Judy K. Brunso-Bechtold

Caloric restriction and age affect synaptic proteins in hippocampal CA3 and spatial learning ability.

Caloric restriction (CR) is a daily reduction of total caloric intake without a decrease in micronutrients or disproportionate reduction of any one dietary component. CR can increase lifespan reliably in a wide range of species and appears to counteract some aspects of the aging process throughout the body. The effects on the brain are less clear, but moderate CR seems to attenuate age-related cognitive decline. Thus, we determined the effects of age and CR on key synaptic proteins in the CA3 region of the hippocampus and whether these changes were correlated with differences in behavior on a hippocampal-dependent learning and memory task. We observed an overall, age-related decline in the NR1, N2A and N2B subunits of the N-methyl-d-aspartate (NMDA)-type and the GluR1 and GluR2 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA)-type ionotropic glutamate receptors. Interestingly, we found that CR initially lowers the glutamate receptor subunit levels as compared to young AL animals, and then stabilizes the levels across lifespan. Synaptophysin, a presynaptic vesicle protein, showed a similar pattern. We also found that both CR and ad libitum (AL) fed animals exhibited age-related cognitive decline on the Morris water maze task. However, AL animals declined between young and middle age, and between middle age and old, whereas CR rats only declined between young and middle age. Thus, the decrease in key synaptic proteins in CA3 and cognitive decline occurring across lifespan are stabilized by CR. This age-related decrease and CR-induced stabilization are likely to affect CA3 synaptic plasticity and, as a result, hippocampal function.

Age and insulin-like growth factor-1 modulate N-methyl-D-aspartate receptor subtype expression in rats.

N-Methyl-D-aspartate (NMDA) receptors have been reported to have an important role in synaptic plasticity and neurodegeneration. Two major subtypes of these receptors, NMDAR1 and NMDAR2, are present in brain and heterogeneity of these receptors have been reported to define specific functional responses. In this study, the effects of age and chronic insulin-like growth factor-1 (IGF-1) administration on NMDA receptor density and subtype expression were investigated in frontal cortex, CA1, CA2/3 and the dentate gyrus of the hippocampus of young (10 months), middle-aged (21 months) and old (30 months) male Fisher 344xBrown Norway (F1) rats. No age-related changes in (125)I-MK-801 binding or NMDAR1 protein expression were observed in hippocampus or frontal cortex. However, analysis of NMDAR2A and NMDAR2B protein expression in hippocampus indicated a significant decrease between 21 and 30 months of age and administration of IGF-1 increased these receptor subtypes. In cortex, NMDAR2A and NMDAR2B protein expression were not influenced by age or IGF-1 treatment, although NMDAR2C protein expression decreased with age and this decline was not ameliorated by IGF-1 administration. These data demonstrate that NMDA receptor subtypes are altered with age in a regional and subtype specific manner. We conclude that both age and IGF-1 regulate the expression of NMDA receptor subtypes and suggest that age-related changes in NMDA receptor heterogeneity may result in functional changes in the receptor that have relevance for aging.

Alterations in Insulin-like Growth Factor-1 Gene and Protein Expression and Type 1 Insulin-like Growth Factor Receptors in the Brains of Ageing Rats

Ageing in mammals is characterized by a decline in plasma levels of insulin-like growth factor-1 that appears to contribute to both structural and functional changes in a number of tissues. Although insulin-like growth factor-1 has been shown to provide trophic support for neurons and administration of insulin-like growth factor-1 to ageing animals reverses some aspects of brain ageing, age-related changes in insulin-like growth factor-1 or type 1 insulin-like growth factor receptors in brain have not been well documented. In this series of studies, insulin-like growth factor-1 messenger RNA and protein concentrations, and type 1 insulin-like growth factor receptor levels were analysed in young (three to four- and 10-12-month-old), middle-aged (19-20-month-old) and old (29-32-month-old) Fisher 344 x Brown Norway rats. Localization of insulin-like growth factor-1 messenger RNA throughout the lifespan revealed that expression was greatest in arteries, arterioles, and arteriolar anastomoses with greater than 80% of these vessels producing insulin-like growth factor-1 messenger RNA. High levels of expression were also noted in the meninges. No age-related changes were detected by either in situ hybridization or quantitative dot blot analysis of cortical tissue. However, analysis of insulin-like growth factor-1 protein levels in cortex analysed after saline perfusion indicated a 36.5% decrease between 11 and 32 months-of-age (P<0.05). Similarly, analysis of type 1 insulin-like growth factor receptor messenger RNA revealed no changes with age but levels of type 1 insulin-like growth factor receptors indicated a substantial decrease with age (31% in hippocampus and 20.8 and 27.3% in cortical layers II/III and V/VI, respectively). Our results indicate that (i) vasculature and meninges are an important source of insulin-like growth factor-1 for the brain and that expression continues throughout life, (ii) there are no changes in insulin-like growth factor-1 gene expression with age but insulin-like growth factor-1 protein levels decrease suggesting that translational deficiencies or deficits in the transport of insulin-like growth factor-1 through the blood-brain barrier contribute to the decline in brain insulin-like growth factor-1 with age, and (iii) type 1 insulin-like growth factor receptor messenger RNA is unchanged with age but type 1 insulin-like growth factor receptors decrease in several brain regions. We conclude that significant perturbations occur in the insulin-like growth factor-1 axis with age. Since other studies suggest that i.c.v. administration of insulin-like growth factor-1 reverses functional and cognitive deficiencies with age, alterations within the insulin-like growth factor-1 axis may be an important contributing factor in brain ageing.

