-old, male C57BL/6 mice were randomly assigned high-fat (42% fat, 43% carbohydrate (CHO), high-sucrose (12% fat, 70% CHO (primarily sucrose) or normal chow (13% kcal fat, 62% CHO) diets. Fecal microbiome analysis, step-down latency, novel object and novel location tasks were performed prior to and 2weeks after diet change. Water maze testing for long- and short-term memory and cognitive flexibility was conducted during weeks 5-6 post-diet change. Some similarities in alterations in the microbiome were seen in both the high-fat and high-sucrose diets (e.g., increased Clostridiales), as compared to the normal diet, but the percentage decreases in Bacteroidales were greater in the high-sucrose diet mice. Lactobacillales was only significantly increased in the high-sucrose diet group and Erysipelotrichales was only significantly affected by the high-fat diet. The high-sucrose diet group was significantly impaired in early development of a spatial bias for long-term memory, short-term memory and reversal training, compared to mice on normal diet. An increased focus on the former platform position was seen in both high-sucrose and high-fat groups during the reversal probe trials. There was no significant effect of diet on step-down, exploration or novel recognitions. Higher percentages of Clostridiales and lower expression of Bacteroidales in high-energy diets were related to the poorer cognitive flexibility in the reversal trials. These results suggest that changes in the microbiome may contribute to cognitive changes associated with eating a Western diet.

The effects of aging on N-methyl-d-aspartate receptor subunits in the synaptic membrane and relationships to long-term spatial memory

There are declines in the protein expression of the NR2B (mouse epsilon2) and NR1 (mouse zeta1) subunits of the N-methyl-D-aspartate (NMDA) receptor in the cerebral cortex and hippocampus during aging in C57BL/6 mice. This study was designed to determine if there is a greater effect of aging on subunit expression and a stronger relationship between long-term spatial memory and subunit expression within the synaptic membrane than in the cell as a whole. Male, C57BL/6JNIA mice (4, 11 and 26 months old) were tested for long-term spatial memory in the Morris water maze. Frontal cortex, including prefrontal regions, and hippocampus were homogenized and fractionated into light and synaptosomal membrane fractions. Western blots were used to analyze protein expression of NR2B and NR1 subunits of the NMDA receptor. Old mice performed significantly worse than other ages in the spatial task. In the frontal cortex, the protein levels of the NR2B subunit showed a greater decline with aging in the synaptic membrane fraction than in the whole homogenate, while in the hippocampus a similar age-related decline was observed in both fractions. There were no significant effects of aging on the expression of the NR1 subunit. Within the middle-aged mouse group, higher expression of both NR2B and NR1 subunits in the synaptic membrane of the hippocampus was associated with better memory. In the aged mice, however, higher expression of both subunits was associated with poorer memory. These results indicate that aging could be altering the localization of the NR2B subunit to the synaptic membrane within the frontal cortex. The correlational results suggest that NMDA receptor functions, receptor subunit composition, and/or the environment in which the receptor interacted in the hippocampus were not the same in the old animals as in younger mice and this may have contributed to memory declines during aging.

Effects of aging on NMDA and MK801 binding sites in mice

[3H]MK801 binding was significantly reduced in three cortical and two subcortical regions in 30-month-old C57Bl mice, as compared to 3-month-olds. NMDA binding sites showed significant reductions with aging in sixteen of nineteen brain regions. These results suggest that, in a majority of cortical and hippocampal regions, decreases in binding to NMDA sites with aging may be due to factors other than cell loss.

Selective Vulnerabilities of N-methyl-D-aspartate (NMDA) Receptors During Brain Aging.

N-methyl-D-aspartate (NMDA) receptors are present in high density within the cerebral cortex and hippocampus and play an important role in learning and memory. NMDA receptors are negatively affected by aging, but these effects are not uniform in many different ways. This review discusses the selective age-related vulnerabilities of different binding sites of the NMDA receptor complex, different subunits that comprise the complex, and the expression and functions of the receptor within different brain regions. Spatial reference, passive avoidance, and working memory, as well as place field stability and expansion all involve NMDA receptors. Aged animals show deficiencies in these functions, as compared to young, and some studies have identified an association between age-associated changes in the expression of NMDA receptors and poor memory performance. A number of diet and drug interventions have shown potential for reversing or slowing the effects of aging on the NMDA receptor. On the other hand, there is mounting evidence that the NMDA receptors that remain within aged individuals are not always associated with good cognitive functioning. This may be due to a compensatory response of neurons to the decline in NMDA receptor expression or a change in the subunit composition of the remaining receptors. These studies suggest that developing treatments that are aimed at preventing or reversing the effects of aging on the NMDA receptor may aid in ameliorating the memory declines that are associated with aging. However, we need to be mindful of the possibility that there may also be negative consequences in aged individuals.

Increased inflammatory response in aged mice is associated with age-related zinc deficiency and zinc transporter dysregulation☆

Aging is a complex process associated with physiological changes in numerous organ systems. In particular, aging of the immune system is characterized by progressive dysregulation of immune responses, resulting in increased susceptibility to infectious diseases, impaired vaccination efficacy and systemic low-grade chronic inflammation. Increasing evidence suggest that intracellular zinc homeostasis, regulated by zinc transporter expression, is critically involved in the signaling and activation of immune cells. We hypothesize that epigenetic alterations and nutritional deficits associated with aging may lead to zinc transporter dysregulation, resulting in decreases in cellular zinc levels and enhanced inflammation with age. The goal of this study was to examine the contribution of age-related zinc deficiency and zinc transporter dysregulation on the inflammatory response in immune cells. The effects of zinc deficiency and age on the induction of inflammatory responses were determined using an in vitro cell culture system and an aged mouse model. We showed that zinc deficiency, particularly the reduction in intracellular zinc in immune cells, was associated with increased inflammation with age. Furthermore, reduced Zip 6 expression enhanced proinflammatory response, and age-specific Zip 6 dysregulation correlated with an increase in Zip 6 promoter methylation. Furthermore, restoring zinc status via dietary supplementation reduced aged-associated inflammation. Our data suggested that age-related epigenetic dysregulation in zinc transporter expression may influence cellular zinc levels and contribute to increased susceptibility to inflammation with age.