Caloric restriction eliminates the aging-related decline in NMDA and AMPA receptor subunits in the rat hippocampus and induces homeostasis

Caloric restriction (CR) extends life span and ameliorates the aging-related decline in hippocampal-dependent cognitive function. In the present study, we compared subunit levels of NMDA and AMPA types of the glutamate receptor and quantified total synapses and multiple spine bouton (MSB) synapses in hippocampal CA1 from young (10 months), middle-aged (18 months), and old (29 months) Fischer 344xBrown Norway rats that were ad libitum (AL) fed or caloric restricted (CR) from 4 months of age. Each of these parameters has been reported to be a potential contributor to hippocampal function. Western blot analysis revealed that NMDA and AMPA receptor subunits in AL animals decrease between young and middle age to levels that are present at old age. Interestingly, young CR animals have significantly lower levels of glutamate receptor subunits than young AL animals and those lower levels are maintained across life span. In contrast, stereological quantification indicated that total synapses and MSB synapses are stable across life span in both AL and CR rats. These results indicate significant aging-related losses of hippocampal glutamate receptor subunits in AL rats that are consistent with altered synaptic function. CR eliminates that aging-related decline by inducing stable NMDA and AMPA receptor subunit levels.

Development of afferent patterns in the inferior colliculus of the rat: Projection from the dorsal nucleus of the lateral lemniscus

The inferior colliculus (IC) receives a variety of layered afferent projections. The purpose of the present study was to determine the development of the projection from the dorsal nucleus of the lateral lemniscus (DNLL) to the IC in rat prior to the onset of hearing (postnatal day 12/13). Lipophilic carbocyanine dye, DiI (1,1′‐dioctodecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate), was used to trace the crossed inhibitory projection of DNLL in a developmental series of rat embryos and pups between ages embryonic day 15 (E15) and postnatal day 12 (P12). Dye‐coated pins were positioned in paraformaldehyde‐fixed brains either unilaterally in DNLL (embryonic cases), or in the commissure of Probst where DNLL fibers cross the midline (postnatal cases). By E15, pioneer fibers have left DNLL and crossed the midline. A few fibers have nearly reached the contralateral IC by E19. At birth (E22 = P0), the projection has invaded ventromedial, high‐frequency layers of the IC. The vast majority of DNLL axons parallel the presumptive IC layers by P4, and by P8 the projection has segregated into a pattern of bands (afferent dense) and interband (afferent sparse) spaces that encompasses the entire frequency axis of the IC. Adult‐like patches, regions along afferent bands that exhibit the heaviest labeling, develop by P12. These results indicate that some mature projection patterns are in place prior to the onset of hearing. Such findings suggest that evoked activity may not be required for the initial organization of patterned projections in the ascending auditory pathway. J. Comp. Neurol. 416:368‐382, 2000.

Age-related decline of presumptive inhibitory synapses in the sensorimotor cortex as revealed by the physical disector.

The synapse, as the site of functional neural interaction, has been suggested as a possible substrate for age‐related impairment of cognitive ability. Using the physical disector probe with tissue prepared for ultrastructural analysis, we find an age‐related decline in the numerical density of presumptive inhibitory synapses in layer 2 of the sensorimotor cortex of the Brown Norway × Fisher 344 rat. This age‐related decline in presumptive inhibitory synapses is maintained when the density of synapses is combined with the numerical density of neurons quantified from the same anatomical space to arrive at a ratio of synapses per neuron. The numerical density of these synapses declines between middle‐aged (18 months) and old (29 months) animals by 36% whereas numerical density of neurons does not change between these ages, resulting in a decline in the ratio of presumptive inhibitory synapses per neuron in this cortical area. This study demonstrates a deficit in the intrinsic inhibitory circuitry of the aging neocortex, which suggests an anatomical substrate for age‐related cognitive impairment. J. Comp. Neurol. 439:65–72, 2001.

Imaging Radiation-Induced Normal Tissue Injury

Technological developments in radiation therapy and other cancer therapies have led to a progressive increase in five-year survival rates over the last few decades. Although acute effects have been largely minimized by both technical advances and medical interventions, late effects remain a concern. Indeed, the need to identify those individuals who will develop radiation-induced late effects, and to develop interventions to prevent or ameliorate these late effects is a critical area of radiobiology research. In the last two decades, preclinical studies have clearly established that late radiation injury can be prevented/ameliorated by pharmacological therapies aimed at modulating the cascade of events leading to the clinical expression of radiation-induced late effects. These insights have been accompanied by significant technological advances in imaging that are moving radiation oncology and normal tissue radiobiology from disciplines driven by anatomy and macrostructure to ones in which important quantitative functional, microstructural, and metabolic data can be noninvasively and serially determined. In the current article, we review use of positron emission tomography (PET), single photon emission tomography (SPECT), magnetic resonance (MR) imaging and MR spectroscopy to generate pathophysiological and functional data in the central nervous system, lung, and heart that offer the promise of, (1) identifying individuals who are at risk of developing radiation-induced late effects, and (2) monitoring the efficacy of interventions to prevent/ameliorate them.

Early-onset GH deficiency results in spatial memory impairment in mid-life and is prevented by GH supplementation

GH levels increase to high concentrations immediately before puberty then progressively decline with age. GH deficiency (GHD) originating in childhood is treated with GH supplementation to foster somatic development during adolescence. It is not clear if or how early GH replacement affects memory in adulthood, or whether it can prevent the cognitive deficits commonly observed in adults with childhood-onset GHD. Rats homozygous for the Dw-4 mutation (dwarf) do not exhibit the normal increase in GH at 4 weeks of age when GH levels normally rise and are used to model childhood or early-onset GHD (EOGHD). One group of these rats was injected with GH from 4 to 14 weeks of age to model GH supplementation during adolescence with GHD beginning in adulthood (adult-onset GHD; AOGHD). Another group received GH from 4 weeks throughout the lifespan to model normal lifespan GH (GH-replete). Age-matched, Dw-4 heterozygous rats (HZ) do not express the dwarf phenotype and were used as controls. At 8 and 18 months of age, spatial learning in the water maze was assessed. At 8 months of age all experimental groups were equally proficient. However, at 18 months of age, the EOGHD group had poor spatial learning compared to the AOGHD, GH-replete, and HZ groups. Our data indicate that GHD during adolescence has negative effects on learning and memory that emerge by middle-age unless prevented by GH supplementation.

Caloric restriction does not reverse aging-related changes in hippocampal BDNF.

Caloric restriction (CR) can attenuate the aging-related decline in learning and memory in rats. Understanding the mechanisms underlying this effect could lead to therapies for human memory impairment. We tested the hypotheses that aging is associated with a decline in hippocampal brain-derived neurotrophic factor (BDNF), a growth factor that enhances learning and memory, and that CR increases hippocampal BDNF. We compared BDNF protein levels in hippocampal subregions of young, middle-aged and old rats fed CR or ad libitum (AL) diets. Mean BDNF levels in the dentate gyrus and CA3 did not differ with diet but increased with age. In CA1, BDNF levels were slightly higher in CR than AL rats at middle and old age but did not change across lifespan. These data suggest that mnemonic impairments with age do not reflect a decrease in hippocampal BDNF. Furthermore, if CRs attenuation of aging-related memory changes is mediated by BDNF, then it must be through a small, CA1-specific increase and does not involve reversal of an aging-related decline in BDNF.

Stereological quantification of GAD-67–immunoreactive neurons and boutons in the hippocampus of middle-aged and old Fischer 344 × Brown Norway rats

The aging process in rodents is associated with learning and memory impairments that are correlated with changes in multiple neurotransmitter systems in the hippocampus. For example, the γ‐aminobutyric acid (GABA)ergic system is compromised in old compared with young rats (Shetty and Turner [ 1998 ] J. Comp. Neurol. 394:252–269; Vela et al. [ 2003 ] J. Neurochem. 85:368–377; Potier et al. [ 1992 ] Neuroscience 48:793–806; Potier et al. [ 1994 ] Brain Res. 661:181–188). The present study investigated the important issue of whether there is a decline of the GABAergic inhibitory system between middle and old age. Five middle‐aged (15–17 months) and five old (25–29 months) Fischer 344 × Brown Norway male rats were perfused, and coronal sections through the dorsal hippocampus were immunoreacted with antibodies either to NeuN, a neuronal marker, or to the 67‐kDa isoform of glutamic acid decarboxylase (GAD), the rate‐limiting enzyme for GABA synthesis. Using the optical dissector technique, NeuN‐immunoreactive (IR) cells, GAD‐IR cells, and GAD‐IR boutons were quantified stereologically in the dentate gyrus, CA3, and CA1. The resulting GAD‐IR cell and GAD‐IR bouton densities then were normalized to NeuN‐IR cell density to exclude the possible confound of tissue shrinkage. The results revealed a significant decline in GAD‐IR cells between middle and old age in CA1 but not in dentate gyrus or CA3. Interestingly, GAD‐IR boutons did not show a decline in CA1, CA3, or dentate gyrus between middle and old age. It is possible that loss of CA1 inhibitory interneurons in the dorsal hippocampus contributes to the learning and memory impairments reported in old rats. J. Comp. Neurol. 478:282–291, 2004.